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MegaBACE - GE Healthcare Life Sciences

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1. Figure 6 1 A section of the Chemistry ini file opened in Notepad Examples of chemistry parameter sets for the sequencing and genotyping applications appear 3 Copy and paste the field names and values from a chemistry parameter set that is similar to the one you are adding Change the values to indicate the e Application R equired field if you are defining a parameter set for an application other than sequencing If you do not specify the application the Instrument Control M anager assumes that the chemistry parameter set is for the sequencing application Only the names of the chemistry parameter sets for the selected application appear in the Plate Setup window figure 6 2 MegaBACE Instrument Administrator s Guide p6 3 Parttwo Understanding and configuring the MegaBACE system SSSR e Beamsplitters The beamsplitter letter indicates the spectral channel BEAM SPLITTER A indicates the beamsplitter used in spectral channels 1 and 2 BEAM SPLITTER B indicates the beamsplitter used in spectral channels 3 and 4 Filters The filter number indicates the spectral channel number For example FILTER 1 indicates the filter used to detect the emissions in spectral channel 1 Bases or dye names R equired field Base names are required for the sequencing applications and dye names are required for the genotyping applications The base number or dye number indicates the channel number used to detect the bas
2. 0 0 00 eee 2 9 4 Light JCAKS 5 coven veri aaa aie eee al teh a ae anes 210 RMTS aa o ioa a detunpetagicie oaae e i dah ache 2 11 Power supply fan module computer and monitor 2 12 System electrical connections saana aaa 2 13 Serial number labels oaa 2 13 1 Instrument serial number label aana 2 13 2 Power supply fan module serial number label 2 14 Service for the MegaBACE instrument 0000005 Part two Understanding and configuring the MegaBACE system Chapter 3 How the MegaBACE instrument works 3 1 Overview of capillary array electrophoresis 0 0 000 3 2 Aboutthe capillaries 0 cee eee 3 2 1 Cathode end of the capillaries 96 capillary instruments 3 2 2 Cathode end of the capillaries MegaBACE 500 and flexible MegaBACE 1000 instruments only 3 2 3 Anode end of the capillaries 0000005 3 2 4 Replaceable sieving matrix 0 00 cece eee vi MegaBACE Instrument Administrator s Guide Table of contents 3 3 Confocal optical system 1 0 te 3 5 3 3 1 Basic confocal optical system 00 0 eee ee eee 3 5 3 3 2 How the system views the capillary array 3 6 3 3 3 How the system views a point in the array 3 6 3 3 4 Advantages of confocal laser scanning 3 8 Chapter 4 Using the Instrument Control Manager windows to configure the system 4 1 Manually set
3. 0 cece eee 1 5 Overview of the Instrument Control Manager features 1 5 1 3 1 About manual or automatic plate setup 1 5 1 3 2 About changing the application 0005 1 6 1 3 3 About data collection with automatic base calling Sequencing ONly cee eee eens 1 6 The flexible MegaBACE instrument 0 0 0 eee eee 1 6 Administrator tasks 22 toia aao a Redad bake Sak ee 1 7 1 5 1 The MegaBACE system initialization files 1 7 1 5 2 The parameter configuration files 0 0005 1 8 MegaBACE Instrument Administrator s Guide v Table of contents Chapter 2 Safety precautions 2 1 General safety precautions 0 ccc eee 2 2 Locations of important labels 0 00 c eee 2 3 Cathode and anode compartments and instrument displays 2 4 Electrophoresis compartment 00 00 cece eee 2 5 Filtercompartment 0 00 c eects 2 6 Internal electronics 0 0 0 eee 27 VONOMIGAIS iid iah cceon ech ica peltemen maids Heeuideulh agd oa ai 2 8 Nitrogen cylinders and pressure regulators 00 e eee 2 8 1 Handling high pressure cylinders and tubing 2 8 2 Regulating the nitrogen pressure 0000 cee 2 9 LAS ONS iiia aioe kaa doin ae da ae eae eae es 2 9 1 Class 1 Laser Productlabel 0 0 00 eee 2 9 2 Laser light warning label 00 00 00 0 cee eee eee 2 9 3 Safety interlock danger label
4. MegaBACE Instrument Administrator s Guide p5 7 Parttwo Understanding and configuring the MegaBACE system 7 If no sample or file names are provided for a plate the Instrument Control M anager uses the well locations well IDs as the sample file names for example A01 rsd You type the file name in the value column to designate the file name The following and figure 5 3 show examples of what you might enter into the psd file to designate a file name that is different from the sample name Field name Value A01 NAME 14660 A01 FILE A01 GenotypingS ample psd Notepad olx File Edit Search Help PLATE ID Genotyping_Demo_Panel INJECTION VOLTAGE 3 INJECTION TIME 45 RUN VOLTAGE 10 RUN TIHE 75 CHEMISTRY GT DyeSet2 ET ROX FAM NED HEX PLATE TYPE SAMPLE PLATE FILE A81 NAHE 14660 FILE n82 File names NAHE 14698 FILE A83 NAHE 11877 FILE no NAHE 16835 FILE Ags Sample NAME 12248 names FILE G6 NAHE 23 01 FILE A87 Field column Value column Figure 5 3 Example of a psd file for genotyping with entries to designate the well ID as the file names 5 3 7 Suppressing rsd file creation for empty or bad capillaries The Instrument Control M anager does not create rsd files for capillaries that are empty broken or clogged You can list the well ID for a defective capillary in the Bad Capillaries section of the M egaBACE ini file This is a
5. MegaBACE Instrument Administrator s Guide 5 11 Parttwo Understanding and configuring the MegaBACE system Important 5 4 2 Value constraints If you type a value that is invalid for example an alpha character when the software requires a number the Instrument Control M anager displays an error message when it loads the template file If this occurs you must correct the value in the tpl file before it can be imported successfully Alternatively if the edit mode is on you can enter the correct value in the Plate Setup window 5 4 3 Creating plate setup template files tpl To create a plate setup template file 1 Use any text editor or Excel to open one of the tpl files provided with your software The available tpl files are located in the M egaBA CE Templates folder Alternatively you can open a new document in any text editor or in Excel 2 Type the fields and values for the plate using the appropriate format section 5 2 N ote If you use spaces in the sample names the Instrument Control M anager converts the spaces to underscore characters in the file name 3 Save the file in the appropriate format N ote If you use Excel save the file as a tab delimited file You must type the tpl file extension and save the file in the M egaBACE Templates folder O therwise the Instrument Control M anager will not recognize the file You can specify a default plate setup template in the MegaBACE ini fil
6. e Configure the software for your laboratory workflow and application sequencing and or genotyping using the system initialization files section 1 5 1 Set up plate definitions that an operator can import automatically at run time section 1 5 2 e Create plate setup and instrument control templates that an operator can import automatically at run time section 1 5 2 Note The system software includes templates of the parameter configuration files for your laboratory s application 1 5 1 The MegaBACE system initialization files To meet the needs of your laboratory you may need to modify the system initialization files The system software includes two initialization files that you can modify e MegaBACE ini file Determines the settings the Instrument Control M anager uses to control the operation of the M egaBACE instrument section 6 4 For example the M egaBACE ini file can specify Thedefault application sequencing or genotyping if your laboratory uses more that one application on the same instrument Whether an operator can manually edit the plate setup or instrument control parameters Sequencing only Whether the software performs base calling automatically after a run is completed e Chemistry ini file Determines the chemistry parameter sets available in the Instrument Control M anager software section 6 3 MegaBACE Instrument Administrator s Guide p1 7 Part One Introduction and s
7. To create an instrument control parameters template 1 Click the Instrument C ontrol tab to display the Instrument Control window figure 4 7 2 To modify an existing template e From the Templates menu point to Instrument Template and choose Select Template T he Select Template window appears and displays the list of available templates icp e Select the template icp you want to modify and click Open The values of the selected template appear in the Instrument Parameters area figure 4 7 3 Modify any of the parameters 4 From the Templates menu point to Instrument Template and choose Save Template T he Save window appears 5 Type anew name and click Save The template is saved in the M egaBACE Templates folder The software adds the icp file extension 4 3 2 Selecting an instrument control parameter template To select a different instrument parameter template 1 From the Templates menu point to Instrument Template and choose Select Template The O pen window appears 2 Select the template name you want to use and click Open The Instrument Parameters area of the Instrument Control window displays the parameters for the selected template p4 14 MegaBACE Instrument Administrator s Guide Important Chapter 4 Using the Instrument Control Manager windows to configure the system 4 4 Using automatic base calling sequencing only For sequencing applications you can use the Instrument
8. Appendix B Plate and instrument parameter setup Figure B 7a shows a basic psd file for genotyping Table B 1 describes the fields You store the psd files in the M egaBAC E Psd folder Table B 1 The field names in a basic genotyping psd file Field name Description PLATE ID The plate ID SAMPLE INJECTION VOLTAGE The range is 1 20 kV For genotyping runs you should start with a value of 3 SAMPLE INJECTION TIME The range is 0 600 s For genotyping runs you should start with a value of 45 RUN VOLTAGE The range is 1 20 kV For genotyping runs you should start with a value of 10 RUN TIME The range is 1 720 min For genotyping runs you should start with a value of 60 to 75 depending on the age of the capillaries For older capillaries you may need a longer run time CHEMISTRY You type the name of the chemistry parameter set for your application The software retrieves the parameters from the Chemistry ini file WELL ATTRIBUTES Reserved for future use The field descriptors that are used internally by the system WELLID NAME Use this well attribute to specify the name or ID of a sample in a well WELLID FILE To make sure the software uses the well location as the file name of the raw sample data file rsd you must use this well attribute Genetic Profiler v1 1 can recognize rsd files only if the well locations are used for the file names for example AO1 rsd Figure B 7
9. Provides quality statistics for M egaBACE data that was base called using Sequence Analyzer e MegaBACE Genetic Profiler ScoreC ard Provides a means for validating and tracking the performance of the M egaBACE genotyping system when used in conjunction with the Genotyping Test Plate kit For more information about the software see the H elp available within the applicable M egaBACE ScoreCard software 1 3 Overview of the Instrument Control Manager features The Instrument Control M anager software allows you to Specify the plate setup definition and the instrument parameters manually or automatically section 1 3 1 e Provide full flexibility to the instrument operator to make changes or limit the operator s ability to modify the plate setup or instrument parameters for running a plate chapter 6 e Change applications The sequencing and genotyping applications are supported section 1 3 2 Sequencing only Automatically call bases on the collected data and export the data to other file formats after base calling section 1 3 3 Collect data 1 3 1 About manual or automatic plate setup During manual plate setup you use the Instrument Control M anager windows to manually enter the plate ID and the plate setup parameters for each plate chapter 4 The manual mode can also be used to enter the instrument control parameters You can create templates of the plate setup parameters and the instrument control pa
10. Each chemistry parameter set includes the names of the dyes for genotyping or bases for sequencing assigned to each spectral channel and lists the filters the beamsplitters and the laser mode Sample Names tab The Sample N ames tab displays the sample names assigned to each well in the plate The Instrument Control M anager uses the following conventions for sample names e If no sample names are provided the software uses the well IDs as the raw sample data file names rsd e If sample names are provided the software uses the sample names as the rsd file names default unless different file names are specified in a plate setup data file psd If duplicate sample names occur for a given plate the software automatically appends the well IDs to the file names to generate unique file names Typically a psd file or a plate setup template file tpl contains a list of sample names and the Instrument Control M anager imports the names from either of these files Alternatively you can edit sample names or type sample names in the Sample N ames tab Genotyping only Genetic Profiler v1 1 cannot recognize rsd files unless well IDs are used for the file names for example A01 rsd Optional Parameters tab N ote You use the O ptional Parameters tab only for unusual plate setups The Optional Parameters tab displays the PM T voltages run temperature and base caller sequencing only that can be specified as a part of the
11. Filter 4 4 1 j ae me U Filter 3 mitte p lightpath Ff I a ge Channel 1 une A oS ae A a po Channel 3 Beamsplitter Filter 1 PMT 1 gt ee 1 PMT1 A Figure A 2 The path of emitted light for the MegaBACE instrument a in the dual laser mode the instrument uses the blue laser 488 nm line during the forward scan and b the green laser 532 nm line during the return scan A 4 1 Emission beamsplitters Dichroic beamsplitters separate light by wavelengths Each beamsplitter has a specified wavelength cutoff e Light with wavelengths longer than the cutoff passes through the beamsplitter figure A 3 e Light with wavelengths shorter than the cutoff is reflected by the beamsplitter figure A 3 Shorter wavelength ye is reflected a Beamsplitter cutoff gt Longer wavelength y light is passed Beamsplitter A a Nooo Wavelength spectrum before filtering Figure A 3 Effect of a dichroic beamsplitter on light encountering the beamsplitter pA 6 MegaBACE Instrument Administrator s Guide Appendix A Fluorescence imaging A 4 2 Emission filters The M egaBACE instrument uses emission filters because e Theseparation of light by the beamsplitter is imperfect e When the sample contains three or four dyes the light leaving the beamsplitter includes the emissions from several dyes The instrument uses two types of emission filters e Band pass R ejects most of the li
12. Parttwo Understanding and configuring the MegaBACE system The Instrument Control M anager allows you to Create a plate setup template W hen you set up a plate definition you can save the plate setup parameters as a template You can also modify an existing template and save it with a new name sections 4 2 2 and 4 2 3 Select a plate setup template Y ou can import a different template section 4 2 4 Select a default plate setup template Y ou can select a template to use as the default for defining multiple plates The software loads the default template automatically when you click the N ew button section 4 2 5 Clear the default plate setup template Y ou can deselect the default template section 4 2 6 You use the Templates menu to select and save plate setup templates and to set and clear default plate setup templates 4 2 2 Creating a plate setup template N ote It is not necessary to enter values in all the parameter fields in the Optional Parameters tab The values entered in the Optional Parameters tab automatically override duplicate parameters specified in the Instrument Control window To create a plate setup template Click the Plate Setup tab to display the Plate Setup window and click New Optional To modify an existing template e From the Templates menu point to Plate Setup Templates and choose Select Template The O pen window appears e Select the template name and click O pen Th
13. arrays according to your instrument model The Instrument Control M anager will reconfigure the INSTALLED ARRAYS field automatically during the Replace Capillaries protocol For more information on the R eplace Capillaries protocol see the M egaBACE Instrument M aintenance and Troubleshooting Guide p6 18 MegaBACE Instrument Administrator s Guide Chapter 7 Bypassing or overriding a protocol This chapter describes how to bypass or override a protocol The topics are Bypassing the selected protocol section 7 1 e Overriding the contents of a protocol section 7 2 In atypical operation the software selects the next protocol in the workflow You may need to use a protocol that is different from the one selected in the List of Protocols in the Instrument Control window figure 7 1 List of Protocols Right click to display the Override button Z Instrument Control Manager MegaBACE Sequencing M13std40_092499Run06 File View Options Templates Cbnfigure Help List of Protocols Instrument Parameters soibannes Matrix Fl HighPressure Time 200 sec Matri Flush Timed 22 sec l Matrix Fill Belaxation Time 20 min Matrix Flush Time2 7 sec Hari ar ee Prerun Time 5 min Low Pressure Time 240 sec rerum uny Prerun Voltage 9 kY User Input Time 120 sec Inject Samples and Run or Apps 7 S Preinjection Voltage 10 kY Preinjection Time 15 sec Freinject Samples PMT1 Voltage 750 hi PMT2
14. ee 5 2 5 3 2 Using a plate ID that is different from the scanned ID 5 4 5 3 3 The psd file format 202 4 ccca0csaw nett een beeen pane 5 5 MegaBACE Instrument Administrator s Guide vii Table of contents 5 4 5 5 5 3 4 The psd file value constraints eee eee 5 3 5 Annotating well specific information in a psd file 5 3 6 Designating a sample file name in the psd file 5 3 7 Suppressing rsd file creation for empty or bad capillaries 5 3 8 Creating a plate setup data file psd 0005 Using plate setup template files tol cc ee 5 4 1 Aboutthe tpl files 0 eee ee 5 4 2 Value constraints 00 eee ee 5 4 3 Creating plate setup template files tpl Using instrument control parameter template files icp 5 5 1 About instrument control parameter template files icp 5 5 2 Value constraints 0 eee 5 5 3 Creating an instrument control parameter template icp Chapter 6 Using the system initialization files 6 1 6 2 6 3 6 4 About the system initialization files 00 0 000s eee eee File format and storage 6 6 eee Modifying the Chemistry ini file 0 0 0 cece eee eee Using the MegaBACE ini file 0 0 c cece eee eee 6 4 1 Modifying the MegaBACE inifile 0 000 6 4 2 Aboutthe edit mode 0 cece eee 6 4 3 About the automatic base calling mode sequen
15. use file extensions N do not use file extensions Cleanup command Allows an operator to enter a command for String None transferring plate records and raw data files to another workstation for analysis or archiving Default plate setup Specifies a default plate setup template String None template Default instrument Specifies a default instrument control String None template parameter template Allow duplicate sample Allows an operator to use duplicate YorN Y names sample names Y allow duplicate names Well IDs are appended to the sample names to create unique file names N do not allow duplicate names p6 10 MegaBACE Instrument Administrator s Guide Chapter 6 Using the system initialization files Table 6 2 The field names and default settings of the MegaBACE ini file continued Field name Description Setting type Default Automatic base call Sequencing only Allows automatic base String None command calling to start right after data collection Installed arrays MegaBACE 500 and flexible Integer 3 or6 MegaBACE 1000 instruments only Defines the number of installed capillary arrays Choices are 1 2 3 or 1 2 3 4 5 or 6 depending on your model 6 4 2 About the edit mode The edit mode determines whether an operator can edit the plate setup and instrument control parameters while they are displayed in the Instrument Control M anager windows The edit mode also determines whether an operator can c
16. 1 excitation modes A 5 export files storage locations 6 14 C 3 C 4 export options sequencing 6 13 to 6 15 extended header E 1 to E 3 F FASTA file creation allow override 6 9 automatic 6 9 options 6 15 file name matching plate ID 4 5 5 2 files APB protocol F 1 to F 4 base called sample data esd C 3 to C 5 Index export options sequencing 6 13 to 6 15 extended header E 1 to E 3 raw sample data rsd C 1 C 2 standard header D 1 storage C 1 to C 7 filter compartment caution 2 8 lid illustrated 2 2 filter configuration genotyping A 10 sequencing A 8 filters A 7 A 8 flare fluorescent 3 8 flexible instrument capillaries 3 3 components 1 2 description 1 6 introduction 1 1 fluorescence dyes in sample 3 6 flare 3 8 imaging 3 1 light pathway 3 5 fluorescent signal recording A 5 fluorochrome defined A 1 focal point 3 5 3 6 folders storage C 1 to C 7 subfolders specifying 6 14 6 15 fuse xiii G genotyping dyes and filters A 10 parameter templates B 8 plate setup data files B 5 to B 7 plate type 5 6 H hardware components 1 2 Header Editor software 1 4 MegaBACE Instrument Administrator s Guide Index 3 Index header file extended E 1 to E 3 standard D 1 high voltage electrophoresis compartment 2 6 internal electronics 2 8 Host Scan Controller software 1 4 imaging fluorescence 3 1 Important statement defined xii importing icp files 5 13 psd files 6 8 6 12 plate setup parameters 5 5
17. 6 4 A yellow arrow appears above an empty capillary which is defined in the psd file Table 6 2 The field names and default settings of the MegaBACE ini file Field name Description Setting type Default Default application Specifies the application to use as the String If no application default when an operator opens the specified Instrument Control Manager Currently sequencing is the choices are sequencing or genotyping the default Allow edit Determines whether an operator can edit YorN Y the parameters in the Plate Setup window Y operator can change parameters N operator cannot change parameters Allow global edit override Determines whether an operator can YorN Y change the Edit Mode command on the Configure menu at any time Y operator can change mode N operator cannot change mode Allow plate edit override Determines whether an operator can YorN Y change the Edit Mode command on the Configure menu for the current plate only Y operator can change mode N operator cannot change mode psd directory Specifies the path for the psd folder Patht MegaBACE psd String A text string tPath The complete path to a file folder MegaBACE Instrument Administrator s Guide p6 7 Parttwo Understanding and configuring the MegaBACE system Table 6 2 The field names and default settings of the MegaBACE ini file continued Field name Description Setting type Default PSD
18. Integer you type a number MegaBACE Instrument Administrator s Guide p6 5 Parttwo Understanding and configuring the MegaBACE system ae 6 4 1 Modifying the MegaBACE ini file Important The Instrument Control Manager can use the MegaBACE ini file only if the file name is MegaBACE ini and the file is stored in the MegaBACE DataSystem folder To modify the M egaBACE ini file 1 Open the M egaBACE ini filein the M egaBA CE D ataSystem folder using any text editor See figure 6 3 for an example of the M egaBACE ini file opened in N otepad 2 Change the appropriate settings 3 Save the file Important After you modify and save the MegaBACE ini file you must close and reopen the Instrument Control Manager in order for the changes to take effect MegaBACE ini Notepad Of x File Edit Search Help General DEFAULT APPLICATION ALLOW EDIT Y ALLOW GLOBAL EDIT OVERRIDE Y ALLOW PLATE EDIT OVERRIDE Li PSD DIRECTORY PSD REQUIRED N PLATE ID LENGTH 8 PLATE ID TERMINATOR DEFAULT BASE CALLER Cimarron 1 53 Slim Phredify AUTOMATIC BASE CALLING ALLOW BASE CALLING OVERRIDE AUTOMATIC ABD FILE CREATION ALLOW ABD OVERRIDE AUTOMATIC FASTA FILE CREATION ALLOW FASTA OVERRIDE AUTOMATIC SCF FILE CREATION ALLOW SCF OVERRIDE AUTOMATIC RAW TEXT FILE CREATION ALLOW RAW TEXT OVERRIDE AUTOMATIC PROCESSED TEXT FILE CREATION ALLOW PROCESSED TEXT OVERRIDE EXPORT IF BASE CALLING FAILED EXPORT GOOD REGION O
19. NANE 16835 AG4 File ABS A185 NAME 12248 AG5 File ABS A186 NAME 23 61 86 File A86 A97 NANE 14668 67 File A07 68 NANE 14698 68 File Ags 69 NANE 11877 A69 File Ago A10 NAME 10835 A10 File A10 14 NANE 23 01 A11 File att tw 4 P b Detailed genotyping psd file CPEE LE Fie Edit Search Help PLATE ID DIAG3PN4126199DS2 INJECTION VOLTAGE 3 INJECTION TIME 45 RUN VOLTAGE GT DyeSet 2 ET ROX FAM NED HEX ELL ATTRIBUTES NAME FILE SAMPLE TYPE COLLECTION DATE GENDER POPULATION PROJECT Diabetes MARKER PANEL SET Mapping Set_II MARKER PANEL Panel 4 SPECTRAL OVERLAP MATRIX MB2_DS2_126199 SIZE STANDARD CHANNEL 1 SIZE STANDARD NAME ET466 R SIZING ALGORITHM Local_3rd_Order LLELE BIN LABELS Alphabetic 61 NANE 14666 A61 FILE A81 61 SAMPLE TYPE string A61 COLLECTION DATE string AG1 GENDER string A61 POPULATION string AG1 AFFECTION STATUS string AG1 LIABILITY CLASS string AG1 PEDIGREE string AG1 PEDIGREE GROUP string AG1 FATHER string AG1 MOTHER string 61 PROBAND string Reserved for future use 4 Reserved for future use Figure B 7 Examples of genotyping psd files a The sample names in the psd file will appear with the data when you analyze the run using Genetic Profiler v1 1 b A psd file with analysis parameters assigned to the plate and multiple attributes assigned to each well pB 6 MegaBACE Instrument Administrator s Guide
20. Voltage 750 v Store Capillaries Flush and Dry Capillaries Run Temperature 44 C Sleep After pie Run E Replace Capilares Sleep Temperature 25 Sleep Time 6 hrs High Pressure Flush Low Fressure Flush oe Janoprint2 for 30 seconds Focus Capilares lpause 5 led janoprint Load 6 lanoprint2 matrix tubes luser 120 ano ignore janoprint Matrix fill lanoprint2 Icatprint2 Until complete Iclamplock close lhipres 200 led janoprint Matrix janoprint2 equilibration Icatprint2 Until complete jpause 20 Icd Iclamplock open Janoprint Prerun in Materials and Instructions Use before the first run of the day Full water tank Full water tubes Icatprint2 Since prerun timer up 1 X l Plate Setup Instrument Control Run Image For Help press F1 27 00 44 00 Run time O00min 00sec Full Run Time 00min 07 25 00 02 08 PM Figure 7 1 The List of Protocols in the Instrument Control window MegaBACE Instrument Administrator s Guide p7 1 Parttwo Understanding and configuring the MegaBACE system The Instrument Control M anager allows you to e Bypass the protocol the software selects You can select any protocol even if it is disabled section 7 1 e Override all or part of the protocol the software selects The software selects the next protocol in the List of Protocols section 7 2 7 1 Bypassing the selected protocol To bypass the selected
21. at 532 nm When the blue or green laser mode is used the laser shutter allows light from only the selected laser to enter the light path during the entire electrophoresis run When the green and blue laser mode is used the movement of the laser shutter is synchronized with the movement of the scan head During the forward scan the 488 nm line excites two dyes and the shutter blocks the 532 nm line During the return scan the shutter moves to block the 488 nm line and the 532 nm line is enabled to scan the remaining two dyes A 4 How the MegaBACE records the fluorescent signal To record four dyes separately the M egaBACE instrument uses two optical sets each consisting of a beamsplitter plus two filters A beamsplitter changer and two filter changers move synchronously with the scan head to place one optical set at a time in the light path figure A 2 During the forward scan figure A 2a the instrument records channels 1 and 2 using beamsplitter A and filters 1 and 2 e During the return scan figure A 2b the instrument records channels 3 and 4 using beamsplitter B and filters 3 and 4 For information on how to care for and change beamsplitters and filters see section 3 4 2 and 3 4 3 in the M egaBACE Instrument M aintenance and Troubleshooting Guide MegaBACE Instrument Administrator s Guide pA 5 Partthree Appendixes D a Forward scan b Return scan Channel 2 Channel 4 PMT 2 PMT 2 Filter 2
22. automatic base calling starts the Instrument Control M anager exports the data files to the formats selected in the Automatic Base Calling window If you specify Y to EX PORT TO SUBFOLDERS in the M egaBACE ini file the software stores the export files in the appropriate subfolder under the analyzed or raw run folder You can also use the M egaBACE ini file to select the file formats you want the Instrument Control M anager to create To do this you specify Y for creation of the desired file format s You use the M egaBACE ini file to specify whether the operator can override the M egaBACE ini file export selections If you specify Y to the ALLOW FILE FORMAT OVERRIDE in the M egaBACE ini file the operator can select or deselect the export options in the Automatic Base Calling window The FASTA file creation has three different options and you can specify one two or all three options You specify the option s for the Automatic Base Call Command field name in the M egaBACE ini file The options are e fa for creation of one seq file for each analyzed esd file These files are stored in the M egaBACE AnalyzedData platelD_runlD folder e fap for creation of one seq file for each base called plate This file is stored in the M egaBACE AnalyzedData platelD_runlD folder This is the default switch used for FASTA in the Automatic Base Call Command in the M egaBACE ini file e fag for global creation of one seq file for a
23. brackets If the element is represented on a single line the brackets appear on the same line Otherwise the brackets are vertically aligned N esting is indicated by indentation e For List elements the number of elements in the List is appended to the L format identified For example L3 denotes a list containing three elements figure F 2 N ote An embedded List counts as a single element for its enclosing List Lists may be nested to any depth lt L8 lt AB Gizmo gt lt 2 414 gt lt L2 Figure F 2 Example of the List format See figure F 1 for notations For character elements A and C formats the number of characters is appended to the format identifier For example A6 denotes a five character ASCII string with its terminating null Strings are not surrounded by quotes unless the quote characters are supposed to be encoded as part of the string MegaBACE Instrument Administrator s Guide pF 3 Part three Appendixes I All other data types are treated as arrays if the element contains more than one item where the array size is shown in square brackets For example U2 4 indicates an array of four 2 byte unsigned integers in the example below figure F 3 lt U2 4 100 200 300 400 gt Figure F 3 Example of an array See figure F 1 for notations eT pF 4 MegaBACE Instrument Administrator s Guide Appendix G Literature references The following references are cited i
24. edit mode in the MegaBACE ini file If you modify the MegaBACE ini file you must restart the Instrument Control Manager for the changes to take effect If you need to edit the chemistry parameters you should do so in the Chemistry ini file before performing plate setup You do not need to restart the Instrument Control Manager after editing the Chemistry ini file section 6 3 MegaBACE Instrument Administrator s Guide p4 1 Parttwo Understanding and configuring the MegaBACE system In the edit mode the Plate Setup window figure 4 1 allows you to Enter or edit electrophoresis parameters Select a chemistry parameter set by name Enter or edit sample names Enter or edit optional parameters Enter or edit comments about the plate You use the following tabs on the Plate Setup window to enter the various plate setup parameters the sample names and the comments Figure 4 1 The tabs in the Plate Setup window The sequencing electrophoresis parameters are displayed Electrophoresis Parameters tab The Electrophoresis Parameters tab displays the conditions for running the plate the sample injection voltage and time and the run voltage and time p4 2 MegaBACE Instrument Administrator s Guide Caution Important Chapter 4 Using the Instrument Control Manager windows to configure the system Chemistry Parameters tab The Chemistry Parameters tab displays a list of names for the chemistry parameter sets
25. for a plate and for individual wells on the plate such as sample names The Instrument Control M anager includes the information from the psd file in the extended header of each raw sample data file rsd rsd files the raw sample data files each of which contains the raw data for a given well on a plate for example A01 The storage location isin a corresponding raw run folder plateID_run ID in the M egaBACE Data folder default or the location you specify Each rsd file contains the plateID run ID well ID plate setup parameters instrument parameters raw electropherogram data current intensities and scan rate scf files sequencing only the base called data files in SCF format Each scf file contains the raw data and electropherogram If analyzed each scf file includes the raw and base called data and the called sequence seq files sequencing only the base called data files in FASTA format Each seq file contains the esd file name esd file location and the called sequence for a given well on a plate You can view these files in another application such as N otepad analyzed run folder sequencing only the folder that contains the base called sample files for a given run The Instrument Control M anager uses the plate ID and base caller ID to name the analyzed run folder The Instrument Control M anager creates an analyzed run folder for the data from each plate for which the Instrument Contr
26. for the base called and exported files N ote that the base called sample data files obtain the file names from the rsd files File contents File name Plate ID well location sample name instrument ID base caller name called sequence sequence starting and ending points read length for the high quality data quality values electrical current information base called electropherogram for the sample Sample_name esd well _ID esd or a combination of both Folder storage PlatelD_basecaller D analyzed run folder Exported ABD All the raw data including electropherogram and spectral separation table If base called contains all the raw and base called data including the called sequence Sample_name abd well_ID abd or a combination of both ABD subfolder default Note In the MegaBACE ini file you can specify that the files be stored at the top level of the run folder section 6 4 Exported SCF Raw data and the electropherograms If base called includes the raw and base called data and the called sequence Sample_name scf well_ID scf or a combination of both Note The scf extension may not display in Windows Explorer SCF subfolder default Note In the MegaBACE ini file you can specify that the files be stored at the top level of the run folder section 6 4 MegaBACE Instrument Administrator s Guide pC 3 Part three Appendixes Table C 2 Naming and stora
27. global setting For every capillary listed a red arrow appears above the capillary button in the Run Image window figure 5 4 p5 8 MegaBACE Instrument Administrator s Guide Chapter 5 Using the parameter configuration files E For plate specific cases you can indicate unused wells by typing NOT USED case insensitive as the sample name specified in the psd file or in the Sample N ames tab in the Plate Setup window figure 4 4 For every unused well a yellow arrow appears above the capillary button in the Run Image window figures 5 4 Bad capillaries Empty capillaries Figure 5 4 The Run Image window for an instrument with bad capillaries and empty capillaries indicated MegaBACE Instrument Administrator s Guide 5 9 Parttwo Understanding and configuring the MegaBACE system Important 5 3 8 Creating a plate setup data file psd When you create a psd file you must type the psd file extension and save it in the MegaBACE Psd folder or the folder you specified as the Psd directory in the MegaBACE ini file otherwise the Instrument Control Manager will not find the file To create a psd file 1 Open anew document or an existing psd file using a text editor such as N otepad or Excel 2 Type the fields and values for the plate figures 5 3 and 5 5 using the appropriate format section 5 2 See table 5 1 for a list of field names N ote If you use spaces in the sample or filenames
28. in Excel 2 If you are modifying an existing icp file edit the fields If you are starting with a new document type two columns separated by a tab figure 5 7 or two columns using Excel figure 5 8 X Microsoft Excel Normal icp File Edit View Insert Format Tools Data Window Help 18 x arial 10 5 OvA A HIGH PRESSURE TIME RELAXATION TIME 20 PRERUN TIME 5 PRERUN VOLTAGE 9 PREINJECTION VOLTAGE 10 PMT1 VOLTAGE 750 RUN TEMPERATURE sal STORE TEMPERATURE 25 FLUSH1 TIME 22 FLUSH12 TIME 7 LOW PRESSURE TIME 240 USER INPUT TIME 120 PREINJECTION TIME 15 PMT2 VOLTAGE 750 STORE TIME 6 4 gt Mh Normal Sheet2 She i Draw j AutoShapesy N a D A O E j I y Figure 5 8 Example of an instrument control parameter template file icp for sequencing created in Excel 3 Save the file in an appropriate format N ote If you use Excel you must save the file as a tab delimited file You must type the icp file extension and save the file in the M egaBACE Templates folder p5 14 MegaBACE Instrument Administrator s Guide Important Chapter 5 Using the parameter configuration files You must store the template files in the MegaBACE Templates folder otherwise the Instrument Control Manager will not be able to find the file You must type the icp file extension You can specify a default instrument control parameter tem
29. laser and a smaller amount of the labeled size standard can be used O ther single R O X labeled standards can be used but because ROX is not excited efficiently by the blue laser the amount of labeled standard must be increased significantly Tables A 1 and A 2 list dye sets that have been validated on the M egaBACE instrument for the genotyping system All the recommended dye sets for genotyping on the M egaBACE include the ET ROX label for the size standard Table A 1 Dye set 1 recommended for genotyping filter set 1 Dye Excitation nm Emission nm FAM 494 522 TET 521 538 HEX 535 553 ET ROX size standard 494 FAM 607 ROX Table A 2 Dye sets recommended for genotyping filter set 2 Dye Set Excitation nm Emission nm a Dye set 2 FAM 494 522 HEX 535 553 NED or TAMRA 560 582 ET ROX 494 FAM 607 ROX b Alternative dye set FAM 494 522 JOE 528 554 TAMRA 560 582 ET ROX 494 FAM 607 ROX pA 4 MegaBACE Instrument Administrator s Guide Appendix A Fluorescence imaging A 3 MegaBACE excitation modes The options for excitation include Blue laser mode Excites up to four dyes at 488 nm Dual laser instrument only G reen laser mode Excites up to four dyes at 532 nm Dual laser instrument only G reen and blue laser mode Uses the blue laser to excite two dyes efficiently at 488 nm and uses the green laser to excite two dyes efficiently
30. mode is selected 3 Select a default base caller section 4 4 2 4 Optional Select one or more check boxes for the file format s you want to use for data export 5 Click OK MegaBACE Instrument Administrator s Guide 4 15 Parttwo Understanding and configuring the MegaBACE system I Automatic Base Caling M Options Default Base Caller Molecular Dynamics bd M Export to ABD Format I Export to SCF Format I Export to FASTA Format I Export RSD files to ASCII format IT Export ESD files to ASCII format Cancel _ Figure 4 8 The Automatic Base Calling window 4 4 2 Specifying base callers for automatic base calling The Instrument Control M anager allows you to specify a e Default base caller for a series of plates e Plate specific base caller which overrides the default base caller e Well specific base caller section 5 3 which overrides the default and the plate specific base caller For each well the Instrument Control M anager looks for a base caller in the following order well specific plate specific and then default If the Instrument Control M anager does not find a base caller and automatic base calling is turned on the software displays an error message Specifying a default base caller You can specify a default base caller in the M egaBACE ini file section 6 4 or you can select a different default base caller to use for analyzing the samples on a series of plates in the Automati
31. protocol 1 In the Instrument Control window select the protocol you want to run and click the right mouse button The O verride button appears figure 7 1 2 Click the Override button A window appears and asks for a password figure 7 2 cancel Figure 7 2 The Enter Password window 3 Type the appropriate password and click OK The O verride Commands window appears figure 7 3 Override Commands Rinse Tips x Select All Deselect All Save Selection Mllaser blue idle laser green on ventp on Mihyv 0 V catstage lower Mcatprintt Load full Mcatprint2 water tank Mluser 120 cat tank Mclamplock open Manostage lower Mlanostage raise x Cancel Figure 7 3 The Override Commands window p7 2 MegaBACE Instrument Administrator s Guide Chapter 7 Bypassing or overriding a protocol 4 Click OK to run all the commands for the selected protocol The window closes In the Instrument Control window figure 7 1 the selected protocol flashes to show that it is running 7 2 Overriding the contents of a protocol You can override all or part of the protocol the software selects as the next logical protocol To do this 1 With the protocol selected click the right mouse button The Override button appears figure 7 1 2 Click the Override button A window appears and asks for a password figure 7 2 3 Type the appropriate password and click OK The O verride Comm
32. required Determines whether the Instrument Control Manager imports a plate setup data file psd when an operator enters a plate ID Y import psd file If no psd file exists the software returns an error N do not import psd file YorN Y Plate ID length Determines the number of characters allowed in the plate ID Used only for bar code entry Integer Plate ID terminator Determines what terminates the entry of the plate ID return or tab Used only for bar code entry String None Default base caller Sequencing only The name of the base caller the laboratory is using String Cimarron 1 53 Slim Phredify Automatic base calling Sequencing only Determines whether the Instrument Control Manager launches automatic base calling Y launch base calling N do not launch base calling YorN Y Allow base calling override Sequencing only Determines whether an operator can override automatic base calling from the Configure menu Y operator can override N operator cannot override YorN Automatic ABD file creation Sequencing only Determines whether the Instrument Control Manager automatically creates ABD files after base calling Y create ABD files N do not create ABD files YorN Allow ABD override Sequencing only Determines whether an operator can override automatic ABD file creation from the Configure menu Y operator can override N
33. s Guide Important Chapter 4 Using the Instrument Control Manager windows to configure the system Electrophoresis Parameters Chemistry Parameters Sample Names Optional Parameters Comments Standard Terminator Figure 4 6 The Comments tab with sequencing comments displayed 12 Type the comments you want to include as a part of the plate setup 13 Click Save in the Plate Setup window figure 4 2 The plate definition is saved and the new plate ID appears in the Plate Catalog N ote Y ou can change the parameters for a plate before it is run After you change parameters you click Save again to save the new parameters You cannot change the parameters after the plate has been run 14 Optional Repeat steps 2 through 13 for each plate definition you want to set up manually 4 2 Creating plate setup templates in the Plate Setup window The edit mode must be turned on to allow you to create edit select or save plate setup templates tpl in the Instrument Control Manager See section 6 4 for information about enabling the edit mode 4 2 1 About plate setup templates You can simplify setting up plates by using plate setup templates tpl In a plate setup template you specify parameters that are common to multiple plates You can create a plate setup template in the Plate Setup window or you can create the template using a text editor or Excel section 5 4 MegaBACE Instrument Administrator s Guide p4 9
34. syntax errors Syntax errors can include e Required field is missing The required fields are laser mode base names sequencing and dye names genotyping Required field has an out of range value For example a base name has a letter other than A G C or T for the value e Duplicate values exist for different fields for example two bases are named A e The parameter set includes a misspelled field name or value for a required field You specify the chemistry parameter set name in the psd file or the tpl file If you turn on the edit mode an operator can select the chemistry name in the Plate Setup window 6 4 Using the MegaBACE ini file The M egaBACE ini file determines the settings the Instrument Control M anager uses to control the instrument The Instrument Control M anager uses only one M egaBACE ini file Figure 6 3 is an example of the M egaBACE ini file configured for sequencing The M egaBACE ini file is in a two column tab separated format section 6 2 The first column field column contains the various modes and conditions that you can customize for the laboratory The second column value column defines the setting Table 6 1 describes the sections in the M egaBACE ini file Table 6 2 provides a description of the field names and the default settings you can modify in the file You type Y for yes or N for no to set the conditions If the setting type is String you type text If the setting type is
35. templates 5 11 initialization system see MegaBACE ini file instrument components 1 2 1 3 displays function of 2 5 electrical rating xiii electrical requirements 2 13 fuse xiii models 1 1 serial number label 2 14 weight 2 2 Instrument Control Manager software overview 1 5 instrument control parameter templates creating in a text editor 5 12 to 5 15 creating in the Instrument Control window 4 12 to 4 14 default setting 5 13 6 10 example genotyping B 8 example sequencing B 4 selecting 4 14 storing C 7 instrument control parameters defined 4 13 selecting 4 14 Index 4 MegaBACE Instrument Administrator s Guide interlock defeat 2 2 2 12 in anode drawer 2 1 in cathode drawer 2 1 in electrophoresis compartment lid 2 1 L label Class 1 Laser Product 2 11 hazardous voltage 2 6 high pressure 2 10 laser light warning 2 11 locations 2 14 low pressure 2 10 pinching hazard 2 6 laboratory procedures for nitrogen 2 9 laser light collimated beam 3 8 hazardous exposure 2 11 2 12 monochromatic 3 8 mW divergence 2 11 power 2 11 warning label 2 11 wavelength emitted 2 11 laser shutter function of 2 8 lasers 2 11 light leaks 2 12 light excitation 3 8 long sequencing run with dye primers B 3 long sequencing run with dye terminators B 3 M MegaBACE Header Editor software 1 4 MegaBACE ScoreCard software 1 5 MegaBACE system hardware components 1 2 to 1 4 software overview 1 4 MegaBACE ini applications multiple 6 16
36. the Instrument Control M anager converts the spaces to underscore characters in the file name 3 Save the file in an appropriate format You must type the psd file extension N ote If you use Excel save the file as a tab delimited file ile Edit View Insert Format Tools Data Window Help 18 xl arial gt 10 G O A z INJECTION VOLTAGE INJECTION TIME 40 RUN VOLTAGE g RUN TIME 100 CHEMISTRY ET Primers RUN TEMPERATURE 44 PMT1 VOLTAGE 750 PMT2 VOLTAGE 750 BASE CALLER Cimarron 1 53 Phat COMMENT M13 Standard Run AD1 Sample_AD1 AD2 Sample_A02 AD3 Sample_A03 AD4 Sample _AD4 Ph m13std1_080899 5 4 a Draw G Autoshapesy OA A E es S E E E Field column Value column Figure 5 5 An example of a psd file for sequencing created in Excel p5 10 MegaBACE Instrument Administrator s Guide Important Chapter 5 Using the parameter configuration files You must use a file name that is the same as the plate ID you enter into the Plate Setup window for example m13std1_060899 psd Ifa bar code is used in the Plate Setup window you must use the bar code as the file name for the psd file for example 10303385858 psd 5 4 Using plate setup template files tpl You can use plate setup template files tpl to assist you when you want to define multiple plates using similar plate setup parameters A tpl file can contain the s
37. the nitrogen pressure to finish flushing the matrix from the capillaries range 1 300 s User Input Time The time Seconds you need to open and close the anode and cathode drawers after the display on the instrument gives the instruction to load the plate tank or tubes in either the anode or the cathode side of the instrument range 10 240 s Preinjection Time The time seconds for preinjecting samples range 1 600 s PMT2 Voltage The voltage for PMT2 range 450 950 V Sleep After This Run The check box that you check if you want the instrument set in sleep mode after the current or next run This parameter is not saved in the template Sleep Time The length of time hours the instrument will be left idle The lasers will be turned off and the temperature in the electrophoresis compartment will be reduced range 1 72 h or 1 16 h if used with Sleep After This Run MegaBACE Instrument Administrator s Guide p4 13 Parttwo Understanding and configuring the MegaBACE system oo 4 3 1 Creating an instrument control parameter template TheM egaBACE software includes instrument control parameter templates that you can use You can modify the existing templates to create new templates Important The edit mode must be turned on to allow creating editing saving or importing instrument control parameter templates See section 6 4 for information about enabling the edit mode in the MegaBACE ini file
38. 0 600 s Run voltage The range is 1 20 kV Run time The range is 1 720 min Chemistry The name of a chemistry parameter set The software retrieves the parameters from the Chemistry ini file Optional Run temperature The temperature used for a special run Note This value will override the value defined in an icp file or the Instrument Control window Optional PMT1 voltage The PMT1 voltage for a special run Note This value will override the value defined in an icp file or the Instrument Control window MegaBACE Instrument Administrator s Guide p5 5 Parttwo Understanding and configuring the MegaBACE system a Table 5 1 The plate setup data file psd field names continued Field name Value Optional PMT2 voltage The PMT2 voltage for a special run Note This value will override the value defined in an icp file or the Instrument Control window Optional Base caller Sequencing only The name of a plate specific base caller that is different from the default base caller specified in the MegaBACE ini file Comment The comments about the plate that you want to include Well_ID or Well_ID NAME for example A01 The sample name that you want to assign to each well location ID and use as the rsd file name default for example Samplel1 Note If you want to use names other than sample names as the file names you must also use the Well_ID FILE field The fo
39. 0 and flexible MegaBACE 1000 instruments only 3 2 3 Anode end of the capillaries Array positions with capillaries installed At the anode end of the capillaries each reservoir contains an array of 16 capillaries and an electrode The instrument applies a voltage pulse to inject all samples simultaneously Each capillary has a clear detection window through which the detection system scans the sample during the electrophoresis run The window is located at a fixed distance from the sample loading point so that the components of a sample are detected at a constant separation distance Empty array positions MegaBACE 500 and flexible MegaBACE 1000 instruments only At the anode end of any empty array position the reservoir contains an anode sleeve and an anode blocker For each empty array position a window blank is installed in place of the missing capillary window The window blanks are required for optimum performance of the instrument confocal optical system section 3 3 SSS p3 4 MegaBACE Instrument Administrator s Guide Chapter 3 How the MegaBACE instrument works 3 2 4 Replaceable sieving matrix The sieving matrix within the capillaries is a flowable noncross linked polymer network Therefore you can replace the matrix without replacing the capillary array You can empty and refill the capillaries rapidly using an automated pressure controlled process 3 3 Confocal optical system The M egaBACE instrument uses a patented con
40. 110 G 2 520DF20 ET ROX C 3 610LP B 595DRLP ET TAMRA A 4 585DF20 Table A 4 lists the dyes beamsplitters and filters for a four color DN A sequencing run using the ET dye primers Table A 4 Beamsplitters and filters for ET dye primers ET primer ET primer dye base order Channel Filter Beamsplitter ET R6G A 1 555DF20 A 540DRLP ET R110 C 2 520DF20 ET ROX T 3 610LP B 595DRLP ET TAMRA G 4 585DF20 The base order changes for the different chemistries but the display colors for the bases in the sequence and the traces in the electropherograms remain fixed The display colors are A green e C blue e T red G yellow or black N ote Although the conventional color representation for G is yellow the trace is displayed in black for ease of viewing Figure A 5 shows the spectral channels used to detect each base for the sequencing dye sets pA 8 MegaBACE Instrument Administrator s Guide a DNA sequencing DYEnamic ET dye terminators Spectral channel 2 ug Spectral channel 4 g Filter 2 520DF20 Filter 4 585DF20 488 nm blue laser Emitted light return scan Beamsplitter B 595DRLP 488 nm blue laser Emitted light forward scan Beamsplitter A 540DRLP b DNA sequencing ET dye primers Spectral channel 2 Spectral channel 4 u ug Filter 2 Filter 4 520DF20 585DF20 488 nm blue laser Emitted light return scan Beamsplitter B 595DRL
41. ATION ALLOW ABD OVERRIDE AUTOMATIC FASTA FILE CREATION ALLOW FASTA OVERRIDE AUTOMATIC SCF FILE CREATION ALLOW SCF OVERRIDE AUTOMATIC RAW TEXT FILE CREATION ALLOW RAW TEXT OVERRIDE AUTOMATIC PROCESSED TEXT FILE CREATION ALLOW PROCESSED TEXT OVERRIDE b Sair ar a aaae Figure 6 5 The MegaBACE ini file with genotyping designated as the default application The Instrument Control M anager displays only the applicable parameters for the selected application For example only the chemistry names for the selected application appear in the Plate Setup window figure 6 2 and only the sequencing plate IDs appear in the Plate Catalog figure 4 2 If you change the default application in the MegaBACE ini file you must close the Instrument Control Manager and restart it in order for the default application change you made in the MegaBACE ini file to take effect Alternatively you can change applications from the Configure menu in the Instrument Control M anager For more information on changing applications using the Instrument Control M anager see the instrument operator s guide MegaBACE Instrument Administrator s Guide 6 17 Parttwo Understanding and configuring the MegaBACE system Ed 6 4 7 Changing the number of arrays MegaBACE 500 and flexible MegaBACE 1000 instruments only Initially M egaBACE System Technical Support sets the number of arrays for your instrument Thereafter you can change the number of installed
42. About the extended header section E 1 Extended header labels in raw sample data files rsd section E 2 About the bar code section E 3 e About the extended header in base called sample data files esd section E 4 E 1 About the extended header The extended header is a variable length variable content data block that is encoded using the Amersham Biosciences self describing data protocol appendix F All entries in the extended header are label value pairs The label is always an ASCII string The values may be in any supported format The value associated with a label may be an array or a list The entries in a list are also packed as label value pairs Therefore the length of any list is always double the number of entries in the list The entries in the extended header are alphabetized by label The entries within a list are also alphabetized The application programming interface API for retrieving values from the extended header accesses items in a list using a dot delimited hierarchical name For example CHEMISTRY CHANNEL1 BASE would return the value associated with the label BASE from the CHAN NEL1 list inside the CHEMISTRY value Labels are treated as case independent Any user defined data imported from a plate setup data file psd appendix B will be included in the extended header MegaBACE Instrument Administrator s Guide pE 1 Part three Appendixes E 2 Extended header labels in raw sa
43. CE Templates folder instrument control parameter file icp is used as a template to specify a set of parameters that control the instrument MegaBACE Instrument Administrator s Guide pC 7 Appendix D Standard header format Figure D 1 in this appendix provides the format of the standard header format for the M egaBACE raw sample files rsd Offset Size Type Variable 0 24 char m_tcSampleName 24 deprecated 24 30 char m_tcOperator 30 unused 54 4 float m_fRunVoltage 58 4 float m_fRunTime minutes 62 4 float m_fInjectionTime 66 4 float m_fInjectionVoltage 70 4 float m_fTemperature 74 4 int m_nLaserIndex deprecated 78 4 float m_fPMTVoltagel 82 4 float m_f PMTVoltage2 86 30 char m_csBeamSplitterA 30 deprecated 116 30 char m_csBeamSplitterB 30 deprecated 146 30 char m_csFilter1 30 deprecated 176 30 char m_csFilter2 30 deprecated 206 30 char m_csFilter3 30 deprecated 236 30 char m_csFilter4 30 deprecated 266 4 float m_fFlushTime 270 4 float m_fFlushTimel 274 4 float m_fFlushTime2 278 4 float m_fFlushTime3 282 4 float m_fPrerunVoltage 286 4 float m_fRelaxationTime 290 4 float m_fPrerunTime 294 64 float m_fSpecSepMx 4 4 358 40 char m_csBaseOrder 4 10 deprecated 398 22 char m_InstID 22 420 30 char m_csParamSetName 30 deprecated 450 4 DWORD m_dMagicNumber 826561349 454 4 DWORD m_dExtHeaderOffset n
44. Control M anager to automatically call bases after each plate has been run and automatically export the base called data files to other file formats ABD FASTA SCF and ASCII text 4 4 1 Manually turning on or off the automatic base calling mode and exporting data You can use the Automatic Base Calling command in the Configure menu to manually turn on or off the automatic base calling mode select a default base caller and to select file formats for exporting data Alternatively you can turn on or off the automatic base calling mode and the automatic file creation export and specify a default base caller in the M egaBACE ini file section 6 4 The default base caller that you specify in the Base Calling window is active only until you close the Instrument Control Manager The next time you open the software the default base caller specified in the MegaBACE ini file is active N ote To use the Automatic Base Calling command in the Configure menu Allow Base Calling O verride must be set to Y yes in the M egaBACE ini file section 6 4 To change the automatic base calling mode and select file formats for data export 1 Choose Automatic Base Calling from the Configure menu The Automatic Base Calling window appears figure 4 8 2 Select the Automatic Base Calling check box to turn on the automatic base calling mode if it is not already selected A check mark appears in the check box to indicate the automatic base calling
45. E 3 Appendix F APB protocol for self describing data This appendix describes a protocol for packing self describing data into a file The topics are Introduction section F 1 e The 1 byte header section F 2 e Length value section F 3 e Data buffer section F 4 e Notes about the protocol section F 5 F 1 Introduction The extended header consists of a list of keyword value pairs H owever the protocol is completely general and can be used to pack data of almost any form In the protocol a data block consists of a series of elements where each element contains e A 1 byte header that contains two fields A length value that consists of 1 to 3 bytes except for Terminator which is always zero as determined by the number of length bytes field in the header e A data buffer that contains the number of bytes specified in the length value F 2 The 1 byte header The 1 byte header contains two fields e A 6 bit format field that defines the type of data contained in the element The format field consists of the most significant 6 bits of the header byte A 2 bit field that states the number of length bytes used to specify the size of the data storage The number of length bytes field consists of the least significant 2 bits of the header byte This field is zero for Terminator For all other formats it must be nonzero Figure F 1 shows the protocol MegaBACE Instrument Administrator s Guide
46. H I Firmware I LogFiles C Psd Contains psd files LJ Scan Sels CI Templates Contains tpl and 8 object s EJ My Comput Z icp files Figure C 1 Storage locations of the system initialization files and the parameter configuration files Table C 4 The system initialization files File name Description Location MegaBACE ini Defines the options available MegaB ACE DataSystem folder in the Instrument Control Manager Chemistry ini Defines the available chemistry _ MegaBACE DataSystem folder parameter sets including the dyes bases filters and laser modes pC 6 MegaBACE Instrument Administrator s Guide Appendix C File Storage C 4 Parameter configuration file storage Figure C 1 shows the system initialization file and parameter configuration file locations Table C 5 lists the parameter configuration files that you can use Table C 5 The parameter configuration files File extension Description Storage location psd Used for a given plate a MegaBACE Psd folder plate setup data file psd allows you to specify plate ID plate setup parameters and plate and well level attributes tpl Used for multiple plates a MegaBACE Templates folder plate setup template file tpl allows you to specify the plate setup parameters The tpl file uses the same field names as the psd file for the plate setup parameters icp For use in multiple runs an MegaBA
47. NLY ADD EXTENSIONS TO EXPORTED FILE NAMES EXPORT TO SUBFOLDERS CLEANUP COMMAND DEFAULT PLATE SETUP TEMPLATE DEFAULT INSTRUMENT TEMPLATE Normal ALLOW DUPLICATE SAMPLE NAMES Y AUTOMATIC BASE CALL COMMAND fAutoBasecall exe fAP etc Z2zeczezezezecc lt Base Callers Molecular Dynamics Sqer d1l Cimarron 1 31 CimBc131_P d11 Cimarron 1 53 Phat CimBc153_beautify d11 Cimarron 1 53 Slim Phredify CimBc153_phredify_noPuff d11 Cimarron 2 1968 Phat CimBc 62918 beautify d11 Cimarron 2 1968 Slim Phredify CimBc62918 phredify_noPuff d11 Bad Capillaries Figure 6 3 An example of the MegaBACE ini file configured for sequencing p6 6 MegaBACE Instrument Administrator s Guide Chapter 6 Using the system initialization files Table 6 1 Sections in the MegaBACE ini file Section name Description General Lists the basic settings the Instrument Control Manager uses to run the instrument Base Callers Lists all the base callers available in the Instrument Control Manager Arrays MegaBACE 500 and flexible MegaBACE 1000 instruments only Defines the number of installed capillary arrays for your instrument model Bad Capillaries In this section you can list the well IDs for bad capillaries The Instrument Control Manager does not create data files for capillaries that are empty broken or clogged For every capillary listed as bad a red arrow appears above the capillary button in the Run Image window figure
48. P 488 nm blue laser forward scan Emitted light Beamsplitter A 540DRLP Appendix A Fluorescence imaging Filter 3 cn 610LP PMT 1 Filter 1 ugh 555DF20 Filter 3 ugr 610LP PMT 1 Filter 1 yr 555DF 20 Spectral channel 3 Spectral channel 1 Spectral channel 3 Spectral channel 1 Figure A 5 The MegaBACE spectral channels with the beamsplitter and filter configurations for the different sequencing chemistries a DYEnamic ET dye terminators and b ET dye primers MegaBACE Instrument Administrator s Guide pA 9 Part three Appendixes A 6 Beamsplitters and filters for genotyping Tables A 5 and A 6 and figure A 6 show the beamsplitters and filters for the genotyping dye sets G enotyping filter set 2 is included as part of the M egaBACE genotyping system Genotyping filter set 1 is available as an additional purchase Table A 5 Genotyping filter set 1 Dye set 1 Channel Filter number Filter Beamsplitter ET ROX size standard Red 1 1 610DF 20 A 540DRLP FAM Blue 2 2 520DF20 HEX Yellow or Black 3 3 565DF 20 B 555DRLP TET Green 4 4 545DF20 Table A 6 Genotyping filter set 2 Dye set 2 Dye set 2 alternatives Channel Filter number Filter Beamsplitter ET ROX ET ROX ET ROX Red 1 1 610DF20 A 540DRLP FAM FAM FAM Blue 2 2 520DF20 NED TMR TMR Yellow or Black 3 3 580DF20 B 570DRLP HEX HEX JOE Green 4 4 555DF20 TE pA 10 M
49. afety Ed 1 5 2 The parameter configuration files The Instrument Control M anager uses two different kinds of parameters Plate setup parameters Include the electrophoresis conditions the chemistry and optionally the sample names for a plate Instrument control parameters A combination of settings defining the instrument run conditions and matrix fill and flush cycles Plate setup parameters You can manually specify the parameters using the Plate Setup window of the Instrument Control M anager chapter 4 Alternatively you can use the following files either in combination or individually Plate setup data file psd Used for a specific plate a psd file can specify plate ID plate setup parameters plate attributes and well attributes such as file name or a well specific base caller See section 5 3 for details on creating a psd file Plate setup template tpl Used for multiple plates a tpl file can specify only the plate setup parameters See section 4 2 for a description of how to create a plate setup template from the Plate Setup window and see section 5 4 for a description of how to create a tpl file using a text editor or Excel Instrument control parameters You can manually specify the instrument parameters using the Instrument Control window chapter 4 Alternatively you can use an instrument control parameter template icp file to specify the parameters for use in multiple runs See se
50. aging Because each of the energy transfer primers in the set is efficiently excited by the laser for example FAM by 488 nm the effective emitted signal strengths are greatly increased compared to the signal from the primers labeled with the corresponding single dyes J ingyue et al 1996 The ET primers can be substituted directly for traditional primers in all dye primer sequencing applications Because the electrophoretic mobility of the ET primers depends on the spacing between the donor and acceptor Ju et al 1995 it is often possible to select a set of four primers with similar electrophoretic mobility When using ET primer chemistry the ET dyes are attached to the primers When using the DY Enamic ET terminators the dyes are attached to the four chain terminators Each of the four dideoxynucleotides ddG ddA ddT or ddC has a different acceptor dye coupled with the fluorescein donor The acceptor dyes then emit light at their characteristic wavelengths The fluorescence is detected by the instrument allowing identification of which nucleotide caused the termination event This results in a sequencing method that is very sensitive and robust The acceptor dyes are the same standard rhodamine dyes used in DY Enamic ET primers rhodamine 110 rhodamine 6 G tetramethyl rhodamine and rhodamine X M aintaining the standard rhodamine dyes as acceptors allows these reaction products to be detected using the same filter set as th
51. ame plate setup parameters as the psd file 5 4 1 About the tpl files You can specify a default tpl file The default tp will be automatically imported when an operator clicks N ew in the Plate Setup window If the edit mode is turned on a tpl file can also be selected from the Plate Setup Templates menu section 4 2 4 You can create and save tpl files in the Plate Setup window sections 4 2 2 and 4 2 3 or you can create a tpl file using any text editor or Excel Appendix B provides examples of sequencing and genotyping tpl files You must use the field names that the Instrument Control M anager recognizes and type them in the first column field column as shown in figure 5 6 You type the values for each field in the second column value column The Instrument Control M anager uses only the values for the field names it recognizes See table 5 1 for a list of the recognized fields for the plate setup parameters N ote If you enter the optional parameters the values will override the values for the same parameters defined in the Instrument Control window StdGenotyping_1 tpl Notepad Ol x File Edit Search Help INJECTION VOLTAGE 3 INJECTION TIME 45 RUN VOLTAGE 16 RUN TIME 75 __ CHEMISTRY GT DyeSet2 ET ROX FAM NED HEX A PMT1 VOLTAGE 750 Optional PMT2 VOLTAGE 750 parameters _lRuN TEMPERATURE 44 COMMENT Genotyping Default Figure 5 6 An example of a plate setup template file tpl for genotyping
52. ands window appears figure 7 3 4 In the Override Commands window Select the commands you want to run You can click Deselect All and select the desired commands Alternatively you can deselect only the commands you do not want to run e Optional Click Save Selection to save a specific set of commands you want to re select again later You can save one set of selected commands for each protocol To re select a previously saved set of selected commands click Load Selection Click OK to close the window In the Instrument Control window figure 7 1 the selected protocol flashes to show that it is running MegaBACE Instrument Administrator s Guide p7 3 Part three Appendixes Appendix A Fluorescence imaging This appendix describes how the fluorescence signal is generated and how the optical system of the M egaBACE instrument records emissions from the fluorescent dyes The topics are e How fluorescence is generated section A 1 e Choosing the dyes for multicolored experiments section A 2 e MegaBACE excitation modes section A 3 e How theM egaBACE records the fluorescent signal section A 4 e Beamsplitters and filters for sequencing section A 5 e Beamsplitters and filters for genotyping section A 6 A 1 How fluorescence is generated Fluorescent dyes absorb light of specific wavelengths The absorbed energy boosts an electron to a higher energy level When the electron falls back to a lower e
53. arameter setup B 1 B 2 B 3 B 4 Plate setup parameters for sequencing 00eee ee eaee B 1 B 1 1 Plate setup data files psd for sequencing B 1 B 1 2 Plate setup parameter template files tpl for sequencing B 3 Instrument control parameters for sequencing 4 B 4 Plate setup parameters for genotyping 00 e ee eae B 5 B 3 1 Plate setup data files psd for genotyping B 5 B 3 2 Plate setup parameter template file tpl for genotyping B 8 Instrument control parameters for genotyping 4 B 8 Appendix C File Storage C 1 C 2 Raw sample data storage 1 eee C 1 C 1 1 Raw sample data file content 0 000 C 1 C 1 2 Naming and storage conventions for raw sample data files and TOES 12 ikea eda sae beneath TERE SAA C 1 Base called sample data storage sequencing only C 3 C 2 1 Naming and storage conventions for the base called and exported files 0 tees C 3 C 2 2 Naming and storage conventions for the analyzed run folders tiv cled a hed eee a ia ena hides Qaida dha a C 5 MegaBACE Instrument Administrator s Guide ix Table of contents C 3 System initialization file storage 6 a C 4 Parameter configuration file storage 00 eee eee Appendix D Standard header format Appendix E Extended header format E 1 About the extended header 0 00sec ee ees E 2 Extended header labels i
54. ase contact M egaBACE System Technical Support for a free replacement label While waiting for the replacement label copy the label from the appropriate figure in this chapter and attach the copy of the label to the instrument 2 3 Cathode and anode compartments and instrument displays When the workflow requires you to access the cathode or anode compartment the system shuts off the high voltage and nitrogen pressure and lowers the cathode or anode stage before unlocking the corresponding drawer Do not overfill the water tank Open and close the cathode drawer slowly Remove any liquid that has been spilled in and around the plate holder Failure to remove the spilled liquid can result in damage to the instrument Figure 2 5 shows the liquid spillage caution label Figure 2 2 shows the location of the label on top of the cathode slider inside the cathode drawer After you open the cathode or anode drawers the displays on the front of the instrument instruct you to perform the next step MegaBACE Instrument Administrator s Guide p2 5 Partone Introduction and safety Warning A Achtung No voltage pressure or laser light can be applied as long as either drawer remains open When you close the cathode or anode drawer the software assumes that you have performed the step displayed on the instrument display The drawer locks and the system raises the stage The system automatically moves to the next step CAUTION To
55. avoid instrument damage Open and close drawer slowly Remove any spilled liquid located in and around plate holder Do not overfill containers Figure 2 5 The liquid spillage caution label 2 4 Electrophoresis compartment You may occasionally need to open the electrophoresis compartment lid When the electrophoresis compartment lid is open do not place your hands on or near the two support bars on each side of the lid If the lid moves your fingers can be pinched WENN DER DECKEL DER ELEKTROPHORESE KAM MER GE FFNET IST FINGER NICHT AN ODER AUF DIE BEIDEN HALTESCHIENEN AUF JEDER SEITE DES DECKELS LEGEN WENN DER DECKEL SICH BEWEGT KONNEN FINGER EINGEKLEMMT WERDEN The label shown in figure 2 6 warns of this pinching hazard Figure 2 2 shows the locations of two pinching hazard labels one on each side of the top portion of the instrument Figure 2 6 The pinching hazard label p2 6 MegaBACE Instrument Administrator s Guide Warnings A Cautions Chapter 2 Safety precautions Under normal operating conditions you are protected from high voltage Nevertheless during the prerun and sample electrophoresis voltages up to 20 kV are present in the electrophoresis compartment The label in figure 2 7 warns of this danger and is located on the left side of the instrument on the side wall inside the electrophoresis compartment and on the photomultiplier tube PM T cover Figure 2 2 shows the lo
56. aw data the data collected by the instrument that have not been sequenced or genotyped The Instrument Control M anager software creates a raw run folder for the raw sample data files rsd for each plate you run raw run folder the folder that contains the raw sample data files rsd for a given run The Instrument Control M anager software uses the plate ID and run ID to name the folder The Instrument Control M anager stores the raw run folder containing the associated rsd files in the M egaBACE Data folder default or the location you specify run the process of injecting and scanning a plate of samples on the M egaBACE instrument and detecting the resulting signal from each capillary Each run has a unique date and user ID MegaBACE Instrument Administrator s Guide Glossary 3 Glossary run folder see analyzed run folder or raw run folder run ID a unique designation the Instrument Control M anager software assigns to each run of a sample plate on the instrument sample files see rsd files For sequencing see also abd files esd files seq files and scf files sample names the designation you provide for a given well in the sample names tab or in a plate setup template or psd file scan number a number representing a sampling of the data during the run and describing the location of a data point Instrument Control M anager samples the data continuously during a run at a rate of 1 75 Hz 105 t
57. b shows a psd file that includes analysis parameters for the plate and for each well The attributes that you include in the psd file become part of the extended header for the rsd file MegaBACE Instrument Administrator s Guide pB 7 Part three Appendixes B 3 2 Plate setup parameter template file tpl for genotyping Figure B 8 shows an example of a plate setup parameter template file tpl that you can use for genotyping This example file StdG enotyping tpl is included with the M egaBACE genotyping system software The file is located in the M egaBACE Templates folder B StdGenotyping tpl Notepad OF x File Edit Search Help INJECTION VOLTAGE INJECTION TIME RUN VOLTAGE RUN TIME CHEMISTRY GT DyeSet2 ET ROX FAM NED HEX COMMENT Genotyping Default Figure B 8 A plate setup parameter template file tpl for genotyping B 4 Instrument control parameters for genotyping Figure B 9 shows an example of a genotyping instrument control parameter file icp The Genotyping icp file shown in figure B 9 is included with the M egaBACE genotyping system software The file is located in the M egaBACE Templates folder E Genotyping icp Notepad Iof x File Edit Search Help HIGH PRESSURE TIME Figure B 9 An instrument control parameter template file icp for genotyping pB 8 MegaBACE Instrument Administrator s Guide Appendix C File Storage This appendix describes Raw sample data
58. bleshooting G uide 2 7 Chemicals Use good laboratory procedures and follow the manufacturer s precautions when working with chemicals Amersham Biosciences is not responsible or liable for any damages caused by or as a consequence of the use of hazardous chemicals 2 8 Nitrogen cylinders and pressure regulators The M egaBACE system requires the use of high pressure nitrogen sources 2 8 1 Handling high pressure cylinders and tubing Always use good laboratory procedures when handling a high pressure cylinder and follow any instructions provided with the cylinder High pressure connection Do not disconnect tubing without bleeding the tubes Disconnecting without bleeding can cause injury HOCHDRUCKVERBINDUNG SYSTEM ENTLUFTEN BEVOR SCHLAUCHVERBINDUNG GELOST WIRD LOSEN DER VERBINDUNG OHNE ENTLUFTEN KANN ZU VERLETZUNGEN F HREN The label in figure 2 8 warns of this danger Figure 2 3 shows the location of the label on the back of the instrument MegaBACE Instrument Administrator s Guide p2 9 Partone Introduction and safety Figure 2 8 The nitrogen pressure general hazard label Warning Make sure a standard cylinder bracket is bolted to a solid permanent structure in A a manner that meets or exceeds all local seismic and safety code requirements 2 8 2 Regulating the nitrogen pressure The regulators on the external nitrogen cylinder s control the amount of nitrogen pressure applied within the instrument The
59. bout creating plate setup and instrument parameter templates using the Instrument Control M anager windows See chapter 5 for details about creating the templates or a plate setup data file using a text editor or Excel B 1 Plate setup parameters for sequencing This section provides examples of plate setup data files psd and plate setup template files tpl that you can use for sequencing These example files are included with the M egaBACE system sequencing software The files are stored in the following locations e The psd files are stored in the M egaBACE Psd folder The tpl files are stored in the M egaBA CE Templates folder B 1 1 Plate setup data files psd for sequencing ET terminators Figure B 1 shows a psd file containing plate setup parameters for sequencing with ET terminator chemistry MegaBACE Instrument Administrator s Guide pB 1 Part three Appendixes Ej Sample1 psd Notepad iof x File Edit Search Help Plate ID FB64 cc_x3 RUN TIME 126 RUN VOLTAGE 8 INJECTION TIME 36 INJECTION VOLTAGE 2 RUN TEMPERATURE 44 COMMENT Mouse BAC Samples PHT2 VOLTAGE 750 PHT1 VOLTAGE 750 CHEMISTRY ET Terminators BASE CALLER Cimarron 1 53 Slim Phredify A81 FBO4_ TSA_A01 x03 B61 FB64 TSA_B61 x63 C61 FBO4 TSA_C61 x63 D81 FB64 TSA_D61 x63 gt Figure B 1 A psd file for sequencing with ET terminators ET primers Figure B 2 shows a psd file containing plate setup parameters for sequencin
60. c Base Calling window The base caller you select in the Automatic Base Calling window is active only until you close the Instrument Control M anager The next time you open the software the default base caller specified in the M egaBACE ini file section 6 4 is active ESSE p4 16 MegaBACE Instrument Administrator s Guide Chapter 4 Using the Instrument Control Manager windows to configure the system To select a default base caller in the Automatic Base Calling window 1 Choose Automatic Base Calling from the Configure menu The Automatic Base Calling window appears figure 4 8 2 Select the Automatic Base Calling check box and then select an appropriate base caller for your application 3 Click OK Specifying a plate specific base caller You can use the plate setup data file psd to specify a plate specific base caller See section 5 3 for details You can also use the O ptional Parameters tab in the Plate Setup window figure 4 5 to specify a plate specific base caller MegaBACE Instrument Administrator s Guide p4 17 Chapter 5 Using the parameter configuration files This chapter describes the parameter configuration files used by the Instrument Control M anager The topics are About the parameter configuration files section 5 1 Parameter configuration file format section 5 2 Using plate setup data files psd section 5 3 Using plate setup template files tpl section 5 4 e Using i
61. cations of the label HAZARDOUS VOLTAGES MAY BE PRESENT IF INTERLOCKS ARE DEFEATED WARNING 5 WARNING HAZARDOUS VOLTAGES MAY BE PRESENT IF INTERLOCKS ARE DEFEATED ACHTUNG GEF HRLICHE SPANNUNG ZUG NGLICH WENN SICHERHEITSVERRIEGELUNG BERBR CKT Figure 2 7 The high voltage warning label The instrument has sensors and interlocks that are designed to protect you from moving parts high pressure hazardous voltage or laser light Do not defeat the sensors or interlocks Do not remove panels for any reason Exposure to these hazards can cause severe or fatal injury Check the operation of the interlock on the electrophoresis compartment lid periodically to make sure the interlock is functioning properly When you replace capillary arrays do not pull on the capillaries to release the cathode bar or the anode plug The capillaries are fine glass tubes and can break leaving sharp ends or fragments which can damage the instrument or cause injury Do not leave any objects inside the electrophoresis compartment or on the stages Metal objects can cause arcing when high voltage is applied during electrophoresis possibly damaging the instrument Always avoid touching the windows of the capillaries Oils and salts from your skin could result in arcing between capillaries during high voltage electrophoresis which could damage the instrument Avoid spills in the chamber and below the cathode stage Cl
62. cing only 6 4 4 Exporting the base called data to other file formats Sequencing only 6 cece eee eens 6 4 5 How automatic file export works sequencing only 6 4 6 Using multiple applications 0 0 00 0c 6 4 7 Changing the number of arrays MegaBACE 500 and flexible MegaBACE 1000 instruments only Chapter 7 Bypassing or overriding a protocol 7 1 7 2 Bypassing the selected protocol 0 0 cece cece eee ees Overriding the contents of a protocol 0 00 cee eee ee viii MegaBACE Instrument Administrator s Guide Table of contents Part three Appendixes Appendix A Fluorescence imaging A l A 2 A 3 A 4 A5 A 6 How fluorescence is generated cece eee eee ens A 1 Choosing the dyes for multicolored experiments 0 00 ee A 2 A 2 1 About energy transfer dyes 0 cece eee A 2 A 2 2 The MegaBACE sequencing ET dye kits A 2 A 2 3 The MegaBACE genotyping dye sets 0 05 A 3 MegaBACE excitation modes 1 0 cece cee eens A 5 How the MegaBACE records the fluorescent signal A 5 A 4 1 Emission beamsplitters 00 0 0 00 cece eee A 6 A 4 2 Emission filters 0 2 cee A 7 AAS P MIS sie ots ee aaa see dad Danae ate eds Gna be A 7 Beamsplitters and filters for sequencing 0 neues A 8 Beamsplitters and filters for genotyping 0 0 ee A 10 Appendix B Plate and instrument p
63. comes the real plate ID in the Plate Setup window figure 5 2b For example The plate ID you scanned into the Plate Setup window 10303385858 The plate setup data file name 10303385858 psd The plate ID opposite the Plate ID field name inside the psd file M13std40_092499 The actual plate ID that appears in the Plate Setup window M13std40_092499 figure 5 2b a Plate setup data file psd psd file name E 10303385858 psd Notepad File Edit Search Help Plate ID RUN TIME RUN VOLTAGE INJECTION TIME INJECTION VOLTAGE RUN TEMPERATURE COMMENT PHT2 VOLTAGE PHT1 VOLTAGE b Plate Setup window Plate ID Plate ID HA Instrument Control Manager MegaBACE Sequencing File View Options Templates Configure Help N13std46_ 692499 126 8 30 2 44 Mouse BAC Samples 750 750 ET Terminators Cimarron 1 53 Slim Phredify FB64 TSA_AGB1 x63 FB64 TSA_B61 x63 FB64 TSA_C61 x63 Plate Catalog FBO4 cc x3 M13std40_092499 Electrophoresis Parameters Cher Sample Injection Voltage 2 E Run Voltage fe Field column Figure 5 2 Replacing the sca the file b The Plate Setup win p5 4 MegaBACE Instrumen Value column nned plate ID a Example of a psd file for sequencing with a plate ID listed in dow with a plate ID that replaced the original bar code that was scanned t Administrator s Guide Chapter 5 Using the parameter configuration file
64. continuously varying signal which is converted to a digital signal that is displayed on the monitor and stored in the sample files for the run Laser Mirror Primary beamsplitter Objective lens Capillary detection window Achromatic lens Aperture PMT oo N Oon A WN b c Figure 3 4 Basic confocal laser light path in the MegaBACE instrument as viewed from above a Elements in the light path b The light path from laser to sample c The light path from sample to PMT MegaBACE Instrument Administrator s Guide p3 7 Parttwo Understanding and configuring the MegaBACE system e 3 3 4 Advantages of confocal laser scanning Confocal detection results in effective elimination of background noise and fluorescent flare from adjacent areas in the capillary The confocal aperture acts as a Spatial filter that allows only the light originating from a volume around the focal point to pass thus rejecting scattered light The collimated beam of the laser light source provides more precise focus for greater resolution The beam is focused down to a small spot for intense illumination of the sample Each laser emits only one wavelength monochromatic light With the monochromatic laser light the excitation light can be separated efficiently from the emitted light p3 8 MegaBACE Instrument Administrator s Guide Important Chapter 4 Using the Instrument Control Manager windows to configure
65. cs e Cp All Cimarron base callers Table C 3 Naming conventions for the analyzed run folders Folder type Folder name Storage location Analyzed PlatelD_Mn_nnn_MD1 MegaB ACE AnalyzedData folder default Molecular Dynamics base caller Analyzed PlatelD_Cpn_nnn_MD1 MegaB ACE AnalyzedData folder default Cimarron 1 53 Phat base caller Analyzed PlatelD_Cpn_nnn_sl_ph_MD1 _ MegaBACE AnalyzedData folder default Cimarron 1 53 Slim Phredify base caller Analyzed Cimarron 1 31 PlatelD_Cpn_nnn_MD1 MegaB ACE AnalyzedData folder default Exported ABD Abd PlatelD_basecallerlD folder default Exported SCF Scf PlatelD_basecallerlD folder default Exported text Text PlatelD_basecallerlD folder default SSS MegaBACE Instrument Administrator s Guide pC 5 Part three Appendixes C 3 System initialization file storage Figure C 1 shows the system initialization file and parameter configuration file locations Table C 4 lists the system initialization files that you can modify to configure the Instrument Control M anager N Exploring C Program Files Molecular Dyn B EG Eie Edit View Go Favorites Tools Help es gt Aly amp Back Forvard Up Cut Copy Address C Program Files Molecular Dynamics MegaBAC x Folders x Ei Molecular Dynamics aj B C MegaBACE H I nalyzedData J Bin H I Data ste Contains Chemistry ini and MegaBACE ini files
66. ction 4 3 for a description of how to create an instrument control parameters template from the Instrument Control window See section 5 5 for a description of how to create an icp file using a text editor or Excel p1 8 MegaBACE Instrument Administrator s Guide Warnings A Chapter 2 Safety precautions The M egaBACE instrument and its accessories have been designed for safe operation It is imperative that you follow the precautions in this chapter The topics are e General safety precautions section 2 1 e Locations of important labels section 2 2 e Cathode and anode compartments and instrument displays section 2 3 Electrophoresis compartment section 2 4 Filter compartment section 2 5 e Internal electronics section 2 6 e Chemicals section 2 7 Nitrogen cylinders and pressure regulators section 2 8 Lasers section 2 9 e PM Ts section 2 10 e Power supply fan module computer and monitor section 2 11 System electrical connections section 2 12 Serial number labels section 2 13 e Service for the M egaBACE instrument section 2 14 2 1 General safety precautions While using the M egaBACE instrument you should follow the laboratory procedures appropriate for the experiments you are performing The operator of the MegaBACE instrument is assumed to be trained in the correct operation of the instrument and the safety issues Throughout the MegaBACE instrument documentation the
67. d 2 14 MegaBACE Instrument Administrator s Guide 2 15 Part two Understanding and configuring the MegaBACE system Chapter 3 How the MegaBACE instrument works This chapter explains how the M egaBACE instrument uses capillary array electrophoresis and a confocal optical system to collect data for DNA analysis The topics are e Overview of capillary array electrophoresis section 3 1 e About the capillaries section 3 2 e Confocal optical system section 3 3 3 1 Overview of capillary array electrophoresis Capillary electrophoresis uses narrow bore capillaries filled with a separation matrix An electric field causes the samples to migrate through the capillaries During electrophoresis the DN A fragments in the sample are separated by size with the shorter fragments moving faster than the longer fragments One advantage of this format is that the large surface to volume ratio of the capillary allows efficient removal of the heat generated during electrophoresis This allows the instrument to apply higher voltages which produces more rapid fragment migration and shorter electrophoresis run times Another advantage is that the capillary requires only a small amount of sample and loading is easily automated 3 2 About the capillaries The M egaBACE instrument uses up to six arrays of 16 capillaries total of 96 to provide rapid parallel separation of multiple samples Depending on your instrument model the capilla
68. default plate setup template You can set a different template as the default template for defining multiple plates To do this 1 From the Templates menu point to Plate Setup Template and choose Set D efault 2 Select the template you want to use as the default and click Set Default The Instrument Control M anager designates the template you selected as the default template The next time you click N ew the values in the new default template appear in the Plate Setup window The template is active only until you close the Instrument Control Manager The next time you open the Instrument Control Manager the default template specified in the MegaBACE ini file section 6 4 is active 4 2 6 Clearing the default plate setup template You can clear the selected plate setup default template To do this from the Templates menu point to Plate Setup Templates and choose C lear D efault The template you cleared is no longer the default template When you click N ew the Instrument Control M anager clears the values in the parameter boxes You can select another default template section 4 2 5 or use the Plate Setup window to create another template save it and then select it as the default template MegaBACE Instrument Administrator s Guide p4 11 Parttwo Understanding and configuring the MegaBACE system a 4 3 Creating instrument control parameter templates in the Instrument Control window The instrument control parameters a
69. e section 6 4 5 5 Using instrument control parameter template files icp You use the instrument control parameter file icp as a template to load a predefined set of parameters into the Instrument Control window The software stores the file in the M egaBA CE Templates folder The icp file contains the parameters that control the instrument Appendix B provides examples of sequencing and genotyping icp files You can specify a default icp file in the M egaBACE ini file The default icp file is loaded into the Instrument Control window when an operator runs a plate If you turn on the edit mode an operator can also select a template from the Instrument Templates menu section 4 3 2 p5 12 MegaBACE Instrument Administrator s Guide Chapter 5 Using the parameter configuration files 5 5 1 About instrument control parameter template files icp You can create and save instrument control parameter templates icp using the Instrument Control window section 4 3 You can also create an icp file in a two column format in Excel or in any text editor using tabs as column separators See section 5 2 for a description of the file format You must use the field names that the Instrument C ontrol M anager recognizes and type them in the first column field column as shown in figure 5 7 You type the values for each field in the second column value column The Instrument Control M anager uses the values only for the fields it rec
70. e optical path The primary beamsplitter directs the beam of light toward the objective lens e The objective lens focuses the laser beam to a point e Atthe focal point the laser light excites the fluorescent dyes in the sample 2 The objective lens collects light emitted from the illuminated point and converts it to a collimated beam figure 3 4a c e Light from the illuminated point travels back through the objective lens The light follows in reverse exactly the same path the laser beam followed The objective lens focuses the point source of light in the sample to a collimated beam directed back toward the mirror on the scan head e Mirrors reflect this light to the beamsplitter where the emitted light is reflected toward a second achromatic lens p3 6 MegaBACE Instrument Administrator s Guide Chapter 3 How the MegaBACE instrument works 3 Theachromatic lens focuses the light to the confocal point and light in focus at the confocal point passes through the aperture figure 3 4a c e Light from the point observed in the sample is brought into focus again by the achromatic lens This second focal point is called the confocal point e The aperture is located at the confocal point The aperture rejects most of the light from points out of focus at the confocal point Light from the point in focus in all three dimensions x y and z passes through the hole to the PM T e ThePMT converts light to an analog
71. e specified with less operator intervention The operator can import the psd file either before the run in the Plate Setup window or during the run in the Select a Plate window M egaBACE Instrument O perator s G uide The parameters that you specify in the psd file become part of the extended header of each raw sample data rsd file created during the run Appendix B provides examples of sequencing and genotyping psd files 5 3 1 About the plate ID When an operator enters the text or bar code for a plate ID in the Plate Setup window or the Select a Plate window described in the instrument operator s guide the Instrument Control M anager looks for a psd file with a file name that matches the plate ID and automatically imports the plate setup parameters that are contained in the psd file An operator can enter a plate ID in the following ways e Type the text in the Plate ID box in the Plate Setup window or the Select a Plate window Scan a bar code into the Plate ID box in the Plate Setup window or the Select a Plate window p5 2 MegaBACE Instrument Administrator s Guide Chapter 5 Using the parameter configuration files SET Important Regardless of the method used to enter the plate ID the text entry or bar code must match the name case insensitive of the psd file in order for a psd file to be imported Figure 5 1 shows an example of a psd file and the Plate Setup window The psd file name matches the pla
72. e DY Enamic ET primers For mobility matching purposes however the ET terminator dyes are attached to different bases A 2 3 The MegaBACE genotyping dye sets In genotyping the simple sequence repeat SSR polymorphisms such as CA repeats and other microsatellite markers are widely used in linkage analysis and other genetic studies Large numbers of these markers have been identified and fluorescently labeled PCR primer sets are commercially available such as the PE Applied Biosystems Linkage M apping Sets or the Weber RG marker screening sets distributed by R esearch G enetics For efficient signal detection the dye sets used for these primers require excitation at two different wavelengths Because of this the M egaBACE dual laser mode is recommended for these marker panels To enhance the detection of the commercially available marker panels you should use a M egaBACE energy transfer ET dye labeled size standard ET400 R ET550 R or ET900 R The label on the M egaBACE ET size standard ET ROX can be efficiently excited by the blue laser The ET ROX label uses FAM as a donor dye and ROX as an acceptor dye The blue laser MegaBACE Instrument Administrator s Guide pA 3 Part three Appendixes emits light at 488 nm which is near the absorption maximum of FAM FAM absorbs the blue laser light and then transfers the energy to exciteROX By using the energy transferred from FAM ROX can be excited very efficiently with the blue
73. e or dye For example BASE 1 indicates the base detected through spectral channel 1 or DYE 1 indicates the dye detected through spectral channel 1 e Laser mode R equired field The available modes include blue green and blue or green Only the names of the chemistry parameter sets for the selected application sequencing or genotyping are available Electrophoresis Parameters Chemistry Parameters Sample Names Optional Parameters Comments Chemistry Name Green And Blue GT DyeSet2 ET ROX FAM NED HEX Laser Mode Channel Base Dye Filter Beamsplitter Eaters A JETRO Je100F20 ane RLP 1 2 3 NED 580DF20 H E al il Figure 6 2 The Chemistry Parameters tab of the Plate Setup window displaying a set of values for the genotyping application 4 Save the file The Instrument Control M anager can use the file only if the file name is Chemistry ini and the file is stored in the M egaBA CE DataSystem folder N ote The changes appear immediately in the Instrument Control M anager p6 4 MegaBACE Instrument Administrator s Guide Chapter 6 Using the system initialization files 5 Verify that the changes appear in the Chemistry Parameters tab of the Plate Setup window figure 6 2 If the name for a new or revised chemistry parameter set does not appear in the Chemistry N ame list for a selected application check the parameters in the Chemistry ini file for
74. e values for the parameters defined in the template appear in the Plate Setup window Enter the parameters or edit the existing values in the Electrophoresis tab Type the values e Chemistry tab Select a name for the chemistry parameters set Optional Sample N ames tab Type the sample names Optional O ptional Parameters tab Type the values Optional Comments tab Type the comments To save the parameters as a template see section 4 2 3 EEE 1 2 3 EEL p4 10 MegaBACE Instrument Administrator s Guide Important Chapter 4 Using the Instrument Control Manager windows to configure the system 4 2 3 Saving the plate setup parameters as a template To save the current plate setup parameters as a template 1 From the Templates menu point to Plate Setup Templates and choose Save Template T he Save window appears 2 Typeanew name and click Save The template is saved in the M egaBACE Templates folder The software adds the tpl file extension 4 2 4 Selecting a plate setup template You can select a different template to use for setting up a plate definition To do this 1 Click New in the Plate Setup window 2 From the Templates menu point to Plate Setup Templates and choose Select Template The O pen window appears 3 Select the template name you want to use and click Open The Plate Setup window displays the parameters from the selected template 4 2 5 Setting the
75. ean all spills immediately and call MegaBACE System Technical Support for information on how to clean any large internal spills below the anode and cathode stages A spill in the high voltage area can cause arcing and damage the instrument MegaBACE Instrument Administrator s Guide p2 7 Partone Introduction and safety Caution Caution Opening the electrophoresis compartment lid during an electrophoresis run interrupts the data recording Open the lid between runs only If you need to open the lid during a run stop the run before opening the lid to protect the data you have already collected N ote T he capillaries become warm during electrophoresis For your protection sensors make sure that when the lid opens e If the electrophoresis voltage is on the high voltage power supply shuts off and the voltage drains e Ifthe laser shutter is open the shutter closes and blocks the laser light from entering the compartment e f nitrogen pressure is present in the anode vessel the pressure shuts off and the pressure vents In addition the temperature control for the electrophoresis compartment turns off You cannot scan until you close the lid 2 5 Filter compartment To make sure data is recorded properly you should check that the appropriate filters and beamsplitters are installed before starting an electrophoresis run For details on changing filters and beamsplitters see the M egaBACE Instrument M aintenance and Tr
76. eate a plate setup template or select or set a default template from the Plate Setup Template menu An operator can also create an instrument parameter template or select a template from the Instrument Templates menu p6 12 MegaBACE Instrument Administrator s Guide Chapter 6 Using the system initialization files 6 4 3 About the automatic base calling mode sequencing only You can turn on the automatic base calling mode in the M egaBACE ini file figure 6 3 to automatically perform base calling after data collection You can also use the M egaBACE ini file to allow an operator to turn on automatic base calling from the Configure menu The settings in the M egaBACE ini file are tied directly to the settings in the Automatic Base Calling window which is accessed from the Configure menu Table 6 4 provides an overview of how you specify the automatic base calling conditions in the M egaBACE ini file and how the Automatic Base Calling command appears in the Configure menu and in the Automatic Base Calling window Table 6 4 The automatic base calling mode conditions in the MegaBACE ini file Automatic Base Calling command Automatic Allow Automatic in the Configure menu and the Base Calling Base Calling Override Automatic Base Calling window Y Y Turned on and checked Can be turned off in the Automatic Base Calling window Y N Turned on and checked Cannot be turned off N Y Turned off and unchecked Can be turned o
77. ed Biosystems 850 Lincoln Center Drive Foster City California 94404 PE Applied Biosystems does not guarantee the performance of this instrument Amersham Biosciences is a licensed vendor for authorized reagents Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Biosciences AB SE 751 84 Uppsala Sweden Amersham Biosciences Corp 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Amersham Biosciences Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany Amersham Biosciences SV Corp 928 East Arques Avenue Sunnyvale CA 94085 4520 USA Table of contents Preface About this guides iinns siana hacia iea a A E alae ax xi Related publications aana aa xi Safn feon mt TO an E iO S a a a a xii Special safety text aaan xii Trained operator 2 eee xii ASSUIMIPUONS ia 5 iiias sicion aada iao ioin a endo aE toh aah iee ict xii Safely standards siririrka E a aA xiii MegaBACE system site requirements anaua xiii Electrical requirements noaua aa xiii Environmental conditions saaa xiii ASSISTANGO ss ei awe ataa a aaa aaa A EE ae AEA and eae xiv Part one Introduction and safety Chapter 1 Introduction to the MegaBACE system 1 1 1 2 1 3 1 4 1 5 System hardware components saana aaa 1 2 The MegaBACE system software ccc ect e eee e ee 1 4 1 2 1 The MegaBACE Header Editor software 0 00000 1 4 1 2 2 The MegaBACE ScoreCard software
78. egaBACE Instrument Administrator s Guide Appendix A Fluorescence imaging a Genotyping filter set 1 and dye set 1 FAM TET Filter 2 m Filter 4 520DF20 PMT 2 545DF 20 532 nm green laser Emitted light Filter 3 HEX return scan M S EAOa 565DF 20 Beamsplitter B 555DRLP PMT 1 488 nm blue laser Emitted light Filter 1 ET ROX forward scan _ __ Rica ssheape errs saeane ners l 610DF20 Beamsplitter A 540DRLP b Genotyping filter set 2 and dye set 2 FAM HEX Filter 2 Filter 4 520DF20 PMT 2 555DF20 532 nm green laser Emitted light Filter 3 NED return scan SSS S E 580DF20 Beamsplitter B 570DRLP PMT 1 488 nm blue laser Emitted light Filter 1 ET ROX forward scan my A l 610DF20 Beamsplitter A 540DRLP Figure A 6 The MegaBACE filter configurations vary for the different genotyping chemistries a genotyping filter set 1 and dye set 1 b genotyping filter set 2 and dye set 2 MegaBACE Instrument Administrator s Guide pA 11 Appendix B Plate and instrument parameter setup This appendix provides application specific examples of plate and instrument parameter setup files The topics are e Plate setup parameters for sequencing section B 1 e Instrument control parameters for sequencing section B 2 e Plate setup parameters for genotyping section B 3 e Instrument control parameters for genotyping section B 4 See chapter 4 for information a
79. ent wavelengths The emission spectra typically overlap but ideally the emission peaks should be separated This section describes the various dye sets that have been validated on the M egaBACE system A 2 1 About energy transfer dyes The sequencing dye kits for the M egaBACE system and the label on the ET size standards ET 400 R use energy transfer ET technology Energy transfer uses two fluorescent dyes attached to the same primer molecule one dye of the pair absorbs light and then transfers or donates the absorbed energy to the second dye of the pair The second dye then emits its characteristic fluorescent light spectrum For efficient energy transfer the emission spectrum of the donor dye must overlap the excitation spectrum of the acceptor dye The donor and acceptor must also be located near each other and in a favorable orientation A 2 2 The MegaBACE sequencing ET dye kits For sequencing on the M egaBACE system the following dye kits are recommended e DYEnamic ET dye terminators e ET dye primers The DNA ET primers use two fluorescent dyes attached to the same primer molecule A set of ET primers for four color sequencing can use the same donor dye on all primers so that all the primers are efficiently excited by a single laser line The set then uses a different acceptor dye for each of the four different base terminator reactions pA 2 MegaBACE Instrument Administrator s Guide Appendix A Fluorescence im
80. ents 1 2 safety 2 13 configuration files parameter configuration defined 1 7 1 8 storage C 7 system initialization defined 1 7 confocal optical system 3 5 confocal point 3 5 3 7 cords supplied 2 13 cover panel checking 2 2 cover removal warning 2 11 2 12 D data recording interruption 2 8 defaults application 6 7 base caller 6 8 instrument control parameter template 6 10 plate setup template 6 10 detectors see PMTs directory see folders documentation user xi duplicate sample names allowing 6 10 DYEnamic ET dye terminators A 8 B 1 dyes see also Chemistry ini file energy transfer A 2 fluorescent A 1 genotyping A 3 A 4 sequencing A 2 edit mode about 6 11 allow edit 6 7 allow global edit override 6 7 allow plate edit override 6 7 specifying conditions 6 11 turned on 4 4 electrical connections 2 13 electrical requirements described 2 13 instrument xiii power supply fan module xiii electronics 2 8 electrophoresis compartment high voltage 2 6 lid illustrated 2 2 Safety 2 6 2 7 Electrophoresis Parameters tab 4 2 4 5 emission beamsplitters A 6 emission filters A 7 emission optical system genotyping A 10 sequencing A 8 emission fluorescent A 1 emitted light 3 8 empty capillaries displayed 5 9 specifying in psd file 5 9 specifying in MegaBACE ini file 5 8 Enter Password window 7 2 environmental conditions xiii ET dye primers A 3 A 8 B 2 ET dye terminators A 3 A 8 B 1 excitation light 3 8 A
81. ers defined 4 4 to 4 9 5 5 saving as a template 4 11 Plate Setup window 4 2 PMT1 and PMT2 voltages 4 8 PMTs photomultiplier tubes function 3 7 A 7 safety 2 13 warning 2 12 power cords 2 13 power supply fan module components 1 2 electrical rating xiii safety 2 13 serial number label 2 14 MegaBACE Instrument Administrator s Guide Index 5 Index precautions safety 2 1 pressure nitrogen 2 10 primary beamsplitter 3 5 3 6 processed text file creation allow override 6 10 protocol for self describing data F 1 to F 4 publications xi R raw text file creation 6 9 recording scan results 3 5 run temperature 4 8 run time 4 6 run voltage 4 6 S safety precautionary statements xii precautions 2 1 switch in the filter compartment lid 2 1 sample injection 4 6 sample names in psd file 5 7 5 8 in tpl file 5 12 in Sample Names tab 4 3 4 6 sample sheet see sample names scanning 3 5 SCF file creation allow override 6 9 automatic 6 9 ScoreCard software 1 5 sensors 2 1 sequencing automatic base calling and file export 4 15 to 4 17 6 13 to 6 15 base called sample data storage C 3 to C 5 dyes and filters A 8 file export options 6 14 6 15 instrument control parameters B 4 plate setup parameters B 1 to B 4 Index 6 MegaBACE Instrument Administrator s Guide serial number label instrument 2 3 2 14 power supply fan module 2 4 2 14 service serial numbers required xiv 2 15 single label scan choosing fl
82. ers 5 091 652 and 5 274 240 and corresponding foreign patents and patent applications including any continuations continuations in part and subdivisions and the like The instrument is also an Authorized DNA Sequencer It is authorized under one or more US Patent Numbers 4 849 513 5 171 534 5 015 733 5 118 800 5 161 507 5 118 802 4 855 225 and 5 366 860 and corresponding foreign patents and patent applications The purchase of this instrument includes limited non exclusive rights under the subject patents to use this instrument for sequencing and fragment length analysis when used with Authorized Reagents The use of this instrument with Authorized Reagents provides a limited license to perform DNA sequencing and fragment length analysis in accordance with the label rights accompanying such reagents Purchase of this instrument does not itself convey to the purchaser a complete license to perform DNA sequencing and fragment length analysis under the subject patents Authorized reagents may be obtained from licensed vendors or reagents may be authorized under separate license arrangements from PE Applied Biosystems The above patent rights are granted solely for research and other uses that are not unlawful No other licenses are granted expressly impliedly or by estoppel Further information on purchasing licenses to perform DNA sequencing and fragment length analysis may be obtained by contacting the Director of Licensing at PE Appli
83. etup parameters for genotyping B 3 1 Plate setup data files psd for genotyping Figure B 7 shows examples of psd files with plate setup parameters for genotyping The M egaBACE genotyping system includes examples of psd files in the M egaBA CE Psd folder If you want to assign sample names for a plate you plan to genotype using Genetic Profiler v1 1 you must use a psd file In the psd file you also specify that the software use the well locations as the file names as figure B 7a shows The Genetic Profiler v1 1 software uses the sample names which you specify in the psd file figure B 7a when you display the analyzed data If you use the Sample Names tab of the Plate Setup window to manually enter the sample names instead of using a psd file the Instrument Control Manager uses the sample names instead of well locations for the file names Genetic Profiler v1 1 cannot recognize rsd files unless well names are used for the file names for example A01 rsd MegaBACE Instrument Administrator s Guide pB 5 Part three Appendixes SSE a Basic genotyping psd file E DIA03PN04120199DS2 psd txt Notepad ox File Edit Search Help PLATE ID DIAG3PNO4126199DS2 a INJECTION VOLTAGE 3 INJECTION TINE 45 RUN VOLTAGE 18 RUN TIME 75 COMMENT CHEMISTRY GT DyeSet2 ET ROX FAM NED HEX WELL ATTRIBUTES NAHE F ILE Sample name 61 NAME 14666 61 FILE A91 62 NAME 14698 File name A62 File A82 83 NAME 11877 A63 File A83 AB4
84. ew 458 4 DWORD m_dExtHeaderLen new 462 4 float m_fPreinjectionTime new 466 4 float m_fPreinjectionVoltage new 470 4 float m_fLowPressureTime new 474 4 float m_fUserInputTime new 478 4 float m_fSleepTime new 482 4 float m_fSleepTemperature new 486 10 char m_csBuildNumber 10 format 19990927A includes 0 496 4 int m_nNumColumns maximum number of letter labels on the plate depends on instrument type 500 4 int m_nNumRows maximum number of number labels on the plate depends on instrument type 504 64 BYTE m_byReserved 64 568 6 char m_csRunID 6 574 18 char m_csPlateID 18 deprecated 592 4 DWORD m_dwPrevRunID unused 596 4 BOOL m_bDWORDFile 1 raw 0 processed 600 4 BOOL m_bSaturated 604 4 float m_fVersion 2 000 608 4 time_t m_tRunStartTime 612 4 time_t m_tRunStopTime 616 4 DWORD m_dwNumberOfLines 620 4 int m_nScanRate 0 1 75 1 3 0 624 1 BYTE m_byFileType reserved 625 1 BYTE m_byNumberOfCapillaries number of arrays 16 626 1 BYTE m_byCapUsage 627 1 BYTE m_byDummy 628 4 UINT m_uiSecurityCode Figure D 1 Standard header format MegaBACE Instrument Administrator s Guide pD 1 Appendix E Extended header format This appendix provides information about the extended header format that is used in the M egaBACE raw sample data files rsd and base called sample data files esd The topics are
85. f bytes specified in length For List the number of elements is specified in length F5 Notes about the protocol The protocol packs the elements as a byte stream In all cases multibyte data are packed with the least significant byte first This means that the packing unpacking software will differ depending on whether the platform implements a big endian or little endian architecture x86 is little endian pF 2 MegaBACE Instrument Administrator s Guide Appendix F APB protocol for self describing data The size of the data block may not be known a priori If the size is indeterminate the Terminator format code can be used to tell the unpacking algorithm where to stop If the size of the data block can be unambiguously determined the use of the Terminator is optional Character strings A and C formats are null terminated See figure F 1 for notations The List format is used for creating complex data structures which are treated as single elements If the value requires multiple parameters of different data types for example character string or int with int to represent the base dye filter center and filter width for each channel in the Chemistry specifier the parameters can be specified inside a List thereby making them a single value associated with the corresponding K eyword H uman readable representation dump format is as follows e By convention the format and data for each element are listed between angle
86. focal optical system to image the fluorescent samples as they electrophorese past the detection windows 3 3 1 Basic confocal optical system The system uses epi illumination which means that the excitation light is focused on the sample by an objective lens and the emitted light is collected by the same objective lens The detection windows of the capillaries lie in the focal plane of the objective lens which focuses the laser light to a point in the focal plane called the focal point figure 3 3 In an array of capillaries only the capillary at the focal point is illuminated and along the length of the capillary only the part of the sample at the focal point is illuminated Laser light with a decreased energy density reaches points in front or in back of the focal point Focal Focal cee Confocal i int plane point light poin Ve g Vi gt e a PMT Microscope Primary Achromatic Aperture objective lens beamsplitter lens Figure 3 3 The focal point and confocal point in a confocal system Fluorescent light from the sample passes back through the microscope objective lens The objective lens and additional lens within the instrument focus the light from the focal point to a second point called the confocal point figure 3 3 The small aperture pinhole located at the confocal point allows light from the focal point to pass through to the PM T Light emitted outside the focal point is not in focus at the con
87. focal point and is therefore rejected by the aperture MegaBACE Instrument Administrator s Guide p3 5 Parttwo Understanding and configuring the MegaBACE system SS 3 3 2 How the system views the capillary array The M egaBACE instrument scans the capillary array by sweeping the scan head horizontally to move the focal point across the array R epeated scanning across the capillary array over time provides a record of the fluorescence emissions as the samples electrophorese past the detection windows Each data point includes the capillary location the time into the electrophoresis run and the value light intensity in each color channel For information on how the different colors are recorded see appendix A At the end of an electrophoresis run the scan results for each capillary are stored separately so that you can access and analyze the data in each of the sample files independently 3 3 3 How the system views a point in the array The M egaBACE instrument can contain one or two lasers The dual laser instrument contains an argon ion laser that emits 488 nm blue light and a solid state laser that emits 532 nm green light The single laser instrument contains the blue laser 488 nm To view a point 1 The objective lens focuses collimated laser light on a point figure 3 4a b e Thelaser emits a collimated beam of light If both lasers are on a shutter allows light from the appropriate laser to proceed down th
88. g with ET primer chemistry Ej Sample2_psd Notepad iof x File Edit Search Help Plate ID INJECTION VOLTAGE 3 INJECTION TIME 40 RUN VOLTAGE 9 RUN TIHE 166 CHEMISTRY ET Primers RUN TEMPERATURE 44 PHT1 VOLTAGE 750 PHT2 VOLTAGE 750 BASE CALLER Cimarron 1 53 Phat COMMENT M13 Standard Run A61 BASECALLER Molecular Dynamics B61 BASECALLER Molecular Dynamics x Figure B 2 A psd file for sequencing with ET primers About the base callers The Cimarron 1 53 Slim Phredify base caller or later is recommended for all sequencing runs The other base callers have not been validated with Phred pB 2 MegaBACE Instrument Administrator s Guide Appendix B_ Plate and instrument parameter setup B 1 2 Plate setup parameter template files tpl for sequencing This section provides examples of plate setup parameter template files tpl that you can use for sequencing Each file defines the parameters you use for a particular type of run e Long run with dye terminators e Long run with dye primers e RunoftheM egaBACE 4 color sequencing standards M 13 standards with ET primers Long sequencing run with dye terminators Figure B 3 shows a tpl file containing the plate setup parameters for a long run with dye terminators E LongDyeT erminator tpl Notepad Iof x File Edit Search Help 368 3 INJECTION TIME 46 INJECTION VOLTAGE 3 COMMENT Standard Terminator CHEMISTRY ET Terminators Figure B 3 A t
89. ge conventions for the base called and exported files continued File type File contents File name Folder storage Exported FASTA File name file location and the called sequence for each well in the base called plate default You can use the MegaBACE ini file to specify other options section 6 4 5 One seq file for each base called esd file One seq file for all base called plates seq PlatelD_basecallerlD folder default Exported ASCII text Plate name sample name instrument ID base caller name sequence starting and ending points electrical current information scan rate instrument parameters the number of bases called Sample_name txt well_ID txt or a combination of both Text subfolder default Note In the MegaBACE ini file you can specify that the files be stored at the top level of the run folder section 6 4 pC 4 MegaBACE Instrument Administrator s Guide Appendix C File Storage od C 2 2 Naming and storage conventions for the analyzed run folders The different types of base called data section C 1 2 are stored in different folders Table C 3 lists the run folders exported data folders and the naming conventions for these folders The software uses the plate ID the base caller ID the base caller version number n_nnn and the base caller method to name the analyzed run folders The base caller IDs are M Molecular Dynami
90. ger allows you to perform continuous base calling and file export after data collection without operator intervention To do this you specify automatic base calling select a default base caller and specify the file formats for the base called data SCF FASTA ASCII text or ABD See section 4 4 for details on how to set up this feature in the Instrument Control M anager windows See section 6 4 for a description of how to set up this feature in the M egaBACE ini file 1 4 The flexible MegaBACE instrument There are two models of the flexible instrument e MegaBACE 500 Depending on your configuration allows you to use 16 32 or 48 capillaries Flexible M egaBACE 1000 Depending on your configuration allows you to use 16 32 48 64 80 or 96 capillaries N ote After starting the H ost Scan Controller downloads the firmware and displays the instrument model number table 1 1 p1l 6 MegaBACE Instrument Administrator s Guide Chapter 1 Introduction to the MegaBACE system Asthe instrument administrator you can configure the system to accommodate the required number of capillary arrays and the corresponding number of array placeholders Array placeholders consist of an anode blocker an anode sleeve a window blank and a cathode plunging tool Array placeholders are used to fill the empty array positions 1 5 Administrator tasks As the instrument administrator you use the tools in the Instrument Control M anager to
91. gh a specific spectral channel but unwanted signal is always present in every channel from the emission of the other dyes in the dye set Glossary 4 MegaBACE Instrument Administrator s Guide Glossary Stokes shift the difference in wavelength between the apex of the excitation spectrum shorter wavelength higher energy and the apex of the emission spectrum longer wavelength lower energy traces the four curves in the electropherogram representing the signals detected through the four spectral channels of the instrument Each trace is displayed in a specific color to represent a corresponding base or dye window blanks M egaBACE 500 and flexible M egaBACE 1000 instruments only the replacement capillary windows installed in place of the missing capillaries for the empty array positions The instrument optical system requires a capillary window or a window blank at each of the six array positions MegaBACE Instrument Administrator s Guide Glossary 5 Index esd files see files base called sample data files icp files see instrument control parameter templates psd files see plate setup data files rsd files see files raw sample data tpl files see plate setup templates A ABD file creation 6 8 absorption wavelength spectrum A 1 achromatic lens 3 5 3 7 administrator tasks 1 7 1 8 allow edit 6 7 allow global edit override 6 7 allow plate edit override 6 7 anode 3 2 anode blocker 1 2 anode compartmen
92. ght with wavelengths shorter than a specified cutoff and longer than a second specified cutoff Allows light of wavelengths between the two cutoffs to pass through figure A 4 e Long pass R ejects light with wavelengths shorter than a specified cutoff and allows light of longer wavelengths to pass through figure A 4 Unfiltered emission spectrum Band pass ie Long pass filter m Wavelengths passed Wavelengths passed Figure A 4 Effect of band pass and long pass filters on light passing through the filter A 4 3 PMTs The instrument uses two photomultiplier tubes PM Ts to collect the filtered light The PM Ts convert light energy into an electrical current Increasing the voltage applied to a PM T increases the signal amplification The PM T voltages are selected for optimal recording of the dyes The operator of the instrument selects the PM T voltages as part of the instrument parameters For information on instrument parameters see chapter 4 MegaBACE Instrument Administrator s Guide pA 7 Part three Appendixes A 5 Beamsplitters and filters for sequencing Table A 3 lists the dyes beamsplitters and filters for a four color DNA sequencing run using the DY Enamic ET dye terminators Table A 3 Beamsplitters and filters for DYEnamic ET dye terminators DYEnamic DYEnamic dye terminator dye terminator base order Channel Filter Beamsplitter ET R6G T 1 555DF20 A 540DRLP ET R
93. hange templates or select templates If the edit mode is turned on a check mark appears next to the Edit M ode command on the Configure menu If the edit mode is turned off no check mark appears The M egaBACE ini file contains the default settings for the edit mode conditions table 6 3 You can use the M egaBACE ini file to enable or disable the edit mode menu command which allows an operator to turn on or off the edit mode using the Configure menu You can also use the M egaBACE ini file to specify whether the edit mode remains on or applies to one plate only If the edit mode is turned on for one plate only the software enables the edit mode after the plate has been set up for this plate only Table 6 3 provides an overview of how to specify the edit mode conditions in the M egaBACE ini file and how the Edit M ode command appears in the Configure menu MegaBACE Instrument Administrator s Guide p6 11 Parttwo Understanding and configuring the MegaBACE system Table 6 3 The edit mode conditions in the MegaBACE ini file Allow Allow Allow Edit Global Edit Override Plate Edit Override Edit Mode command in the Configure menu Y N A N A Turned on and checked Can be turned off N Y N A Turned off and unchecked Can be turned on at will N N Turned off and unchecked Can be turned on for one plate only newly created plate and then turns off automatically Turned off and unchecked No editi
94. hose size the hose characteristics and the fittings inside the instrument are designed to withstand the working pressures Warnings Do not attempt to adjust the regulators to pressure settings above those described A in this guide If you are using separate cylinders for high and low pressure make sure that the correct pressure is applied to each line The nitrogen pressure in the high pressure line must not exceed 6 89 x 103 kPa 1000 psi of pressure Never apply high pressure to the low pressure line This can damage the instrument or the low pressure line and can cause injury Figure 2 9 shows the labels that are placed on the back of the instrument next to the high and low pressure nitrogen line connections Figure 2 3 shows the locations of the labels Warning Use only hose types with ratings that exceed the required operating pressures Do A not use a frayed or damaged hose which can rupture and cause injury 1000 PSI 100 PSI 6 89 X 10 kPa 6 89 X 10 kPa Figure 2 9 The high and low pressure nitrogen labels p2 10 MegaBACE Instrument Administrator s Guide Warning Warning A Chapter 2 Safety precautions 2 9 Lasers Changing controls making adjustments or performing procedures other than those specified in the MegaBACE instrument documentation can resultin hazardous laser light exposure 2 9 1 Class 1 Laser Product label The M egaBACE instrument satisfies the Class 1 requireme
95. iated with a given base A green C blue T red G black Although the conventional color representation for G is yellow the trace and letter are displayed in black for ease of viewing calibration run see spectral calibration run cathode the negative end of the capillaries where each capillary tip is inserted into one of the wells on the microplate along with an electrode The cathode stage is located on the left side of the instrument cathode plunging tools M egaBACE 500 and flexible M egaBACE 1000 instruments only the component used to fill the wells for the empty capillary array positions channels see spectral channel chemistry parameters the names of the dyes the base order or dye to channel mapping the names of the filters and beamsplitters and the laser mode used for a given application sequencing or genotyping dye set the dyes used to label your experiment The M egaBACE system is capable of detecting and separating the emissions of four dyes of different colors in a single capillary electropherograms the digitized graphs the system produces from the fluorescent intensity detected from the capillaries during the run The system produces an electropherogram for each capillary Each electropherogram consists of four colored traces that represent the signals detected through the four spectral channels Glossary 2 MegaBACE Instrument Administrator s Guide Glossary electrophoresis pa
96. imes per minute sieving matrix the sieving substance used to separate the DNA fragments in the sample by size for example LPA spectral calibration run genotyping only a genotyping run of a sample plate that contains the spectral matrix standards for the selected dye set You use the data collected from the calibration run to create a spectral overlap matrix in Genetic Profiler spectral channel the combination of laser beamsplitter optical filter and PM T the system uses to detect the emission signals of a given dye The M egaBACE instrument has a total of four spectral channels The output of each spectral channel is represented by a different colored trace in the electropherogram spectral overlap matrix the dye to channel mathematical matrix that defines which dye is detected through each channel and measures the amount of unwanted signal spectral overlap present in each spectral channel from the other dyes in the dye set The Instrument Control M anager creates a real time view of the spectrally separated data for display only e For sequencing the base callers automatically perform spectral separation during data analysis e For genotyping the operator uses the Genetic Profiler software to create a matrix that the software can use to perform spectral separation spectral separation the software process that removes the unwanted signals that are present in each spectral channel Each dye is detected throu
97. k on the electrophoresis compartment The label is located on the left side of the instrument on the side wall inside the electrophoresis compartment Figure 2 2 shows the location of the label Do not defeat the interlocks or try to gain access to the interior of the MegaBACE instrument through any other opening Exposure to laser light can cause injury DANGER Laser light when open and interlock defeated AVOID DIRECT EXPOSURE TO BEAM DANGER LASER LIGHT WHEN OPEN AND INTERLOCK DEFEATED AVOID DIRECT EXPOSURE TO BEAM GEFAHR LASERSTRAHLUNG WENN ABDECKUNG GEOFFNET UND SICHERHEITSVERRIEGELUNG UBERBRUCKT Figure 2 12 The interlock defeat danger label 2 9 4 Light leaks If a panel becomes damaged and the M egaBACE instrument is no longer light tight do not continue to use the instrument Ambient light can damage electrical components in the MegaBACE instrument such as the PMTs Call MegaBACE System Technical Support immediately to arrange for repair See Assistance in the preface for contact information p2 12 MegaBACE Instrument Administrator s Guide Warning A Warning A Important Warning A Chapter 2 Safety precautions 2 10 PMTs The PM Ts are covered by a protective housing and are not accessible by the operator During a prerun or electrophoresis run the PM Ts carry a high voltage which can cause injury if you touch them Do not try to gain access to the PMTs or re
98. ke sure you follow the precautionary statements presented in this guide Indicates a possibility of severe or fatal injury to the user or other persons if the precautions or instructions are not observed Indicates that damage to the instrument loss of data or invalid data could occur if the user fails to comply with the advice given Highlights information that is critical for optimal performance of the system N ote Identifies items of general interest Trained operator The operator of the MegaBACE instrument is assumed to be trained in the correct operation of the instrument and the safety issues Throughout the MegaBACE instrument documentation the word you refers to this trained operator Assumptions The software related instructions in this user s guide assume you have basic computer skills You should be familiar with the M icrosoft Windows NT or Windows 2000 graphical user interface If you do not have these skills refer to the Windows NT or Windows 2000 documentation or refer to the H elp for Windows NT or Windows 2000 MegaBACE Instrument Administrator s Guide MegaBACE system site requirements Safety standards The M egaBACE instrument complies with CE and other applicable standards such as UL CSA and FDA For the latest conformity information contact M egaBACE Technical Support See the Assistance section for contact information MegaBACE system site requirements Electrical requiremen
99. le in the electrophoresis compartment you should immediately turn off the instrument and call MegaBACE System Technical Support See Assistance in the preface for contact information Do not attempt to lift the instrument The MegaBACE instrument weighs approximately 272 kg 600 Ib Lifting the instrument can cause severe or fatal injury p2 2 MegaBACE Instrument Administrator s Guide Chapter 2 Safety precautions 2 2 Locations of important labels The locations of important labels on the M egaBACE instrument are shown in figures 2 2 and 2 3 Figure 2 4 shows the location of the serial number certification label on the power supply fan module MegaBACE instrument left side I Top of cathode drawer Top of PMT cover Laser light warning High voltage warning Pinching hazard Serial number certification Class 1 Laser product Liquid spillage caution D DIRECT EXPOSURE TO BEAM HAZARDOUS VOLTAGES MAY BE PRESENT IF INTERLOCKS ARE DEFEATED lt 4 Amersham e K Biosciences MODEL MegaBACE 1000 200 240V 6A _50 60Hz SERIAL 928 E Arques Ave Sunnyvale CA 94086 CE ELALLA AISSA SISSA SSS E This product conforms to the applicable requirements of Z1CFR subchapter atthe date of manufacture MANUFACTURED To avoid instrument damage Open and close drawer slowly Remove any spilled liquid located in and ar
100. ler provides the communication between the M egaBACE instrument and the Instrument Control M anager The Instrument Control M anager contains the graphical user interface that allows an operator to control the M egaBACE instrument See section 1 3 for an overview of the Instrument Control M anager features e Viewing and analyzing the collected data T he system software includes the analysis software for your application sequencing or genotyping See the corresponding user s guide for details The software also includes the M egaBACE Header Editor and the M egaBACE ScoreCard 1 2 1 The MegaBACE Header Editor software M egaBACE H eader Editor allows you to view and edit the data in the extended file header of the raw sample data files rsd For sequencing data you can also view and edit the data in the extended file header of the base called sample data files esd M egaBACE H eader Editor also allows you to view the data in the standard file header of these files For more information about M egaBACE H eader Editor see the H elp available within M egaBACE H eader Editor p1l 4 MegaBACE Instrument Administrator s Guide Chapter 1 Introduction to the MegaBACE system 1 2 2 The MegaBACE ScoreCard software The M egaBACE ScoreCard software provides a variety of ways to view the quality of the analyzed data in each well of a plate There are two versions of the M egaBACE ScoreC ard software e MegaBACE Sequencing ScoreC ard
101. ll the analyzed plates This file is stored in the M egaBACE AnalyzedD ata platelD_runID folder MegaBACE Instrument Administrator s Guide 6 15 Parttwo Understanding and configuring the MegaBACE system 6 4 6 Using multiple applications Instrument Control M anager supports the use of multiple applications Currently sequencing and genotyping are the two applications available under separate licenses The name of the selected application appears in the title bar of each of the Instrument Control M anager windows figure 6 4 Application name pinc XE MegaBACE Gen Figure 6 4 The Plate Setup window The name of the selected application appears in the title bar of each of the Instrument Control Manager windows p6 16 MegaBACE Instrument Administrator s Guide Important Chapter 6 Using the system initialization files When an operator starts the Instrument Control M anager the application sequencing or genotyping is selected based on the default application specified in the M egaBACE ini file figure 6 5 Application name MegaBACE ini Notepad File Edit Search Help General DEFAULT APPLICATION genotyping ALLOW EDIT Y ALLOW GLOBAL EDIT OVERRIDE Y ALLOW PLATE EDIT OVERRIDE Y PSD DIRECTORY PSD REQUIRED N PLATE ID LENGTH 8 PLATE ID TERMINATOR DEFAULT BASE CALLER Cimarron 1 53 Slim Phredify AUTOMATIC BASE CALLING ALLOW BASE CALLING OUERRIDE AUTOMATIC ABD FILE CRE
102. llowing fields either well specific in a psd file or plate specific can be defined only Well_ID FILE for example AO1 FILE The rsd file name that you want to assign for a given well location For example A01 if you want to use the well location as the file name This parameter overwrites the sample name as the file name Well_ID BASE CALLER for example A01 BASE CALLER The name of the base caller that you want to use for a specified well Plate type Genotyping only Recognized by the MegaBACE Genetic Profiler ScoreCard software The options for the field are e Genotyping Test Plate e Sample Plate User defined fields Fields that can be imported but are not used by the software These data are stored in the extended header of the files p5 6 MegaBACE Instrument Administrator s Guide Caution Chapter 5 Using the parameter configuration files 5 3 4 The psd file value constraints If you type a value that is invalid for example an alpha character when the software requires a number or a value that is out of range the Instrument Control M anager displays an error message when you import the psd file If this occurs you must correct the value in the psd file before it can be imported successfully Alternatively if the edit mode is on you can enter the correct value in the Plate Setup window figure 4 1 5 3 5 Annotating well specific information in a psd file The values for
103. lso acts as a power line regulator line conditioner and surge suppressor and works to protect against all power line problems MegaBACE Instrument Administrator s Guide p2 13 Partone Introduction and safety Cautions In the event of a power failure see the MegaBACE Instrument Operator s Guide a UPS might not contain enough stored power to finish the run and allow the capillaries to be flushed for storage Contact MegaBACE System Technical Support for information about a qualified UPS See Assistance in the preface for contact information Plug the computer and monitor into the UPS Make sure the voltage selection switch on the back of the computer correctly matches the voltage at the outlet 2 13 Serial number labels 2 13 1 Instrument serial number label You can find the serial number and model number of your M egaBACE instrument on the serial number label figure 2 13 The label is located on the lower right side of the M egaBACE instrument Figure 2 2 shows the location of the label You will need the serial number when contacting M egaBACE System Technical Support about your instrument Amersham 928 E Arques Ave e Y Biosciences Sunnyvale CA 94086 ro oa moo C Eck ce contorty SERIAL I nort AS ORYRKRAY SAAS e ven AAMSAS S V BRAAA NRL This product conforms to the applicable requirements of 21CFR subchapter J at the date of manufacture MANUFACTURED Figure 2 13 The MegaBACE i
104. meters tab appears figure 4 5 N ote Generally you set the PM T voltages and run temperature in the Instrument Parameters area of the Instrument Control window section 4 3 1 MegaBACE Instrument Administrator s Guide p4 7 Parttwo Understanding and configuring the MegaBACE system Important Electrophoresis Parameters Chemistry Parameters Sample Names Optional Parameters Comments PMT1 Voltage 750 Y Run Temperature 44 iB PMT2 Voltage 750 Y Base Caller Cimarron 1 53 Slim Phredify x Figure 4 5 The Optional Parameters tab with sequencing parameters displayed 10 Type the optional parameters you want to use Valid ranges for the fields in the Optional Parameters tab are e PMT1 and PMT 2 Voltages 450 950 V e Temperature 27 44 C The PMT voltages are instrument specific and are set by the MegaBACE Instrument Specialist at installation Contact MegaBACE System Technical Support before you alter the PMT voltages N ote T he values entered in the Optional Parameters tab automatically override duplicate parameters specified in the Instrument Control window Sequencing only If you want to use a specific base caller for the plate you are setting up include the base caller The base caller you specify in the Optional Parameters tab overrides the default base caller for this plate only 11 Click the Comments tab The Comments tab appears figure 4 6 p4 8 MegaBACE Instrument Administrator
105. move the protective panels for any reason Exposure to high voltage from the PMTs can cause severe or fatal injury 2 11 Power supply fan module computer and monitor Voltages are exposed inside the power supply fan module computer and monitor See the computer manufacturer documentation for the high voltage hazard warning M ake sure you follow the instructions for the safe operation of the computer Do not open the power supply fan module or monitor Open the computer only by following the computer manufacturer s instructions 2 12 System electrical connections The M egaBACE system includes four devices that require electrical power the instrument the power supply fan module the computer and the monitor A total of four electrical power cords are supplied with each system one for each of these main components See your M egaBACE Planning G uide for detailed electrical requirements You must locate the right side of the MegaBACE instrument within 2 5 m 8 ft of the electrical outlets Use only the power cords supplied Make sure the cords are in good condition and are not frayed Use of incorrect power cords can cause damage to the instrument Use of frayed or damaged power cords can cause injury You should use an uninterruptible power supply UPS rated for at least 4 kVA to protect the instrument the capillaries and your data from damage or loss caused by unexpected power failures surges or AC line fluctuations A UPS a
106. mple data files rsd The following labels will always appear in raw sample data files rsd N ote The indentations within CHEMISTRY denote nested lists BAR CODE BASE CALLER CHEMISTRY APPLICATION BEAM SPLITTER A BEAM SPLITTER B CHANNEL1 BASE DYE FILTER CHANNEL2 BASE DYE FILTER CHANNEL3 BASE DYE FILTER CHANNEL4 BASE DYE FILTER LASER MODE NAME COMMENT MACHINE ID PLATE ID SAM PLENAME WELL ID pE 2 MegaBACE Instrument Administrator s Guide Appendix E Extended header format E 3 About the bar code BAR CODE is the string entered into the Plate ID field on the Plate Setup screen either by keyboard entry or with a bar code scanner If there is no psd file associated with the name or if there is no PLATE ID entry in the psd file the plate ID will match the bar code E 4 About the extended header in base called sample data files esd N ote To view the esd filein a M egaBACE application other than Sequence Analyzer see the applicable analysis user s guide All entries in a raw sample data file rsd are preserved in the corresponding base called sample data file esd The following additional entries appear in base called sample data files BASES CALLED END GOOD SEQUENCE END SEQUENCE END SIGNAL END TRACE PROCESSING PEAK POSITIONS QUALITY INDEX QUALITY SCORES SEQUENCE START GOOD SEQUENCE START SEQUENCE START SIGNAL START TRACE PROCESSING MegaBACE Instrument Administrator s Guide p
107. n at will in the Automatic Base Calling window N N Turned off and unchecked Cannot be turned on 6 4 4 Exporting the base called data to other file formats sequencing only You can use the M egaBACE ini file figure 6 3 to set up automatic export to other file formats after the automatic base calling The format options for export are ABD FASTA SCF and ASCII text The file formats you choose are created in addition to the base called sample data files esd See section C 2 for a description of each file format MegaBACE Instrument Administrator s Guide 6 13 Parttwo Understanding and configuring the MegaBACE system Settings in the MegaBACE ini file The settings in the M egaBACE ini file are tied directly to the settings in the Automatic Base Calling window which is accessed from the Configure menu If you specify Y in the M egaBACE ini file for automatic creation of any of the file formats the check box es for the specified file formats contain a check mark in the Automatic Base Calling window N ote Automatic base calling must be turned on to export the selected file format s If you specify N in the M egaBACE ini file for automatic creation of any of the file formats the check box es for the specified file formats do not contain a check mark in the Automatic Base Calling window If you specify Y to any of the automatic file format overrides an operator can use the Automatic Base Calling window to
108. n combination or individually to automate the parameter setup required for defining and running a plate For example if you use a psd file to specify all the plate setup parameters you do not MegaBACE Instrument Administrator s Guide p5 1 Parttwo Understanding and configuring the MegaBACE system SSF need to use a tpl file Similarly if you use a tpl file to specify all the plate setup parameters for multiple plates you do not need to use a psd file unless you want to specify additional parameters for a particular plate The parameters that you enter last whether entered manually in the Instrument Control M anager windows or using a psd or tpl file overwrite any previously entered parameters 5 2 Parameter configuration file format You can open the existing files or create new files using a text editor that supports a tab separated format for example N otepad or Excel The parameter files consist of two columns field column and value column For files created using a text editor the two columns can be separated by any number of tabs The order of the fields rows is arbitrary and the file can include blank rows The field names are case insensitive 5 3 Using plate setup data files psd You can define a plate ID the plate setup parameters and other plate and well level attributes in a psd file so that an operator can automatically import the plate definition With automatic import the plate setup parameters ar
109. n raw sample data files rsd E 3 Aboutthe barcode eee E 4 About the extended header in base called sample data files esd Appendix F APB protocol for self describing data Fl INtOdUCHON s a cfc oe eed dees asia Goad ew ii a wba ii F 2 Thel byteheader 02 ees F3 Lengi yale retinian a wane weenie Mace teen ed F4 Dataibufier sriciria curosa tiraria gaai Gem dea Be a F 5 Notes about the protocol 1 2 eee ee Appendix G Literature references Glossary X MegaBACE Instrument Administrator s Guide Preface About this guide The M egaBACE Instrument Administrator s G uide provides software configuration information for the MegaBACE DNA Analysis System Part one Introduction and safety Provides introductory and safety information about the M egaBACE instrument Part two Understanding and configuring the M egaBACE system Describes how the instrument works and how to manually set up plate definitions create plate setup and instrument parameter templates and use the configuration files Part three Appendixes Provides application specific information about dyes filters beamsplitters examples of plate setup and instrument parameter files file storage description the M egaBACE extended header and standard header file formats and literature references Related publications In addition to the M egaBACE Instrument Administrator s G uide the following publications are a
110. n this manual Understanding fluorescence M olecular D ynamics Technical N ote 57 Jingyue J A N Glazer and R A M athies 1996 Energy transfer primers A new fluorescence labeling paradigm for DNA sequencing analysis N ature Medicine 2 246 249 Ju J Kherttepul J R Scherer C Ruan C W Fuller A N Glazer and R A M athies 1995b Design and synthesis of fluorescent energy transfer dye labelled primers and their application for DNA sequencing and analysis Annal Biochem 231 131 140 MegaBACE Instrument Administrator s Guide pG 1 Glossary abd files sequencing only the base called data files that can be viewed using ABD software and used by the Phred application Each abd file contains the data for a given well on a plate for example A01 including the raw and analyzed electropherogram data esd files sequencing only the base called data files Each esd file contains the called sequence read length sequencing starting and ending points quality values current profile and the analyzed electropherogram for a given well on a plate for example A01 The sequenced data can be generated automatically by the Instrument Control M anager after a sequencing run or by the Sequence Analyzer software N ote that the Instrument Control M anager stores the esd files for each run in an analyzed run folder psd files the plate setup data files that you can use to automatically import attributes
111. nergy level the dye emits light of specific wavelengths figure A 1 488 laser line 490 520 al we is T T a x 7 r NY 400 450 500 550 600 Wavelength nm Figure A 1 Absorption and emission spectra of fluorescein The wavelength with maximum absorption or emission is shown above the curves These curves are approximations To generate fluorescence the excitation light must be within the absorption wavelength spectrum of the fluorescent dye The closer the exciting wavelength is to the peak excitation wavelength of the dye the stronger will be the fluorescent emission MegaBACE Instrument Administrator s Guide pA 1 Part three Appendixes The width and location of the absorption and emission spectra are specific for each dye In each case the apex of the emission spectrum is of lower energy longer wavelength than the apex of the excitation spectrum This effect is called the Stokes shift For more information on how fluorescence is generated see M olecular Dynamics Technical N ote 57 Understanding Fluorescence A 2 Choosing the dyes for multicolored experiments The M egaBACE system is capable of detecting and separating the emissions of four dyes of different colors in a single capillary To allow accurate detection and separation of the signals a set of fluorescent dyes must be used that e Areexcited by the 488 nm or 532 nm laser lines e Emit light of significantly differ
112. ng is allowed An operator can create a new plate definition by importing a psd file A more detailed description of these conditions follows Allow Edit Specifying Y turns the edit mode on and allows an operator to edit any of the plate setup or instrument parameters The Edit M ode command on the Configure menu has a check mark to indicate that it is turned on Specifying N turns off the edit mode but an operator can turn on the edit mode if a Y is specified for the ALLOW GLOBAL EDIT OVERRIDE field Allow Global Edit O verride Specifying Y allows an operator to turn on or off the edit mode on the Configure menu at will While in the edit mode an operator can edit the plate setup parameters or instrument parameters Specifying N turns off the edit mode which prevents the operator from turning on the edit mode unless a Y is specified for the ALLOW PLATE EDIT OVERRIDE field Allow Plate Edit O verride Specifying Y allows an operator to choose the edit mode for the current plate only newly created plate The edit mode becomes unavailable when the operator selects another plate Specifying N disables the edit mode feature If all the above conditions are set to N in the M egaBACE ini file an operator cannot edit any of the plate setup or instrument parameters in the Instrument Control M anager windows An operator can set up plate definitions only by importing psd files When you turn on the edit mode an operator can cr
113. nstrument control parameter template files icp section 5 5 5 1 About the parameter configuration files The parameter configuration files are optional files that can be used to define the plate setup parameters and instrument control parameters that an operator can import at run time The software includes files that are ready for you to use or modify or you can create new ones The Instrument Control M anager uses three kinds of parameter configuration files Plate setup data file psd Used for a specific plate a psd file can specify plate ID plate setup parameters and other plate and well level attributes such as sample names and file names section 5 3 You can create psd files using a text editor or Excel e Plate setup template file tpl Used for multiple plates a tpl file can specify only the plate setup parameters See section 4 2 for a description of how to create a plate setup template from the Plate Setup window or see section 5 4 for a description of how to create a tpl file using a text editor or Excel Instrument control parameter template file icp You can use an icp file to specify the instrument control parameters for multiple runs See section 4 3 for a description of how to create an instrument control parameter template from the Instrument Control window and see section 5 5 for a description of how to create an icp file using a text editor or Excel These files can be used either i
114. nstrument serial number certification label 2 13 2 Power supply fan module serial number label You can find the serial number and model number of the power supply fan module on the serial number certification label figure 2 14 The label is located on the back of the power supply fan module Figure 2 4 shows the location of the label You will need the serial number when contacting M egaBACE System Technical Support about your instrument p2 14 MegaBACE Instrument Administrator s Guide Chapter 2 Safety precautions Amersham 928 E Arques Ave e Y Biosciences Sunnyvale CA 94086 r MODEL MegaBACE P S FAN MODULE 208 220 1230 240 10A 50 60Hz iC CE conformity I N Uf SERIAL ran KAAOS AIS e OO Saee MANUFACTURED MADE IN USA An Figure 2 14 The power supply fan module serial number certification label 2 14 Service for the MegaBACE instrument To protect your warranty and for proper operation the instrument should be serviced only by an authorized service representative If the instrument is not working correctly call M egaBACE System Technical Support See Assistance in the preface for contact information When you call M egaBACE System Technical Support be prepared to give the serial number of your instrument or power supply fan module You can find the serial numbers on the serial number certification labels figures 2 2 2 4 2 13 an
115. nts of IEC 825 1 1993 and EN 60825 1 Figure 2 10 shows the Class 1 Laser Product label Figure 2 2 shows the location of the label on the lower right side of the instrument CLASS 1 LASER PRODUCT Per IEC 825 Figure 2 10 The Class 1 Laser Product label 2 9 2 Laser light warning label The instrument does not allow operator exposure to laser light N evertheless the instrument contains a blue argon ion laser with power up to 25 mW at 488 nm with a 0 95 mrad divergence The instrument can also contain a green solid state laser with power up to 50 mW at 532 nm with 1 2 mrad divergence Do not remove any of the inner covers of the MegaBACE instrument The laser power specified in the paragraph above could be accessible if you remove the inner covers The label in figure 2 11 warns of laser light danger Figure 2 2 shows the locations of the label on the PM T cover and in the electrophoresis compartment of the instrument MegaBACE Instrument Administrator s Guide p2 11 Partone Introduction and safety Warning A Caution DANGER Laser light when open AVOID DIRECT EXPOSURE TO BEAM DANGER LASER LIGHT WHEN OPEN AVOID DIRECT EXPOSURE TO BEAM GEFAHR LASER NACH DEM OFFNEN DIREKTEN KONTAKT ZUM LASERSTRAHL VERMEIDEN Figure 2 11 The laser light warning label 2 9 3 Safety interlock danger label The label in figure 2 12 warns of the laser danger from defeating the interloc
116. o Ora MegaBACE Instrument Administrator s Guide Version 2 4 Amersham Gn 375 691 5 e Biosciences DYEnamic MegaBACE and ScoreCard are trademarks of Amersham Biosciences UK Limited Amersham and Amersham Biosciences are trademarks of Amersham plc Microsoft Windows and Windows NT are trademarks of Microsoft Corporation The polymerase chain reaction PCR is covered by patents owned by Roche Molecular Systems and F Hoffmann La Roche Ltd A license to use the PCR process for certain research and development activities accompanies the purchase of certain reagents from licensed suppliers such as Amersham Biosciences and affiliates when used in conjunction with an authorized thermal cycler The PCR process for amplifying DNA is covered by US patent numbers 4 683 195 and 4 683 202 assigned to Hoffman La Roche Inc and F Hoffmann La Roche Ltd Patents are pending or issued in other countries The MegaBACE DNA Analysis System is for research purposes only It is not intended or approved for diagnosis of disease in humans or animals All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group that supplies them A copy of these terms and conditions is available on request Amersham Biosciences Corp 2002 All rights reserved June 2002 Notice to purchaser limited license The MegaBACE instrument is a confocal scanning system licensed under US Patent Numb
117. odified MegaBACE 1000 that accepts only one two or three 16 capillary arrays for a capacity of 16 32 or 48 capillaries Flexible MegaBACE 1000 A modified MegaBACE 1000 that accepts up to six 16 capillary arrays for a capacity of 16 32 48 64 80 or 96 capillaries MegaBACE 1000 The standard model with 96 capillary capacity grouped in six 16 capillary arrays MegaBACE Instrument Administrator s Guide p1 1 Part One Introduction and safety 1 1 System hardware components The M egaBACE system consists of the following hardware components figure 1 1 e MegaBACE instrument Electrophoresis components and temperature regulation system lasers and light collection system and scanner electronics Power supply fan module Blue laser power source and fan for cooling the laser e Computer Computer monitor keyboard and mouse Other components included with the instrument Cathode water tank anode plugs emission beamsplitters and filters and capillary arrays MegaBACE 500 and flexible M egaBACE 1000 instruments only Additional components M egaBACE 500 anode cover and for both modes array placeholder which consists of an anode blocker an anode sleeve a window blank and a cathode plunging tool figure 1 2 e Accessory kit H oses cables fittings tools documents and software CD e Optional items not included Bar code reader Uninterruptible power supply UPS with batte
118. ognizes See table 4 1 for a list of the parameter field names and a description of the expected values File Edit Search Help PRERUN TIME STORE TEMPERATURE LOW PRESSURE TIME RELAXATION TIME RUN TEMPERATURE STORE TIME PRERUN VOLTAGE PHT2 VOLTAGE FLUSH2 TIME PREINJECTION TIME FLUSH1 TIME PHT1 VOLTAGE PREINJECTION VOLTAGE USER INPUT TIME HIGH PRESSURE TIME Field column Value column Figure 5 7 A sequencing example of an instrument control parameter template file icp created using Notepad 5 5 2 Value constraints If you type a value that is invalid for example an alpha character when the software requires a number or a value that is out of range the Instrument Control M anager displays an error message when you import the icp file If this occurs you must correct the value in the icp file before it can be imported successfully Alternatively if the edit mode is on you can enter the correct value for the current run in the Instrument C ontrol window MegaBACE Instrument Administrator s Guide 5 13 Parttwo Understanding and configuring the MegaBACE system 5 5 3 Creating an instrument control parameter template icp To create an icp file 1 Use any text editor or Excel to open one of the icp template files provided with your software The available icp files are located in the M egaBA CE Templates folder Alternatively you can open a new document in any text editor or
119. ol M anager performs automatic base calling The software stores the resulting analyzed run folder in the M egaBACE AnalyzedD ata folder default or the location you specify MegaBACE Instrument Administrator s Guide Glossary 1 Glossary anode the positive end of the capillaries where each reservoir tube contains an array of 16 capillaries and an electrode The anode reservoir is located on the right side of the instrument The negatively charged DNA ions migrate toward the anode M egaBACE 500 and flexible M egaBACE 1000 instruments only Each empty anode reservoir contains an anode sleeve to prevent you from loading a tube for this array position array placeholders M egaBACE 500 and flexible M egaBACE 1000 instruments only the components that are installed in the empty array positions Each array placeholder consists of a cathode plunging tool that is used to fill the empty wells of the sample plate an anode blocker that is installed in place of an anode plug an anode sleeve that is installed in the anode reservoir and a window blank that is installed in place of the capillary window band pass filters optical filters that allow a band of selected wavelengths to pass through while rejecting both shorter and longer wavelengths base callers sequencing only the software that identifies the candidate peaks and calls the bases in a sequence base colors the display color of the base letter and trace assoc
120. operator cannot override YorN Integer A number p6 8 MegaBACE Instrument Administrator s Guide Chapter 6 Using the system initialization files Table 6 2 The field names and default settings of the MegaBACE ini file continued Field name Description Setting type Default Automatic FASTA file Sequencing only Determines whether the YorN N creation Instrument Control Manager automatically creates FASTA files after base calling Y create FASTA files N do not create FASTA files Allow FASTA override Sequencing only Determines whether YorN Y an operator can override automatic FASTA file creation from the Configure menu Y operator can override N operator cannot override Automatic SCF file creation Sequencing only Determines whether the YorN N Instrument Control Manager automatically creates SCF files after base calling Y create SCF files N do not create SCF files Allow SCF override Sequencing only Determines whether an YorN Y operator can override automatic SCF file creation from the Configure menu Y operator can override N operator cannot override Automatic raw text file Sequencing only Determines whether the YorN N creation Instrument Control Manager automatically creates raw text files after base calling Y create raw text files N do not create raw text files Allow raw text override Sequencing only Determines whether an YorN Y operator can override automatic raw te
121. oubleshooting G uide Opening the filter compartment lid during an electrophoresis run interrupts data recording Open the lid between runs only If you need to open the lid during a run stop the run before opening the lid to protect the data you have already collected When you open the filter compartment lid the system shuts off the voltage to the PM Ts which protects the PM Ts and stops the data collection 2 6 Internal electronics Under normal operating conditions you are protected from high voltage within the instrument electronics N evertheless voltages up to 20 kV are present in the instrument during a scan The label in figure 2 7 warns of this danger Figure 2 2 shows the locations of the label on the left side of the instrument on the side wall inside the electrophoresis compartment and on the PM T cover p2 8 MegaBACE Instrument Administrator s Guide Warning A Warning Warning A Achtung Chapter 2 Safety precautions The instrument has sensors and interlocks that are designed to protect you from moving parts high pressure hazardous voltage or laser light Exposure to such hazards can cause severe or fatal injury Do not remove panels for any reason Do not defeat the sensors or interlocks or try to gain access through any other opening N ote You can however remove the air filter panel on the left side of the instrument to clean the air filter see the M egaBACE Instrument M aintenance and Trou
122. ound plate holder Do not overfill containers Figure 2 2 Locations of important labels on the MegaBACE instrument side views MegaBACE Instrument Administrator s Guide p2 3 Partone Introduction and safety 8 License agreement 9 Made in U S A 10 Nitrogen pressure general hazard 11 Low pressure nitrogen 12 High pressure nitrogen Pe ts may be obtained endors of reagents may be authorized under rangements fan ossi 50 Lincoln Center Deive Foster City California S404 PE Applied Biosystems does not guarantee the performance of this instrument AMERSHAM 1S A LICENSED VENDOR FOR AUTHORIZED REAGENTS Figure 2 3 Locations of important labels on the MegaBACE instrument back view p2 4 MegaBACE Instrument Administrator s Guide Caution Chapter 2 Safety precautions 5 Amersham 928 E Arques Ave Y Biosciences Sunnyvale CA 94086 MODEL MegaBACE P S FAN MODULE ze 208 220 1230 240 10A 50 60Hz DE Trka e monte A S W y HAUARD ma AAAA AA NORE MADE IN USA BL Serial number certification Back of power supply fan module Figure 2 4 Location of the serial number certification label on the power supply fan module If a label becomes illegible for any reason ple
123. ow the MegaBACE instrument works 3 2 2 Cathode end of the capillaries MegaBACE 500 and flexible MegaBACE 1000 instruments only The instrument scans and collects data from the number of capillaries installed in the instrument 16 32 48 64 80 or 96 The software saves the data in a raw run folder which contains a corresponding number of rsd files 16 32 48 64 80 or 96 When a plate is in position at the cathode end of the capillaries only the active wells for the array positions with installed capillaries contain a capillary tip and an electrode For any empty array positions a cathode plunging tool is used to fill the inactive wells Table 3 1 and figure 3 2 show the active well positions for the number of capillaries installed in the instrument Table 3 1 The active well positions for the number of installed arrays Number of arrays installed in instrument Total capillaries Active well positions 16 A01 through H02 2 32 A01 through H04 3 48 A01 through H06 4 64 A01 through H08 5 80 A01 through H10 6 96 A01 through H12 MegaBACE Instrument Administrator s Guide p3 3 Parttwo Understanding and configuring the MegaBACE system SSS 6 arrays 5 arrays 4 ee 3 arrays 2 arrays NF a N i OO QIO QO Q IOOO CHO CO eee WO OW OrO C0 C100 OO OO O O10 O10 O O OO O Figure 3 2 The active wells for the number of installed capillary arrays MegaBACE 50
124. p Instrument Control Run Image For Help press F1 27 00 44 00 Run time 000min 00sec Full Run Time 000 min 04 11 00 01 56 PM Figure 4 7 The Instrument Control window with the sequencing instrument parameters displayed p4 12 MegaBACE Instrument Administrator s Guide Chapter 4 Using the Instrument Control Manager windows to configure the system Table 4 1 Instrument control parameters Parameter Description Matrix Fill High Pressure Time The time seconds for applying high pressure to fill the capillaries with matrix range 1 600 s Matrix Fill Relaxation Time The time minutes to allow the matrix to equilibrate range 0 120 min Prerun Time The time minutes for the electrophoresis prerun range 0 120 min Prerun Voltage The voltage for the prerun range 1 20 kV Preinjection Voltage The voltage for preinjecting samples range 1 20 kV PMT1 Voltage The voltage for PMT1 range 450 950 V Run Temperature The electrophoresis compartment temperature for the run range 27 44 C Sleep Temperature The temperature you want to maintain in the electrophoresis compartment while the instrument is idle 25 2C Matrix Flush Timel The time Seconds for the first matrix flush range 0 60 s Matrix Flush Time2 The time Seconds for the second matrix flush range 0 60 s Low Pressure Time The time seconds for
125. p4 5 Parttwo Understanding and configuring the MegaBACE system EEE 4 In the Electrophoresis Parameters tab type the values you want to use for the following parameters N ote See section B 1 2 for examples of sequencing parameters and see section B 3 2 for a genotyping example e Sample Injection Voltage T he voltage you use for the sample injection range 1 20 kV Sample Injection Time T he length of time seconds you want to inject the samples range 0 600 s Run Voltage T he voltage you use for the run range 1 20 kV e Run Time The length of time minutes for the run range 1 720 min 5 Click the Chemistry Parameters tab The Chemistry Parameters tab appears figure 4 3 Electrophoresis Parameters Chemistry Parameters Sample Names Optional Parameters Comments Chemistry Name ET Terminators x Laser Mode Blue Channel Base Dye Filter Beamsplitter A 1 M Erec E55DF20 540DRLP 2 fa feat E200F20 3 E JETRO j OLP E 595DALP 4 je 5 Figure 4 3 The Chemistry Parameters tab with sequencing parameters displayed 6 Select the name of the chemistry parameter set you want to use from the Chemistry N ame list The list contains the names of chemistry parameter sets for the selected application only N ote You can edit the chemistry parameters in the Chemistry ini file only section 6 3 The Chemistry ini file specifies the chemistry parameter sets available for each a
126. pF 1 Part three Appendixes Format octal Symbol Description 00 Ln List where nis a number 01 An ASCII 02 Cn Unicode 10 B Boolean 1 byte 1 true 0 false 31 Ul 1 byte unsigned integer unsigned char 32 U2 2 byte unsigned integer unsigned short 34 U4 4byte unsigned integer unsigned long 40 F8 8 byte floating double 44 F4 Abyte floating float 51 sl l byte signed integer char 52 S2 2 byte signed integer short 54 54 4byte signed integer Jong 7 T Terminator Figure F 1 The protocol for self describing data N otes to figure F 1 The symbol that represents a format is used only in readable dumps of the data block The symbol is not embedded in the data structure itself The lines delimited with square brackets indicate format codes that are not allowed in the extended header because those data types are not supported by the COM interface that enables the header F3 Length value The length value consists of 1 to 3 bytes except for Terminator as determined by the number of length bytes field in the header If the format is List this field contains the number of elements in the list Otherwise it contains the number of bytes of data storage used in the element N ote The number of individual data values in the element that is the array size is the length divided by the size of each value F 4 Data buffer For all formats except List the data buffer contains the number o
127. pl file for a long sequencing run with ET terminators Long sequencing run with dye primers Figure B 4 shows a tpl file containing the plate setup parameters for a long sequencing run with dye primers E LongDyePrimer tpl Notepad ioj x File Edit Search Help RUN TIME 366 RUN VOLTAGE 3 INJECTION TIME 46 INJECTION VOLTAGE 3 COMMENT Standard Primer CHEMISTRY ET Primers Figure B 4 A tpl file for a long sequencing run with ET primers MegaBACE Instrument Administrator s Guide pB 3 Part three Appendixes Standard M13 sequencing run Figure B 5 shows a tpl file containing the plate setup parameters for a standard APB M 13 run E StdAPBm13 tpl Notepad Iof x File Edit Search Help INJECTION TIME 46 INJECTION VOLTAGE 3 COMMENT APB M13 Standard CHEMISTRY ET Primers Figure B 5 A tpl file for a standard APB M13 run B 2 Instrument control parameters for sequencing Figure B 6 shows the N ormal icp file containing the instrument parameters you typically use for a sequencing run The N ormal icp file is included with the M egaBACE system software The file is stored in the M egaBACE Templates folder 8 Normal icp Notepad Iof x File Edit Search Help HIGH PRESSURE TIME Figure B 6 A typical instrument control parameter template file icp for sequencing pB 4 MegaBACE Instrument Administrator s Guide Caution Appendix B Plate and instrument parameter setup B 3 Plate s
128. plate in the MegaBACE ini file section 6 4 The default template is loaded when the Instrument Control Manager starts MegaBACE Instrument Administrator s Guide 5 15 Chapter 6 Using the system initialization files This chapter describes the system initialization files that you can modify to configure the system for your laboratory workflow and applications The topics are About the system initialization files section 6 1 File format and storage section 6 2 M odifying the Chemistry ini file section 6 3 Using the M egaBACE ini file section 6 4 6 1 About the system initialization files The system initialization files are M egaBACE ini file Determines the settings the Instrument Control M anager uses to control the operation of the M egaBACE system The Instrument Control M anager reads this file only on startup Chemistry ini D etermines the chemistry parameter sets available in the Instrument Control M anager A chemistry parameter set specifies the names of the dyes the dye to channel or base to channel mapping the names of the filters and beamsplitters and the laser mode used for a given application sequencing or genotyping The Instrument Control M anager re reads this file every time you choose a Chemistry N ame in the Plate Setup window section 4 1 3 You can open the files in a text editor that supports a tab separated format for example Notepad or Excel The system initialization file
129. plate setup parameters The parameters specified on this tab automatically override the same parameters specified in the Instrument Parameters area of the Instrument Control window The PMT voltages are instrument specific and are set by the MegaBACE Instrument Specialist at installation Contact MegaBACE System Technical Support before you alter the PMT voltages Sequencing only The base caller displayed in the O ptional Parameters tab overrides the default base caller section 4 4 2 Comments tab The Comments tab allows you to include descriptive information about the plate with no limitation on the number of characters MegaBACE Instrument Administrator s Guide p4 3 Parttwo Understanding and configuring the MegaBACE system Important 4 1 2 About the plate ID An operator identifies a plate by typing a plate ID or scanning a bar code into the Plate Setup window figure 4 1 Because the plate ID along with the run ID is used to name the folder in which the Instrument Control M anager stores the rsd files for the run a plate ID should be a manageable size such as 32 characters or fewer 4 1 3 Using the Plate Setup window to manually define a plate setup The edit mode must be turned on for you to edit the parameters in the Plate Setup window See section 6 4 for information about enabling the edit mode in the MegaBACE ini file When you set up a plate definition you provide a plate ID or plate bar code and specif
130. pplication 7 Click the Sample N ames tab The Sample N ames tab appears figure 4 4 p4 6 MegaBACE Instrument Administrator s Guide Caution Chapter 4 Using the Instrument Control Manager windows to configure the system Electrophoresis Parameters Chemistry Parameters Sample Names Optional Parameters Comments Figure 4 4 The Sample Names tab N ote The tab changes dynamically depending on the instrument model and the number of capillary arrays installed If no sample names are provided the software uses the well IDs for the sample names default To provide sample names that are different from the well IDs type the names you want to use to identify the samples in each well Alternatively you can use a plate setup template tpl or plate setup data file psd chapter 5 N ote You can use the arrow buttons to expand the size of the columns to allow for viewing longer names Genotyping only The Instrument Control Manager uses sample names entered in the Sample Names tab as the file names of the raw sample data files rsd Genetic Profiler v1 1 cannot recognize rsd files unless well IDs are used for the file names for example A01 rsd 9 O ptional If you want to include the PM T voltages and run temperature as part of the plate setup parameters or if you want to specify a different base caller click the O ptional Parameters tab The O ptional Para
131. rameters the settings for the sample injection voltage and time and the run voltage and time The electrophoresis parameters are displayed in the Electrophoresis tab in the Plate Setup window of the Instrument Control M anager software empty array positions M egaBACE 500 and flexible M egaBACE 1000 instruments only the instrument array positions that contain array placeholders instead of capillaries instrument parameters a combination of settings defining the instrument run conditions and matrix fill and flush cycles The instrument parameters are displayed in the Instrument Control window of the Instrument Control M anager software long pass filters optical filters that allow light of wavelengths longer than a specified cutoff to pass through to the PMT while rejecting light of wavelengths shorter than the specified cutoff LPA linear polyacrylamide see sieving matrix matrix see sieving matrix or spectral overlap matrix Phred sequence read editor program used to verify the accuracy of sequenced DNA The program was developed by Phil Green at the University of Washington plate definition includes the plate ID and plate setup parameters plate ID the name you give to the plate when you create a plate definition in the Instrument Control M anager plate setup parameters a combination of electrophoresis parameters chemistry parameters sample names and optional parameters that define a plate r
132. rameters that you enter manually You can set up plate definitions and templates for running the plate that an operator can import automatically at run time To run a plate in the automatic mode the operator types a plate ID or scans a bar code MegaBACE Instrument Administrator s Guide p1 5 Part One Introduction and safety Ss The Instrument Control M anager can automatically perform the following tasks when the operator enters the plate ID e Load the plate setup and instrument control parameters from the default template files section 1 5 2 Import the parameters and attributes unique to a given plate from a plate setup data file section 1 5 2 1 3 2 About changing the application The Instrument Control M anager currently supports sequencing and genotyping If your laboratory s instrument is configured for both applications and has the software licenses for both applications the Instrument Control M anager allows you to change between applications at any time N ote For genotyping the instrument must contain both the blue and green lasers and you must have the appropriate filter sets required for each application You can use the system initialization files to specify the default application chapter 6 and the parameter configuration files to specify the corresponding parameters for the application chapter 5 1 3 3 About data collection with automatic base calling sequencing only The Instrument Control M ana
133. re displayed in the Instrument Parameters area of the Instrument Control window figure 4 7 Table 4 1 describes the instrument control parameters You can create a template from the values entered in the Instrument Control window or you can create a template using a text editor or Excel section 5 5 Instrument control parameters ZE Instrument Control Manager MegaBACE Sequencing plate1Run01 Fie View Options Templates Configure Help List of Protocols p Instrument Parameters Prager Capea Matrix Fil High Pressure Time 200 sec Matrix Flush Timel 22 ses Rinse Tips Matrix Fill Belaxation Time 20 min Matrix Flush Time2 7 sec Matie Fil and Frerun ay ae Prerun Time Eo min Low Pressure Time 240 sec 4 Buin Pretun Voltage 3 kv User Input Time 120 see SIDES Preinjection Voltage 70 KV Preinjegtion Time T5 sec f PMTI Voltage z0 V Store Capillaries Okage 750 eae 750 v Flush and Dry Capillaries eatae 44 C Sleep After oe Replace Capilares Sleep Temperature 25 c Sleep Time 6 his High Pressure Flush LowFFressue Fish Workflow Activity Le tmpr 44 Janoprint Prerun in janoprint2 progress catprint2 Until complete Focus Capitanes Materials and Instructions Use to inject the samples and begin electrophoresis Full and clean water tank Sample plate anoprint Prerun lanoprint2 complete catprint2 Since prerun timer up 1 Plate Setu
134. ries may have one of two configurations e MegaBACE 1000 instrument section 1 1 Flexible M egaBACE instrument configuration section 1 4 The anode end of the capillaries is the same for all configurations 3 2 1 Cathode end of the capillaries 96 capillary instruments The instrument scans and collects data from all capillaries in one run The software saves the data in a run folder which contains raw sample data files rsd When a plate is in position at the cathode end of the capillaries each well contains both a capillary tip and an electrode figure 3 1 MegaBACE Instrument Administrator s Guide p3 1 Parttwo Understanding and configuring the MegaBACE system d Cathode array stand Window platform Anode reservoir holder An array of l 16 capillaries Capillary f i detection window Cathode bar i Anode cover kw HL E top view Q aN E looooc00e Anode plug 7 N 0000000 N oH jooooooco A Q T o B aaee ae Cathode assembly al a H e2 00000 top view pi 9 Ap TA A afl eo Figure 3 1 The capillary path p3 2 MegaBACE Instrument Administrator s Guide Chapter 3 H
135. ry storage recommended The system uses a nitrogen pressure source cylinder with regulators or multiunit manifold The MegaBACE Planning Guide provides the specifications for the nitrogen system For more information on the nitrogen pressure source see the M egaBACE Instrument M aintenance and Troubleshooting Guide pl 2 MegaBACE Instrument Administrator s Guide Chapter 1 Introduction to the MegaBACE system MegaBACE instrument Power supply fan module Computer Nitrogen cylinders Note In addition to the components shown an uninterruptible power supply UPS is recommended Capillary array HHD Anode plug Cathode bar Cathode water tank Emission beamsplitters Emission filters Figure 1 1 The MegaBACE system and components MegaBACE Instrument Administrator s Guide p1 3 Part One Introduction and safety A Array placeholder EN fe l QV EHD Q MegaBACE 500 Anode Anode Window Cathode anode cover blocker sleeve blank plunging tool Figure 1 2 Additional components MegaBACE 500 and flexible MegaBACE 1000 instruments only 1 2 The MegaBACE system software The M egaBACE system includes software for Instrument control The instrument control software includes the Instrument Control M anager and the H ost Scan Controller The H ost Scan Control
136. s 5 3 3 The psd file format Table 5 1 lists the psd file field names that are recognized by the Instrument Control M anager For the plate setup parameters you want to import into the Plate Setup window you must use the field names that the Instrument Control M anager recognizes You must type the field names in the first column the field column You type the values for each field name in the second column the value column For additional plate information you want included in the output file that will not appear in the Plate Setup window you can use field names the Instrument Control M anager does not recognize for example O perator You must also provide a name in the value column for example M ary Smith Table 5 1 The plate setup data file psd field names Field name Value Plate ID The string for the plate ID If the plate ID entered here is different from the bar code of the psd file name the plate ID entered here becomes the real plate ID that appears in the Plate Setup window The following fields are for plate setup parameters that will be imported into the Plate Setup window These fields can be defined in either the psd file or a plate setup parameters template tpl If both files are used to define the same parameter the values provided in the file imported last overwrite the values for any identical fields Injection voltage The range is 1 20 kV Injection time The range is
137. s a raw run folder that includes the rsd files for the run The M egaBACE system uses the plate ID and the run ID to name the raw run folders platelD_runlD You assign the plate ID when you set up the plate in the Instrument Control M anager software The run ID defaults to Run1 or RunOl Table C 1 summarizes the conventions for naming and storing the raw sample data files and folders Table C 1 Naming and storage conventions for the raw sample data File or folder type Name Location Raw sample data file Sample_name rsd PlatelD_runID raw run folder rsd well_ID rsd or a combination of both Raw run folder PlatelD_runID MegaBACE Data unless you select a different location The File Storage command in the O ptions menu allows you to change the storage location of the data at any time This feature only affects the data storage of subsequent runs It does not change the location of data already stored on the computer See the M egaBACE Instrument O perator s Guide for description of how to change the file storage location pC 2 MegaBACE Instrument Administrator s Guide Table C 2 Naming and storage conventions for the base called and exported files File type Base called Sequencing only Appendix C File Storage C 2 Base called sample data storage C 2 1 Naming and storage conventions for the base called and exported files Table C 2 provides an overview of the naming and storage conventions
138. s are located in the M egaBA CE DataSystem folder MegaBACE Instrument Administrator s Guide p6 1 Parttwo Understanding and configuring the MegaBACE system Important Important 6 2 File format and storage The initialization files consist of two columns field column and value column separated by any number of tabs The order of the fields is arbitrary and the field names are case insensitive The Instrument Control M anager ignores any unrecognized fields or any blank rows between fields The system initialization files must be stored in the MegaBACE DataSystem folder The software will not function properly unless these files are stored properly 6 3 Modifying the Chemistry ini file The Chemistry ini file contains all the chemistry parameter sets that are available in the Instrument Control M anager The Instrument Control M anager uses only one Chemistry ini file to define all the available chemistry parameter sets See section 6 2 for a description of the file format If you are defining a parameter set for an application other than sequencing you must specify the application If you do not specify the application the Instrument Control Manager assumes that the chemistry parameter set is for the sequencing application For sequencing you must specify the bases Optionally you can specify both the bases and the dye names To add an additional chemistry parameter set to the Chemistry ini file 1 Open
139. shambiosciences com xiv MegaBACE Instrument Administrator s Guide Latin America Tel 55 11 3667 5700 Fax 55 11 3667 87 99 M iddle East and Africa Tel 30 1 96 00 687 Fax 30 1 96 00 693 Netherlands Tel 0165 580 410 Fax 0165 580 401 Norway Tel 2318 5800 Fax 2318 6800 Portugal Tel 21 417 70 35 Fax 21 417 31 84 Russia amp other C 1 S amp N I S Tel 7 095 232 0250 956 1137 Fax 7 095 230 6377 Southeast Asia Tel 60 3 8024 2080 Fax 60 3 8024 2090 Spain Te 93 594 49 50 Fax 93 594 49 55 Sweden Tel 018 612 1900 Fax 018 612 1910 Switzerland Tel 01 802 81 50 Fax 01 802 8151 UK Tel 0800 616928 Fax 0800 616927 USA Te 1 800 526 3593 Fax 1 877 295 8102 Part one Introduction and safety Chapter 1 Introduction to the MegaBACE system TheM egaBACE DNA Analysis System is a high throughput automated gene analysis system This chapter describes System hardware components section 1 1 The M egaBACE system software section 1 2 Overview of the Instrument Control M anager features section 1 3 The flexible M egaBACE instrument section 1 4 Administrator tasks section 1 5 Table 1 1 lists the available models of the M egaBACE instrument Throughout this guide some section titles and some paragraphs use the model name to highlight model specific topics Table 1 1 Available models of the MegaBACE instrument Model Description MegaBACE 500 A m
140. storage section C 1 Base called sample data storage sequencing only section C 2 e System initialization file storage section C 3 e Parameter configuration file storage section C 4 C 1 Raw sample data storage This section describes how the sample data files are stored and what the files contain C 1 1 Raw sample data file content For each run the Instrument Control M anager creates a raw sample data file rsd for the data collected from each well Each rsd file contains e Plate ID Well location Sample name if provided e Run data run ID instrument ID instrument control parameters plate setup parameters and date Information about the electrical current e Raw electropherogram C 1 2 Naming and storage conventions for raw sample data files and folders By default the software uses the sample names that you provide as the file names for the rsd files If the samples names that you assign for a given plate are not unique the Instrument Control M anager appends the well ID to the file name sample_name_well_ID If no sample names are provided the software uses the well ID as the file name for the rsd files Alternatively you can use a plate setup data file psd to specify the file names for the rsd files section 5 3 MegaBACE Instrument Administrator s Guide pC 1 Part three Appendixes as Each time you run a plate on the M egaBACE instrument the Instrument Control M anager create
141. t accessing 2 5 drawer illustrated 2 2 Safety 2 5 anode cover MegaBACE 500 1 4 anode plug 1 2 1 3 3 2 anode sleeves 1 2 1 4 aperture illustrated 3 5 location 3 7 application changing 1 6 6 16 to 6 17 default 6 7 argon ion laser 2 11 array placeholders 1 2 1 4 assistance xiv automatic base calling see base calling automatic MegaBACE Instrument Administrator s Guide Index 1 Index background noise elimination of 3 8 bad capillaries displayed 5 9 specifying in MegaBACE ini file 5 8 specifying in Sample Names tab 5 9 bar code 4 5 5 2 E 3 base call command automatic 6 11 base callers Cimarron 1 53 Slim Phredify B 2 default 4 16 4 17 specifying plate specific 4 17 base calling automatic see also base callers configuring MegaBACE ini 6 13 to 6 15 file storage C 3 to C 5 overview 1 6 beamsplitters for ET dye terminators A 8 functional description A 6 illustrated 3 5 C capillary windows avoid touching 2 7 illustrated 3 2 cathode 3 2 cathode assembly configuration flexible instrument 3 2 3 3 standard instrument 3 1 3 2 cathode compartment 2 2 2 5 cathode plunging tool 1 2 1 4 Caution statement defined xii CE declaration xiii Chemistry Parameters tab 4 3 4 6 Chemistry ini file configuring 6 2 to 6 5 storage C 6 Class 1 Laser Product label 2 11 cleanup command 6 10 collimated beam 3 8 Index 2 MegaBACE Instrument Administrator s Guide COM interface F 2 Comments tab 4 3 4 9 computer compon
142. te ID typed in the Plate Setup window a Plate setup data file psd b Plate Setup window psd file name Plate ID M13std1_060899 psd Notepad of x HA Instrument Control Manager MegaBACE Sequencing File Edit Search Help File View Options Templates Configure Help PLATE ID INJECTION VOLTAGE 3 INJECTION TIME 46 RUN VOLTAGE 9 RUN TIME 166 CHEMISTRY ET Primers RUN TEMP 44 PHT1 VOLTAGE 756 PHT2 VOLTAGE 756 BASE CALLER Cimarron 1 53 Phat COMMENT M13 Standard Run A81 Sample1 A81 Base Caller Molecular Dynamics AG1 MATRIX LPA Plate Catalgg M13std1_060899 M13std1_060899 Electrophoresis Parameters Cha Sample Injection Voltage Run Voltage Figure 5 1 Using a psd file a Example of a psd file for sequencing b The Plate Setup window with a plate ID that matches the psd file name MegaBACE Instrument Administrator s Guide p5 3 Parttwo Understanding and configuring the MegaBACE system 5 3 2 Using a plate ID that is different from the scanned ID f the bar code is not what you want to use as the plate ID you can use the psd file to replace a bar code scanned into the Plate ID box in the Plate Setup window To do this you type a plate ID in the psd file opposite the Plate ID field figure 5 2a The plate ID you typed opposite the Plate ID field in the psd file replaces t he bar code and be
143. the Chemistry ini file using a text editor or Excel The Chemistry ini file is located in the M egaBA C E D ataSystem folder Figure 6 1 shows a section of the Chemistry ini file The name of each chemistry parameter set appears in brackets for example TSll Version 2 Terminators The field names appear in the left column and the values appear in the right column The columns are separated by one or more tabs N ote that the field names vary slightly between the sequencing example and the genotyping example figure 6 1 2 Scroll to the end of the file and type a name for the new chemistry parameter set You must provide a unique name for each chemistry parameter set M ake sure you put the name in brackets p6 2 MegaBACE Instrument Administrator s Guide Chemistry parameter set for sequencing Chemistry parameter set for genotyping i Chemistry ini Notepad File Edit Search Help TSII Version 2 Terminators BEAMSPLITTER A S46DRLP BEAMSPLITTER B 595DRLP A G C T SSSDF26 526DF26 616LP 585DF26 R6G R116 ROX TAMRA Blue GT DyeSet2 ET RO0X FAM NED HEX APPLICATION Genotyping BEAMSPLITTER A 54 DRLP BEAMSPLITTER B 576DRLP 616DF26 526DF26 S86DF28 SSSDF26 ET ROX FAM NED HEX GREEN AND BLUE Chapter 6 Using the system initialization files Biles Name of the chemistry parameter set Name of the chemistry parameter set You must specify the application name if other than sequencing
144. the field names that begin with a well ID for example A01 are well specific For example you can specify the following types of information for a given well e Sample name which becomes the rsd file name by default e Filename and sample name where the rsd file name is different from the sample name section 5 3 6 Base caller User defined information If you want additional well specific information for your records the Instrument Control M anager allows you to include a dot character after the well ID followed by an additional name to annotate the well ID The following and figure 5 2 show examples of what you might enter in the psd file for annotation of well specific information Field name Value A01 BASE CALLER Molecular Dynamics A01 SAMPLE TYPE Tissue The Instrument Control M anager writes the additional well specific information only into the rsd file header for the corresponding well The well specific annotation does not appear in the Plate Setup window 5 3 6 Designating a sample file name in the psd file Genetic Profiler v1 1 cannot recognize rsd files unless the well locations are used for the file names for example A01 rsd If sample names are provided for a plate the software uses the sample names as the file names for example samplel rsd H owever you can provide sample names and designate file names that are different from the sample names To do this you use the fidd name Well_ID FILE
145. the system The Instrument Control M anager allows you to manually set up the plate definitions that will be used in the laboratory In addition you can save the frequently used plate setups and instrument parameters as templates so that you do not have to reenter the parameters You can set up the parameter templates so that an operator can perform automatic plate setup at run time The topics in this chapter are e Manually setting up plate definitions section 4 1 e Creating plate setup templates in the Plate Setup window section 4 2 e Creating instrument control parameter templates in the Instrument Control window section 4 3 e Using automatic base calling sequencing only section 4 4 See chapter 5 and the M egaBACE Instrument O perator s G uide for instructions on automatic plate setup 4 1 Manually setting up plate definitions The plate definition includes the plate ID electrophoresis parameters chemistry parameters sample names optional parameters if any and comments You can enter the information manually in the Plate Setup window Alternatively you can set up plates automatically by using a plate setup template section 4 2 and or importing a plate setup data file psd See chapter 5 for instructions on creating psd files 4 1 1 About the Plate Setup window The edit mode must be turned on to allow you to edit the parameters in the Plate Setup window See section 6 4 for information about enabling the
146. ting up plate definitions 0 00 05 4 1 4 1 1 About the Plate Setup window 00 0 cece ee eee 4 1 4 1 2 Aboutthe plate ID naaa 4 4 4 1 3 Using the Plate Setup window to manually define a plate SetiD ianea cate emia ARA RA DA 4 4 4 2 Creating plate setup templates in the Plate Setup window 4 9 4 2 1 About plate setup templates nauau 4 9 4 2 2 Creating a plate setup template aana 4 10 4 2 3 Saving the plate setup parameters as a template 4 11 4 2 4 Selecting a plate setup template nanana aaa 4 11 4 2 5 Setting the default plate setup template 4 11 4 2 6 Clearing the default plate setup template 4 11 4 3 Creating instrument control parameter templates in the Instrument Control WiNdOW 2 eee 4 12 4 3 1 Creating an instrument control parameter template 4 14 4 3 2 Selecting an instrument control parameter template 4 14 4 4 Using automatic base calling sequencing only 4 15 4 4 1 Manually turning on or off the automatic base calling mode and exporting data 2 eee 4 15 4 4 2 Specifying base callers for automatic base calling 4 16 Chapter 5 Using the parameter configuration files 5 1 About the parameter configuration files 000 0 eeu 5 1 5 2 Parameter configuration file format 00 000 cece 5 2 5 3 Using plate setup data files psd 2 0 cece cee ees 5 2 5 3 1 Aboutthe plate ID l a 2
147. to 6 17 automatic base calling and file export 6 13 to 6 15 description of field names 6 7 to 6 11 edit mode configuring 6 11 6 12 example 6 6 modifying 6 6 sections in the file 6 7 storage C 6 mirror location of 3 6 monitor safety 2 13 multiple applications Chemistry ini file 6 2 configuring MegaBACE ini 6 16 to 6 17 N nitrogen 2 9 2 10 nitrogen system components 1 2 safety 2 9 Note statement defined xii 0 objective lens function of 3 6 optical system 3 5 Optional Parameters tab 4 3 4 7 Override Commands window 7 2 P parameters see instrument control parameters or plate setup parameters pinching hazard 2 6 pinhole defined 3 5 plate defining 4 1 4 4 setting up 4 4 plate ID about 4 4 and psd file 4 5 length 6 8 Index replacing bar code 5 4 terminator 6 8 plate setup about manual or automatic 1 5 configuring for automatic import 5 2 to 5 12 manual 4 1 to 4 9 plate setup data files psd annotation of well specific information 5 7 creating 5 2 to 5 11 designating a sample name 5 7 examples genotyping B 5 to B 7 examples sequencing B 1 B 2 field description 5 5 5 6 B 7 importing 6 8 6 12 storage C 7 using 5 2 value constraints 5 7 plate setup parameter templates creating in a text editor 5 11 5 12 creating in Plate Setup window 4 9 to 4 11 default setting 4 11 6 10 example genotyping B 8 examples sequencing B 3 B 4 selecting 4 11 storage C 7 using 5 11 plate setup paramet
148. ts MegaBACE instrument e Fuserating Total of 6 fuses 2A 250V quantity 2 and 5A 250V quantity 4 e Fuse type TypeT slow acting e Electrical rating 200 240V 6A 50 60Hz Power supply fan module Electrical rating 180 229V or 230 264V 10A 50 60Hz Environmental conditions Ambient temperature range 20 25 C 68 77 F e Humidity condition lt 80 noncondensing e Pollution degree 2 e Installation category II MegaBACE Instrument Administrator s Guide xiii Preface Assistance When calling for assistance be prepared to supply the serial number of your instrument The serial number is located on the lower right side of the M egaBACE instrument figure 2 2 For contact by phone or fax please use one of the numbers below Asia Pacific Tel 852 2811 8693 Fax 852 2811 5251 Australasia Tel 61 2 9899 0999 Fax 61 2 9899 7511 Austria Tel 01 576 0616 22 Fax 01 576 0616 27 Belgium Tel 0800 73 888 Fax 03 272 1637 Canada Tel 1 800 463 5800 Fax 1 800 567 1008 Central East and Southeast Europe Tel 43 1 982 3826 Fax 43 1 985 8327 Denmark Tel 45 16 2400 Fax 45 16 2424 Finland amp Baltics Tel 358 0 9 512 39 40 Fax 358 0 9 512 17 10 France Tel 01 69 35 67 00 Fax 01 69 41 96 77 Germany Tel 0761 4903 291 Fax 0761 4903 405 Italy Tel 02 273221 Fax 02 27302 212 Japan Tel 81 3 5331 9336 Fax 81 3 5331 9370 Web site http www amer
149. turn on or off exporting of the data files to the selected format s Using the file export options If you turn on the automatic base calling feature the Instrument Control M anager provides the following export options e EXPORT IF BASE CALLING FAILED Default is N e EXPORT GOOD REGION ONLY DefaultisN ADD EXTENSIONSTO EXPORTED FILE NAM ES Default is Y You turn on or turn off the options in the M egaBACE ini file by typing Y or N Using the storage locations for exported files If you turn on the automatic base calling feature the software stores the exported files in subfolders within the raw or analyzed run folder as appropriate default You enable this feature in the M egaBACE ini file by specifying Y to EXPORT TO SUBFOLDERS The software creates subfolders for the specified export files Each subfolder has the name of the export file format Abd Scf Fasta Text See section C 2 for a description of each file format If you specify N for EXPORT TO SUBFOLDERS the raw or base called data will reside in the top level raw or analyzed run folder respectively p6 14 MegaBACE Instrument Administrator s Guide Chapter 6 Using the system initialization files 6 4 5 How automatic file export works sequencing only After automatic base calling the Instrument Control M anager creates files in the formats requested in the Automatic Base Calling window For more detail see the instrument operator s guide When
150. uorochrome A 1 site requirements xiii software 1 4 1 5 solid state laser emission wavelength 2 11 spills avoiding 2 7 standard header format D 1 standard M13 sequencing run B 4 Stokes shift A 2 subfolders export files 6 14 T tab delimited file 5 10 temperature 4 8 trained operator defined xii 2 1 U uninterruptible power supply UPS 2 13 user documentation xi using Automatic Base Calling window 4 15 Ww Warning statement defined xii wavelength emitted by lasers 2 11 weight of instrument 2 2 window blanks 1 2 1 4 window platform illustrated 3 2
151. vailable for the M egaBACE system M egaBACE Instrument O perator s G uide describes how to use the M egaBACE DNA Analysis System to automatically set up plate definitions and perform runs M egaBACE Instrument M aintenance and Troubleshooting G uide provides instructions on maintaining the instrument and guidelines on troubleshooting M egaBACE Planning Guide provides instructions for setting up the installation site for the M egaBACE instrument R eading the planning guide is a prerequisite for the installation of the M egaBACE system M egaBACE analysis software user s guides provide instructions on how to use the software to analyze the data collected from the M egaBACE instrument MegaBACE Instrument Administrator s Guide xi Preface xi Warning A Caution Important Warning A e TheM egaM anual provides detailed troubleshooting guidelines for the sequencing application The M egaM anual is posted in the M egaBACE User Zone at the following Web site www amershambiosciences com You need the serial number of your instrument to obtain a password to the User Zone Electronic versions of most of the documents listed above are available on the software CD provided with your M egaBACE system Safety Chapter 2 in this guide provides important safety information to be used in conjunction with your training Read and understand it thoroughly before you begin operating the instrument Special safety text M a
152. word you refers to this trained operator Using controls making adjustments or performing procedures other than those specified in this guide may result in hazardous exposure to laser light high voltage high pressure or moving parts Such exposure can cause severe or fatal injury Under normal operating conditions you are protected from laser light high voltage high pressure and moving parts The cathode and anode drawers and the electrophoresis compartment lid are fitted with sensors and interlocks The access lid of the filter compartment has a safety switch Figure 2 1 shows the locations of the drawers and lids used during routine operation of the instrument MegaBACE Instrument Administrator s Guide p2 1 Partone Introduction and safety Warnings A Filter Air filt i ir filter oponim Su compartment lid Cathode drawer Anode drawer Figure 2 1 Locations of the drawers and lids used during routine operation of the MegaBACE system The air filter opening is used infrequently Do not defeat the sensors and interlocks or try to gain access to the interior of the instrument through any other opening Do not remove panels for any reason Exposure to laser light high voltage high pressure or moving parts inside the instrument can cause severe or fatal injury To prevent hazardous exposure to laser light check the cover panels all around the instrument regularly If laser light is visib
153. xt file creation from the Configure menu Y operator can override N operator cannot override Automatic processed text Sequencing only Determines whether the YorN N file creation Instrument Control Manager automatically creates processed text files after base calling Y create processed text files N do not create processed text files MegaBACE Instrument Administrator s Guide p6 9 Parttwo Understanding and configuring the MegaBACE system Table 6 2 The field names and default settings of the MegaBACE ini file continued Field name Description Setting type Default Allow processed text Sequencing only Determines whether an YorN Y override operator can override automatic processed text file creation from the Configure menu Y operator can override N operator cannot override Export if base calling failed Sequencing only Allows an operator to YorN N specify whether to export files in which base calling failed Y export files N do not export files Export good region only Sequencing only Allows an operator to YorN N specify whether to export only the good region of sequence Y export good region N export all Add extensions to Sequencing only Adds standard YorN Y exported file names extensions to exported file names Y add extensions N do not add extensions Export to subfolders Sequencing only Exports specified files to YorN Y subfolders named with the file extensions Y
154. y the setup parameters for the plate To manually set up a plate definition 1 M ake sure the edit mode is on In the Configure menu a check mark appears in front of the Edit M ode command to indicate the mode is on If no check mark appears choose Edit M ode from the Configure menu and make sure a check mark appears 2 Click the Plate Setup tab to display the Plate Setup window figure 4 2 and click N ew If a default plate setup template has been specified the Instrument Control M anager loads the values of the template into the parameter boxes on the plate setup parameters tabs If no default plate setup template has been specified the Instrument Control M anager leaves the boxes blank p4 4 MegaBACE Instrument Administrator s Guide Chapter 4 Using the Instrument Control Manager windows to configure the system 2 Typea plate ID or 3 Enter parameters scan a bar code in the tabs YE Instrument Control Manager MegaBACE Sequencing 1 Click New 4 Click Save Figure 4 2 The Plate Setup window with the Electrophoresis Parameters tab displayed 3 Inthe Plate ID box figure 4 2 provide a plate ID type the text or scan the bar code N ote If a psd file exists section 5 3 the Instrument Control M anager loads the values The text or bar code in the plate ID box must match the file name of the psd file in order to import the file MegaBACE Instrument Administrator s Guide

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