Home

Applied Biosystems 3500/3500xL Genetic Analyzer User Guide (PN

1. Setup Define Plate Properties Sample Filename Asy Name PAProtocolName Basg ff Version MoBjifFie E assign Piete Contents aa RE d i ns Entei BDTv3 1 PA Protoc B 14 18 KB 3500 POP7 BD AA Smpl BDTv3 1 PA Protocol POPT 24 AA Seq Assay KB 3500 POP7 BD 1 2 Run instrument AA Smpl BDTv3 1 PA Protocol POP4 AA Seq Assay KB 3500 POP7 BD 17 LORA PIRES ORIN AA Smpl BDTv3 1 PA Protocol PGPT 9 AA Seq Assay s KB_3500_POP7_BD 1 RM AA Seq Assay pe KB 14 18 KB 3500 POP7 BD J dii AA Seq Assay KB LAL8 KB_3500_POP7_BD 1 TENE Monitor Run KBLALS KB 3500 POP7 BD li Review Fans ABT v3 1 PAProtoc KB L4 18 KB 3500 POP7 BD BDTv3 1 PA Protoc KB 1 4 1 8 KB 3500 POP7 BD View Sequencing Results bets BDTv3 1 PA Protoc KB 1 4 1 8 KB 3500 POP7 BD View Fragment HID Results pq Assay BDTv3 1 PA Protoc KB 1 4 1 8 KB 3500 POP7 BD hs AA Seq Assay BDTv3 1 PA Protoc KB 1 4 1 8 KB 3500 POP7 BD 17 r SS MM HB OAR Ed aw di gt Tokyo lp PP Oo LI A o w N N nor ooo 420 490 560 630 700 770 CAACACTGCA 840 201 Navigate the views here d a y a AGTATTTA 4 Analyzed Raw Analyzed Raw Annotation Sequence EPT i 276 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Auto analysis with MicroSegO ID
2. 48 Assign assay file name convention and results group in the Plate View 49 PAINTING plate da yQU sariari cenut ie Dp ERE HIP RUE GP ade dot dete dug qi dte dass iri pus 50 Prepare and load sample plates 0 0 0 eee tenes 51 Prepare sample plates nerd sd ae Be SAR ee Re ee C acd 52 Prepare the plate assembly 00 cc ce ee eee 53 Load the plate in the instrument 0000 cc eee 53 Check InStrument StalUS uad acia eos a wa ga ce 53 Hate late ase andes ate aaa aaa aaa a 54 QUICK Start TUN A E e A week aid 55 Load plates for run and create the injection list o ooooooooooooo 56 Review and modify the injection list in Preview Run o oo oocoonoono noo 59 Start the TU basal erase o en in da edo d enr 61 MONMOF TS TUN caaea paina e A Rn de De de Bo A ed M en A Be 61 Check sequence or sample quality and specify re injections 62 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Contents Check sequence or sample quality 0 0 0 cc eee ee ee eee eae 62 Specify TE INJSCTIONS ia ans e ROT Co oe wae ORC a ac a CR TR E an ae 65 Review completed injections in Review Results 0 000 ees 69 Stake SMG Stob AUN ASAS AAA eae oe Se Se X arg ies 69 More features in Assign Plate Contents 0 000 cee eee eee 70 WSe ine Plate VIEW uu vac ps diei rig A hee paraiso heen ad ewes 4 70 Use the Table VIEW xs uos ded eaa
3. Select Table PENA MET ue Select Analysis Results Run Information Data Collection Information E Metrics Analysis Results b i Trace Identification 2 Click the Table Settings button then specify the columns to show or hide 3 Double click column headers to sort columns Multi column sorting is supported see Sort on page 97 4 Review the results Select your table display preferen Available Columns to Display Result Description Trace Score The average basecall quality value QV of bases in the clear range sequence of a trace The clear range is the region of the sequence that remains after excluding the low quality or error prone sequence at the o and 3 ends The clear range is calculated by the KB basecaller using QVs CRL QV20 Trace Score Quality CRL Quality QV20 Quality PUP Score 5 Review warnings The longest uninterrupted segment of bases with a Quality Value QV 20 In addition to evaluating the QV of a base call the software considers the QV of adjacent bases within 20 bases before including a base in the continuous read length The total number of bases in the entire trace that have basecaller quality values equal to or greater than 20 Pass fail check determined by the settings in the Basecalling protocol QV Settings tab A measure of noise as calculated as the ratio of the fluorescence signal of the highest seco
4. Filter 4 iit Note You can set the default plate type for this filter in Preferences See Specify the default plate type for the Open Plate dialog box on page 76 b Find templates by selecting an attribute entering the text to search for then clicking Go Click Clear to clear the field and enter different search criteria 3 Select the template then click Open 4 In the Define Plate Properties screen select the plate type Plate Details Mame Enter plate name Number of Wells 96 96 FastTube 2384 Capillary Length cm Polymer IPOP e 96 Supports 96 well standard reaction plate 8 strip standard tubes are also supported with appropriate retainers e 96 Fast Tube Supports 96 well Fast reaction plate 8 strip fast tubes are also supported with appropriate retainers 5 Setremaining plate properties then select Save 6 Click Assign Plate Contents then go to Assign plate contents on page 46 Import a plate 1 Do either of the following Create a plate on another 3500 Series Data Collection Software system then export see Import and export a plate on page 75 Create a plate import file see Create a plate import file on page 74 44 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Create a plate 2 Access the Assign Plate Contents screen Click the iB Main workflow arrow in the Dashboard then ile sele
5. 6 Name your new fragment analysis protocol and optionally enter a description Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 285 Appendix C Secondary Analysis Fragment 7 Select the panel s you previously created in GeneMapper then click Apply to Assay Setup a GeneMapper Protocol Protocol Name Your Secondary Analysis Protocol F Locked Description Application Type Fragment Analysis Secondary Analysis Software GeneMapper Secondary Analysis Software Instance GeneMapper 3500 FVTEST 4 Properties Size Standard GS500 35 250 340 ROX SNPlex_48plex_Panel_3130 Panel11 HD5 V2 5 3 Panel02 HD5 V2 5 a Panel62 HD5 V2 5 Apply to Assay Save to Library Panel53 HD5 V2 5 bg Note For more instruction on setting up a secondary analysis protocol see Create a new fragment analysis protocol on page 193 8 Click Close when you are finished applying all the panels to the assay 9 Click Apply to Plate then close the Setup an Assay dialog box Actions Y Your Fragment Assay A El 286 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Set up a GeneMapper plate in the 3500 Series Data Collection Software 10 Name your samples by highlighting the number of wells in your plate and naming the sample in Customize Sample Info box Fi Plate View BE Table View Show In Wells
6. Chemical safety General chemical safety Chemical hazard N WARNING CHEMICAL HAZARD Before handling any chemicals refer warning to the Material Safety Data Sheet MSDS provided by the manufacturer and observe all relevant precautions WARNING CHEMICAL HAZARD All chemicals in the instrument including liquid in the lines are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument Chemical safety To minimize the hazards of chemicals guidelines 328 Read and understand the Material Safety Data Sheets MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About MSDSs on page 329 Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the MSDS Comply with all local state provincial or natio
7. 83 Chapter 4 Review Results Understand Quality Values QVs Quality value ranges Pure base versus mixed base QVs Quality values QV and probability of error Pe Display thumbnails 84 Applied Biosystems recommends the following ranges for QVs set in Preferences see Set sequencing preferences on page 36 Pure bases Low QV 15 Medium QV 15 to 19 High QV 20 default Mixed bases Low QV 5 Medium QV 5 to 10 High QV gt 10 investigate to determine the best range for your application Note The predicted probability of error for a basecall is high QV gt 10 Note You can set the software to trim set the clear range using quality values in the basecalling protocol see Basecalling protocols library primary analysis sequencing on page 174 Pure bases and mixed bases have the same probability of error for the associated basecall 10 19 Note the following e High quality pure bases typically have QVs of 20 or higher The distribution of quality values for mixed bases differs dramatically from that of pure bases e For mixed bases quality values greater than 30 are rare Good mixed bases may be assigned quality values as low as 5 because the probability of error with mixed bases is higher Review mixed bases with QVs between 5 and 10 QV Pe QV Pe 1 79 0 30 0 10 o 32 0 35 0 032 10 10 0 40 0 010 15 3 2 45 0 0032 20 1 0 50 0 0010 25
8. ia Read Length Start Start Run stocker of Wels 06 1 96 FastTubs 384 Plate Postion HA OB gs Read Length End In Creep Spectral Calibration Data PR ae PE mia Capillary Run Data o Spectral Calibration Run Contiguous Ford Length CAL CRL Pass Fail Comparison with Ref Sequence CRL Basepalr Accuracy Basepar Accuracy Read Length Bl Passed B ru Borrowed Not Calibrated Quality Value Condition F Status Message Intensity vs Scan Number Ri Dista P ELEJE 3 3 B ES ES EE 3000 LR 1206 Lon LLE 000 ZODUU 32000 Inter s Scan Hierbas bk Sequence Comparison to Sample Capillary 1 k Intensity va Piel Number Figure 9 Sequencing Install Standard screen Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Spectral calibration 4 Optional If you have not already run a spectral calibration select Keep Spectral Calibration Data to save the sequencing install standard run if it passes as a spectral calibration General Sequencing with BDTv3 1 Install standard and POP 7 polymer generates a Z dye set spectral calibration Note he spectral calibration record will only be saved if Keep Spectral Calibration Data option 1s checked on the screen If you decide to uncheck the option create a separate spectral calibration from the Maintenance menu e MicroSeq with BDTv1 1 Install Standard and POP 6 polymer generates an E dye set spectral calibration 5 Click Start Run
9. 2 Create a plate Select one of the following topics e Create a new plate on page 144 Create a plate from a template on page 43 e Import a plate on page 44 e Orselect Open Plate gt Edit Existing Plate 3 Click Show In Wells to specify the attributes to display ENS E Show In Wells s aj in wells Le Assay Mame a Figure 5 on page 47 shows the Plate View of the Assign Plate w Assay Color L w Assay Icon Results Group Marne Results Group Color Contents screen File Convention Marne File Convention Color Sample Mame Sample Type Well Position 46 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Assign plate contents Show well attributes E Show In Wells Select Wells Array Selection E Zoom In FE Zoom Cut F E Row Column Customize Sample Info Property Assays E 1 Sample Assign sample types and user defined fields Name samples Assign assays file Name Barcode name conventions actions 7 Sample Mame Sample Type Sample amp Std Seq Assay xL POP A El El 2 Custom User Defined Fie User Defined Fie lil Link the plate and results groups Figure 5 Assign Plate View of the Assign Plate Contents screen Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 47 Chapter 3 Set Up and Run Name samples and assign sample types in the plate view This section provides
10. Actions 7 Action e X FMC A EJ e v v Sequencing Results Gro A El is 4 Select Save Plate 5 Go to Print the plate layout on page 50 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 49 Chapter 3 Set Up and Run How file location If you do not specify a file name convention data files are named in this format in file name lt sample name well conventions and results groups work If you do not specify a results group files are stored in the location specified in the file name convention or in Preferences User gt Run see User preferences on page 34 If you specify both a file name convention and a results group files are stored in the location specified in the results group Print the plate layout 1 In the Assign Plates for Run screen click View Plate Grid Report um gu kis J Find Replace gt sie Plate Grid Report ea Pi fa ch oe C lS r i TI Plate View ES Table view Page lofl 346 amr v E Es zd S B qu Applied Report Created on 11 Jan 2009 01 2200 PM B zi E im Biosystems 3500 Instrument Plate Grid Report Plate Details Plae Name Sequencing Plate 1 Owner Number of Wells 96 Barcode Plae Type Sequencing Capillary Length 50 Palymer POP Note A 384 well report displays the plate layout in four quadrants on four pages 2 Select Print Preview or Print as needed 3 To save the report electronic
11. Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 213 Section 1 Administrators system Configuration History Summary Report H Date User Name User Full Name Record Type Record Name Action 1 28 Jan 2009 05 01 08 Administrator Administrator Security Settings Update PM 2 28 lan 2008 05 00 57 Administrator Administrator security Settings Update PM System Configuration History Detailed Report 1 Date 26 Jan 2009 05 01 08 PM Action Update User Name Administrator UserFullName Administrator Record Type security Settings Record Name H Record Type Object Name Old Value Curent Value Action 1 security Settings security On Security DISABLED ENABLED Update Off Date 26 Jan 2009 05 00 57 PM Action Update User Name Administrator UserFullName Administrator Record Type security Settings Record Name Record Type Object Name Old Value Curent Value Action 1 security Settings Security On Security EMABLED DISABLED Update Off 4 In the Report screen click toolbar Page 1 of 1 Aly options to manipulate the report as L1 needed Place the mouse pointer over an item for a description of the iem k Modify report settings D08 Feb 2 Font settings 5 To print the report click Print 6 To save the report electronically pdf print the report and select CutePDF Writer as the printer 7 Close the report Page 1 of 1 Archive purge and restore audit records The audi
12. Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 187 Chapter 6 Manage Library Resources Setup a OC Protocol E Protocol Name is a required Field Provide a unique value Protocol Name Locked Description Size Standard G5600LIZ Y Sizecaller SizeCaller v1 Hi aM Analysis Settings QC Settings o Size Quality Fail if Value is Suspect Range Pass if Value is pe Ec raa 0 25 0 75 Broad Peak Activate Broad Peak Flag if value E 5 Figure 29 Create New QC Protocol Analysis Settings IMPORTANT Normalization is not applied to samples with PY Size Quality flags The 3500 Series Data Collection Software does not support re analyzing data with new settings Table 17 QC Protocol QC Settings Setting Description Size Quality Enter the Pass Range and the Low Quality Range for the SQ flag displayed in View HID Results Results that are within the Pass range are flagged as Pass Results that are within the Low Quality range are flagged as Low Quality Results that are between the Pass and Low Quality ranges are flagged A Check For example with a Pass Range of 0 75 to 1 0 and a Low Quality Range of 0 0 to 0 25 any result above 0 75 is ua any result at 0 25 or lower is 2 and any result between 0 26 to 0 74 is Size Quality How Size Quality is determined The Size Quality algorithm evaluates the similarity between the fragment pattern for the size sta
13. LA Hun Instrumont page 59 from Load Pistes for Run The Load Plates for Run screen by clicking AF g Ad Previews Run Create Injection List Monitor Run e The navigation pane by selecting Preview Run mE The Dashboard by clicking the Main workflow arrow then selecting Preview Run in the MM As navigation pane hM 55aw oor 2 Cli k h b h 1 if h ib Hssay Icon He t e 1con above t e plate to specity the attributes to Results creup Mamie display in the plate view Results Group Color File Convention Mame 3 Click the plate tabs to display Plate A or Plate B File Convention Color w Sample Mame Sample Type Well Position w Injection dM Move Down in List E Delete EJ Duplicate 2 Inieckinn List 4 injections crested 4 in Plate A Din Plate E IF Horm POP x li 2 04 2 IF horm_POR4_sl HIDS P P4x Gt 3 09 Run 2 i 2 3 5 amp 3 IFNorm POP d HID36 POP 5 3 09 Run z sample sample sample ample sample sample sample sample sample sample sample ample 4 IF4 amp orm POP4 x HID36 POP4xl 5 3 09 Run 2 j 3 j 3 9 4 ba E sample sample sample sample sample sample sample sample sample sample sample sample 1 1 l g 2 z 3 3 3 4 4 4 i i 1 Fi z 2 3 3 3 4 4 4 i 1 1 z 2 2 3 3 3 4 4 4 sample sample sample sample sample sample sample ample sample sample sample sample 1 1 1 2 2 2 3 3 3 4 4 4 1 1 1
14. ooooooooooomomoo 261 MO Analy SIS Ve SOS suo irene Meee ed a pr heres Bee d EE LH EON PE ES 262 RUN Moqules PM c r 263 Capillary array and polymer sequencing analysis run modules 263 Capillary array and polymer fragment and HID analysis run modules 264 Secondary Analysis Sequencing o ooooooooo 267 Perform secondary analysis on sequencing experiments 267 Auto analyze projects in the sequencing analysis software 267 Set up an auto analysis project in SeqScape n nana aaan ee 268 Set up a SeqScape plate in the 3500 Series Data Collection Software 270 Auto analysis with MicroSeq ID oooocococconeon eee 217 Secondary Analysis Fragment o ooooooooo 279 Perform secondary analysis on fragment experiments 219 Auto analyze projects in the fragment analysis software 219 Set up an auto analysis project in GeneMapper amp ee 280 Set up a GeneMapper plate in the 3500 Series Data Collection Software 284 Auto Analysis with GeneMapper ID X llle 289 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Contents Appendix D Remote Auto Analysis Setup sl 291 Remote auto analysis configuration llle eres 291 Remote auto analysis installation llle 291 Greate a
15. 3 Select the ROI Regions of Interest tab then click Add Ref Segment Reference Sequence Add Ref Segment Paste Ref Segment 268 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Set up an auto analysis project in SeqScape 4 Select the file you want to use as your reference file then click Import 5 Inthe NT Variants tab RDG Properties select the NT Variants that you want to add to the reference sequence then click Import to import a tab delimited variants file or a multi aligned sequence fsta file Note When importing an amino acid variants file use a tab delimited format 6 Click OK Define settings 1 Open Tools SeqScape Manager 2 Select the Analysis Protocols tab then click New to enter a name 3 Select the Basecalling tab then select your Basecaller and Dye Primer files Analysis Protocol Editor General Basecalling Mixed Bases Clear Range Filter Basecalling Basecaller allas DyeSet Primer DT3730POP BDv3 mob X Processed Data True Profile at Profile Note Unless a project requires a custom setting keep the Processed Data Ending Base and Quality Threshold settings at their default values 4 Inthe Mixed Bases tab specify the secondary analysis peak threshold for mixed base identification 5 Keep the default settings for the other parameters listed in the Clear Range and Filter tabs then click OK 6
16. ART Technology Co Ltd timto prohlasuje e tentn 4514000 95 4651 je ve shod se zakladnimi po adavky a dal mi prislusnymi ustanovenimi sm mice 199 SES Dansk Danish Undertegnede ART Technology Co Ltd erkl rer herred at felqende udstyr A514000 96 6851 overholder de veesentlige krav og synge relevante krav i direkty 1999 5 EF Eest E stonian Kaesolevaga kinnitab ART Technology Co Ltd seadme 4514000 95 B5 1 vastavust direktivi 1999 5 EU p hin uetele ja nimetatud direktivist tulenevatele teistele asjakohastele satetele EAA IE Greek ME THN NAPOY244RT Technology Co Ltd AHAONE OTI A514000 98 851 2 YMMOPOONE TAI MPO TE OY IOAEIS ANAITHEELE KA TIE AOINE gt AE TIKE AIATA EI TH OAHIL TA 1999f5 ER Latviski Latvian Arso ART Technology Co Ltd deklare ka ASI4000 96 651 atbilst Direktvas 1999 S EK butiskajam prasibam un citem arto saisttaiem noteikumiem Lietuvi Lithuanian iuo ART Technology Co Ltd deklaruoja kad is ASI4000 98 89 1 atitinka esminius reikalavimus ir kitas 1888 5 E B Direktyvos nuostatas Malt Maltese Hawnhekk ART Technology Co Ltd jiddikjara li dan 4514000 95 851 jikkonforma mab fic Dirrettva 1999 S EC 326 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Instrumentation safety Magyar Hungarian Alulirott ART Technology Co Ltd milatkozom hogy a ASIADOC S8 B S1 megfelel a vonatkoz alapvet kovetelmenyeknek s az 1999 EC ir
17. F Fragment Name Test Fragment Plate Assays A Your Fragment Assay Actions Y 2 Y Property Customize Sample Info 1 Sample User Defined User Defined User Defined User Defined User Defined Sample Type Sample 2 Custom Value m Test Smpl Test Smpl F F Test Smpl Test Smpl F F B Test Smpl Test Smpl F F Test Smpl Test Smpl i ou i a Test Smpl Test Smpl F F g Test Smpl Test Smpl lat a Test Smpl Test Smpl Le La Test Smpl Test Smpl Note For more information on naming samples see Name samples in the Plate View on page 70 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Test Smpl F Test Smpl F Test Smpl F Test Smpl F Test Smpl F Test Smpl F Test Smpl F Test Smpl 287 Appendix C Secondary Analysis Fragment Specify FNC and 1 288 RG Specify a File Name Convention FNC and a Results Group RG to associate with your project Note You can create a FNC with the specimen name as a part of your sample file name Highlight the wells of your plate configuration Plate View and check the box next to the appropriate FNC to apply it to your project Repeat for the Results Group Note For more information on setting up a FNC see Create a new file name convention on page 151 For more information on setting up a RG see Create a new results group on page 156 Click
18. IMPORTANT The software does not display the well location of allelic ladder samples in this dialog box To identify allelic ladder samples for re injection include the well position in the allelic ladder sample name when you assign plate contents 2 Select whether to collect data for the remaining samples in the allelic ladder re injection Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 67 Chapter 3 Set Up and Run 3 Select whether to apply a modified instrument protocol to the allelic ladder re injections or whether to use the original instrument protocol for the allelic ladder re injection s You will select the modified protocol in the next screen IMPORTANT Allelic ladders that are injected under the same conditions are recommended to accurately genotype samples in the secondary analysis software GeneMapper D X Software v1 2 or later 4 Click OK 5 Specify the remaining re injection settings as described in Specify re injections on page 65 Two re injections are added to the injection list The first re injection collects data for the selected sample The second re injection collects data for the allelic ladder 68 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Review completed injections in Review Results Review completed injections in Review Results You can review results for any completed injections Select the injection then click Review Results The samples for the injection
19. Numbers in the column headers reflect sort order Customize tables You can customize any table in the software Click the Table Settings button then specify the columns to show or hide Wi Table Preferences Select your table display preferer Available Columns to Display 72 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide More features in Assign Plate Contents Add assays file name conventions and results groups to a plate 1 If no assay is listed at the bottom of the Assign Plate Contents screen add at least one assay You can specify different assays for different wells Assays File Mar Actions Add From ks Ade Create Mew Assay S Add Assay From Library Create Mer Instructions Select rows From table and click on Add To Plate button 1 Fragment Analysis Fragment FragmentAnalysis50 POP 2 Optional If no file name conventions or results groups are listed at the bottom of the Assign Plate Contents screen add as needed File name conventions and results groups are optional but are very useful for naming and organizing data files Create a plate for importing Create a plate The 3500 Series Data Collection Software allows you to import plate information import template from files that you create in an application other than the 3500 Series Data Collection Software To create a template for importing plate information set up a plate in the 3500 Series Data Collec
20. SA lr OM Amo Intensity vs Pixel Number View previously run sequencing install standards Select History View then select an install standard to view the associated calibration information View and print a sequencing install standard report IMPORTANT Ensure that all dyes are selected before viewing the report The report may contain incomplete data 1f all dyes are not selected Note the following Install standard reports include the most recent install date if a capillary array was removed then re installed on the instrument Spatial and spectral calibration reports include the date on which a capillary array is installed on the instrument for the first time Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 127 Section 2 Performance check Close the report The sorting in the Install Standard screen is not applied to the report You can generate a report for a failed installation standard run before you click Reject Results Click E View Summary Report or t View Detail Report In the Report screen click toolbar options to manipulate the report as needed Place the mouse pointer over an item for a description of the item Page 1 of 1 Select the font to be used in reports v Cancel To print the report click 24 Print To save the report electronically pdf print the report and select CuteP DF Writer as the printer Page 1 of 1 Save historical performance check
21. on page 200 8 Click Save Settings Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 199 Section 1 Administrators The new settings are applied to the logged in user the next time the user logs in Spaces in user If you allow spaces in user names be aware of the following issues AMES e Leading and trailing spaces in user names are difficult to detect on the screen or in printed reports The number of consecutive spaces in a user name 1s difficult to determine on the screen or in printed reports Spaces in user names may cause confusion when searching for an audit or E Sig record associated with a user name To find a record associated with a user name you must specify the user name exactly including leading consecutive and trailing spaces Set up messaging notifications 1 In the Security screen Figure 34 on page 198 click Messaging Notifications to display the Setup Notifications dialog box k Setup Notification Set Up Notifications EN of Failed Authentications over specified Time interval A Session Timeout For a User Account Suspension For Failed Authentication Notification For SAE Activation Close 2 Select the events for notification failed authentications over specified time interval A user attempts to log in with an incorrect password The message indicates the number of failed authentications Session timeout for a user No activity occurred in a user account for th
22. on page 210 Note You cannot delete factory provided items Select an item then click 1 Delete Deleting a library entry does not affect existing items that contain the entry When you select an item to include in a higher level item a copy of that item is included in the higher level item For example when you select an instrument protocol to include in an assay a copy of the instrument protocol is included in the assay If you delete the instrument protocol the copy of the instrument protocol in the assay remains intact For information on how deleted items are tracked in auditing see Audit action on page 210 Edit a library entry IMPORTANT Auditing of an item depends on whether it 1s edited directly from the library or from within another item for example you can edit an assay directly from the library or within a plate in the Assign Plate Contents screen For more information on auditing see Review the object audit history on page 210 1 Select an item then click Edit 2 Modify parameters as needed 3 Click Save Import and export a library entry You can import and export xml files for use with other 3500 or 3500xL analyzer instruments Import Click i Import then select the xml file to import If any items in the import file exist 1n the library the software displays a message and gives you the option to replace or skip the item e Export Select one or more entries then cli
23. Applied Biosystems 3500 3500xL Genetic Analyzer User Guide User Guide For Research Use Only Not for use in diagnostic procedures AS Boece ms HITACHI For Research Use Only Not for use in diagnostic procedures Copyright 2009 2010 Life Technologies Corporation All rights reserved Information in this document is subject to change without notice APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE TO THE FULLEST EXTENT ALLOWED BY LAW IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUEN TIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES NOTICE TO PURCHASER The purchase price of this Instrument includes a grant of a limited non transferable license under U S patents and method claims of its foreign counterparts and element clams of its foreign counterparts to use this particular instrument for electrophoresis methods employing fluorescence as a means of detection No other licenses or rights are hereby conveyed either expressly by implication or estoppel including but not limited to any
24. Audi Repons ministrakor Administrat Manual Commands 1 4d E E Sinnsture Reports Manage Lers 2 Click ECreate to display the New User dialog box Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 201 Section 1 Administrators 202 R New User Setup a User amp 3 User First name Both User First name and User last name can nat be empty User Mame ERA Created By Admin On Last Updated On Password f Pre expired Re Enter Password Password Expires On User Role Administrator Status Electronic Signature CO Enable 2 Disable Em Pone 0 Enter user name password first name middle initial optional and last name Click a field to display the field limits which are specified in Security settings Note First name MI middle initial and last name are used to create User Full Name which is displayed as the name of the logged in user Note You cannot change the user name after you save the user account Select Pre expired to require the user account to specify a new password at first log in The Password Expires On date 1s specified in Security settings Select the user role described in Create or edit a user role on page 203 and the electronic signature state determines 1f a user account has permission to electronically sign objects Leave the status set to Active Optional Enter email for information only phone and comments Click Save If
25. Limit Scans To 3250 Sensitivity 0 4 Minimum Quality Score Applied Biosystems Figure 22 Create New Dye Set Table 11 Dye set settings Setting Description Dye Set Name Name of the dye set Names must be unique Locked When enabled allows the entry to be unlocked and modified only by the user who created it the administrator or another user with unlock permissions Useful when your system includes the SAE module described in Chapter 7 Use Security Audit and E Sig Functions SAE Module on page 197 Chemistry The standard for which you are creating the dye set Sequencing Standard or Matrix standard Dye Set Template Factory provided template upon which to base the dye set The Any Dye template can be used for applications that do not use all of the dye colors contained in the matrix standard kits used for spectral calibration Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 169 Chapter 6 Manage Library Resources Table 11 Dye set settings Setting Description Arrange Dyes Displays the dyes and the peak order for the dye set template selected Editable only for AnyDye template e Dye Selection Specifies the dyes to use for calibration e Reduced Selection Specifies the dyes used in the samples For example if you use the 5 dye kit and have samples with only blue peaks you can reduce or deconvolute with blue and orange size standard
26. normalization parameters circled in red are displayed for fragment analysis and HID applications only Instrument protocol settings Table 10 Instrument protocol settings Setting Description Application Type e Sequencing e Fragment analysis e HID Capillary Length Polymer Capillary length polymer type and dye set with which the protocol will be used Dye set Run module Factory provided modules that specify instrument control parameters For more information see Run modules on page 263 Protocol name Name of the protocol Names must be unique Locked When enabled allows the entry to be unlocked and modified only by the user who created it the administrator or another user with unlock permissions Useful when your system includes the SAE module described in Chapter 7 Use Security Audit and E Sig Functions SAE Module on page 197 Description Optional text entry 166 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Table 10 Instrument protocol library Instrument protocol settings continued Setting Description Oven temperature C Temperature setting for main oven throughout run Run voltage kVolts Final sample electrophoresis separation run voltage Prerun voltage kVolts Pre run voltage setting before sample injection Injection voltage kVolts Injection voltage setting for sample injection Run time sec Len
27. on page 257 Quarterly maintenance tasks Task Frequency For information see Run performance check Every three months Chapter 5 Calibrate and Check Performance Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 231 Chapter 8 Maintain the Instrument Annual planned maintenance tasks Call your Applied Biosystems representative to schedule annual planned maintenance As needed instrument maintenance tasks Task Frequency For information see Change the tray As needed Routine instrument cleaning on page 242 Remove dried polymer from the capillary tips with a lint free wipe moistened with deionized water Archive and purge library objects Chapter 6 Manage Library Resources Dashboard Manage gt Archive or Purge Use the maintenance calendar The Maintenance calendar is a monthly or daily view of the routine maintenance tasks scheduled for your instrument When a task 1s due to be performed it is listed in the Maintenance Notifications list in the Dashboard see Review maintenance notifications on page 229 View the calendar To go to the Schedule from the Dashboard 1 In the Dashboard click Maintain Instrument toggle key The Planned Maintenance options appear on the left hand yp prainenance d pane highlighted below The Dashboard provides you with a list of current Mm lr Calibrat mainten
28. z i 3 d 4 4 4 sample sample sample sample sample sample sample sample sample sample sample sample 1 1 Fi Zz z 3 3 3 4 4 4 Jti 1 1 r4 2 2 3 3 3 4 4 4 Legend Mame 30 Run 2 Duplicate Injection feg Re Injection Consumables Information Consumable Mame ratus Lus ioni Bnesbrument Expur ation Liste Polymer POP 384 Samples Remaining 65 D1 Jan 2010 11 514007 4315930 Anode Buffer AB 3xxx Buffer 299 Danes Remaining 66 Ol Jen 201002 51 8 34007 7213931 Cathode Buffer AB 3xxx Buffer 299 Daye Remaining 66 l Jan 2010 02 8751 6TH B CB 4314 01 Capillary Array S6cm 24 eap 105 Injections Remaining 66 Ol Jan 2010 11 804005 4319893 Serial BDK2450 Start the run CG Run Figure 7 Preview Run screen Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 59 Chapter 3 Set Up and Run 60 The Preview Run screen contains an injection list and a plate view The injection list is linked to the plate view Click an injection to select the associated wells in the plate view IMPORTANT If the injection list 1s blank make sure that you clicked Create Injection List on the Load Plates for Run screen To modify the injection list at any time before a run or during a run select an injection then click 4 Move Up Ay Move Down and 1 Delete as needed Note Samples with assays that specify more than one instrument protocol are listed one time in the injection list for each instrument protocol To specify a duplicate i
29. Calibrate and Check Performance Section 1 Calibration Spatial calibration The 3500 Series Data Collection Software uses images collected during the spatial calibration to establish a relationship between the signal emitted by each capillary and the position where that signal falls on and is detected by the CCD camera When to perform a spatial calibration Perform a spatial calibration after you Remove or replace the capillary array Open the detector door or move the detection cell Move the instrument Perform a spatial calibration IMPORTANT Do not open the instrument door during a spatial calibration run Doing so will stop the run and require you to restart the 3500 Series Data Collection Software 1 Access the Spatial Calibration screen ss Select Maintenance then select Library Maintenance Tools Manage Spatial Calibration in the navigation pane Calibrate Note The screen does not display results unless you have previously Spectral performed a spatial calibration r Performance Check Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 99 Section 1 Calibration y Print 7 o Status Idle 20000 16000 12000 8000 4000 c la R iti Y 5 S Int D Options Fill No Fill Co N Fal n2rform QC Checks Start Calibration 2 Select No Fill or select Fill to fill the array with polymer before starting the calibration Optional Select Perfo
30. Confirm run When the run successfully transfers for downstream analysis the Autoanalysis completion Manager displays the project as successfully processed File Edit Help SeqScape Job Queue Job Project Arrival Date Status Injection 2009 04 30 09 51 46 950 Auto Analysis RG BDTv3 1 PA ProtocoPOP7 2009 Apr 30 09 55 53 AA Testeri Project Apr 30 20099 55 53 AM Complete Project successfully processed 4 Details Resubmit Edit Properties Stop Processing Auto delete Jobs Delete You can now launch SeqScape and review the analyzed project Note For guidelines on reviewing data and results see the SegScape Software v2 7 Workflow Quick Reference Guide PN 4401740 or the SeqScape9 Software User Guide PN 4359442 Auto analysis with MicroSeq ID For instructions detailing how to set up a MicroSeq ID analysis protocol see Create a new MicroSeq ID analysis protocol on page 191 For installation information on setting up the MicroSeq ID Software to work with the 3500 Series Data Collection Software see the MicroSeq ID v2 2 Getting Started Guide Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 277 Appendix B Secondary Analysis Sequencing 278 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide secondary Analysis Fragment Perform secondary analysis on fragment experiments The Applied Biosystems 3500 3500xL Genetic Analyzers and 3500 Series Data Collection Softw
31. E Run Module Type e amp a Run Module Name Contiguous Capillary Polymer nun Read Length Time 3500 3500xL Type Length cm min CRL S Rapid sequencing 50 POP 7 40 2280 2840 2500 RapidSeq50_POP7 Standard sequencing 50 POP 6 lt 135 gt 80 gt 240 gt 600 StdSeq50_POP6 Fast sequencing 50 POP 7 65 2168 2504 2700 FastSeq50 POP7 Standard sequencing 50 POP 7 lt 125 gt 88 gt 264 gt 850 StdSeq50_POP7 Short read sequencing 50 POP 7 lt 30 gt 368 gt 1104 gt 300 ShortReadSeqPOP7 Rapid sequencing BigDye XTerminator 50 POP 7 lt 40 gt 280 gt 840 gt 500 RapidSeq BDX 50 POP7 Standard sequencing BigDye XTerminator 50 POP 6 lt 140 280 2240 2600 StdSeq BDX 50 POP6 Fast sequencing BigDye XTerminator 50 POP 7 65 2168 2504 2700 FastSeq BDX 50 POP7 Standard sequencing BigDye XTerminator 50 POP 7 125 288 2264 2850 StdSeq BDX 50 POP7 Short read sequencing BigDye XTerminator 50 POP 7 30 2368 21104 2300 ShortReadSeq BDX POP7 Microbial Sequencing 50 POP 7 lt 125 gt 88 gt 264 gt 850 MicroSeq_POP7 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 263 Appendix A Application Reagents and Run Modules Table 33 Capillary array and polymer sequencing analysis run modules continued Q O S Configuration 23 hours Throughput E Run Module Type e amp a Run Module Name Contiguous Capillary Polymer nun Read Length Time 3500
32. E Signature Type Prompt After Approve MicroSeq ID Protocol Save Approve Assay Save Approve Plate Template Save Approve Plate Save Approve Sample Save Approve Sequencing Install Standard Accept Results Approve MicroSeg ID Install Standard Accept Results Approve Fragment Install Standard Accept Results Approve HID Install Standard Results Accept Table 26 E signature settings to check before Function to Signatures and E Signature Type Check Before editan dei rA Approve Spatial Calibration Start Run 1 signature any authorities any user any user role Approve Spectral Calibration Approve Spatial Calibration Approve Spectral Calibration Approve Plate Approve Sequencing Install Standard Results Approve MicroSeq ID Install Standard Results Approve Fragment Install Standard Results Approve HID Install Standard Results Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 219 Section 1 Administrators How the software Ifthe system is configured to check that data is signed fore starting a run and the data prompts forthe run is not signed a message is displayed when the user clicks Start Run electronic signature before Example a run The e sig system is configured to require signatures from two users one from the user account named Administrator and the other from any user account with a scientist user role for a spatial calibration before it can
33. IMPORTANT Do not accept a sequencing installation standard run until you examine the data Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 123 Section 2 Performance check What you see during a run The system performs one run then evaluates e Spectral data if you specified to keep spectral data e Sequence data The Capillary Run Data display Figure 10 on page 124 updates after the run is complete The spectral calibration status 1s displayed in the first row of the run results table Passing and failing capillaries in the performance run are shown in green and red respectively for the CRL criteria Borrowed capillaries spectral only are shown in yellow with an arrow indicating the adjacent capillary from which results were borrowed The spectral result for each capillary 1s displayed below the run results table Note Clicking a borrowed capillary displays borrowed not failed data For information on why a capillary failed look in the Sequencing Install Standard Detail Report The sequencing install standard status 1s displayed in the third row of the run results table CRL Pass Fail The Quality Value and Condition Number for each capillary is displayed below the table Capillary Run Data m E dl Spectral Calibration Run a Z esto 12 0 doo 711 0 705 0 z03 0 708 0 704 0 704 0 10 CRL Basepair Accuracy Basepair Accuracy 100 0 rr rn er rr ma
34. If the Sizing Quality is Ei normalization is not applied even if the Normalization Factor is within the normalization range Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 89 Chapter 4 Review Results Flag Symbols Description Sizing Quality Ej o MEE The Sizing Quality is in the Fail or Suspect range Place the mouse pointer X over a flag to display the Sizing Quality value for the sample See Chapter 6 Manage Library Resources Note If the Sizing Quality is hormalization is not applied even if the Normalization Factor is within the normalization range 3 Click a flag in the samples table or select samples in the samples table to display the associated data in the Plot View and Sizing Table View 4 Optional Modify the sample view e Right click the Size Standard field to view the size standard for a sample e Click Minimize and Restore to collapse and expand the samples table Review normalized data Normalization corrects for instrument capillary and injection variability When specified in the primary analysis protocol the software calculates a normalization factor for each sample The normalization factor is used as a multiplier to adjust the peak height of the sample peaks relative to the GS600 LIZ V2 size standard peaks A sample is normalized if it is collected with a normalization size standard specified in the primary analysis protocol sizecalling or QC in the assay Note If the Sizin
35. Provide a unique value Analysis Settings e Settings Size Quality Fail if Value is Suspect Range Pass if Value is Hz se 025 0 75 Assume Linearity From bp E To bp 80 Pull Lip Actuate Pull Up Flag if Pull Up Ratio 0 05 and Pull Up Scan _ Close Figure 27 Sizecalling Protocol QC Settings IMPORTANT Normalization is not applied to samples with E Size Quality flags The 3500 Series Data Collection Software does not support re analyzing data with new settings Table 15 Sizecalling protocol QC settings Setting Description Size Quality Enter the Pass Range and the Low Quality Range for the SQ flag displayed in View Fragment Results Low Quality range are flagged as Low Quality Results that are between the Pass and Results that are within the Pass range are flagged os Pass Results that are within the Low Quality ranges are flagged t For example with a Pass Range of 0 75 to 1 0 and a Low Quality Range of 0 0 to 0 25 any result above 0 75 is ud any result at 0 25 or lower is 2 and any result between 0 26 to 0 74 is dh How Size Quality is determined The Size Quality algorithm evaluates the similarity between the fragment pattern for the size standard dye specified in the size standard definition and the actual distribution of size standard peaks in the sample calculates an interim SQ a value between O and 1 Assume Linearity Defines the expected linea
36. User The name of the user Acknowledge Date Time The date and time when the event was acknowledged Description The description for the event Notification time is determined in the Preferences From the Dashboard click Preferences to open the Preferences dialog box click Scheduler Preference and follow the prompts Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Review the Maintenance Notifications Log El Preferences type filter text Scheduler Preference System Date Format Maintenance reminders trigger time Instrument Settings Scheduler Preference a Sequencing Settings iui Export Spectral Calibration B ser Plate Setup 2 Reports Settings Run Setup me Sequencing Settings 1 Trace Trace Print Trace Quality Trace Quality Repor Restore Defaults Apply Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 235 Chapter 8 Maintain the Instrument Instrument operational procedures The day to day operation of the instrument involves performing the following tasks Check consumables on the Dashboard Change the Anode Buffer Container ABC Change the Cathode Buffer Container CBC Change the polymer e Use the Conditioning Reagent e Fill Capillary Array with fresh polymer Remove bubbles The Quick View section of the Dashboard provides the necessary information that you need to operate the instrument The information shown within the Quick View is
37. View instrument sensor details Click View Instrument Sensor Details in the Dashboard to display instrument information View Instrument Sensor Details Run status of the instrument is displayed while a run is in progress sensor States Sensor Values Laser n EP voltage kv EP Current fa Laser Power irm Laser Current m EP en 19 oe 4t20 0 200 500 0 20 0 3000 0 Oven Off Oven Door Open 15 0 375 0 48193 450 2250 0 13 4 gt Messages 10 0 250 0 10 0 1500 0 S L 1198 125 0 S L 750 0 0 0 0 0 0 0 121 gt 0 0 Oven Temp PC CCD Camera Temp C Ambient Temp c Detection Cell Temp C 70 0 0 0 40 0 70 0 n a za 60 0 535 9e f 50 0 n 50 0 5 0 24 0 40 0 40 0 112 gt 255 20 39 20 0 30 0 4t30 0 30 0 15 0 16 0 23 5 20 0 10 0 20 0 10 0 20 0 S L 10 0 Figure 37 Instrument sensor details Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 305 Appendix E Troubleshoot Data electropherogram troubleshooting Symptom Possible cause Action Signal too high Sample concentration is too high Dilute the sample Decrease the injection time Too much DNA added to the reaction resulting in uneven signal distribution Optimize reaction conditions No signal Failed reaction Repeat reaction Blocked capillary Refill capillary array You may have to install a fresh array or consider that capillary non usable for purposes of planning your runs
38. amp Click inisa Click create Injection List then click OK after the instrument performs its validations Start the auto Click Start Run to begin your auto analysis analysis run The 3500 3500xL analyzer displays a progress indicator while it checks the level of consumables on the instrument Progress Information O Preparing Instrument Run Starting Instrument Run Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Auto Analysis with GeneMapper ID X Confirm run When the run successfully transfers for downstream analysis the Autoanalysis completion Manager displays the project as successfully processed c amp mim File Edit Help General GeneMapper v4 1 GeneMapper Job Queue c Job Project User of Samples Arrival Date Completed Date Status a Analysis completed successfully Analysis completed successfully Configure Schedule Edit Properties Requeue Job You can now launch GeneMapper and review your analysis Note For guidelines on reviewing fragment data and results see the GeneMapper v4 1 Quick Reference Guide PN 4362816 or refer to the specific Getting Started Guide for your application Auto Analysis with GeneMapper D X For instructions detailing how to set up a GeneMapper D X analysis protocol see Create a new HID analysis protocol on page 195 For installation information on setting up the GeneMapper D X Software v1 1 to work with the 3500 Ser
39. and consumables information is listed as Unknown Instrument is not connected message After you start 3500 Series Data Collection Software Internal buffer data overflow message Connection between the computer and instrument Check connection between the instrument and computer and restart both the instrument and computer Set up the injections again and started the runs Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 313 Appendix E Troubleshoot Reset the instrument Reset the instrument when There is a fatal error as indicated by the red status light The instrument does not respond to the Data Collection software Reset with the 1 Shut down the computer Reset button Close the instrument doors 3 Reset the instrument with the Reset button as shown Note The Reset button is accessible through a small hole to the left of the Tray button Reset button pd Shut down the computer Reset by powering down Close the instrument doors 3 Power off the instrument by pressing the on off button on the front of the instrument 4 Power on the instrument and wait until indicator light turns solid green 5 Power on the computer 6 Launch the Data Collection software Service Console applications start automatically IMPORTANT Wait until the computer has completely restarted before proceeding 314 Applied Biosystems 3500 3500xL Gene
40. capillary to the buffer position of the CBC Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 5 Chapter 1 Instrument and Software Description Results Ramps the voltage up to a constant voltage A high electric field is created between the ground end of the Anode Buffer Container ABC and the negative voltage applied to the load header of the capillary array This field pulls the negatively charged DNA through the separation polymer The smaller fragments migrate faster than the larger fragments and reach the detector first To insure optimal separation and maintain denaturation of the DNA the capillaries are thermally controlled in the oven and in the detection cell The oven has a Peltier heat unit and fan circulated air The Peltier can heat and cool the oven to maintain sub ambient temperatures which are useful for non denaturing applications such as SSCP Single strand conformation polymorphism In the detection cell the dyes attached to DNA are excited by a narrow beam of laser light The laser light is directed into the plane of the capillaries from both the bottom and top A small amount of laser light is absorbed by the dyes and emitted as longer wavelength light in all directions Captures the fluorescent light on the instrument optics while blocking the laser light The light passes through a transmission grating which spreads the light out The light is imaged onto a cooled scientific grade CCD array For eac
41. claims to a composition This instrument incorporates technology subject to one or more patents licensed from Hitachi Ltd as well as patents and patented technology owned by or under control of Applied Biosystems This instrument is Authorized for use in DNA sequencing and fragments analysis only This Authorization is included in the purchase price of the instrument and corresponds to the up front fee component of a license under process claims of U S patents and under all process claims for DNA se quence and fragment analysis of U S patents now or hereafter owned or licensable by Applied Biosystems for which an Authorization is required and under corresponding process claims in foreign counterparts of the foregoing for which an Authorization is required The running royalty component of licenses may be purchased from Applied Biosystems or obtained by using Authorized reagents purchased from Authorized suppliers in accordance with the label rights accompanying such reagents Purchase of this instrument does not itself convey to the purchaser a complete license or right to perform the above processes This instrument is also licensed under U S patents and apparatus and system claims in foreign counterparts thereof No rights are granted expressly by implication or by estoppel under composition claims or under other process or system claims owned licensable by Applied Biosystems For more information regarding licenses please contact the Director of
42. create new item from 140 file name conventions 151 filter 142 GeneMapper ID X protocols 195 import entry 141 locked 139 locked create new item from 140 overview 196 search 142 SeqScape protocols 189 sort 142 template 139 template create new item from 140 view audit records 142 view e sig records 142 linearity large fragment size standards 183 link plate 54 57 link plate error message 304 link plate troubleshooting 309 load plate for run 56 load plate troubleshooting 309 locked library item 139 log files search and use 305 login 26 225 logged in user name determining 226 display 203 in user account 202 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide M Main workflow access from Dashboard 19 maintenance calendar create entries 233 default entries 233 F entries 233 FR entries 233 recommended entries 233 view 232 maintenance notifications complete a task 28 229 Dashboard 28 229 dismiss a task 28 229 log 234 257 trigger time setting 33 maintenance wizards change polymer type 247 fill array with polymer 251 install capillary array 252 instrument shutdown 253 overview 244 remove bubbles 251 replenish polymer 245 wash pump and channels 249 maintenance computer data files archive 255 defragment 257 disk space monitor 256 library items archive purge and restore 254 uninstall software 254 maintenance consumables anode buffer change 237 capillary array change 252 cap
43. empty and replace the Anode Buffer Cup 3 Remove the pouch from the supply connection Set aside the pouch for reinstallation or discard 4 Rinse the pouch connection with purified e g distilled or deionized water and dry with a lab wipe 5 Attach a new Conditioning Reagent Pouch to the pouch Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 249 Chapter 8 Maintain the Instrument Use the conditioning reagent For details see Instrument reagents and consumables on page 9 IMPORTANT Expired pouches cannot be used on the instrument Once installed on the instrument the pouch is good for a one time use only The use of the conditioning reagent is dictated by the instrument wizards Contamination might cause poor quality data To prevent the contamination use genuine packaged polymer anode buffer cathode buffer and conditioning reagent Use genuine parts and reagent The use of inappropriate parts or reagents causes poor quality data or damage the instrument Refer to Chapter 3 Set Up and Run for instructions on priming the pump and initiating the run The Quick View section of the Dashboard provides the necessary information that you need for using the Conditioning Reagent Note Install the pouch only when requested to do so by the wizard To place the conditioning reagent on the instrument 1 Check for expiration date on the label to make sure it is not expired prior to use IMPORTANT
44. fragment analysis protocols create 193 defined 193 export 141 import 141 in assay 150 fragment analysis results See review results fragment HID fragment HID install performance check analyze data in secondary analysis software 136 estimated run time 129 evaluate data 135 examples 137 historical reports 138 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide history 137 instrument prepare 129 plate prepare 129 recommended monthly schedule 231 report 137 run 132 signing 228 standards 130 troubleshoot 136 303 what occurs 134 when to perform 129 G GeneMapper ID X protocols See also HID analysis protocols create 195 GeneMapper protocols See also fragment analysis protocols create 193 green indicator light 23 GS600 LIZ v2 0 size standard 171 260 GS600 60 600 LIZ Normalization 171 GS600 80 400 LIZ Normalization 171 GS600LIZ Normalization 171 guidelines chemical safety 328 chemical waste disposal 330 chemical waste safety 330 H hazard icons See safety symbols on instruments 315 hazard symbols See safety symbols on instruments hazards See safety Help system accessing 336 HID analysis allelic ladder validation 52 dye sets 262 reagents part numbers storage conditions 260 results group for one allelic ladder per run folder 52 results See review results fragment HID run modules 264 HID analysis protocols create 195 defined 195 export 141 import 141 inassay 150 HID install performance
45. library items 254 results See review results See review results fragment HID analysis See review results sequencing results groups assign to plate 49 73 create 156 defined 155 export 141 folder 159 for HID applications 52 155 161 import 141 store samples by plate name 160 resume instrument run 69 review results currently running plate fragment HID 88 currently running plate sequencing 80 import sample files 81 import sample files fragment HID 89 previously run plate 81 previously run plate fragment HID 89 rename 97 show hide columns 81 sort 81 review results fragment HID access 88 label peaks 93 modify data in secondary analysis software 98 normalized data 90 overlay plots 93 plots 91 quality flags 89 re injections 95 sample quality 89 scale 92 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide sizing 94 sizing curve overlay 95 thumbnails 94 troubleshoot 311 zoom 92 review results sequencing 87 See also sequence quality access 80 base colors 37 export defaults 36 metric analysis 81 modify data in secondary analysis software 98 QV colors and ranges 37 re inject 85 thumbnails 84 trace defaults 36 trace quality reports 85 RFID error message 304 troubleshoot 304 run module 166 run modules fragment HID analysis 264 sequencing analysis 263 run name default 57 enter 57 run voltage 167 run see instrument run S SAE module See audit See electronic signature See security safety bar cod
46. page 6l Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 57 Chapter 3 Set Up and Run If a plate is not Ifyou access the Load Plates for Run screen from the navigation pane a plate may linked not be linked indicated by the active Link button SS Plate Name Run Information You can edit the Run Mame by entering text V setup Run Mame Run 2009 03 04 15 33 16 629 Can Plates on Instrument Plate A Link Plate Unilink Define Plate Properties Assign Plate Contents A Run instrument Previews Run Monitor Fun ilii Review Results fist ue m e m E a E iH To link a plate 1 Click Link Plate to display the Select Plate from Library dialog box S Select Plate From Library Instructions Select row From table and click on Link Plate button Search All Sequencing Sequencing Link Plate 2 Select a plate then click Link Plate 3 Do either of the following Click Create Injection List then go to Review and modify the injection list in Preview Run on page 59 Or Click Start Run then go to Monitor the run on page 61 58 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Review and modify the injection list in Preview Run Review and modify the injection list in Preview Run The Preview Run screen allows you to modify the injection list before you start the run 1 Access the Preview Run screen Figure 7 on
47. pouch originally on a 3500 8 capillary instrument do not subsequently use that same polymer pouch on a 3500xL 24 capillary instrument or vice versa Doing so may result in a lower number of samples injections than specified If a partially used pouch 1s removed for later use use the suggested Pouch Cap to plug the fitment opening and store the pouch under recommended storage conditions The Pouch Cap is sold separately 4412619 If you remove a polymer pouch for storage place a Pouch Cap PN 4412619 onto the pouch then place an empty pouch or conditioning reagent on the connector to prevent desiccation of any residual polymer on the connector If the polymer dries on the fitment or in the pouch opening the dried polymer prevents the pouch fitment from closing the internal cap properly If that happens the polymer pouch is no longer usable IMPORTANT Follow the instructions in the wizard to ensure the proper installation and operation of the pouch and the instrument Wash the pump chamber and channels Note The Wash Pump and Channels wizard takes over 40 minutes to complete 1 From the Maintenance Wizards screen click Wash Pump and Channels WASH the pump chamber and channels 2 Follow the prompts in the Wash Wizard Wash Pump Chamber Step 1 window Install a Conditioning Reagent Pouch To wash the pump chamber and channels you will need New Conditioning Reagent Pouch 1 Open the main door 2 Remove
48. v1 1 BigDye Terminator Rapid DNA sequencing Z 3 1 BigDye Terminator DNA sequencing Fragment analysis dye sets for all applications Note For reagent or consumable shelf life expiration date see the package label The following table shows all the dye sets for fragment analysis Table 31 Fragment analysis dye sets Dye Set Application E5 SNaPshot kit G5 DNA sizing for 5 dye chemistry J6 DNA sizing for 6 dye chemistry F DNA sizing for 4 dye chemistry Any dye DNA sizing Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 261 Appendix A Application Reagents and Run Modules HID analysis dye sets Table 32 AmpF STR Kit Table Dye set use with HID Fragment Analysis P AmpF STR Kits 36_POP4 run module 4 dye F e COfiler e Profiler Plus e Profiler Plus ID e SGM Plus e Other 4 dye kits 5 dye G5 e dentifiler9 e Minifiler e SEfiler Plus e SinoFiler e Yfiler e Other 5 dye kits 262 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Run modules Run modules Capillary array and polymer Sequencing analysis run modules Decide what combination of capillary array and polymer matches your resolution and performance specifications from the table below Table 33 Capillary array and polymer sequencing analysis run modules o O Configuration 23 hours Throughput
49. 4 Check the Quick View section of the Dashboard for updated status after filling of the Capillary Array with fresh polymer Remove bubbles from the polymer pump Remove bubbles from the polymer pump fluid path before each run See Daily instrument maintenance tasks on page 230 for more information IMPORTANT Wear gloves while handling polymer the capillary array septa or CBC 1 To remove bubbles from the polymer pump fluid path that travel from the polymer pouch through the pump array port and the Anode Buffer Container click Remove Bubbles Note The Bubble Remove Wizard takes 5 to 15 minutes to complete Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 251 Chapter 8 Maintain the Instrument 2 Follow the prompts in the Bubble Bubble Remove Wizard Remove Wizard window eee ea Welcome to the Bubble Remove maintenance wizard 3 Check the Quick Vi CW S ection of the ul z NS i Run this wizard to remove bubbles from the polymer pump fluid path Click Next to continiue Dashboard for updated status of the polymer pouch after removing bubbles from the polymer pump fluid path To change the capillary array CAUTION SHARP The load end of the capillary array has small but blunt ends and it could lead to piercing injury IMPORTANT Check the loading end header to ensure that the capillary tips are not crushed or damaged For details see Instrument reagents and consumables o
50. 4 44h B 2 4 2 4 2 4 6 86 8 6 8 10 12 10 12 10 12 14 16 14 16 14 16 ELSE 51919 po 15 efi fa fila it is s 71s 715 7 2 le ae i ss i 13 15 13 25 ON Faso CA E Wl eel i E A Ss k p 2 4 2 4 2 4 6 8 6 8 6 amp 10 12 10 12 10 12 14 16 14 16 14 16 ililzizlzil3l3l3141414 TUN eM EZ 20 ad E E ee E enm ei 3 fi f3 i fs 5 7151715 7 3 3 9 it 13 5 13 15 13 15 WR ET ERI Co E ee F 2 4 2 4 2 4 6 8 6 8 6 8 10 12 10 12 10 12 14 16 14 16 14 16 iar te te to lot eke tae to l 1 s 1r s 1 a3 s 715 7 5 7 se u u u 9 i 13 5 13 15 13 15 H 2 4 2 4 2 4 6 8 6 8 6 8 10 12 10 12 10 12 14 16 14 16 14 16 r 3131 35757579 119 119 11 13 15 13 15 13 15 J 2 4 2 4 2 4 6 8 6 8 6 8 10 12 10 12 10 12 14 16 14 16 14 16 kiz spris 1 s s z s v s 7 e u o u 9 it 13 15 13 15 13 25 L 2 4 2 4 2 4 6 8 6 8 6 8 10 12 10 12 10 12 14 16 14 16 14 16 mji jeji fe pi els F s z e ue u at 1 r5 13 is 13 25 N 2 4 2 4 2 4 6 8 6 8 6 8 10 12 10 12 10 12 14 16 14 16 14 16 e risir sir a s z s 7 s z g un fs 11 i 15 aa is 13 05 P 2 4 2 4 2 4 6 8 6 8 6 8 10 12 10 12 10 12 14 16 14 16 14 16 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 51 Chapter 3 Set Up and Run Allelic ladder run requirements Applied Biosystems recommends that you inject one allelic ladder for each set of 24 samples e 8 capillary instruments One allelic ladder per 3 injections e 24 capillary instruments One allelic ladder pe
51. 5 Save the results group If you are creating the results group from the Library click Save If you are creating the results group from the Assign Plate Contents screen click Apply to Plate or Save to Library The Results Group file location overrides the File Name Convention file location Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 157 Chapter 6 Manage Library Resources S Create New Results Group Setup a Results Group e Mame is a required Field Provide a unique value Name Po Eo Color Select Results Group Attributes Preview of Results Group Name lt Results Group Name Selected Attributes Results Group Name Available Attributes Assay Mame Injection Mumber TP Mame Delimiters Select a delimiter Dash V add between attributes Enter a custom value as either the Prefix or Suffix optional Select Reinjection Folder Option 2 Store reinjection sample files in a separate Reinjection folder same level as Injection Folders store reinjection sample Files with original sample files same level Select Folder Option Default file location C Applied Biosystems 3500 Datal lt IR Folder Results Group Name Folder gt lt Inj Folder gt Custom file location Include an Instrument Run Mame Folder Include a Result Group Mame Folder Include an Injection Folder Close Figure 14 Create New Results Group Table 9 Results group settings Setting
52. Appendix B Secondary Analysis Sequencing Specify FNC and 2 4 RG Customize Sample Info Property Value Assays File Name Conventions 1 Sample Actions v i Sample Name samplel00 M BDx Fast Seq Assay A EY V gt General File Name 2 B ae Type Sample a Custom User Defined Fie User Defined Fie User Defined Fie User Defined Fie User Defined Fie m Note For more information on naming samples see Name samples in the Plate View on page 70 Specify a File Name Convention FNC and a Results Group RG to associate with your project Note You can create a FNC with the specimen name as a part of your sample file name Highlight the wells of your plate configuration Plate View and check the box next to the appropriate FNC to apply it to your project Repeat for the Results Group Note For more information on setting up a FNC see Create a new file name convention on page 151 For more information on setting up a RG see Create a new results group on page 156 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Set up a SegScape plate in the 3500 Series Data Collection Software E Show In Wells v Select Wells y E Array Selection Column Ra adsavad RA a ora M Afr P Asioy4 S M Assays M Af S h Assaya S M Assayas M Assays M Asad S RA Assay S ha Assay M Assay S RA Assay S M Assay S M Assay S M Assay S M Assay S M Ati S M Ati S M Assays M Ass
53. Audit disable or enable Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 207 Section 1 Administrators Select objects to audit 1 Select the objects and actions to audit and the mode for each enabled item Object Type audit records displayed in Object Audit History Action Type audit records displayed in Action Log Dye set Size standard Instrument protocol PA protocol primary analysis SA protocol Secondary analysis QC protocol Assay Plate template File name convention Results group Plate Sample files Export assay Export plate record Note For a list of items that the system audits silently in addition to the configurable items listed above see Generate audit reports on page 209 2 Set the Audit Mode for each item you enable for auditing Prompt The event is audited a reason prompt is displayed but the user can cancel and continue without entering a reason Required The event is audited a reason prompt is displayed and the user must specify a reason Silent The event is audited no reason prompt is displayed 3 Click Save Settings Create audit reason settings You can create modify and delete the reasons that are available for selection in the Audit Reason dialog box displayed when a user performs an audited action 208 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 1 To require users to f Audit Reason Settings sele
54. Bent capillary array tips Replace the capillary array Cracked or broken capillary array Visually inspect the capillary array including the detector window area for signs of breakage Low signal strength Degraded Formamide Use a fresh aliquot of Hi Di Formamide Not enough sample Pipetting error Increase the amount of DNA added Sample has high salt concentration Dilute with distilled or deionized water Desalt using a column purification method Insufficient mixing Vortex the sample thoroughly and then centrifuge the tube to condense the sample to the bottom of the tube Weak amplification of DNA Reamplify the DNA Check DNA quality Autosampler out of calibration Check the volume of your samples If still low signal strength call your Applied Biosystems representative Elevated baseline Possible contaminant in the polymer path Use the conditioning reagent for washing the polymer pump Possible contaminant or crystal deposits in the polymer Bring the polymer to room temperature Replace the polymer if it has expired Poor spectral calibration Perform new spectral calibration Loss of resolution Too much sample injected Dilute the sample and re inject Poor quality water Use distilled or deionized water Degraded polymer Use a fresh supply of polymer Capillary array used for more than 160
55. Biosystems 3500 3500xL Genetic Analyzer User Guide Assign plate contents 8 Optional For sequencing assays Property Value specify amplicon and specimen a sample Sample Mame 9 Repeat to assign the Sample Type for Sample Type Sample ll d lls 2 2 Custom all named wells User Defined Field 1 User Defined Field 2 10 Goto Assign assay file name User Defined Field 3 User Defined Field convention and results group in the E ELILIC Ic Plate View on page 49 3 Analysis Amplicon Specimen Note For HID applications include the well position in the allelic ladder sample names Well position is needed to identify the position of allelic ladder samples during re injection Assign assay file name convention and results group in the Plate View Note Ifan assay file name convention or results group is not listed for the plate go to Add assays file name conventions and results groups to a plate on page 73 1 Select the wells for which to specify an assay 2 Enable the checkbox next to the A 5 5dy5 assay name to assign it to the i selected wells amp BDx Fast Seg Assay xL P A El Note To normalize fragment E analysis or HID data select an assay that contains a sizecalling protocol or a QC protocol that specifies a normalization size standard Actions 3 Optional Repeat for file name conventions and results group File Name Conventions Results Groups
56. Bubble Remove Wizard to clear the bubbles Possible contaminant or crystal deposits in the polymer Properly bring the polymer to room temperature do not heat Replace the polymer if it has expired Elevated baseline Poor spectral calibration Perform new spectral calibration Spectral calibration history does not If you change polymer type spectral No action display previously run calibration calibrations for the original polymer type are not retained Pull down mirror image peaks The first time you perform a spectral No action calibration for each dye set after installing a new capillary array you may notice pull down peaks or mirror image peaks These pull down peaks will eventually correct themselves once the run completes Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 301 Appendix E Troubleshoot Sequencing install standard troubleshooting Symptom Possible cause Action No signal Incorrect preparation of sample Replace samples with fresh samples prepared with fresh Hi Di Formamide Bubbles in sample wells Centrifuge samples to remove bubbles The capillary tips may not be Check the volume of your samples If touching the samples no results call your Applied Biosystems representative The capillary tips may be hitting the Call your Applied Biosystems bottom of the wells Autosampler representative not correctly aligned I
57. Contents screen E Setup Define Plate Properties Assign Plate Contents N k 3500 Data Collection Software E Dashboard Library Resources Edit ibrary Mainter Plates Azssays ALL ITEMS i dd Identif Minifile File Name Conventions Results Groups Actions Y MiniFiler My Sequencing Resul A My Fragment Analvysi A A Add From Library Save To Library A SVE Import From File Select Related Wells 3 In the Create Results group dialog box Figure 14 on page 158 select attributes and delimeters see Table 9 on page 158 As you select attributes the software displays a preview of the results group name 156 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Result group library Available Attributes Selected Attributes PA Protocol Mame Results Group Mame Plate Mame 4 gt Remove MT gt z Remove Select Results Group Attributes Preview of Results Group Name lt Results Group Name gt lt PA Protocol Name gt lt Plate Name gt Available Attributes Selected Attributes Prefix Add Results Group Name 4 To add delimiters between items in the Selected Attributes list a Ctrl click or Shift click to select two or more attributes b Select a delimiter c Select the Add between attributes check box d Click Add
58. Create New Sizecalling Protocol Analysis Settings IMPORTANT Normalization is not applied to samples with PY Size Quality flags Specify analysis settings that accurately detect and size the size standard and QC settings with appropriate pass fail ranges The 3500 Series Data Collection Software does not support re analyzing data with new settings Table 14 Sizecalling protocol Analysis settings Setting Description Protocol Name Name of the protocol Names must be unique Description Optional text entry Size standard Size standard definition in the software that corresponds to the dye set used in the chemistry To apply normalization select a normalization size standard see Normalization size standards provided on page 171 180 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Sizecalling protocols library primary analysis fragment Table 14 Sizecalling protocol Analysis settings continued Setting Description Analysis Range Specify the range in data points to analyze Full Range to analyze the entire scan region as collected by the genetic analysis instrument including the primer peak Partial Range to analyze only data points within a specified range Enter Start Point in data points after the primer peak and before the first required size standard peak Enter a Stop Point after the last required size standard fragment Start and Stop points may vary from
59. Degree Slope Threshold Peak Start Slope Threshold Peak End Figure 28 Create New QC Protocol Analysis Settings IMPORTANT The default values in the QC protocol templates other than peak amplitude threshold values have been optimized for each kit You must optimize and validate peak amplitude threshold values during internal HID validation If you modify other settings ensure that the size standard 1s accurately detected and sized with the new settings Normalization is not applied to samples with 9 Size Quality flags The 3500 Series Data Collection Software does not support re analyzing data with new settings Table 16 QC protocol Analysis settings Setting Description Protocol Name Name of the protocol Names must be unique Description Optional text entry Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 185 Chapter 6 Manage Library Resources Table 16 QC protocol Analysis settings continued Setting Description Size standard Size standard definition in the software that corresponds to the dye set used in the chemistry To apply normalization select a normalization size standard see Normalization size standards provided on page 171 Analysis Range Select Full to collect data points for the entire scan region including the primer peak You can specify a limited analysis range in the GeneMapper D X Software Note If you select Partial ensure that t
60. Description Name Name of the results group Names must be unique The Results Group Name is a required attribute you cannot remove this attribute from the Selected Attribute list Locked When enabled allows the entry to be unlocked and modified only by the user who created it the administrator or another user with unlock permissions Useful when your system includes the SAE module described in Chapter 7 Use Security Audit and E Sig Functions SAE Module on page 197 158 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Result group library Table 9 Results group settings continued Setting Description Color Color code for the results group when it is displayed in the Assign Plate Contents screen if Results Group Color is selected for Show In Wells Results Groups C Minifiler p My Fragment Analvsi F My Sequencing Resul A Preview of name Interactively displays the attributes you select Available attributes e Results Group Name required e PA Protocol Name primary analysis e Assay Name e Plate Name e Injection Number e Prefix e P Name instrument protocol e Start Instrument Run Date time Stamp e Logged in User Name available only e Suffix when security is enabled in the SAE module Delimiters Symbols you can include in the results group name Dash Dot Underscore _ Plus Dollar Prefix suffix text Text
61. EME SHUTDOWN the instrument Instrument Replenish polymer IMPORTANT Do not use a polymer pouch that has been installed on one type of instrument on another type of instrument For example if you install a new polymer pouch originally on a 3500 8 capillary instrument do not subsequently use that same polymer pouch on a 3500xL 24 capillary instrument or vice versa Doing so may result in a lower number of samples injections than specified For the following hazard s see the complete safety alert descriptions in Specific chemical alerts on page 333 WARNING CHEMICAL HAZARD POP 4 POP 6 and POP 7 polymers For details see Instrument reagents and consumables on page 9 If you are replacing the same polymer type only follow the procedures below IMPORTANT If you remove a polymer pouch for storage place a Pouch Cap PN 4412619 onto the pouch then place an empty pouch or conditioning reagent on the connector to prevent desiccation of any residual polymer on the connector Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 245 Chapter 8 Maintain the Instrument 1 In the Maintenance Wizards screen click Replenish Polymer Replenish Palymer Note The Replenish Polymer Wizard takes 10 to 20 minutes to complete 2 Follow the prompts in the Replenish Replenish Polymer Wizard Replenish Polymer Introduction Po lymer Wiz ard win dow Welcome to the Replenish Polymer maintenance
62. From the SeqScape Manager select the Analysis Defaults tab then click New and enter an Analysis Defaults Name 7 Go to the Sample tab and select the Analysis Protocol you just created in the drop down list 8 Keep the default settings in the Project and Specimen tabs then click Save In most cases you will want to keep the default Display Settings and continue with creating a project template in the SeqScape Software Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 269 Appendix B Secondary Analysis Create a project 1 template Sequencing Open Tools gt SeqScape Manager Select the Project Templates tab then click New and enter a name for the 4 template In the Reference Data Group and Analysis Defaults drop down lists select the RDG and Analysis Default that you previously created Keep the default Display Settings then click OK With the project template created continue with adding your sample files Create an empty 1 project In the SeqScape Software select File New Project Name the project In the Project Template list select the project template that you previously created When the project opens click Add Specimen Tools New Specimen to create as many blank specimens as you have in your project then click OK Close the SeqScape Software You are now ready to set up a run in the 3500 Series Data Collection Software specifying a SeqScape Protocol as y
63. HID results 90 fragment analysis 183 fragment analysis difference from GeneMapper ID X 183 HID analysis 188 monitor run 64 weighting 188 stand alone installation of software 19 standard plate plate base 53 plate type 44 51 105 120 145 standards EMC 322 fragment HID install performance check 130 safety 322 sequencing install performance check 120 spectral calibration 104 start instrument run 69 start the system 21 status consumables in Dashboard 26 front panel indicator lights 23 software icon in Windows tray 24 stop instrument run 69 symbols safety 315 system configuration history contents 211 Index display 209 system defaults date format 18 system description 1 system startup 21 T Table of Acronyms xvi table view assign plate contents show hide columns 72 sort 72 use 71 user defined fields 48 tables results show hide columns 81 settings 72 settings default 35 show hide columns 72 sort 97 sort multi column 72 technician user role 203 template create plate from 43 library 139 plate 75 plate import 73 terminate instrument run 69 theory of operation 5 thumbnails fragment HID 94 sequencing 84 timeout session 199 226 trace quality reports 85 Trace Score 81 178 Trace Score report 85 training information on xvii troubleshoot anode buffer container 303 audit 312 cathode buffer container 304 Dashboard 309 data 306 electropherogram 306 fragment HID install performance check 303 instrument 2
64. Instrument and Software Description 20 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide start the System Workflow Start the system 1 Start the instrument page 22 Start the computer page 24 Check maintenance notifications in the Dashboard page 28 Check consumable status in the Dashboard page 29 Replenish consumables page 31 Set up and run 1 Prepare the instrument page 42 2 Preheat the oven page 42 3 Check instrument status page 53 Create or import a plate page 43 Assign plate contents page 46 Print the plate layout page 50 Quick Start a run page 55 Prepare and load sample plates page 51 Load plates for run and create the injection list page 56 Review and modify the injection list in Preview Run page 59 Start the run page 61 Monitor the run page 61 check sequence or sample quality and specify re injections page 62 Review sequencing results Review fragment HID results 1 Review sequence quality page 81 1 Review sample quality page 89 2 Specify re injections page 95 2 Specify re injections page 85 3 Review quality reports page 85 3 Review quality reports page 95 4 Export sequencing results page 87 4 Export sizing results page 96 Optional print or save pdf calibration and performance check reports to save with results Spatial calibration page 99 Spectral calibration page 103
65. Instrument interior components lel 3 Instrument parts and functions 0 enn 4 Theory OF ODSKATION sica esd 8 0 5 3 27 0 8 on eot o e Doe d Rd eod Aene ode e A ot 5 Preparing Samples sera dar oi at eate wha ne Ao d teu Set a lit at al caede ra i 5 Preparing the instrument 2 0 eens D PUAN APU a TREE ESO S EET UNT INIT DRITTE UT 5 RESURS adas dro ide eg ders dc ete di det ira io 6 NOMMAIZANON 26446008 rodea LT da eeu a de de T Overview of the normalization feature unana anana aa 7 When to use the normalization feature 2 0 0 00 ce es 7 Materials for routine operation llle eens 8 External barcode scanner a an anaana aeaa eee ee eee 8 Uninterruptible Power Supply UPS 2 0000 8 Instrument reagents and consumables 2 0000 ccc eee 9 Anode buffer container ABC 0 0 ee ee ees 9 Cathode buffer container CBC o o ooococoononn eee eee 9 OW MNCS 2 acai RR eerie een ed ad GAO eae wees See Tcu 10 Conditioning reagent 0 cc ee eee teens 11 Hi Di Formamlide 2 24x cte a anoo e or dead ede badd bead ema 12 Caplan arrays Es setos Sof sun aoe ath nailed alt ah Baha E ea ak he eee a 12 Overview of the 3500 Series Data Collection Software 14 About th software a a a 3 o HEN RO O AAA AAA 14 Pans Ol dle SOR WOO ed dri ads tac E 15 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide iii Contents Ch
66. Log Out The full name of the logged in user is also 7 displayed in the Load Plates for Run screen and the Monitor Run screen Create or edit a user role User roles determine the permissions associated with a user account Three default user roles are included in the software You can modify two of them and can create your own roles with customized settings as needed Administrator cannot be edited or deleted Scientist e Technician Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 203 Section 1 Administrators To determine the permissions for these roles or to edit these roles select the role then click Edit Create a user role 1 Access the Roles screen Dashboard Edit 7 Settings Resources Library Maintenance Tools Manage Preferent Audit vo Manage Reports Audit Reports E Sgghnature Reports Manage teers 2 Click Create E Signature Change Password View Logs Manual Commands 3 Enter a role name and optional comment 4 Select permissions see Table 22 on page 204 To select all permissions in a category select the checkbox next to the category 5 Click Save Role Table 22 User role permissions Category Permissions Setup Create plate plate template Run e Edit default instrument name e Manage injection list e Duplicate injection e Re inject Primary Analysis Edit sample names Export sequencing r
67. Protocol Approve Microseq ID Protocol Approve Assay Approve Plate Template 1 Any Select the minimum signatures that must exist For data to qualify as being signed w Approve Plate Approve Sample Approve Sequencing Install Standard Results Approve MicroSegID Install Standard Results Approve Fragment Install Standard Results Approve HID Install Standard Results 3 Click Save Settings Function Mame Run Stark Signatures Required A Authorities Required Ary Cancel By default no E Signature types are enabled Table 25 E signature settings to prompt after Function to E Signature Type Prompt After Approve Dye Set Save Approve Size Standard Save Approve Spatial Calibration Accept Approve Spectral Calibration Accept Approve Instrument Protocol Save Approve Sizecall Protocol Save Approve Basecall Protocol Save Approve QC Protocol Save Approve Size Standard Save Approve Spatial Calibration Accept Approve Spectral Calibration Accept Approve Instrument Protocol Save Approve Sizecall Protocol Save Approve Basecall Protocol Save Approve QC Protocol Save Approve GeneMapper Protocol Save Approve GeneMapper IDX Protocol Save Approve SeqScape Protocol Save Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Manage electronic signature Table 25 E signature settings to prompt after continued Function to
68. Quality Report pane Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 39 Chapter 2 Start the System S Preferences not used Trace Quality Reports System Date Format Instrument Settings Scheduler Preference Sequencing Settings These settings affect all reports Sort data based on This setting applies to the Trace Score Report CRL Report OV20 Report and Signal Strength Report Export e Run Mame Spectral Calibration Capillary Number User Plate Setup Reports Settings Signal based on Run Setup This setting applies to the QC Report and Signal Strength Report Sequencing Settings Trace Trace Print e Average Raw Signal Intensity Trace Quality Q Average Raw Signal to Noise Ratio Trace Quality Reports Display well image by 2 Wider thumbnail without the File name C2 Smaller thumbnail with the File name 2 Specify the following settings Setting Description Sort data Sort data in Trace Score CRL QV20 and Signal Strength reports based on e Run Name e Capillary Number Signal based on Base signal in QC and Signal Strength reports based on e Average Raw Signal Intensity e Average Raw Signal to Noise Ratio Display well Specify the thumbnail option for Plate reports image by e Wider thumbnail without file name e Smaller thumbnail without file name 3 Click Apply to save the user preferences see User preferences on page 34 40 Appli
69. Secondary Analysis Sequencing Monitor the run Monitor the run by checking the status icons in the Injection Details section Monitor Run screen Check Status icons here Library Maintenance Tools Y Manage Y Preferences Help Log Out 4 Move Up in List d Move Down in List fi Date ic ate Re Inject m Pause Run gt gt Resume Run Eg Abort Injection EY Terminate Injection List Connection Status Connected Run Name Run 2009 04 53 Last Login Time 28 Apr 2009 12 43 51 PM Estimated Time Remaining 03 05 54 lc Setup Define Plate Properties Assign Plate Contents Run histrumont Plate A Plate B Load Plates for Run AA Assay 4 SeqScape RapidSeq50 POP7xl 1 AA Seq Plate 4 Se A AA Assay 4 SeqScape RapidSeq50_POP7xi_L AA Seq Plate 4 Se 1 AA Assay 4 SeqScape RapidSeq50 POP7xl 1 AA Seq Plate 4 Se AA Assay 4 SeqScape RapidSeq50_POP7x_L AA Seq Plate 4 Se lidia Review View Sequencing Results View Fragment HID Results 4 n m Legend Sef Not Started a Active m Paused Aborted E Completed P4 Re Injection E Duplicate Flags C View sequencing You can view the Sequencing Results in the 3500 Series Data Collection Software by results going to the View Sequencing Results screen and selecting the tab of interest ab Trace Quality View Cerere Analysis Rests y ate Al macs O 7 i
70. Select the report type Reports are displayed in the Sizing Table View at the bottom of the screen 4 Modify report settings as needed MM Modify report settings Font settings Select the Font En be used in reports 5 To print the report click 24 Print then preview or print 6 To save the report electronically pdf print the report and select CutePDF Writer as the printer 7 Close the report Page lofl A P Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 95 Chapter 4 Review Results Report options Sizing One page per selected sample that shows the quality ranges set in the sizecalling or QC protocol the quality values for the sample and the electropherogram for the sample Plot zooming is not retained in the report Overlay One page for all selected samples that shows the size standard dyes overlaid with the size standard curves e Plate One page per plate for all selected samples that shows the well location thumbnail traces with color coded headers that reflect sizing quality Plot zooming 1s not retained in the report Applied potiiis Applied Biosystems Borse ms Applied Bioriste ms H Export sizing results 1 Set up the sizing table as described above All rows and columns displayed in the sizing table are exported 2 Click Export Results 96 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide More features in Review Results More
71. Things to consider before lifting the computer and or the monitor Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 317 Appendix F Safety Make sure that you have a secure comfortable grip on the computer or the monitor when lifting Make sure that the path from where the object is to where it is being moved is clear of obstructions Do not lift an object and twist your torso at the same time Keep your spine in a good neutral position while lifting with your legs e Participants should coordinate lift and move intentions with each other before lifting and carrying Instead of lifting the object from the packing box carefully tilt the box on its side and hold it stationary while someone slides the contents out of the box Operating the Ensure that anyone who operates the instrument has instrument Received instructions in both general safety practices for laboratories and specific safety practices for the instrument Read and understood all applicable Material Safety Data Sheets MSDSs See About MSDSs on page 329 Cleaning or CAUTION Before using a cleaning or decontamination method other than decontaminating those recommended by the manufacturer verify with the manufacturer that the the instrument proposed method will not damage the equipment Physical hazard safety Moving parts WARNING PHYSICAL INJURY HAZARD Keep hands clear of moving parts while operating the instrument Disconnect powe
72. Window Size setting is limited to odd numbers To increase peak detection sensitivity Increase polynomial degree decrease peak window size To decrease peak detection sensitivity Decrease polynomial degree increase peak window size Slope Thresholds Peak Start and End e Peak Start The peak starts when the first derivative slope of the tangent in the beginning of the peak signal before the inflection point becomes equal to or exceeds the Peak Start value This threshold is set to O by default which means that the peak will normally start at the leftmost point where the slope of the tangent is closest to O horizontal line A value other than O moves the peak start point toward its center The value entered must be non negative e Peak End The peak ends when the first derivative slope of the tangent in the end of the peak signal after the inflection point becomes equal to or exceeds the Peak End value This value is set to O by default which means that the peak will normally end at the rightmost point where the slope of the tangent is closest to 0 horizontal line A value other than O moves the peak end point toward its center The value entered in this field must be non positive 182 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Sizecalling protocols library primary analysis fragment ll Create New Sizecalling Protocol Setup a Sizecalling Protocol y e Protocol Name is a required Field
73. analysis method size standard and panel to use for auto analysis 194 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide HID analysis protocols library secondary analysis HID analysis protocols library Secondary analysis HID analysis protocol overview An HID analysis protocol GeneMapper ID X protocol is the optional secondary analysis auto analysis protocol for GeneMapper D X Software v1 2 or later for HID applications An HID analysis protocol defines the Secondary analysis software GeneMapper D X Software location e GeneMapper D X Software analysis method size standard and panel that the GeneMapper D X Software will use during auto analysis When you create an HID assay you can optionally add an HID analysis protocol to the assay If you add this 1tem from the library a copy of the item 1s added to the assay and can be modified independently from the original item stored in the library For information on how changes are tracked if auditing 1s enabled see Audit action on page 210 Create a new HID analysis protocol 1 Access the HID Analysis B 3500 Data Collection Software Protocols library 3 Dashboard Edit Library Mainter Library Resources A be Filter A Manage Plates Asssys m Protocol Marr File Mame Conventions 2 Click zn Create 3 In the Create New HID Analysis Protocol dialog box Figure 33 on page 196 specify settings see Table 21
74. are assigning assays file name conventions and contents results group to be associated with your auto analysis Set up an assay 1 In the Assign Plate Contents screen go to the Assays box and select either Create New Assay or Add From Library Assays Actions Add fram Library Create New Assay 2 Name your new assay in the Setup an Assay dialog box Note Optional Select a color for this assay to display with in the Plate View 3 Select an Instrument Protocol to apply to the assay Note For more instruction on setting up an instrument protocol see Create a new instrument protocol on page 165 4 Select a Sizecalling Protocol to apply to the assay Note For more instruction on setting up an instrument protocol see Create a new sizecalling protocol on page 179 5 Create a new fragment analysis protocol GeneMapper Protocol to apply to the samples by clicking Create New Test Fragment Plate Create New Assay Setup an Assay Assay Name Your Fragment Assay Locked Color Dark Blue z Application Type Fragment Protocols Do you wish to assign multiple instrument protocols to this assay No Yes Instrument Protocol FragmentAnalysis50_POP7x_1 Edit Create New Sizecalling Protocol Fragment Analysis PA Protocol Edit Create New a ae NE a a Microsatellite SA Protocol SNPlex_3730 SA Protocol Apply to Plate Save to Library
75. are loaded in the Samples Table in Review Results For more information see Review Results on page 79 Start and stop a run Start a run You can start a run in the Load Plates for Run screen see Load plates for run and create the injection list on page 56 e Preview Run screen see Start the run on page 61 Pause and As needed click resume a run jj Pause Pauses the run after the current injection completes the i symbol is not displayed in the injection list because the injection continues to completion Resume Resumes the run Abort or As needed click terminate Abort Stops the current injection Do not click Delete to stop an injection IMPORTANT You can stop the current injection only when the front panel indicator is blinking green If you click Abort when the front panel indicator is solid green the physical injection is already completed although the software is still processing the information and a message is displayed indicating that there 1s no injection in process e Terminate Stops the instrument run Terminate is active only when a run is paused Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 69 Chapter 3 Set Up and Run More features in Assign Plate Contents Use the Plate View Name samples in To name samples in the Plate View the Plate View To name one sample e Click a well then type a sample name directly into the field
76. are selected before viewing the report The report will contain incomplete data if all dyes are not selected Note the following Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Section 2 Performance check Install standard reports include the most recent install date if a capillary array was removed then re installed on the instrument Spatial and spectral calibration reports include the date on which a capillary array 1s installed on the instrument for the first time The sorting in the Install Standard screen is not applied to the report To generate a report for a failed installation standard run you must do so before you Click Reject Results Click View Detail Report In the Report screen click toolbar options to manipulate the report as needed Place the mouse pointer over an item for a description of the item Page 1 of 1 3I gO LER x kh Modify settings For this report S Modify report settings E 0D8 Feb 2 Font settings Select the Font to be used in reports v 10 w ox JPL cancel 3 To print the report click 2 Print 4 Close the report Page lofi Save historical performance check reports pdf for record keeping IMPORTANT After performing a performance check save the performance check report electronically for record keeping The software does not save historical calibration results Only the most recent spectral calibration for each dye set
77. auditing 1s enabled see Audit action on page 210 Create a new file name convention If factory provided file name conventions do not suit your needs you can create new file name conventions 1 Access the File Name Conventions library k 3500 Data Collection Software 3 Dashboard Edit 2 Click A Create i Library Resources ibrary Mainben I Wieate Filter ae Manage Plates Note You can also create a file name convention from the Assign Plate Contents screen Assays 1 FNC Example Micrast Flle Name Conventions Results Group Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 151 Chapter 6 Manage Library Resources V Setup BO how in ela Sol We c Dj Define Plate Properties a h Hn i m Hi m n n r 3 ES x li Barm Peas prb vem Sauer omy Repas Lr y A Mes E E _File Name Conventions ney Actions a Ao A Hi Te F a A ramos PODa GhaMorrsalis part dw Add Fram Library Import From File 3 In the Create New File Name Conventions dialog box Figure 13 on page 153 select attributes and delimeters see Table 8 on page 153 As you select attributes the software displays a preview of the file name Available Attributes Selected Attributes Amplicon Mame Sample Marne Analysis Protocol Mame A Assay Mame Capillary Number Custom Texti Custom TexEz Custom Texts Date of Run Injection Number Instrument Mare
78. change these settings drag the sliding bar to the desired setting Plate Setup Alternatively right click on Ehe bar and enter the desired setting A AS Trace Score Color and Threshold Setting een Serup The Trace Score is the average of basecall quality values For bases in the clear range B Sequencing Settings Trace D 15 3 100 ag Trace Quality Trace Quality Reports Contiguous Read Length Color and Threshold Setting Full Sequence Drop In Quality Values q pene Ae Contiguous Read Length CRL The Contiguous Read Length CRL is Ehe longest uninterrupted stretch of bases with quality higher than a specified limit It is the length of the sequence that remains after trimming only using quality values Evaluation of the quality of each base is determined using the quality value of the base and the quality value of adjacent bases within a specified window 0 100 300 1000 2 Set colors and ranges a Click a color bar to select a new color b Place the mouse pointer over a slider then drag to set a new range 15 30 100 3 Click Apply to save the user preferences see User preferences on page 34 Trace Quality Trace quality Report preferences determine the content and formatting used in QC Report user Plate Trace Score CRL QV20 and Signal Strength reports preference 1 In the Preferences dialog box click Trace Quality Report under User Sequencing settings to display the Trace
79. continue POP 4 960 4393710 3500 8 capillary Lower of 7 days or 960 Expiry date 7 day limit gg samples or 120 Injections Sample limit a 260 ooo n T and or Injections POP 7 960 4393714 9900xL 24 capillary Lower of 7 days or 960 iene samples or 50 Injections POP 4 384 4393715 3500 8 capillary Lower of 7 daysor384 Expiry date 7 day limit _a tt samples or 60 Injections Sample limit om 3500xL 24 capillary Lower of 7 days oraga 2 orlnjections POP 7 384 4393708 xL 24 capillary Lower of 7 days or limit samples or 20 Injections The polymer pouch includes additional volume to accommodate a limited number of installation and wizard operations However if the number of wizard operations exceeds a certain limit the number of remaining samples or injections will be reduced For example if you run the total bubble remove option in the bubble remove wizard more than four times or run other wizards operations excessively the number of remaining samples or injections will be reduced Refer to the polymer gage on the dashboard for the up to date number of remaining samples or injections at any given point Replace the pouch before proceeding further Applied Biosystems has verified the polymer for a maximum of 7 days on the instrument Ambient temperature must be in the range of 15 C to 25 C Sustained use at higher temperatures may result in shorter read lengths
80. dyes only Parameters Specifies the Quality Value Condition Number Scan and Sensitivity requirements for the dye set Notes Optional text entry 170 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Size standards library Size standards library Size standard overview A size standard defines the sizes of known fragments It is used to generate a standard curve The standard curve is used to determine the sizing of unknown samples When you create a sizecalling fragment or QC HID protocol you add a size standard to the protocol If you add this item from the library a copy of the item 1s added to the protocol and can be modified independently from the original items stored in the library For information on how changes are tracked if auditing is enabled see Audit action on page 210 Normalization size standards provided The library contains factory provided normalized size standards that you can use to normalize fragment analysis and HID data Fragment analysis GS600LIZ Normalization GS600 60 600 LIZ Normalization For applications that have primer peaks that obscure the 20 and 40 mer peaks of the GS600 size standard HID GS600 80 400 LIZ Normalization Normalization corrects for instrument capillary and injection variability For each sample the software calculates a normalization factor based on a threshold setting The normalization factor is used as a multiplier
81. ee ee e 227 EIGGIONIC SIGN AUIS 22 5 afud um CR eg e Dg mee CY erat ecu AS oes ni 221 Chapter8 Maintain the Instrument o ooooooooooooo 229 Maintenance schedule sibarita 229 Review maintenance notifications 0 eee 229 Daily instrument maintenance taSkS 0 000 cee eee eee 230 Weekly instrument maintenance tasks ees 231 Monthly instrument maintenance tasks 0000 cee eee ee ees 231 Quarterly maintenance taSkS 0 00 cee eee ee eee ees 231 Annual planned maintenance tasks 0 0000 cece ee ees 232 As needed instrument maintenance tasks len 232 Use the maintenance calendar llle 232 NICW Me calenda al tom Re ies ad Ce Seed wee a ae Aerie a Se 232 Default calendar entries 2 0 0 0c eee 233 Create calendar entries 0 ae e a i A eee eee ee 233 Review the Maintenance Notifications Log llle 234 Instrument operational procedures 0 cee 236 Check consumables on the Dashboard 000 cee eee 236 Change the anode buffer container ABC 0 02 0 cee eee ee eee 237 Change the cathode buffer container CBC 0 000 eee eee eee 238 Check stored capillary arrays 0 00 240 Flush the water trap pump trap 0 ee ee ees 241 Routine instrument cleaning 0 00 ccc eee ees 242 Move and level the instrument 2 0 00 ce eee 243 Applied Bios
82. evaluates the Quality Value and Condition Number of all capillaries If all capillaries pass the calibration is complete and injections 2 and 3 are not performed If any capillaries fail the software borrows from an adjacent capillary If after borrowing gt 1 or gt 3 capillaries fail injection 2 is performed Injection 2 The software evaluates the quality values between adjacent capillaries in injection 2 and for each capillary across injections 1 and 2 and the information with the highest Quality Value for each capillary If all capillaries pass the calibration is complete and injection 3 is not performed If after borrowing gt 1 or gt 3 capillaries from injection 1 or 2 do not pass injection 3 is performed Injection 3 The software evaluates the quality values between adjacent capillaries in injection 3 and for each capillary across injections 1 2 and 3 then the information with the highest Quality Value for each capillary If all capillaries now pass the calibration passes If after borrowing gt 1 or gt 3 capillaries from injection 1 2 or 3 do not pass the calibration fails Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 113 Section 1 Calibration Example spectral calibration data Dye Set E created from Sequencing Standard Intensity vs Scan Number Calibrated Data v E 7 0 4000 8000 12000 16000 ll il i MU M EU m n ll Intensity vs Scan Nu
83. features in Review Results Use Rename Note Changes to sample names are tracked only if your system includes the SAE module and auditing 1s enabled on your system 1 In the Sample Name column select the samples to rename or click the Sample Name column header to select the entire column 2 Click 4 Rename 3 In the Search field enter the sample name to change 4 Inthe Rename field enter the new name 5 Click Search then click Rename Sort Double click column headers to sort Multi column sorting is supported e Double click a column header to sort the column e Alt Shift click another column header to sort another column e Alt Shift click a third column header to sort a third column Numbers in the column headers reflect sort order Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 97 Chapter 4 Review Results Modify sequence fragment analysis or HID data To edit modify or further analyze sequence fragment analysis or HID data import the sample data files into a secondary analysis software application such as Sequencing SeqScape Software v2 7 or later MicroSeq ID Analysis Software v2 2 or later Variant Reporter Software v1 1 or later and Sequence Analysis SeqA Software v5 4 or later Fragment analysis GeneMapper Software v4 1 or later HID GeneMapper D X Software v1 2 or later 98 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide
84. in instrument 53 load in software 56 overview 143 owner 145 pending 76 print 50 processed 76 properties 44 results group 49 73 sample plate prepare 51 select for run 54 setup error 309 signing 228 type 145 unlink 57 plate base standard versus fast and 8 tube strip 44 51 53 105 120 145 plate definition 143 plate grid report 50 plate import template 73 plate layout pdf 50 print 50 saving for future use 75 plate map print 50 plate properties 145 plate record See plate plate report fragment HID 95 sequencing generate 85 plate report sequencing quality ranges 38 plate template create 75 create plate from 43 save well attributes 75 plate type default 34 set default 76 specify 44 51 105 120 145 plate view Applied Biosystems 3500 3500xL Genetic Analyzer User Guide assign plate contents 48 customize assign plate contents 71 monitor run 62 plate to capillary map diagram 51 display in software 71 plots fragment HID display plots 91 label peaks 93 overlay 93 scale 92 settings 92 settings default 35 zoom 92 polymer ambient temperature requirements 30 change type 247 described 10 expiration 11 fill array 251 on instrument limits 30 oven temperature POP 4 POP 6 POP 7 42 part numbers 11 replenish 245 storage conditions 10 storing partially used pouch 249 Polynomial Degree 182 POP polymer See polymer positive control 48 preferences borrowing event limits 34 date format 33 file location 35 ins
85. injection Create a Re injection Instrument Protocol Options Reuse the existing protocol Modify the existing protocol CO Create a new protocol using the template HID36_POP4x CO Reuse a protocol in Ehe library HID36 POP4xl F v Placement of Re injections 5 Following all injections CQ After original injection If you select a protocol other than the original If you select a protocol other than the original the software e Creates a copy of the assay specified for the re injected well Original_Assay 1 e Adds the new or modified instrument protocol to Original_Assay 1 e Assigns Original_Assay 1 to the re injected well only e Saves the plate the software does not save the copy of the assay to the library How re injections are displayed in the plate view If the Injection Number attribute is selected for display in the plate view the number of the original injection and the re injection are shown Sample 1 selected for re injection Re injection number listed for all samples in the re injection Note If you select only specific wells for the re injection which physically re injects all samples for the capillary array but collects data only for the selected wells the re injection number is displayed for all samples in the re injection not just the samples selected for data collection 66 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Check sequence or sample quality a
86. injections Replace with new capillary array Degraded formamide Prepare fresh Hi Di Formamide and re prepare samples High salt concentration in samples Use a recommended protocol for salt removal Dilute salts with water 306 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Data electropherogram troubleshooting Symptom Possible cause Action Poor resolution in some capillaries Insufficient filling of capillary array Refill the capillary array and look for polymer leakage If problem persists call your Applied Biosystems representative Re inject the same samples Poor quality samples Check the sample preparation Leak in system Tighten the connectors and array lever No current Not enough buffer in ABC Ensure that the buffer is filled up to the fill line Bubble s present in the lower polymer block and or the array and or channels Pause the run and inspect for bubbles hidden in the tubing connectors Select the Bubble Remove Wizard to remove the bubbles Elevated current Degraded polymer Open fresh supply of polymer and use Replenish Polymer Wizard Arcing in the lower polymer block Inspect the lower polymer block for discoloration or damage Replace the lower polymer block if necessary Fluctuating current Bubble in polymer block Pause run and inspect for bubbles hidden in the tub
87. m nce E E Panel Manager Kit Name Kit Type Comment Ge LMS HDS 42 5 PMS HDS 2 5 Microsate none H aia LMS MD10 V2 5 Microsate none B MCN Kit iiy Ercole 9 3 SNPlex 48plex 3130 Microsatellite Kit Microsate none H O SNPlex_48plex_3730 SNPlex_48plex_3130 SMPlex none SMPlex 48plex 3730 SMPlex none 5 Import the bin sets that are associated with the panels you just imported above Click Import for each bin set Note You have to associate a bin set to every panel that you imported e Panel Manager File Edit Bins View TM X g E E g Bin Set SNMPlex 48plex Bin 3130 S Ey Panel Manager Panel Name Comment t CQLMS HD5 v2 5 SNPlex 48plex Panel 31 none H CL MS MD10 V2 5 SH LOH kit GE Microsatellite Kit ig eet SNPlex_48plex_3130 E O SNPlex_48plex_3730 6 Click OK to save and close the Panel Manager Note For more information on how to create panels and bin sets see the GeneMapper v4 1 Quick Reference Guide PN 4362816 or refer to the specific Getting Started Guide for your application Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 281 Appendix C Secondary Analysis Fragment Create a new l project Select the Analysis Method tab then click New GeneMapper Manager 2004 09 21 11 39 02 0 2008 04 17 14 59 07 0 jig O SNaPshotie H SNPlex Rules 3730 5 OLA Analysis O SMPlex Model 3730 5 SMPlex Rules 3130 AFLP
88. of runs or days remaining the number of days on the instrument the expiration date lot number and part numbers Quick View Gauges POP Polymer AB 3500 Buffer Anode AB 3500 Buffer Cathode 50cm 24 cap Array 384 576 3 4 3 4 64 96 QA E 2 AE 5 2 ein Fa 5 a Y G i lt Es 7 C Lo ar A 3 Ts F 28 auges ix J ab a E 144 y 4960 0 A 0 On 1 160 634 Samples Remaining 7 Days Remaining 7 Days Remaining 43 Injections Performed 34 Injections Remaining 96 Injections Remaining 96 Injections Remaining Pre Heat the Oven Instrument 3500 Instrument State Idle View Instrument Sensor Details Set Temperature En Instrument Laser On Owen OFF information EP On Oven Door Open oven Temperaiive 995 eo co aaa i o 1 EE E E moss Detection Cell Temperature C 23 5 Consumables Information Refresh o Polymer POP 634 Samples Remaining 1 26 Mar 2009 11 514007 4315930 Consumables Anode Buffer 46 356 Buffer 7 Days Remaining 1 28 Mar 2010 11 518007 4315931 Cathode Buffer AB 356 Buffer 7 Days Remaining 1 26 Mar 2009 11 516007 4315931 Capillary Array SOcm 24 cap 117 Injections Remaining 80 31 Dec 2009 11 80K005 4319899 Serial 80K2450 Maintenance Notifications o Replace cathode buffer c HIGH 22 Mar 2009 1 Replace c v X Clean Drip Tray HIGH 22 Mar 2009 1 Clean Drip X Maintenance Ol cations Clean Aritecampler HIGH 22 Mar 2009 1 Clean Aut E 3 Re
89. of this guide Audience Assumptions The Applied Biosystems 3500 3500xL Genetic Analyzer User Guide provides step by step instructions for preparing and analyzing a sample It 1s designed to help you learn how to use the instrument N CAUTION The protection provided by the equipment may be impaired if the instrument is operated outside the environment and use specifications the user provides inadequate maintenance or the equipment is used in a manner not specified by the manufacturer Applied Biosystems This user guide is written for principle investigators and laboratory staff who are planning to operate and maintain the Applied Biosystems 3500 3500xL Genetic Analyzers The Applied Biosystems 3500 3500xL Genetic Analyzer User Guide assumes that your 3500 or 3500xL analyzer has been installed by an Applied Biosystems service representative This guide also assumes you have the following background Familiarity with Microsoft Windows Vista operating system Knowledge of general techniques for handling DNA samples and preparing them for electrophoresis Ageneral understanding of hard drives data storage file transfers and copying and pasting Applied Biosystems 3500 3500xL Genetic Analyzer User Guide XV Preface How to use this guide Text conventions Xvi User attention words Table of Acronyms This guide uses the following conventions Bold text indicates user action For example Type 0 then
90. one way to name samples and assign sample types For other ways to name samples see Use the Plate View on page 70 and Use the Table View on page 71 Procedure 1 Click a well then type a sample name directly into the well then press Enter 2 Click drag multiple wells ample 3 Right click and select Fill or Fill i ee Series to populate the selected fields Yt To use Fill Series type a number as the last character of the named well O l gd PERS You can copy and paste sample D Copy Ctrl names instead of using fill 2 SN Fill Ctrl commands ace Fill Series F Fill Sample Mame Type Delete G Rename Sample 4 At the bottom right of the Assign Plate Contents screen expand the Recalts Grape Customize Sample Info pane Actions E E E m un iu a E 2 ma Add From Library 5 In the plate view click drag te New Results Group to select wells of interest 6 Specify the Sample Type for 00 Customize Sample Info the selected wells then press Enter Property Value 7 Optional Specify User Defined El 1 Sample Sample Marne Fields and Comments User Defined Sere Fields contain additional attributes E 2 Custom A i Positive Contro a you can assign to a plate and are See En e coin User Defined Allelic Ladder A displayed only in Table View User Defined lic 3 User Defined User Defined 3 Misc Comments 48 Applied
91. or re injections are part of the same instrument run An injection 1s an instance of 8 or 24 samples depending on instrument configuration processed simultaneously under the same conditions IMPORTANT It takes approximately 10 seconds for the instrument to initialize after the instrument door 1s closed Do not start a run until the instrument status light 1s green When you access the Load Plates for Run screen by clicking Load Plates for Run on the Assign Plate Contents screen the plate is automatically linked indicated by the active Unlink button Plate Marne Run Information You can edit the Run Mame by entering text Run Mame Run 2009 03 04 15 33 16 629 Can Plates on Instrument A Setup Detine Plate Properties Assign Plate Contents s Raw Imstruneont Plate A 96 wells ink Plate P Mame Plate 01 h Preview Run Type HID Monitor Run Barcode mM Review Results Mr Dean ima Dm be 4 Ifneeded click Unlink then follow the steps in If a plate 1s not linked below 5 As needed click Switch Plates position in the autosampler to assign the plate to the other 6 Click either of the following Create Injection List Displays the Preview Run screen where you can modify the injection list before starting the run Go to Review and modify the injection list in Preview Run on page 59 e Start Run Displays the Monitor Run screen Go to Monitor the run on
92. other advanced sharing optics PEER al 7 3 On Advanced Sharing window click Permissions Ivancedl Sharing Share this Felder Settings Share name Add Remesve Lime the number oF Simubaneous users bo 10 al Comments Permissions Caching Ok Cancal Apply Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 295 Appendix D Remote Auto Analysis Setup 4 Inthe Permissions for the lt shared folder name gt dialog box select the checkbox for Full Control in the Allow column M Permissions far Remate Autoanalysis Shae Permesions Group or user names FA 2500 40 MIN 2500 P0 425010001 IN aa Permiesiore for S500 4DM IM Alo Deru Ful Contre Change v El Bead P Lean eboul access contol and permesions Lok Caneel Apply 5 Click OK twice 6 Click Close Turn off password IMPORTANT Before starting Remote Auto analysis you must make sure that the protected sharing password protected sharing settings on the Data Collection computer are turned off 1 On the Data Collection computer select Start Control Panel Network and Sharing Center 2 Click the expand button v for Password protected sharing 3 Select Turn off password protected sharing 296 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Set up the 3500 Series Data Collection Software v1 0 Network and Sharing Center View full map jy ss Q 3500 PE applera net In
93. page 299 Also check the status light in the bottom left hand corner of the data collection window to see if it flashes red Review the Maintenance Notifications Log The Notifications Log is a history of all notifications messages and the action taken for the task completed or dismissed You can use this option to review a previous run information The Dashboard provides you with a list of current routine and maintenance notifications as explained below Multi column sorting is supported see Multi column sorting on page 72 To go to the Notifications Log from the Dashboard 1 Click Maintain Instrument 2 From the Left hand pane under Planned Maintenance click Notifications Log Click on the top left hand corner of the Notification Log for more information Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 257 Chapter 8 Maintain the Instrument The Notification Log provides the following information on each event Notification Description Name The name of the event Priority The event priority Notification Date The date of notification Status The current status of the event User The name of the user Acknowledge Date Time The date and time when the event was acknowledged Description The description for the event Notification time is determined in the Preferences From the Dashboard click Preferences to open the Preferences dialog box click Scheduler Preference and fol
94. press Enter for each of the remaining fields e Italic text indicates new or important words and is also used for emphasis For example Before changing reagents always determine what chemicals have been used in the instrument e A right arrow symbol gt separates successive commands you select from a drop down or shortcut menu For example Select File Open gt Spot Set Right click the sample row then select View Filter View All Runs Two user attention words appear in Applied Biosystems user documentation Each word implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product IMPORTANT Provides information that 1s necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical The following table explains the acronyms used in the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Acronym Definition ABC Anode Buffer Container BDT BigDye Terminator Kit BDX BigDye Xterminator Cap Capillaries CBC Cathode Buffer Container CV Fitting Check Valve pouch attachment fitting EPT ElectroPhoresis Telemetry FR Factory Repeating MicroSeq kit Microbial Sequencing or other product name NIC Network Interface Card NT Nucleoid Type Nucleotide Base Color A G C T Pe Probability of error QV Quality Value Ap
95. protocol If you add these items from the library a copy of the items 1s added to the assay and can be modified independently from the original items stored in the library For information on how changes are tracked if auditing 1s enabled see Audit action on page 210 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 147 Chapter 6 Manage Library Resources Create a new assay If factory provided assays do not suit your needs you can create new assays j v Maintena Bate Filter all 1 Access the Assays library R 3500 Data Collection Software 2 Click A Create 3 Dashboard Edit Note You can also create an Library Resources assay from the Assign Plate Contents screen e Setup Define Plate Properties Assays Actions 35 Normaliz Add From Library Import Fram File 3 In the Create New Assays dialog box select an application type Sequencing Fragment or HID The screen changes depending on the application type you select Figure 12 on page 149 shows the sequencing screen 4 Specify settings see Table 7 on page 149 5 Save the assay e Ifyou are creating the assay from the Library click Save e Ifyou are creating the assay from the Assign Plate Contents screen click Apply to Plate or Save to Library 148 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Table 7 Assay settings Assays
96. report Page toT Export e sig records As needed you can export e sig records to a txt file for additional manipulation and reporting outside the 3500 Series Data Collection Software 1 Display the records of interest as described above 2 Click 33 Export E Sig Records UY Specify a name and location for the export txt file 4 Click Save Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 223 Section 1 Administrators Export and import user accounts security audit and electronic signature settings Export 1 In any screen in the SAE module click m Export in the navigation pane j Manage Users Users 2 Select the items to export Manage Settings Security Select Security Configurations to Export Audit E Signature User Profiles system Configuration User Profiles Contains all settings in the following screens Edit User All user accounts with Active status User Role All user roles and associated permissions in case a user account specifies a user role that does not exist on the system into which you import the profiles System Configuration Contains all settings in the following screens Security Account setup and security policies Audit Objects selected for auditing audit modes and reasons E Signature Settings Objects selected for E Signature functions number of signatures and authorities User Roles All use
97. reports pdf for record keeping IMPORTANT After performing a performance check save the performance check report electronically for record keeping The software does not save historical calibration results Only the most recent spectral calibration for each dye set 1s maintained in the software 128 Click E View Summary Report or E Lm 2 E View Detail Report Click a Print In the Printer dialog box select CutePDF Writer as the printer Specify a name and location for the report Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Run the fragment analysis or HID Install standard performance check Run the fragment analysis or HID Install standard performance check When to perform When your instrument is installed the service engineer runs a fragment analysis or HID install standard install performance check Applied Biosystems recommends that you run the fragment or HID install standard performance check monthly to verify that the instrument conforms to fragment analysis sizing precision sizing range and peak height specifications IMPORTANT The performance check is application specific If you change polymer and capillary length you must perform a new performance check Estimated run 30 minutes time Prepare for the fragment or HID install standard performance check Prepare the l instrument If you have not already done so perform a spatial calibration see Spatial calibra
98. safety Safety labels on instruments The Applied Biosystems 3500 3500xL Genetic Analyzers contain warnings at the locations shown below Locations of laser warnings On the detection cell as shown below General instrument safety N WARNING PHYSICAL INJURY HAZARD Use this product only as specified in this document Using this instrument in a manner not specified by Applied Biosystems may result in personal injury or damage to the instrument WARNING PHYSICAL INJURY HAZARD Using the instrument in a manner not specified by Applied Biosystems may result in personal injury or damage to the instrument Moving and lifting AN CAUTION PHYSICAL INJURY HAZARD The instrument is to be moved the instrument and positioned only by the personnel or vendor specified in the applicable site preparation guide If you decide to lift or move the instrument after it has been installed do not attempt to lift or move the instrument without the assistance of others the use of appropriate moving equipment and proper lifting techniques Improper lifting can cause painful and permanent back injury Depending on the weight moving or lifting an instrument may require two or more persons Moving and lifting N WARNING Do not attempt to lift or move the computer or the monitor stand alone without the assistance of others Depending on the weight of the computer computers and and or the monitor moving them may require two or more people monitors
99. see during a spectral calibration A spectral calibration automatically sets up three injections The number of injections performed depends on The number of capillaries that pass or fail during an injection Whether you select the Allow Borrowing option Note The first time you perform a spectral calibration for each dye set after installing a new capillary array you may notice pull down peaks or mirror image peaks These pull down peaks will eventually correct themselves once the run completes Capillary A spectral calibration can share capillary information information Between injections If a capillary in an injection does not meet the spectral sharing Quality Value and Condition Number limits shown on page 109 the software automatically uses the information from that capillary in a different injection Within an injection If a capillary in an injection does not meet the spectral Quality Value and Condition Number limits shown on page 109 and the Allow Borrowing option is selected the software can also use the information from a capillary to the left or the right of that capillary if the values are higher than those for that capillary in a different injection Spectral When Borrowing is disabled all capillaries must pass meet the spectral Quality calibration with Value and Condition Number limits for the calibration to pass Borrowing Injection 1 e The software evaluates the Quality Value and Condit
100. sig See electronic signature export assays 141 audit records 215 audit settings 224 basecalling protocols 141 dye sets 141 e sig records 223 e sig settings 224 file name convention 141 fragment analysis protocols 141 HID analysis protocols 141 instrument protocols 141 plate 75 141 protocol 141 QC protocols 141 results groups 141 security settings 224 sequencing analysis protocols 141 sequencing results reports traces 87 size standards 141 sizecalling protocols 141 spatial calibration 101 spectral calibration 115 user account settings 224 340 F factory provided library items 139 fast plate plate base 53 plate type 44 51 105 120 145 file location default 35 in file name convention 154 with file name convention 50 151 with results group 50 155 without file name convention 50 151 without results group 50 155 file name conventions assign to plate 49 73 create 151 defined 151 export 141 file location 154 import 141 in plate 151 file name format default format 50 with file name convention 151 without file name convention 50 151 file name maximum length 154 fill and fill series sample name 48 71 72 Fill Array with Polymer maintenance wizard 251 first run migration effects 53 flags display flag table in monitor run 62 monitor run 62 re injecting samples with 65 Flat Profile 176 formamide sample type for 48 fragment analysis dye sets 261 reagents part numbers storage conditions 260 run modules 264
101. the Save button is grayed it indicates an invalid entry in a field Click a field to display the limits for the field then enter a valid entry The Users screen displays the following information for each user account e Full Name Password Change Date by e Role either user or administrator e Status e Email for records only e Password Expired true yes Phone false no Comments ast Modified On Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Manage user accounts Edit a user 1 In the Users screen select a user account then click Edit account Note If you select multiple users only Status and Role will be changed EA Create 4 3 Delete E ViewReport Prnt 1 Adm Adminis Ad Active False Maser Userl 4d Active true 12 Feb 2009 1 12 Feb 2700 12 Feb 2009 12 28 2 Edit settings as needed You cannot edit the user name of an existing user 3 Click Save Activatea 1 Select the user suspended user account 2 Click Edit 3 Change the status from Suspended to Active Delete inactivate You cannot delete a user because user records are required for auditing To disable a a user account user account inactivate it 1 Select the user 2 Click Edit 3 Change the status from active to inactive 4 Click Save Determine the name of the logged in user To display the full name of the logged in user place the mouse pointer on the Logout menu zii Help 7
102. the base position is considered a potential mixed base Adjust this parameter by dragging the bar in the display or typing in a numeric value Use Mixed Base Identification Do not assign a mixed base when the secondary peak height is lt to Hy Analyzed Data Scaling Determines scaling of the processed traces This parameter does not affect the accuracy of the basecalling e True Profile The processed traces are scaled uniformly so that the average height of peaks in the region of strongest signal is about equal to a fixed value The profile of the processed traces will be very similar to that of the raw traces e Flat Profile The processed traces are scaled semi locally so that the average height of peaks in any region is about equal to a fixed value The profile of the processed traces will be flat on an intermediate scale gt about 40 bases Clear range methods Use clear range minimum and maximum Specifies the first and last base in the range to consider or trims the specified number of bases from the 3 end e Use quality values Sets a window with a specified number of allowed low quality bases by removing bases until there are lt X number of bases per Z number of bases with QV lt Y e Use identification of N cells Sets a window with a specified number of allowed ambiguous base calls Ns by removing bases until there are lt X number of Ns per Y number of bases 176 Applied Biosystems 35
103. to continue 160 injections 24 Capillary 50 cm 4404689 aoe cas Applied Biosystems has verified the array for 160 injections Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 13 Chapter 1 Instrument and Software Description Overview of the 3500 Series Data Collection Software About the software Table 5 3500 Series Data Collection Software applications supported Application Supports Sequencing e Direct sequencing for mutation detection e Comparative sequencing with and without references e Microbial sequence identification Fragment analysis Microsatellite e AFLP amplified fragment length polymorphism e SNaPshot kit e LOH loss of heterozygosity e MLPA Multiplex ligation dependent probe amplification HID m Forensic DNA casework e Databasing e Paternity testing During a run the software Controls the instrument and generates sample data files Sequencing abl Fragment analysis fsa HID hid Performs primary analysis and reporting that evaluate the quality of the data Sequencing Basecalling and trimming Fragment analysis and HID Peak detection and sizing Optional Performs secondary analysis auto analysis with the following Applied Biosystems software applications Sequencing SeqScape Software v2 7 or later or MicroSeq ID Analysis Software v2 2 or later Fragment analysis GeneMapper S
104. 0 32 60 0 00010 1 Click View Thumbnails to display results as thumbnails Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Review Sequencing Results AQ4 AD4 abl AD5_A05 abl A06_A06 abl B 4 BO4 abl C06_C06 abl 004_D04 ab1 DOS _DO5 abl D06 D 6 abl F 5 F 5 abl F06 F 6 abl G04 G 4 abl G05 G 5 abl 2 Sort as needed 3 To compare signal across all samples on a plate select Uniform Y Scaling Trace Mame Average Raw Signal Intensity 4 Click View Tables to close the thumbnail pane Filter All Traces RC Run Module Mame sampled aun Mame Instrument Mame Well ID Capillary Results Group o Specify re injections Before the run is complete you can select a sample then click fig Re 1nject View print and save pdf trace quality reports View Trace 1 Click View Trace ele MR F rr 7 Reports Reports to see the available piures ns es 9090 HO QC Report reports for traces and print the reports you want You can set defaults for the 09 AM reports in Preferences see Set sequencing preferences on page 36 E Plate Report i QVY2U4 Report e Signal Strength Report 2 Select the report type and review the content of each report See Report options on page 86 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 85 Chapter 4 Review Results 86 Report options 3 Modify report settings as needed You can specify addit
105. 0 3500xL Genetic Analyzer User Guide 115 Section 1 Calibration 2 Specify an export file name and location 3 Click Save The export file contains the following results e Capillary Number e Quality Value Condition Number Peak Height e Scan Number e Reason For Failure e Borrowed From Capillary e Run From Injection View and print a spectral calibration report Note Spatial and spectral calibration reports include the date on which a capillary array 1s installed for the first time on the instrument Install standard reports use the most recent install date if a capillary array was removed and re installed on the instrument 1 Click View Spectral Calibration Report 2 In the Report screen click toolbar options to manipulate the report as needed Place the mouse pointer over an item for a description of the item Page 1 of 1 O a B x k Modify settings For this repart k Modify report settings E 0D8 Feb 2 Font settings Select the font to be used in reports 3 To print the report click Print 4 Close the report Page 1 of 1 Save historical calibration reports pdf for record keeping IMPORTANT After performing a calibration save the calibration report electronically for record keeping The software does not save historical calibration results Only the most recent spectral calibration for each dye set 1s maintained in the software 116 Applied Biosystems 3500 3500xL Genetic
106. 0 Cd Under normal operating conditions the instrument is categorized as a Class I laser product When safety interlocks are disabled during certain servicing procedures the laser can cause permanent eye damage and therefore is classified under those conditions as a Class 3B laser CAUTION LASER Use of controls or adjustments or performance of procedures other than those specified herein may result in hazardous radiation exposure Laser safety To ensure safe laser operation requirements The system must be installed and maintained by an Applied Biosystems Technical Representative e All instrument panels must be in place on the instrument while the instrument is operating When all panels are installed there 1s no detectable radiation present and the instrument is Class I If any panel is removed when the laser is operating during service with safety interlocks disabled you may be exposed to laser emissions in excess of the Class 3B rating e Do not remove safety labels or disable safety interlocks Additional laser Refer to the user documentation provided with the laser for additional information on safety government and industry safety regulations information T Also note the laser warnings provided in Safety labels on instruments on page 317 AS WARNING LASER HAZARD Lasers can burn the retina causing permanent blind spots Never look directly into the laser beam Remove jewelry and other items that can
107. 00 1500 500 Export spatial calibration results To export spatial calibration results aes 1 Click Export OF No Fill Perform QC Checks 2 Enter an export file name Start Calibration 3 Select the export file type Save az hype Ca Comma delimited csv ES Comma delimited cs Export Results di Note Do not open Ehe instrument bs during a 4 Click Save spatial calibration run Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 101 Section 1 Calibration The export file contains the following results e Capillary Number Spacing Position pixels Intensity View and print a spatial calibration report Note Spatial and spectral calibration reports include the date on which a capillary array is installed for the first time on the instrument Install standard reports use the most recent install date if a capillary array was removed and re installed on the instrument 1 Click View Spatial Calibration Report 2 In the Report screen click toolbar options to manipulate the report as needed Place the mouse pointer over an item for a description of the item Page 1 of 1 3I gO LER x k Modify settings For this report S Modify report settings E 08 Feb 2 Font settings Select the font to be used in reports v 3 To print the report click 54 Print 4 Close the report Page 1 of 1 Save historical calibration reports pdf for record
108. 00 3500xL Genetic Analyzer User Guide Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Basecalling protocols library primary analysis sequencing S Create New Basecalli ng Protocol Setup a Basecalling Protocol E Protocol Mame is a required Field Provide a unique value Protocol Mame SSs Locked Basecaller KB1 4 1 7 Analysis Settings QY Settings Sequence Quality Contiguous Read Length Trace Score QV2Z04 Clase save Figure 25 Create New Basecalling Protocol QV Settings QV settings are quality value ranges used in the following screens Monitor Run screen The state of the QV flag If all three values are in the pass range the QV flag in Monitor Run is set to Ils green Ifany values are in the suspect range the QV flag in Monitor Run is set to yellow If any value fails are in the fail range the QV flag in Monitor Run is set to El red View Sequencing Results Metric Analysis Results table The pass check fail status for Trace Score Quality CRL Quality and QV20 Quality results 177 Chapter 6 Manage Library Resources Table 13 Basecalling protocol QV settings Setting Description Contiguous Read Length The longest uninterrupted segment of bases with an average Quality Value QV gt 20 In addition to evaluating the QV of a base call the software considers the QV of adjacent bases within a 20 bp m
109. 00 cc eee 88 Access the View Fragment HID Results screen 0 0 0 cee es 88 Review sample quality s 23 ts C2tiaee ees EU Oe eee eed doe Cee ek EEG Re 89 Review normalized data 000 cc ees 90 REVIEW DOIS z ri Ii an es Bee ura a eek 91 mirror 94 Specify r InJectlorns 5 3 x scs pp d eL a vas VQ Poe caeso E cca 95 View print and save pdf sample quality reports 000 cee eee 95 EXPOFrESIZINO TeSultS cada ass oleae ett Veri NEU eBid Mee etal se a eRe ae 96 More features in Review Results 0 0 0 0 ee ees 97 Nis cS M ROUEN ETT ak eats ek A a Ben UE TM 97 SOM gm cpr tte a dias dia 97 Modify sequence fragment analysis or HID data o oooooooo 98 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide V Contents Chapter 5 vi Calibrate and Check Performance 99 section Calibrallom sick adele es Shaw Ec kao aca a 99 Spatial Calibration 2 todo seed eds gant bode geet de ACERO 99 When to perform a spatial calibration lel 99 Perform a spatial calibration celle 99 Evaluate the spatial calibration profile leen 101 Example spatial profiles eee ene 101 Export spatial calibration results llle 101 View and print a spatial calibration report 2 0 0 ee 102 Save historical calibration reports pdf for record keeping
110. 01 B02 B03 B04 B05 B06 B07 B08 B09 B10 B11 B12 S S S S S S S S S S S S 3 samplel02 samplel10 samplel18 samplel26 samplel34 samplel42 samplel50 sample158 samplel66 samplel74 samplel82 samplel90 Monitor Run aye ase C03 eae de C06 eye ra coo aon ay C2 rm ple103 e ple111 H le119 2 ple127 ple135 a le143 3 ple151 ple159 A le167 ple175 ple183 le191 i sam sample sam sam sample sample sam sample sam sam sample sample19 tid Review Resuts D01 D02 Dos D04 D05 Dos D07 Dos DOS D10 D11 D12 S S S S S S S S S B S S c samplel04 samplel12 samplel20 samplel28 samplel36 samplel44 samplel52 samplel60 samplel68 samplel76 samplel84 samplel92 E01 m E02 pl E03 E04 E05 E06 E07 E08 E09 e E10 E11 E12 View Fragment HID Results S S S S S S S S S S S S x sample105 sample113 sample121 sample129 sample137 samplel45 sample153 sample161 sample169 sample177 sample185 sample193 BrP za Fos BP gn FOG ehe e Ege Eph gr Fa S S S S S S S S S S S S samplel06 samplell4 samplel22 samplel30 samplel38 samplel46 samplel54 samplel62 samplel70 sample178 samplel86 samplel94 Sef eye le aos gP a Goe eye en G9 aor G11 G2 S S S S S S S S S S S S H rr sud samplel15 sample123 samplel31 sample139 samplel47 samplel55 sample163 sample171 samplel79 sample187 sample 9 Name your samples by highlighting the number of wells in your plate and naming the sample in Customize Sample Info box Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 273
111. 09 1 Clean Drip HE Maintenance S Clean Aiuitecampler HIGH 22 Mar 2009 1 Clean Aut X notifications z Restart computer Inskru MEDIUM 22 Mar 2009 1 Restart co X 13 Defragment Hard Driwe MEDIUM 22 Mar 2009 1 Defragme e E 236 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Instrument operational procedures Change the anode buffer container ABC For the following hazard s see the complete safety alert descriptions in Specific chemical alerts on page 333 a WARNING CHEMICAL HAZARD Anode Buffer Container ABC For details see Instrument reagents and consumables on page 9 Contamination might cause poor quality data To prevent the contamination use genuine packaged polymer anode buffer cathode buffer and conditioning reagent l Za Remove the ABC from storage Check for expiration date on the ABC label to make sure 1t 1s not expired prior to or during intended use Allow refrigerated ABC to equilibrate to ambient temperature prior to first use Do not remove the seal until you have completed step 5 below IMPORTANT Ensure that all the buffer is moved to the larger side of the ABC prior to removing the seal Verify that buffer level is at or above the fill line and check that seal 1s intact IMPORTANT Do not use if buffer level is too low or seal has been compromised A fill tolerance of 1 mm is acceptable Tilt the ABC slightly as shown in th
112. 1 defined 171 export 141 import 141 in QC protocol 186 in sizecalling protocol 180 large fragment non linearity 183 normalization 171 part numbers 260 sizecalling protocols create 179 defined 179 export 141 import 141 in assay 150 sizing curve overlay 95 sizing quality See SQ sizing quality Sizing Range fragment analysis 181 HID analysis 186 sizing report 95 sizing results fragment HID label peaks 94 Slope Thresholds Peak Start and End 182 187 Smoothing 181 186 software without instrument 19 software See 3500 Series Data Collection Software solvents safety 318 sort library 142 multi column 142 spatial calibration export 101 historical reports 102 perform 99 purpose 99 signing 228 troubleshoot 300 when to perform 99 specimen specifying 49 spectral calibration AnyDye 108 borrowing event limits 34 capillary sharing 112 condition number 109 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide estimated run time 103 evaluate data 110 examples 114 export 115 historical reports 116 history 117 instrument prepare 104 not retained for new polymer type 106 perform 106 plate prepare 104 pull down peaks 112 purpose 103 quality value 109 report 116 required well positions 105 signing 228 standards 104 troubleshoot 301 what occurs 112 when to perform 103 zoom 111 spectral pull up flag 89 SQ sizing quality and broad peaks 188 and normalization 183 188 flag in monitor run 64 flag in view fragment
113. 102 Spectral caliDEFAt rnt x vu ce Pee a a RS EN AC Boe a ee UL 103 When to perform a spectral calibration llle 103 Prepare for the spectral calibration llle 104 Perform a spectral calibration 0 0 cc ee eee 106 Evaluate the spectral calibration data 0 0 ccc es 110 What you see during a spectral calibration o o oooooooooooo 112 Example spectral calibration data 0 000 ces 114 Export spectral calibration results llle 115 View and print a spectral calibration report ooooooonomoooooo 116 Save historical calibration reports pdf for record keeping 116 View the spectral calibration history 0 000 ee ees 117 Section 2 Performance check 0 0 ee eee eee 119 Run the sequencing install standard performance check 119 Prepare for the sequencing install standard performance check 119 Run the sequencing install standard performance check 122 What you see duringarun Le tees 124 How the software determines passing and failing capillaries for the spectral calibration 125 How the software determines passing and failing capillaries for the sequencing performance CHOCOK sti atm Ac nde EE a Seve ao eit CAS toa CR LIAC OR CURA Pe 125 Evaluate sequencing install standard data lllsn 126 Example sequencing install standard re
114. 13_G5 Sample GS600LIzZ TM13_G5 m 6 E03 fsa mm SERE i EET 7 EXE mem Data au 120 160 200 40 2a 320 360 400 au 120 160 360 400 3000 3000 000 ing 8 CO02 fsa Analyzed Data i Ing 9 C03 Fsa Analvzed Data 120 160 60 320 360 400 120 160 00 40 ea 320 360 400 B 52 1ng 7 CO1 fsa 327 21 1246 7962 B 55 1ng 9 CO3 fsa 327 18 1369 8360 B 55 ing 6 B03 fsa 327 16 1218 7066 Y 68 1ng 8 CO2 fsa 325 75 59 424 G 57 ing 6 B03 fsa 325 5 1190 8002 8 57 1ng 8 CO2 fsa 325 49 1230 8275 ss 6 57 1ng 7 C01 fsa 325 45 1224 8036 88 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Review previously run samples Review Fragment HID Analysis results If you access the View Fragment HID Results screen when no run is in progress and no plate is linked no samples are listed If the plate from the most recent run is linked the results from that plate are displayed To view results for samples other than those from the most recent run click Note By default the Fragment Samples view is selected If you 7 ia Print gt li Import e are importing HID files click HID Samples Review sample quality 1 In the samples view click the Table Settings button then specify the columns to show or hide Table Preferences 2 Double click Offscale Pull Up fragment Broad Peak HID and SQ columns to sort suspect and failing flags to the top of the table Available Columns Fa Mier
115. 14 18 4 COSBAC2 v3 lfwd8 A1 v3 1ShortReadS v3 1 Sequencing KB 1 4 1 4 KB 3500 POP7 BDT 14 01 5 B04 hCV11168429 StdSeq50 POP6 v3 1 BDTv3 1 PA Protocol KB 1 4 1 6 KB 3500 POP6 BDT 13 81 6 B09 hCV1119233 9 StdSeq50 POP6 v3 1 BDTv3 1 PA Protocol KB 1 4 1 6 KB 3500 POP6 BDT 13 76 7 CO1BAC7 v3 1fwd7 A2 v3 1RapidSeq50 v3 1 Sequencing KB 1 4 1 4 KB 3500 POP7 BDT 14 18 8 COSBAC2 v3 lfwd8 A1 v3 1ShortReadS v3 1 Sequencing KB 1 4 1 4 KB 3500 POP7 BDT 14 01 3 i ms i Mm gt T ab Trace fal Al EE CY S dl a i Set Tab Key to LI 43 p ab BO4 h 68429 1 53 ab Copy 10 of BO9_ ab Copy 10 of CO1B ab Copy 10 of COSB KE m Coordi nates x y 1151 9 1650 1700 1750 1800 1850 1900 1950 2000 2050 2100 2150 2200 2250 2300 2350 A A TT Analyzed Raw Analyzed Raw Annotation Sequence EPT Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Review Sequencing Results Review Ifyou access the View Sequencing Results screen when no run is in progress and no previously run plate is linked no samples are listed If the plate from the most recent run is linked samples _ the results from that plate are displayed To view results for samples other than those from the most recent run click Review sequence quality 1 Display Metric Analysis results to review sample basecalling and trimming results
116. 2 This feature attenuates signal variations associated with instrument capillary array sample salt load and injection variability between capillaries and instruments Normalization can be applied during primary analysis of the data Not Normalized Normalized 2000 2000 Instrument A 1500 1500 Instrument A Instrument B 1000 1000 Instrument B 500 500 UL U Figure 2 Comparison of fragment analysis results with and without the normalization feature To use the normalization feature prepare each sample with the GS600 LIZ v2 size standard then specify the appropriate normalization size standard for file primary analysis The GS600 LIZ v2 reagent can function as an internal standard for signal height normalization as well as a size standard for peak sizing When to use the normalization feature The 3500 Series Data Collection Software provides three normalization size standard definition files that you can specify for primary analysis of samples prepared with the GS600 LIZ v2 size standard e Fragment GS600LIZ Normalization GS600 60 600 LIZ Normalization For applications that have primer peaks that obscure the 20 and 40 mer peaks of the GS600 LIZ v2 size standard HID GS600 80 400 LIZ Normalization Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 7 Chapter 1 Ins
117. 2000 E ables Informati ansumables Information o Polymer POP4 384 Samples Remaining 56 Ol Jan 2010 11 514007 4315930 Anode Buffer AB boo Buffer 299 Dans Remaining amp 6 01 Jan 2010 02 51 B 34007 72Y8931 Cathode Buffer AB 3xxx Buffer 299 Danes Remaining amp 6 01 Jan 2010 02 8751 6TH B CE 431A 01 Capilsry Array 36cm 24 cap 105 Injections Remaining amp 6 01 Jlan 2010 11 B0K006 4319899 Serial E 8062450 Calibration Information Capillary Array 90K7450 o Spatial ID Spakia Foun Z003 03 03 14 43 X2 Calibration Date 03 Mar 2005 02 53 38 PM Spectral Sendwd 04 Mar 2009 08 09 26 PM Run 2009 03 04 20 07 29 198 6 1 reseau astra Figure 6 Load Plates for Run 2 Review the consumables information and the calibration information and ensure the status is acceptable for a run 56 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Load plates for run and create the injection list 3 Enter a Run Name or use the default run name Start Instrument Run Date Time Stamp gt Y Y Y Y MM DD hh mm ss SSS milliseconds for example Run 2009 02 05 15 03 42 096 where the run start date is February 5 2009 and the run start time 1s 15 03 42 096 Note An instrument run begins when you click Start Run on the Load Plates for Run screen and ends when the last injection on the last plate has completed For example 1f you link two plates then start the run both plates and any duplicate injections
118. 3 HID e QC Protocol Protocol for primary analysis peak detection and sizing and quality determination For information see QC protocols library primary analysis HID on page 184 e GeneMapper ID X Protocol Optional protocol for secondary analysis auto analysis For information see HID analysis protocols library secondary analysis on page 195 150 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide File name conventions library File name conventions library File name convention overview A File Name Convention FNC specifies the naming convention for sample data files It is an optional component in a plate If you do not specify a file name convention data files are named in this format lt sample name gt _ lt well gt The file extension is determined by the application you run Sequencing abl you can also set Preferences to export additional file formats See Set sequencing preferences on page 36 Fragment analysis fsa e HID hid Note The file location specified in a file name convention is used only if a results group is not specified for a well When you set up a plate for a run you can optionally add file name conventions to the plate If you add this item from the library a copy of the item is added to the plate and can be modified independently from the original item stored in the library For information on how changes are tracked if
119. 3500 3500xL Genetic Analyzer User Guide 329 Appendix F Safety Chemical waste safety Chemical waste hazards CAUTION HAZARDOUS WASTE Refer to Material Safety Data Sheets and local regulations for handling and disposal A WARNING CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical waste To minimize the hazards of chemical waste safety guidelines Waste disposal 330 Read and understand the Material Safety Data Sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage Minimize contact with chemicals Wear appropriate personal pr
120. 3500 3500xL Genetic Analyzers and 3500 Series Data Collection Software provide integration between the instrument and secondary sequencing analysis software applications specifically SeqScape Software v2 7 and MicroSeq ID Software v2 2 Using auto analysis samples are loaded sequencing data is generated and basecalling along with secondary analysis 1s performed according to the protocols assigned to the plates prior to the run Software Purpose SeqScape A comprehensive resequencing tool designed to detect SNPs profile mutations perform medical sequencing identify haplotypes subtype pathogens and confirm clone constructs MicroSeq ID LA comparative sequencing tool for microbial identification of bacteria and fungi Auto analyze projects in the sequencing analysis software Auto analysis can only be performed on the same computer that collects the sample files therefore SeqScape or MicroSeq ID Software must be co installed and configured with the 3500 Series Data Collection Software on a Windows Vista operating system Automated basecalling occurs with KB Basecaller v1 4 1 calls pure or mixed bases with quality values and secondary analysis occurs with SeqScape or MicroSeq ID Software This procedure initially describes how to set up panels and bin sets in SeqScape and then describes how to auto analyze samples using the 3500 Series Data Collection Software Once a run is complete your data is seaml
121. 3500xL Type 3 Length cm min CRL S Microbial Sequencing 50 POP 6 lt 135 gt 80 gt 240 gt 600 MicroSeq_POP6 Throughput Samples Day The total number of samples run in 23 hours 0 5 hour for User interaction and 0 5 hour for warm up time The maximum number of contiguous bases in the analyzed sequence with an average QV gt 20 calculated over a sliding window 20 base pairs wide from an AB Long Read Standard sequencing sample This calculation starts with base number 1 The read length is counted from the middle base of the 1st good window to the middle base of the last good window where a good window is one in which the average QV gt 20 Capillary array and polymer fragment and HID analysis run modules Table 34 Capillary array and polymer fragment and HID analysis run modules Configuration 23 hours Throughput Performance Run Modules Type Sizing Precision amp Capillary Polvmer Run Length Time 3500 3500xL Range Run Modules Name fem Type min Je 5Obp 401bp 601bp 400bp 600bp 1200bp Fragment analysis 50 POP 7 x40 2280 2840 lt 40to 0 15 0 30 NA FragmentAnalysis5O_POP7 nel Fragment analysis 50 POP 6 lt 100 2112 2336 lt 20to lt 0 15 lt 0 30 NAH FragmentAnalysis50_POP6 eM Long fragment analysis 50 POP 7 lt 125 gt 88 2360 lt 40to 0 15 0 30 0 45 LongFragAnalysis5O0 POP7 be
122. 5 04 15 acceptable 10 51 A lied Report created on 04 May 2009 03 14 16 PM Biosyste S 3500 Data Collection Sotware Version 1 0 0 E Signature Detailed Report 1 User Name Administrator User Full Name Administrator Object Type Approve Spatial Calibration Object Name Spatial_Run 2009 05 04 15 10 51 Date 04 May 2009 03 11 47 PM Comments Spatial calibration is acceptable Eai t Detail Esignature Type Approve Spatial Calibration Signed By Administrator Full Name Administrator Signed On 04 May 2009 03 11 47 PM Authority User Account Administrator User Role Administrator Object Details 5 Intensity 165 551328912 1 1736 11 74485 2092 30 6 6 27 44 9 3 8 38 742 1 3 5 21 3 456530201 12513230 1304 34 0 5 7 5 7 314 6 RunID Spatial Run 2009 05 04 15 10 51 7 Number of Capillaries 24 8 Locked false 9 Instrument 13527 029 10 Capillary Array 80K0850 3 In the Report screen click toolbar options to manipulate the report as needed Place the mouse pointer over an item for a description of the Page 1 of 1 GO00 Modify settings For this report k Modify report settings x item Font settings 14 l 4 To print the report click Print Select the Font to be used in reports 5 To save the report electronically pdf print the report and select CutePDF Writer as the printer 222 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Generate e signature reports 6 Close the
123. 75 specify settings see Table 12 on page 175 Assays Basecalling Pr BOT 1 BDTv3 BOT ya MicrosE File Mame Conventions 4 Click QV Settings In the QV Settings tab of the Create New Basecalling Protocol dialog box Figure 25 on page 177 then specify settings and Table 13 on page 178 Results Group ili Arratyze Instrument Protocols Dye Sets Size Standards 5 Click Save in m um fae P E e Basecalling Protocols Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Basecalling protocols library primary analysis sequencing ES Create New Basecalling Protocol Setup a Basecalling Protocol E Protocol Mame is a required field Provide a unique value Basecaller KB1 4 1 Ch co Analysis Settings Q Settings t Mobility File k Quality Threshold k Mixed Bases Threshold Analyzed Data Scaling k Clear Range Methods Summary of current settings Mobility File KB 3500 POP BDTv3 Quality Threshald Do not assign N s to Basecall Mixed Base Threshold 25 0 Scaling True Profile Clear Range Methods Use quality values Close Figure 24 Create New Basecalling Protocol Analysis Settings Table 12 Basecalling protocol Analysis settings Setting Description Name Name of the protocol Names must be unique Locked When enabled allows the entry to be unlocked and modified only by the user who create
124. 80 400 Normalization 1233868687454 hid C I 3 Example S aL11 65_15 80 400 Normalization 1233868687454 hid E amp gt Run 2009 02 05 14 56 03 468 E sample G5 LS 80 400 Narmalization 123 3868687454 2 hid E Run 2009 02 05 14 59 56 703 El sample G5 L5 80 4300 4 Marmalizatian 1233868687454 hid t R Example 2009 02 05 15 03 42 4dministrator E sample 65_L5 80 400 Normalization 1233868687469 2 hid c3 Inji 2009 02 05 15 03 29 610 Ej sample i 5 L 5 80 400 4 Marmalizakion 1233868687469 3 hid 5 Inj 2009 02 05 15 03 29 626 s sample G5_LS S80 400 Mormalization 1233868687465 hid E Inja 2009 02 05 15 03 29 626 Injd 2009 02 05 15 03 29 62 E 5 Injs 2009 02 05 15 03 29 62 y Inj amp 2009 02 05 15 03 22 626 Y Inj 2009 02 05 15 03 29 642 Figure 20 Folder hierarchy and file naming example File location from results group 2 custom file location CilExample Instrument Run Name folder from results group Include an Instrument Run Mame Foldet 1 2 3 Results group Name folder from results group Include a Result Group Name Folder 4 Injection folder from results group Include an Injection folder Duplicate injections indicated with _n where n is the number of duplicates Run name default or user defined from injection list Run Mame Run 2009 02 05 14 59 56 70 6 Results group name syntax from results group RG Example 4 lt Start Instrument Run Date Time Stamp gt
125. 99 313 link plate 304 309 load plate 309 log files search and use 305 manual commands 313 monitorrun 311 red indicator status light 23 review results fragment HID 311 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 349 Index RFID 304 sequencing install performance check 302 spatial calibration 300 spectral calibration 301 view instrument sensor details 305 True Profile 176 U uninstall the software 254 Uninterruptible Power Supply 8 unlink plate 57 user account activate suspended 203 create or edit 201 delete 203 inactivate 203 permissions 203 user attention words described xvi user role create 203 user defined fields displayed in table view only 48 including in file name 154 specifying for samples 48 V validation allelic ladder 52 verification runs See fragment HID performance check sequencing performance check view fragment HID results See review results fragment analysis results HID analysis results view run results See review results view sequencing results See review results sequencing results W WARNING description xiii warnings metric analysis 81 Wash Pump and Channels maintenance wizard 249 waste disposal guidelines 330 waste profiles description 330 weekly instrument maintenance 231 weighting SQ 188 well attributes assign plate contents 46 save in template 75 workflow diagram 21 79 workstation safety 321 Z ZOOM 350 fragment HID plots 92 plate vi
126. Analyzer User Guide Spectral calibration 1 Click View Spectral Calibration Report 2 Click Print 3 In the Printer dialog box select CutePDF Writer as the printer 4 Specify a name and location for the report View the spectral calibration history Select History View then select a dye set to view the associated calibration history E Calibration Run 9 Hi Calibration Information uy Dye Set hemistry Standard calibration Date wi Capillary n F Matrix Standard 20 Jan 2009 11 38 52 AM BOK 2450 35 Matrix Standard 13 Jan 2009 10 11 36 PM SOK 2450 T Capillary Run Data Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 117 Section 1 Calibration 118 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Spectral calibration Section 2 Performance check The Performance check allows you to periodically self check the instrument system using Applied Biosystems standard Run the sequencing install standard performance check When to perform When your instrument is installed the service engineer runs a sequencing install standard performance check Applied Biosystems recommends that you run the sequencing install standard performance check monthly to verify that the instrument meets read length specifications The Sequencing Install Performance check has an option to include and save the spectral calibration If you select this option and you accept the sequencing inst
127. BC is stable at this temperature until the expiration date shown on the label Once the seal is peeled the running buffer is stable at ambient temperature for up to 7 days Ensure that the seal remains in place until just prior to use on the instrument To ensure optimal performance the use of the CBC is limited to either 7 days after the first installation or 120 injections on a 3500 8 capillary 50 injections on a 3500xL 24 capillary whichever comes first When notified of the limit by the instrument software you have to replace the ABC with a new one before you can proceed further For more details see the product insert included in the product package See Change the cathode buffer container CBC on page 238 for instructions on how to change the CBC The polymer for 3500 or 3500xL analyzer is available as a ready to use pouch with either POP 4 POP 6 or POP 7 polymer as the separation matrix IMPORTANT Do not use a polymer pouch that has been installed on one type of instrument on another type of instrument For example if you install a new polymer pouch on a 3500 8 capillary instrument do not use that polymer on a 3500xL 24 capillary instrument The pouch has adequate polymer to support the stated number of samples 384 or 960 or injections and additional volume to handle a limited number of installations and setup related wizard operations Incorporated into the label 1s a radio frequency identification RFID t
128. Biosystems instruments see Safety symbols on page 315 MSDSs The MSDSs Material Safety Data Sheets for any chemicals supplied by Applied Biosystems or Ambion are available to you free 24 hours a day For instructions on obtaining MSDSs see MSDSs on page 329 IMPORTANT For the MSDSs of chemicals not distributed by Applied Biosystems or Ambion contact the chemical manufacturer Applied Biosystems 3500 3500xL Genetic Analyzer User Guide xiii Preface Safety labels on The following CAUTION WARNING and DANGER statements may be displayed instruments on Applied Biosystems instruments in combination with the safety symbols described in the preceding section Hazard symbol English Francais ZN CAUTION Hazardous chemicals Read the Material Safety Data Sheets MSDSs before handling ATTENTION Produits chimiques dangereux Lire les fiches techniques de s ret de mat riels avant toute manipulation de produits CAUTION Hazardous waste Refer to MSDS s and local regulations for handling and disposal ATTENTION D chets dangereux Lire les fiches techniques de s ret de mat riels et la r gulation locale associ es la manipulation et l limination des d chets CAUTION Potential slipping hazard ATTENTION Risque potentiel d avoir un sol glissant CAUTION Hot surface ATTENTION Surface br lante PEE DANGER High voltage DANGER Haute tension WARNING To r
129. C protocol an audit record for the change considered a deletion is listed in the Object Audit History 210 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Display the object history Generate audit reports To display the history for an object select the object then click Show Object History The object history shows the audit history for the object and for all objects contained in the selected object For example when you create an assay a copy of the instrument protocol the primary analysis protocol and therefore dye set and size standard and the secondary analysis protocol are included in the assay object The objects contained within an object have audit histories distinct from the audit history of the objects stored in the Library Review the system configuration history Table 23 Audit system configuration history The System Configuration History lists SAE configuration records Note The Reason field in System Configuration History is not used Record Type Action Corresponds To Security settings Update Enable security Disable security Modify security policies Session timeout settings Account settings Audit reason for change Audit settings Audit type Audit type Update Update Create Delete Update Update Update Modify user name settings Modify password settings Modify security policies Password expiration Account suspension Mo
130. CBC a Align the buffer septa the part that is symmetrical over the 24 holes of the CBC b Push the septa lightly into the holes to start and then push firmly to seat the septa Install the CBC on the autosampler Note When properly installed 1t will click on the autosampler as the tabs are snapped in place Close the instrument door to re initialize Click Refresh from the Dashboard to update the screen Check the Quick View section of the Dashboard for updated status after changing the CBC Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 239 Chapter 8 Maintain the Instrument Check stored capillary arrays 240 IMPORTANT Wear appropriate protection including gloves laboratory goggles and coat whenever you work with the fluids used on this instrument or parts that may come into contact with these fluids When the capillary array is installed electrodes at the bottom are inserted on the CBC The electrodes at the top connect with the polymer delivery pump Applied Biosystems recommends you keep the electrodes on the bottom in the tray with 1X running buffer For details see Instrument reagents and consumables on page 9 IMPORTANT Keep the loading end of the capillary array in 1X running buffer to prevent the polymer from drying in the capillaries If fluid level 1s low add distilled water DI to buffer solution Refer to the Install capillary wizard for instructions on how t
131. Click Create Library Resources p date 3 In the Create New MicroSeq ID Protocol dialog box Figure 31 on page 192 specify settings see T Filter Table 19 on page 192 Assays El Protocol Marr File Mame Conventions 4 Select the remaining secondary analysis items then click Save Results Group Note If the project or specimen of interest 1s not displayed in a list re select the secondary analysis software instance to update the list ilii Analyze Instrument Protocols Dye Sets size Standards Basecalling Protocols Sizecalling Protocols IMPORTANT The auto analysis settings you specify for the plate to run with this protocol must contain the same secondary software and location settings For 3 more information see Create a new plate on page 144 Gui Protocols Sequencing Analysis Protocols a bant m Fr ae rA j 2 MicroSequn Protocols Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 191 Chapter 6 Manage Library Resources S Create New MicroSeqID Protocol Setup a MicroSeq ID Protocol e Protocol Name is a required Field Provide a unique value Protocol Mame Description Application Type Seo Secondary Analysis Software Secondary Analysis Software Instance we Properties Close Figure 31 Create New MicroSeq ID Protocol Table 19 MicroSeq ID Analysis protocol settings Setting Description Protocol N
132. Click GeneMapper Manager to open the GeneMapper Manager 4 Name your Analysis Method General tab ver el Analysis Method Description General Peak Detector Peak Quality Quality Flags uU Instrument 3500xL Analysis Type Microsatellite pt Bin Set Marker Repe Use SNPlex_48plex_Bin_3130 SNPlex_48plex_Bin_3730 Values For dinucleotide repeats are calculated automatically 282 Mono Tri Tetra Penta Hexa Cut off value 0 25 o2 j 0 25 Plus ratio 10 0 0 95 0 95 0 95 PlusA distance oo 16 he fe Stutter ratio oo 095 015 0 15 Stutter distance From 0 0 00 0 0 oo foo To 00 3 5 45 ls es Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Set up an auto analysis project in GeneMapper 6 Select your Peak Detector Algorithm as Basic Advanced or Classic Peak Detector tab IMPORTANT If you want to enable size standard normalization you must select Advanced Analysis Method Editor Microsatellite General Allele Peak Detector Peak Quality Quality Flags Peak Detection Algorithm Bag Minimum Peak Height Advanced Classic Automatic 5 User specified rfu 50 50 50 50 50 50 7 Customize your Peak Quality and or Quality Flag settings in the appropriate tab then close the Analysis Method Edit
133. D Status To check the status go to My Computer gt right mouse click on C drive gt Select Properties Click General tab 256 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Review the Maintenance Notifications Log Local Disk C Properties PRIX General Tools uota Note The Data Collection software will prompt you when it is 70 75 full At 78 full the software will not start a run If there is insufficient space e Archive the sample files e Delete the sample file data from the drive D and empty the contents of the Recycle Bin Defragment the This option can be set as a reminder in the scheduler The fragmentation of files computer hard decreases the performance of both the Data Collection software and the computer drive operating system Programs take a longer time to access files by performing multiple search operations of the fragments Go to Start Programs Accessories System Tools Disk Defragmenter and follow the prompts Note You can click Analyze to see if you should defragment or not Check available Before arun the Data Collection software checks free disk space If adequate free space on all disk space is not available to store the data the Data Collection software displays the drives following message Remove data the drive is getting full View the errors that appear for generated errors and in the Event Log window See Appendix E Troubleshoot on
134. D in the File Convention Color Fail or Suspect range Red marks are displayed for e Sample Name wells with offscale data du ee ell Position e Place the mouse pointer over a well to display sample Injection details CED Sampasd Samp 6z Samp 9 Samp Sampes5 same olaa 3 3 4 4 4 1 1 Samp4 well cna Sample Mame SampA5 Sample Type Sample Samp Assay Mame HID36 POP4 65 Normalization_LS File Convention Mame General File Mame 3 Result Group Mame IdentiFiler Injection 3 Check sequence or sample quality and specify re injections When an injection is complete it is flagged with in the Injection and Analysis columns If the software detects a problem with offscale data or low quality samples the injection is also flagged with Es Paw Tois 00 00 00 Tr mn1 o m EFI cA cec Note If the Injection Analysis or Flags columns are not displayed you can click the Table Settings button then show them in the injection list Check sequence or sample quality 62 1 Expand the Flag pane at the bottom right of the screen Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Check sequence or sample quality and specify re injections Connection Status Connected User Name Administrator Last Login Time OF felar 20009 11 27 35 AM Injection List Details o 8 injections created E in Place A Qin Place E Be same sam sam sang same SARE 3535 3 5 4 6 4 6 4 6 Sa
135. D X Software which considers broad peaks in sizing quality Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 311 Appendix E Troubleshoot Review error message details Error messages in the 3500 Series Data Collection Software include a Details button Click Details to display more information about an error message E Instrument Run zi a Pre run validation check failed Consumable Anode Buffer has exceeded recommended time on Instrumeri l Installation date lan 1 009 Recommended replacement Jan 8 2009 Consumable Cathode Butter has exceeded recommended time on instrument Audit troubleshooting Symptom Possible Cause Action Export did not complete You exported records for samples Return sample data files to their successfully that are not in their original location original location then export again samples have been deleted or moved Electronic signature troubleshooting Symptom Possible Cause Action Electronic signature prompt is Electronic signature prompt is No action displayed when you edit sample displayed for sample comments comments regardless of the electronic signature setting 312 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Manual commands troubleshooting Manual commands troubleshooting Symptom Possible Cause Action When you select Tools Manual Commands Set defined command for Consumables t
136. Do not use if pouch label is damaged or top seal 1s missing 2 Peel off the seal at the top of the conditioning reagent pouch fitment 3 Insert the pouch fitment on to the slot of the pump lever mechanism Push the lever up to snap the pouch into the connector end of the instrument pump Note The RFID label must be facing the instrument and not you to ensure that the RFID information is read accurately by the instrument 4 Follow the wizard for further instructions HFID label must 5 Click Refresh from the Dashboard to face the instrument update the screen 6 Check the Quick View section of the Dashboard for updated status after changing the Conditioning Reagent 250 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Use the Maintenance Wizards to perform operations Fill capillary array with fresh polymer For the following hazard s see the complete safety alert descriptions in Specific chemical alerts on page 333 WARNING CHEMICAL HAZARD POP 4 POP 6 and POP 7 polymers For details see Instrument reagents and consumables on page 9 The filling of the capillary array with fresh polymer is dictated by the instrument wizards 1 To fill capillary array with fresh polymer same type of polymer click Fill the Array with fresh Polymer 2 Follow the prompts in the Fill Array m Fil Array Wizard Wizard window jm s 3 Click Refresh from the Dashboard to update the screen
137. For longer durations optional battery cabinets can be added to the base PPS unit A base unit PPS rated for 800W can provide over 20 minutes of backup protection and over 2 hours when a single battery cabinet was added Note Battery output can be affected by temperature and the age of battery so these backup times are not guaranteed 8 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Instrument reagents and consumables Instrument reagents and consumables For application specific reagents consumables and run modules see Appendix A Application Reagents and Run Modules Anode buffer container ABC The ABC PN 4393927 contains 1X running buffer to support all electrophoresis applications on the 3500 or 3500xL analyzer The ABC is made in a ready to use disposable container with a radio frequency identification RFID tag incorporated into the label It has a built in overflow chamber to maintain constant fluid height For the following hazard s see the complete safety alert descriptions in Specific chemical alerts on page 333 AN WARNING CHEMICAL HAZARD Anode Buffer Container ABC Store the ABC at 2 C to 8 C until ready to use The sealed ABC 1s stable at this temperature until the expiration date shown on the label Once the seal is peeled the running buffer 1s stable at ambient temperature for up to 7 days Ensure that the seal remains in place until just prior to use on the instrument To ens
138. GeneMapper D X Software may identify alleles not identified by the 3500 Series Data Collection Software because of the bin offsetting feature which uses the observed alleles in the allelic ladder samples to adjust the reference bin locations for samples If the alleles are not properly called Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Run the fragment analysis or HID Install standard performance check Optional Click t View Detail Report to save a record of the failed run To save the report electronically pdf print the report and select CutePDF Writer as the printer For more information see Save historical performance check reports pdf for record keeping on page 138 Click Reject Results Rerun the install standard to determine 1f the problem may be caused by sample preparation a poor injection a capillary issue or a system problem which may require instrument service For more information see Fragment HID install standard troubleshooting on page 303 Example fragment install standard results 28 30 3230 3630 1630 2030 2430 Example HID install standard results 250 3350 3750 4150 4550 4950 3350 3000 000 1000 View previously run install standards Select History View then select an install standard to view the associated calibration information View and print a fragment or HID install standard detail report IMPORTANT Ensure that all dyes
139. General library Proc dures vencio ecards ecards 140 ACCOSS DEMOS E ET 140 Create a new entry from a factory provided template or locked entry 140 Delete aldibrary entry iaa e AA ERR AAA vg d 141 EGIL AID FAR ONU wer PERCULIT 141 Import and export a library entry 0 es 141 View audit and e signature histories for library entries 142 Sort filter and search library entries llle 142 Custornize a librany Table ae a eee eS ad 143 ES A O O A Bog ts Oe 143 Plate OVelVIGW uos di A aa A 143 Create a NEw plate 4 53 33 das e qd eg dd A ee cm ee ed od 144 ASSAS IDAN ad o Mo id 147 ASSAY OVEIMICW AAA tadas 147 Create a new ASSAY ee eee eee etna 148 File name conventions library 2 0 0 0 ee ees 151 File name convention overview 1 6 ees 151 Create a new file name convention 0 0 0 ccc ee ees 151 FR SUI Group Dra E A Sgn as et Aa 155 Results Group OvervieW 239 A S ad p ee Oe ee A 155 Create a new results group 6 eens 156 Results group example 1 store files by plate name oooooooo 160 Results group example 2 store one allelic ladder per run folder 8 capillary NSTUMENS ua esa se one gs a as eater de Sra Ata S a eua A ao eter 161 Results group example 3 store re injections in separate folders 162 Instrument protocol library 2 0 eee ees 165 Instrument protocol overview eee ete 165 Create a new instrument pr
140. HID 36 POP 4 lt 35 2912 2936 60 to 0 15 NA NA HID36_POP4 2400 HID 36 POP 7 26 2424 21272 60 to 0 15 NA NA HID36_POP7 un SNaPshot 50 POP 7 lt 30 29 6 21104 40 to 0 50 NA NA SNaPshot50_POP7 mae Throughput Samples Day The total number of samples run in 23 hours 0 5 hour for User interaction and O 5hr for warm up time 264 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Run modules Resolution Range The range of bases over which the resolution peak spacing interval divided by the peak width at half max in a GS600 or GS1200 LIZ size standard sample sized with a third order fit is 21 The table shows the resolution range in 29096 of samples Sizing Precision Standard deviation of sizes for one allele in the DS 33 install standard sized with the GS600 LIZ size standard across multiple capillaries in the same run For one injection to pass 10096 of the alleles in that injection must meet the intra run sizing precision specifications The table shows the sizing precision of 10096 of alleles in 29096 of samples t Not applicable because of the size of the fragments collected in the run Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 265 Appendix A Application Reagents and Run Modules 266 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide secondary Analysis Sequencing Perform secondary analysis on sequencing experiments The Applied Biosystems
141. I l E a to C a Is ibrary Mainten liftate Filter ml Protocol Narn 193 Chapter 6 Manage Library Resources MM Create New Fragment Analysis Protocol Setup a GeneMapper Protocol B Protocol Name is a required field Provide a unique value Protocol Name C Locked Description Application Type Fragment Analysis Secondary Analysis Software Secondary Analysis Software Instance Properties Analysis Method Size Standard Panel Figure 32 Create New Fragment Analysis Protocol Table 20 Fragment Analysis protocol settings Setting Description Protocol Name Name of the protocol Names must be unique Description Optional text entry Lock When enabled allows the entry to be unlocked and modified only by the user who created it the administrator or another user with unlock permissions Useful when your system includes the SAE module described in Chapter 7 Use Security Audit and E Sig Functions SAE Module on page 197 Application Type Automatically set to Fragment analysis Secondary Analysis Software IMPORTANT The secondary analysis software must be installed and properly configured with the 3500 Series Data Collection Software before it is listed as a selection in this screen Secondary Analysis Software Instance Computer on which the secondary analysis software is running Properties GeneMapper software
142. IF Norm_POP4_ xl HID36 POP4xl G5 Plate 01 IF Norm POP4 xl HID36 POP4xl G5 Plate 01 6 IF4 Morm POP4 xl HIDS6 POP4x G5 Plate 01 y IF Norm_POP4_ xl HID36_POP4d_G5 Plate 01 Figure 18 Injection list example Figure 19 on page 164 shows an example file name convention that specifies a sample name syntax of sample name primary analysis protocol name unique time stamp integer The numbers in the figure relate the elements in the file name convention with the files created by a run that uses file name convention Figure 20 on page 164 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 163 Chapter 6 Manage Library Resources Mame FAC Example Locked Select File Mame Attributes Preview of File Mame lt Sample Name gt lt Analysis Protocol Name gt lt Unique Time Stamp Integer Available Attributes Selected Attributes Amplicon Marne Sample Mare Assay Mame Dot gt Capillary Number ss Analysis Protocol Mame Custom Textl as Doti Custom Text Unique Time Stamp Integer Custom Texts E Mate nf Riim Figure 19 File name convention example Figure 20 on page 164 shows the folders and files generated by the results group file name convention run name and injections shown in Figure 17 on page 163 Figure 18 on page 163 and Figure 19 on page 164 9 CH Example Run 2009 02 05 14 59 56 7031RG Example 2009 02 05 15 03 42 4dministrator Inj 2009 02 05 15 03 29 642 X EJaL10 65_15
143. If the Bo c reagents of any well contain bubbles or are not located at the bottom of the well briefly centrifuge the plate remove the plate from the centrifuge and verify that each sample is positioned correctly in the bottom of its well Sample is at the bottom of the well 4 Store the plate on ice until you prepare the plate assembly and load the plate in the instrument Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Prepare and load sample plates Prepare the plate assembly IMPORTANT Prepare the plate assembly on a clean level surface Do not heat plates that are sealed with septa 1 Align the holes in the septa strip with the wells of the plate then firmly press downward onto the plate Plate retainer 2 Place the sample plate into the Plate with plate base septa strip IMPORTANT Make sure to use the correct plate base for Plate base standard plates versus 8 tube strips and fast plates Using the wrong plate base may affect performance 3 Snap the plate retainer cover onto the plate septa and plate base 4 Verify that the holes of the plate retainer and the septa strip are aligned If holes are not aligned re assemble and then assemble the plate assembly IMPORTANT The array tips will be damaged if the plate retainer and septa strip holes do not align correctly Load the plate in the instrument 1 Place the plate in the autosampler with the labels facing you or
144. Instrument Protocol Move Lp ror ee flores Select File Mame Attributes Preview of File Name lt Sample Name gt lt Capillary Number gt Available Attributes Selected Attributes 4 To add delimiters between items in the Selected Attributes list a Ctrl click or Shift click to select two or more attributes b Select a delimiter c Select the Add between attributes check box d Click Add 5 Save the file name convention e Ifyou are creating the file name convention from the Library click Save e Ifyou are creating the assay from the Assign Plate Contents screen click Apply to Plate or Save to Library 152 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide File name conventions library k Create New File Name Convention Setup a File Name Convention E Mame is a required field Provide a unique value Select File Mame Attributes Preview of File Mame lt Sample Name gt Available Attributes Selected Attributes Amplicon Marne Sample Marne Analysis Protocol Mame E Assay Mame Capillary Number Custom Texti Custom Text Custom Texts Date of Run Injection Number Instrument Mame Instrument Protocol Owner Mame ls Delimiters Select a delimiter Plus Add between attributes Add a custom value to available attributes optional Select File Location 5 Default File Location C Applied Eiosystems 3500 Data O custom Fis Location A Eres Figure 13 Create New File Na
145. N_Injfolder_R61Inj1 2009 03 22 13 19 21 211 X Ejalo1_401 hid E eration oc E E 5 Applied Biosystems Es test A02 hid ame _Inifolder I muon M oem a 6 3500 EM test BID1 hid m a helt Gen tan SES 4 El Ej Data m teme cd Press Grove fes case Rame P5 Inlfolder RG ea E test_BO2 hid a Selected Attributes pa A P lt t BOS hid la jinji 2009 03 22 13 19 21 211 te pe Plate Name E test CO1 hid 4 Dash C3 Injz 2009 03 22 13 19 21 227 ca Results Group Name 3 Inj3 2009 03 22 13 19 21 243 El Peste Sic a dot Du Inj 2009 03 22 13 19 21 243 ES test C03 hid ladd betwee attributes sen z Es test DO1 hid Legs A pg nne ad pp tue thee Peet cur us opor mp un Tebi ee m fella hime a 5 Store reinjection sample Files with original sample Files same level Default File location C Applied Biosystems 3500 Datal Plate Name PN Injfalder Ri lt Inj Folder gt Cuan Ple canon Include an Instrument Run Mame Folder Include a Result Group Mame Folder Lv Include an Injection folder Figure 15 PN_Injfolder_RG results group 160 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Result group library Me Edi Results Group PN Injfolder AG O a R nia larini E E C Applied Biosystems 3500 Data Plated Ph RG X es AL 01_A01 hid l el E Applied Biosystems Ag Es test_A02 hid Name PN RG Nis mia a 5 3500 E test A03 hid re Dep P
146. Outlicensing at Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA TRADEMARKS Trademarks of Life Technologies Corporation and its affiliated companies Applied Biosystems AB Design AmpF STR BigDye BigDye Xterminator COfiler GeneMapper GeneScan Hi Di Identifiler KB LIZ MicroSeq Minifiler POP 4 POP 6 POP 7 Profiler Profiler Plus SEfiler Plus SeqScape SGM Plus SNaPshot Variant Reporter Yfiler AFLP is a registered trademark of Keygene N V MLPA is a registered trademark of MRC Holland All other trademarks are the sole property of their respective owners Part Number 4401661 Rev C 06 2010 Contents IAC stc ato ace os io as dota acia der cacao rd eee are xiii Saleb IM ONMaANOMN scu a duce ds da A pe A ee Ve s Med ur hee an xiii ADOUT TNE DFOQOUGL Vii A a eae Ir aa Qe E XV Purpose or dillscellllele idet uio a oa a E EEA eee can XV leac PCT rp XV ASSUMPTIONS 22 4 5 Eos e 2789 13 a ea RE ga De a Aet Men adio Mag 3 4 d acea MEL edo Mte Bo XV HOW 1o Use this QUide diia da dd uid e OR dom Du Pe eec ceu Xvi HOW to ODEIaII SUDDOEE caine d i 3 Ie be ne pages bl eee ce PE ee e xvii Chapter 1 Instrument and Software Description 1 System descripHOFi sis seti is B las em aee sow dae EE iude board lcgi ice dS pA ews 1 Instrument CESCHDUON os d uester eom uy gera vos acte e E Cc OS eM RESCUE Bod aca eic 2
147. ROCOL Mame F_LSt 5 400 F L5 75 450 G5 3rd 8D 400 2 Mrc G5 3rd 8n 400 G5_LS 80 400 Nor 65 1550 4001 File Mame Conventions Results Group li Analyze Instrument Protocols Dye Sets Size Standards Bazecalling Protocols Sizecalling Protocals QC Protocols Taare Mali Deed ols 2 Click Create 3 In the Analysis Settings tab of the Create New QC Protocol dialog box Figure 28 on page 185 specify settings see Table 16 on page 185 4 Click QC Settings In the QC Settings tab of the Create New QC Protocol dialog box Figure 29 on page 188 specify settings Table 17 on page 188 5 Click Save 184 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide QC protocols library primary analysis HID R Create New QC Protocol Setup a OC Protocol B Protocol Name is a required Field Provide a unique value Protocol Mame Description Size Standard 65600 _L1Z Normalzation 80 400 y Sizecaller SizeCaller v1 1 0 v Analysis Settings ac Settings o Analvsis Range Ful v Sizing Range Full we Size Calling Method I Local Southern Analysis Start Point g Sizing Start Size O Analysis Stop Point 000000 Sizing Stop Size LOCODOL 7 Blue Green Yellow Red Purple Orange Peak Amplitude im 1 1175 Threshold l a Common Settings Use Smoothing Use Baselining Baseline Window Pts Minimum Peak Half Width Peak Window Size Polynomial
148. Restore Archive restore and purge are Purge described in Chapter 8 Maintain the Instrument Select Preferences in the menu ary Maintenance Tools Manage Preferences Hel bar to access the parameters for which you can set defaults Preferences allow you to set system and user defaults for settings such as the date format sample data file storage location export file formats for k Preferences hot used System Select one of tH E System Date Format sequencing data and a variety of Instrument Settings jon ilt sequencing specific settings Scheduler Preference gt Sequencing Settings System defaults apply to all users EE p Ppty Spectral Calibration El User User defaults apply to sie R ts Setti All users If your system does not am include the SAE module B Sequencing Settings Trace Each logged in user If your ucc ere a system includes the SAE module Trace Quality Ex Trace Quality Reports Preferences are described in Chapter 2 Start the System on page 21 Select Help in the menu bar to access 3500 Series Data Collection Software Help Preferences Help Help Contents About 3500 Data Collection Software Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Navigate the Software Overview of the 3500 Series Data Collection Software The Help provides quick access to brief information about how to perform tasks on a screen For details about tasks and othe
149. Select a color for this assay to display with in the Plate View 3 Select an Instrument Protocol to apply to the assay Note For more instruction on setting up an instrument protocol see Create a new instrument protocol on page 165 4 Select a Basecalling Protocol to apply to the assay IMPORTANT Make sure your basecalling settings match the Analysis Settings specified in SeqScape Note For more instruction on setting up a Basecalling protocol see Create a new basecalling protocol on page 174 5 Create a new sequencing analysis protocol to apply to the samples by clicking Create New Create New Sequencing Analysis Protocol Setup a Sequencing Analysis Protocol 2 Protocol Name Test already exists in the Library Q Protocol Name Test Locked Description Application Type Sequencing Secondary Analysis Software SeqScape Secondary Analysis Software Instance SegScape fospradhaaaD03 Properties Project p53 v2 Project Template p53 exon Specimen 12586 x Close 272 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Set up a SegScape plate in the 3500 Series Data Collection Software 6 Name your new sequencing analysis protocol then select your specimens one by one clicking Save after each specimen IMPORTANT Each SeqScape protocol has one specimen so you will need to create multiple protocols for multiple spe
150. Sequencing install standard performance check page 122 Fragment or HID install standard performance check page 132 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 21 Chapter 2 Start the System Start the instrument 1 Verify that the instrument is connected to the appropriate power supply CAUTION Do not unpack or plug in any components until the Applied Biosystems service representative has configured the system for the proper operating voltage See the Applied Biosystems 3500 Series Genetic Analyzer Site Preparation Guide 4401689 for details Note The purpose of the Site Prep Guide is to help you prepare your site for installation of the 3500 or 3500xL analyzer For specific details about your system please refer to this user guide IMPORTANT Do not rename the computer after the 3500 Series Data Collection Software has been installed The instrument computer has been assigned a unique name Changing the name may cause the 3500 Series Data Collection Software to malfunction 2 Inspect instrument interior Ensure that a The oven door is closed b No objects are left inside the instrument IMPORTANT Misplaced objects left inside the instrument can cause damage 3 Close instrument door 4 Turn on the instrument Press the power on off button on the front of the instrument and wait for the green status light to turn on a Press the Tray button on the outside of the instrument to bring the a
151. T CAUTION WARNING and DANGER see Safety alert words on page xiii General alerts for N WARNING Wear appropriate protection including gloves laboratory all chemicals goggles and coat whenever you work with the fluids used on this instrument or parts that may come into contact with these fluids IMPORTANT Use the cleaning agents as described in this manual only Use of cleaning agents not described in this manual can impair the instrument Please contact your local Life Technologies sales office if you have any questions e Read the MSDS for this product and follow the handling instructions e Avoid inhalation contact with eyes skin clothing and prolonged or repeated exposure Consumables have a limited lifetime Overusing the parts might result in poor quality data 332 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Safety alerts Specific WARNING CHEMICAL HAZARD POP 4 POLYMER Causes eye chemical alerts skin and respiratory tract irritation Avoid breathing vapor Use with adequate ventilation WARNING CHEMICAL HAZARD POP 6 POLYMER Causes eye skin and respiratory tract irritation Avoid breathing vapor Use with adequate ventilation N WARNING CHEMICAL HAZARD POP 7 POLYMER Harmful by inhalation and if swallowed Causes eye skin and respiratory tract irritation Do NOT taste or swallow Avoid breathing vapor or dust Keep container tightly closed Use only with adequate ve
152. Table 16 QC protocol Analysis settings continued Setting Description Peak Amplitude IMPORTANT Optimize these thresholds during internal HID validation Thresholds Specify the threshold RFU for peak detection for each dye color Peaks below the threshold are not detected For example if you use the default values of 175 peaks with heights equal to or greater than 175 are detected Peaks with heights below 175 are still displayed in the electropherogram plots but are not detected or labeled Note Ensure that the same peak amplitude thresholds are used in secondary analysis software such as GeneMapper v4 1 or later Min Peak Half Width Specify the smallest half peak width at full height for peak detection The range is 2 to 99 data points J Adjust to affect the sensitivity of peak detection You can adjust this parameter to detect a single base pair difference while minimizing the detection of shoulder effects and or noise 7 Polynomial Degree The peak detector calculates the first derivative of a polynomial curve fitted to the data within a window that is centered on each data point in the analysis range Using curves with larger polynomial degree values allows the curve to more closely approximate the signal and therefore captures more of the peak structure in the _ electropherogram Peak Window Size Enter a window width in data points for peak detection sensitiv
153. Troubleshoot l If the expected number of alleles and size standard peaks are found click Accept Results If the expected number of alleles and size standard peaks are not found troubleshoot as described below Click a capillary with fewer than the expected number of peaks to display detailed information for each allele in the table below the plot Double click the Size column to sort results and identify the alleles that were not found A 0 Size value indicates that an allele falls outside the expected size window Nominal x apilar Fun Gate Espected size standard Peak ir 21 Dsperted Abele Peak 7105 i iz i l amp ih i i m io IVIL 1i i 1 ty i i xe zt x n x J x x a L pesen oo Yoo L2 B osson oo Jos Lj jpssesamepyio 104 49 867 0 s jae js 93t 0 Size 0 7 bp or 0 5 for THOI Troubleshoot failing data a Analyze the install standard data files in your secondary analysis software GeneMapper Software v4 1 or later GeneMapper ID X Software Software v1 2 or later using Identifiler kit panels and bins b Evaluate the failed data and examine the alleles not found by the 3500 series Data Collection Software c Ifthe alleles are properly called in the secondary analysis software you can Deselect the Include checkmark for a capillary Click Recalculate Accept the install standard results Note The
154. Version 4 1 Choose the setup type that best suite pour needs Remote auto analysis option wall instal Ene auiccanalysis manager Lhilty to automaticaly analyze data collected from Dala Collecton system loo dant have a Osta Colection system available you should choose stand alone option D Stand alone E eel E Remote Autnanalysis Lf r BS itin Back Mex gt Cancel In the GeneMapper Client setup window type the server name full computer name for the Data Collection computer see step 4 on page 292 select ABI 3500 then click Next Read the release notes then click Next Note For other installation and configuration setup instructions see Chapter 3 of the GeneMapper v4 1 Installation and Administration Guide Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 293 Appendix D Remote Auto Analysis Setup Create a shared folder Create a shared l folder Windows Vista 294 Select Start Computer then double click the drive on which you want the shared folder to reside Select File New Folder Name the folder for example Remote_Autoanalysis Right click the new shared folder then select Properties Select the Sharing tab click Share n Remote_Autoanalysis Properties Shain Secu Merwnrk File and Folder Sharing i Remote _Autboanalysis A Hot Shared Network Path Hot Shared In the Choose people to share with dialog box click the
155. a Select your table display preferen Multi column sorting is supported see Sort on page 97 Flag Symbols Description Offscale i Spectral Pull Up fragment analysis only mA Broad Peak HID analysis only a At least one data point in the analysis range has saturated the CCD camera Note In the Monitor Run screen an offscale sample is flagged with EJ At least one peak contains a pull up peak A pull up peak is identified when the peak height of the minor peak is lt X of and within Y data point of the major peak where X and Y are values you specify See Chapter 6 Manage Library Resources A At least one peak exceeds the Broad Peak threshold Broad peaks affect Sizing Quality See Chapter 6 Manage Library Resources Note The value displayed when you place the mouse pointer over a Broad Peak flag is an internal value and does not reflect the peak width Normalization Limit a e ua Sample was collected with a normalization size standard sample Normalization Factor is within range Sample was collected with a normalization size standard sample Normalization Factor is not within range e No Data Normalization is enabled but Sizing Quality is x e NO Sample was not collected with a normalization size standard e N A Sample was not collected on a 3500 or 3500xL analyzer instrument For more information see Review normalized data on page 90 Note
156. abled disabled the events that are audited and the reasons available to users when audit mode is set to Prompt or Required Auditing is enabled by default IMPORTANT If you disable security you inactivate audit and electronic signature functions No audit record is generated for the inactivation of audit and electronic signature functions when you disable security 1 Access the Audit screen intenance Tools Manage Preferen 2 Click Disable or Enable Figure 35 on security Audit page 207 E Signature Change Password Note When auditing is disabled the is not active in lower parts of the screen View Logs Manual Commands k 3500 Data Collection Software Dashboard Edt Library Plaintbenance Tio Manage T Preferences Help Log Cot Settings Resouces l 9 fudit Settings Audit Settings 4 audting should be enabled PFU DUCI Audi Reports v Object Type E Signature Reports Dye Set v Size Standard J Menage Users Instrument Protocol PA Protocol 5A Probocol QC Protocol Assay Piste Template v File Name Convention v Result Group Plate v Sample Files amp Action Type Export Assay v Export Plate Record EY FESFEEEEEEEE E Pl Select a reason From Ehe let For your change Creste Reason Manually edited Entry emor Well anomaly Calculation error 5 Need to change threshold Need to reanalyze Figure 35
157. abled on your system you must provide a user 3500 Log In name and password to access the l Provide your user name and password to login software Your access to functions in the User Name software 1s based on the 2 Password permissions associated with your user account Functions for which you do not have permissions are grayed out Se L If your system is configured for password expiration you will periodically be prompted to change your password If your system is configured to monitor failed log in attempts you will be locked out of the software if you incorrectly enter your user name or password for a specified number of times Permissions If your user account does not have permission to perform any function in the software menu commands are grayed Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 225 Section 2 Users Determine the name of the logged in user Change your password when it expires Account suspension Session timeout 226 To display the full name of the logged in user place the mouse pointer on the Logout menu gt Help Log Out The full name of the logged in user 1s also displayed in the Load Plates for Run screen and the Monitor Run screen When your password is about to expire a message 1s displayed when you log in To change your password select Tools Change ntenance Tools Manage Preferer Password Security Enter your current password the
158. ag Note The top part of the pouch fitment is sealed with a plastic film which should be removed prior to direct installation on to the instrument For the following hazard s see the complete safety alert descriptions in Specific chemical alerts on page 333 WARNING CHEMICAL HAZARD POP 4 POP 6 and POP 7 polymers Store the polymer at 2 C to 8 C until ready to use The sealed polymer is stable at this temperature until the expiration date shown on the label For more details see the product insert included in the product package Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Instrument reagents and consumables See Change polymer type on page 247 for instructions on how to change polymers IMPORTANT If you remove a polymer pouch for storage place a Pouch Cap PN 4412619 onto the pouch then place an empty pouch or conditioning reagent on the connector to prevent desiccation of any residual polymer on the connector Follow the instructions in the wizard to ensure the proper operation of the pouch and the instrument Applications POP 6 and POP 7 polymers are recommended for sequencing and fragment analysis applications POP 4 polymer is recommended for HID Forensic applications Table2 Polymers used for all applications Pouch limits Polymer type cant Instrument used On instrument ite or User y P number whichever comes first Cannot 8 option to exceed iie _
159. alibration El User Plate Setup Reports Settings Run Setup Sequencing Settings Trace Trace Print Trace Quality Trace Quality Reports Restore Defaults Apply Note The type filter text field at the top of the dialog box is not used System preferences These settings apply to all users Date format Instrument settings instrument name e Scheduler preference trigger time for maintenance notifications e Sequencing export settings e Spectral calibration number of allowed borrowing events 32 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Set preferences User preferences These settings apply to all users if your system does not include the SAE module but are saved individually per user if your system includes the SAE module Note For information on the SAE module see Chapter 7 Use Security Audit and E Sig Functions SAE Module on page 197 e Plate setup e Reports settings e Run setup e Sequencing review and report settings Set general preferences System 1 In the Preferences dialog box click the following items preferences Date Format to set the date and time format for the software k Preferences type Filter text not used Date Format en 7 System ao Default Format 09 Feb 2009 10 11 55 AM Instrument Settings Choose date Format to use 09 Feb 2009 fdd romm yyyy v Scheduler Preference February 09 2009 rmmmm dd yvy Sequencing Settings Exp
160. all standard results you do not need to run the spectral calibration described in Spectral calibration on page 103 for E and Z dye sets You still need to run spectral calibrations for other dyes sets The performance check is application specific If you will run general sequencing applications with POP 7 polymer and MicroSeq ID applications with POP 6 polymer install the appropriate polymer and perform separate performance checks Estimated run General sequencing 45 minutes times MicroSeq ID 2 hours Prepare for the sequencing install standard performance check Prepare the 1 In the Dashboard check consumable status Check consumable status on instrument page 29 Ensure that Consumables are not expired Adequate injections remain for consumables 2 Ensure that the buffer levels are at the fill lines Check buffer fill levels on page 31 3 Setthe oven temperature then click Start Pre heat 60 C General sequencing POP 7 polymer e 50 C MicroSeq ID POP 6 polymer Pre heat the oven and detection cell while you prepare for a run detection cell temperature is set by the software Preheating helps mitigate subtle first run migration rate effects The pre heat function automatically turns off after 2 hours Applied Biosystems recommends that you pre heat the oven for at least 30 minutes before you start a run if the instrument is cold Applied Biosystems 3500 3500xL Genetic Analy
161. ally pdf print the report and select CutePDF Writer as the printer 4 Close the report Pagelofl 5 Goto Prepare and load sample plates on page 51 50 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Prepare and load sample plates Prepare and load sample plates tg ce eave T IMPORTANT Do not use warped or damaged plates Capillary to plate The capillary to plate mapping for the default injection order is shown below If you mapping change the injection order in the injection list mapping differs from the examples shown below e 96 Supports 96 well standard reaction plate 8 strip standard tubes are also supported with appropriate retainers e 96 Fast Tube Supports 96 well Fast reaction plate 8 strip fast tubes are also supported with appropriate retainers 8 capillary 96 well plate 8 capillary 384 well plate Not supported on the 3500 Dx Genetic Analyzers 8 capillary c d e e TT To Tae A 4 3 4 5 6 FT 4 9 10 li 2 Cap B 2 3 45 6 7 8 9 il p sj iS j4 S C 3 3 4 5 6 7 8 9 10 1 12 13 EY o eg 5 8 17 8 9 en iun E Z 4 4 EIL 3 4 5 6 7 8 9 140 ll D c 495 5 2 6 Fli 2 3 4 5 6 7 8 9 1011 2 a 1 2 3 4 5 6 7 8 9 1 1 12 123 4 5 67 8 9 0 il 1 24 capillary 96 well plate 24 capillary 384 well plate LEE f TC T fe Jr Je T T i ie os Tis is ss oz Tus T oo es fe es Jas 3 1 3 1 3 B 7 D 7 5 7 9 1d 9 1i 9 11 13 15 13 15 13 15 1 Capi 2 3 A 45 6n 8 lt 2 3 3 3
162. alysis protocols Sequencing analysis fragment analysis and HID analysis Items you select when you create instrument sizecalling and QC protocols Dye sets Size standards You can click Main Workflow or select Dashboard or any other menu item at any time to advance from the Library workflow Create a new entry from a factory provided template or locked entry 140 IMPORTANT Auditing of an item depends on whether it is created directly from the library or from within another item for example you can create an assay directly from the library or within a plate in the Assign Plate Contents screen For more information on auditing see Review the object audit history on page 210 1 Select the factory provided entry in the library 2 Click j Duplicate The software creates a Copy of the item you duplicated 3 Select the Copy of item then click Edit 4 Enter a name for the item 5 Modify parameters as needed see the appropriate section for information Applied Biosystems 3500 3500xL Genetic Analyzer User Guide General library procedures 6 Click Save Delete a library entry IMPORTANT Auditing of an item depends on whether it is deleted directly from the library or from within another item for example you can delete an assay directly from the library or within a plate in the Assign Plate Contents screen For more information on auditing see Review the object audit history
163. ame Name of the protocol Names must be unique Description Optional text entry Lock When enabled allows the entry to be unlocked and modified only by the user who created it the administrator or another user with unlock permissions Useful when your system includes the SAE module described in Chapter 7 Use Security Audit and E Sig Functions SAE Module on page 197 Application Type Automatically set to Sequencing Secondary Analysis Software IMPORTANT The secondary analysis software must be installed and properly configured with the 3500 Series Data Collection Software before it is listed as a selection in this screen For information on setting up the MicroSeq D Analysis Software for auto analysis see the MicroSeq ID Analysis Software Getting Started Guide Secondary Analysis Software Instance Computer on which the secondary analysis software is running Project MicroSeq ID software project and specimen to create Project Template Project template to use Specimen Specimen in which to save the sample data files 192 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Fragment analysis protocols library secondary analysis Fragment analysis protocols library secondary analysis Fragment analysis protocol overview A fragment analysis protocol GeneMapper protocol is the optional secondary analysis auto analysis protocol for GeneMap
164. ampler Array head lock mechanism Polymer Delivery Pump PDP Water Trap Waste Container Lever to install and remove polymer pouch Polymer pouch Check Valve CV Fitting Drip Tray Anode Buffer Container ABC Buffer Pin Valve Instrument interior components Figure 1 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Chapter 1 Instrument and Software Description Instrument parts and functions Table 1 Instrument parts and functions Part Function Autosampler Holds the sample plates and Cathode Buffer Container CBC and moves to align the plates and CBC with the capillaries Oven Maintains uniform capillary array temperature Oven condensation reservoir Collects condensation from the oven Pump block Includes the displacement pump chamber polymer chambers piston water seal syringe fitting array attachment point array port the lower polymer block and the CV Fitting Check Valve pouch attachment fitting Detection cell heater block Holds the detection cell in place for laser detection and maintains the detection cell temperature of 50 C Polymer Delivery Pump PDP Pumps polymer into the array and allows for automated maintenance procedures Lower polymer block Contains the buffer valve anode electrode buffer gasket and holds the anode buffer container Radio Frequency Identification RFID RFID tags to read the following informat
165. analyze in the 3500 Series Data Collection Software see GeneMapper ID X v 1 1 User Guide Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 279 Appendix C Secondary Analysis Fragment Set up an auto analysis project in GeneMapper IMPORTANT When using GeneMapper Software to auto analyze results data from the 3500 3500xL analyzer you must have v4 1 installed on the same computer as the 3500 Series Data Collection Software The fragment analysis workflow for auto analysis is summarized in this flow chart Auto GeneMapper analysis Software Manager Data Collection Set up a project in the secondary analysis software before starting a run on the 3500 3500xL analyzer All analysis in GeneMapper occurs in a project Specify a kit a 1 Open GeneMapper v4 1 by double clicking xw panel and a bin pun set for the project 2 Click to open the Panel Manager 3 Select the Panel Manager node in the Navigation pane to highlight uw Panel Manager File Edit Bins View ix NEIN a Panel Manager a US Bin set 280 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Set up an auto analysis project in GeneMapper 4 Select the panel then Import to import a previously created kit folder with panel marker information Note You have to import panels one by one repeat this step for each panel Panel Manager File Edit Bins View i x ee
166. ance notifications as shown Click for LOT A Spatial information Spectral 2 From the Left hand pane under Planned Maintenance F Performance Check click Schedule Sequencing Install Standard Fragment Install Standard 3 Click on the top left hand corner of the Schedule for HD Install Standard more intormation s Maintenance Wizards Planned Maintenance Additionally Applied Biosystems suggests that you add the regular maintenance tasks listed below to the maintenance calender Notifications Log Service Log Schedule 232 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Use the maintenance calendar Default calendar entries A set of Applied Biosystems recommended tasks are scheduled in the calendar flagged with FR Factory Repeating in the monthly view and F Factory in the daily view User specified repeating tasks are flagged with R Repeating in the monthly view see picture below You can change the priority of factory tasks but you cannot remove them from the calendar or alter the frequency at which the notifications for the tasks are displayed Additionally Applied Biosystems suggests that you add to the maintenance calendar e The regular maintenance tasks A maintenance task to replace a consumable based on its installation date for example create a task to replace the polymer for two days before the polymer will expire Create calendar entries To create a new schedule
167. and follow the prompts Load Plate Plate loaded successfully Load Plate for Run Current injection list if any will be cleared Please select ane of the options below amp One or more positions on the instrument stacker are already occupied amp Place this plate onto the stacker in position A replacing the plate that is already in that position Load this plate onto the stacker in position B OK J Cancel 310 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Monitor run troubleshooting Monitor run troubleshooting Symptom Possible Cause Action Re inject button is dimmed when you select an injection Injection contains samples with assays that specify more than one instrument protocol Select in the injection list the injection with the instrument protocol of interest select in the array view the capillary that corresponds to the well of interest then click Re inject The instrument run goes into pause state unexpectedly RFID read write process Check the Dashboard Conduct an RFID refresh if it does not refresh restart both the computer and instrument Start run does not respond The instrument has not initialized dt takes approximately 10 seconds for the instrument to initialize after the instrument door is closed Do not start a run until the instrument status light is green Fragment performance chec
168. ane Quick iew Gauges POP Polymer AB 3500 Buffer Anode AB 3500 Buffer Cathode 50cm 24 cap Array 384 576 ep be r 634 Samples Remaining 7 Days Remaining Days Remaining 43 Injections Performed 34 Injections Remaining 96 Injections Remaining 96 Injections Remaining IMPORTANT Applied Biosystems recommends that you add a maintenance notification to your calendar for polymer and buffer replacement Set the notification to display two days before the polymer should be replaced Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 29 Chapter 2 Start the System On instrument limits the first limit met applies Notes Consumable Polymer 8 cap 960 sample pouch 960 samples or 120 injections Use within 7 days of installation on ES inst t 384 sample pouch 384 samples or 60 injections Hii The software allows you to continue 24 cap 960 sample pouch 960 samples or 50 injections running past 7 days However 384 sample pouch 384 samples or 20 injections Applied Biosystems has verified the polymers for up to 7 days only on the instrument Buffers 8 cap 7 days or 120 injections To ensure optimal buffer performance the software requires 24 cap 7 days or 50 injections buffer replacement after 7 days Capillary Array 160 injections The software allows you to continue running after 160 injections However Applied Biosystems has verified the arrays
169. ane the instrument Note You do not create a plate for the performance check The software uses predetermined positions for the run You cannot specify standard location on the plate 3 Click Start Run Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Select the plate type and plate position in Library Maintenance Tools Manage Fj Performance Check Sequencing Install standard Fragment Install Standard a HID Install Standard Library Maintenance Tools Manage Pi Performance Check Sequencing Install Standard Fragment Install Standard HID Install Standard Run the fragment analysis or HID Install standard performance check writ 8 ren Calibration Settings Cumert Instrument Consumables o Polymer Type POP Cap lsry Length 36mm 2 Humber of Welk 6 96 96 FastTube 7384 Stork Run Plate Postion MA OB Tow Status Ready Capillary Run Dat 3 apillary Run Data o Albee Peaks 5te Standard Peaks Iriclude j YA MA YM AM A Ys a AM e III Capillary Information BEC E 73 E ES ER 4000 8000 12000 16000 20000 24000 26000 32 50000 40000 k Run Information All capillaries Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 133 Section 2 Performance check What you see during a run The system performs one run and indicates the number of observed allele and size standard peaks The Capillary Run Data display updates after the ru
170. ange red yellow green blue Extraneous None E E aW Note The E5 profile may include extraneous peaks outside the matrix peak region which can be ignored 110 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Spectral calibration Attribute Acceptance Criteria Example Peak m No gross overlaps dips or other morphology in irregularities the spectral Peaks separate and distinct profile Note The profiles of G5 shown to the right F and J6 may not be as smooth as the profiles for other dye sets shown above due to the effect of variable binning a feature that reduces signal variation between dyes of different fluorescent efficiencies 3 As needed zoom on the spectral profile traces to determine if the data meet the criteria a Place the pointer above the top of the plot or to the va left of the plot at the start of the area you want to zoom then click to turn the pointer to C 28000 b With the Q still above the plot or to the eft of the plot click drag to the end of the area you want to f zoom Do not drag the inside the plot area Doing so changes EN 4 If the data for all capillaries meet the criteria above click Accept Results 5 Ifany capillary data does not meeting the criteria above click Reject Results then go to Spectral calibration troubleshooting on page 301 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 111 Section 1 Calibration What you
171. apter 2 Chapter 3 Use the software without an instrument lille 19 olart the SVSterfi esa ose die ae eee PORE ee eee d 21 odio pcr dd dre 21 Start the Inst merit se etai ada e eC e CRUDO de lio D Mae MOS e e dede 22 Start The computer escisiones us 24 LOG ONTO WINGOWS uc sg Hur Dea EURO fre eae Ree ee tes Beate RON UR DERE A E 24 Launch the applICallOki s son ogg desnudo eR ages s epa i RR REN dob A ds 24 Eod NER lE TET TN 26 Check system status in the Dashboard llli 26 Dashboard a quick glance sse a e ue Eu Re dex qoe s 26 Check maintenance notifications l c 28 Check consumable status 44 ver DA Ra UICE US CAUTE AD ua 29 Check buffer fill levelS ei EE RRRRRRRRR 31 Replenish consumables iie mi Ra Ve PE A RE 31 Sel DI elerel 6S a suc d cp daras Sew ees tdg oe eee eis cR 32 OVEIVICW M Pcr 32 oyster prelerelloes ia eS vele sce dta Pa sS dee arae dts oe ele 32 User preterell68S 4 ow sea ws E xor rd oe CR ab Re CRI e nl 33 Set general preferences 0 0c eee eee ees 33 Set sequencing preferences 0 ccc ee eee ees 36 SPUD 3nd RUN sara ari Bae we Waa ee Ree f 41 vus APT PIPER 41 Prepare the Instrulment 2252 9a d Ahmed Ron he Rogge dee dae peo C do e e d 42 Credte a plate auti secte aoa cda ORE AUR Odes ho Msi acia Rel ace aser ir ctos 43 ASSIGN plate cortells Vii ciar pre E SOR a ES 46 Name samples and assign sample types in the plate view
172. ard and want to run a performance check and a spectral calibration you can skip this process and run the Sequencing Install Standard performance check If you select Keep Spectral Calibration Data in the Performance Check the software runs a spectral calibration for dye set E or Z during a sequencing check and allows you to save the spectral calibration data For information see Run the sequencing install standard performance check on page 119 Standard Polymer Type Run Time min Matrix standard Any lt 30 Sequencing standard POP 7 polymer 40 POP 6 polymer 135 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 103 Section 1 Calibration Prepare for the spectral calibration Prepare the l instrument If you have not already done so perform a spatial calibration see Spatial calibration on page 99 In the Dashboard check consumable status page 29 Ensure that Consumables are not expired e Adequate injections remain for consumables Ensure that the buffer levels are at the fill lines Check buffer fill levels on page 31 Set the oven temperature then click Start Pre heat e 60 C POP 7 polymer e 50 C POP 6 polymer Pre heat the oven and detection cell while you prepare for a run detection cell temperature is set by the software Preheating helps mitigate subtle first run migration rate effects The pre heat function automatically turns off after 2 h
173. are provide integration between the instrument and secondary fragment analysis software applications specifically GeneMapper Software v4 1 and GeneMapper D X Software v1 1 Using auto analysis samples are loaded fragment data is generated and allele calling is performed according to the protocols assigned to the plates prior to the run Software Purpose GeneMapper A high performing and versatile software package for all fragment analysis and genotyping applications GeneMapper D X A software for use in Human Identification testing databasing casework and paternity applications and used in conjunction with AmpF STR kit and the 3500 3500xL analyzer Auto analyze projects in the fragment analysis software Auto analysis can only be performed on the same computer that collects the sample files therefore GeneMapper or GeneMapper D X Software must be installed and configured with the 3500 3500xL analyzer on a Windows Vista operating system Secondary analysis occurs within the GeneMapper or GeneMapper D X Software This procedure initially describes how to set up panels and bin sets in GeneMapper Software v4 1 and then describes how to auto analyze samples using the 3500 Series Data Collection Software Once a run is complete your data is seamlessly transferred into GeneMapper for analyzing processing and reporting Note For detailed information on setting up a GeneMapper D X analysis to auto
174. array is Sequencing Settings Export E Spectral Calibration 2 Click Apply to save the system preferences see System preferences on page 32 User preferences 1 In the Preferences dialog box click the following items as needed Plate setup to set the default settings for Plate type and attributes when you create a plate Plate type in the Open Plate dialog box k Preferences type filter text not used Plate Setup IA Choose the default application type polymer type and capillary length to use when the instrument settings cannot be detected El User Flate Setup Reports Settings Application Polymer Type Capillary Length H Run Setup E Sequencing Settings Trace j Choose the default Assign Plate Contents view Trace Print Trace Quality Trace Quality Reports Assign Plate Contents View Plate vw Reports settings to set the default font and size reports Note You can override this setting in each report view 34 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Set preferences Preferences type Filter text not used Reports Settings it System These settings affect all reports i User Font settings Plate Setup y Select the font En be used in reports Reports Settings Sequencing Settings 10 Trace Trace Print Trace Quality Trace Quality Reports e Run Setup to set the default storage location for data files in file name conventi
175. ate the report No signal Incorrect preparation of sample Replace samples with fresh samples Bubbles in sample wells prepared with fresh Hi Di Formamide Centrifuge samples to remove bubbles The capillary tips may not be touching the samples Check the volume of your samples If no results call your Applied Biosystems representative The capillary tips may be hitting the bottom of the wells Autosampler not correctly aligned Call your Applied Biosystems representative If the Fragment HID install standard Performance check fails Blocked capillary Refill capillary array You may have to install a fresh array or consider that capillary non usable for purposes of planning your runs Insufficient filling of array Check for broken capillaries and refill the capillary array Expired matrix standards or old reagents Check the expiration date and storage conditions of the matrix standards and or reagents If necessary replace with a fresh lot Expired polymer Replace the polymer with a fresh lot using the Replenish Polymer Wizard Bubbles in the polymer system Select the Bubble Remove Wizard to clear the bubbles Possible contaminant or crystal deposits in the polymer Properly bring the polymer to room temperature do not heat Replace the polymer if it has expired Anode buffer container troubleshooting Also see Data electroph
176. audit and e sig disabled e Allow only certain users to create or modify protocols e Allow only certain users to approve reviewed samples Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 197 Section 1 Administrators Configure the security system Access the Security screen and enable or disable security The Security screen allows you to disable and enable security control restrictions and security policies for all user accounts and set up notifications when certain security events occur Security is enabled by default IMPORTANT If you disable security you inactivate audit and electronic signature functions However when you disable security no audit record 1s generated to indicate that audit and electronic signature functions are disabled 1 Access the Security screen ntenance Tools Manage Preferer 2 Click Disable or Enable Figure 34 on Security page 198 Note the following Audit E Signature e Disabling Security inactivates Auditing Change Password and E signature View Logs e The Disable and Enable commands are Manual Commands grayed when a run is in process The software requires you to enter your user name and password when you enable security When security is disabled the is not active in lower parts of the screen t Disable System Security Account Setup like hane lie Pad The length ci usse rie mat ba eee d acd JI a The length of use pee mags b e
177. ay S RA Assay S RA Assad M Assay dS M asas RA Assay dS M Ais S RA Assay S M Aj S RA Aste S M Assay S M Ate S M Assad S M Assay S RA Assay M Assay dS M Atty S RA Assay dS M Afi RA Assay S M Aj RA Assay S M Ajay S M Atty S M Assay S M Assay S RA Assad M Assay dS M Atty S RA Assay dS M Atia RA Assay S M Aj S RA Assad S M Atl S M Atty S M Assay S M Assay S M Assay M Assay dS M Atty S RA Assay dS M Afia RA Assay S M Ajay S RA Assad S M Assay S M Atty S M Assay dS M Atty S M Assay M Assay dS M Atty Ra Assay S M Assay RA Assay S M At S RA Atty S M Assay dS M Atty S RA Assay S M Af S RA Assay Ba Altay S Ba A S BO A a S BO APA S Ba Anu S Bo Ala a S Ba Ala a S Ra Altay S Ba Alas S Ba Alleys S BS Asa S BA Alava 7 n p s Sequencing Name AA Seq Plate 4 SeqScape Barcode sqa Assays File Name Conventions JL Results Groups Actions Y Actions Y Action oc y Li O M TEST FNC 2H O A TEST RG 2H Customize Sample Info Link Plate for Run 5 Click Link Plate for Run 6 Click Create Injection List then click OK after the instrument performs its validations Start the auto Click Start Run to begin your auto analysis analysis run The 3500 3500xL analyzer display a progress indicator while it checks the level of consumables on the instrument o Preparing Instrument Run y b p N Starting Instrument Run Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 275 Appendix B
178. be used in a run The spatial calibration has not been signed A user starts a run The following message is displayed S E Signature Check The Spatial Calibration Spatial Run 2009 01 29 09 59 13 record must be signed by 2 users to be utilized For this Start Run but is mot signed by any Users The Following signatures are missing Approve Spatial Calibration by a user with User Account Administrator authority Approve Spatial Calibration by a user with User Role Scientist authority Would vau like bo sign now Before the run can start the following users must sign e The Administrator user e Any other user with the Scientist role specified and electronic signature enabled in their user account If a user that does not meet the specified criteria signs this message 1s displayed again 220 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Generate e signature reports Display e signature records 1 Access the E Signature Reports screen 2 Optional Specify filters date range user name action object type object name then click Go Select a record then click Show Object History In the history dialog box select a record then click Show E Signature Details Double click column headers to sort Multi column sorting is supported see Multi column sorting on page 72 Generate e signature reports ltenance Tools Manage Preferen Security Audit Change Pas
179. bels b Under Labelling Options Enable Show Peak Labels To label all peaks with the selected labels click Label Peaks make sure All is selected To label selected peaks select the category from the Label Peaks list Height Area Size specify the range to label for the selected category for example if you select Height specify the height range of the peaks to label then click Label Peaks Enable Retain Labels a Click Save to Preferences to save these settings for future use You can change preferences at any time b Click Apply Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 93 Chapter 4 Review Results View thumbnails Click View Thumbnails to display the traces for the samples selected in the samples view and the sepas v Ws lu Em7 dyes selected in the plot view Tan B3 Thumbnail View elim Thumbnail View 2 G0430 fsa G0538 fsa GO0646 fsa H0431 fsa Review sizing The Sizing Table View displays For fragment samples All dyes For HID samples Size standard dye only orange or red Set up the sizing 1 Select the samples of interest in the samples table to display plots table E 2 In the sizing table click the Table Settings button then specify the columns to show or hide Select your table display preferen Aw ailahla Calin Fo Mienlas Show Show All Peaks m ve 3 Filter the table a
180. brary l Instructions Select row from table and click on Open button Filter HID Search an Go dear Date Modified Plate 011 12 4pr 2009 05 15 13 PM test well attributes 13 Apr 2009 09 58 39 4M 228 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Maintain the Instrument Maintenance schedule This section lists the common tasks required to maintain your Applied Biosystems 3500 3500xL Genetic Analyzers in good working condition The Dashboard in conjunction with the data entered in the schedule section of the Planned Maintenance provide a comprehensive outline of maintenance tasks AN WARNING Wear appropriate protection including gloves laboratory goggles and coat whenever you work with the fluids used on this instrument or parts that may come into contact with these fluids IMPORTANT Use the cleaning agents as described in this manual only Use of cleaning agents not described in this manual can impair the instrument For the instrument troubleshooting issues see Appendix E Troubleshoot on page 299 Review maintenance notifications Review maintenance notifications list in the Dashboard daily then perform the scheduled tasks Maintenance Notifications Perform Performance Check HIGH 26 Jan 2009 12 00 00 AM Performance Check wv E Clean Drip Tray HIGH 26 Jan 2009 12 00 00 4M Clean Drip Trav e E Clean Autosampler HIGH 26 Jan 2009 12 00 00 4M Clean Autosampler e E Rep
181. cR Mo ioe EY eode oo o ee bid di RON ee Bes ae 71 Sort and customize tables lille 72 Add assays file name conventions and results groups toa plate 13 Create a plate for importing 0 0 cece ee eens 13 EGIL a plate a 4 d pot ace a E oie ee th ere QUE SUE Rab CA Ka Bosco te aed 74 Import and export a plate 0 eee 15 Create a plate template 0 ee eee eas 15 Specify the default plate type for the Open Plate dialog box 76 Save electronic version of reports anaa aa eee ees 76 More features in Load Plate for Run 0 0 0 ee es 76 Link a plate from the recent plates or recent runs tab o oooooo 76 More features in Monitor Run 1 2 ee ees 17 Review the Instrument Run views llle 17 Chapter 4 REVIEW ResullS usos ic eeiclon d brad id doe E d Ene oid 79 Review Sequencing Results 0 0 00 cc eee eee eee 80 Access the View Sequencing Results screen 0000 ee eee 80 Review sequence quality l l 81 REVISWITAaces 5 d bed ua dae tie da o Up us ae n iie dio ei does 82 Understand Quality Values QVs lellllelee ee 84 Spec re Injec HOFIS erise Are aio tk NE aur Bead rau e O uad cat s ad SOR os CY a 85 View print and save pdf trace quality reports lessen 85 Export sequencing results cir Soe iow DG NEED E E ee 87 Review Fragment HID Analysis results 0 0 0 0
182. ch is removed for later use use the suggested cap to plug the fitment opening and store the pouch under recommended storage conditions From the Maintenance Wizards screen click Change Polymer Type CHANGE the type of polymer installed on the instrument with the option to change arrays IMPORTANT This feature allows you to change the type of polymer installed on the instrument with the option to change the Capillary Arrays Note The Change Polymer Type Wizard takes 60 to 70 minutes to complete Follow the prompts in the Change m Change Polymer Type Wizard Polymer Type Wizard window urrently installed polymer information Note Changing polymer re pe Por requires the use of a Conditioning ec hate ate Nm rc ee C onditioning re agent y on se Use the Replenish Polymer Wizard to despite id of polymer of any type with polymer of the same typ p age 2 5 0 Click Next to install a new polymer type Click Cancel to exit this wizard artnumber 4315930 Click Refresh from the Dashboard to update the screen Check the Quick View section of the Dashboard for updated status after changing the polymer Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Use the Maintenance Wizards to perform operations Partially used polymer IMPORTANT Do not use a polymer pouch that has been installed on one type of instrument on another type of instrument For example if you install a new polymer
183. check See fragment HID install performance check Hi Di Formamide on instrument limits 12 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Index part number 12 Hi Di sample type 48 import assays 141 audit settings 224 basecalling protocols 141 dye sets 141 e sig settings 224 file name convention 141 fragment analysis protocols 141 HID analysis protocols 141 instrument protocols 141 plate 44 75 141 QC protocols 141 results group 141 sample files 81 sample files fragment HID 89 security settings 224 sequencing analysis protocols 141 size standards 141 sizecalling protocols 141 user account settings 224 IMPORTANT description xiii Information Development department contacting 336 injection defined 57 duplicate 60 folder 159 re injection 65 replicate 60 specify new instrument protocol 65 injection list abort injection 69 blank 60 columns in table 62 completed injections 62 duplicate injection 60 modify 60 monitor run 61 preview run 59 re injection 65 replicate injection 60 samples with assays that specify more than one instrument protocol 61 terminate 69 Install Capillary Array maintenance wizard 252 installation category 319 instrument error messages 313 load plate 53 maintenance See maintenance 341 Index move and level 243 name specifying 33 prepare forrun 42 reset 314 routine cleaning 242 shutdown 253 start up 22 status in
184. cimens If you have multiple protocols you will have multiple assays as each assay is associated with one secondary analysis protocol Note For more instruction on setting up a secondary analysis protocol see Create a new sequencing analysis protocol on page 189 T Setup an Assay vx Q Assay Name New SeqScape Assay Locked Color Dark Cyan x Application Type Sequencing Protocols Do you wish to assign multiple instrument protocols to this assay No 0 Y es Instrument Protocol BDxFastSeq50 POP7 1 Y Basecalling Protocol 3500 MDC Basecaling Protocol z Edit AA SeqScape SA Protocol AA SA Protocol AA SeqScape SA Protocol o Plate Save to Library SA Protocol 4 SeqScape 7 Click Apply to Plate then Save to Library if you want to use this assay again 8 Click Close TW Plate View ES Table View 2 Setup B Show In Wells y Select Wells El Array Selection gt Column Zoomin Zoom Out EN Fit G Define Plate Properties s Run Instrument le100 t ple108 le116 ple124 ple132 le140 3 ple148 ple156 ple164 ple172 ple180 lel un meni sam sam sam sample sam sam sample sam sam sam sample188 P A02 A03 A04 A05 A06 A07 A08 A09 A10 All A12 Load Plates for Run S S S S S S S S S B S S samplel01 samplel09 samplel17 samplel25 samplel33 samplel41 samplel49 samplel57 samplel65 samplel73 samplel81 samplel89 we cct B
185. ck box When enabled allows the entry to be unlocked and modified only by the user who created it the administrator or another user with unlock permissions Useful when your system includes the SAE module described in Chapter 7 Use Security Audit and E Sig Functions SAE Module on page 197 e Enter a description Assays File Mame Conventions Results Group uli Analyze Instrument Protocols Dye Sets Sire Standards hs Basecalling Protocols Mi ee ee Pe ee ee 5 Select a dye color 6 Enter sizes in the list on the left Separate sizes with a comma space or return 7 Click Add Sizes 172 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Size standards library 8 Click Save R Create New Size Standard Setup a Size Standard B Size Standard Name is a required field Provide a unique value Enter sizes in tha fier below separated by a comma space or rebun bhen otick fe dati Size 2 button fo add tham fo fhe current size standard definition aS Current Size Standard definition Delete Selected Sizes Enter new Size Standard definition e g 11 0 34 2 55 Add Sizelsj gt gt Close Figure 23 Create New Size Standard Modify a factory provided normalization size standard 1 Select a factory provided normalization size standard indicated in the name with Normalization 2 Click Duplicate 3 Edit the copy of the normalized siz
186. ck pm Export then specify a location for the export file To select multiple entries Shift click to select contiguous entries Ctrl click to select non contiguous entries Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 141 Chapter 6 Manage Library Resources View audit and e signature histories for library entries Note An administrator can also view audit and e signature histories in the SAE module For information see Chapter 7 Use Security Audit and E Sig Functions SAE Module on page 197 To view the audit or e signature history for a library entry 1 Select the 1tem in the library 2 Click View Audit History or View E Signature History active only if the selected item 1s enabled for e sig Note Factory provided items do not list creation date in the audit history If you duplicate a factory provided item the new item contains an audit history that starts with the duplication date listed as the creation date 3 For more information see Display audit histories on page 209 Sort filter and search library entries Sort Double click column headers to sort Multi column sorting is supported e Double click a column header to sort the column e Alt Shift click another column header to sort another column e Alt Shift click a third column header to sort a third column Numbers in the column headers reflect sort order Filter You can select an application type from the Filter lis
187. comes your comments and suggestions for improving its user documents You can e mail your comments to techpubs appliedbiosystems com IMPORTANT The e mail address above is for submitting comments and suggestions relating only to documentation To order documents download PDF files or for help with a technical question see How to obtain support on page xvii Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Index Symbols annotation txt 36 87 csv 75 fsta 36 87 html 87 pdf 87 action log 209 audit reports 214 e sig report 222 fragment HID install performance check 138 plate layout 50 sample quality reports 95 sequencing install performance check 128 spatial calibration report 102 spectral calibration 116 trace quality reports 85 user report 206 phd 1 36 87 qual 36 87 scf 36 87 seq 36 87 txt 75 87 xls 75 87 xml 75 Numerics 3500 Series Data Collection Software 3500 Software 25 compatible secondary analysis applications 14 Daemon 24 Dashboard 15 default settings 32 33 files generated 14 Library workflow 17 login 26 Main workflow 16 Maintenance workflow 17 navigate 19 overview 14 parts of 15 required procedure to start 24 Server Monitor 25 status icon in Windows tray 24 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide use without an instrument 19 8 tube strip plate base 53 plate type 44 51 105 120 145 96 Fast tube plate type 145 A ABC See anode b
188. ct select samples that specify the same results group To collect data for all wells in an injection 1 Select the injection in the injection list 2 Click MY Re inject To collect data for only specific wells 1 Select the injection Samples with assays that specify more 2 Select in the array view the capillary than one instrument protocol are listed that corresponds to the well or one time in the injection list for each sample of interest see Array view instrument protocol on page 77 Note You can also specify re injections 3 Click 4 Re inject for specific samples in Review Results To collect data for only samples that 1 Select the samples in the flag table contain flags see Check sequence or sample quality on page 62 2 Click 3 Re inject Note If you are running an HID plate see Re injections of HID allelic ladder samples on page 67 In the Re injection dialog box select options then click OK The protocol to use for the re injection original modified new or one from the library When to make the re injection Note Sample data files for each re injection can be saved in a separate folder in the results group folder if specified in the results group For more information see Results group example 3 store re injections in separate folders on page 162 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 65 Chapter 3 Set Up and Run AS Re injection Re
189. ct Assign Plate Contents in the navigation pane Estike Rae t pies Assign Plate Contents 4 Click Assign Plate Contents Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 45 Chapter 3 Set Up and Run Assign plate contents You assign the following information to the wells in a plate before you can run the plate e Sample names and sample types required Identifies the well positions of each sample for data collection and processing Assay required Specifies the parameters that control data collection and primary analysis basecalling or sizing All named wells on a plate must have an assigned assay For more information on assays see Assays library on page 147 Filename convention optional Specifies file naming For more information on assays see File name conventions library on page 151 e Results group optional Specifies sample data file storage For more information on assays see Result group library on page 155 Before you assign 1 Access the Assign Plate Contents screen Figure 5 Dis plate contents on page 47 from The Define Plate Properties screen by clicking Assign Plate Contents described above Define Plate Properties Assign Plate Contents The navigation pane by selecting Assign Plate Contents in the navigation pane e The Dashboard by clicking the Main workflow arrow then selecting Assign Plate Contents in the navigation pane
190. ct a pre defined Generate audit reports reason in the Audit Select a reason From the list For your change Reason dialog box E displayed when a Creato user performs an audited action enable the User must select a reason checkbox Users are not permitted to enter a reason Fa Manually edited Fa Entry error Well anomaly Calculation error nm Need to reanalyze 2 As needed click N Reason Need to change threshold Audit Reason Setup Audit Reason ES Audit Reason is a required Field Provide a un value Enter an Audit Reason in E LB Close Create or select a reason then click XE Edit or 1 Delete Generate audit reports Display audit histories 1 Access the Audit Reports screen Note To access the Audit Reports screen the user role for an account must specify the Configure SAE permission Users without the Configure SAE permission can view object audit histories for individual entries in the libraries by selecting entries then clicking View Audit History see View audit and e signature histories for library entries on page 142 2 Select a tab to display intenance Tools Manage Preferen Security E Signature Change Password View Logs Manual Commands vo Manage Reports Audit Reports E Signature Reports Object Audit History The most recent audit for all user objects samples and objects in the Library that have be
191. d it the administrator or another user with unlock permissions Useful when your system includes the SAE module described in Chapter 7 Use Security Audit and E Sig Functions SAE Module on page 197 Description Optional text entry Basecaller Basecalling algorithm used to identify bases Note The basecaller version listed in the basecalling protocol is a 3 digit number The version listed in sequencing results is a 4 digit number The fourth digit is an internal number used by the software Mobility file Compensates for mobility differences between dyes and primers correcting the color code to the chemistry used to label the DNA during instrument processing Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 175 Chapter 6 Manage Library Resources Table 12 Basecalling protocol Analysis settings continued Setting Description Quality Threshold e Basecall Assignment ambiguous bases Do not assign N s to basecalls Assign N s to basecalls with QV 15 Bases with a QV less than the threshold display N instead of the base letter e Ending base Last base on which to perform basecalling AtPCR Stop After X number of Bases After X number of Ns in X number of Bases After X number of Ns Note If you have short PCR products select the At PCR Stop check box Mixed bases threshold When enabled allows the software to determine the secondary peak height where
192. d specimen to use for auto analysis When you create a sequencing assay you can optionally add a sequencing analysis protocol to the assay If you add this item from the library a copy of the item 1s added to the assay and can be modified independently from the original item stored in the library For information on how changes are tracked if auditing is enabled see Audit action on page 210 Create a new sequencing analysis protocol 1 Access the Sequencing Analysis Protocols library 3500 Data Collection Software Dashboard Edit ibrary Mainten Library Resources ate Filter 2 Click Create 3 In the Create New Sequencing Analysis Protocol dialog box Figure 30 on page 190 specify settings see Table 18 on page 190 H 4 Manage Plates Assays al Protocol Mame 4 Select the remaining secondary File Mame Conventions analysis items then click Save Results Group Note If the project project ili Amatyze template or specimen of interest is not displayed in a list re select the secondary analysis software instance to update the list Instrument Protacols Dye Sets size Standards Bazecalling Protocols Sizecalling Protocols xc Protocols IMPORTANT The auto analysis settings you specify for the plate to run with this protocol must contain the same secondary software and location settings For more information see Create a new plate on page 144 Se
193. d task click Create and follow the prompts The following is an example of scheduled events in the calender 12 FR Restart PC Instrum FR Defragment Hard Dr The Month and Day tabs allow you to view your schedule in different formats Click Detach to move the calendar window Dashboard Edit 7 Maintenance ER Create 4 Edit fl Delete A Detach a D bay Calibrate Spatial Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 233 Chapter 8 Maintain the Instrument Review the Maintenance Notifications Log 234 The Notifications Log is a history of all notifications messages and the action taken for the task completed or dismissed You can use this option to review a previous run information The Dashboard provides you with a list of current routine and maintenance notifications as explained below Multi column sorting is supported see Multi column sorting on page 72 To go to the Notifications Log from the Dashboard 1 Click Maintain Instrument 2 From the Left hand pane under Planned Maintenance click Notifications Log Click on the top left hand corner of the Notification Log for more information The Notification Log provides the following information on each event Notification Description Name The name of the event Priority The event priority Notification Date The date of notification Status The current status of the event
194. date The auditing of updates depends on whether an object is modified or overwritten Modified A record is created when an object is modified Updated A record is not created when an object is overwritten in the library Example You create a plate then create a results group from within the plate and save it to the library You then open the plate edit the results group from within the plate then save it to the library A message indicates that the results group already exists and asks if you want to overwrite it You click Yes This action is considered a creation of anew results group not a modification of the existing results group No Update record is created a Create record is created Create A record is created when you Create an item in the library Create an item from within another item Modify an item from within another item then overwrite the item in the library when you save it as described in the Updated bullet above Delete The auditing of deletions depends on the item deleted Items in the library A record is retained until it is deleted from the library The deletion of the item from the library is not audited For example if you delete a size standard from the library no audit record for the deletion is listed in the Object Audit History Items within other items The deletion of an item from within another item is audited For example if you change the size standard in a Q
195. dicator lights 23 status in Dashboard 53 theory of operation 5 troubleshoot 299 313 verification See fragment HID performance check sequencing performance check instrument operation 236 instrument operation safety 318 instrument protocols create 165 defined 165 export 141 import 141 in assay 150 normalization settings 167 instrument run abort injection 69 defined 57 monitor 61 pause 69 preview 61 resume 69 start 61 69 start with existing plate 55 stop 69 terminate injection list 69 instrument run name See also run name folder name 159 instrument run views 77 instrument sensor details view 305 Instrument Shutdown maintenance wizard 253 IQ 00 runs See fragment HID performance check sequencing performance check is signed field 228 italic text when to use xvi L label peaks 93 94 laser classification 320 321 laser safety 320 bar code scanner 321 requirements 320 321 libraries 342 assays 147 basecalling protocols 174 dye sets 168 file name conventions 151 fragment analysis protocols 193 HID analysis protocols 195 instrument protocols 165 MicroSeq ID analysis protocols 191 QC protocols 184 results group 156 sequencing analysis protocols 189 size standards 171 sizecalling protocols 179 library AB 139 access 140 archive 254 create new item 140 delete entry 141 delete entry auditing 141 edit entry 141 export entry 141 factory provided 139 factory provided
196. dify reason for change Create reason for change Delete reason for change Enable auditing Disable auditing Modify audit settings Modify audit settings Create reasons for change Delete reasons for change E Signature function E Signature settings E Signature type Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Update Update Update Modify the number of signatures or the authorities for a prompt before function Modify the Enable state of either a check before or prompt before function Enable e signature Disable e signature Modify e signature settings Modify the enable state of an E Signature Type 211 Section 1 Administrators Table 23 Audit system configuration history continued Record Type continued Action Corresponds To Role assignment Create e Create a new user account e Assign a different user role to an existing user account Delete Assign a different user role to an existing user account Role permissions Update Modify user role permissions Create Create a user role creates one role assignment record for each permission in a role Delete Delete a user role creates one role delete record for each permission in the deleted role User account Update e Edit e Suspend Create Create new user account User role Update Modify user role Create Create user role Delete Delete user role Review the action log The Action log lists system specified audit events All ite
197. drag to the end of the area you want to zoom Do not drag the EN inside the plot area Doing so changes C 28000 back to a pointer and does not zoom as expected i Plot Settings in the Plot View toolbar For information on plot settings Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Review Fragment HID Analysis results If the T button is grayed it indicates that the Plot Settings dialog 1s open Click the 3500 task bar icon then select Plot Settings Overlay samples 1 Label peaks l Select samples from the Samples View to display the plots Click Overlay All When Combine Dyes is selected the plot view displays one plot with all samples and all dyes When Separate Dyes is selected the plot view displays on plot per dye Each dye plot contains all samples Select samples from the Samples View to display the plots Plot Settings in the Plot View toolbar jasepairs ul ul s In the Plot Settings dialog box select the Labels tab If you have already specified default labeling preferences under Labelling Options a Enable Show Peak Labels b Click Label Peaks c Click Apply IMPORTANT You must open Plot Settings each time you access the View Results screen then click Label Peaks Labelling settings are not automatically applied when you access this screen or when you click Apply If you have not specified default label settings a Under Labels to Show select the needed la
198. drop down and select Everyone All users in this list Choose people to share with People must have a user account and password for this computer to access files you have shared To change this setting use the Network ancl Sharing Center 000000000 6 Add 7 3500 4017 3500 USER Level ABSanyice Acirministrator Everyone All users m thas hist Create a new user Tell me about different ways to share in Windows 8 share Cancel Click Add In the Permission Level column change the value from Reader to Co owner Click Share then click Done and Close Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Create a shared folder Your folder is shared You may e mail these links to notify people that you have shared these files or copy the links onto the Windows clipboard where you can paste them into any program you choose d Remote Autoanalysis 5 3500 P ewe 22900 P C Remote Autoanalysi Show me all the network shares an this computer CHE 9 10 Click OK Set security 1 Right click the shared folder then select Properties preferences for the shared folder 2 Select the Sharing tab then click Advanced Sharing Gereial shang Securtu Previous Versions Customize Network File and Folder Sharing a7 Remote_Autoanalysis Shared Netewoik Path 44 3500 PC Remote A teanalysis Advanced Sharing S el custom permissiars cale mulbple shares and set
199. ds IMPORTANT If you modify peak detection settings ensure that the size standard is accurately detected and sized with the new settings Normalization is not applied to samples with Size Quality flags The 3500 Series Data Collection Software does not support re analyzing data with new settings For more information on peak detection parameters see the GeneMapper D X Software Reference Guide Smoothing l Baseline Window Specify a window to adjust the baseline signals of all detected dye colors to the same level Select an option to smooth the outline of peaks and reduce the number of false peaks detected e None to apply no smoothing Best if the data display sharp narrow peaks of interest e Light default to provide the best results for typical data Light smoothing slightly reduces peak height e Heavy for data with very sharp narrow peaks of interest Heavy smoothing can significantly reduce peak height for an improved comparison of relative signal intensity Note the following e Asmall baseline window relative to the width of a cluster or grouping of peaks spatially close to each other can result in shorter peak heights e Larger baseline windows relative to the peaks being detected can create an elevated baseline resulting in peaks that are elevated or not resolved to the baseline 186 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide QC protocols library primary analysis HID
200. e Note If this setting does not allow detection of the 20 and 40 mer peaks for samples that use the GS600 LIZ size standard running samples with the GS600 60 600 LIZ Normalization may allow detection of the peaks Peak Amplitude Thresholds Specify the threshold RFU for peak detection for each dye color Peaks below the threshold are not detected For example if you use the default values of 175 peaks with heights equal to or greater than 175 are detected Peaks with heights below 175 are still displayed in the electropherogram plots but are not detected or labeled Note Set the peak amplitude thresholds to 175 in your GeneMapper Software analysis method l Smoothing Select an option to smooth the outline of peaks and reduce the number of false peaks detected e None default to apply no smoothing Best if the data display sharp narrow peaks of interest e Light to provide the best results for typical data Light smoothing slightly reduces peak height e Heavy for data with very sharp narrow peaks of interest Heavy smoothing can significantly reduce peak height Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 181 Chapter 6 Manage Library Resources Table 14 Sizecalling protocol Analysis settings continued Setting Description Baseline Window Specify a window to adjust the baseline signals of all detected dye colors to the same level for an improved comparison of relativ
201. e specified period of inactivity Account suspension for failed authentication The user exceeds maximum number of allowed failed authentications login attempts with an incorrect password Notification for SAE activation Security has been enabled or disabled 200 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Manage user accounts 3 Select the notification method e Pop up dialog The software immediately displays a pop up message to the current user if an event is triggered by the current user The message instructs the user to inform a system administrator of the event Message when Admin logs in If an event triggers notification the next time any user with an Administrator role logs in the software displays a list those events indicating the time each event occurred and the user who triggered the event The Administrator has the option of acknowledging the event which removes it from the notification list 4 Click OK Manage user accounts Create or edit a user account The software includes a default Administrator user account with permissions defined by the account user role to perform all functions in the software You cannot modify this account Create a user 1 Access the Users screen account Dashboard Edit Library Maintenance Tools Manage Prefere Audit Settings Resources ER Create A ii E Signature Change Password vo Manage Reports View Logs
202. e which the system will check for required electronic signatures see Table 26 on page 219 This selection presents an e sig prompt to users when they start a run if the required signatures have not previously been made Users must sign before they can continue For check before functions you can also Change the number of signatures required e Seta special authority for a signature click the Authorities Required field then select the user account or the user role to require for electronic signature of this function By default each required signature needs no special authority any user can sign Click Apply Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 217 Section 1 Administrators 218 2a 2b E Signature Settings Select the electronic signature types that should be allowed E Signature Type Approve Dye Set Approve Size Standard Approve Spatial Calibration Approve Spectral Calibration C Approve Instrument Protocol Approve Sizecall Protocol Approve Basecall Protocol Approve QC Protocol Select the Functions after which the system will prompt Far an electronic signature of th Function Accept Spatial Calibration Select the Functions before which the system will check Far required electronic signature Function Signatures Required Authorities Required Approve Gene Mapper Protocol Approve Gene Mapper IDx Protocol Approve Seg5cape
203. e at Name Number Conditions Environmental Temperature BigDye Terminator BDT v3 1 Cycle Sequencing Kit 4337454 15 C to 25 C 24 hours 24 reactions BigDye Terminator BDT v3 1 Cycle Sequencing Kit 4337455 15 C to 25 C 24 hours 100 reactions BigDye Terminator BDT v3 1 Cycle Sequencing Kit 4337456 15 C to 25 C 24 hours 1000 reactions BigDye Terminator BDT v3 1 Cycle Sequencing Kit 4337457 15 C to 25 C 24 hours 5000 reactions BigDye Terminator BDT v1 1 Cycle Sequencing Kit 4337449 15 C to 25 C 24 hours 24 reactions BigDye Terminator BDT v1 1 Cycle Sequencing Kit 4337450 15 C to 25 C 24 hours 100 reactions BigDye Terminator BDT v1 1 Cycle Sequencing Kit 4337451 15 C to 25 C 24 hours 1000 reactions BigDye Terminator BDT v1 1 Cycle Sequencing Kit 4337452 15 C to 25 C 24 hours 5000 reactions Table 28 Sequencing standards On instrument Part Storage Shelf life at Name Number Conditions Environmental Temperature BigDye Terminator BDT v3 1 Sequencing Standard long read 4404312 15 C to 25 C 24 hours Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 259 Appendix A Application Reagents and Run Modules Table 28 Sequencing standards On instrument None Part Storage Shelf life at Number Conditions Environmental Temperature BigDye Terminator BDT v1 1 Sequencing Standard long read 4404314 15 C to 25 C 24 hours BigDye T
204. e figure below to make sure most of 1X buffer is in the MENS larger side of the container There should be less d t d e N than ml of 1X buffer remaining in the smaller 9 side of the container Verify that the buffer 1s at the fill line Peel off the seal at the top of the ABC Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 237 Chapter 8 Maintain the Instrument 8 Place the ABC into the Anode end of the instrument below the pump IMPORTANT The RFID label must be facing the instrument and not you to ensure that the RFID information is read accurately by the instrument 9 Close the instrument door to re initialize Note If you do not close the instrument door to re initialize you need to click Refresh from the Dashboard 10 Click Refresh from the Dashboard to update the screen 11 Check the Quick View section of the Dashboard for updated status after changing the ABC Change the cathode buffer container CBC For the following hazard s see the complete safety alert descriptions in Specific chemical alerts on page 333 a WARNING CHEMICAL HAZARD Cathode Buffer Container CBC For details see Instrument reagents and consumables on page 9 Contamination might cause poor quality data To prevent the contamination use genuine packaged polymer anode buffer cathode buffer and conditioning reagent Use genuine parts and reagent The use of inappropriate parts or r
205. e in an instrument protocol adjust the Normalization Target proportionately For example for an instrument protocol with an injection time of 10 seconds and a Normalization Target of 2000 if you change the injection time to 15 seconds 50 increase change the Normalization Target to 3000 50 increase Normalization Factor Thresholds The passing range for Normalization Factor default range is 0 3 to 3 0 IMPORTANT Increasing the factor threshold above 3 0 may cause amplification of noise If the calculated Normalization Factor is outside the Normalization Factor range the software multiplies the peak heights of the sample by the low or high Normalization Factor threshold setting for example if the Normalization Factor range is 0 3 to 3 0 and the calculated Normalization Factor is 5 the software applies a Normalization Factor of 3 0 Normalization Factor Average peak height of the subset of peaks in the GS600 LIZ v2 size standard used for normalization divided by the Normalization Target Samples are flagged with ua in results if Normalization Factor is within threshold range or with if it is out of threshold range Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 167 Chapter 6 Manage Library Resources Dye sets library Dye set overview A dye set defines the following for an instrument protocol e Dye color s e Order of the dye peaks in the standard e Spectral analysis paramete
206. e scanner 321 before operating the instrument 317 biological hazards 331 chemical 328 chemical waste 330 electrical 319 ergonomic 321 guidelines 328 330 instrument operation 318 laser 320 321 moving and lifting computers and monitors 317 moving and lifting instrument 317 moving parts 318 physical hazard 318 pressurized fluids See also compressed gases safety repetitive motion 321 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Index solvents 318 standards 322 workstation 321 safety labels on instruments xiv 317 safety standards 322 safety symbols on instruments 315 sample data file storage file name convention location 50 injection folder 159 instrument run name folder 159 results group location 50 results group name folder 159 sample data file storage location 18 sample data files archive 255 sample info enter 48 sample name allelic ladder 49 assign 48 71 fill and fill series 48 71 sample plate See plate sample quality See also review results fragment HID in monitor run 62 in review results 89 sample type plate view 48 table view 71 sample view 77 scale plots 92 scientist user role 203 secondary analysis fragment 193 GeneMapper 193 GeneMapper ID X 195 HID 195 MicroSeq ID 191 SegScape 189 sequencing 189 191 security policies 199 security administrators account setup 199 disable effect on audit and e sig 198 enable or disable 198 export settings 224 export user account settings 224
207. e signal intensity Note the following e Asmall baseline window relative to the width of a cluster or grouping of peaks spatially close to each other can result in shorter peak heights e Larger baseline windows relative to the peaks being detected can create an elevated baseline resulting in peaks that are elevated or not resolved to the baseline Min Peak Half Width l Specify the smallest half peak width at full height for peak detection The range is 2 to 99 data points Polynomial Degree E Polynomial Degree cannot be greater than Peak Window Size Adjust to affect the sensitivity of peak detection You can adjust this parameter to detect a single base pair difference while minimizing the detection of shoulder effects and or noise The peak detector calculates the first derivative of a polynomial curve fitted to the data within a window that is centered on each data point in the analysis range Using curves with larger polynomial degree values allows the curve to more closely approximate the signal and therefore captures more of the peak structure in the electropherogram For information on optimizing Polynomial Degree and Peak Window Size see Peak Window Size Enter a window width in data points for peak detection sensitivity If more than one peak apex is within the window all are labeled as a single peak Note the following e The maximum value is the number of data points between peaks e The Peak
208. e standard The size standard peaks used to normalize the data are displayed in gray and are not editable 4 Click Save Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 173 Chapter 6 Manage Library Resources Basecalling protocols library primary analysis sequencing Basecalling protocol overview A basecalling protocol 1s the required primary analysis protocol for sequencing applications A basecalling protocol defines the settings used by the sequencing basecallers to assign base calls to each detected peak and assign a quality value e Analysis settings e Ranges for the sequencing quality flags displayed in View Results When you create a sequencing assay you add a basecalling protocol to the assay If you add this item from the library a copy of the item is added to the assay and can be modified independently from the original item stored in the library For information on how changes are tracked if auditing 1s enabled see Audit action on page 210 Create a new basecalling protocol 1 4 If factory provided basecalling protocols do not suit your needs you can create new basecalling protocols 1 Access the Basecalling Protocols library k 3500 Data Collection Software l Dashboard Edit Library Mainten 2 Click g Create Library Resources Filter 4 Manage Plates 3 Inthe Analysis Settings tab of the Create New Basecalling Protocol dialog box Figure 24 on page 1
209. e with the same settings but different names for example Injection 4 6 Injection 7 9 and Injection 10 12 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 161 Chapter 6 Manage Library Resources S Create New Results Group Mame Injection 1 3 Locked Select Results Group Attributes Preview of Results Group Mame Injection 1 3 Available Attributes Selected Attributes IP Mame Add gt Results Group Mame Logged in User Name 3 PA Protocol Name ere liil gt PRENDE Select Reinjection Folder Option Store reinjection sample files in a separate Reinjection Folder same level as Injection fo P Store reinjection sample Files with original sample Files same level Select Folder Option Default File location C Applied Biosystems 3500 Datal lt IR Folder AInjection 1 31 Custom file location Include an Instrument Run Name Folder Include a Result Group Mame Folder Close Save Results group example 3 store re injections in separate folders Figure 17 on page 163 shows an example results group that specifies a sample file storage location of C Example instrument run IR folder result group name folder results group name start instrument run date time stamp logged in user name Nnjection name or re injection name folder The numbers in the figure relate the elements in the results group with the elements in the file hierarchy created by a run that uses t
210. ead the details Click Details to determine the cause of the error When the plate is successfully loaded the Load Plates for Run screen is displayed MM Link Unlink Plate X Plate Setup Error es N No sample name has been assigned to the Following well GO1 HO1 G02 HO GOS ABA To proceed please assign a sample name to the wells Incompatible Polymer type Configured on plate POP Currently installed POP4 Incompatible Capillary length Configured on plate 50 cm Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 309 Appendix E Troubleshoot Symptom Possible Cause Action No plate in position A message You physically loaded plate in position B plate B position and try to link plate Click Link Plates and link the plate directly to position B plate B position Follow the prompts No plate detected message The plate is in position B Place the plate in position A Fragment performance check is required message Running fragment modules after loading the plate Change polymer to POP 7 Run fragment analysis performance check Sequencing performance check is required message After loading the plate Running sequencing modules POP 6 after loading the plate Change polymer to POP 7 Run sequencing performance check Load plate or Load Plate for Run message Performance issues Click OK
211. eagents causes poor quality data or damage the instrument 1 Remove the CBC from storage 2 Check for expiration date on the CBC label to make sure it is not expired prior to or during intended use 3 Allow refrigerated CBC to equilibrate to ambient temperature before use 4 Wipe away condensation on the CBC exterior with a lint free lab cloth 5 Verify that buffer level is at or above the fill line and check that seal is intact IMPORTANT Do not use if buffer level 1s too low or seal has been compromised A fill tolerance of 0 5mm 1s acceptable Note The meniscus must be at or above the fill line Fill line es m 238 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 10 11 12 bs 14 Wipe off any buffer on top of the CBC with a IMPORTANT Failure to perform this action may Instrument operational procedures Tilt the CBC back and forth gently and carefully to ensure that the buffer 1s evenly distributed across the top of the baffles Note If you do not tilt the CBC back and forth the buffer sticks to the baffles due to surface tension Verify that the buffer 1s at or above the fill line When ready to install CBC place the container on a flat surface such as a lab bench and peel off the seal lint free cloth Ensure that the top of the container 1s dry result in an arcing event and termination of the run Place the appropriate septa on both sides of the
212. eason when you make certain changes in the software S Audit Reason Setup Audit Reason Based on your system configuration you can either select a reason or enter a reason for change Select From List of Reasons Comments Calculation error Close Electronic signature If your system is configured for electronic signature you may be prompted to provide your user name and password when you perform certain actions in the software ES Electronic Signature Electronic Signature Enter your user name password and any comments If an item is set to require two signatures the signers are not required to sign at the same time When the first signer signs the E Sig status 1s set to Partially Signed When the second signer signs the E Sig status is set to Signed User Marne Password Comments You may also be permitted to sign objects such as plates calibrations or other library items If electronic signature is enabled for items any of the following may apply The 5 E Signature button is enabled in the library or the calibration e You are prompted to sign as described in How the software prompts electronic signature before a run on page 220 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 227 Section 2 Users The Open Plates dialog box or the library displays an Is signed column that reflects the electronic signature status of an item S Open Plate From Li
213. ectrical equipment for measurement control and laboratory use Part 1 General EMC requirements Group 1 Class B For the Reader Writer unit in the Applied Biosystems 3500 3500xL Genetic Analyzers CE Notice European Union Marking by the symbol indicates compliance of this ASI4000 98 BS1 RFID R W Module to the Electromagnetic Compatibility Directive and the Low Voltage Directive of the European Union Such making 1s indicative that this RFID R W Module meets the following technical standards EN 300330 Electromagnetic compatibility and Radio spectrum Matters ERM Short Range Devices SRD Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 323 Appendix F Safety Europe CE declaration of conformity Reader Writer 324 e EN 301489 Electromagnetic compatibility and Radio spectrum Matters ERM ElectroMagnetic Compatibility EMC standard for radio equipment and services EN 60950 Safety of Information Technology Equipment EN 300 330 1 V1 5 1 2006 04 EN 300 330 2 V1 3 1 2006 04 EN 301 489 3 V1 4 1 2002 08 EN 301 489 1 V1 6 1 2005 09 EN 60950 1 2006 English Hereby ART Technology Co Ltd declares that this ASI4000 98 BS 1 is in compliance with the essential requirements and other relevant provisions of Directive 1999 5 EC Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Instrumentation safety Fran ais French Parla pr sente ART Technolo
214. ed Biosystems 3500 3500xL Genetic Analyzer User Guide cathode buffer change 238 described 9 on instrument limits 30 part number 9 storage conditions 9 troubleshoot 304 CAUTION description xiii CBC See cathode buffer Change Polymer Type maintenance wizard 247 check valve 28 chemical safety 328 chemical waste safety 330 Clear range 176 computer maintenance See maintenance computer name requirement 22 start up 24 condition number spectral calibration 109 conditioning reagent described 11 part number 11 place on instrument 250 consumables anode buffer 9 237 capillary array 240 252 cathode buffer 9 238 conditioning reagent 11 250 fragment analysis and HID 260 Hi DiTM Formamide 12 on instrument limits 30 polymer 10 245 247 249 sequencing analysis 259 status in Dashboard 29 236 contiguous read length See CRL controls specifying 48 conventions bold text xvi for describing menu commands xvi IMPORTANTS xvi italic text xvi Notes xvi user attention words xvi CRL ambient temperature dependence 30 defined 81 distribution report 85 performance check 125 QV settings 178 report 85 result 81 threshold 178 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Index CRL Basepair Accuracy 125 CRL Median and SD 125 custom dye set See dye sets custom customer feedback on Applied Biosystems documents 336 customize sample info 48 D daily instrument maintenance 230 231 DANGER description xiii Das
215. ed Biosystems 3500 3500xL Genetic Analyzer User Guide set Up and Run Workflow Start the system 1 Start the instrument page 22 Start the computer page 24 Check maintenance notifications in the Dashboard page 28 Check consumable status in the Dashboard page 29 Replenish consumables page 31 Set up and run Prepare the instrument page 42 Preheat the oven page 42 Check instrument status page 53 Create or import a plate page 43 Assign plate contents page 46 Print the plate layout page 50 Quick Start a run page 56 Prepare and load sample plates page 51 Load plates for run and create the injection list page 56 Review and modify the injection list in Preview Run page 59 Start the run page 59 Monitor the run page 61 check sequence or sample quality and specify re injections page 62 Review sequencing results Review fragment HID results 1 2 Specify re injections page 85 3 4 Export sequencing results page 87 Review sequence quality page 81 1 Review sample quality page 89 2 Specify re injections page 95 Review quality reports page 85 3 Review quality reports page 95 4 Export sizing results page 96 Optional print or save pdf calibration and performance check reports Spatial calibration page 102 Spectral calibration page 116 Sequencing install standard performance check
216. educe the chance of electrical shock do not remove covers that require tool access No user serviceable parts are inside Refer servicing to Applied Biosystems qualified service personnel AVERTISSEMENT Pour viter les risques d lectrocution ne pas retirer les capots dont l ouverture n cessite l utilisation d outils L instrument ne contient aucune pi ce r parable par l utilisateur Toute intervention doit tre effectu e par le personnel de service qualifi venant de chez Applied Biosystems CAUTION Class 2 lIl visible and or invisible radiation present Do not stare directly into the beam or view directly with optical instruments ATTENTION Rayonnement visible ou invisible d un faisceau Ne pas regarder le faisceau directement ou au travers d un instrument optique DANGER Class 3B Ill visible and or invisible radiation present Avoid exposure to beam DANGER Rayonnement visible ou invisible d un faisceau de Classe 3B lll en cas d ouverture Evitez toute exposition au faisceau CAUTION Sharp object AVERTISSEMENT Objet Sharp XIV Applied Biosystems 3500 3500xL Genetic Analyzer User Guide About the product About the product The Applied Biosystems 3500 3500xL Genetic Analyzers is an automated 8 and or 24 capillary instrument designed for a wide range of sequencing and fragment analysis applications IMPORTANT For Research Use Only Not for use in diagnostic procedures Purpose
217. ee dd amd So chee Debra uS Specie Dira ae Se Cli ts D Cowesckwe H basing GF ming x Conmendne cael es Sarre A A cee tee bard Ad Elda F e perce Scere paria iha rrai Security Policies Password Eap r bon dico pribor Sarak Tirren ral Libor Fury X nup wr fear F Patbeesr is vell expare 5 ere Che Logs athens sih an noel par edi apar the or acon SPR REI Here i n bane ae ier pepinos val b imed ad e Cree C1 ever dO dwa de le Eire J daeya bore agar Pore reed Mr i es 50 zx TI in ete run i mox considered coner actis velitw arre a Setup Messaging Notifications save Settings b Figure 34 Security disable or enable 198 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Configure the security system Set account setup and security policies Security policies apply to all user accounts 1 Under Account Setup specify user name limits Account Setup User Password User Name The length of user passwords must be between and characters The length of user names must be between and characters Define password spacing Define name spacing Leading Trailing Consecutive Leading Trailing C Consecutive cad Define password characteristics Define name characteristics o Alpha o Numeric o Uppercase o Lowercase o Special Alpha Numeric Uppercase Lowercase Special User may not reuse the previous passwords IMPORTANT The software allows spaces i
218. eee ee eee 313 MISCOlanedUsS dra del ad 313 ResettneIMStUMEAE cia AA 314 ADPDENdIX F A 315 Instrumentation safety a na naana naana eee eee eas 315 SymMDOISOMINS MEN S 0 cies o ng REE eee A 315 Safety labels on instruments 0 0 000 cee eee es 317 General instrument safety 0 0 0 000 een 317 Physical NaZara Salcly airis de Duelo A Bes REED dc de grece Debt iri ent 318 EISCTIC ASAI OLY ROLLE 319 B cleccus a a TP RTL ERIT 320 Bar code scanner laser safety 0 0 0 0 eee 321 Workstation Safely cnn Eas denen Wh ce No Gee shone RR eld b RERO DEL t eli oi i 321 Safety and electromagnetic compatibility EMC standards 322 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide xi Contents xii Olhemical Saleby urns dear nea wow Da dee e a eee eb cr 328 General chemical safety aa 328 MSUSS N E caera os als A EEN 329 Chemical waste safety 0 ccc eee ee eee eee 330 Biological hazard salely subas ra ead de ds 331 SA INT 332 ChiemiGal alens 5 23 rd da ah ub io erbe Dow cea a ede ek 332 Instrumentation alerts paris tee CERO 3 ee Bo ae es eck ace x eee Das 334 DOCUMENtaCI N arranca E ond EC Io eee des 335 Related documentation 000 ee eee ees 335 Obtaining information from the Help system 00 000 eee eee 336 oend us your COMMONS esla EE ER SERA Ib E ded es s RE 336 lae CLIP TP TIR E 33 Applied Biosystems 3500 3500xL Genetic Analyze
219. een click toolbar options to manipulate the report as needed Place the mouse pointer over an item for a description of the item 3 To print the report click a Print Close the report Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Page 1 of 1 Page 1 of 1 205 Section 1 Administrators Save electronic To save the report electronically pdf print the report and select CutePDF Writer copies pdf of as the printer the report Example reports User Report Last Password Ae Role Email Phone Status Uan Modified Change Hoe epe Comments User Name Date On Date Expired Administra Administr Adminis Active false tor ator trator technician First Technici Active 28 Jan 01 Feb 31 Jan false Mame MI an 200910 200810 200810 12 Liser2 1357 AM 10 48 AM 48 AM scientist Firsthlam Scientist Active 29 Jan 01 Feb 31 Jan e MI 200911 200910 2009 03 36 User3 30 12AM 10 22 AM 58PM Analystl First Log in 01 Feb 01 Feb 01 Feb Mame MI to 200910 200810 200910 21 Analysti timed 21 26 AM 21 26 AM 26 AM out session User Role Report Role Name Description Last Updated Date User Counts Administrator Technician Scientist Log in to timed out session 01 Feb 2009 10 12 18 AM 206 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Manage auditing Manage auditing Access the Audit screen and enable or disable auditing The Audit screen controls the auditing state en
220. en audited System Configuration History SAE configuration records including audit history for each user account e Action log System specified audit events 3 Optional e Sort the table See Multi column sorting on page 72 e Specify filters date range user name action object or record type object or record name reason then click Go Note The Reason field in System Configuration History is not used Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 209 Section 1 Administrators e Select a record then click Show Object History p or Show Audit Details e In the history dialog box select a record then click Show Audit Details Click Table Settings then specify the columns to show or hide Select your table display preferen Available Columns to Display Review the object audit history Audit records The Object Audit History lists the most recent audit for the user objects listed below samples and objects in the libraries that have been audited e Dye set e Assay e Size standard Plate template Instrument protocol File name convention PA protocol primary analysis Results group SA protocol secondary analysis Plate QC protocol e Sample files Audit action Possible actions for all objects are update create and delete Audit records are generated under the following conditions Action Description Up
221. en view a 5 dye sample you must manually select the fifth dye It is not automatically selected when you switch to a 5 dye sample plots Apply scaling settings to CE du m K Plot Settings Setup Plot Settings Enter the range for Y axis and X axis then Display Labels Scaling and Zooming click the Zoom buttons IMPORTANT You must Zooming open Plot Settings each time you Zoom Y axis relative to selected peak access the View Results screen then click Zoom Scaling settings are not automatically applied when you Zagm axis From T access this screen or when you click Apply To apply scaling settings to all samples in the samples table select all of the samples in the samples table to display them in the plot view specify the scaling settings click Zoom then click Page Up and Page Down in the plot view to move through the samples 2 button is grayed it indicates that the Plot RSE Settings dialog is open Click the 3500 task bar KS 3500 Data Collection Software icon then select Plot Settings Display multiple plots as needed in the Plot checker Board Settings Display tab select Checkerboard oce 2 w peaks on page 93 Place the mouse pointer above the top of the plot or to the left of the plot at the start of the area you want to zoom then click to turn the pointer to C With the a still above the plot or to the left of the plot 0 0 al click
222. enetic Analyzer User Guide 125 Section 2 Performance check Evaluate sequencing install standard data When a sequencing install standard run completes successfully the CRL Pass Fail row displays green or red results For each capillary 1 Click a capillary to display the spectral and raw data profiles for a capillary 2 Check that the data meet the following criteria Attribute Acceptance Criteria Example Order of the peaks in the 4 dye blue green yellow red Sle Green Wellow Red spectral profile intensity vs pixel from left to right Extraneous peaks in the raw None data profile intensity vs scan Peak morphology in the spectral No gross overlaps dips or other profile intensity vs pixel Note The E5 profile may include extraneous peaks outside the matrix peak region which can be ignored irregularities e Peaks separate and distinct e Peak apexes are separate and distinct the tails will overlap 126 3 Optional Review the CRL accuracy to determine discrepancies from the reference sequence General sequencing 40 to 539 bp e MicroSeq ID 20 to 619 bp If you observe large discrepancies for example 5 to 10 contiguous miscalled bases in the middle of a sequence review the data If you see a raw data peak larger than the adjacent peaks with baseline pull up in all 4 dye color channels it may indicate the presence of a bubble Check the pump run the Remove Bubble
223. entry Lock When enabled allows the entry to be unlocked and modified only by the user who created it the administrator or another user with unlock permissions Useful when your system includes the SAE module described in Chapter 7 Use Security Audit and E Sig Functions SAE Module on page 197 Application Type Automatically set to HID Secondary Analysis Software IMPORTANT The secondary analysis software must be installed and properly configured with the 3500 Series Data Collection Software before it is listed as a selection in this screen For information on setting up the GeneMapper D X Software for auto analysis see the GeneMapper D X Software v1 2 Installation Guide Secondary Analysis Software Computer on which the secondary analysis software is running Instance Properties GeneMapper D X analysis method size standard and panel to use for auto analysis Note If you are running a stand alone version of the 3500 Series Data Collection Software a version that is not installed on the instrument computer you can create plates and protocols then export them for use on the instrument computer 196 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Use Security Audit and E Sig Functions SAE Module Section 1 Administrators Administrators overview of system security auditing and electronic signature The SAE Security Audit E Signature module 1s an optional component of t
224. er different search criteria 3 Select a plate then click Load Plate 4 Click Start Run from the Load Plates on Run Screen IMPORTANT It takes approximately 10 seconds for the instrument to initialize after the instrument door is closed Do not start a run until the instrument status light 1s green Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 55 Chapter 3 Set Up and Run Load plates for run and create the injection list Load the plate in the instrument see Load the plate in the instrument on page 53 and link the plate Link the plate on page 54 before proceeding 1 Access the Load Plates for Run screen Figure 6 on LA Hun Instrumont page 56 from The Assign Plate Contents screen by clicking bs Or Link Plate for Run Monitor Run The navigation pane by selecting Load Plates for Run in the navigation pane The Dashboard by clicking the Main workflow arrow 4 then selecting Load Plates for Run in the navigation pane Run Information You can edit the Run Mame by entering best 3 T PARU a Run 2003 03 08 13 28 46 903 7 Connection Stabus Connected Leer Mame Administr abor Plates on Instrument o Plate A 96 wells Plate B Recent Pates Recent Runs CA T 770708 3 09 Run 2 DB Mar 200 4 Tena HID 3 07 09 Run 2 07 Mar 2000 Barcode 39 07 Mar 2300 z template 04 Mar 2700 best reinpections 03 Mar
225. erage quality value See also quality value monitor run 64 QV settings sequence basecalling protocol 177 color and ranges 37 monitor run screen 177 recommended ranges 84 QV20 report 85 result 81 setting 178 R radioactive waste handling 331 Read Length 125 recent plates 76 runs 76 red indicator light 23 re injection See also duplicate injection allelic ladder 67 collect data for specific wells 65 defined 65 file location 159 in view sequencing results 85 instrument protocol selecting 65 monitor run 65 order 65 samples all in injection 65 samples individual 65 re injection button grayed 65 re injections in view fragment HID results 95 Remove Bubbles maintenance wizard 251 rename samples in review results 97 repetitive motion safety 321 Replenish Polymer maintenance wizard 245 replicate injection See injection list report settings fragment HID 95 sequence 86 reports action log 209 212 audit 213 CRL and CRL distribution 85 default settings 34 default settings sequencing 38 e sig 222 346 export sequencing 87 fragment HID install performance check 137 fragment HID results 95 object audit history 209 210 plate grid 50 sample quality 95 sequencing defaults 38 sequencing install performance check 127 sequencing results 85 spatial calibration 102 spectral calibration 116 system configuration history 209 211 trace score 85 user 205 reset instrument 314 restore audit records 214
226. erence _ anmotation txt Sequencing Settings C phd 1 Spectral Calibration scf User Plate Setup Reports Settings Entire Sequence Post trim Sequence Only Run Setup B Sequencing Settings fsta Trace O Trace Print Trace Quality qual Trace Quality Reports O seq 2 Select the file types to export Exported files are stored in the same directory as the abl files File type Description annotation txt Information from the Annotation tab in the sequencing trace view such as data collection time run time start finish phd 1 scf Sequencing files fsta qual seq Reference files specify Entire Sequence or Post trim Sequence Only 3 Click Apply to save the system preferences see System preferences on page 33 Trace user The Trace preference settings determine the default settings for color representation preference ofnucleotide and quality value bars in the Trace View in View Sequencing Results 1 In the Preferences dialog box click Trace under User Sequencing settings to display the Trace pane 36 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Set preferences k Preferences type Filter text not used Trace 7 E System These settings affect the Trace panel polis MT Base Color Pure Base QV Colors Plate Setup Reports Settings MT Base Foreground Background n 1520 100 Sequencing Settings 0 c mm 31 Tao odis s m
227. erminator BDT v3 1 Matrix Standard 4336974 2 C to 8 C 24 hours BigDye Terminator BDT v1 1 Matrix Standard 4336824 2 C to 8 C 24 hours Fragment and HID analysis reagents Note For reagent or consumable shelf life expiration date see the package label The following table shows all the reagents for fragment and HID analysis Table 29 Fragment analysis HID standards On instrument Part Storage Shelf life at Name Number Conditions Environmental Temperature Fragment Analysis Matrix Standards 5 Dye DS 02 4323014 2 to 8 C 24 hours Fragment Analysis Matrix Standards 4 dye DS 32 4345831 2 to 8 C 24 hours Fragment Analysis Matrix Standards 5 Dye DS 33 4345833 2 to 8 C 24 hours Fragment Analysis Installation kit 5 Dye DS 33 4376911 2 to 8 C 24 hours GS120LIZ Size Standard 4322362 2 to 8 C 24 hours GS500ROX Size Standard 401734 2 to 8 C 24 hours GS600 LIZ Size Standard v2 for Normalization 4408399 2 to 8 C 24 hours GS1200 LIZ Size Standard 4379950 2 to 8 C 24 hours 260 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Sequencing analysis dye sets for all applications Sequencing analysis dye sets for all applications Note For reagent or consumable shelf life expiration date see the package label The following table shows all the dye sets for various applications Table 30 Dye Sets for various applications Dye Set Application Name E
228. erogram troubleshooting on page 306 Symptom Possible cause Action Electrophoresis failure Buffer below fill line inadequate amount of buffer Ensure that buffer level is at or above the fill line Do not use if buffer level is too low or seal has been compromised Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 303 Appendix E Troubleshoot Cathode buffer container troubleshooting Also see Data electropherogram troubleshooting on page 306 Symptom Possible cause Action Electrophoresis failure Buffer below fill line inadequate amount of buffer Ensure that buffer level is at or above the fill line Do not use if buffer level is too low or seal has been compromised RFID troubleshooting Symptom Possible cause Action Unable to read RFID information Failure to Read from RFID tag Consumable package is improperly installed or defective label Polymer Conditioning reagent pouch mis oriented Ensure that the RFID label is not visibly damaged and consumable package is properly installed Ensure that label is close and parallel to the instrument Reposition or re install consumable and click Refresh on the dashboard If no results restart the instrument and the computer If no results install a new consumable if available and call your Applied Biosystems representative for a replace
229. essly transferred into SeqScape for analyzing processing and reporting Note For detailed information on setting up a MicroSeq ID project to auto analyze in the 3500 Series Data Collection Software see the MicroSeq ID v2 2 Getting Started Guide Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 267 Appendix B Secondary Analysis Sequencing Set up an auto analysis project in SeqScape IMPORTANT When using SeqScape Software to auto analyze results data from the 3500 3500xL analyzer you must have v2 7 installed on the same computer as the 3500 Series Data Collection Software Set up a project in the secondary analysis software before starting a run on the 3500 3500xL analyzer All analysis in SeqScape occurs in a project Create a project by following these steps 1 Create a RDG by importing a Reference Sequence 2 Define Analysis and Display Settings 3 Create a Project Template 4 Create an empty Project with blank Specimens e Importa 1 Start the SeqScape Software B then select Tools SeqScape Manager reference r Select the Reference Data Group tab then click New and enter a name RDG Properties General ROI NT Variants AA Variants Variant Style Reference Data Group Description Reference Data Group Name YourRDG Created Modified Created By Modified By Source General Settings Codon Table vertebrate mitochondrial w Comments
230. esults e Assay e File name convention e Results group e Instrument protocol e PA protocol primary analysis basecalling and sizecalling e SA protocol secondary analysis sequencing fragment analysis HID analysis e QC protocol primary analysis HID e Size standard e Dye set e Create e Edit e Delete e Import e Export 204 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Manage user accounts Table 22 User role permissions continued Category Permissions Plates and templates e Edit e Delete e Import e Export Locking Unlocking e Assay e File name convention e Results group e Instrument protocol e PA protocol e SA protocol e QC protocol e Size standard e Dye set Preferences e Edit system preferences e Export system preferences e Import system preferences e Export user preferences all Calibrations e Perform spatial calibration e Perform spectral calibration Performance check Run performance check install standards Archiving e Archive e Purge e Restore SAE configuration e Configure SAE e Log in to timed out user sessions Edit a user role l In the Roles screen select a user role then click Edit 2 Edit settings as needed You cannot edit the Administrator user role 3 Click Save View and print a user report 1 Select the User or Roles tab Click View Report 2 In the Report scr
231. etwork you do not need to log on to the network before starting the instrument c Click OK Wait until the computer finishes booting IMPORTANT The status icon on the right lower corner of your screen shows when the 3500 Server Monitor 1s active by displaying the icon shown here IMPORTANT Do not close this icon Doing so will prevent proper functioning of the software Log on to Windows Follow the prompts to log on to the Windows operating system Launch the application Step one Launch Ifthe Daemon does not start automatically launch the Daemon the Daemon Start Programs Applied Biosystems gt 3500 Daemon Daemon Note It will take approximately 15 seconds for Daemon to populate 24 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Start the computer Step two Launch _ Ifthe Server Monitor does not start automatically launch the Server Monitor the Server Start Programs Applied Biosystems gt 3500 Server Monitor Monitor k Server Monitor Note It will take approximately 2 minutes for the Server Monitor to set up During this time you will see CAG 2 45 PM the status icon transition from a red circle with an X in L the middle indicating that not all 3500 services are loaded to the shape of an hour glass on your desktop next to the clock 2 45 PM When Server Monitor set up is complete the icon in the shape of an hour glass will disappear and a 2 47 PM c
232. ew 71 spectral calibration 111 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Part Number 4401661 Rev C 06 2010 A ie d Headquarters International Sales Bi t 850 Lincoln Centre Drive Foster City CA 94404 USA For our office locations please call the division lOSys ems Phone 650 638 5800 Toll Free 800 345 5224 headquarters or refer to our Web site at www appliedbiosystems com www appliedbiosystems com about offices cfm
233. f the Create New Sizecalling Protocol dialog box Figure 26 on page 180 specify settings see Table 14 on page 180 4 Click QC Settings In the QC Settings tab of the Create New Sizecalling Protocol dialog box Figure 27 on page 183 then specify settings and Table 15 on page 183 5 Click Save Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Library Resources 3500 Data Collection Software 3 Dashboard Edit ibrary Mainken Bate 4 Manage Plates Assays Sizecalling Pr File Name Conventions Fragm Results Group Long f SNaPsl ili Arratyze Instrument Protocols Dye Sets Size Standards Basecalling Protocols eizecaliing Protocols 179 Chapter 6 Manage Library Resources MM Create New Sizecalling Protocol Setup a Sizecalling Protocol Px Protocol Name is a required Field Provide a unique value Protocol Mame Description Size Standard G5600LIZ Sizecaller SizeCaller v1 1 0 Analysis Settings Qc Settings Analysis Range Ful w Sizing Range Full hal Size Calling Method Local Southern Analysis Start Point Sizing Start Size X Primer Peak Present v Analysis Stop Point 1000000 Sizing Stop Size LOoooo 7 Blue Green Yellow Red Purple i Drange Minimum Peak Height l ums 1175 um ms Common Settings Polynomial Degree Slope Threshold Peak Start Slope Threshold Peak End Figure 26
234. f the Sequencing install standard Blocked capillary Refill the capillary array You may have Performance check fails to install a fresh array or consider that Fail capillary capillary non usable for purposes of planning your runs e UE fale capiat Incorrect chemistry file dye set Correct the files and rerun the and or run module selected calibration e f more than three failed capillary for 24 capillary Insufficient filling of array Check for broken capillaries and refill the capillary array Accept button is not active but Reject button is active Expired matrix standards or old Check the expiration date and storage reagents conditions of the matrix standards and or reagents If necessary replace with a fresh lot Expired polymer Replace the polymer with a fresh lot using the Replenish Polymer Wizard Bubbles in the polymer system Select the Bubble Remove Wizard to clear the bubbles Possible contaminant or crystal Properly bring the polymer to room deposits in the polymer temperature do not heat Replace the polymer if it has expired 302 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Fragment HID install standard troubleshooting Fragment HID install standard troubleshooting Symptom Possible cause Action Fragment HID report contains blank pages or incomplete information All dyes are not selected before you generate the report Select all dyes then gener
235. faulr 06 45 48 933 BemoryBoniror Min Freee memor No errors displayed 4 Obtain the host name full computer name a Right click Computer on the desktop then select Properties b Locate the full computer name You will need to enter the name when you install the GeneMapper Software Windows Vista Computer name domain and workgroup settings Computer name ServerName Full computer name ServerName Computer description c Close the dialog box 292 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Install GeneMapper v4 1 for remote auto analysis Remote auto analysis installation Insert the GeneMapper v4 1 Software Full Installation DVD into the DVD drive to start the installer If the installer does not start automatically a Right click Computer then select Explore b Expand the DVD drive then select the GeneMapper v4 1 folder to display its contents c Double click Rz to start the installer GeneMapper v InstallShield Wizard Es Ol Before proceeding with the installation please close all running applications and its windowsa But i Data Collection application as installed in this system please ensure that it is running In the Welcome window click Next Review the installation requirements status then click Next Select Remote Analysis for type of installation then click Next GeneMapper i Fm GeneMapper Software Sabai
236. fer to prevent the polymer from drying in the capillaries If fluid level is low add DI water to buffer solution Install the new CBC when ready to resume runs for more than 2 weeks Long term See below for long term instrument shutdown Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 253 Chapter 8 Maintain the Instrument Computer maintenance This section lists the common tasks required to maintain the computer for your 3500 or 3500xL analyzer in good working condition For the computer troubleshooting issues see Appendix E Troubleshoot on page 299 Uninstall the software When you uninstall the software you are prompted to back up the datastore the directory that contains all library items you created such as plates and protocols IMPORTANT Do not back up the datastore to the installation directory The installation directory 1s deleted during the uninstall Archive purge and restore data e Archive Makes a copy of the data in an external file that you can save in another location Purge Allows you to delete purge user created items stored in the library Factory provided items are not purged You have an option to archive the items also Restore Restores archived data back to the system IMPORTANT These functions affect items stored in the library datastore These functions do not affect sample data files Frequency Applied Biosystems recommends that you purge t
237. flow Tools Manage Preferences Help Lior ar Maintenance The Library workflow contains the screens where you manage assays protocols and other items that you use to acquire and process data The Library workflow contains e tems that you select when you set up for a run plates assays filename conventions and results groups e tems that you select when you create an assay Instrument protocols Primary analysis protocols Basecalling sequencing sizecalling fragment analysis QC HID analysis Optional secondary analysis protocols a Manage Assay File Mame Conventions Results Group iii Analyze Instrument Protocols Dye Sets size Standards Basecalling Protocols Sizecalling Protocals 2C Protocols Sequencing Analysis Protocols MicroSeqiD Protocols Fragment Analysis Protocols HID Analysis Protocols Y Sequencing analysis fragment analysis and HID analysis e Items that you select when you create instrument sizecalling and QC protocols Dye sets and size standards You can click Main Workflow or select Dashboard or any other menu item at any time to advance from the Library workflow The Library workflow 1s described in Manage Library Resources on page 139 Select Maintenance in the menu bar to access the Maintenance workflow ary Maintenance Tools Manage Preferences Help The Maintenance workflow contains the screens where you calibrate check ins
238. for up to 160 injections t 30 The Polymer Sample Counter decrements only for wells that contain sample but the Polymer Injection counter decrements for each injection regardless of whether all wells contain sample The sample limit and the corresponding injection limit may not coincide Note that the initial injection limit is higher than the initial sample limit Example 960 sample pouch on 24 cap If all wells contain sample for all injections 960 24 40 injections If all wells do not contain sample for all injections 960 lt 24 40 injections up to a maximum of 50 injections and a maximum of 960 samples A polymer pouch includes additional volume to accommodate the volume used during installation and by wizards However excessive use of wizards reduces the number of remaining samples and injections based on how many times specific wizards are run For example if you run the total bubble remove option in the Remove Bubbles wizard more than four times or run other wizards excessively including multiple pouch installations the number of remaining samples and injections is reduced Ambient temperature must be in the range of 15 C to 25 C POP 6 Sustained use at higher temperatures may result in shorter read lengths than specified Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Check buffer fill levels Check system status in the Dashboard Check the fill levels on buffers Verify that buffer
239. four traces at a time m c ile Viewers 1x1 3429 1DA MI e Set trace colors in Preferences see Set sequencing preferences on page 36 Tile Viewers 1x2 sd Tile Viewers 2x1 fea Tile Viewers 3x1 1 Ey a b illl 15 ye fife Tile Viewers 2x2 mm mE m NM NH ES AAA ES EE S TGCCAA GOA CTAAATCT CT ESE 82 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 4 Set the category of base for the Tab key 5 Review traces press Tab to review bases from left to right in a trace Shift Tab to move right to left Bases Coordinates xu 152 481 350 a0 245 250 1000 Gry bar MM oo val cL Analyzed Raw Analyzed Raw Annotation Sequence EPT Review Sequencing Results HI Ls EL 7 SetTabKeyte e pr Low GV Base lil am Med Qv Base ia High QV Base lil M MN Base a Mixed Base TA TTT Place mouse pointer on Mixed base bar to display Gs value rj I 3050 9100 3200 41 55 56 30 56 56 58 55 55 55 56 55 58 54 111111111 GGGRCT GG MA Cccc WN T View ing options analyzed data of the data Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Click the tabs at the bottom of the trace pane for different views Peak Place mouse pointer in trace to zoom Move slider to scale vertically Analyzed Raw Analyzed cRaw Annotation Sequence EPT
240. g Quality is EJ normalization is not applied even 1f the Normalization Factor is within the normalization range Ensure that you use the normalization size standard appropriate for your application For more information see Normalization size standards provided on page 171 90 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Review Fragment HID Analysis results How Tonormalize the software normalization is P applied 1 Determines if the data was collected on the 3500 or 3500xL analyzer instrument 2 Determines if the sample was collected with a normalization size standard definition file normalization 1s enabled 3 f normalization 1s enabled the software calculates a Normalization Factor for the sample using multiple size standard fragments The Normalization Factor is calculated by dividing the Normalization Target by the observed average peak height of the size standard fragments in the samples 4 Compares the sample Normalization Factor to the thresholds set in the instrument protocol 5 Ifthe calculated Normalization Factor 1s within the Normalization Factor range multiplies the peak heights of the sample by the calculated Normalization Factor If the calculated Normalization Factor 1s outside the Normalization Factor range multiplies the peak heights of the sample by the maximum or minimum Normalization Factor threshold setting for example if the Normalization Factor range is 0 3 to 3 0 and t
241. generated automatically via the Radio Frequency Identification RFID reader Use the information presented to you in the Quick View section before and after performing a maintenance task Quick View Gauges POP Polymer AB 3500 Buffer Anode AB 3500 Buffer Cathode 50cm 24 cap Array 384 576 3 4 3 4 64 96 t RE 2 41 1 T E B 2 ntir Fap 8 DARE PE G I2 3 5s I D Es a Pa 32 7 128 e a 4 gt auges TS 26 s E sdi y a96C o a 0 gm On bu 634 Samples Remaining 7 Days Remaining 7 Days Remaining 43 Injections Performed 34 Injections Remaining 96 Injections Remaining 96 Injections Remaining Pre Heat the Oven Instrument 3500 Instrument state Idle view Instrument Sensor Details Set Temperature to Instrument Laser On Owen Off o 1 information EP On Oven Door Open Oven Temperature C 53 5 C Start Pre Heat I o 1 masas Detection Cell Temperature C 23 5 Consumables Information o Palymer POP 634 Samples Remaining 1 20 Mar 2009 11 514007 4315930 Consumables Anode Buffer 46356 Buffer 7 Days Remaining 1 28 Mar 2010 11 518007 4315931 Cathode Buffer AB 356 Buffer 7 Days Remaining 1 26 Mar 2009 11 518007 4315931 Capillary Array 5 cm 24 cap 117 Injections Remaining 80 31 Dec 2009 11 80K005 4319899 Serial 80K2450 Maintenance Notifications o Replace cathode buffer c HIGH 22 Mar 2009 1 Replace c X Clean Drip Tray HIGH 22 Mar 20
242. gment Plate Number of Wells O 96 96 FastTube 384 Plate Type Fragment z Capillary Length 150 cm Polymer POP7 gt 4 Select the Number of Wells Plate Type as Sequencing Capillary Length and Polymer associated with this plate for the current run Specify auto 1 Check Perform Auto Analysis right side of the Plate Details section to analysis for expand the Secondary Analysis section secondary analysis y Analysis ero Auto Analysis o Software Type GeneMapper Software Location GeneMapper 3500 FVTEST 2 Confirm GeneMapper auto populates as the Software Type Note If GeneMapper does not appear in the drop down list under Software Type check your installation Secondary analysis software must be installed correctly before GeneMapper is automatically listed as a selection 3 Confirm Your computer name auto populates as the Software Location IMPORTANT For auto analysis to be successful the secondary analysis protocol must match the software location set here 4 Enter your Username and Password for auto analysis access to the secondary analysis software 5 Click F SavePlate p Save to save your plate with these settings then Assign Plate Contents to advance to the next screen 284 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Set up a GeneMapper plate in the 3500 Series Data Collection Software Assign plate When assigning plate contents you
243. gth of time data is collected after voltage is ramped up to the run voltage and the run starts PreRun time sec Prerun voltage time Injection time sec Sample injection time Data delay sec Time from the start of separation to the start of sample data collection Advanced options Do not change unless advised otherwise by Applied Biosystems support personnel Voltage tolerance kVolts Maximum allowed voltage variation Voltage of Steps nk Number of voltage ramp steps to reach Run Voltage Voltage step interval sec Dwell time at each voltage ramp step First read out time ms The interval of time for a data point to be produced First ReadOut time should be equal to Second ReadOut time Second read out time ms The interval of time for a data point to be produced Second ReadOut time should be equal to First ReadOut time Fragment and HID protocols only Normalization parameters Leave at default settings for information on how these parameters are used see Review normalized data on page 90 Normalization Target The expected average RFU for the subset of peaks in the GS600 LIZ v2 size standard used for normalization The default value for each run module has been experimentally determined based on the average peak height of selected peaks in the GS600 size standard with a specific injection time IMPORTANT If you change the injection tim
244. gy Co Ltd d clare que l appareil A4514000 95 651 est conforme aux exigences essentielles et aux autres dispositions pertinentes de la directrve 1999 S CE Deutsch German Hiermit erklart ART Technology Co Ltd dass sich das Gerat 4514000 95 651 in bereinstimmung mit den grundlegenden Anforderungen und den brigen einschlagigen Bestimmungen der Fichtlinie 1888 5 E G befindet Italiano Italian Con la presente ART Technology Co Ltd dichiara che questo ASI4000 96 651 conforme ai requisiti essenziali ed alle altre disposizioni pertinenti stabilite dalla direttrea 1999 53 CE Espanol Spanish Por medio de la presente ART Technology Co Ltd declara que el A4514000 95651 cumple con los requisitos esenciales y cualesquiera otras disposiciones aplicables o exigibles de la Directiva 1839 5 C E Portugues Portuguese ART Technology Co Ltd declara que este 4514000 95 85 1 est conforme com os requisitos essenciais e outras disposigoes da Directrva 199 Y 5fC E Suomi Finnish ART Technology Co Ltd Vakuuttaa taten etta 514000 586551 tyyppinen larte on direkorain 1999 5EY oleellisten vaatimusten ja sita koskevien direkbiran muiden ehtojen muk amen Nederlands Dutch Hierbij verklaart ART Technology Co Ltd dat het toestel 4514000 96 85 1 in overeenstemming is met de essentiele eisen en de andere relevante Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 325 Appendix F Safety Cesky Czech
245. h capillary 20 zones on the CCD are collected to provide20 color data for each capillary Converts the 20 color data into multi dye data for the entire run For sequencing applications 4 different dyes are used to determine the 4 bases A G C and T For fragment and HID analysis applications up to 6 dyes can be used in a single run for higher throughput The software generates an electropherogram intensity plot for each dye based on the migration of DNA fragments over the run and generates primary analysis results For sequencing applications the electropherogram is adjusted to compensate for slight mobility differences due to the dyes then basecalling is performed and quality values are assigned For fragment and HID analysis the software uses the internal size standard to assign a fragment size and a sizing quality value to each peak If the autoanalysis functionality has been set up the system transfers the sample data to a secondary analysis software application for further processing Alternatively you can manually transfer the sample data to a secondary analysis software application for further processing Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Normalization Normalization Overview of the normalization feature For fragment analysis and HID applications the 3500 Series Data Collection Software includes a normalization feature for use with the GeneScan 600 LIZ Size Standard v2 0 GS600 LIZ v
246. hboard 29 access from Main workflow 19 consumables status 236 instrument status 53 troubleshoot 309 data collection software See 3500 Series Data Collection Software data files See sample data files data troubleshooting 306 datastore back up during uninstall 254 date format setting 33 default settings specifying 32 33 define plate properties 44 detection cell temperature 42 104 119 129 documentation related 335 duplicate injection See also re injection defined 60 preview run 60 duplicate library item 140 dye set overview See dye sets dye sets calibrate 103 create 168 custom calibrate 108 custom create 169 defined 168 export 141 fragment analysis 261 HID analysis 262 import 141 sequencing 261 E electrical safety 319 electromagnetic compatibility standards See EMC standards electronic signature records actions 221 view in library 142 electronic signature administrators 339 Index actions that allow e sig 217 enable or disable 216 export 223 export settings 224 functions that require e sig 217 import settings 224 is signed field 228 overview 197 report 222 when security is disabled 216 electronic signature users is signed field 228 overview 225 signing 227 electropherogram troubleshooting 306 EMC standards 322 EPT view 78 ergonomics safety 321 error messages display detail 312 instrument 299 313 link plate 304 RFID 304 spatial calibration 300 spectral calibration 301 e
247. he 3500 Series Data Collection Software The SAE module provides the following functionality System security Controls user access to the software A default Administrator user account is provided and additional user accounts and permissions can be user defined System security can be enabled or disabled globally Auditing Tracks changes made to library items actions performed by users and changes to the SAE settings The software automatically audits some actions silently You can select other items for auditing and specify the audit mode Provides reports for audited library items SAE changes and actions Auditing can be enabled or disabled globally and by record type It is enabled globally by default Electronic signature e sig Determines if users are permitted prompted or required to provide a user name and password when performing certain functions Can be configured so that a predefined list of functions can be performed only if the data used for the functions 1s signed for example you can run a plate only if the calibration data for the system has been signed Can be configured to require multiple signatures and to require specific users or users with specific permissions to sign Electronic signature can be enabled or disabled globally and by e sig type It 1s enabled globally by default Example You can configure the SAE module in a variety of ways applications Require users to log in and leave
248. he Analysis Range contains all size standard fragments included in the Sizing Range specified below Sizing Range Size Calling Method Select Partial then specify 80 to 400 to limit the fragment sizes evaluated for the size standard If you specify sizes outside this range the Sizing Quality may fail Select the method to determine the molecular length of unknown fragments appropriate for the AmpF STR kit you use Local Southern default Determines the fragment sizes using the reciprocal relationship between fragment length and electrophoretic mobility The unknown fragment is surrounded by two known sized fragments above and one below then two below and one above The results are averaged and the size of the allele is determined dentifiler kit Profiler Plus9 kit SEfiler Plus kit COfiler kit Sinofiler kit Profiler9 kit Yfiler kit SGM Plus kit e 3rd Order Least Squares Uses regression analysis to build a best fit size calling curve MiniFiler reagent Size calling options for kits other than those listed above are e 2nd Order Least Squares Uses regression analysis to build a best fit size calling curve e Cubic Spline Interpolation Forces the sizing curve through all the known points of the selected size standard e Global Southern Method Compensates for standard fragments with anomalous electrophoretic mobility similar to least squares metho
249. he calculated Normalization Factor is 5 the software applies a Normalization Factor of 3 0 6 Indicates the normalization state of the sample in the Normalization Limit column in the Samples View Normalization If normalization is applied in the 3500 Series Data Collection Software the factor in calculated Normalization factor is stored with the raw data and is applied to the raw secondary data in the GeneMapper ID X Software v4 1 and the GeneMapper ID X Software analysis Software v1 2 secondary analysis software You can turn normalization off and on in the analysis method used in the GeneMapper v4 1 and GeneMapper D X Software v1 2 secondary analysis software If normalization is not applied in the 3500 Series Data Collection Software either a normalization size standard was not used or Sizing failed E3 normalization cannot be applied in the secondary analysis software Review plots 1 Select the samples of interest in the samples table 2 Select items from the plot toolbar to manipulate the plot as needed Place the mouse pointer over a button for the description of the button a MEDS me tem wl TO EE Show Thumbnail View Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 91 Chapter 4 Review Results Zoom Change plot settings 92 l Click click in the plot settings tabs Click a peak to label it to label all peaks see Label IMPORTANT If you first view a 4 dye sample th
250. he holes in the septa to avoid damaging capillary tips Ensure that the plate assemblies and the cathode buffer container are positioned on the plate deck properly They should sit securely on the deck Ensure the array locking lever on the capillary array is secured Before each run Check consumables on the Dashboard on page 236 Change the cathode buffer container CBC on page 238 Prepare the plate assembly on page 53 Load the plate in the instrument on page 53 Chapter 1 Instrument and Software Description Check for bubbles in the pump block and channels Note Use the Remove Bubble wizard to remove bubbles Check the loading end header to ensure that the capillary tips are not crushed or damaged Daily or before each run Remove bubbles from the polymer pump on page 251 To change the capillary array on page 252 Ensure that the pump block is in pushed back position Clean the instrument surfaces of dried residue spilled buffer or dirt Check for leaks and dried residue around the Buffer Pin Valve check valve and array locking lever IMPORTANT If leaks persist contact Applied Biosystems Daily Chapter 1 Instrument and Software Description Routine instrument cleaning on page 242 Check maintenance notifications on page 28 230 Applied Biosystems 3500 3500xL Genetic Analyzer User Gu
251. he library objects once every three months Archive library This function archives items stored in the library To archive audit records see Items Archive purge and restore audit records on page 214 1 Access the Archive screen als 7 Manage Preferen Restore Purge 2 Specify the date category and range then click OK 254 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Computer maintenance Date Created Modified All Date Range from CS to E cancel 3 Specify a location and file name for the archive dsz file then click Save A message 1s displayed when the archive 1s complete IMPORTANT Do not specify x Applied Biosystems 3500 datastore as the archive location If you do so your archive can be deleted 1f you uninstall the software and do not back up the datastore If you specify a location to which you do not have permission to save a warning message is displayed and gives you the option to save in another location Archive data files There are two ways to archive the data files 1 Start gt Control Panel System and Maintenance Backup and Restore Center OR Programs Accessories System tools Backup 2 Use either Back up File folder or Back up Computer options Note If you export audit records for samples that are not in their original location samples have been deleted or moved an error message is displayed Return sample data files to t
252. he plate import template under a new name UY Enter sample names required Optional Enter information for the remaining columns Note If you specify assay results group or file name convention names the names you enter must exactly match the names of existing items in the library N Save the plate import file Edit a plate You can edit a plate from e Library Select a plate then click Edit Dashboard Click Edit Existing Plate Define Plate Properties screen Select Open Plate Edit Existing Plate Assign Plate Contents screen Select Open Plate Edit Existing Plate 74 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide More features in Assign Plate Contents Import and export a plate You can import and export plates from Plates library Plates in xml format for use on another 3500 or 3500xL analyzer instrument See Import and export a library entry on page 141 Define plate properties Plates in txt csv and xls format files you create that contain plate information in a specific format Assign Plate Contents Plates in txt csv and xls format files you create that contain plate information in a specific format Create a plate template A plate template contains default settings that you can edit when you create a plate from the template l d Create a plate see Create a new plate on page 144 Optional Add sample na
253. heckmark icon appears indicating that the 3500 Server Monitor has started and all 3500 services loaded Step three Launch the application Launch the 3500 application Start Programs Applied Biosystems gt 3500 E sso j Splash screen After you launched the 3500 application the 3500 Series Data Collection Software splash screen appears This screen will remain active for a few seconds until the 3500 Log In dialog box opens Ke Applied KS Biosystems 3500 Series Data Collection Software V1 0 The programs included herein are subject to a restricted use license and can only be used in conjunction with this application FOR RESEARCH USE ONLY Not for use in diagnostic procedures Copyright 2009 Applied Biosystems All rights reserved After the 3500 Series Data Collection Software splash screen disappears one of the following occurs e The Dashboard is displayed go to Check system status in the Dashboard on page 26 The Login dialog box is displayed go to Log In on page 26 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 25 Chapter 2 Start the System Log In Security Audit and E Signature The Security Audit E Signature SAE module is an optional component of the 3500 Series Data Collection Software Researchers have the option to purchase this feature and enable disable the functionality for SAE If the SAE feature is enabled see Chapter 7 Use Security Audit and E Sig Fu
254. heir original location then export again Restore This function restores items stored in the library To restore audit records see Archive purge and restore audit records on page 214 1 Access the Restore function als Manage Preferenc Archive Purge 2 Select the archive dsz file to restore then click Open If the archive file contains items that exist in the system a message 1s displayed Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 259 Chapter 8 Maintain the Instrument ltem exists in datastore Datastore item PlateRecord 1 29 09 already exists 3 Select an option to continue A message 1s displayed when the restore 1s complete Purge This function purges deletes items stored in the library To purge audit records see Archive purge and restore audit records on page 214 1 Access the Purge function as Manage Preferenc Archive Restore CE 2 Click Yes in the Purge warning message stating that you are about to permanently delete all files in the library 3 Specify the date category and range then click OK 4 Click Yes in the Purge warning message A message 1s displayed when all records are deleted Monitor disk space Ensure that you have sufficient drive space by regularly Archiving data e Deleting unneeded files e Emptying the trash Defragmenting the drives Hard disk and Manually check available disk space on Drive
255. hen select a Read Command the information displayed is not readable The feedback from Consumables Read Tag commands does not display valid information Refer to the Dashboard for consumables RFID tag information Miscellaneous Symptom Possible Cause Action Polymer crystals on the Buffer Pin Valve Buffer valve leakage Clean the Buffer Pin Valve Ensure that the maintenance schedule is followed per 3500 Series Data Collection Software notifications Fluid does not move through the pump and into the ABC from polymer or conditioning pouch Not applicable Call your Applied Biosystems representative Electric discharge message during runs ABC may be low Replace the ABC Ensure that the ABC is being replaced per 3500 Series Data Collection Software notifications Leak detected during bubble compression during run or while filling the array Leak in system Run the Bubble Removal wizard Ensure that there are no bubbles in the pump If problem persists use conditioning pouch for water wash Use Replenish Polymer wizard to fill pump and array with polymer Only some injections from a series of injections are completed 3500 Series Data Collection Software never moves on to the next injection Injection failed message After some of the injections complete Capillary RFID cannot be read When you click Refresh on the dashboard
256. here 1s no overlap in a dye set the Condition Number is 1 0 ideal conditions the lowest possible value The condition number increases with increasing peak overlap The ranges that the software uses to determine if a capillary passes or fails are Dye Set Quality Value Minimum Condition Number Maximum AnyDye 0 8 default 20 0 default 0 95 5 5 E5 0 95 6 0 F 0 95 8 5 G5 0 95 13 5 J6 0 95 8 0 Z 0 95 5 5 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 109 Section 1 Calibration Evaluate the spectral calibration data IMPORTANT Do not accept a spectral calibration until you examine the data for all capillaries When a spectral calibration completes successfully the Overall row displays green red or yellow results For each capillary 1 Click a capillary to display the spectral and raw data for a capillary 2 Check that the data meet the following criteria Attribute Acceptance Criteria Example Order of the e 4 dye blue green yellow red lie Green vel Bed peaks in the p spectral profile from left to right Order of the peaks in the raw data profile from e 5 dye blue green yellow red orange e Sequencing matrix standard only 4 dye red yellow blue green ELS araci folor Hod range Fg elk nv Euge een Trance Rar Mal Green Rie left to right e Fragment analysis HID 4 dye red yellow green blue 5 dye or
257. his results group Figure 20 on page 164 162 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Result group library Mame R Example Selected Attributes Results Group Name Plus 4 Start Instrument Run Date Time Stamp Plus 4 Logged in User Mame Select Reinjection Folder Option Store reinjection sample Files in a separate Reinjection Folder same level as Injection Folders CO Store reinjection sample Files with original sample files same level Select Folder Option Default file location C Applied Biosystems 3500 Datal 2 Custom file location C Example 4 Include an Instrument Run Mame Folder Include a Result Group Mame Folder Include an Injection Folder Figure 17 Results group example Figure 18 on page 163 shows the injection list for a run that specifies duplicate and re injections The numbers in the figure relate the elements in the injection list with the elements in the file hierarchy created by this run Figure 20 on page 164 Connection Status Connected User Name Administrator Run Mame Run 009 02 05 14 59 56 703 a Run Status Running Injection List Details name used for logged in user name Instrument Run 7 injections created 7 in Plate A Din Plate B Name folder laca pecu IF Narm POP4 xl HID36 POP4x G5 Plate 01 ijedne IF Norm POP4 xl HID36 POP4xl G5 Plate 01 kg ES IF Norn_POR4_xl HID36 POP4x G5 Plate 01
258. ide Weekly instrument maintenance tasks Maintenance schedule Task Frequency For information see Check the storage conditions of the used arrays to ensure the Weekly Check stored capillary array tip is covered in the reservoir arrays on page 240 Run the Wash Pump and Channels wizard Wash the pump chamber and channels on page 249 Use a lab wipe to clean the anode buffer container valve pin Chapter 1 Instrument assembly on the polymer delivery pump and Software Description Restart the computer and instrument Reset the instrument on page 314 Monthly instrument maintenance tasks Task Frequency For information see Flush the pump trap Empty the condensation container and the water trap waste container The waste container is to the right of the pump block Replace cathode buffer container septa Run a performance check Clean the autosampler Clean the drip tray Check disk space Monthly or as needed Flush the water trap pump trap on page 241 Chapter 1 Instrument and Software Description Change the cathode buffer container CBC on page 238 Chapter 5 Calibrate and Check Performance Routine instrument cleaning on page 242 Monitor disk space on page 256 Defragment the hard drive Monthly Before fragmentation reaches 10 Defragment the computer hard drive
259. ier A im LL l o Preferences Help Logout Select Wells gt E Arr E Run Instrument i E l 44 E l Load Plates for Run an ms Previews Fun E Sequencing Monitor Fun Mame Sequencing Plate 1 3 NM Review Results View Sequencing Results View FragmentHiD Results OO Customize Link Plate Fa Run 2 Go to Load plates for run and create the injection list on page 56 Note By default the plate in position A 1s selected 54 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Quick Start a run Quick Start a run You can start a run in the Dashboard by selecting a plate with plate contents already assigned Load the plate in the instrument before proceeding see Load the plate in the instrument on page 53 gt gt 1 In the Dashboard click Quick Start Run to display ue the Select Plate from Library dialog box ES Select Plate From Library Instructions Select row From table and click on Link Plate button Search All Sequencing Sequencing 2 Optional Filter the plates listed a Select a plate type you can set the default plate type in Preferences see Specify the default plate type for the Open Plate dialog box on page 76 Sequencing Filter b Find a plate based on an attribute by selecting an attribute entering the text to search for then clicking Go Click Clear to clear the field and ent
260. ies Data Collection Software see the GeneMapper ID X Software v1 1 User Guide Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 289 Appendix C Secondary Analysis Fragment 290 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Remote Auto Analysis Setup Remote auto analysis configuration Local Computer Remote Computer Remote Auto analysis Applied Biosystems 3500 Series Instrument Data Collection ae aris Software For remote auto analysis the 3500 Series Data Collection Software resides on the instrument computer and the GeneMapper Software resides on a different computer In this configuration you can set up both softwares so that GeneMapper connects to a remote computer running the 3500 Series Data Collection Software obtains sample files from the remote 3500 Series Data Collection Software database performs analysis of the generated sample files automatically Hemote auto analysis installation Install the remote auto analysis configuration when you want to auto analyze data and you plan to connect to a separate computer running the 3500 Series Data Collection Software Installing GeneMapper Software as a remote auto analysis configuration requires that you 1 Start the Data Collection services on the remote Data Collection computer 2 Install GeneMapper Software v4 1 on the local computer IMPORTANT Before installing GeneMapper start
261. illary array check stored 240 cathode buffer change 238 polymer change type 247 polymer fill array 251 polymer replenish 245 polymer store partially used pouch 249 maintenance instrument annual 232 as needed 232 daily 230 monthly 231 move and level the instrument 243 planned 232 pump flush water trap 241 pump remove bubbles 251 pump wash chamber and channels 249 quarterly 231 routine cleaning 242 schedule 229 Index shutdown 253 weekly 231 workflow 17 manual commands troubleshoot 313 matrix standard part numbers 260 menu commands conventions for describing xvi metric analysis display results 81 flag settings 177 warnings 81 MicroSeq ID analysis protocols create 191 defined 191 migration effects first run 53 Min Peak Half Width 182 187 mixed base color 37 QV range 37 settings 84 threshold 176 Mobility file 175 monitor run currentrun 61 flag settings 177 flags in injection list 62 flags in plate view 62 injection list 61 injection list modify 61 re injections 65 start stop resume run 69 troubleshoot 311 monthly instrument maintenance 231 move the instrument 243 moving and lifting safety computers and monitors 317 instrument 317 moving parts safety 318 MSDSs about xiii description 329 obtaining xvii 329 N negative control 48 non linearity in large fragment size standards 183 normalization and SQ sizing quality 183 188 factor 91 167 factor in secondary analysis software 91 factor th
262. import settings 224 import user account settings 224 notification 199 200 overview 197 security policies 199 spaces in user names 200 user accounts 201 347 Index user report 205 user role 203 username restrictions 199 security users account suspension 226 login 225 overview 225 password change 226 permissions 225 session timeout 226 SeqScape protocols See also sequence analysis protocols create 189 sequence quality See also review results sequencing in monitor run 62 in review results 81 mixed base settings 84 pure base settings 84 recommended ranges 84 trace quality reports 85 sequencing analysis dye sets 261 run modules 263 sequencing analysis protocols create 189 defined 189 export 141 import 141 in assay 150 sequencing analysis reagents part numbers and storage conditions 259 sequencing install performance check evaluate data 126 examples 127 historical reports 128 history 127 instrument prepare 119 plate prepare 120 recommended monthly schedule 231 report 127 required well positions 120 run 122 signing 228 standards 120 thresholds 125 troubleshoot 302 what occurs 124 when to perform 119 sequencing results See review results sequencing Server Monitor 25 service log 258 session timeout 199 226 348 shutdown 253 signal strength report sequencing 85 signing electronic signature 227 Size Calling Method fragment analysis 181 HID analysis 186 size standard plot 94 size standards create 17
263. ing connectors Select Bubble Remove Wizard to remove the bubbles A slow leak may be present in the system Check polymer blocks for leaks Tighten all fittings Not enough buffer in ABC Ensure that the buffer is filled up to the fill line Arcing Check for moisture in and around the septa the CBC the oven and the autosampler Poor performance of capillary array used for fewer than 100 runs Poor quality samples possible cleanup problems Desalt samples using a recommended purification protocol Poor quality formamide Prepare fresh Hi Di Formamide and re prepare samples Leak in system Tighten the connectors and array lever Migration time becomes progressively slower Leak in system Tighten the connectors and array lever Improper filling of the system with polymer Polymer delivery pump may need to be serviced If the issue persists call your Applied Biosystems representative Migration time becomes progressively faster Water in polymer system resulting in diluted polymer Use Bubble Remove Wizard to add polymer to system Buffer valve leakage Check the Buffer Pin Valve and see if it closes correctly Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 307 Appendix E Troubleshoot Symptom Possible cause Action Extra peaks in the electropherogram Data off scale Dilute the sample and re in
264. instrument to instrument and platform to platform Display raw data to determine the appropriate analysis range Data points outside the specified analysis range are ignored Note Ensure the Analysis Range contains all size standard fragments included in the Sizing Range specified below Sizing Range Specify the size range in base pairs appropriate for the kit you are using e All Sizes for the software to analyze fragments of all sizes in the Analysis Range e Partial Sizes for the software to analyze only fragments within a specified range Enter a Start Size and a Stop Size appropriate for the size standard used Size Calling Method Local Southern default Determines the fragment sizes using the reciprocal relationship between fragment length and electrophoretic mobility e 3rd Order Least Squares Uses regression analysis to build a best fit size calling curve e 2nd Order Least Squares Uses regression analysis to build a best fit size calling curve e Cubic Spline Interpolation Forces the sizing curve through all the Known points of the selected size standard e Global Southern Method Compensates for standard fragments with anomalous electrophoretic mobility similar to least squares methods Primer Peak If the primer peaks in your application obscure peaks of interest select Present Selecting Present instructs the algorithm to ignore primer peaks Primer peaks are still displayed in the trac
265. ion Number of all disabled capillaries Allow Borrowing e fall capillaries pass the calibration is complete and injections 2 and 3 are not performed e f any capillaries fail injection 2 is performed Injection 2 The software evaluates the Quality Value for each capillary across injections 1 and 2 and uses the information from the capillary with the highest Quality Value e fall capillaries now pass the calibration is complete and injection 3 is not performed e fthe same capillary fails in both injection 1 and 2 injection 3 is performed Injection 3 e The software evaluates the Quality Value for each capillary across injections 1 2 and 3 and the information from the capillary with the highest Quality Value e lf all capillaries now pass the calibration passes e lf the same capillary fails in injection 1 2 or 3 the calibration fails 112 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Spectral calibration Spectral When Borrowing is enabled all capillaries have to pass meet the spectral Quality calibration with Value and Condition Number limits within the borrowing limits Borrowing enabled Allow Borrowing e 8 capillary instruments One adjacent capillary borrowing event allowed e 24 capillary instruments Up to three adjacent capillary borrowing events allowed the number of allowed borrowing events can be decreased in Preferences Injection 1 e The software
266. ion for primary instrument consumables e Lot numbers e Serial numbers e Dates expiration e Capacity usage The primary consumables are e Capillary Array e Cathode Buffer Container CBC e POP Polymer e Anode Buffer Container ABC Capillary Array Enables the separation of the fluorescent labeled DNA fragments by electrophoresis It is a replaceable unit composed of 8 or 24 capillaries 50 cm and 36 cm length Note The 36 cm capillary is for HID applications only Anode Buffer Container ABC The Anode Buffer Container ABC contains 1X running buffer to support all electrophoresis applications on the instrument It has a built in overflow chamber to maintain constant fluid height Cathode Buffer Container CBC The Cathode Buffer Container CBC contains 1X running buffer to support all electrophoresis applications on the instrument Polymer pouch Supplies polymer to the Polymer Delivery Pump Conditioning reagent The pouch is used for priming the polymer pump washing the polymer pump between polymer type changes and during instrument shut down It has adequate volume for a one time use Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Theory of operation Theory of operation The 3500 or 3500xL analyzer is a fluorescence based DNA analysis system that uses proven capillary electrophoresis technology with 8 or 24 capillaries The 3500 or 3500xL analyzer i
267. ional report settings in Preferences see Trace Print user preference on page 38 Trace Quality user preference on page 38 and Trace Quality Report user preference on page 39 Select Report Type in Pagelof2 5 cx o0 Ol Elx gt MN Modify report settings Sort data based on This setting applies to Ehe Trace Score Report CRL Report OV20 Report and Signal Strength Report Oo Run Mame C Capillary Number Signal based on This setting applies to Ehe QC Report and Signal Strength Report Average Raw Signal Intensity O Average Raw Signal to Noise Ratio Font settings Select the Font to be used in reports Arial 110 Double click different elements in the report to open the Trace view and display the associated sample To print the report click Print then preview or print To save the report electronically pdf print the report and select CutePDF Writer as the printer Close the report Pagelohl QC One page bar chart that shows trace score statistics and results for each selected sample Plate One page per plate for all selected samples that shows the well location thumbnail raw data traces with color coded headers that reflect Trace Score quality Trace Score CRL and QV20 One page bar chart that shows trace score CRL or QV20 statistics and results for each selected sample CRL Distribution One page bar chart that shows CRL statistics and CRL res
268. is maintained in the software 138 Click View Detail Report Click Print In the Printer dialog box select CutePDF Writer as the printer Specify a name and location for the report Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Manage Library Resources Overview of libraries The Library workflow contains the screens where you manage assays protocols and other items that you use to acquire and process data The Library workflow contains e tems that you select when you set up a run Plates Assays Optional filename conventions Optional results groups e tems that you select when you create an assay Instrument protocols Primary analysis protocols Basecalling sequencing sizecalling fragment analysis QC HID analysis Optional secondary analysis protocols Sequencing analysis fragment analysis and HID analysis tems you select when you create instrument sizecalling and QC protocols Dye sets Size standards Factory provided The 3500 Series Data Collection Software libraries include factory provided items template and that are optimized for different applications for example instrument protocols with locked items specific run modules and primary analysis protocols with specific settings You can use the factory provided items directly If the factory provided items do not suit your needs you can modify the factory provided items or c
269. istrator Last Login Time 07 Mar 2009 11 27 35 AM Foun Name Run 2009 035 06 13 26 46 900 Foun Status Runrig Estimated Time Remaining 03 16 Injection List Details a injections created 4 in Mate 4 0 in Plate B F Morm POP xl F Norm POP xl BIOS POP GS 3 09 Run 2 F4 Norm POP xl HID3 amp POP4x G5 3 08 Run 2 4 DF Norm POP xd HIDI POP4 G5 3 09 Run 2 l a 2 2 3 3 3 2 m Tm mu Sos p Li UEM S ore we ie is ia ta o E m e EN a rok cuis ruby e suele reme coe or sek l Legend Li Mot Started EB Active T paused ES Aborted Id Completed EJ Re 1njection ES Duplicate Fn Flags Figure8 Monitor Run screen Note Samples with assays that specify more than one instrument protocol are listed one time in the injection list for each instrument protocol 1 Click the Table Settings button then specify the columns to show or hide in the injection list Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 61 Chapter 3 Set Up and Run 2 Optional e Click the icon above the plate to specify the attributes to display in the plate view In addition to the attributes available in Preview Run a Flag attribute is Assay Name ilabl Assay Color ae Assay Icon If you select the Flags attribute yellow or red marks Results Group Name Results Group Color are displayed for wells with an Average QV value Ei ile Convention Mame sequencing or an SQ value fragment HI
270. ity If more than one peak apex is within the window all are labeled as a single peak Note the following e he maximum value is the number of data points between peaks e The Peak Window Size setting is limited to odd numbers To increase peak detection sensitivity Increase polynomial degree decrease peak window Size To decrease peak detection sensitivity Decrease polynomial degree increase peak window size Slope Thresholds Peak Not recommended for use with AmpF STR kit data Start and End Peak Start The peak starts when the first derivative slope of the tangent in the beginning of the peak signal before the inflection point becomes equal to or exceeds the Peak Start value This threshold is set to O by default which means that the peak will normally start at the leftmost point where the slope of the tangent is closest to 0 horizontal line A value other than O moves the peak start point toward its center The value entered must be non negative e Peak End The peak ends when the first derivative slope of the tangent in the end of the peak signal after the inflection point becomes equal to or exceeds the Peak End value This value is set to O by default which means that the peak will normally end at the rightmost point where the slope of the tangent is closest to O horizontal line A value other than O moves the peak end point toward its center The value entered in this field must be non positive
271. ity Value sequencing Ga El green yellow red 3 yellow or EK red The Average Quality Value based on CRL Trace Score fe QV20 results is in the Suspect or Fail range For information see Basecalling protocol QV settings on page 178 Sizing Quality fragment HID a al El El green yellow red yellow or B red The Sizing Quality is in the Suspect or Fail range For information see Table 15 on page 183 or Table 17 on page 188 IMPORTANT Normalization is not applied to samples with El red Sizing Quality le EPT 3 Click a row in the flag table then click the Sample tab in Instrument Run Views to display the associated data in the Sample view Raw Data 500 n abd i Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Specify re injections Check sequence or sample quality and specify re injections You can specify a re injection before the run completes A re injection physically re injects all samples in the capillary array You can select a different instrument protocol than the original injection and can specify whether to collect data for all or only selected samples in the array l Select the injections or wells to re inject Note 5j Re inject is grayed if you select an injection that contains more than one results group or if you select flags in the flags table that correspond to samples with different results groups To enable j Re inje
272. ject the sample Possible contaminant in sample Re amplify the DNA Sample re naturation Heat denature the sample in good quality formamide and immediately place on ice Peaks exhibit a shoulder in GeneMapper D X Software applications Sample re naturation Heat denature the sample in good quality formamide and immediately place on ice Error messages e Leak detected during polymer delivery e Leak detected during bubble compression The run aborts Bubbles in the polymer system Select the Bubble Remove Wizard to clear bubbles Leak in the polymer system Check for evidence of leaks If polymer leak occurred conduct a water wash and wash the pump trap using the cleaning kit supplied Buffer valve leakage Check the Buffer Pin Valve and see if it closes correctly Clean the Buffer Pin Valve Ensure that the maintenance schedule is followed per 3500 Series Data Collection Software notifications Filling the array during install array Run Fill the Array with fresh Polymer wizard or run Change Polymer Type wizard Detection cell stuck It is difficult to remove when changing the capillary array Improperly placed detection cell To loosen the detection cell 1 Undo the array lever and pull the polymer block towards you to first notch 2 Hold both sides of the capillary array around the detection cell area and apply gentle pressure equall
273. k is required message Running fragment modules after loading the plate Change polymer to POP 7 Run fragment analysis performance check Sequencing performance check is required message After loading the plate Running sequencing modules POP 6 after loading the plate Change polymer to POP 7 Run sequencing performance check Heview results troubleshooting Symptom Possible Cause Action sample files are not displayed when imported You imported hid files and you did not click HID Samples Click HID Samples Peaks are not labeled when you access the screen Labels are not automatically applied See Label peaks on page 93 Click Zoom x and y scaling plot settings are not Scaling settings are applied only applied when you click Apply when you click Zoom The sizing quality result reported in You imported fsa files instead of No action the 3500 Series Data Collection Software differs from the sizing quality result for reported in the GeneMapper D X Software hid files into the GeneMapper D X Software The 3500 Series Data Collection Software does not consider the presence of broad peaks when determining sizing quality for fragment analysis data therefore the sizing quality result reported in the 3500 Series Data Collection Software will differ from the sizing quality result reported in the GeneMapper
274. keeping IMPORTANT After performing a calibration save the calibration report electronically for record keeping The software does not save historical calibration results Only the most recent spatial calibration 1s maintained in the software 1 Click View Spatial Calibration Report 2 Click 5 Print 3 In the Printer dialog box select CutePDF Writer as the printer 4 Specify a name and location for the report 102 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Spectral calibration Spectral calibration A spectral calibration creates a de convolution matrix that compensates for dye overlap reduces raw data from the instrument in the 4 dye 5 dye 6 dye or AnyDye data stored in each sample file When to perform a spectral calibration Estimated run times Perform a spectral calibration for each dye set polymer type combination you will use e Sequencing dye set polymer type Fragment dye set polymer type e HID dye set polymer type Perform a spectral calibration when you e Use a dye set that you have not previously calibrated Change the capillary array Change the polymer type e Have a service engineer perform an optical service procedure such as realigning or replacing the laser or CCD camera or mirrors on the instrument e See a decrease in spectral separation pull up pull down in peaks in the raw or analyzed data Note If you are using the v3 1 sequencing standard or v1 1 sequencing stand
275. lace Reservoir Sepa HIGH 26 Jan 2009 12 00 00 AM Replace Reservoir Septa af E Wash Pump Trap HIGH 26 Jan 2009 12 00 00 AM Wash Pump Trap af E When you complete a task click to mark it as complete click to mark it as dismissed Note Completed and dismissed tasks are removed from the Maintenance Notification section and they do not appear again unless they are repeating tasks Dismissed tasks can be logged in the Notifications Log All actions are recorded in the Notification Log See Review the Maintenance Notifications Log on page 257 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 229 Chapter 8 Maintain the Instrument Daily instrument maintenance tasks Clean the assemblies anode buffer container and cathode buffer container and ensure that the outside of the assemblies 1s dry IMPORTANT Use the cleaning agents as described in this manual only Use of cleaning agents not described in this manual can impair the instrument Task Frequency For information see Check consumables on the Dashboard Refer to the gauges on the Dashboard to see the status for anode buffer container cathode buffer container and polymer Visually inspect the level of fluid inside the anode buffer container and the cathode buffer container The fluid must line up with the fill line Ensure that the plate assemblies are properly assembled IMPORTANT Align the holes in the plate retainer with t
276. late hame gt P _inficider_RG E Data res tera SAN disse bores rosa E TE RZ test_B02 hid mor Selected Attributes Oe Eb t BOS hid an 5 Cj Plate03 PN Ra iem gt Dash n Debian E test_02 hid E test_c03 hid E test_D01 hid E test_DO2 hid Ej test_DO3 hid Results Group Name A a i MILII akbribuues Li wears atiribuben i amp Lees a 0 one ad ee situe thee Pas cur Ru Depor Ig mur Vai cr em pma de cor 5 Store reinjection sample Files with original sample Files same level l Default file location C 4pplied Biosystems 3500 Datal lt Plate Mame PN Rl Cua s localizan Include an Instrument Run Mame Folder Include a Result Group Mame Folder LE Include an Injection Folder Figure 16 PN RGresults group Results group example 2 store one allelic ladder per run folder 8 capillary instruments Applied Biosystems recommends that you run one allelic ladder for each set of 24 samples see Allelic ladder location HID analysis on page 155 To store one allelic ladder per run folder on an 8 capillary instrument create one results group for each set of three injections on the plate Each results group specifies a results group name folder Because you assign one results group to a set of three injections all 24 sample data files including the allelic ladder are stored in the same results group folder The example below shows one results group for a full 96 well plate create three mor
277. les on page 9 IMPORTANT If the polymer dries on the fitment or in the pouch opening the dried polymer prevents the pouch fitment from closing the internal cap properly If that happens the polymer pouch is no longer usable When the pouch is removed cover the fitment with a new empty or a conditioning pouch To prevent drying the pouch fitment must be covered with Pouch Cap PN 4427991 Note Expired pouches cannot be used on the instrument 1 Remove the polymer from storage 4 C 2 Allow refrigerated polymer to equilibrate to ambient temperature before use 3 Check for expiration date on the pouch label to make sure it is not expired prior to use IMPORTANT Do not use if the pouch and or the label 1s damaged or the top seal is missing 4 Peel off seal at the top of the pouch fitment Note You may occasionally notice a tiny droplet of polymer inside the fitment residual from the pouch filling process This is not expected to cause any performance issues Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 247 Chapter 8 Maintain the Instrument 10 248 Slide the pouch fitment on to the slot of the lever assembly Push the lever up to snap the pouch into the connector end of the instrument pump Note The RFID label must be facing the instrument and not you to ensure that the RFID information is read accurately by the instrument RFID label must face the instrument If a partially used pou
278. level is at the top of the fill line and check that Fill line seal is intact IMPORTANT Do not use if the buffer level is too low or the seal has been compromised Ensure that the buffer level is at or above the fill line and the seals 1s intact Replenish consumables As needed see Replenish polymer on page 245 Change polymer type on page 247 IMPORTANT Wear gloves while handling polymer the capillary array septa or CBC Change the anode buffer container ABC on page 237 Change the cathode buffer container CBC on page 238 Fill capillary array with fresh polymer on page 251 To change the capillary array on page 252 Go to Chapter 3 Set Up and Run on page 41 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 31 Chapter 2 Start the System Set preferences Overview Preferences are user definable default settings To access the Preferences dialog box select Preferences in Manane 7 Preferences Hel the toolbar You can optionally set any or all preferences k Preferences type Filter text not used System e System Select ane of the sub categories to set its properties You may choose En reset all Date Format iei system preferences bo the Factory installed defaults IF your user permissions enable vou to do so vou may import or export the system preferences Instrument Settings Scheduler Preference Sequencing Settings Export Spectral C
279. library S Create New Assay Setup an Assay E Basecalling Protocol cannot be empty Application Type Protocols Do you wish En assign multiple instrument protocols to this assay No Yes Basecalling Protocol Edit Segscape Microseglb Protocol Edit Figure 12 Create New Assay sequencing the highlighted area changes based on the Application Type Setting Description Assay Name Name of the assay Names must be unique Locked When enabled allows the entry to be unlocked and modified only by the user who created it the administrator or another user with unlock permissions Useful when your system includes the SAE module described in Chapter 7 Use Security Audit and E Sig Functions SAE Module on page 197 Color Color code for the assay when it is displayed in the Assign Plate Contents screen if Assay Color is selected for Show In Wells _Assays HID 356 POP4 Ge rMo HID 38 POP4 as L5 HID36 POP4 G5 3rd HIDS6_POP4 Ge HMo e ETHAN EJ Application Type e Sequencing e Fragment analysis e HID Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 149 Chapter 6 Manage Library Resources Table 7 Assay settings continued Setting Description Do you wish to assign When you select multiple instrument Yes allows you to POSES protocols to this assay select or create Do vou wish Eo assign multiple instrument protoc
280. lick 2 New Plate then select an option Select a plate click Open then specify settings Create Mew Plate From Template Create Mew Plate From an Existing Plate Create Mew Plate From a Standard Format File For information on other Create New Plate options see e Create a plate from a template on page 43 e Create a plate for importing on page 73 for the Create New Plate from a Standard Format File option Select a Save option Save Save As Save As Template Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Plates library Mew Plate A Close Plate Plate Details o Wumber of Wels Plate Mame is a required Field Provide a unique value Brenes Plate Type Capillary Length cm Description Polymer POP4 3 Secondary Analysis Perform Auto Analysis E Software Type Software Location Auto Analysis is performed Only when the results group is complete When every injection completes Figure 11 Define Plate Properties Table 6 Define Plate Properties Setting Description Plate Details Name Plate name Names must be unique Number of Wells e 96 well For standard 96 well plates 96 Supports 96 well standard reaction plate 8 strip standard tubes are also supported with appropriate retainers e 96 Fast tube For Fast 96 well plates and 8 strip tubes 96 Fast Tube Supports 96 well Fast reaction plate 8 st
281. ll plate A C E G I K g9 gmg gm gm m m Note 384 well M O plates are not NH NH NH NH NH NM E a supported on 8 capillary EH HN EN EN NH N instruments i e 96 Supports 96 well standard reaction plate 8 strip standard tubes are also supported with appropriate retainers e 96 Fast Tube Supports 96 well Fast reaction plate 8 strip fast tubes are also supported with appropriate retainers 3 Bnefly centrifuge the plate containing the standards 4 Verify that each sample is positioned correctly PC in the bottom of its MEER ES well SEO IMPORTANT Ifthe AREA 0 reagents of any well contain bubbles or are not located at the bottom of the well briefly centrifuge the plate remove the plate from the centrifuge and verify that each sample is positioned correctly in the bottom of its well sample is at the bottom of the well 5 Store the plate on ice until you prepare the plate assembly and load the plate in the instrument Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 105 Section 1 Calibration Prepare the plate assembly Load the plate in the instrument IMPORTANT Prepare the plate assembly on a clean level surface Do not heat plates that are sealed with septa pa Align the holes in the septa strip with the wells of the plate then firmly press downward onto the plate Place the sample plate into the plate base IMPORTANT Make sure to SS u
282. llation of the detection cell Detection cell on the array is not properly seated Unistall then re install the array Reinstall the detection cell to reposition and make sure it fits in the proper position If the calibration fails again 1 Fill the capillaries with polymer 2 Repeat the spatial calibration The instrument may need more time to reach stability An unstable instrument can cause a flat line with no peaks in the spatial view Repeat the spatial calibration Broken capillary resulting in a bad array fill Check for a broken capillary particularly in the detection cell area If necessary replace the capillary array using the Wizard Persistently bad spatial calibration results Bad capillary array Replace the capillary array and then repeat the calibration Call your Applied Biosystems representative if the results do not improve Spatial Calibration Error message The instrument cannot perform Spatial Calibration with Array fill Conditioning reagent is installed Replace the Conditioning reagent with an appropriate Polymer 300 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Spectral calibration troubleshooting Spectral calibration troubleshooting Symptom Possible cause Action No signal Incorrect preparation of sample Replace samples with fresh samples prepared with fresh Hi Di Formamide Bubbles in sample wells Cen
283. llowing two conditions 1 This device may not cause interference and 2 This device must accept any interference including interference that may cause undesired operation of this device Changes or modifications not expressly approved by the party responsible for compliance could void the user s authority to operate the equipment NOTICE This equipment has been tested and found to comply with the limits for a Class B digital device pursuant to part 15 of the FCC Rules These limits are designed to provide reasonable protection against harmful interference in a residential installation This equipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instructions may cause harmful interference to radio communications However there 1s no guarantee that interference will not occur in a particular installation If this equipment does cause harmful interference to radio or television reception which can be determined by turning the equipment off and on the user 1s encouraged to try to correct the interference by one or more of the following measures e Reorient or relocate the receiving antenna 322 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Instrumentation safety e Increase the separation between the equipment and receiver Connect the equipment into an outlet on a circuit different from that to which the receiver 1s connected Consult the dealer o
284. low the prompts Service Log The Service Log is a record of instrument service and it is used and completed by the Applied Biosystems service engineer at the time of service To go to the Service Log from the Dashboard 1 Click Maintain Instrument 2 From the Left hand pane under Planned Maintenance click Service Log Click on the top left hand corner of the Service Log for more information The Service Log screen contains a history of all the service events that have occurred on the system starting with the most recent event and provides the following information on each event Event Description Ticket Number The number assigned to the event Service Type The type of service requested Event Occur Date The date that the event took place Service Start Date The date that the service started Service End Date The date that the service ended Service Engineer The name of the service engineer Reason The reason for logging the event Comments Any additional comments 258 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Application Reagents and Run Modules Sequencing analysis reagents Note For more details see the product insert included in the product package The following table shows all the reagents for sequencing analysis Table 27 Sequencing analysis reagents On instrument Part Storage Shelf lif
285. lt Logged in User Name gt T File name syntax from file name convention lt Sample Name lt Analysis Protocol Name gt lt Unique Time Stamp Integer gt 164 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Instrument protocol library Instrument protocol library Instrument protocol overview An instrument protocol contains the parameters that control the instrument during data acquisition An instrument protocol is a required element of an assay for all applications When you create an assay you add one or more instrument protocols to the assay If you add these items from the library a copy of the items 1s added to the assay and can be modified independently from the original items stored in the library For information on how changes are tracked 1f auditing 1s enabled see Audit action on page 210 Create a new instrument protocol If factory provided instrument protocols do not suit your needs you can create new instrument protocols 1 Access the Instrument Protocols library R 3500 Data Collection Software Dashboard Edit 2 Click A Create Library Mainter m ate Filter E Library Resources 3 Inthe Create New Instrument Protocol dialog box Figure 21 on page 166 select an application type Sequencing Fragment or HID The run module selection list is filtered based on the application you Select ae Manage a Plates Assays Instrument F
286. mables and indicates in red if any consumable is about to expire based on RFID tags Maintenance notifications Lists the scheduled maintenance tasks Help icon Displays a help topic specific to a screen or an area of the screen For more information see Check system status in the Dashboard on page 26 Main workflow Click the main workflow arrow at the top left of the Dashboard to access the Main workflow Main workflow The Main workflow contains the screens where you set up load and run plates and view results The Main workflow navigation pane is designed as a task workflow Each screen contains a button that you can click to advance to the next screen in the workflow e Setup Define Plate Properties Assign Plate Contents Select a task in the navigation pane to access each 8 Run Instrument screen Load Plates for Run You can select Dashboard or any other menu item at Preview Run any time to advance from the Main workflow Monitor Run iii Review Results R 3500 Data Collecti View Sequencing Results Dashboard Edit View FragmentHiD Results The Main workflow is described in Chapter 3 Set Up and Run on page 41 and Review Results on page 79 16 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Library workflow Maintenance workflow Overview of the 3500 Series Data Collection Software Select Library in the menu bar to access the Library work
287. mamide is used for sequencing analysis fragment analysis and HID Forensic applications To determine the exact and necessary volume of formamide for each specific application follow the provided protocols and product inserts Table 3 Hi Di Formamide used for all applications Hi Di Formamide name Instrument Part number On instrument life and usage Hi Di Formamide 5 ml bottle 3500 8 capillary 4440753 24 hours Pack otrou 3500xL 24 capillary Capillary arrays 12 The capillary array for 3500 or 3500xL analyzer 1s installed on the instrument and ready to use CAUTION SHARP The load end of the capillary array has small but blunt ends and it could lead to piercing injury See To change the capillary array on page 252 for instructions on how to change the capillary array Applications e The 36 cm capillary array is used for HID Forensic applications The 50 em capillary array is used for sequencing and fragment analysis applications Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Table 4 Capillary arrays used for all applications Instrument reagents and consumables RFID controlled limits Part Instrument P Capillary array name ieee used nm User option to continuet 8 Capillary 36 cm 4404683 3500 160 Under user option to continue 160 injections 8 Capillary 50 cm 4404685 IDISCHOnS are exp y sae 24 Capillary 36 cm 4404687 3500xL Under user option
288. mber v Intensity vs Pixel Number s 4 7 53 E ESI D 10 20 30 40 50 60 70 60 90 100 110 120 130 140 150 160 170 150 190 200 210 220 230 240 250 0 8 0 6 0 4 0 2 iio ene Dye Set Z created from Sequencing Standard Intensity vs Scan Number Calibrated Data y E 7 0 4000 8000 12000 16000 j lan ull 1 l Wht NN r M Intensity vs Scan Number Intensity ys Pixel Number Is 4 7 53 E ES d 0 10 20 30 40 50 60 70 60 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 0 8 0 6 0 4 0 2 R e 0 0 114 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Spectral calibration Dye Set G5 created from Matrix Standard Set DS 33 v Intensity vs Scan Number Calibrated Data Y 1 2 400 800 1200 1600 2000 2400 2600 3200 2400 2000 1600 1200 800 400 Intensity vs Scan Number v Intensity vs Pixel Number E Y 4 E E BE Dye Set E5 created from Matrix Standard Set DS 02 Calibrated Data Y E ER 0 400 600 1200 1600 2000 2400 2800 I 5000 4000 3000 2000 1000 Intensity vs Scan Number w Intensity ys Pixel Number E El 0 10 20 30 40 50 60 70 60 90 100 110 120 130 140 150 160 170 180 190 200 210 220 Export spectral calibration results To export spectral calibration results 1 Click Export Spectral Calibration Results Applied Biosystems 350
289. me Convention Table 8 File name conventions settings Setting Description Name Name of the file name convention Names must be unique Locked When enabled allows the entry to be unlocked and modified only by the user who created it the administrator or another user with unlock permissions Useful when your system includes the SAE module described in Chapter 7 Use Security Audit and E Sig Functions SAE Module on page 197 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 153 Chapter 6 Manage Library Resources Table 8 File name conventions settings continued Setting Description Color Color code for the file name convention when it is displayed in the Assign Plate Contents screen if File Name Convention Color is selected for Show In Wells A E EE File Name Conventions 7 Actio i General File Mame A E iJ My FNC A E Preview of name Interactively displays the attributes you select Available attributes e Amplicon Name from Customize e Polymer Type Sample Info in sequencing assays e run name e Analysis Protocol Name primary e Sample Type analysis protocol e Specimen Name from Customize Sample e Assay Name Info in sequencing assays e Capillary Number e Time of Run run start time Custom Text fields up to 3 e Unique Time Stamp Integer numeric string e Date of Run in milliseconds that does not correspond to e Injection Number the current
290. ment Link a plate troubleshooting Symptom Possible cause Action Plate does not link Plate was linked but now it is unlinked Spatial Spectral calibration was not performed If you access the Load Plates for Run screen from the navigation pane a plate may not be linked indicated by the active Link button 1 Perform spatial calibration 2 Relink the plate s Access the Load Plates for Run screen from the navigation pane and click Link Plate No plate in position A message You physically loaded plate in position B plate B position and try to link plate Click Link Plates and link the plate directly to position B plate B position Follow the prompts No plate detected message The plate is in position B Place the plate in position A 304 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide How to search and use the log files How to search and use the log files The 3500 Series Data Collection Software generates the following log files that you view using a text editor such as Wordpad e 3500UsageStatistics txt Provides a summary of the number of plates run as well as number of run types sequencing fragment and HID Stored in x Applied Biosystems 3500 UsageData e 3500ConsumableUpdates txt Provides a summary of consumables installation information and dates Stored in D Applied Biosystems 3500 LogFiles
291. mes and sample types see Name samples and assign sample types in the plate view on page 48 Optional Add the assays file name conventions and results groups appropriate for this plate template s application see Add assays file name conventions and results groups to a plate on page 73 Adding assays file name conventions and results groups to the plate template automatically displays these items in the Assign Plate Contents screen when you open the plate template You do not have to add these items from the library for each plate you create Optional Click Show In Wells to specify the attributes to display in wells in the template Show In Wells kos q Assay Mame a Select Save Plate Save As Template The software _ v Assay Color L displays the template icon below the plate layout ena Results Group Marne Results Group Color File Convention Name File Convention Color e Sample Name Sample Type Well Position Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 75 Chapter 3 Set Up and Run Specify the default plate type for the Open Plate dialog box Specify the default plate type for the Open Plate dialog box in Preferences Library Maintenance Tools Manage 7 Preferences Help 7 Log Out AS Preferences type filter text po sse Plate Setup s El System Choose the default application type palvmer type and capillary length to use when the ins Dake Format Instrume
292. mm 3 mmm gt Trace Quality Reports T Mixed Base OV Colors 0 1015 100 we a Preview LONE ERA EAE EXT ETRARAR TER TERRAE 2 Specify the following settings Setting Description NT nucleotide Click an NT or Mixed base Foreground or Background color NTBase Foreground Background Base Color block then select a color for the letter annotation or the highlight color for the letter annotation m A Pure Base and Sets the colors and ranges for pure and mixed base quality value Mixed Base QV indicators QVs displayed in the Trace View the default settings are Colors recommended 150 a Click a pure base or mixed base color bar to select a new color b Place the mouse pointer over a slider then drag to set a new range Applied Biosystems recommends that you set the following ranges for QVs e Pure bases Low QV lt 15 Medium QV 15 to 19 High QV 20 default e Mixed bases Low QV lt 5 Medium QV 5 to 10 High QV gt 10 investigate to determine the best range for your application Note The predicted probability of error for a basecall is high QV gt 10 3 Click Apply to save the user preferences see User preferences on page 34 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 37 Chapter 2 Start the System Trace Print user Trace Print preferences determine settings for sequencing trace reports preference 1 In the Preferences dialog bo
293. mplates In addition to pre defined plate parameters a plate template can also contain a list of the appropriate assays file name conventions and results groups for an application For more information see Create a plate template on page 75 1 In the Dashboard click Create Plate From Template to display the Open Plate Template from Library dialog box Create Plate From Template AS Open Plate Template From Library Instructions Select row From table and click on Open button Sequencing A BDx Rapid Seq A 4 Std_Seq POP H Short Read Seq T Y Std Seq xL POPE Y Std Seq xL POP7 A Fast Seg xL POP7 4 BDx Fast Seq xL T 4 BDx Std Seg POP7 BENE EE Illi Sequencing Sequencing Sequencing Sequencing Sequencing Sequencing Sequencing Sequencing Applied Biosystems 3500 3500xL Genetic Analyzer User Guide For use with samples purified with BigDye Termin For read lengths of 850 bp or greater and a run til For read lengths of 300 bp and a run time of 30 m For read lenghts of 600 bp or greater and a run til For read lengths of 850 bp or greater and a run til For read lengths of 700 bp and a run time of 65 m For use with samples purified with BigDye Termin For use with samples purified with BigDye Termin Cee oaa ike see lee ee ified akh Din VUT 43 Chapter 3 Set Up and Run 2 Optional Filter the templates listed a Select a template type Sequencing
294. ms in the action log are audited silently except for the items noted as configurable Configurable items may include comments in the action log Table 24 Audit action log Category Action Assay Assay exported successfully Note Only one audit record is generated if you export multiple assays Log In e User logged in e Login failed e User logged out Maintenance Wizards e Remove Bubbles Wizard started e Flush Array Port Wizard started e Change Polymer Type Wizard started e Change Array Wizard started e Replenish Polymer Wizard started e Perform Fill Polymer Wizard e Perform Water Wash Wizard Plate Plate exported successfully Note Only one audit record is generated if you export multiple plates 212 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Table 24 Audit action log continued Generate audit reports Category Action Run Start Pause Resume Stop Abort injection Terminate injection list SAE Configuration Export System Audit Records Archive Purge Restore System Action Records Archive Purge Restore User Profile Export View and print audit reports 1 Display the records of interest as described above 2 Filter the list to decrease the time required to generate reports IMPORTANT You cannot cancel a report after you click a view button 3 Click View Audit Summary Report or View Audit Detailed Report
295. n enter the new Audit E Signature password two times then click OK Change Password View Logs Manual Commands If your system is configured to suspend a user account for failed logins and you enter an incorrect user name and password for more than the allowed number of times your user account is suspended and the Log In dialog box indicates that your account is inactive S 3500 Log In 3500 Log In E The user is not active User Name technician Password There are two ways to activate a suspended account e You can wait until the suspension period ends e An administrator can change the account status from Suspended to Active Note While a user is suspended another user can click Reset then log in and replace the suspended user If your system is configured to timeout and there is no user activity for the specified time the Log In dialog box indicates that your user session has timed out You must enter your user name and password to access the software R 3500 Log In The user session has timed out Please provide your user name and password to unlock your session 3 User Name Administrator Note The administrator or another user with permission to log in to timed out sessions can click Reset then log in gt Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Audit Audit If your system is configured for auditing you may be prompted to specify a r
296. n is complete The number of observed size standard and allele peaks 1s shown Results for each allele are shown at the bottom of the screen in the Run Information table Note The example shown below is for the HID install standard Number of peaks per capillary Plot and allele size height information for the selected capillary Allele results for all capillaries 134 MIME 100 32 1934 0 2 L osseo 104 53 1736 0 3 L misss 108 63 1871 0 4 Lj osseo 112 74 1429 0 5 L lorsszamepl12 2 114 72 1605 0 3 it llele Peaks 5ize Standard Peaks Include 3000 Run Information AIl capillaries included Filter the results by dye color e Dye Allele L Morninal Size Mean Avg Peak Helght PE 7 p1ssasstuep Tte 100 35 100 38 2346 08 O Dissen 104 5 104 56 2121 79 O pissasastuEpT 108 65 108 7 2270 08 D195433 MED 12 META Wema 1730 55 D195433 MED 12 2 114 74 114 6 Harea ipiesassruEDT3 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Run the fragment analysis or HID Install standard performance check How the software determines passing and failing capillaries for the fragment HID performance check The software evaluates peaks in the data for each capillary To be identified as a possible allele peaks must be within the following ranges nominal allele size or reference bin size 1s hard coded e All markers except THOI 0 7 bp of nominal
297. n page 9 1 From the Maintenance Wizards screen click Install Capillary Array INSTALL a capillary array Note The Install Capillary Array Wizard takes 15 to 45 minutes to complete 2 Follow the prompts in the Install TEMPI Capillary Array Wizard window TORT Welcome to the Install Capillary Array maintenance wizard 3 Check the Quick View section of the m q B eem Dashboard for updated status of the Y capillary array 252 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Use the Maintenance Wizards to perform operations To shutdown the instrument Use the Instrument Shutdown Wizard for short and long term shutdown 1 From the Maintenance Wizards screen click Shutdown the Instrument SHUTDOWN the instrument t Note The Instrument Shutdown Wizard takes 60 minutes to complete 2 Follow the prompts in the Instrument m Instrument Shutdown Wizard Shutdown Wizard window l This wizard will help you prepare the instrument for an extensive Perform the appropriate shutdown procedure id based on the information in the following i table Array port plug Click Next to continiue IMPORTANT Place a conditioning reagent pouch onto the instrument when performing instrument shutdown If the instrument will be unattended for Perform this shutdown procedure no more than 1 week No action is required 1 to 2 weeks IMPORTANT Keep the load end of the capillary array in 1X buf
298. n user names Define name spacing Use spaces in user names with caution For information see Spaces in user names on page 200 2 Specify the allowed characters in user names spaces and alpha numeric upper lower case and special characters commas periods semicolons dashes underscores and tildes 3 Specify password limits 4 Specify the required characters in passwords spaces and alpha numeric upper lower case and special characters any non space non alpha or non numeric characters 5 Specify password reuse You cannot disable the password reuse restriction 6 Under Security Policies specify password expiration account suspension and session timeout settings Password Expiration Account Suspension Session Timeout Passwords will expire es Mo Login attempts with an incorrect password will Oves On User sessions will be timed out if suspend the user account ES a there is no user activity ves O every days For the next z4 Hours w F or minutes Notify User days before expiration iF consecutively Failing ls tires An instrument run is not considered user activity within any eo minute Note A session times out while a run is in progress if the timeout period is exceeded and there is no other user activity 7 Click Setup Messaging Notification Settings to specify when and how to notify the administrator of certain security events For information see Set up messaging notifications
299. nal laws and regulations related to chemical storage handling and disposal Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Chemical safety MSDSs About MSDSs Chemical manufacturers supply current Material Safety Data Sheets MSDSs with shipments of hazardous chemicals to new customers They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated MSDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a new MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your files Obtaining The MSDS for any chemical supplied by Applied Biosystems is available to you free MSDSs 24 hours a day To obtain MSDSs 1 Goto www appliedbiosystems com click Support then select MSDS 2 In the Keyword Search field enter the chemical name product name MSDS part number or other information that appears in the MSDS of interest Select the language of your choice then click Search 3 Find the document of interest right click the document title then select any of the following Open To view the document Print Target To print the document e Save Target As To download a PDF version of the document to a destination that you choose Note For the MSDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer Applied Biosystems
300. nctions SAE Module for user configurations After the 3500 Series Data Collection Software splash screen disappears log in from the Dashboard 1 Enter the User Name and Password in the 3500 Log In dialog box l 3500 Log In 3500 Log In Provide your user name and password to login 2 Click OK The 3500 Series Data Collection Software splash screen re appears This screen will remain active for a few seconds and the 3500 Series Data Collection Software opens The 3500 Series Data Collection Software launches and the Dashboard appears IMPORTANT If you accidentally close any of the services via 3500 Server Monitor the system will not work To open a closed service place the cursor on the status icon click the right mouse button go to Services and click the service that 1s closed Check system status in the Dashboard Dashboard a quick glance 26 The first screen that 1s displayed when you start the 3500 Series Data Collection Software is the Dashboard Figure 4 The Dashboard displays gauges instrument information consumable information and maintenance notifications that provide a quick overview of the usage of each consumable and the status of the instrument Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Check system status in the Dashboard Consumable containers include radio frequency identification RFID tags that identify the consumable and allow the software to monitor the number
301. nd specify re injections Re injections of Ifyou select to re inject a sample that includes an allelic ladder in its results group HID allelic ladder but the allelic ladder is not part of the injection the software prompts you to select samples one or more allelic ladder samples to re inject For example e You are running an 8 capillary instrument and you have specified one results group for each set of three injections for more information see Results group example 2 store one allelic ladder per run folder 8 capillary instruments on page 161 e The allelic ladder sample is in Injection 1 e You select for re injection a sample that is in injection 2 The software prompts you to select one or more allelic ladder samples to re inject The allelic ladders available to select are from the same plate and within the same results group as the original injection If the results group does not contain an allelic ladder sample the software does not prompt you to select one for re injection S Add Allelic Ladder to Re injection Add Allelic Ladder to Re injection Select zero 0 or more Allelic Ladder samples A01_allelic ladder Deselect All Allelic Ladder Options Collect data For all other wells in the selected allelic ladder Apple modified instrument protocol to all wells in the selected allelic ladder s inj In the Add Allelic Ladder to Re injection dialog box 1 Select one or more allelic ladder samples
302. ndard dye specified in the size standard definition and the actual distribution of size standard peaks in the sample calculates an interim SQ a value between 0 and 1 Weighting The Broad Peak BD threshold specified in the QC Protocol QC Settings tab affects the SQ To determine the final SQ value the software e Evaluates size standard peak widths in the sample in the dye color specified in the size standard definition e fthe width of any size standard peak in the sizing range exceeds the broad peak threshold applies a 0 5 weighting factor Interim SQ x 1 0 5 Note The GeneMapper D X Software allows you to set broad peak weighting For more information see the GeneMapper D X Software Reference Guide Broad Peak Enter the maximum peak width in base pairs When a peak width is greater than the threshold the A Check flag is displayed for the BD Broad Peak quality flag in View HID Results 188 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Sequencing analysis protocols library secondary analysis Sequencing analysis protocols library secondary analysis Sequencing analysis protocol overview A sequencing protocol is the optional secondary analysis auto analysis protocol for SeqScape Software v2 7 or later sequencing applications A sequencing analysis protocol defines the Secondary analysis software SeqScape Software location SegScape Software project template an
303. ndary Analysis Software Instance Computer on which the secondary analysis software is running Project SeqScape software project to create Project Template Project template to use Specimen Specimen in which to save the sample data files Note For each specimen a sequencing analysis protocol is required 190 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide MicroSeq ID protocols library secondary analysis MicroSeq ID protocols library secondary analysis MicroSeq ID analysis protocol overview A MicroSeq ID protocol is the optional secondary analysis auto analysis protocol for MicroSeq JD Analysis Software v2 2 or later sequencing applications A MicroSeq ID analysis protocol defines the Secondary analysis software MicroSeq JD Analysis Software location e MicroSeq ID Analysis Software project and specimen to use for auto analysis When you create a sequencing assay you can optionally add a MicroSeq ID analysis protocol to the assay If you add this item from the library a copy of the item is added to the assay and can be modified independently from the original item stored in the library For information on how changes are tracked if auditing is enabled see Audit action on page 210 Create a new MicroSeq ID analysis protocol l Access the MicroSeq ID Protocols library 3500 Data Collection Software 3 Dashboard Edit Library Mainten 2
304. ndary peak to the fluorescent signal of the main called base a Scroll to the right of the Metric Analysis table to display the Warning column b Display the Analysis Status legend Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 81 Chapter 4 Review Results Library Maintenance Tools Manage Preferences Help Analysis Status Legend Success LJ Success with warning Fail Error Unclassified 3 c Review warnings Result Description E Success Basecalling and trimming successful Success with Basecalling successful trimming not successful warning Warning messages are listed in the Warning Error Message column default position is the last column in the table A Fail Basecalling and trimming failed no results generated Basecalling and trimming failed due to internal software Error error no results generated No analysis performed LJ Unclassified 6 Optional Click Minimize and Restore to collapse and expand the samples table Review traces 1 Select the samples of interest in the samples table then click gt Open Trace 2 Select items from the trace toolbar to manipulate the trace as needed Place the mouse pointer over a button for the description of the button aca i ps Set Tab Key bo en is E B Select Tie Viewers 3 Optional Modify trace display e Use the Tile Viewer options to display Eur d ES up to
305. ne gamp sag zag Camp Lang co pit pre pron P Eg Y Y Legend E Not started J active I paused ES aborted FJ Completed Re Injection ES Duplicate Fal Flags Instrument Run Yiews and Flags AA Time Elapsed seconds B eter Z Flags Found The flag table displays a quick preview of HH 2Flags Found sample quality and identifies samples that may need investigation sample 3 id El 18 sample The flag table is linked to the plate view Click mera omina mma ug n a flag to select the associated well in the plate View Note If no samples are listed in this pane no flags were found and the samples have passed quality checks Display o E All samples passed At least one sample is in the suspect range and requires review oO Flags Found Bj At least one sample is offscale or is in the suspect range Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 63 Chapter 3 Set Up and Run 64 Array Injection 3 2 To filter the flag table select a flag type To display HID flags select All To sort the table double click column headers The flags you may see in the flag table are Flag Symbols Description Offscale green or red El red At least one data point in the analysis range has aturated the CCD camera Note In the View Results screen an offscale sample is hi with Average Qual
306. ng fragment mixed or HID Number of wells capillary length and polymer type When you set up a plate for a run you add assays optional file name conventions and optional results groups to wells in the plate If you add these items from the library a copy of the items 1s added to the plate and can be modified independently from the original items stored in the library For information on how changes are tracked if auditing is enabled see Audit action on page 210 Plate templates The Plates library includes templates that are optimized for different applications for example plates defined with the appropriate polymer and capillary length that you can use to create new plates Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 143 Chapter 6 Manage Library Resources Create a new plate l 2 144 To create a new plate specify settings Access the Plates library y 3500 Data Collection Software Click Create The software switches to the Main workflow and displays the Define Plate Properties screen Figure 11 on page 145 Dashboard Edit Library Maintenan Em Library Resources Filter al Note You can also access the Define Plate Properties screen from the Dashboard and the Assign Plate Properties screen K ASSES te Mam File Marne Conventions 1 4 Table 6 on page 145 E Create Mew Plate To create a new plate based on an existing plate c
307. nic signature Access the E Signature Settings screen and enable or disable e sig IMPORTANT If you disable security you inactivate audit and electronic signature functions No audit record is generated for the disabling of audit and electronic signature functions when you disable security 1 Access the E Signature Settings screen ntenance Tools Manage Preferenc screen Security Audit 2 Click Disable or Enable Figure 36 on prp E pag Change Password W Note When e sig is disabled the is not Mend Manual Commands active in lower parts of the screen k 3500 Data Collection Software e Ek Deshibaard ER 7 Library Maintenance Took Manage 7 Preferences Help Log Out Be ngsHesowces d We Enable E 5ig jj Disable E Sig T E Sipnature Settings Select the electronic signature types that Nn Menge agora E Signature Type Audit Reports L Approve Dye Set Approve Site Standard E Signature Reports A Approve Spatial Calibration Approve Spectral Calibration Manage Users 11 GO Approve instrument Protocol re O Approve Sizecall Probocel E Mawage Settings Approve Basecall Protocol Approve QC Protocol Approve Gene Mapper Protocol Security Approve Gene Mapper IDx Protocol Audi C Approve SeqScape Protocol Approve MicroSeq ID Protocol Approve Assay rds E Plate Templat An Mi import a tee Plate i Select the functio
308. njection a replicate injection that uses the same instrument protocol as the original injection select an injection then click EX Sample data files for each duplicate injection can be saved in a separate folder in the results group folder 1f specified in the results group For more information see Results group example 3 store re injections in separate folders on page 162 Note To use a different protocol for a replicate injection specify a re injection in the Monitor Run screen after you start the run Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Start the run Start the run When the injection list is configured click Start Run The Monitor Run screen is automatically displayed IMPORTANT You must specify re injections before the run completes Note It takes approximately 10 seconds for the instrument to initialize after the instrument door is closed Do not start a run until the instrument status light is green Monitor the run The Monitor Run screen Figure 8 on page 61 1s automatically displayed when you click Start Run in the Load Plates for Run screen or the Preview Run screen The current injection is highlighted in green in the plate view The injection list 1s linked to the plate view Click an injection to select the associated wells in the plate view A selected injection is highlighted in yellow in the plate view Abort Injection A Connection Stetus Connected User Name Admin
309. ns before which the system will check C Approve Sample l C Approve Sequencing Install Standard Results Function F ignatur Approve Micro5eqlD Install Standard Results Approve Fragmerk Install Standard Results Approve HID Install Standard Results Figure 36 E Sig disable or enable 216 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Manage electronic signature Select the actions that allow signature IMPORTANT Do not change electronic signature settings during a spectral calibration 1 Select the checkbox next to an item in the E Signature Type list to identify Approve Size Standard i C Approve Spatial Calibration events for which Approve Spectral Calibration to allow nd electronic signature see Table 25 on page 218 This selection activates the E Sig button for the selected items it does require an electronic signature for these selections E Signature Settings Select the electronic signature types that should be allowed Select the Functions after v E Signature Type Function E Save Dye Set 2 Optional For each item that you select a From the top right of the screen select a function after which the system will prompt for electronic signature This selection presents an e sig prompt to users when they perform a function Users can sign or can continue without signing b From the bottom right of the screen select a function start run befor
310. nt Settings Scheduler Preference Application Sequencing v Polymer Type POP Capillary Length 50 B Sequencing Settings Export Spectral Calibration El User Reports Settings Run Setup Sequencing Settings Trace c Trace Print Choose the default Assign Plate Contents View Trace Quality Trace Quality Reports 5 Assign Plate Contents View Table Plate Table zu Save electronic version of reports When you print any report you can select CutePDF Writer as the printer to save the report to pdf More features in Load Plate for Run Link a plate from the recent plates or recent runs tab Instead of clicking Link to select a plate you can click drag a plate from the Recent Plates tab pending plates or the Recent Runs tab processed plates ment Link Plate Unilink Plate B Link Plate Unlint 76 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide More features in Monitor Run More features in Monitor Run Review the Instrument Run views Select an injection then click an instrument run view tab As needed e Click E e to zoom in and out e Click to isa a view and display it in a una separate window that you can move around on the ri dl 3500 Data Collection Software screen 32 Java gt To locate a detached view click the 3500 task bar icon Array view The Array view shows the color data based on the dominant fluorescence c
311. nt status light is blinking red Instrument error 1 Power off the instrument 2 Power on the instrument 3 Restart the computer An error has been detected from the instrument Instrument monitor circuit failure Restart the computer 3500 Series Data Collection Software status icon is e instead of em MA E EA zs sinn i epee oci A eee One or more of the services are stopped Right click the status icon then select Services If any item does not display a checkmark click the item to start the service E SOUL Integration Services wv Webmethods Broker Server Settings About Exit Unable to transmit measurement data Internal data buffer overflow Communications error Restart instrument and computer Electric discharge message during runs The ABC buffer may be low Replace the ABC Ensure that the ABC is being replaced per 3500 Series Data Collection Software notifications Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 299 Appendix E Troubleshoot Spatial calibration troubleshooting Symptom Possible cause Action Start Spatial Calibration button is disabled Communication failure between the Data Collection Software and instrument Restart instrument and computer Check the NIC cable connection Unusual peaks or a flat line for the spatial calibration Improper insta
312. ntilation Wash thoroughly after handling WARNING CHEMICAL HAZARD Hi Di Formamide Causes eye skin and respiratory tract irritation Possible developmental and birth defect hazard Avoid breathing vapor Use with adequate ventilation WARNING CHEMICAL HAZARD Anode Buffer Container ABC May cause eye skin and respiratory tract irritation Avoid breathing vapor Use with adequate ventilation d WARNING CHEMICAL HAZARD Cathode Buffer Container CBC May cause eye skin and respiratory tract irritation Avoid breathing vapor Use with adequate ventilation WARNING CHEMICAL HAZARD 1X GA Buffer EDTA May cause eye skin and respiratory tract irritation Avoid breathing vapor Use with adequate ventilation Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 333 Appendix F Safety Instrumentation alerts General instrumentation alerts Specific instrumentation alerts 334 A WARNING Wear appropriate protection including gloves laboratory goggles and coat whenever you work with the fluids used on this instrument or parts that may come into contact with these fluids WARNING Wear appropriate protection including gloves laboratory goggles and coat whenever you work with the fluids used on this instrument or parts that may come into contact with these fluids The instrument uses a Solid state laser Under normal operating conditions the instrument is categorized as a Class I laser LED prod
313. nyDye selection in the Dye Set list contains default settings It does not correspond to custom dye sets created with the AnyDye dye set template Optional Select Allow Borrowing Selecting this option instructs the software to automatically replace information from failed capillaries with information from an adjacent passing capillary with the highest Quality value For more information see What you see during a spectral calibration on page 112 Click Start Run The following occurs The system sets up three injections see What you see during a spectral calibration on page 112 for information on the number of injections performed The Capillary Run Data display updates after each injection is complete The status bar updates during Run 1 IMPORTANT The status bar does not update during Run 2 or Run 3 e Passing and failing capillaries are shown in green and red respectively Borrowed capillaries are shown in yellow with an arrow indicating the adjacent capillary from which results were borrowed To display the result for each capillary spectral data Quality Value and Condition Number below the run results table click a capillary in the table Note The results displayed when you click a borrowed capillary are the passing results borrowed from the adjacent capillary To determine the reason that a capillary fails view the spectral calibration report See View and print a spectral calibration report on
314. nyelv egy b eloirasainak Polski Polish Niniejszym ART Technology Co Ltd o wiadcza ze 4514000 90 651 jest zgodny z zasadniczymi wymogami oraz pozostahymi stosownymi postanowieniami Dyrektywy 1999 50 Slovensko Slovenian ART Technology Co Ltd izjavlja da Je ta 4514000 95 85 1 y skladu z bistvenimi zahtevami in ostalimi relevantnimi dolo ili drektve 1999 54 85 Slovensky Slovak ART Technology Co Ltd t mto vyhlasuje e ASIMOUD 88 BS 1 spl a z kladn po iadavky a v amp etky prislu amp n ustanovenia Smemice 199WY HE S Svenska Swedish Harmed intygar ART Technology Co Ltd att denna 45 14000 88 B51 star I overensstammelse med de vasentliga egenskapskrav och ovnga relevanta best mmelser som framg r ay direkty 19994 EG slenska Icelandic Her me l sir ART Technology Co Ltd yhr ri ad 451400036851 eri samr mi vi grunnkrofur og a rar krofur sem ger ar eru i tlskipun 199S 4 EC Norsk Norwegian ART Technology Co Ltd erkl rer herred at utstyret AS 4000 96 65 1 er i samsvar med de grunnleggende kray og evrige relevante krav 1 direktry 199S S EF Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 327 Appendix F Safety Australian EMC This instrument has been tested to and complies with standard AS NZS 2064 Standards Limits and Methods Measurement of Electromagnetic Disturbance Characteristics of Industrial Scientific and Medical ISM Radio frequency Equipment
315. o s 5 M j hd 1 1 e Click Zoom In Zoom Per 7 Select Wells Array Selection 6 Sample Type Gem 4 Out and Fit as Well Position oaks dni needed Results Group Name File Convention Name Well Position TI Samples Samples Sample View the Click Array Selection to select wells by capillary plate injection Click again to turn off array Select wels gt 27 Array Selection r Row E Column selection ini a A Use the Table View 1 Click Table View 2 Click the Sample Name field then type a name well oc 401 3 Click next to each field then BOL 3 Col select a setting 4 DO Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 71 Chapter 3 Set Up and Run 4 Right click a column header then select Fill or Fill Series to populate the selected fields to use Fill Series type a number as the last character of the named well Ckri x Copy Ckri c Fill Ctrl D Fill Series BEN No Data al Note You can double click column headers to sort columns Multi column sorting is supported see Multi column sorting below Sort and customize tables Multi column You can sort any table in the software Multi column sorting is supported sorting e Double click a column header to sort the column e Alt Shift click another column header to sort another column e Alt Shift click a third column header to sort a third column
316. o help you prepare your site for installation of the 3500 or 3500xL analyzer For specific details about your system please refer to this user guide electrical requirements needed to support the Applied Biosystems 3500 3500xL Genetic Analyzers Portable document format PDF versions of this guide as well as the Quick Reference Card and the Warranty statement are also available on the Applied Biosystems 3500 3500xL Genetic Analyzers the software installation CD which will be shipped with the system Note For additional documentation see How to obtain support on page xvii Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 335 Documentation Obtaining information from the Help system The 3500 Series Data Collection Software interface has instructions guiding the user through basic tasks of the workflow and expanded help information for complex decisions and operations Users can access these instructions by clicking the help icon Y The 3500 or 3500xL analyzer has a Help system that describes how to use each feature of the user interface Access the Help system by doing one of the following Click in the screens of the 3500 Series Data Collection Software window e Select Help gt Help Contents You can use the Help system to find topics of interest by Reviewing the contents e Searching for a specific topic e Searching an alphabetized index Send us your comments 336 Applied Biosystems wel
317. o store the capillary array 1X running buffer and distilled water Dl Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Instrument operational procedures Flush the water trap pump trap The water trap must be flushed once per month to prolong the life of the pump and to clean any diluted polymer Flush with either distilled or deionized water and ensure that the water flows into the overflow container Dispose the excess water inside the overflow container See General chemical safety on page 328 Note Leave the trap filled with either distilled or deionized water l Open the Luer fitting by grasping the body of the Fill the supplied 20 mL all plastic Luer lock syringe in the PDP Cleaning kit 4359572 with distilled or deionized water Expel any bubbles from the syringe IMPORTANT Do not use a syringe smaller than 20 mL Doing so may generate excessive pressure within the trap Attach the syringe to the forward facing Luer fitting at the top of the pump block Hold the fitting with one hand while threading the syringe onto the fitting with the other hand fitting and turning it to loosen Attached syringe and turn counterclockwise approximately one half turn IMPORTANT DO NOT USE EXCESSIVE FORCE when you push the syringe plunger as this may damage the trap seals Take approximately 30 seconds to flush 5 mL of either distilled or deionized water through the trap Note Because the wa
318. oftware Description System description The 3500 or 3500xL analyzer is shipped with the following system components Capillary Electrophoresis instrument 3500 8 capillary or 3500xL 24 capillary array and POP polymer DNA sequencing or fragment analysis reagents and other consumables for system qualification Dell computer workstation with flat screen monitor Integrated software for instrument control data collection quality control and basecalling or sizing of samples Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Chapter 1 Instrument and Software Description Instrument description The Applied Biosystems 3500 3500xL Genetic Analyzers are fluorescence based DNA analysis instrument using capillary electrophoresis technology with 8 or 24 capillaries For detailed dimensions of the instrument refer to the Applied Biosystems 3500 Series Genetic Analyzer Site Preparation Guide 4401689 Note The purpose of the Site Prep Guide is to help you prepare your site for installation of the 3500 or 3500xL analyzer For specific details about your system please refer to this user guide AS Applied ns HITACHI 3500 Genetic Analyzer 2 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide System description Instrument interior components Oven door Capillary Array frame Detection cell heater block Oven condensation reservoir Plate Cathode Buffer Container CBC Autos
319. oftware v4 1 or later HID GeneMapper D X Software v1 2 or later Note You can also manually import sample data files in to the secondary analysis software applications above Sample data files generated by the 3500 series Data Collection Software are also compatible with Applied Biosystems Variant Reporter Software v1 1 or later and Sequence Analysis SeqA Software v5 4 or later 14 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Overview of the 3500 Series Data Collection Software Parts of the software Dashboard The first screen that is displayed when you start the 3500 Series Data Collection Software 1s the Dashboard Figure 3 Library Maintenance Tools Manage Preferences Help Log Cut Common Operations E e Create Create Plate New Plate From Template Quick Start Run Main workFlow Quick iew Gauges POP Polymer AB 356 Buffer Anode AB 356 Buffer Cathode 50cm 384 576 3 4 3 4 5 bug 2 At S 2 Gt 3 192 u js S P T e 32 4 96 as 0 l 2960 0 eo al 0 eo al On 634 Samples Remaining 7 Days Remaining 7 Days Remaining 43 In 34 Injections Remaining 96 Injections Remaining 96 Injections Remaining Consumables Information Refresh Polymer POP 634 Samples Remaining 1 26 Mar 2009 11 514007 4315930 Anode Buffer AB 356 Buffer 5 Days Remaining 1 28 Mar 2010 11 51B007 4315931 Ca
320. olor for each capillary as a function of instrument scan number time Adjust the brightness and color by using the slider bars above the view ay Sample EPT Injection 3 Brightness d ES Color lt EZ adam ni i mns iin dd mist wii mum im is Rudd slide aaa daa d iaa RCM dum aw alm ian ia ix 32000 28000 24000 20000 16000 12000 Bn Sample view The Sample view shows the relative dye concentrations as a function of instrument scan number time for the selected capillary You can select and deselect the dye colors to display Array ample EPT eth E pe Injection 3 7 E a Raw Data 500 mc bla M tcs bno sion Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 77 Chapter 3 Set Up and Run EPT view The EPT view ElectroPhoresis Telemetry shows instrument data conditions laser power temperatures electrophoresis voltage as a function of time In the legend to the right of the EPT view you can select and deselect the traces to display in the view Array Sample EpvVolkaget ky EpCurrentimid OvenTempi C LaserPower mW ambientTemp C CCDTempr Cc LaserCurrent 4 CellHeater C Select All eG wee 1000 z000 000 4000 S000 6000 000 so00 Time Elapsed seconds 78 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Review Results Workflow Start the system 1 Start the instrument page 22 Star
321. ols to this assay No 9 Yes additional Instrument Protocols instrument protocols for the Instrument Protocols M soot assay The software creates one 0 Instrument Protocol s Assigned to this Assay injection for each instrument protocol Edit Remove Move Up Move Down specified in an assay MOTE Ord isk o ocols in the Es Oe oy Hd order vou v Instrument Protocol Instrument protocol for data collection For information see Instrument protocol library on page 165 Sequencing e Basecalling Protocol Protocol for primary analysis basecalling and trimming and quality determination For information see Basecalling protocols library primary analysis sequencing on page 174 e SegScape software MicroSeq software D Protocols Optional protocol for secondary analysis auto analysis For information see e Sequencing analysis protocols library secondary analysis on page 189 e MicroSeq ID protocols library secondary analysis on page 191 Fragment e Sizecalling Protocol Protocol for primary analysis peak detection and sizing and quality determination For information see Sizecalling protocols library primary analysis fragment on page 179 e GeneMapper software Protocol Optional protocol for secondary analysis auto analysis For information see Fragment analysis protocols library secondary analysis on page 19
322. on page 196 4 Select the remaining secondary analysis items then click Save Results Group Note If the analysis method size standard or panel of interest is not displayed in a list re select the secondary analysis software instance to update the list lii Analyze Instrument Protocols Dye Sets size Standards Basecalling Protocols Sizecalling Protocols IMPORTANT The auto analysis settings you specify for the plate to run with this protocol must contain the same secondary software and location settings For more information see Create a new plate on page 144 GC Protocols Sequencing Analysis Protocols MicroSeqiD Protocols Fragment Analysis Protocols Du n haeret eai nes T A 1 iL A na I l a HPF al tor al 15 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 195 Chapter 6 Manage Library Resources MM Create New HID Analysis Protocol Setup a GeneMapper ID X Protocol Px Protocol Name is a required Field Provide a unique value Protocol Mame n l l mE C Locked Description Application Type HIC Secondary Analysis Software Secondary Analysis Software Instance Froperties Analysis Method Size Standard Panel Close Figure 33 Create New HID Analysis Protocol Table 21 HID Analysis protocol settings Setting Description Protocol Name Name of the protocol Names must be unique Description Optional text
323. ons and results groups Note You can override this setting in file name conventions and results groups A Preferences type Filter text not used Run Setup WD T System Choose the default File location where sample Files will be stored El User Plate Setup DC 4pplied Biosystems 3500 Data Reports Settings az Run Setup El Sequencing Settings 2 Click Apply to save the user preferences see User preferences on page 33 Table and plot Users can also save user preferences while viewing tables and plots settings user Table settings dialog box Determines the columns displayed in a table and the preferences order of the columns Plot settings dialog box Determines the settings applied to plots Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 35 Chapter 2 Start the System Set sequencing preferences Export system Export preferences set the defaults for the file types to automatically export during a preference sequencing run Exported files are stored in the same directory as the ab1 files 1 In the Preferences dialog box click Export under System Sequencing settings to display the Export pane A Preferences type Filter text not used Export ems System Date Format Instrument Settings Select the file types to export during analysis then check each file type and specify to export Ehe entire sequence or post trim only Click Apply File Type Scheduler Pref
324. or Analysis Method Editor Microsatellite General Allele Peak Detector Peak Quality Quality Flags Quality weights are between O and 1 r Quality Flag Settings Spectral Pull up SPU 0 5 Control Concordance CC 0 5 Broad Peak BD 0 5 Low Peak Height LPH 0 5 Single Peak Artifact SPA 0 5 Off scale OS 0 5 Sharp Peak SHP 0 5 Peak Height Ratio PHR 0 5 Cross Talk XTLK 0 5 One Basepair Allele OBA 0 5 Out of Bin Allele BIN 0 8 Split Peak SP 0 5 PQY Thresholds Sizing Quality From 0 75 to 1 0 From0 Dto 0 25 Genotype Quality From 0 75 to 1 0 From0 0to 0 25 Assume Linearity From bp 0 To bp 800 8 Click OK to save then click Done to close GeneMapper IMPORTANT Close GeneMapper v4 1 before performing the auto analysis run on the 3500 3500xL analyzer Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 283 Appendix C Secondary Analysis Fragment Set up a GeneMapper plate in the 3500 Series Data Collection Software Set up a fragment analysis run in the 3500 Series Data Collection Software by assigning an Assay a File Name Convention and a Results Group Start the 3500 1 Start the Auto Analysis Manager before starting the 3500 Series Data Series Data Collection Software Collection E Software 2 Start the 3500 Series Data Collection Software then go Dashboard MESS New Plate 3 Name your new plate Plate Details Mame Test Fra
325. ort Spectral Calibration User Plate Setup Choose time Format to use Reports Settings 10 11 55 AM 12 hour SS Run Setup 10 11 55 24 hour Sequencing Settings Trace All dates in the user interface display in the Formats that you choose ATA Examples reports imported files and exported Files Instrument Settings to set the instrument name appears in the Dashboard reports file name conventions instrument sensor details view sequencing results R Preferences type Filter text not used Instrument Settings System Date Format Instrument Settings Instrument name 3500 Instrument Scheduler Preference to set the time to trigger maintenance notifications displayed in the Dashboard see Check maintenance notifications on page 28 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 33 Chapter 2 Start the System Preferences type Filter text not used Scheduler Preference System gt Date Format Maintenance reminders trigger time 12 00 PM Instrument Settings Z2 Scheduler Preference Spectral Calibration to decrease the number of allowed borrowing events for spectral calibration see What you see during a spectral calibration on page 112 S Preferences type Filter text not used Spectral Calibration uem System Date Format Instrument Settings Scheduler Preference Choose the maximum number of borrowing events Far a 24 Cap
326. otective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Handle chemical wastes in a fume hood After emptying a waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations If potentially hazardous waste is generated when you operate the instrument you must Characterize by analysis 1f necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Chemical safety Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Biological hazard safety General WARNING BIOHAZARD Biological samples such as tissues body fluids biohazard infectious agents and blood of humans and other animals have
327. otocol 0 0c eee ees 165 DYE SEIS IDAN 4 5 O x a Ls att oe eee REL AL 168 Dve Set OVerlvIOW itunes Cecio Sud wear a eee Sone eS Ge Rad 168 Create anew dye Set celle eee 168 Size Standards library aa isda ca sa cub Sal oes EMS ee a 171 Size standard overview lese hh 171 Normalization size standards provided eee 171 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide vil Contents Chapter 7 viii Create a new size standard n nananana aa aaa 172 Modify a factory provided normalization size standard 173 Basecalling protocols library primary analysis sequencing 174 Basecalling protocol overview leeren 174 Create a new basecalling protocol llle 174 Sizecalling protocols library primary analysis fragment 179 Sizecalling protocol overview leer 179 Create a new sizecalling protocol llle 179 QC protocols library primary analysis HID llle 184 QC protocol overview 4 m x IRR P C C CR TC Seah D ad ww DR un DR D nx 184 Create d new OG DEIOIOCOl iss adobe edad hc Exe dob ea 184 Sequencing analysis protocols library secondary analysis 189 Sequencing analysis protocol overview celles 189 Create a new sequencing analysis protocol eee 189 MicroSeq ID pr
328. otocols library secondary analysis o o ooooooooooo 191 MicroSeq ID analysis protocol overview ee ne 191 Create a new MicroSeq ID analysis protocol 0 00 cece eee eee 191 Fragment analysis protocols library secondary analysis 193 Fragment analysis protocol overview lees 193 Create a new fragment analysis protocol llle 193 HID analysis protocols library secondary analysis 0000 cece eee eee 195 HID analysis protocol overview 0 0 ce eee ees 195 Create a new HID analysis protocol 0 00 eee 195 Use Security Audit and E Sig Functions SAE Module 197 Section 1 Administrators ius ROC WO deen eee Sense MURA 197 Administrators overview of system security auditing and electronic signature 197 Configure the security system naaa 198 Access the Security screen and enable or disable security 198 Set account setup and security policies llle 199 Set up messaging notifications celle 200 Manage user accounts 3 33 99 a Vau obe East ueni x Da d oa dr 201 Create or edit a user account co 201 Determine the name of the logged in user 0000 c eee eee es 203 Create or edita user role 0 0 te eee nes 203 View and print a user report 00 0 ees 205 Manage auUdilNd ce st ca es ee Sener te es sy aes ieee darias 207 Access the Audi
329. our secondary analysis method Set up a SegScape plate in the 3500 Series Data Collection Software Start the 3500 l Series Data Collection Software 2 3 4 5 2 0 Start the Auto Analysis Manager before starting the 3500 Series Data Collection Software E Start the 3500 Series Data Collection Software Dashboard gt Cees New Plate Name your new plate Select the Number of Wells Plate Type as Sequencing Capillary Length and Polymer associated with this plate for the current run Optional Enter your name as Owner a barcode and description for the plate Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Set up a SegScape plate in the 3500 Series Data Collection Software Specify auto 1 Check Perform Auto Analysis right side of the Plate Details section to analysis for secondary an alys is 3500 Data Collection Software Dashboard Edit v E Setup Define Plate Properties O SA Assign Plate Contents Run Instrument Load Plates for Run expand the Secondary Analysis section Plate Details Name New SeqScape Run Number of Wells 96 96 FastTube 384 Plate Type Sequencing x Capillary Length cm ose ute fessa Library Maintenance Tools Manage v Preferences Help Log Out Owner tester Barcode first sequence on 3500 Description ili Review Results Preview Run Polymer POP7 Monitor Run v Secondar
330. ours Applied Biosystems recommends that you pre heat the oven for at least 30 minutes before you start a run if the instrument is cold Check the pump assembly for bubbles and run the Remove Bubble wizard if needed see page 251 A ln Prepare the IMPORTANT Do not use warped or damaged plates standard calibration plate 1 104 Prepare the calibration standard as described in the standard product insert See Table 28 on page 259 and Table 29 on page 260 for standard part numbers Dye set Standard E BigDye Terminator BDT v1 1 Sequencing Standard BigDye Terminator BDT v1 1 Matrix Standard Z BigDye Terminator BDT v3 1 Sequencing Standard BigDye Terminator BDT v3 1 Matrix Standard F DS 32 Matrix Standard E5 DS 02 Matrix Standard G5 DS 33 Matrix Standard Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Spectral calibration 2 Load the standards in injection position 1 in the spectral calibration plate IMPORTANT You do not create a plate for the calibration The software uses predetermined positions for the calibration You cannot specify standard location on the plate If you do not place calibration standards in the positions indicated the calibration will fail 8 capillary A1 through H1 TTATATRTGTA 96 well plate muHEHHER 24 capillary A1 through H1 A2 through H2 and 96 well plate A3 through H3 ee eee 384 we
331. oving average to determine a contiguous read length based on quality values the software starts from the 5 end and calculates the average QV across a moving window size of 20 sliding 1 bp at a time to the 3 end The resulting longest contiguous segment is determined as the CRL Trace Score The average basecall quality value QV of bases in the clear range sequence of a trace QV20 The total number of bases in the entire trace with quality values gt 20 178 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Sizecalling protocols library primary analysis fragment Sizecalling protocols library primary analysis fragment Sizecalling protocol overview A sizecalling protocol is the required primary analysis protocol for fragment applications A sizecalling protocol defines peak detection sizing and quality values When you create a fragment assay you add a sizecalling protocol to the assay If you add this item from the library a copy of the item 1s added to the assay and can be modified independently from the original item stored in the library For information on how changes are tracked if auditing 1s enabled see Audit action on page 210 Create a new sizecalling protocol If factory provided sizecalling protocols do not suit your needs you can create new sizecalling protocols 1 Access the Sizecalling Protocols library 2 Click A Create 3 In the Analysis Settings tab o
332. page 116 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Spectral Quality Values and Condition Numbers Spectral calibration Capillary Run Data Run E Run 3 Overall 1 Passed m FS Capillary 1 Run 1 For all spectral calibration injections even capillaries that are green in the Overall row evaluate the data as described in the next section Spectral Quality Value A spectral Quality Value reflects the confidence that the individual dye emission signals can be separated from the overall measured fluorescence signal It is a measure of the consistency between the final matrix and the data from which it was computed A Quality Value of 1 0 indicates high consistency providing an ideal matrix with no detected pull up pull down peaks In rare cases a high Quality Value can be computed for a poor matrix This can happen if the matrix standard contains artifacts leading to the creation of one or more extra peaks The extra peak s causes the true dye peak to be missed by the algorithm and can lead to a higher Quality Value than would be computed with the correct peak Therefore it is important to visually inspect the spectral calibration profile for each capillary see Evaluate the spectral calibration data on page 110 Condition Number A Condition Number indicates the amount of overlap between the dye peaks in the fluorescence emission spectra of the dyes in the dye set If t
333. page 127 Fragment or HID install standard performance check page 127 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 41 Chapter 3 Set Up and Run Prepare the instrument 1 In the Dashboard check consumable status page 29 Ensure that Consumables are not expired Adequate buffer levels are at the fill lines 2 Set the oven temperature then click Start Pre heat e 60 C POP 7 and POP 4 polymers e 50 C POP 6 polymer Pre heat the oven and detection cell while you prepare for a run detection cell temperature is set by the software Preheating helps mitigate subtle first run migration rate effects The preheat function automatically turns off after 2 hours of instrument inactivity Applied Biosystems recommends that you pre heat the oven for at least 30 minutes before you start a run if the instrument is cold 3 Check the pump assembly for bubbles and run the Remove Bubble wizard if needed see page 251 42 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Create a plate Note If you are running a stand alone version of the 3500 Series Data Collection Create a plate Software a version that is not installed on the instrument computer you can create plates then export them for use on the instrument computer Create a plate The software includes factory provided plate templates that you can use as a starting from a template point to create a plate you can also create your own plate te
334. per Software v4 1 or later fragment applications A fragment analysis protocol defines the Secondary analysis software GeneMapper Software location e GeneMapper software analysis method size standard and panel that the GeneMapper software will use during auto analysis Create a new fragment analysis protocol 1 Access the Fragment Analysis Protocols library 2 Click p Create 3 In the Create New Fragment Analysis Protocol dialog box Figure 32 on page 194 specify settings see Table 20 on page 194 4 Select the remaining secondary analysis items then click Save Note Ifthe analysis method size standard or panel of interest is not displayed in a list re select the secondary analysis software instance to update the list IMPORTANT The auto analysis settings you specify for the plate to run with this protocol must contain the same secondary software and location settings For more information see Create a new plate on page 144 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide K 3500 Data Collection Software 3 Dashboard Edit Library Resources 4 Manage Plates Assays File Mame Conventions Results Group ilii Analyze Instrument Protocols Dye Sets Size Standards Bazecalling Protocols Sizecalling Protocols 2C Protocols Sequencing Analysis Protocols MicroSeq Protocols HID Analysis Protocols Tuum JE ria Cle EF ad ment A ne
335. plied Biosystems 3500 3500xL Genetic Analyzer User Guide How to obtain support Acronym Definition GM GeneMapper Software GMIDx GeneMapper IDx POP Polymer Brand name of the polymer PPS Power Protection System SAE Security Administration Electronic signature How to obtain support For the latest services and support information for all locations go to www appliedbiosystems com At the Applied Biosystems web site you can e Access worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities e Search through frequently asked questions FAQs e Submit a question directly to Technical Support e Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents e Download PDF documents e Obtain information about customer training Download software updates and patches How to obtain For detailed information on preparing for installation refer to the Applied Biosystems more information 3500 Series Genetic Analyzer Site Preparation Guide 4401689 Note The purpose of the Site Prep Guide is to help you prepare your site for installation of the 3500 or 3500xL analyzer For specific details about your system please refer to this user guide Applied Biosystems 3500 3500xL Genetic Analyzer User Guide XVII Preface xviii Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Instrument and S
336. port sequencing results 1 Filter the table of interest 2 Select an export option Results Reports Select Filter All Traces EN E l All Traces zu Failed Traces 3 Select the export options and the location for the export file then click OK The file s are exported to the specified location with the following naming conventions Results export ReportName txt Reports ReportName is the format you selected txt xls pdf html e Traces FileName is the export format you selected annotation txt phd 1 scf fsta qual seq Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 87 Chapter 4 Review Results Review Fragment HID Analysis results Access the View Fragment HID Results screen Access the View Fragment HID Results screen from The Monitor Run screen by clicking Review illi Review Resuits Results View Sequencing Results eye Fragment AIO Results e The navigation pane by selecting View Sequencing Results e The Dashboard by clicking View Run Results v View Run Results Review results for If you access the View Fragment HID Results screen while an instrument run is in the currently progress the samples table lists results for completed injections in the current run running plate Select one or more samples in the samples table to display their data in the plot view and sizing table view Sample GS6DOLIZ 65 Sample GS600L1zZ TM
337. pos poo ps pao mo pas Figure 10 Sequencing install standard capillary run data 124 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Spectral calibration How the software determines passing and failing capillaries for the spectral calibration The software evaluates the Quality Value and Condition Number for each capillary for more information see Spectral Quality Values and Condition Numbers on page 109 Borrowing is automatically enabled 1 borrowing event 1s allowed for 8 capillary instruments up to 3 borrowing events for 24 capillary instruments For more information see Capillary information sharing on page 112 The number of borrowing events can be decreased see User preferences on page 34 Thresholds used by the software for pass fail are Condition Number Dye Set Quality Value Minimum Maximum E 0 95 5 5 Z 0 95 5 5 How the software determines passing and failing capillaries for the sequencing performance check The software calculates the Contiguous Read Length for each capillary Capillaries that are below the threshold fail The remaining results that the software displays are for information only Result Description Contiguous Read The longest uninterrupted segment of bases with an average Quality Value QV gt 20 Length CRL In addition to evaluating the QV of a base call the software considers the QV of adjacent bases within a 20 bp moving average
338. quencing Analysis Protocols 3 MicroSegID Protocols Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 189 Chapter 6 Manage Library Resources a Create New Sequencing Analysis Protocol Setup a Sequencing Analysis Protocol S Protocol Name is a required field Provide a unique value Protocol Name Description Application Type Sequencing Secondary Analysis Software 5eq5cape Secondary Analysis Software Instance Seqscape_sanghahs pc Properties Project p53_v2 1 Project Template p53 Specimen 12586 Figure 30 Create New Sequencing Analysis Protocol Table 18 Create New Sequencing Analysis Protocol Setting Description Protocol Name Name of the protocol Names must be unique Description Optional text entry Lock When enabled allows the entry to be unlocked and modified only by the user who created it the administrator or another user with unlock permissions Useful when your system includes the SAE module described in Chapter 7 Use Security Audit and E Sig Functions SAE Module on page 197 Application Type Automatically set to Sequencing Secondary Analysis Software IMPORTANT The secondary analysis software must be installed and properly configured with the 3500 Series Data Collection Software before it is listed as a selection in this screen For information on setting up the SeqScape Software for auto analysis Seco
339. r 1 injections Allelic ladders that are injected under the same conditions are recommended to accurately genotype samples in the secondary analysis software GeneMapper ID X Software v1 2 or later IMPORTANT Variation in laboratory temperature can cause changes in fragment migration speed that can in turn cause sizing variation Applied Biosystems recommends the frequency of allelic ladder injections described above to account for normal variation in fragment migration speed However during internal HID validation studies verify the required allelic ladder injection frequency to ensure accurate genotyping of all samples in your laboratory environment Results group for one allelic ladder per run folder For a 24 capillary instrument create a results group that specifies an injection folder then select this results group for all injections on the plate For an 8 capillary instrument create one results group for each set of three injections on the plate each results group specifies a results group name folder For more information see Results group example 2 store one allelic ladder per run folder 8 capillary instruments on page 161 Prepare sample plates 52 1 Pipette samples into the plate according to the plate layout see Print the plate layout on page 50 2 Briefly centrifuge the plate 3 Verify that each sample is positioned correctly in the bottom of its well B fo e a gih IMPORTANT
340. r User Guide Preface Safety information Note For general safety information see this Preface and Appendix F Safety on page 315 When a hazard symbol and hazard type appear by a chemical name or instrument hazard see Appendix F Safety on page 315 for the complete alert on the chemical or instrument Safety alert Four safety alert words appear in Applied Biosystems user documentation at points words in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical CAUTION Indicates a potentially hazardous situation that 1f not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word 1s to be limited to the most extreme situations Except for IMPORTANTS each safety alert word in an Applied Biosystems document appears with an open triangle figure that contains a hazard symbol These hazard symbols are identical to the hazard symbols that are affixed to Applied
341. r an experienced radio TV technician for help Canadian EMC This instrument has been tested to and complies with ICES 001 Issue 3 Industrial standard Scientific and Medical Radio Frequency Generators Cet appareil numerique de la classe B est conforme a la norme NMB 001 du Canada Canadian Department of Communications Industry Canada IC Notice This device complies with RSS Gen of IC Rules Operation is subject to the following two conditions 1 This device may not cause interference and 2 This device must accept any interference including interference that may cause undesired operation of this device European safety Safety and EMC This instrument meets European requirements for safety Low Voltage Directive standards 2006 95 EC This instrument has been tested to and complies with standards C EN 61010 1 2001 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements EN 61010 2 010 Particular Requirements for Laboratory Equipment for the Heating of Materials EN 61010 2 081 Particular Requirements for Automatic and Semi Automatic Laboratory Equipment for Analysis and Other Purposes EN 60825 1 Radiation Safety of Laser Products Equipment Classification Requirements and User s Guide EMC The 3500 or 3500xL analyzer meets European requirements for emission and immunity EMC Directive 2004 108 EC EN 61326 1 2006 El
342. r before servicing the instrument Solvents and a WARNING PHYSICAL INJURY HAZARD Always wear eye protection pressurized fluids when working with solvents or any pressurized fluids 318 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Instrumentation safety Electrical safety AN WARNING ELECTRICAL SHOCK HAZARD Severe electrical shock can result from operating the 3500 3500xL analyzers without its instrument panels in place Do not remove instrument panels High voltage contacts are exposed when instrument panels are removed from the instrument Power A WARNING ELECTRICAL HAZARD Grounding circuit continuity is required for the safe operation of equipment Never operate equipment with the grounding conductor disconnected WARNING ELECTRICAL HAZARD Use properly configured and approved line cords for the voltage supply in your facility WARNING ELECTRICAL HAZARD Plug the system into a properly grounded receptacle with adequate current capacity Overvoltage The 3500 3500xL analyzers system has an installation overvoltage category of II rating and is classified as portable equipment Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 319 Appendix F Safety Laser safety Laser The 3500 or 3500xL analyzer uses a a solid state laser The laser specifications are classification Wavelength 505nm Output power 20mW The LED specifications are e Emitting color Natural White Luminous Intensity 25
343. r information refer to the chapters in this user guide From the Dashboard To advance from the Dashboard to e Main workflow Click e e Other screens in the software Select items from the menu bar G 7500 Data Collection So bware From the Main workflow To advance from the Main workflow to Dashboard Dashboard Click Dashboard mon E e Other screens in the Main workflow Select items in the E navigation pane gates Other screens in the software Select items from the menu Enee bar Preview Run From the Library or Maintenance workflows To advance from the Maintenance or Library ES ye workflow to e Dashboard Click Dashboard Other screens in the workflow Select items in the navigation pane M Menger ey Poel Se e Main workflow Click Main Workflow in the Frgeri ratu Sartre navigation pane HD Filmi terete e Other screens in the software Select items from the menu bar Sein Loy Use the software without an instrument You can install the 3500 Series Data Collection Software on a computer that is not connected to an instrument You can use this stand alone version of the software to create plates protocols and other library items and to review completed results IMPORTANT Do not select instrument related functions in the stand alone version of the software Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 19 Chapter 1
344. r range Useful in large fragment size standards where non linearity might be expected Pull Up Enter the pull up ratio and tolerance for pull up peak identification A pull up peak is identified when the peak height of the minor peak is e lt X pull up ratio of the major peak and e Within Y data point pull up scan of the major peak When at least one peak is identified as a pull up peak the Check flag is displayed for the Spectral Pull Up quality flag in View Fragment Results Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 183 Chapter 6 Manage Library Resources QC protocols library primary analysis HID QC protocol overview A QC protocol is the required primary analysis protocol for HID applications A QC protocol defines peak detection sizing and quality values When you create an HID assay you add a QC protocol to the assay If you add this item from the library a copy of the item is added to the assay and can be modified independently from the original item stored in the library For information on how changes are tracked if auditing is enabled see Audit action on page 210 Create a new QC protocol If factory provided QC protocols do not suit your needs you can create new QC protocols 1 Access the QC Protocols library R 3500 Data Collection Software Dashboard Edit 7 Library Maintenance Tools Library Resources Filter All Xx Manage Plates Assays PPO
345. r roles and associated permissions 3 Click Export 4 Specify the name and location for the exported dat file then click Save A message 1s displayed when the export completes Import 1 In any screen in the SAE module click j Import in the navigation pane 2 Select the dat file to import then click Open A message is displayed asking if you want to overwrite the current system configuration Click Yes If any imported user accounts already existon V fuser overwrite skp the system you are prompted to overwrite or skip each account Please resolve conflicts 224 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Users overview of System Security Audit Trail and E Signature Section 2 Users Users overview of System Security Audit Trail and E Signature The Security Audit E Signature SAE module is an optional component of the 3500 Series Data Collection Software The SAE module provides the following functionality System security Controls user access to the software Auditing Tracks changes made to library items actions performed by users and changes to the SAE settings Electronic signature e sig Requires users to provide a user name and password when performing certain functions Depending on the way that your administrator configures these features you may see the following dialog boxes and prompts when you use the software Security Log in If security is en
346. ragment amp r HID 3 HID 3 HID 3 HID 3 HID 3 re m File Mame Conventions Results Group 4 Specify settings Table 10 on page 166 iii Analyze Dye Sets 5 Save the assay e Ifyou are creating the assay from the Library click Save e If you are creating the assay from the Assign Plate Contents screen click Apply to Plate or Save to Library Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 165 Chapter 6 Manage Library Resources Create New Instrument Protocol x Setup an Instrument Protocol B Protocol Mame FragmentAnalysis50_ POP7x 1 already exists in the Library Application Type Fragment Capillary Length 50 cm Polymer por7 Dye Set 65 Instrument Protocol Properties Run Module FragmentAnalysis50_POP x Protocol Mame Fragmentanalysiss0_POP7_1 Locked Description Oyen Temperature C 60 Run Voltage kvolt 19 5 PreRun Yoltage kvolts 15 Injection Voltage kvols 1 6 Run Time sec 1330 PreRun Time sec 180 Injection Time sec 4 15 Data Delay sec 1 Advanced Options Following values are not recommended to be changed Voltage Tolerance kvolts Voltage of Steps mk Voltage Step Interval sec First Read o ime ims o0B econd Read ime ims ovn Normalization Target 4500 0 Normalization Factor Threshold Min Normalization Factor Threshold Max Figure 21 Create New Instrument Protocol
347. rary 142 audit administrators action log 209 212 archive records 214 audit actions 210 212 audit mode 208 audit reason settings 208 audit records object 210 audited objects and actions 208 enable or disable 207 export records 215 export settings 224 import settings 224 object audit history 209 210 overview 197 purge records 214 restore records 214 system configuration history 209 211 troubleshoot 312 when security is disabled 207 338 audit users enter reason for change 227 overview 225 view audit records 142 auto analysis plate setting 146 average quality value QV monitor run 64 B barcode 145 barcode scanner 8 basecall probability of error 84 Basecaller 175 basecaller version in basecalling protocol and View Sequence results 80 basecalling protocols create 174 defined 174 export 141 import 141 inassay 150 Basepair Accuracy 125 BigDye Terminator kits and standards 259 biohazardous waste handling 331 bold text when to use xvi borrowing defined 112 limits 34 Broad Peak BD and SQ 188 flag in view fragment HID results 89 threshold 188 buffer anode See anode buffer buffer cathode See cathode buffer Buffer Pin Valve 28 C calibration See spatial calibration See spectral calibration capillary array change 252 check stored 240 described 12 fill with polymer 251 on instrument limits 30 part number 13 storage conditions 240 capillary to plate map diagram 51 display in software 71 Appli
348. rds To activate the Maintenance Wizards from the Dashboard click Maintain Instrument toggle key The Maintenance Wizards feature of the Data Collection software allows you to perform operations necessary for sustaining the instrument In no particular order these operations include the following e Install a Capillary Array e Remove bubbles from the polymer pump Wash the pump chamber and channels e Fill the array with fresh polymer e Replenish the polymer installed on the instrument Change the type of polymer installed on the instrument with the option to change the capillary array e Shutdown the Instrument IMPORTANT Once started Wizard operations cannot be canceled IMPORTANT After performing a conditioning wash ensure that the buffer level inside the ABC 1s at or above fill line before proceeding to the next step except for the wash pump and channels wizard 244 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Use the Maintenance Wizards to perform operations Fill Install Capillary Array Array INSTALL a capillary array with FILL the array with fresh polymer Polymer Remove Replenish Bubbles REMOVE bubbles from the polymer pump Polymer REPLENISH the polymer installed on the instrument BEEN Pump and WASH the pump chamber and channels Channels Change Polymer CHANGE the type of polymer installed on the instrument Type with the option to change arrays Shutdown the EM
349. reate new items Entries in the library may be flagged with the following symbols e Factory provided Cannot be edited or deleted Y Template e Locked If your system includes the SAE module can be unlocked and modified only by the user who created it the administrator or another user with unlock permissions For information see Chapter 7 Use Security Audit and E Sig Functions SAE Module on page 197 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 139 Chapter 6 Manage Library Resources General library procedures Access libraries Select Library in the menu bar to access the Library workflow Library Maintenance Tools Manage Preferences Hel 1 Azzayz File Mame Conventions Results Group a Manage The Library workflow contains the screens where you manage assays protocols and other items that you use ili Analyze to acquire and process data Instrument Protocols Dye Sets The Library workflow contains Size Standarda e Items that you select when you set up for a run plates assays filename conventions and results groups Basecalling Protocals Sizecalling Protocals iac Protocols Items that you select when you create an assay RT nstrument protocols MicroSeqiD Protocols Primary analysis protocols Basecalling Fragment Analysis Protocols sequencing sizecalling fragment analysis HDD Analysis Protocols QC HID analysis Optional secondary an
350. reflect the beam into your eyes Do not remove the instrument top or front panels Wear proper eye protection and post a laser warning sign at the entrance to the laboratory 1f the top or front panels are removed for service 320 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Instrumentation safety Bar code scanner laser safety Laser classification Laser safety requirements Using a bar code scanner is optional The bar code scanner must be categorized as a Class 2 II laser Class 2 II lasers are low power visible light lasers that can damage the eyes Never look directly into the laser beam The scanner is designed to prevent human access to harmful levels of laser light during normal operation user maintenance or during prescribed service operations Ke WARNING LASER HAZARD Class 2 II lasers can cause damage to eyes Avoid looking into a Class 2 II laser beam or pointing a Class 2 II laser beam into another person s eyes Workstation safety Correct ergonomic configuration of your workstation can reduce or prevent effects such as fatigue pain and strain Minimize or eliminate these effects by configuring your workstation to promote neutral or relaxed working positions CAUTION MUSCULOSKELETAL AND REPETITIVE MOTION HAZARD These hazards are caused by potential risk factors that include but are not limited to repetitive motion awkward posture forceful exertion holding static unhealthy position
351. resholds 91 167 flag in view fragment HID results 89 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 343 Index GS600 LIZ v2 0 size standard 171 how the software normalizes 91 overview 7 review data 90 size standards 171 target 91 167 notifications log for maintenance actions 234 257 maintenance in Dashboard 28 229 SAE module 200 O object audit history contents 210 display 209 offscale flag monitor run 64 view fragment HID results 89 online Help See Help system oven temperature in instrument protocol 167 pre heat in Dashboard 42 overlay report 95 overvoltage category rating 319 owner plate 145 P part numbers anode buffer 9 capillary array 13 cathode buffer 9 conditioning reagent 11 fragment HID analysis standards 260 Hi Di Formamide 12 sequencing analysis reagents 259 password change your own 226 expiration 199 restrictions 199 pause instrument run 69 Peak Amplitude Thresholds 181 187 Peak Window Size 182 pending plates 76 performance check See fragment HID install performance check See sequencing install performance check permissions user account 203 225 physical hazard safety 318 planned maintenance 232 plate See also assign plate contents 344 assay 49 73 assembly 53 base for standard versus fast and 8 strip 53 create 43 create for import 73 create from template 43 edit 74 edit existing 46 export 75 file name convention 49 73 import 44 75 link 54 57 load
352. rip fast tubes are also supported with appropriate retainers e 384 well 384 well plates Plate Type e Sequencing e Fragment analysis e Mixed Seq Frag e HID Capillary Length and Polymer Capillary length and polymer type with which the plate will be used Owner Barcode Description optional Optional text entries You can use these entries to search for plates in the Plates library and in run logs Tools View Logs Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 145 Chapter 6 Manage Library Resources Table 6 Define Plate Properties continued Setting Description Secondary Analysis Note The secondary analysis protocol settings you specify in an assay must match the auto analysis settings for the plate For more information see e Sequencing analysis protocols library secondary analysis on page 189 e MicroSeq ID protocols library secondary analysis on page 191 e Fragment analysis protocols library secondary analysis on page 193 e HID analysis protocols library secondary analysis on page 195 Perform Auto Analysis Enables the plate for use with auto analysis with a supported secondary software Software Type Supported secondary software Software Location Computer on which the supported secondary software is installed Username User name and password required by the secondary analysis software Password Auto Analysis is performed Dete
353. rm QC Checks if you want the system to check each capillary against the specified range for spacing and intensity During the calibration the software calculates Attribute Calculation Threshold Average peak height sum of all peak heights e 8 cap 6400 RFU 24 cap 3000 RFU number of peaks DITOFmity peak height standard deviation we similarity Z ak See eae a average peak height Capillary spacing max spacing min spacing 2 pixels 3 Click Start Calibration The display updates as the run progresses 13500 icd pe pun 11500 9500 7500 5500 3500 1500 500 0 100 200 300 400 500 If the average of any of the QC values exceeds the threshold a Spatial QC Check error message 1s displayed 100 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Spatial calibration Evaluate the spatial calibration profile When the run is complete 1 Evaluate the spatial calibration profile to ensure that you see Onesharp peak for each capillary Small shoulders are acceptable One marker at the apex of every peak No off apex markers An even peak profile all peaks about the same height 2 Ifthe results meet the criteria above click Accept Results If the results do not meet the criteria above click Reject Results then go to Spatial calibration troubleshooting on page 300 Example spatial profiles 8 capillary 13500 11500 as 7o00 5500 3500 1500 24 capillary 5500 35
354. rmines when data is sent to the secondary analysis software QUPD BIL Only e Only when the results group is complete e When every injection completes 146 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Assays library Assays library Assay overview An assay contains the instrument protocol dye set and run configuration and primary analysis protocol needed to collect data and basecall or sizecall a sample Assays File Name Conventions and Results Groups may already be listed in the plate template when you create a plate from a template Note If no assay is listed add at least one assay An assay contains Oneor more instrument protocols appropriate for the sample type dye set for which the assay will be used A primary analysis protocol that depends on your application Sequencing Basecalling protocol Fragment Sizecalling protocol HID QC protocol Optional A secondary analysis protocol that depends on your application Sequencing SeqScape Software v2 7 or later or MicroSeq ID Analysis Software v2 2 or later Fragment analysis GeneMapper Software v4 1 or later HID GeneMapper D X Software ID X Software v1 2 or later Assays are required by all application types You must assign an assay to all named sample wells on a plate before you can link a plate and run it When you create an assay you add one or more instrument protocols and a primary analysis
355. rs e 96 Fast Tube Supports 96 well Fast reaction plate 8 strip fast tubes are also supported with appropriate retainers 3 Bnefly centrifuge the plate containing the standards 120 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Prepare the plate assembly Load the plate in the instrument 4 Verify that each sample Spectral calibration is positioned correctly pc in the bottom of its puc C well NT pau aT e eT x od IMPORTANT Ifthe HSE pag 97 reagents of any well contain bubbles or are not located at the bottom of the well briefly centrifuge the plate remove the plate from the centrifuge and verify that each sample is positioned correctly in the bottom of its well Sample is at the bottom of the well Store the plate on ice until you prepare the plate assembly and load the plate in the instrument IMPORTANT Prepare the plate assembly on a clean level surface Do not heat plates that are sealed with septa pa Align the holes in the septa Place the sample plate into the strip with the wells of the plate then firmly press downward onto the plate Plate retainer plate base Adel septa strip IMPORTANT Make sure to use the correct plate base for Plate base standard plates versus 8 tube strips and fast plates Using the wrong plate base may affect performance snap the plate retainer cover onto the plate septa and plate base Verify that
356. rs When you create an instrument protocol you add a dye set to the protocol If you add this item from the library a copy of the item is added to the assay and can be modified independently from the original item stored in the library For information on how changes are tracked if auditing 1s enabled see Audit action on page 210 Create a new dye set If factory provided dye sets do not suit your needs you can create new dye sets 1 Access the Dye Sets library 2 Click A Create 3 In the Create New Dye Set dialog box Figure 22 on page 169 4 Specify settings Table 11 on page 169 5 Click Save 168 3500 Data Collection Software Dashboard Edit 7 v Mainten MFeate Filter 4 Manage Plates Library Resources Azzsym File Mame Conventions Results Group lili Analyze Instrument Protocols Dye Sets Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Dye sets library Create New Dye Set F x Setup a Dye Set B Dye Seb Mame is a required Field Provide a unique value Dye Set Name Locked Chemistry Matrix Standard Dye Set Template RAEE Arrange Dyes Dye Selection Reduced Selection Calibration Peak Order 5 Parameters The parameters will be used Far instruments configured with 50cm capillary array and polymer POP Matrix Condition Number Upper Limit 13 5 Locate Start Point After Scan 1000 Before Scan
357. s e z data click History View e History View Calibration Settings Current Instrument Consumables o Polymer Type POP4 Capillary Length 36cm 2 mo of Wells 296 D 96 FastTube 384 Chemistry Standard w 3 Plate Position 214 2B Dye Set hu 095 4 Allow Borrowing Status Ready 5 Capillary Run Data o D Passed B Failed Borrowed Not Calibrated Quality Value Condition Status Message Intensity vs Scan Number 0 4000 ann 12000 16000 20000 4000 5000 32000 40000 0000 Intensity vs Scan Number t Intensity ws Pixel Number A nl 2 Select the number of wells in the spectral calibration plate and specify the plate location in the instrument Note You do not create a plate for the calibration The software uses predetermined positions for the calibration You cannot specify standard location on the plate Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 107 Section 1 Calibration 108 3 Select the chemistry standard and the dye set that you are running the calibration for Note If the dye set list is empty ensure that your instrument is configured with a compatible polymer type and capillary length for the selected chemistry standard IMPORTANT To calibrate a custom dye set using AnyDye first create the dye set see Create a new dye set on page 168 then select the name of the custom dye set from the Dye Set list The A
358. s contact pressure and other workstation environmental factors To minimize musculoskeletal and repetitive motion risks Use equipment that comfortably supports you in neutral working positions and allows adequate accessibility to the keyboard monitor and mouse Position the keyboard mouse and monitor to promote relaxed body and head postures Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 321 Appendix F Safety Safety and electromagnetic compatibility EMC standards This section provides information on e U S and Canadian safety standards on page 322 Canadian EMC standard on page 323 European safety and EMC standards on page 323 Australian EMC Standards on page 328 U S and The 3500 or 3500xL analyzer has been tested to and complies with standard UL 61010 1 CSA C22 2 No 61010 1 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General C UL is Requirements UL 61010 2 010 Particular Requirements for Laboratory Equipment for the Heating of Materials The 3500 or 3500xL analyzer has been tested to and complies with the 21 CFR 1040 10 and 1040 11 except for deviations pursuant to Laser Notice No 50 dated June 24 2007 as applicable For the Reader Writer unit in the Applied Biosystems 3500 3500xL Genetic Analyzers FCC WARNING This device complies with Part 15 of FCC Rules Operation is subject to the fo
359. s fully automated from sample loading to primary data analysis for sequencing fragment analysis and HID human identification analysis Note In this document primary analysis for sequencing is referred to as basecalling Primary analysis for fragment or HID procedures is referred to as sizing Preparing samples When DNA samples are prepared for sequencing fragment analysis or HID analysis on the 3500 or 3500xL analyzer fluorescent dyes are attached to the DNA For most applications the sample is denatured so that only single strand DNA is present Preparing the instrument Two calibrations are required to prepare the instrument for sample runs e Spatial calibration Determines the position of the image from each capillary on the CCD array For more information refer to Spatial calibration on page 99 Spectral calibration Generates a matrix for each capillary that compensates for dye overlap and is used to convert the 20 color data into 4 5 or 6 dye data For more information refer to Spectral calibration on page 103 During a run During a run the system e Prepares the capillary by pumping fresh polymer solution under high pressure from the polymer delivery pump to the waste position in the Cathode Buffer Container CBC Electrokinetically injects the sample into the capillary using a low voltage for a few seconds e Washes the capillary tips in the rinse position of the CBC then returns the
360. s needed AAA AA M 4 Double click column headers to sort columns Multi column sorting is supported see Sort on page 97 5 Selecting rows in the sizing table then click Label Selected Peaks pad Examine the size In the Plot View toolbar deselect all dye colors except the size standard dye standard plot color red or orange 2 In the sizing table select the size standard peaks of interest 94 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Review Fragment HID Analysis results 3 Click Label Selected Peaks to label the size standard peaks in the Plot View Note If labels are not displayed click Plot Settings in the Plot View toolbar then select Show Labels in the Labels tab Click Save to Preferences to retain this setting 4 Ensure that all size standard peaks are present and correctly labeled Overlay the 1 Click 1 sizing curve Plot Settings in the Plot View toolbar 2 Select Overlay Sizing Curve in the Display tab Specify re injections Before the run completes select a sample with suspect or failing flags then click fi Re inject View print and save pdf sample quality reports 1 Select the samples of interest in the samples table 2 Click 5 Reports to see the available reports for traces and print S Views Reports E Punt TB the reports you want Samples vie EY Sizing Redit View Overlay Report View Plate Report 3
361. s wizard see Remove bubbles from the polymer pump on page 251 then repeat the run as needed 4 Ifthe data for all capillaries meet the criteria above click Accept Results 5 Ifthe data for the required number of capillaries do not meet the criteria above 7 capillaries for 8 capillary instruments 21 capillaries for 24 capillary instruments a Optional If you want to generate a report for the failed calibration click View Summary Report or t View Detail Report before you click Reject Results To save the report electronically select CutePDF as the printer Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Spectral calibration b Click Reject Results For troubleshooting information see Sequencing install standard troubleshooting on page 302 IMPORTANT If you reject results the spectral calibration 1s not saved Example sequencing install standard results Intensity ys Scan Number Calibrated Data v 1000 000 3000 4000 S000 6000 000 12000 apg 4000 a Intensity vs Scan Number Sequence Comparison to Sample Capillary 1 Base Position ee Reference caaTTCCCTGCAGGCGTGGCTGCAGCCTGGTTATGATTACTGTTAATGTTGCTACTACTGCTGACAATGCTGCTGCTGH Sample E Coulo Ta AGL CTiaTTATGATTACTITTAAT GTT GCTACTACT GCTGACAATGCT cT acT at Intensity vs Pixel Number Capillary 1 il a el El El rid 90 100 110 120 150 140 150 160 170 o 10 20 30 40 50 60 0 a
362. se the correct plate base for standard plates versus 8 tube strips and fast plates Using the wrong plate base may affect performance Plate retainer Plate with septa strip Plate base Snap the plate retainer cover onto the plate septa and plate base Verify that the holes of the plate retainer and the septa strip are aligned If not aligned re assemble and then assemble the plate assembly IMPORTANT The array tips will be damaged if the plate retainer and septa strip holes do not align correctly Place the plate in the autosampler with the labels facing you or the instrument door and the notched corner of the plate in the notched corner of the autosampler Close the instrument door to re initialize the instrument Perform a spectral calibration 106 AR 96 Woll Fast PIN 4404434 96 Woll Fast 5 A PIN 2404434 orios IMPORTANT Do not change electronic signature settings during a spectral calibration IMPORTANT If you change polymer type spectral calibrations for the original polymer type are not retained Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Spectral calibration 1 Access the Spectral Calibration screen Select Maintenance then select Library Maintenance Tools Manage Spectral Calibration in the navigation pane ie Calibrate Note The screen does not display results until you perform a spectral calibration To view previous calibration
363. shared 10lder ua ues vacio etx veut vesper a ugue Bo Reet dae et 294 Set up the 3500 Series Data Collection Software v1 0 lesen 297 Appendix E Troubleshoot llle 299 Instrument troubleshooting 0 0 ce ee mn 299 Spatial calibration troubleshooting 000 ce eee eee 300 Spectral calibration troubleshooting 0 0 00 ccc eee ees 301 Sequencing install standard troubleshooting eee eee 302 Fragment HID install standard troubleshooting 00 cece eee eens 303 Anode buffer container troubleshooting llle 303 Cathode buffer container troubleshooting llle 304 RFID TOUDIGSMOOUING visitado a tarde aote 304 Link a plate troubleshooting ee eee ees 304 How to search and use the log files llle 305 View instrument sensor details llle 305 Data electropherogram troubleshooting llle 306 Dashboard troubleshooting 0 0 cc ee eee nnn 309 Load plate troubleshooting serra ei AA ea OR 309 Monitor run troubleshooting lecce rrr 311 Review results troubleshooting llle ens 311 Review error message details 2 0 0 cee nnn 312 AUGIETOUDIGSHOOUNG 1 2 ata ed ia Eder ow Boos ee orbe ie ra do Ple a RR te 312 Electronic signature troubleshooting llli llle 312 Manual commands troubleshooting 0 00 c
364. size for the allele e THOI 0 5 bp of nominal size for the allele For all peaks that are within the nominal size range the software calculates the Average Peak Height and the Sizing Precision Peaks that meet the thresholds below pass Result Description Threshold Avg Peak Height Average of peak heights for observed allele peaks of the Fragment gt 175 RFU included capillaries e HID gt 400 RFU Sizing Precision Standard deviation of the observed allele fragment lt 0 15 for expected alleles sizes Pass Fail Alleles with a sizing precision and average peak height that do not meet thresholds fail Note Review the data for failed alleles as described below Result Description For information only Nominal Size Expected allele fragment peak size bp Mean Average fragment size for the observed allele peaks Peak Height gt Min Percentage of observed allele peaks with a peak height above the minimum threshold Sizing Accuracy Difference between the allele size and the mean allele size Evaluate fragment HID install standard data 1 Examine the number of size standard and allele peaks found for each capillary Note The number of expected peaks shown below is for the HID install standard Expected Allele Peaks 5ize Standard Peaks Observed Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 135 Section 2 Performance check 136
365. start computer Inskru MEDIUM zz Mar 2009 1 Restart co e E a Defragment Hard Drive MEDIUM 22 Mar 2009 1 Defragme e E wi Figure 4 Dashboard Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 27 Chapter 2 Start the System Check maintenance notifications The Maintenance Notification section displays reminders for the tasks scheduled in the maintenance calendar see Use the maintenance calendar on page 232 You can set the time to trigger maintenance notifications in Preferences see Set general preferences on page 33 1 Review the Maintenance Notifications pane r Maintenance Notifications Perform Performance Check HIGH Clean Drip Tray HIGH Clean Aubosampler HIGH Replace Reservoir Sepa HIGH wash Pump Trap HIGH 26 Jan 2009 12 00 00 AM 26 Jan 2009 12 00 00 AM 26 Jan 2009 12 00 00 AM 26 Jan 2009 12 00 00 AM 26 Jan 2009 12 00 00 AM Performance Check Clean Drip Tray Clean Aubosampler Replace Reservoir Septa Wash Pump Trap 2 Perform any scheduled maintenance tasks then click to mark it as complete or click to mark it as dismissed if you do not perform the task Actions are recorded in the Notifications log for more information see Review the Maintenance Notifications Log on page 257 3 Perform any daily monthly or quarterly maintenance tasks that are not listed in the Maintenance Notifications pane see Chapter 8 Maintain the Instrument 4 Inspect the in
366. strument interior See Start the instrument on page 22 a If you see any spills clean immediately b If you see any leaks and dried residue around the Buffer Pin Valve check valve and array locking lever If leaks persist contact Applied Biosystems 28 CV Check Valve Fitting Buffer Pin Valve Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Check system status in the Dashboard Check consumable status IMPORTANT The Days Remaining for buffers updates only when you click Refresh or start a run As part of daily startup click Refresh to update consumable status 1 Click Refresh to update consumable status The Consumables pane displays expiration dates and lot numbers read from the RFID tags on the consumable containers Consumables Information o Palvmer POP amp 34 Samples Remaining 26 Mar 2009 11 514007 4315930 node Buffer AB 356 Buffer 5 Days Remaining 26 Mar 201011 518007 4315931 Cathode Buffer AB 356 Buffer 5 Days Remaining 1 28 Mar 2010 11 518007 4315931 Capillary Array SOcm 24 cap 117 Injections Remaining S0 3l Dec 2009 EE 43519699 Serial GOK2450 i 2 Check the consumables gauges for the number of injections samples or days remaining for a consumable Table below lists specifications for each consumable When lt 10 of the specified use of the consumable remains the gauge moves into the red warning range The consumable also displays in red in the Consumables p
367. sults s 127 View previously run sequencing install standards 127 View and print a sequencing install standard report l l 127 Save historical performance check reports pdf for record keeping 128 Run the fragment analysis or HID Install standard performance check 129 Wenta PENON is neh den oc Eu ob ERE IUS EO OO Dor a o Dew e ac dea cn 129 Prepare for the fragment or HID install standard performance check 129 Run the fragment analysis or HID install standard performance check 132 Vial you see during d TUN d datos Or ia 134 How the software determines passing and failing capillaries for the fragment HID pertortridoe GDe6 i saos aci aon Ra Ea RO REGERE pop ee a mena ks 135 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Contents Evaluate fragment HID install standard data ooooooomomoo 135 Example fragment install standard results 0000 ees 137 Example HID install standard results 0 0 cee ees 137 View previously run install standards llle 137 View and print a fragment or HID install standard detail report 137 Save historical performance check reports pdf for record keeping 138 Chapter6 Manage Library Resources sL 139 Overview of libraries x xdes ge e ash der is e odor dee Ri wpe a BS RR o di t o Pa 139
368. sword S View Logs Manual Commands vo Manage Reports Audit Reports Esigrature Reports Customize the table see Customize tables on page 72 3 The records that are displayed if they are specified in E Signature settings are Approve Dye Set Approve Size Standard Approve Spatial Calibration Approve Spectral Calibration Approve Instrument Protocol Approve Sizecall Protocol Approve Basecall Protocol Approve Qc Protocol Approve Genemapper Protocol Approve Genemapper ID X Protocol Approve Seqscape Protocol Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Approve Microseq ID Protocol Approve Assay Approve Plate Template Approve Plate Approve Sample Approve Sequencing Install Standard Results Approve Microseq ID Install Standard Results Approve HID Install Standard Results Approve HID Install Standard Results 221 Section 1 Administrators View and print e signature reports 1 Display the records of interest as described above Note Filter the list to decrease the time required to generate reports 2 Click View E Sig Summary Report or View E Sig Detailed Report Report created on 04 May 2009 03 13 09 PM Ded ms 3500 Data Collection Software Version 1 0 0 E Signature Summary Report Date UserName UserFull Name Object Type Object Name Comments 1 04 May 2009 03 11 Administrator Administrator Approve Spatial X Spatial Run Spatial calibration is Calibration 2009 0
369. t feet to display only plates for the selected application Search In each library you can select a category to search then enter the text to search for The list of categories corresponds to the column headers in each library Click Go to search Click Clear to remove the search criteria 142 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Plates library Customize a library table Click the Table Settings button then us specify the columns to Aser Preferences show or hide EE TI 1 E PO E na Select your table display preferences below Click Available Columns to Display j Apply To use Column os the settings for Colum 8 this session only Col ra C Add Selected gt gt e Save to COE add all gt Preferences To save for future use by all users If your system includes the SAE module preferences are saved for the logged in user Restore Defaults To restore factory default settings Plates library The Plates library contains all plates that have been saved in the software plates that have been run and plates that have not yet been run Plate overview Plate definition A plate associates sample attributes sample information and analysis information with a well position A plate defines how samples are analyzed during capillary electrophoresis and how sample files are named and stored after analysis When you create a plate you specify Plate type sequenci
370. t archive function makes a copy of audit records Purge makes a copy of audit records and then deletes them You can use the Restore function to restore purged audit records For information on archiving library items datastore see Archive purge and restore data on page 254 Archive and To selectively archive or purge delete system configuration or action audit records purge 1 Select records in the appropriate screen 214 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Generate audit reports 2 Click Archive Audit Records or I Purge Audit Records 3 Ifyou select Archive specify a location and name for the asz archive file Restore To restore system configuration or action audit records click Restore then select the asz file to restore Export audit records As needed you can export audit records to a txt file for additional manipulation and reporting outside the 3500 Series Data Collection Software 1 Display the records of interest as described above 2 Click l Export Audit Records 3 Specify a name and location for the export txt file 4 Click Save Note If you export audit records for samples that are not in their original location samples have been deleted or moved an error message is displayed Return sample data files to their original location then export again Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 215 Section 1 Administrators Manage electro
371. t screen and enable or disable auditing 207 Select objects to audit 2 0 ce ee eee eee ees 208 Create audit reason settings 0 ccc eee eee 208 Generate audit reports ri AAA AA A Er TEE gu 209 Display audit histories s xs roza ario ci Hem oc EE dee di elm etr e oan c Rosle od RR 209 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Contents Review the object audit history ccc ees 210 Review the system configuration history 0 000 cece eee eee 211 Review tne action log zs hee area A AAA Aad ee 212 View and print audit reports 0 00 cc eas 213 Archive purge and restore audit recordS 0 00 cee 214 EXPO audit FeCOFGS i ded d HIE E ieee dora too ee Debe sees 215 Manage electronic signature llle 216 Access the E Signature Settings screen and enable or disable e sig 216 Select the actions that allow signature llle 217 Generate e signature reports 0 0 cc eer 221 Display e signature records o oooocococononn nmn 221 View and print e signature reports 0 0 ees 222 EXPO C SIG TECOS id a beens A td AA 223 Export and import user accounts security audit and electronic signature settings 224 SECUN 2 US Sims ais a a a e 225 Users overview of System Security Audit Trail and E Signature 225 DECU rd id edi a ak Gp Aa a idad Set dla 225 AU sure B dor debuit uci td daa taria a
372. t the computer page 24 Check maintenance notifications in the Dashboard page 28 Check consumable status in the Dashboard page 29 Replenish consumables page 31 Set up and run 1 Prepare the instrument page 42 2 Preheat the oven page 42 3 Check instrument status page 53 Create or import a plate page 43 Assign plate contents page 46 Print the plate layout page 50 Quick Start a run page 55 Prepare and load sample plates page 51 Load plates for run and create the injection list page 56 Review and modify the injection list in Preview Run page 59 Start the run page 61 Monitor the run page 61 check sequence or sample quality and specify re injections page 62 Review sequencing results Review fragment HID results 1 Review sequence quality page 81 1 Review sample quality page 89 2 Specify re injections page 85 2 Specify re injections page 95 3 Review quality reports page 85 3 Review quality reports page 95 4 Export sequencing results page 87 4 Export sizing results page 96 Optional print or save pdf calibration and performance check reports to save with results Spatial calibration page 99 Spectral calibration page 103 Sequencing install standard performance check page 122 Fragment or HID install standard performance check page 132 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 79 Chap
373. te for the performance check The software uses predetermined positions for the performance check run You cannot specify standard location on the plate If you do not place standards in the positions indicated the calibration will fail 8 capillary A1 through H1 TTETATSTISTA 96 well plate mEHEHHEEH 24 capillary A1 through H1 A2 through H2 and 96 well plate A3 through H3 SS HEHEHEHEHE 384 wellpate C E GLK 89 gm m gH m m HN Note 384 well M O plates are not NH NH NE NH NH NH N a supported on 8 capillary E NH NENNEN NEN HS instruments e 3 Bnefly centrifuge the plate containing the standards 4 Verify that each sample is positioned correctly Pa in the bottom of its poo C wel OEO LOLOS IMPORTANT Ifthe Sie 9 reagents of any well contain bubbles or are not located at the bottom of the well briefly centrifuge the plate remove the plate from the centrifuge and verify that each sample 1s positioned correctly in the bottom of its well sample is at the bottom of the well 130 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Prepare the plate assembly Load the plate in the instrument Run the fragment analysis or HID Install standard performance check 5 Store the plate on ice until you prepare the plate assembly and load the plate in the instrument IMPORTANT Prepare the plate assembly on a clean level surface Do not heat pla
374. te met This computer A applera net Public network Customuze Access Local and Internet Connection AB Instrument Motherboard NIC View status E Sharing and Discovery Network discovery a On ve File sharing a On iv Public folder sharing a On password required v Printer sharing a Off v j Password protected zhanng amp On ES can access shared files printers attached to this computer and the Public folder To give other people access you must tum off password protection E rd protected sharing V Media sharing a OH e 4 Click Apply Set up the 3500 Series Data Collection Software v1 0 Complete auto To complete your remote auto analysis setup you must create a new Results Group analysis setup on the Data Collection computer 1 Select the Results Group node in the navigation pane 2 Click New to open the Results Group editor 3 Complete the selections in the General tab by a Enter the new Results Group name b Enter the Results Group owner c Optional Enter the Results Group comment d Check Results Group Entry Completed 4 Complete the selections in the Analysis tab by a Select the GeneMapper instance GeneMapper computer name from the drop down list Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 297 Appendix D Remote Auto Analysis Setup b Check Do Autoanalysis Note If you plan to perform an auto analysis for every Results Group Complete instead of each r
375. ter 4 Review Results Review Sequencing Results Access the View Sequencing Results screen Access the View Sequencing Results screen from The Monitor Run screen by clicking Review ilii Results Review results for 80 the currently running plate View Sequencing Resuits Results f g View Fragment HID Results The navigation pane by selecting View Sequencing Results The Dashboard by clicking View Run Results y View Run Results If you access the View Sequencing Results screen while an instrument run is in progress the Trace Quality View lists results for completed injections in the current run Select one or more samples then click Open Trace to display their data in the Trace pane Note The basecaller version listed in the basecalling protocol 1s limited to a 3 digit number The version listed in sequencing results is a 4 digit number The fourth digit is an internal number used by the software Analysis Results v Select Filter All Traces M a Trace Quality Reports Select Table ab Trace Quality View B04 hCV11168429 StdSeq50 POP6 v3 1 BDTv3 1PA Protocol KB 1 4 1 6 KB 3500 POP6 BDT 13 8 2 B09 hCV1119233 9 StdSeq50 POP6 v3 1 BDTw3 1 PA Protocol KB 1 4 1 6 KB 3500 POP6 BDT 13 76 3 CO1BAC7 v3 1fwd7 A2 v3 1RapidSeq50 v3 1 Sequencing KB 1 4 1 4 KB 3500 POP7 BDT
376. ter trap volume is approximately 325 uL a relatively small volume of water 1s adequate for complete flushing However a larger volume only improves flushing as long as force and flow rate are kept within the limits given above Remove the syringe from the Luer fitting Hold the fitting with one hand while turning the syringe counterclockwise with the other hand Close the Luer fitting by lightly turning clockwise until the fitting seals against the block Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 241 Chapter 8 Maintain the Instrument Routine instrument cleaning 242 IMPORTANT Wear appropriate protection including gloves laboratory goggles and coat whenever you work with the fluids used on this instrument or parts that may come into contact with these fluids Ensure the oven and instrument doors are closed Press the Tray button on the front of the instrument to move the autosampler to the forward position IMPORTANT Use the cleaning agents as described in this manual only Use of cleaning agents not described in this manual can impair the instrument Please contact your local Life Technologies sales office 1f you have any questions Wipe off any liquid on or around the autosampler using a lint free tissue Clean off any polymer build up crystals on the instrument including the capillary tips with deionized water and lint free tissue Clean the array plug Clean out the drip tra
377. tes that are sealed with septa pi Align the holes in the septa strip with the wells of the plate then firmly press downward onto the plate Align the holes in the septa strip with the wells of the plate then firmly press downward onto the plate Plate retainer Place the sample plate into the plate base ciate Wit septa strip IMPORTANT Make sure to use the correct plate base for Plate base standard plates versus 8 tube strips and fast plates Using the wrong plate base may affect performance Snap the plate retainer cover onto the plate septa and plate base Verify that the holes of the plate retainer and the septa strip are aligned If not aligned re assemble and then assemble the plate assembly IMPORTANT The array tips will be damaged if the plate retainer and septa strip holes do not align correctly Place the plate in the autosampler with the labels facing you or the instrument door and the notched corner of the plate in the notched corner PIN 4404434 of the autosampler Close the instrument door to re initialize the instrument Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 131 Section 2 Performance check Run the fragment analysis or HID install standard performance check 132 1 Access the Fragment Install Standard or the HID install standard screen Select Maintenance then select Fragment Install Standard or HID Install Standard in the navigation p
378. than specified Conditioning reagent The conditioning reagent PN 4393718 for 3500 or 3500xL analyzer is available as a ready to use pouch It is used for priming the polymer pump washing the polymer pump between polymer type changes and during instrument shut down It has adequate volume for a one time use Note Use of the conditioning reagent is dictated by the instrument wizards Install the pouch when requested to do so by the wizard CAUTION Expired pouches cannot be used on the instrument Once installed on the instrument the pouch is good for a one time use only Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 11 Chapter 1 Instrument and Software Description For more details see the product insert included in the product package See Use the conditioning reagent on page 250 for instructions on how to use the conditioning reagent Hi Di Formamide Hi Di Formamide pack of four 5 ml tube PN 4440753 is a highly deionized formamide formulated with a stabilizer ready for use as an injection solvent for all applications on the 3500 or 3500xL analyzer For the following hazard s see the complete safety alert descriptions in Specific chemical alerts on page 333 N WARNING CHEMICAL HAZARD Hi Di Formamide For more details see the product insert included in the product package N CAUTION Expired Hi Di Formamide cannot be used on the instrument Applications The Hi Di For
379. the Data Collection services on the remote Data Collection computer Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 291 Appendix D Remote Auto Analysis Setup Start the 3500 1 On the Data Collection computer select Start All Programs Applied data collection Biosystems Data Collection Run Data Collection version 1 0 services Note If the services do not start automatically click Start All STOPPING POINT Wait until all services have changed to green before continuing 2 Ifthe 3500 Series Data Collection Software requires a password type the login name and password then click OK 3 Verify that Data Service started without errors a In the Service Console right click the graphic next to each service listed and select Show Console to display the Data Service output window b Verify that no errors are displayed then close the Data Service dialog box L Data Service output bee IMFO De fault getCurrent 3tatusTypes IBHFO Deraulr O B 49 13 411 For testing only ICFGerManua INFO Detault Dakar ins trumenrTryzpeNsme gaJ 100 ivant LIHFO Default getCurrentZStatusTygpez INFO Default OB 42 13 873 Spatial Eun Dara Manager bear IHMFO Isfault 0B 29 13 8B89 WEENING Ho valid spatial cal CINFO Default Spectral Bun Bata Manager bean has been tere INFO Default OB 45 18 HH7 BemoryHenitor Nin Free memor IMFO Default 06 49 33 910 Memory eonitor Min Fres memor IHFO IDe
380. the holes of the plate retainer and the septa strip are aligned If not aligned re assemble and then assemble the plate assembly IMPORTANT The array tips will be damaged if the plate retainer and septa strip holes do not align correctly Place the plate in the autosampler with the labels facing you or the instrument door and the notched corner of the plate in the notched corner Tus of the autosampler Close the instrument door to re initialize the instrument Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 121 Section 2 Performance check Run the sequencing install standard performance check 122 a Bb WP 1 Access the sequencing install standard screen Figure 9 on page 122 Select Maintenance then select Sequencing Install Standard in the navigation pane Library Maintenance Tools Manage Fj Performance Check 2 Select the chemistry type General Sequencing install Standard Sequencing or MicroSeq ID Fragment Install Standard 3 Select the plate type and plate position in Apta Standard the instrument Note You do not create a plate for the performance check The software uses predetermined positions for the run You cannot specify standard location on the plate E ph mx Lo Calibration Settings Scoring Settings o sFal The j Current Iretrument Consumables CEL Pas pies hok Polymer Type POP Capdiary Length 360m eamistry Type General Sequencing
381. the instrument door and the notched corner of the plate in the notched corner PIN 4404434 of the autosampler 2 Close the instrument door to re initialize the instrument Check instrument status Check instrument status in the Dashboard Temperatures are displayed in red as they warm to the set points When temperatures are at the set point they are displayed in green Temperatures may fluctuate slightly when they reach the set point as they stabilize Applied Biosystems recommends that you pre heat the oven for at least 30 minutes before you start a run if the instrument is cold Pre heating mitigates subtle first run migration rate effects If you start the run when red indicators are shown the run does not start until all indicators are green Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 53 Chapter 3 Set Up and Run Instrument 3500 Instrument State Idle Laser On Oven Off EP in Oven Door Open Instrument Door Close Pre Heat the Oven View Instrument Sensor Details Set Temperature Ea Oven Temperature Ch 53 5 Detection Cell Temperature C 23 5 C3 Start Pre Heat Link the plate 1 In the Assign Plates for Run screen click Link Plate for Run MM 3500 Data Collection Software Dashboard Edit Library Maintenance Plate Name sequencing Plate 1 ls e Setup Define Plate Properties E IE Assign Plate Contents Tools Manage E Show In Wells A
382. the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 bmbl od nih gov e Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html e Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines 1s available at www cdc gov Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 331 Appendix F Safety Safety alerts For the definitions of the alert words IMPORTANT CAUTION WARNING and DANGER see Safety alert words on page xiii Chemical alerts For the definitions of the alert words IMPORTAN
383. then press Enter or e Copy and paste a name from another well ES Table view To set the direction for the cursor when you press Enter Click Row to set the Enter key to move the cursor vertically to the next row e Click Column to set the Enter key to move the cursor horizontally to the next column To name multiple 1 Click a named well Samples 2 Click drag multiple wells 3 Right click and select Fill or Fill Series to populate the selected fields Note To use Fill Series type a number as the a 1 last character of the named well You can also copy and paste sample names Cut Ctrl D an Chrl4 E nr Ctrl I Fill Series F Fill Sample Mame Type i Delete G Rename Sample To name all wells at 1 Select all wells one time 2 Select assays file name conventions and results group for the plate 3 Enter name and select sample type in the Customize Sample Info pane for the whole plate a B D 70 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide More features in Assign Plate Contents Customize the Click Show In Wells plate view to specify the EE showin wels py El attributes to display in 7 Assay Name _ w Assay Color wells ow Assay Icon Click Select Wells to Results Group Name Results G Col select wells with a EME ieee cus File Convention Mame specific attribute File Convention Co
384. thode Buffer AB 356 Buffer 5 Days Remaining 1 28 Mar 2009 11 51B007 4315931 Capillary Array SOcm 24 cap 117 Injections Remaining 80 31 Dec 2009 11 80K005 4319899 Serial 80 i Maintenance Notifications Replace cathode buffer c HIGH 22 Mar 2009 1 Replace c X Clean Drip Tray HIGH 22 Mar 2009 1 Clean Drip Clean Autosampler HIGH 22 Mar 2009 1 Clean Aut K Restart computer Instru MEDIUM 22 Mar 2009 1 Restarbtco X 4 E Defragment Hard Drive MEDIUM 22 Mar 7009 1 Defragme Figure 3 3500 Series Data Collection Software Dashboard The Dashboard gives you quick access to the information and tasks you need to set up and run d e Main workflow arrow Advances to the screens where you set up load and run plates and view results e Menu bar Accesses all other parts of the software The menu bar is displayed on all screens Common operations Allows you to quick start load a plate that 1s set up create or edit plates view results and access the Maintenance workflow Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 15 Chapter 1 Instrument and Software Description Quick view Displays gauges that show the remaining usage of consumables and gives the status of instrument conditions Consumable usage is automatically tracked by the instrument by radio frequencing identification RFID tags Consumables information Gives details for the installed consu
385. tic Analyzer User Guide Safety Instrumentation safety Symbols on instruments Electrical The following table describes the electrical symbols that may be displayed on symbols on Applied Biosystems instruments instruments Symbol Description Light switch Indicates the On position of the circuit breaker Indicates the Off position of the circuit breaker Indicates a standby switch by which the instrument is switched on to the Standby condition Hazardous voltage may be present if this switch is on standby Indicates the On Off position of a push push main power switch Indicates a terminal that may be connected to the signal ground reference of another instrument This is not a protected ground terminal Indicates a protective grounding terminal that must be connected to earth ground before any other electrical connections are made to the instrument l D OGO Ww Indicates a terminal that can receive or supply alternating current or voltage Safety symbols The following table describes the safety symbols that may be displayed on Applied Biosystems instruments Each symbol may appear by itself or with text that explains the relevant hazard see Safety labels on instruments on page 317 These safety symbols may also appear next to DANGERS WARNINGS and CAUTIONS that occur in the text of this and other product support documents Applied Biosystems 3500 3500xL Genetic Analy
386. time e Instrument Name e User defined Fields up to 5 specified in Assign Plate Contents see page 48 e User Name available only when security is Owner Name plate owner enabled in the SAE module e Plate Name e Well Position e nstrument Protocol IMPORTANT The maximum allowed length of a file name including the path is 240 characters The software warns you if your selections will possibly exceed the maximum but allows you to save the file name convention However you will see a pre check validation error when you start a run if the file name will exceed 240 characters Delimiters Symbols you can include in the file name Dash Dot Underscore Plus Dollar Custom text Text to display for the custom text attribute fields File location The file location in the file name convention is used only if no results group is specified for a well f The Results Group file location overrides the File Name Convention file location 154 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Result group library Result group library Results group overview A Results Group is used to name sort and customize the folders in which sample data files are stored It is an optional component in a plate Note The file location specified in a results group overrides the file location in the file name convention specified for a well When you set up a plate for a run you can optionall
387. tion on page 99 In the Dashboard check consumable status page 29 Ensure that Consumables are not expired e Adequate injections remain for consumables Ensure that the fluid levels are at the fill lines Check buffer fill levels on page 31 Set the oven temperature to 60 C then click Start Pre heat Pre heat the oven and detection cell while you prepare for a run detection cell temperature is set by the software Preheating helps mitigate subtle first run migration rate effects The pre heat function automatically turns off after 2 hours Applied Biosystems recommends that you pre heat the oven for at least 30 minutes before you start a run if the instrument is cold Check the pump assembly for bubbles and run the Remove Bubble wizard if needed see page 251 RN Prepare the IMPORTANT Do not use warped or damaged plates installation standard plate Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 129 Section 2 Performance check 1 Prepare the installation standard as described in the product insert See Table 29 on page 260 for standard part numbers Application Installation Standard Fragment analysis GeneScan Installation Standard DS 33 G5 dye set POP 7 polymer 50 cm capillary HID AmpF STRS Identifiler Allelic Ladder G5 dye set POP 4 polymer 36 cm capillary 2 Load the standards in injection position in the plate IMPORTANT You do not create a pla
388. tion Software then export 1t to create a file that contains the correct header and column information for importing 1 In the Dashboard click Create Plate from Template 2 Inthe Open Plate Template from Library dialog box Create Plate From Template a Selecta filter to display the plate template type of interest b Select a plate template then click Open 3 Enter a name for the plate then specify the capillary length and polymer type for the plate 4 Click Assign Plate Contents Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 73 Chapter 3 Set Up and Run Note Before you click Export you can assign other plate elements to the plate import template as described in Assign plate contents on page 46 6 Select a file type for the plate import teni aes evel a a 7 Enter a name and location for the plate record File name S 50 em POP F import template sl template Save as type FUN 8 Click Save The figure below shows the format of the exported plate Plate Mame Application Type Capillary Length cm Polymer Number of Wells Owner Mame Barcode Number Comments plate import template Sequencing 50 POP 36 B Vell Sample Mame Assay Results Group File Mame Convention Sample Type User Defined Field 1 User Defined Field 2 User Deti 201 H co Create a plate Open a plate import template see Create a plate import template on page 73 import file p save t
389. to adjust the peak height of the sample peaks relative to the GS600 LIZ v2 0 size standard peaks IMPORTANT Normalization is not applied to samples with failing sizing quality Select a size standard definition file appropriate for your application that accurately sizes samples For example if your application includes small fragments that may be obscured by primer peaks or large fragments that may not be present due to slower migration rates specify a size standard definition file that eliminates these fragments from sizing Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 171 Chapter 6 Manage Library Resources Not Normalized Normalized 2000 2000 Instrument A 1500 1500 Instrument A Instrument B 1000 1000 Instrument B 500 500 U U For more information see Review normalized data on page 90 Create a new size standard If factory provided size standards do not suit your needs you can create new size standards 1 Access the Size Standards library R 3500 Data Collection Software Librarw Maintena Create Filter ae Manage Plates 2 Click Ey Create 3 Dashboard Edit 3 In the Create New Size Standard dialog box Figure 22 on page 169 enter a size standard name Library Resources 4 Optional e Select the Locked che
390. to determine a contiguous read length based on quality values the software starts from the 5 end and calculates the average QV across a moving window size of 20 sliding 1 bp at a time to the 3 end The resulting longest contiguous segment is determined as the CRL CRL Pass Fail e General sequencing Capillaries with a CRL lt 500 bp fail e MicroSeq ID Capillaries with a CRL lt 600 bp fail For information only Based on alignment of the base called sample sequence with the known reference of the sequencing install standard CRL Basepair CHRL accuracy is determined by base pair comparison between the base called sample and Accuracy the known reference sequence for the install standard within the contiguous read length region calculated as described in the CRL definition above Read Length The length of read in bases at which base calling accuracy is 298 596 The read length value for this information is derived from basecall accuracy not from quality value Basepair Accuracy Basepair Accuracy is determined by base pair comparison between the sample and the i Read Length known reference sequence for the install standard in the read length range see the Scoring Accuracy settings at the top of the screen for read length range with 298 596 accuracy in the called sequence when compared to the reference sequence CRL Median and SD Median and standard deviation for all capillaries Applied Biosystems 3500 3500xL G
391. to display for the prefix or suffix text attribute fields Select re injection folder option e Store reinjection sample files in a separate reinjection folder same level as injection folders e Store reinjection sample files with original sample files same level Select folder option Location e Default file location specified in Preferences User Run Setup e Custom location Sub folder options e Include an instrument run name folder run name can be user defined in the Load Plates for Run screen e Include a results group name folder e Include an injection folder Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 159 Chapter 6 Manage Library Resources Results group example 1 store files by plate name Two default factory provided results groups are provided that store sample data files by plate name Figure 15 on page 160 shows the factory provided PN_Injfolder_RG results group and the folders created when it is used This results group creates a folder for each injection Figure 16 on page 161 shows the factory provided PN_RG results group and the folders created when it is used This results group does not create a folder for each injection All samples for a plate are stored in the same folder If you include two plates in a run a separate folder 1s created for each plate Ik Edit Results Group PH Injflder RG O a Hr bring 5 CiApplied Biosysterms3500 DataPlate01 P
392. trifuge samples to remove bubbles The capillary tips may not be touching the samples Check the volume of your samples If no results call your Applied Biosystems representative The capillary tips may be hitting the bottom of the wells Autosampler not correctly aligned Call your Applied Biosystems representative If the spectral calibration fails or if a message displays No spectral files found Blocked capillary Refill the capillary array You may have to install a fresh array or consider that capillary non usable for purposes of planning your runs Incorrect chemistry file dye set and or run module selected Correct the files and rerun the calibration Insufficient filling of array Check for broken capillaries and refill the capillary array Expired matrix standards or old reagents Check the expiration date and storage conditions of the matrix standards and or reagents If necessary replace with a fresh lot Data Error One or more peaks fall below the minimum required amplitude of 750 One or more peaks fall below the minimum required amplitude of 750 Rerun the spectral standards and if necessary increase the amount of spectral standard added Spikes in the data or Bad dye order detected error message Expired polymer Replace the polymer with a fresh lot using the Replenish Polymer Wizard Bubbles in the polymer system Select the
393. trument and Software Description Materials for routine operation Contact your local Applied Biosystems service representative or go to www appliedbiosystems com then click Products to order the materials for 3500 or 3500xL analyzer External barcode scanner An external barcode scanner can be used with the 3500 or 3500xL analyzer to scan the plate template Applied Biosystems recommends the Symbol LS 1203 handheld barcode scanner shown which connects to the instrument computer r The scanner allows you to scan barcodes into any text box in the 3500 Series Data Collection Software For details on installation use decoding capabilities and product specifications see the product documentation Uninterruptible Power Supply UPS Loss of power during a run can result in lost data To address concerns with loss of power in the laboratories Applied Biosystems recommends the use of an Instrumentation Power Protection System IPPS with the 3500 or 3500xL analyzer If your laboratory has a backup power generator a battery powered backup Power Protection System PPS is required to provide power for the amount of time that it takes for your laboratory s backup power to begin generating power and stabilize Note The instrument computer and monitor must all be connected to the PPS If your laboratory does not have a backup power generator a PPS can provide protection from power disruptions of a limited duration
394. trument name 33 maintenance notification time 33 overview 32 plate type 34 plot settings 35 reports 34 reports sequencing 38 sequencing export 36 sequencing trace 36 system 32 table settings 35 user 33 pre heat duration 42 POP 4 POP 6 POP 7 polymer 42 pressurized fluids safety 318 preview run duplicate injection 60 injection list 59 start instrument run 61 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Index primer peak eliminating 181 print action logs 209 audit reports 213 e sig report 222 fragment HID install performance check 137 plate layout 50 sample quality reports 95 sequencing install performance check 128 spatial calibration report 102 spectral calibration 116 trace quality reports 85 user report 205 processed plates 76 Pull Up peak flag 89 settings 183 pump flush water trap 241 remove bubbles 251 wash chamber and channels 249 PUP Score 81 pure base color 37 QV range 37 pure base settings 84 purge audit records 214 library items 254 Q QC protocols create 184 defined 184 export 141 import 141 in assay 150 QC report sequencing generate 85 quality ranges 38 quality flags fragment HID 62 89 sequencing 62 81 quality settings fragment analysis 183 HID analysis 188 sequencing analysis 177 quality value See also QV probability of error 84 spectral calibration 109 quarterly instrument maintenance 231 345 Index Quick Start 55 QV average quality value See also av
395. trument performance run maintenance procedures and access records about instrument maintenance and service You can click Main Workflow or select Dashboard or any other menu item at any time to advance from the Maintenance workflow The Maintenance workflow is described in Chapter 8 Maintain the Instrument on page 229 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Calibrate Spectral Fi Performance Check Sequencing Install Standard Fragment Install Standard HID Install Standard Maintenance Wizards Planned Maintenance Notifications Log Service Log Schedule 17 Chapter 1 Instrument and Software Description 18 Tools menu Manage menu Preferences menu Help menu Select Tools in the menu bar to access 3500 Series Data Collection P Maintenance e or MM LL MN Software tools Securit Audit Tools provided are E Signature e Security Audit and View Logs E signature if your system includes the SAE module Change Password that allows you to change passwords Manual Commands View Logs that provides reports of instrument runs e Manual Commands that you can use to troubleshoot instrument performance The SAE module is described in Chapter 7 Use Security Audit and E Sig Functions SAE Module on page 197 Select Manage in the menu bar to ary Maintenance Tools Manage ferences Helg access archive restore and purge Archive functions
396. uct When safety interlocks are disabled during certain servicing procedures the laser can cause permanent eye damage and therefore is classified under those conditions as a Class 3B laser The instrument has been tested to and complies with 21 CFR 1040 10 and 1040 11 except for deviations pursuant to Laser Notice No 50 dated June 24 2007 as applicable The instrument has been tested and complies with standard EN60825 1 2001 Radiation Safety of Laser Products Equipment Classification Requirements and User s Guide CAUTION Use of controls or adjustments or performance of procedures other than those specified herein may result in hazardous radiation exposure Laser Parameter Wave length 505nm Output power 20mW LED Parameter Emitting color Natural White Luminous Intensity 250 Cd Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Documentation Related documentation The following related documents are shipped with the system Document Fart Description number Applied Biosystems 3500 3500xL 4401662 E Provides a flowchart on how to run your samples and Genetic Analyzers Quick Reference instrument Card e Provides a table of maintenance tasks and e Contains Data Collection Software reference guide Applied Biosystems 3500 Series 4401663 Provides information about the space environmental and Genetic Analyzer Site Preparation Guide 4401689 Note The purpose of the Site Prep Guide is t
397. uffer abort injection 69 account setup 199 suspended activate 203 suspension 199 226 user 201 Acronyms xvi action log contents 212 display 209 Administrator user role 203 allelic ladder internal validation 52 re injection 67 requirements for accurate genotyping 52 68 results group for one allelic ladder per run folder 52 run requirements 155 sample name for re injection 49 sample type 48 amber indicator light 23 amplicon specifying 49 Analysis Range fragment analysis 181 HID analysis 186 anode buffer change 237 described 9 on instrument limits 30 part number 9 storage conditions 9 troubleshoot 303 AnyDye calibrate spectral 108 create dye set 169 Applied Biosystems customer feedback on documentation 336 337 Index Information Development department 336 archive audit records 214 data files 255 library items 254 array See also capillary array view 77 array selection assign plate contents 71 as needed instrument maintenance 232 assays assign to plate 49 73 create 147 defined 46 147 instrument protocol 150 normalize data 49 assign plate contents amplicon and specimen 49 array selection capillary to plate map 71 assay 49 73 file name convention 49 73 name samples 48 70 overview 46 parts of screen 46 plate view 48 70 plate view customize 71 results group 49 73 sample type 48 table view 71 template for 75 Assume Linearity 183 audit records factory provided items creation date 142 view in lib
398. ults distribution for all selected samples Signal Strength One page graph that shows with average sequencing dye signal strength for all selected samples Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Review Sequencing Results pplied Repat Created On 19 Mar 2009 03 49 27 PM ENS Fst Bosystenis 3500 Data Calisction Software Version 1 0 0 A T AP pplied Fepat m Quality Control Report Blosystemis Contiguous Read Length Trace Score Legend Number af traces IN 43 Numberof Tr TM MEME C 0 5 0 15 20 25 3 35 4 45 5 O trace have medium CRL 4 4 4 n 4 4 1 48 traces have bng CRL Mean 685 Median 696 B wea 100300 Shat 100 BU hCV11168420 1Q Mg f B04 m NM 515 450 267 470 27 t BO hCV1118233 9A MB i BUY 6 563 589 324 539 392 ES pplied AN Applied enira Conus arson mace ma Biosystems Trace Score Report Signal Strength Report Trace Scare is the avemige basecal quality value af bases in the pat Average Raw Signal Stengt is the average retative Suarescent unit riu far each dye Trace score N 48 Range 50 55 Median 54 Mean 53 Signal N 48 Range 104 589 Medim 232 Mean 271 StandardDev 155 55 600 50 550 45 500 y 450 n E 400 350 3 5 300 is g 20 20 amp 200 15 2 150 10 100 5 50 0 0 ESSE PS ODOAOOHODOAY VDD MOO NODA OOAMAOO OAD A ESANA ESOS SAO NO AS NUS S greene a PRR ROOD pg POORER O OOUE OED ye E ue ES oe SF IES die e ofS e Ex
399. un individually check Results Group Entry Completed c Enter the GeneMapper Login ID and password 5 Complete the selections in the Destination tab by a Check Use Custom Location b Enter the Destination using the format Remote analysis computer name Shared folder name for example myPC Remote_Autoanalysis c For remote auto analysis specifically Establish a connection with the remote analysis computer by e Select Start Run Enter the destination path then click OK e Click Test to test the Location path name connection STOPPING POINT If the test Passes the message displays Path Name test successful If the test Fails the message displays Could not make the connection Please check that the Path Name 1s correct In this case click Browse then select the correct location 298 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Troubleshoot If you encounter any unforeseen and potentially hazardous event while operating the instrument turn off the power switch unplug the instrument and call your Applied Biosystems representative IMPORTANT See the Safety appendix for instrumentation and chemical safety information and guidelines Instrument troubleshooting Symptom Amber light blinking Possible cause Action Run paused Resume run Door open Close the instrument door Run failure that doesn t require restart of instrument Conduct another run Instrume
400. ure optimal performance the use of the ABC is limited to either 7 days after the first installation or 120 injections on a 3500 8 capillary 50 injections on a 3500xL 24 capillary whichever comes first When notified of the limit by the instrument software you have to replace the ABC with a new one before you can proceed further For more details see the product insert included in the product package See Change the anode buffer container ABC on page 237 for instructions on how to change the ABC Cathode buffer container CBC The CBC PN 4408256 contains 1X running buffer to support all electrophoresis applications on the 3500 or 3500xL analyzer The CBC is made in a ready to use disposable container with a radio frequency identification RFID tag incorporated into the label It has two separate sides The side containing 24 holes provides the cathode buffer for electrophoresis The side that contains 48 smaller holes provides the liquid for wash and waste functionality for rinsing the capillary tips and collecting wash waste between injections Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 9 Chapter 1 Instrument and Software Description Polymers 10 For the following hazard s see the complete safety alert descriptions in Specific chemical alerts on page 333 N WARNING CHEMICAL HAZARD Cathode Buffer Container CBC Store the CBC at 2 C to 8 C until ready to use The sealed C
401. utosampler to forward position Wait until the autosampler stops at the forward position Note When the door is open the yellow status light blinks while the instrument performs self check and the autosampler adjusts 22 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Start the instrument b Check the instrument status Ensure the green status light 1s on and not flashing before proceeding The table below explains the status indicator lights for the instrument Indicator Status All lights off Instrument off Green light Operational awaiting run Pause run terminate run stop injection button in SW pressed by user Note You can only abort an injection when the green light is flashing not when it is solid green Green light blinking Operational Run in progress Amber light blinking Power up self test in progress Run paused Door open Run failure that doesn t require restart of instrument Amber light Standby Red light Self test failed Instrument failure Requires a restart of the instrument and computer Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 23 Chapter 2 Start the System Start the computer 1 Power on the computer 2 Power on the monitor 3 In the Log On to Windows dialog box a Enter the user name b If applicable enter a password Note If the computer is connected to a n
402. wizard 3 Click Refresh from the Dashboard to Shh update the Screen t Y Expiration Date 30 Aug 2009 Lot Number 514007 Part Number 4315930 E 2 IMPORTANT NOTE 7 3 Thi be changed with this wizard thi 4 Check the Quick View section of the 7 NEG bles a ba tag td I a other than POP7 Dashboard for updated status after E da ai Pouch of POP polymer replenishing the polymer e ah crea tn di Cancel 246 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Use the Maintenance Wizards to perform operations Change polymer type IMPORTANT Do not use a polymer pouch that has been installed on one type of instrument on another type of instrument For example if you install a new polymer pouch originally on a 3500 8 capillary instrument do not subsequently use that same polymer pouch on a 3500xL 24 capillary instrument or vice versa Doing so may result in a lower number of samples injections than specified For the following hazard s see the complete safety alert descriptions in Specific chemical alerts on page 333 WARNING CHEMICAL HAZARD POP 4 POP 6 and POP 7 polymers IMPORTANT If you remove a polymer pouch for storage place a Pouch Cap PN 4412619 onto the pouch then place an empty pouch or conditioning reagent on the connector to prevent desiccation of any residual polymer on the connector For details see Instrument reagents and consumab
403. x click Trace Print under User Sequencing settings to display the Trace Print pane k Preferences type Filter text not used Trace Print Ee System These settings affect the printed Trace Reports User l Select data to be printed For each trace Plate Setup Reports Settings Analyzed data Entire sequence C Post trim sequence only Run Setup E Sequencing Settings L Annotation Trace Sequence Trace Print Trace Quality Select Settings Trace Quality Reports Panels per page Points per panel 1000 Set Y Scale For analyzed data 2 Individual CO Uniform with a maximum sc 4000 Print QV information 2 Specify the type of trace data specific print settings and Y Scale preference to display in the Trace Report 3 Click Apply to save the user preferences see User preferences on page 34 Trace Quality Trace Quality preferences control the quality ranges for user preference QC report Trace Score and CRL e Plate report Trace Score 1 In the Preferences dialog box click Trace Quality under User Sequencing settings to display the Trace Quality pane 38 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Set preferences AS Preferences E type Filter text not used Trace Quality Ga E 2e These settings define thresholds For the Trace Quality Reports Trace scores and contiguous read length settings can also control isl User the colors used within the Trace Quality Reports To
404. y on both sides 3 Release Electrophoresis current is unstable Bubbles in the polymer system Select the Bubble Remove Wizard to clear bubbles Electrophoresis failure Buffer below fill line Ensure that the buffer has not split into the overflow If so move the buffer back to main reservoir 308 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Dashboard troubleshooting Dashboard troubleshooting Symptom Possible Cause Action The Days Remaining value for buffer polymer does not automatically update The Days Remaining for buffers updates only when you click Refresh or Start A Run As part of daily startup click Refresh to update buffer status Load plate troubleshooting Symptom Possible Cause Action Pre run validation check does not display a date for a consumable The software does not display a date if it is identical to the preceding date In the example below the installation and recommended replacement dates for cathode buffer are identical to the dates for anode buffer No action Instrument Run P Pre run validatian check failed Reason Consumable 4node Buffer has exceeded recommended time on instrument Installation date lan 1 2009 Recommended replacement Jan 8 2009 Consumable Cathode Buffer has exceeded recommended time on instrument Link Unlink Plate error message R
405. y Analysis Y Perform Auto Analysis View Sequencing Results View Fragment HID Results No Username eds Password 3 WG LS 44 0 NS Confirm SeqScape auto populates as the Software Type Note If SeqScape does not appear in the drop down list under Software Type check your installation Secondary analysis software must be installed correctly before the 3500 Series Data Collection Software is automatically listed as a selection Confirm Your computer name auto populates as the Software Location IMPORTANT For auto analysis to be successful the secondary analysis protocol must match the software location set here Enter your Username and Password for auto analysis access to the secondary analysis software Click F Save Plate p Save to save your plate with these settings then Assign Plate Contents to advance to the next screen Assign plate When assigning plate contents you are assigning assays file name conventions and contents results group to be associated with your auto analysis Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 271 Appendix B Secondary Analysis Sequencing Set up an assay 1 In the Assign Plate Contents screen go to the Assays box and select either Create New Assay or Add From Library Assays Actions Y Add fram Library Create New Assay 2 Name your assay in the Setup an Assay dialog box Note Optional
406. y add results groups to wells in the plate If you add this item from the library a copy of the item 1s added to the plate and can be modified independently from the original item stored in the library For information on how changes are tracked if auditing 1s enabled see Audit action on page 210 Allelic ladder To accurately genotype samples the GeneMapper D X Software requires at least location HID one allelic ladder sample per run folder Multiple allelic ladder samples in a single analysis run folder can also be used for analysis Applied Biosystems recommends that you run one allelic ladder for 24 a set of samples e 8 capillary instruments One allelic ladder per 3 injections e 24 capillary instruments One allelic ladder per 1 injection Note Run HID validation studies to determine the required number of allelic ladders for your application See Results group example 2 store one allelic ladder per run folder 8 capillary instruments on page 161 for a results group example that places three injections in each run folder for 8 capillary instruments Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 155 Chapter 6 Manage Library Resources Create a new results group If the factory provided results groups do not suit your needs you can create new results groups 1 Access the Results Groups library 2 Click Create Note You can also create a results group from the Assign Plate
407. ys with deionized water or ethanol absolute and lint free tissue Note The drip tray can be removed Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Instrument operational procedures Move and level the instrument CAUTION PHYSICAL INJURY HAZARD Do not attempt to lift the instrument or any other heavy objects unless you have received related training Incorrect lifting can cause painful and sometimes permanent back injury Use proper lifting techniques when lifting or moving the instrument Two or three people are required to lift the instrument depending upon instrument weight 1 Remove the following components from the instrument Any plate assemblies from the autosampler e CBC from the autosampler e Capillary array click Shutdown the Instrument in the Maintenance Wizards See To shutdown the instrument on page 253 e Anode buffer reservoir 2 Switch off the circuit breaker on the back of the instrument 3 Disconnect the power cord and the Ethernet cable IMPORTANT While moving the instrument avoid any shock or vibration 4 Move the instrument 5 Turn the instrument legs to level the instrument To move the instrument corner Turn the leg up right clockwise down left counterclockwise Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 243 Chapter 8 Maintain the Instrument Use the Maintenance Wizards to perform operations About Maintenance Wiza
408. ystems 3500 3500xL Genetic Analyzer User Guide ix Contents Appendix A Appendix B Appendix C Use the Maintenance Wizards to perform operations 0000 eee ee eee 244 About Maintenance Wizards 0 00 eee 244 Replenish DONNE va were dias uox eR CREE o gU Pe AP ee eae 245 Change polymer type sirios EE A dees Lee esas Ss 247 Partially used polymer ri a EE RARE oie Side ete eh ae e eds 249 Wash the pump chamber and channels llle 249 Use the conditioning reagent 0 0 cc ee es 250 Fill capillary array with fresh polymer leen 251 Remove bubbles from the polymer pump 0 00 cece ee es 251 To change the capillary array 0 0 0 ce te ees 252 To shutdown the instrument 2 0000 cee ee ees 253 Computer maintenance clle leer hrs 254 Uninstall the software llle 254 Archive purge and restore data 2 0c cee ees 254 WONMOF GISKSOACE Bako i tint Popes hose eerie es Coe eee eee ee ee hi 256 Review the Maintenance Notifications Log 2 00 0 cee eee 257 SENICE EOG oara ranae e NN 258 Application Reagents and Run Modules 259 Sequencing analysis reagents 0 0 cc ee ee eee 259 Fragment and HID analysis reagents 000 ce ee ees 260 Sequencing analysis dye sets for all applications o ooooooomooo 261 Fragment analysis dye sets for all applications
409. zer User Guide 119 Section 2 Performance check 4 Check the pump assembly for bubbles and run the Remove Bubble wizard if needed see page 251 i AISA ESSER AGEL GR AA Prepare the IMPORTANT Do not use warped or damaged plates installation standard plate 1 Prepare the sequencing install standard as described in the product insert See Table 28 on page 259 for standard part numbers Application Standard General sequencing BigDye Terminator BDT v3 1 Standard POP 7 polymer 50 cm capillary MicroSeq ID applications BigDye Terminator BDT v1 1 Standard POP 6 polymer 50 cm capillary 2 Load the standards in injection position 1 in the spectral calibration plate IMPORTANT You do not create a plate for the performance check The software uses predetermined positions for the performance check run You cannot specify standard location on the plate If you do not place standards in the positions indicated the calibration will fail 8 capillary A1 through H1 96 well plate mEHEHEEE 24 capillary A1 through H1 A2 through H2 and 96 well plate A3 through H3 24 capillary Columns 1 3 and 5 in rows 384 well plate A C E G l K Note 384 well M O plates are not NH NH NN NE NH N gt supported on 8 capillary E HN NE NH NEN N instruments m e 96 Supports 96 well standard reaction plate 8 strip standard tubes are also supported with appropriate retaine
410. zer User Guide 315 Appendix F Safety Description Indicates that you should consult the manual for further information and to proceed with appropriate caution Indicates the presence of an electrical shock hazard and to proceed with appropriate caution Indicates the presence of a hot surface or other high temperature hazard and to proceed with appropriate caution Indicates the presence of a laser inside the instrument and to proceed with appropriate caution Indicates the presence of a biological hazard and to proceed with appropriate caution Indicates the presence of sharp object and piercing injury and to proceed with appropriate caution PEEP PD i Environmental The following symbol applies to all Applied Biosystems electrical and electronic symbols on products placed on the European market after August 13 2005 instruments Symbol Description Ne Do not dispose of this product as unsorted municipal waste Follow local municipal waste ordinances for proper disposal provisions to reduce the environmental impact of waste electrical and electronic equipment WEEE European Union customers Call your local Applied Biosystems Customer Service office for equipment pick up and recycling See www appliedbiosystems com for a list of customer service offices in the European Union i A eee i 316 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Instrumentation

Applied Biosystems 3500/3500xL Genetic Analyzer User Guide (PN

Contents

Download Pdf Manuals

Related Search

Related Contents