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GeneAtlas System User's Guide

1. G Command Console Viewer File View Help EF Open File la Save Help Properties laz 24 E Array Information EREBE TE INN Appendix A Using the Viewer 109 Figure A 41 Correct alignment of corner grid D Repeat steps A through C for the other corners of the grid Continue manually aligning all misaligned grids 5 After you align the grid click the Save button or select File Save from the menu bar The DAT file is saved and new CEL file data is generated Changing the Manual Grid Adjustment Setting You can apply the coordinate adjustments made to one exposure time or channel to the other exposure time or channel To change settings for the manual grid adjustment 1 From the View menu select Options The Options dialog box opens Figure A 42 Figure A 42 Options dialog box Options JPEG Creation Compression Ratio 2 Sampling Ratio 3 Review Window Enable Review window on startup C Show only items with grid alignment errors Remove item upon close Polling Interval sec Grid Lines Grid line colar Manual Grid Adjustment C Apply adjusted coordinates to all channels and exposures Saving Image display options Select or deselect the Manual Grid Adjustment checkbox 3 Click OK to close the dialog box and enable the changes 110 GeneAtlas System User s Guide Regenerating Intensity Va
2. 2 In the Fluidics tab Figure 5 8 click the Strip button eS z FLUIDICS Q Utility Actions v Start No Strips Added The Add Strip Window appears Figure 5 9 Add Strip Scan or enter the bar code Bar Code strip Name Protocol 3 Scan or manually enter the Bar Code required of an Array Strip you have registered The Strip Name field is automatically populated The appropriate protocol will be selected by the application If you wish to run a custom protocol you may select it from the dropdown list 50 GeneAtlas System User s Guide 4 From the Instrument drop down box select the proper instrument if more than one is installed Add Strip Scan or enter the bar code Bar Code 5600030000000121212000 Strip Name Test 1 Rasa mE 5 Click the Add button in the Add Strip dialog box Figure 5 10 The array strip information is displayed on the left side of the Fluidics tab and a notice to close the fluidics door before starting the procedure is displayed Figure 5 11 IYO Affymetrix FLUIDIC S o Utility Actions Start 38C 20m Wash B Temperature 38 C 38 C Please close the fluidics door before starting the protocol Chapter 5 Fluidics Wash and Stain 51 6 Gently open the cover of the Fluidics station Figure 5 12 Figure 5 12 Ope
3. Autoscale F 3 Cul Date 2Z ga dat d b X I AQ QQ Ol Ot ew j Ga U g Se TI ggi 1800ms Zoom Color Contrast Copy Contrast Brightness Grid and Exposure Intensity Display CEL File tool bar y xj Ctrl Date 2 ga cel ilm QQ Bj Ot Ga Ji M Color Contras Mf Copy aM Bea Contrast Brightness iapa a Select file types Part of the tool bar may be hidden if the GeneAtlas Viewer is too small To display the hidden controls Click on the Hidden Tool Bar button at the right of the toolbar Figure A 17 ii Cui Date 2 05 dat NN Click here to display idden controls GeneChir HT Human Ger The hidden controls are displayed below the tool bar Figure A 17 Examining Different Parts of the Image These functions work on DAT CEL and JPG files The Zoom controls are at the left end of the tool bar 98 GeneAtlas System User s Guide Figure A 18 Zoom controls Zoom buttons To zoom in on a selected area of the image 1 Click on the Zoom Select button i1 2 Click and drag around the area you want to examine in more detail Figure A 19 Figure A 19 Click and drag g Ctrl Date 2 ga dat ila 2 Q EO Oi a 23 3 Release the mouse button 4 The selected area is displayed in the GeneAtlas Viewer Figure A 20 t Lil Date 2 ga dat n Q D2 G OLA JE u iii xe xo JE F 7 IEEE E B BI ELEM T TIN MIN BL
4. Gripper Arm in Home Position Hybridization Tray with Array Strip Wash B Tray Wash A Stain Tray Imaging Tray Figure 5 22 Closing the lid on the Fluidics station 56 GeneAtlas System User s Guide After loading the Fluidics Station 14 Click the OK button in the Close Fluidics Door notice Figure 5 23 Please close the fluidics door before starting the protocol 15 Click the Start button in the Fluidics tab Figure 5 24 FLUIDIC 5 t The Start notice appears Figure 5 25 A Start are you sure Chapter 5 Fluidics Wash and Stain 57 16 Click Yes After clicking the Yes button the LED light on the front of the Fluidics Station flashes green to indicated that the unit has begun performing the protocol Protocol progress 1s shown in the Protocol view Figure 5 26 Affymetrix FLUIDIC 5 O Utility Actions Pause WashB is preheating Time remaining 00 05 13 38C 20m The Protocol View displays Wash B temperature range and actual temperature Steps in protocol a Time duration for each step Status of each step in the protocol 58 GeneAtlas System User s Guide The Clock View Figure 5 27 displays the time left for the overall fluidics step Affymetrix 14 40 Q FLuiDiCs n Utility Actions 5500030000000121212000 Fluidics 1 ji 01 14 10 1 38 C 38 C 37 8 C The List view Figure 5 28 shows a
5. Failed to Find the arid corner pattern Please use the Command Console Viewer to verify that the corner locations do not have any smudges or crossover patterns that may cause the algorithm to Fail If you see the error message you can manually adjust the grid by using the following procedure The boundaries of a grid are indicated by the alignment patterns at the four corners of the grid Figure A 35 Grid borders 3 Command Console Viewer File View Help EF Open File la Save E Help Properties Failed_Grid_2_ HT_HG U133_Plus_PM _A04 DAT ez ie QQ G Olin 2a 0 El Array Information Pixel X 218 Pixel Y 2 1005 Intensity 22 Navigating from Grid to Grid The failed grids are marked with an X in the Grids box Figure A 36 Misaligned grids are marked with an X Appendix A Using the Viewer 107 Highlighting Selected grids are highlighted in black a Selected misaligned grids are highlighted in yellow Modified grids are highlighted in green You can step through grids using the right and left buttons Figure A 37 Figure A 37 Grid box toolbar grid step buttons All Grid Left Right Bad Grid Only HH Active Grid BE TATRA EE Grid Corner Buttons To step through all grids a Click the left and right E buttons to step through the grids To step only through the misaligned grids 1 Toggle the Step button to the misaligned position 2 Click the left
6. les 80 Download Ubrany FES sas sk saa yee k ese A kak k een ab ala klan d kud et eye de E h 4 ead db ah 2 el ade GO09E CEANA AMAMAMNHHHHHHHEHnHun nNnRHEnRnEnRnEnNnNRnE HnEHnEEI HHEHEHRDNKERn 84 GeneAtlas System User s Guide Appendix A Using the Viewer i ned 9o ac eee ww eeu pde d 3 deeem nae wa 87 Opening the Image Viewer in the GeneAtlas System kk KK KR KI ee 88 y an Grid TYDES bina cs 2k Gay ou en lr pee Ee di a anka hall ka das beds ee kee ewe 90 Introduction to the Viewer kk kk kk kk KK ls 91 Displaying Multiple Files AW kk kk kk kK KK KK eee 95 Changing the Display of the Image us kk kK KK KK KK KIRI KI K K KI KII K KI KIRI KIR KI KI KII K K KI 96 Learning about the Image File Ak kk kk kk KK KK KK KK KI KK KI KI K KIRI K KIRI KI K K KI KI KI KIRI KI KIRI KI 102 Checking the Gild AOBEEIBEIE iu dos s sa a na Pues Gout d l h lk 6 9 79 ol dk dl Al nt la 103 Exporting Images in Other Formats kk kk kk kk kK KK KI KK KII K KI K K KIRI KII KI KI KI KI KIRI KK KK KI 110 Appendix B Instrument Care kk kk cies kK KK KK kK kK KK kk kk kk kk kk kk kk kk ka 113 GeneAtlas Instrument Care kk kk kk kk KK KK K KI KK KI KI KI KI KI KI KIRI K KI KI KI KI KI KI KI KI KIR KI KK KI 113 Maintenance sorser bod Hadad x n din ne ande r av al di di dikek pnts ai ake a di t n db nb oda dr diy a aes il a 113 Index The GeneAtlas Instrument Overview I
7. 141 096 6 262 838 6 294 327 6 407 858 6 597 000 7 406 391 7 682 782 and other U S or foreign patents Software Products may be covered by one or more of the following patents Products may be protected by one or more of the following patents U S Patent Nos 6 090 555 6 611 767 6 687 692 6 829 376 7 130 458 7 451 047 7 634 363 7 674 587 and other U S or foreign patents Arrays Strips Products may be covered by one or more of the following patents U S Patent Nos 5 445 934 5 744 305 6 399 365 6 733 977 7 790 389 and other U S or foreign patents Products are manufactured and sold under license from OGT under 5 700 637 Copyright 2010 2012 Affymetrix Inc All Rights Reserved Contents i Table of Contents Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 The GeneAtlas Instrument Overview 0 002 cee YKY eee 1 lese mPCET 1 I O osaevenece bated cee ooo sea sewed aXas eee de eae RES EEYEEFS E NEA 3 User DOCUMIeNIQUON S d sS sa du Ak qb AV x SIE PETAT Id MESS ee bet eee es 4 Safety Information and Warnings llle 4 GeneAtlas Instrument Specifications lise e 6 Required Lab Equipment and Supplies 20 0 00 00 anaana ee 8 Technical Support kk kk kK KK KI KK KI KK KI KI KI KI KI KI KI es 9 Regulatory and Conformity kk kk kk kk KK KK KK KI KI KI KK KI KI K K KI KIR KI KI K KI
8. 3 Registering Samples 25 weka Affymetrix REGISTRATION R Utility Actions v Import Template gt Import Data i Save and Proceed gt gt mtm gt mmm text text text Sample 1 HT HG U133 Plus PM AOI HT HG U133 Plus PM HT HG U133 Plus PM HT HG U133 Plus PM Remove Strip ane P Are you sure you want to remove the strip Exp Date 1292 2002 Sample 8 56000300000001212120017 9 To add a sample attribute to the data grid A Click the Add Column button Add Column Alternately click a column header and select Add Column After or Add Column Before from the drop down list Figure 3 10 The Add Column dialog box appears REE Affymetrix REGISTRATION Import Template v Import Data i Save and Proceed gt gt ee Add Column After Column Name Gender Data Type Required Default Value New column will be New column can be added at the right side added before or after a of the data grid selected column B Enter the column name and select a data type text number or date from the drop down list C If an attribute value is required put a check mark next to Required If applicable enter a default attribute value 26 GeneAtlas System User s Guide D Click OK The new column appears in the data grid Figure 3 11 XC Affymetrix REGISTRATION Utility Actions v Import Template Import Data 1 Save and Proceed gt gt Add Column Sample File Name Probe Array T
9. 48 PM 10 13 2009 1 49 52 PM Completed Stopped Imager Imager Unknown 10 13 2009 3 56 39 PM 10 13 2009 4 06 03 PM 10 13 2009 4 30 49 PM Completed Completed Completed Completed Completed Stopped Array Information Hybridization Information Fluidics Information Imager Information 82 GeneAtlas System User s Guide Hybridization Information Fluidics Figure 7 11 Hybridization Information Process Step Instrument Name Start Time Stop Time Run Stop Time Temperature Processing Status Run Status Hybridization Hyb 10 13 2009 1 37 06 PM 10 13 2009 1 39 06 PM 10 13 2009 1 39 10 PM 45 C Completed Stopped Figure 7 12 Fluidics Information Process Step Instrument Mame VerificaBion Status Protocol Wash B MIN temperature Wash B MAX temperature Start Time Stop Time Run Step Time Pracessing Status Run Status Imager Information Fluidics Fluidics Unknown FluidicsTest orc 40 C 10 13 2009 1 42 15 PM 10 13 2009 1 49 48 PM 10 13 2009 1 49 52 PM Completed Stopped Figure 7 13 Imager Information Process Step Instrument Name Verification Status Start Time Stop Time Run Stop Time Imaging Status Gridding Status JPEG Generation Status CEL Generation Status QC Status Run Status Imager Imager Unknown 10 13 2009 3 56 39 PM 10 13 2009 4 06 03 PM 10 13 2009 4 30 49 PM Completed Completed Comple
10. E amp J and right amp buttons to step through the grids To manually align a failed grid 1 Click on the grid you wish to align in the Grid box 2 A zoomed in view of the grid appears in the Image window Figure A 38 File View Help Open File Help Properties i Failed Grid HT HG U133 Plus PM A04 DAT i ESPA BuNcWcWeW Ol ja 2 x4 000 28 El Array Information Pixel X 463 Pixel Y 977 Intensity 14 108 GeneAtlas System User s Guide 3 Align the grid at each corner A Click the Go To Corner 7 11 4 button for the corner you wish to align A zoomed in view of the corner of the grid appears Figure A 39 Figure A 39 The upper right grid corner Command Console Viewer File View Help E Open File a Save St E ZE EZ Pixel x 1199 Pixel Y 93 Intensity B Place the mouse arrow over the grid perimeter the arrow becomes a double arrow T The diagonal orientation of the double arrow along the perimeter of a corner probe cell indicates horizontal and vertical adjustments can be made simultaneously using the click and drag method or by using the keyboard arrow keys C Use the click and drag method or the keyboard arrow keys to adjust the horizontal or vertical position of the grid so that it 1s aligned over the outside corner of the small checkerboard pattern Figure A 40 Figure A 40 Dragging the grid boundary
11. GAPDH probe set signals Ratio lt 3 e Ratio is gt 3 The QC Parameters The QC Report displays information on a Signal Value u Hybridization Controls 78 GeneAtlas System User s Guide u Labeling Controls a Sample Quality Housekeeping Genes Signal Value This is a measure of the average brightness of the probe sets on the array minus the background noise The Signal to Noise ratio must be above a certain value for the array to pass QC Hybridization Controls Biotin labeled controls added to hybridization cocktail The 20X Eukaryotic Hybridization controls are high quality controls for monitoring array hybridization washing and staining for reproducible results The 20X Eukaryotic Hybridization Controls are composed of a mixture of biotinylated and fragmented aRNA of bioB bioC and bioD from E coli in staggered concentrations The premixed controls are ready to be added directly to the hybridization cocktail The 20X Eukaryotic Hybridization Controls are spiked into the hybridization cocktail independent of RNA sample preparation and are thus used to evaluate sample hybridization efficiency on eukaryotic gene expression arrays Labeling Controls Poly A Controls are added to RNA Sample prior to using IVT express kit Four independent poly A RNA controls derived from the lys phe and dap genes of B subtilis are provided conveniently in a pre mixed stock solution at staggered concentrations After s
12. KIRI KI K eee 10 Getting Started With the GeneAtlas System J RR RR KI 13 Array Processing Workflow kk kk kk kK KK KK KK KII KK KI KII KI K KIRI KI K K KI KI K K KI KK KK KK 14 File Types in the GeneAtlas System kk kk kk KK KK KI KI KI KI KI K KI KI K KI KI KI K KI KIRI KI KI RIK KI KIR KK 16 Data Organization in the GeneAtlas System kk kk kK KK K KIRI K K KI KIIR K KIRI KII KI KI K 18 Introduction to the Software Interface kk kk kk kK KK KK KK KI KI K K KI KII K KI KIRI KII KIRI KIRI K K 18 Registering Samples kak kK KK KK KK KK KI K KIR KI KI KI KI KI KK KK KK KK KK ka 21 Sample Registration A kk kk kk kk kK KK KK KK KK KI KI KI KI el 2 Using Templates tuo 6 bbe s eh nen Rede ee odd owe a ede KI KI K KI KI KI KI KI KI K KI KI K KI ee KK KK 28 Iiportng Sample DAL a s a decas Y ierat iet eR eae d l Seek eed dln oe a 29 Editino Sample EBBS a y i a arbe tenes a bb anal lk s on b dk db deal eb ete A le Qa alla la alal da 31 PVDMGIZAUOM cageeeenenda cee wk a heb a 35 Fluidics Wash and Stain a a naaa kk KIRI KIR eee RIK RI RIK KI KIR KK kk ka 43 Preparing Reagents and Filling Trays kk kk KK KK KK K K KI KII KI KIR KI KI KII KIRI K KI KI KK K 45 Loading Fluidics Station and Setting Up Run KK eee 49 la M T IPC O T 63 GeneAtlas Utilities llli 73 viewnga OG o Lr 74 Viewing Audit Files 20
13. MA ER MA EA M MET See Hi Ta E BE RA ME MI MAMA MI BI MI HES SERRE FA SAT SENE BEREBERE h a EEMEB BEE Bee BEBEPTNEEFNNEN mB mr SRE eee BET Eee Bee hill n ET EH See eee eee ee grDESREERHE et Lt e tt el a TT TT fot Tel TT TT rr HEHEHBNHEEN SEBE e e aa a AATAS a T a ET TTT te oT Tl Te Bis LEE Tae TT YY E wet tt sR EE miu BEL WE n rubr Ee eee BI DESPRE ee ee ee EBMEBEFKEHEEBIFES To zoom in or out on the whole image a Click on the Zoom In button or the Zoom Out button a To view a different area in magnified zoom a Click and drag the image to view the area of interest To zoom out u Click the Zoom Reset button Appendix A Using the Viewer 99 NOTE You can also use the Grid box controls to select a particular corner or grid for examination see Checking the Grid Alignment on page 103 Adjusting the Colors and Contrast These functions work on DAT CEL and JPG files Figure A 21 Color and Contrast controls Pseudo Auto Color Contrast Date 2 qa dat Po 2 BO ota a0 lt Gray Set Scale Contrast Contrast Slide Bars To switch between Gray Scale or Pseudo Color display u Click the Gray Scale or Pseudo Color buttons To adjust the contrast range for the image 1 Click the Set Contrast button lA The Set Contrast dialog box opens Figure A 22 Figure A 22 Set Contrast dialog box 5et Contrast E Intensity scale must be to 65535 Minimum Contrast Maximum Contrast 1
14. Strip Self Installation Instrument Chapter 1 The GeneAtlas Instrument Overview Component Barcode Reader Hybridization Station Fluidics Station Imaging Station Description Scan the array strip barcode The application enters the names into the computer workstation Add target samples to the hybridization tray and insert the array strip into the hybridization tray Scan the array strip barcode The application automatically populates the naming information Place the array strip on the hybridization station and click start on the user interface The computer workstation will track and log the hybridization time for each strip Scan the array strip barcode The application automatically populates the naming information Load wash imaging and hyb trays Scan the array strip barcode The application automatically populates the naming information Click the Open Imaging Station button load a array strip and Click the Close Imaging Station button The Imaging Station images the fluorescent labeled array and converts the intensity information into cel files These cel files contain a single signal value for each array feature 3 We have designed the GeneAtlas System for simple set up and operation A single computer workstation which has the pre installed instrument control software connects and controls the GeneAtlas Instrument components The GeneAtlas System Setup and Verificat
15. array Sample is re imaged one time the new data files will be named Sample 1 The original data files will not be overwritten Affymetrix jO MAGER Utility Actions v Open Imaging Station Start Imaging 1 Imaging 1 l Sample 1 lj Sample 2 W Sample 3 Sample 4 7 Click Open Imaging Station in the Imager tab The Imaging Station door opens Figure 6 7 Pm WARNING Do not open the Imaging Station door manually Doing so will damage the Imaging Station door Chapter 6 Imaging 67 Figure 6 7 Open Imaging Station door Be sure that the barcode on the Test Strip is facing to the left and that the oval on the imaging tray is facing out towards you 9 Click the Close Imaging Station button on the Imager tab Figure 6 9 68 GeneAtlas System User s Guide AGE Affymetrix IMAGER Utility Actions Close Imaging Station Start Imaging 1 lj Sample 1 iz Sample 2 W Sample 3 Sample 4 The Imaging Station door closes and the Start button is activated Figure 6 10 eR MAGER Utility Actions Open Imaging Station Start 10 Click the Start button in the Imager tab The Start Imaging Confirmation dialog box appears Figure 6 11 Chapter 6 Imaging 69 A Start are you sure 11 Click Yes to start imaging You can track the progress of imaging in the Progress view Figure 6 12 Affymetrix KT IMAGER Utility Actions Y Open Imaging Station Stop Test 1 55DOO DO0000
16. well plates Ambion AM10050 96 well Magnetic Stand or AM10027 Magnetic Stand 96 Orbital shaker for 96 well plates multiple e g Barnstead Lab Line Titer Plate Shaker Vacuum Centrifuge Concentrator Optional multiple Spectrophotometer NanoDrop ND 8000 e g NanoDrop ND 8000 UV Vis Spectrophotometer Technologies Reagents and apparatus for preparation and electrophoresis multiple of agarose gels Optional Pipette for 0 1 to 2 uL Rainin Pipette for 2 to 20 uL Rainin Pipette for 20 to 200 uL Rainin Table 1 7 Lab Equipment and Supplies Continued Material Pipette for 100 to 1000 uL Sterile barrier RNase free Pipette Tips Bioanalyzer optional Non stick RNase free microfuge tubes 0 5 mL Non stick RNase free microfuge tubes 1 5 mL Network Cable Or equivalent Technical Support Chapter 1 The GeneAtlas Instrument Overview Source Rainin multiple Agilent Ambion N12350 Ambion multiple When to Contact Technical Support Under any of the following conditions unplug the instrument from the power source and contact Affymetrix Technical Support when the power cord is damaged or frayed a if any liquid has penetrated the instrument Table 1 4 and Table 1 5 If the instrument must be returned for repair call Affymetrix Technical Support ES IMPORTANT Make sure you have the model and serial number Affymetrix provides technical support to all licensed use
17. 000 E Set the minimum and maximum contrast range Click OK to use the settings or Click Default to return to the default settings or Click Cancel to close the dialog box without changing the settings You can also use the slide bars in the tool bar Figure A 21 to set the contrast without opening the Set Contrast dialog box Using the Autoscale Function The autoscale function takes the image area you are currently viewing and calculates the intensity to find a better minimum and maximum contrast To use the Autoscale function Click the Autoscale button k The contrast and brightness are automatically adjusted 100 GeneAtlas System User s Guide Changing the Grid and Intensity Display These functions only work when DAT files are displayed Figure A 23 Figure A 23 Image window tool bar grid and intensity controls for DAT files only Chi Date 2 ga dat Autoscale Intensity Cell Centers Displaying Cell Intensity Data If you have a DAT file open with the associated cell intensity data CEL file available you can view the intensity data in the DAT file Image window To display or hide the cell intensity data a Click the Cell Intensity button ES The cell intensity data for the array 1s displayed Displaying Different Exposures GeneAtlas DAT Files Only Each GeneAtlas array is imaged twice with different exposure times The image data from both exposures are in the GeneAtlas DAT file Yo
18. 0121212000 Imaging 1 Green check marks appear next to the array names that have passed the internal QC parameters If an array fails internal QC parameters a red x mark and View Image link appears next to the array name Figure 6 13 Click the View Image link to view the array in the Image Viewer 70 GeneAtlas System User s Guide See a list of the arrays being imaged in the List view Figure 6 14 KM Affymetrix Do Utility Actions 1 Open Imaging Station Stop Sample File Mame Gender Probe Array Type Probe Array Position Test 1 Sample 1 Male HT HG U133 Plus PM A01 Imaging 1 Sample 2 Female HT HG U133 Plus PM CO1 Sample 1 Sample 2 W Sample 3 w Sample 4 Sample 3 Female HT HG U133 Plus PM E01 Sample 4 Male HT HG U133 Plus PM GOL A notice appears when imaging 1s finished and the QC Report button appears Figure 6 15 x b AN Affymetrix Utility Actions Open Imaging Station Stop Test 1 Imaging 1 NS Imaging 1 Sample 1 Sample 2 W Sample 3 Sample 4 You can click on the View QC Report to see a QC report on the arrays see Viewing a QC Report on page 74 Chapter 6 Imaging 71 12 Click on the Stop button 13 The Confirm Stop dialog box opens Figure 6 16 A Stop are you sure 14 Click Yes The Open Imaging Station button appears in the Imaging tab Figure 6 17 T Utility Actions Y Open Imaging Sta
19. 12 5 2008 Editing Sample Files sample files ARR can be edited You can a Import a template Edit sample data not all types of data can be modified Import data a Add user specified columns to the data grid 1 To open a sample file s select Edit Files from the Utilities drop down list 32 GeneAtlas System User s Guide Figure 3 23 Select one or more sample files to open AX Affymetrix REGISTRATION i Utility Actions v Save and Proceed gt gt Edit Files I Open File Current Directory C NGeneAtlasNData Up One Level A Name Pr Probe Array Type Gen A lal 5600001234567113018000 EO1 ar E01 FluidicsTest lal 5600001 234567113018000_G01 ar G01 FluidicsTest lai 5600011234567113018000_A01 ar A 1 AutoFocus Array lai 600011234567113018000_CO1 ar C01 AutoFocus_Array laj 5600011234567113018000_E01 ar E01 AutoFocus_Array laj 5600011234567113018000_G01 ar B 1 AutoFocus Array Sample 1 arr HT_HG U133_Plus_PM laj Sample 10 arr HT_HG U133_Plus_PM Male lai Sample 11 arr HT_HG U133_Plus_PM Male laj Sample 12 arr HT_HG U133_Plus_PM Female Sample 2 arr HT_HG U133 S Sample 3 arr HT_HG U M Female Sample 4 arr HT_HG U133_Plus_PM Male HT_HG U133_Plus_PM gt File name Open o amme Oe S UI Files of type Sample Files Cancel Female 2 In the Open File dialog box that appears select the sample files that you want to modify and click Open The Edit Files window appear
20. 1s complete a notice Figure 7 20 appears with the file name based on the date and time of collection and location Please send the GeneAtiaslog_20091025102721 zip from CACommand Consolevwip logs to supportiaffymetrix com 2 Click OK to close the notice You can now send the file to Affymetrix Support 86 GeneAtlas System User s Guide Using the Viewer After the array has been imaged the GeneAtlas System a Aligns a grid on the Image DAT file to identify the probe cells Computes the probe cell intensity data for the array and creates a CEL file The GeneAtlas Viewer displays DAT CEL and JPG files and enables you to a View the files for quality control purposes a Perform grid alignment for sub images that have a gridding failure See Checking the Grid Alignment on page 103 a Create a JPG version of a DAT file for archiving See Exporting Images in Other Formats on page 110 This chapter describes the operation of the GeneAtlas Viewer in the following sections a Opening the Image Viewer in the GeneAtlas System Array and Grid Types on page 90 Introduction to the Viewer on page 91 a Displaying Multiple Files on page 95 a Changing the Display of the Image on page 96 Learning about the Image File on page 102 a Checking the Grid Alignment on page 103 a Exporting Images in Other Formats on page 110 88 GeneAtlas System User s Guide Opening the Image Viewer in the GeneAtlas System If an array fails to
21. 28 GeneAtlas System User s Guide Using Templates In the data grid the default columns are File Name Probe Array Type and Probe ArrayPosition Templates provide additional column headers A template can also be applied after the sample files are created for more details see Editing Sample Files on page 31 The Express template is provided with the GeneAtlas application This template contains a set of attributes that might be useful while analyzing CEL files using the Partek Express Software You can assign this template to sample files during registration This automatically adds all the attributes in the template to the Sample file You can then enter values for each attribute This allows you to standardize the attributes that are assigned to samples The attributes are s Subject ID a Gender m Age Disease State Treatment Dose Time a Tissue a RNA extraction date a RNA extraction method Sample prep date Sample prepped by Sample source Importing a Template 1 In the Registration window add an Array Strip s to the data grid maximum of four strips For details on adding a strip see Step 3 to Step 4 on page 23 Click Import Template and select a template from the drop down list 3 Click Yes in the confirmation message that appears Figure 3 17 AX Affymetrix REGISTRATION Import Template Import Data Probe Array Type y Probe Array Position text HT HG U133 Plus PM HT HG U13
22. 3 Plus PM HT HG U133 Plus PM HT HG U133 Plus PM Are you sure you want to import the ExpressTemplate template xn aA The template columns appear in the data grid Figure 3 18 PV A Affymetrix REGISTRATION Import Template gt Import Data Add Column Sample File Name text HT HG U133 Plus PM HT HG U133 Plus PM HT HG U133 Plus PM HT HG U133 Plus PM Probe Array Position v text AQI coi E01 G01 Chapter 3 Registering Samples 29 Utility Actions v Save and Proceed gt gt NOTE Follow Step 9 on page 25 to add a user specified column header to the data grid Importing Sample Data You can import sample data to the data grid for more convenient sample registration 1 In Microsoft Excel create a data file xls that has the column Sample File Name in addition to any attributes you want to add The Probe Array Type and Probe Array Position columns do not need to be included in the data file Figure 3 19 shows an example data file IMPORTANT If you are using Excel 2007 be sure to save the file in Excel 1997 2003 Compatibility mode with an xls extension rather than Excel 2007 mode with an xlsx extension 30 GeneAtlas System User s Guide Gender Sample Type Tissue Date ofsample prep 25 Male RNA Blood 12 15 2009 14 Male RNA Kidney 12 8 2009 54 Male RNA Lung 11 28 2009 36 Female RNA Brain 12 5 2009 In the Registration window add an Array Strip s to the data grid ma
23. 3 Sample3 ga cel include v o Sample4 Sample4 ga cel Include 7 en gt v v 9 Table 7 2 QC Report Summary view Item Description ARR File Name Sample name CEL File Name File name automatically generated by the system for the intensity data Summary Include Sample data meets QC thresholds Exclude Sample data does not meet QC thresholds It is recommended that you exclude the sample from downstream analysis Intensity data CEL meet QC thresholds x Intensity data do not meet QC thresholds Hybridization Controls Control data meet QC thresholds Labeling Controls e3 Control data do not meet QC thresholds Sample Quality e GAPDH meets sample quality thresholds 3 GAPDH does not meet sample quality thresholds Chapter 7 GeneAtlas Utilities 77 Detail View The Detail View provides more information about the QC metrics This information can help you evaluate problems if they arise Figure 7 5 Figure 7 5 QC Report Detail view XWe Affymetr x QC Report Detail 9 qese E2 Print Signal Hybridization Controls Labeling Controls Sample Quality Sample File Name CEL File Name Summary Mean background Signal Noise ratio BioB less Bio C BioCless BioD Lys less Phe Phe less Dap Signal GAPDH GAPDH 3 5 ratio Samplei Samplet ga cel Include 98 63919 40 0 2448158 True True True 91 98 1 100272 Sample2 Sample2 ga cel Include 9627876 40 0 2381125 I True True True 90
24. 31875 1 105188 Sample3 Sample3 ga cel Include 91 68285 39 3 0 2307434 I True 86 09542 1 134278 Sample4 Sample4 ga cel Include 88 79778 39 3 0 2220342 I True 84 59669 1 065883 Table 7 3 QC Report Detail view Item Description ARR File Name Sample file name CEL File Name Intensity data file name v Labeling hybridization and sample quality controls meet QC thresholds x A labeling hybridization or sample quality control does not meet QC threshold Signal Mean The average signal of the probesets on the array after removing background signal Signal background The average value of the background signal Signal signal noise ratio Signal to noise ratio for the array log2 mean signal log2 background log2 background Hybridization Controls BioB less BioC If False the table cell for the array and item is highlighted in red BioC less BioD If False the table cell for the array and item is highlighted in red If both thresholds are not met the hybridization controls are marked J in the summary report a Labeling Controls Lys less Phe If False the table cell for the array and item is highlighted in red Phe less Dap If False the table cell for the array and item is highlighted in red If both thresholds are not met the labeling controls are marked in the summary report j Sample Quality Signal GAPDH Intensity signal for GAPDH GAPDH 3 5 ratio The ratio of 3 GAPDH probe set signals to 5
25. A Using the Viewer sample on page 87 Manage Instrument Enables qualification of the Fluidics Station or Imager Select and Name Instruments Station and displays the qualification information on page 19 of the GeneAtlas Setup and Verification Manual Download Library Files Click to select and download library files from Download Library Files on affymetrix com page 82 Collect Logs Creates a system log of all activity on the GeneAtlas Collect Logs on page 84 system including a log files for the Fluidics Station and Imager Station Troubleshooting Not available Help Opens the GeneAtlas Online Help About Opens the GeneAtlas About screen with information on the software To start a utility 1 Click Utility Actions 2 Make a selection from the drop down list 74 GeneAtlas System User s Guide XY Affymetrix HOME N c Utility Actions v View QC Report l Hybridization Fluidics Viewing a QC Report To help researchers establish quality control processes for gene expression analyses Affymetrix has developed several controls which enable researchers to monitor assay data quality These include but are not limited to a hybridization controls a labeling controls internal control genes The QC report displays the results of these controls When analysis results are viewed within the software the metrics for the report controls are compared to a set of pre defined thresholds Any results identified as bei
26. CAN CSA C22 2 No 61010 1 04 Canada as harmonized to IEC 61010 1 UL 61010 1 USA as harmonized to IEC 61010 1 EMC Directive 89 336 EEC Low Voltage Directive 73 23 EEC UL 61010 1 2004 R7 05 USA CAN CSA C22 2 No 61010 1 2004 Canada GeneAtlas Hybridization Station ICES 003 Industry Canada Interference Causing Equipment Standard Digital Apparatus Class A Canada FCC Part 15 Radio Frequency Emissions for Class A Equipment USA CAN CSA C22 2 No 61010 1 04 Canada as harmonized to IEC 61010 1 UL 61010 1 USA as harmonized to IEC 61010 1 Getting Started With the GeneAtlas System The GeneAtlas System provides tools for processing arrays and extracting the intensity data for use by the probe level analysis software To fully use the capabilities of the GeneAtlas System you need to understand a The array processing workflow that the GeneAtlas components perform The types of files that the GeneAtlas System produces and uses The structures that the GeneAtlas System uses to organize the resulting data This chapter introduces those concepts in a Array Processing Workflow on page 14 a File Types in the GeneAtlas System on page 16 a Data Organization in the GeneAtlas System on page 18 This chapter also includes an introduction to the GeneAtlas Instrument Control user interface see Introduction to the Software Interface on page 18 NOTE See the Affymetrix GeneAtlas Setup and Verification User s Guide
27. Chapter 4 Hybridization on page 35 to finish the hybridization 44 GeneAtlas System User s Guide Figure 5 2 The GeneAtlas Fluidics Station Chapter 5 Fluidics Wash and Stain 45 Preparing Reagents and Filling Trays You first need to prepare reagents and fill the wells in the various trays for the Fluidics processing These steps are described in the following sections u Types of trays a Required Supplies and Instruments a Preparation of Trays Types of trays You need to prepare the following trays before beginning the Fluidics processing a GeneAtlas Wash B Tray P N 2022256 a GeneAtlas Wash A Stain Tray P N 202257 46 GeneAtlas System User s Guide a GeneAtlas Imaging Tray P N 5600004 Figure 5 5 Imaging Tray with cover Required Supplies and Instruments Table 5 1 Reagents Reagent List No Spec Product Factory Storage Wash and Stain Kit 900720 30 rxns Affymetrix 4 Table 5 2 Instruments Instruments Type Product Factory Microcentrifuge 5415D Multiple sources Vortex Mixer TDX 1 Multiple sources Micropipettes P 2 P 20 P 200 P 1000 Multiple sources Sample Tube Rack 0 2 mL 0 5 1 5 mL Multiple sources GeneAtlas Fluidics Station Affymetrix GeneAtlas Imaging Station Affymetrix Table 5 3 Consumables Consumable Type Product Factory Disposable Tip 0 5 10 uL 2 200 uwL 100 1000 uL Multiple sources Disposable Tube 0 2 mL 1 5 mL Multiple sources Imaging tray Affymetrix
28. GeneAtlas Imaging Station and the GeneAtlas Instrument Control software as described in Chapter 6 maging on page 63 Tracking Gridding and CEL file Generation After the array has been imaged the GeneAtlas system a Aligns a grid on the Image DAT file to identify the probe cells Computes the probe cell intensity data for the array and creates a CEL file The Image Viewer enables you to manually correct gridding problems if necessary The Viewer and its use are described in Using the Viewer on page 87 16 GeneAtlas System User s Guide File Types in the GeneAtlas System Different types of information are collected by the GeneAtlas System in different types of files Information about the sample and experiment are collected in Sample files see Sample Files below Probe array data generated during imaging and processing are collected in Data files of various types see Data Files on page 16 a Audit and Log files contain information about array processing and other processes see Other File Types on page 17 Sample Files Data Files The Sample ARR file Figure 2 2 collects two types of information a Sample Attributes information that you can use to interpret the experimental data It can include information about the sample itself the experimental conditions or other information you may find useful Some attributes can be used by the probe level analysis software during analysis You can use template
29. J El Array Information Array ID 24cc 5al 11aa 438d 8 Array Mame Barcode DAT File Mame Design Type Expression Probe Array Type HT_HG U133_Plus_Ph Cel Cel Calumns 44 Cel Rows 44 File Id File Version 1 Folder Location C GeneAtlas D ata hd asimum Intensity Minimum Intensity Modified Date 9 8 2009 4 24 28 PM Cel Summary Report Bright features Dim features Maon synthesized fea Fluidics Actual Time Fluidics Serial Humb Cell X 440 Cell Y 2 740 Intensity 51 Each array is imaged twice with different exposure times Each image produces a single DAT file This DAT file can be viewed in the Image Viewer the file has all the image and gridding data for each grid and each exposure and allows you to check the gridding independently for each exposure The data from the DAT file is used to determine the cell intensity data for the grid for that exposure Only one CEL file is produced per array A CEL file is automatically generated after a DAT file is successfully gridded Introduction to the Viewer You can learn more about the basic functions of the GeneAtlas Viewer in u File Display Differences on page 91 u Moving the Components Out of the Viewer on page 92 u Moving the Component Borders in the GeneAtlas Viewer on page 94 File Display Differences The GeneAtlas Viewer has different types of functions and options for the different image file types that it displays DAT Files DAT files are the image data fi
30. KYO Affymetrix User S Gttide GeneAtlas System User s Guide For research use only Not for use in diagnostic procedures P N 08 0306 Rev B 0 GeneAtlas System User s Guide Trademarks Affymetrix Axiom Command Console CytoScan DMET GeneAtlas GeneChip GeneChip compatible GeneTitan Genotyping Console myDesign NetAffx OncoScan Powered by Affymetrix PrimeView Procarta and QuantiGene are trademarks or registered trademarks of Affymetrix Inc All other trademarks are the property of their respective owners Limited License Notice Limited License Subject to the Affymetrix terms and conditions that govern your use of Affymetrix products Affymetrix grants you a non exclusive non transferable non sublicensable license to use this Affymetrix product only in accordance with the manual and written instructions provided by Affymetrix You understand and agree that except as expressly set forth in the Affymetrix terms and conditions that no right or license to any patent or other intellectual property owned or licensable by Affymetrix is conveyed or implied by this Affymetrix product In particular no right or license is conveyed or implied to use this Affymetrix product in combination with a product not provided licensed or specifically recommended by Affymetrix for such use Patents Scanner Products may be covered by one or more of the following patents U S Patent Nos 6
31. La prise de courant doit tre situ e ou install e a proximit du materiel et tre facile d acc s ACHTUNG Zur sicheren Trennung des Ger tes vom Netz ist der Netzstecker zu ziehen Vergewissern Sie sich da die Steckdose leicht zug nglich ist Safe Operation The GeneAtlas Instrument 1s intended for indoor laboratory use in a controlled environment Do not attempt to service the instruments Any attempt at unauthorized service may result in injury or damage the instrument and or void the warranty Failure to properly support the instruments may cause serious damage or injury and may void the warranty The instruments must be surrounded by adequate airspace Slots and openings in the instruments and the electronics compartment covers are for ventilation Do not block or cover them Never push an object into the instrument ventilation slots equipment damage or injury may result Do not set liquids on top of the instrument a The instrument has an AC receptacle with a safety ground appropriate for the country of destination The plug is designed to connect only to a 3 prong ground receptacle This safety feature should not be compromised in any way If the instrument AC plug does not mate with the available power source receptacle consult a licensed electrician to install one that does Do not open the instrument electrical cabinets These contains electrical hazards A WARNING Users are not allowed to gain access t
32. TION a ccar ala Import Data Print Chapter 3 Registering Samples 23 3 Click the Strip button GES The Add Strip dialog box appears Figure 3 4 Add Strip Bar Code Strip Name 4 Enter or scan the Array Strip bar code and enter a strip name optional Figure 3 5 Add Strip Bar Code 5600030000000121212000 Strip Name Test 1 5 Click Add The Array Strip 1s added to the data grid in the Registration window Figure 3 6 eS Affymetrix REGISTRATION Import Template Import Data 1 Save and Proceed gt gt 6 Click a box under the Sample File Name column enter the sample name and press the Enter key Enter a unique name for each of the four samples on the Array Strip Figure 3 7 24 GeneAtlas System User s Guide pe P Affymetrix REGISTRATION Import Template Imgort Data i Save and Proceed gt gt text text text Sample 1 HG U133 Plus PM A01 T HG U133 Plus PM Col HG U133 Plus PM G01 7 To edit the Array Strip name or a sample name select the name and enter a new name Figure 3 8 ERNE Affymetrix REGISTRATION Import Template Import Data i Save and Proceed gt gt Add Column Sample File Name Probe Array Position text A01 col G01 8 To remove an Array Strip from the data grid A Click the Array Strip name and select Remove Strip from the drop down list Figure 3 9 B In the prompt that appears click Yes Chapter
33. Types The Alignment algorithm uses the checkerboard image of the control probes located at the corners of the probe array to superimpose a grid on the image The algorithm aligns the grid so that each square in the grid delineates a probe cell The alignment of the grids usually takes place automatically after imaging the array If the alignment algorithm fails you can perform a manual alignment of the grids The GeneAtlas Arrays use grids Figure A 5 File View Help E Open File lt Seve Help El Array Information Array ID 54be1dd3 5a7a 483b a Array Name Barcode Desig Pro r Type 1 be Array Type Expression HT HG U133 Plus Plv ui dics Wash B Min Fluidics Wash B Set El Grid Alignment Grid Algorithm Versic 3 0 0 157 arid Corners 150 73 1143 83 1 Pixel X 544 Pixel Y 0 Intensity 106 An array strip has four arrays Each array on the array strip has multiple grids Some arrays use a 7 x 7 grid for a total of 49 grids while other array types use a 7 x 6 grid for a total of 42 grids These sub grids are similar to the sub grids used for some cartridge arrays but on an array strip array the sub grids are not aligned to a main grid Appendix A Using the Viewer 91 Figure A 6 GeneAtlas array Full Image displayed as CEL file A Command Console Viewer File View Help E Open File lal save Help Properties gj EUl ga cel QQ Q BIO OL
34. WashA Stain tray Affymetrix WashB tray Affymetrix Chapter 5 Fluidics Wash and Stain 47 Preparation of Trays 3 NOTE You can use the GeneAtlas IVT WT Fluidics Station Quick Reference Card P N 08 0310 or the GeneAtlas miRNA Fluidics Station Quick Reference Card P N 703113 as a convenient reference when filling the trays To prepare trays for a fluidics run 1 Prepare wash and stain reagents twenty minutes before running the fluidics module NOTE The reagents need to be acclimatized to room temperature before using 2 Take the following reagents from Stain Module Box 1 and shake gently taking care to avoid bubble formation u Stain Cocktail 1 u Stain Cocktail 2 a Array Holding Buffer NOTE You may use a repeating pipettor to speed up the process NOTE If you are careful you can fill the trays without having bubbles form If you get bubbles you can use a pipet tip to pop them this works if you have 1 bubble in the corner for example NOTE For WT and IVT array products use tray layout map shown in Figure 5 6 on page 48 For miRNA array products use tray layout map shown in Figure 5 7 on page 48 3 Add 850 uL of Wash B to the corresponding positions on the Wash B Tray Figure 5 6 or Figure 5 7 Add 200 uL of Array Holding Buffer to the corresponding positions in the Imaging tray Figure 5 6 or Figure 5 7 5 Add 850 uL of Wash A to the corresponding positions in the Wash A Strain Tray Figu
35. abeled with a Triangle Figure 5 17 The label with the barcode should be facing left and the Triangle on the Hybridization Tray should be facing out toward you Figure 5 17 Loading Hyb tray and array strip The Imaging Tray Figure 5 18 has been filled with array holding buffer 54 GeneAtlas System User s Guide Figure 5 18 Imaging Tray and Protective cover Imaging Tray Protective Cover Discard NOTE Imaging Trays should always be handled with care Do not touch the glass bottom of the tray 11 Slide the Imaging Tray on the deck of the Fluidics Station in the Position labeled with a rectangle Figure 5 19 The label with the barcode should be facing left and the oval on the Imaging Tray should be facing out toward you Figure 5 19 Loading Imaging Tray 12 Close the retaining lever in front of the Hybridization Tray and Imaging Tray locations by rotating the lever in the clockwise direction Figure 5 20 Chapter 5 Fluidics Wash and Stain 55 WARNING Improper alignment of the trays and array strip can cause instrument damage Please ensure that all consumables are aligned properly To ensure proper alignment the trays should be pushed into the right hand inner corner of their position on the deck of the Fluidics Station Be sure that labels and notched corners on both trays are facing to the left as shown in the image below Figure 5 21 Figure 5 21 Fluidics Station Tray Deck
36. alog box for JPG creation My Recent Documents File name Falled Grid 2 HT Ha LI133 Plus PM AU4 My Network Files of type DAT Files dat v 2 Select the DAT file you wish to copy 3 Click Open The JPG file 1s created Viewing the JPG File To open the JPG file in the GeneAtlas Viewer 1 Click File Open File from the main menu or Click the Open File button in the GeneAtlas Viewer tool bar The Open dialog box opens Figure A 45 Figure A 45 Open dialog box Look in Data a 401 ga cel a CO1 ga cel My Recent 8 01 9a cel Documents 9 Failed_Grid_2_ HT_HG U133_Plus_PM _A04 CEL ES GO1 ga cel A01 ga dat 8 co1 ga dat 3it EO1 ga dat 4 Failed Grid 2 HT HG U133 Plus PM A04 DAT ii G01 ga dat I A01 ga jpg Ij C01 ga jpg IF E01 ga ipg Failed_Grid_2_ HT_HG U133_Plus_PM _A04 JPG E GO1 ga jpg a ie Of 4 I 2 5 File name lFailed Grid 2 HT HG U133 Plus PM A04J My Network Files of type Supported types dat cel mccel jpa htda If necessary use the dialog box tool bar to navigate to the directory with the file 3 Select the file you wish to view Click Open The selected image file is displayed in the GeneAtlas Viewer 112 GeneAtlas System User s Guide Changing Settings for the JPG Conversion To change settings for the JPG conversion 1 From the View menu select Options The Options dialo
37. ature 45 C 7 Hit the Add button in the Add Strip dialog box Chapter 4 Hybridization 37 The strip is displayed on the left side of the screen You can enter up to four arrays for hybridization z A HYBRIDIZATION o Utility Actions v Start Hyb 1 16 00 00 i Set temperature to 45 C 8 Set the Hybridization Station to the specified temperature Before proceeding be sure that the Hybridization Oven has reached the correct temperature 9 Open the hybridization station by pushing the front of the strip clamp towards the rear of the station and applying downward pressure on the top Figure 4 6 10 Place the hybridization tray with the array strip into a clamp inside the Hybridization Station Refer to the Assay Manual for details 11 Close the cover over the Array Strip 38 GeneAtlas System User s Guide 12 Click the Start button on the Hyb tab Figure 4 7 z GA Affymetrix A HYBRIDIZATION Utility Actions v Start Hyb 1 16 00 00 4 Set temperature to 45 C NOTE Please label the hybridization station cover to identify the location of a specific array strip The software does not track the location of a specific array strip and premature removal of an array strip will affect results The software displays the hybridization time countdown This time is displayed with a white background Figure 4 8 When the countdown has completed the display turns yellow and the time be
38. ave the following reagents instruments and supplies available in order to perform a run on the GeneAtlas Instrument Table 1 6 and Table 1 7 list the GeneChip 3 IVT Express kit and components required reagents and other supplies Table 1 6 Reagents Material Source P N GeneChip 3 IVT Express Kits Affymetrix 901228 10 Rxn 901229 30 Rxn GeneAtlas Hybridization Wash and Stain Kit for 3 IVT Arrays Affymetrix 901531 60 Rxn containing Box 1 of 2 o GeneAtlas 1X Pre Hybridization Mix o GeneAtlas 1 3X Hybridization Mix Solution A o GeneAtlas 1 3X Hybridization Mix Solution B n Nuclease free water n GeneAtlas Stain Cocktail 1 o GeneAtlas9 Stain Cocktail 2 o GeneAtlas Array Holding Buffer Box 2 of 2 n GeneAtlas Wash Buffer A n GeneAtlas Wash Buffer B 100 ethanol ACS reagent grade multiple The 10 reaction GeneChip 3 IVT Express Kit will produce 20 reactions on the GeneAtlas System The 30 reaction GeneChip 3 IVT Express Kit will produce 60 reactions on the GeneAtlas System Or equivalent Table 1 7 Lab Equipment and Supplies Material Source Thermal Cycler with heated Lid multiple capable of holding 0 2 mL tubes for reaction incubations and with appropriate adaptors to accommodate strip tubes Vortex Mixer with flat top adaptor for strip tubes multiple Microcentrifuge with an adapter for the PCR strip tubes or multiple plates supplied with the kit Magnetic Stand for 96
39. d_Console Data Defaul Up One Level Probe Array Type Er HT_HG U133_Plus_PM HT_HG U133_Plus_PM HT_Hu LI133 Plus PM HT_Hu LI133 Plus PM bull Files af type Sample Files Cancel 2 Select the file s to open and click Open The QC report is displayed Figure 7 3 Figure 7 3 QC Report Summary view Aff metr QC Report Summary Sample File Name CEL File Name Summary Signal Hybridization Controls Labeling Controls Sample Quality Samplei Samplel ga cel Include 7 N p p g v v Sample2 Sample2 gacel Include Sample3 Sample3 ga cel include v v Sample4 Sample4 ga cel Include v A Chapter 7 GeneAtlas Utilities 75 76 GeneAtlas System User s Guide Buttons at top of the QC Report allow you to Show the Summary view for a quick check of the QC status see Summary View below Show the Detail view that includes additional information to help you evaluate the quality of your results and locate problems see Detail View on page 77 Close the QC report Save the QC report txt Print the QC report Summary View The Summary view displays the basic pass fail status of the arrays Figure 7 4 PNE eee GeneAtlas TM Instrument KO Affymetrix QC Report Summary Sample File Name CEL File Name Summary Signal Hybridization Controls Labeling Controls Sample Quality Samplei Samplel ga cel Include gt Aa o e e Sample2 Sample2 ga cel Include v D v v Sample
40. displays information on all of the array processing workflow steps that have been started a Array Information on page 81 u Hybridization Information on page 82 a Fluidics on page 82 a Imager Information on page 82 If a workflow process has not been started the information will not be displayed in the Audit file If a workflow process has been run more than one time the information will be displayed separately Chapter 7 GeneAtlas Utilities 81 XGA Affymetrix Utility Actions Audit File Sample Name testi Strip name Barcode Probe array type Lot number Expiration date Date created Process Step Instrument Name Start Time Stop Time Run Stop Time Temperature Processing Status Run Status Process Step Instrument Name Verification Status Protocol Wash B MIN temperature Wash B MAX temperature Start Time Stop Time Run Stop Time Processing Status Run Status Process Step Instrument Name Verification Status Start Time Stop Time Run Stop Time Imaging Status Gridding Status JPEG Generation Status CEL Generation Status QC Status Run Status Array Information 5600030000000121212000 HT HG U133 Plus PM 12 12 2012 10 13 2009 1 31 17 PM Hybridization Hyb 10 13 2009 1 37 06 PM 10 13 2009 1 39 06 PM 10 13 2009 1 39 10 PM 45 C Completed Stopped Fluidics Fluidics Unknown FluidicsTest orc 40 C 10 13 2009 1 42 15 PM 10 13 2009 1 49
41. e nlla C Selection pi z ge 2 Select a printer and other options and click OK The report is printed Saving the QC Report Chapter 7 GeneAtlas Utilities 79 NOTE The Print function only saves the content displayed on the QC Report screen at the time Summary or Detail To save the QC Report 1 Click the Save button The Print dialog box opens Figure 7 6 Figure 7 7 Print dialog box Save As Save in B Data D hy Recent Documents Mu Hetwork File name Places Save as type Test Documents v 2 Enter a file name for the file and click Yes The report is saved in a text format file Cancel 80 GeneAtlas System User s Guide Viewing Audit Files An audit file Figure 7 9 displays information about the m Array a Hybridization a Fluidics processing Imaging 1 From the Utility Actions menu select View Audit Files The Open dialog box appears Figure 7 8 Open File dialog box for audit files Open File Current Directory C Command_Console Data Defaul Up One Level Probe Array Type Er al Sample 1 ar HT HG U133_Plus_PM al Sample 2 ar HT HG U133_Plus_PM al Sample 3 ar HT HG U133_Plus_PM ul Sample 4 arr HT HG U133_Plus_PM File name ni US Files of type Sample Files Cancel 2 Select the sample file that you want to view the audit file for and click Open The audit file is displayed Figure 7 9 The file
42. enance 113 O Overview 1 Q QC Report printing 78 saving 79 register samples 21 registering array names 15 registering samples 15 registration editing sample files 31 importing sample data 29 Registration tab 22 Regulatory 10 Regulatory approval 11 S Safe operation 4 Safety information 4 Sample ARR files creating 21 introduction 16 sample attributes 15 16 Save dialog box 23 scanner log 17 selecting subgrids 106 subgrid alignment 103 subgrid alignment algorithm 104 subgrids selecting 106 Systemlog XML 17 T template attributes 15 templates 28 Express 28 U user attributes 15 UserSettings XML 17 V Viewer 15 adjusting colors and contrast 99 Autoscale function 99 changing display of image 96 displaying grid 100 hiding components 93 moving components 92 zoom functions 97 Voltage fluctuations 6 W workflow see array processing workflow Working environment 6 Z zoom functions 97
43. ent Control Software After that GeneAtlas aligns a grid on the DAT files and computes the Cell Intensity data CEL file The CEL files can then be used for downstream data analysis The workflow steps are described in more detail in the following sections u Registering Samples and Arrays below u Hybridizing Arrays and Samples on page 15 a Performing Fluidics on page 15 a Running Imager on page 15 a Tracking Gridding and CEL file Generation on page 15 The GeneAtlas Sample and data files that are created during the workflow are described in more detail in File Types in the GeneAtlas System on page 16 Chapter 2 Getting Started With the GeneAtlas System 15 Registering Samples and Arrays In the GeneAtlas System the Sample file is the beginning of the data chain for a given experiment The sample information is stored in a Sample file with an ARR extension The arrays used in analysis and data files produced by analysis are linked to this Sample File The information about the sample and experiment are collected as attributes The attributes are described in more detail below The links between the sample and data files and the GeneAtlas tools used to generate the sample files are described in more detail in File Types in the GeneAtlas System on page 16 Template and User Attributes There are two types of sample attributes in the GeneAtlas System Template Attributes A template in the GeneAtlas System 1s a list of attribu
44. er 92 changing colors for grid display 101 Cleaning and maintenance 113 clipboard copying an image to 110 colors adjusting 99 changing for grid display 101 Compliance 10 Conformity 10 declaration 12 contrast adjusting 99 copying an image to the computer clipboard 110 D DAT file JPG conversion 110 DAT files 17 aligning grid during array processing workflow 15 manually in viewer 103 in Viewer 91 data files 16 see also CEL files CHP files DAT files parent child file relationships data organization 18 displaying grid 100 editing sample files 31 Electrical supply 6 exporting images 110 Express template 28 F file types 17 files data 16 log 17 see also CEL files DAT files Sample ARR files fluidics log 17 FS log 17 G GeneAtlas Instrument compliance 10 conformity 12 specifications 6 GeneAtlas System data organization 18 GeneAtlas Viewer see Viewer grid aligning subgrid 103 displaying 100 grid display changing colors for 101 gridding algorithm for subgrids 104 118 GeneAtlas System User s Guide Grids box 106 GUIDs 18 Hazards electrical 5 ergonomic 5 mechanical 5 highlighting subgrids 107 l Image DAT files see DAT files images exporting 110 imaging 63 importing sample data 29 Instrument specifications 6 Intensity CEL data files see CEL files J JPG conversion changing settings 110 JPG files 92 creating 110 in Viewer 92 L log files 17 Maint
45. f the window Figure A 10 94 GeneAtlas System User s Guide Figure A 10 Hidden Grid Box and tab Command Console Viewer File View Help EF Open File W Help EH Properties n x 78 Ctl Date 2 ga dat i Fall m HEBE TZA L Q FO alca El Array Information To display a hidden box temporarily a Place your cursor on the tab To restore a hidden box Display the box and click on the AutoHide button J Moving the Component Borders in the GeneAtlas Viewer You can change the relative size of a component in the Viewer by moving the borders of that component To change the size of the component 1 Move the cursor over the border until it changes to a double arrow 12 Or iii Figure A 11 Placing cursor on boundary between two components 3 Command Console Viewer File View Help EF Open File 9 5 z Help Properties 1 X Ctl_Date_2 ga dat F r 1 amp Q Q JTO La Ea El Array Information El Fluidics El Grd Alignment eei He E Wa Lnd Ol Image Processing Pixel X 0 Pixel Y 2 724 Intensity 14 2 Click and drag the cursor to change the size of the area Figure A 12 Figure A 12 Grid box enlarged ii Command Console Viewer File View Help Ex Open File be Save E Help Properties AN Ctrl Date 2 ga dat im Q agoa 2a El Array Information El Fluidics TS E I Full Image HH Active Grid ERE B Ta E La OP Ima
46. fied in reference drawing 99 027384 01 530 590 nm for Revision B serial numbers 91000210 91001960 617 nm for Revision C serial numbers 91001970 and up 2 um 0 1flors um2 lt 1 hour 12 bits DAT Windows 7 Windows 2000 with Service Pack 4 or Windows XP with Service Pack 2 15 3 389 mm L x 6 4 164 mm W x 11 9 303 mm H 56 11 Voltage100 240 V x 1096 Voltage Current 6 2 2 6 A Line Frequency 50 60 Hz Table 1 5 Specifications of the GeneAtlas Fluidics Station Specifications Supported Array Formats Supported Imaging Tray Supported Hybridization Tray Supported Wash A Tray Supported Wash B Tray Wash stain Time one array strip Operating System Dimensions Noise dB Weight Kg Power Supply Description Array strip mated with the scan tray specified in reference drawing 99 027384 01 Specified in reference drawing 99 027384 01 Peter Joyce Consumables DIR Rev1 doc Aug 26 2006 Affymetrix Peter Joyce Consumables DIR Rev1 doc Aug 26 2006 Affymetrix Peter Joyce Consumables DIR Rev1 doc Aug 26 2006 Affymetrix About 2 hours Windows 7 Windows 2000 with Service Pack 4 or Windows XP with Service Pack 2 16 8 428 mm L x16 5 420 mm W x 14 3 364 mm H lt 56 12 Voltage100 240 V 10 Voltage Current 6 2 2 6A Line Frequency 50 60 Hz 7 8 GeneAtlas System User s Guide Required Lab Equipment and Supplies You must h
47. for information on setting up and running the verification for the GeneAtlas system NOTE Before running the GeneAtlas System for a particular GeneChip Array you must have the library files for that array type installed on your computer For more information see Download Library Files on page 82 14 GeneAtlas System User s Guide Array Processing Workflow The GeneAtlas Systemis used to process the arrays used in your experiment The recommended workflow for array strips Figure 2 1 enables you to include data about the sample and experiment and to easily track the processing steps for the array strip Figure 2 1 Array Strip Processing Workflow in the GeneAtlas System GeneAtlas Array Steps performed using GeneAtlas Processing Workflow Instrument Control Software Register Sample s and Assign S 4 Registration Arrays in Sample ARR files m J Hybridize Array Strip Hybridization and Sample Perform wash and stain steps a Fluidics on array strip Image arrays on array strip and Autosave data in DAT files e Imaging Autogenerate intensity data Import CEL files into downstream data analysis software programs In the recommended array processing workflow for GeneAtlas Array strips you create sample files for all the arrays on the strip as the first step You then perform hybridization wash and stain and imaging on the strip using the GeneAtlas Fluidics Station the GeneAtlas Imaging Station and the GeneAtlas Instrum
48. g box opens Figure A 46 Figure A 46 Options dialog box Options JPEG Creation Compression Ratio Sampling Ratio 4 Review Window Enable Review window on startup C Show only items with grid alignment errors Remove item upon close Polling Interval sec Grid Lines Grid line colar Manual Grid Adjustment C Apply adjusted coordinates to all channels and exposures Saving Image display options 2 Change the values for Compression Ratio and Sampling Ratio Increasing either of these values increases the resolution of the JPG image but also increases the size of the JPG file 3 Click OK to close the dialog box and enable the changes Instrument Care GeneAtlas Instrument Care This chapter provides instructions on caring for and maintaining the instrument and on troubleshooting if problems arise a The GeneAtlas Instrument instruments should be positioned on a sturdy level bench away from extremes in temperature and away from moving air E IMPORTANT Before performing maintenance turn off power to the station to avoid injury in case of a pump or electrical malfunction Cleaning and Maintenance The GeneAtlas instruments require little in the way of customer maintenance The instruments must be kept clean and free of dust Dust buildup can degrade performance Wipe the exterior surfaces clean using a mild dish detergent solution in water Do not use ammonia based cleaners or organic s
49. ge Processing Pixel X 10 Pixel 821 Intensity 27 Displaying Multiple Files You can open more than one file in the GeneAtlas Viewer Figure A 13 Figure A 13 Displaying multiple image files Appendix A Using the Viewer Image Window Ctrl Date xd d i a Cul Date 3 ga dat title bar oid 95 96 GeneAtlas System User s Guide To display a particular image when you have more than one open Click the tab at the top of the Image Window Use the lt and gt scroll buttons in the Image title bar to scroll through the tabs if necessary Figure A 14 Figure A 14 Image window title bar Different icons for DAT and CEL files Scroll buttons E click to close C8 Ctrl Date 2Z ga dat Ls Chl Date 2 ga cel 000 APX image Different icons are used for DAT and CEL files To display the full path to a displayed file a Place your cursor on the file s title bar tab The full path is displayed below the title bar Figure A 15 Figure A 15 Displaying the path to a file SU zz B Ct Date 2 g cel PECI C GeneAtlas D ata Ctrl_ Date 2 ga dat Changing the Display of the Image This section explains how to use the Image tool bar controls Figure A 16 for a Examining Different Parts of the Image on page 97 u Adjusting the Colors and Contrast on page 99 a Changing the Grid and Intensity Display on page 100 Appendix A Using the Viewer 97 DAT File tool bar
50. gins to count up Chapter 4 Hybridization 39 KRY Affymetrix HYBRIDIZATION Utility Actions v Pause Hyb 1 12 25 10 j Set temperature to 45 C You can also click the List button to display a list of the samples being processed on the selected array strip Figure 4 9 pe vs Affymetrix HYBRIDIZATION Utility Actions Sample File Name Probe Array Type Probe Array Position Ctri 09 10 1 HT HG U133 Pius PM A01 Ctri 09 10 2 Ctri 09 10 3 HT HG U133 Plus PM 01 Ctri 09 10 4 HT HG U133 Pius PM G01 When the timer begins counting the area around the timer is white When hybridization is complete the area around the timer changes to yellow Two hours after hybridization is complete the time changes from yellow to red Figure 4 10 40 GeneAtlas System User s Guide Figure 4 10 Color changes with time in Hybridization tab Start Hybridization 15 59 47 Set temperature to 45 C Hybridization Hyb 1 Complete Set temperature to 45 C Hybridization process is complete Please remove the strip from the hybridization station Two Hours after Hyb 1 Set temperature to 45 C Hybridization process is complete Please remove the strip from the hybridization station 13 When hybridization has completed click the Stop button in the upper right corner A confirmation message box appears Figure 4 11 Figure 4 11 Confirmation Message A Stop are you sure 14 Click Yes to co
51. grid or generate a CEL file a view image link is available on the User Interface Clicking on the link will launch the image viewer v View QC Report Imaging process is complete Please remove the strip from the imaging station You can also open image files for inspection that did not fail gridding using the Open dialog box To open image files for inspection 1 From the Utilities menu select View Image Figure A 2 OG 5 Affymetrix HOME x ry Actions View Image Ww Hybridization Fluidics The Open dialog box opens Figure A 3 Current Directory C Genedtlas Data Up One Level Name Pr Probe Array Type lai 5600060000000121212001_A401 ar 01 AutoFocus_Array lai 5600060000000121212001_CO1 ar C01 AutoFocus_Array 5600060000000121212001_E01 ar E01 AutoFocus Array lai 5600060000000121212001_GO01 ar B 1 AutoFocus Array lai 5600080000000121212000_A01 ar A01 FluidicsTest lai 5600080000000121212000_CO1 ar C01 FluidicsTest 5600080000000121212000_E01 ar E01 FluidicsTest lai 5600080000000121212000_G01 ar B 1 FluidicsTest lai Ct l_Date_1 arr A01 HT HG U133 Plus PM lai Ctrl Date 2 arr C01 HT HG U133 Plus PM Ctrl Date 3 arr E01 HT HG U133 Plus PM Ctrl Date 4 arr G01 HT HG L133 Plus PM Files of type Sample Files Cancel Appendix A Using the Viewer 2 Select the array file that you wish to inspect the DAT file for and click Open The Image Viewer opens with the DAT file fo
52. idics processing To manually remove an array strip from the Fluidics tab 1 Click on the downward facing arrow in the Strip Information box on the left of the screen and select Remove Strip from the menu Chapter 5 Fluidics Wash and Stain 61 Remove Strip The Remove Strip Notice appears Figure 5 33 Are you sure you want to remove the strip 5600030000000121212000 7 2 Click Yes The array strip is removed from the system 62 GeneAtlas System User s Guide Imaging Imaging the arrays is the fourth step in the array processing workflow Figure 6 1 Figure 6 1 Array Processing Workflow GeneAtlas Array Processing Workflow Register Sample s and Assign Arrays in Sample ARR files Hybridize Array Strip and Sample Perform wash and stain steps on array strip Image arrays on array strip and Autosave data in DAT files Autogenerate intensity data Import CEL files into downstream data analysis software programs Steps performed using GeneAtlas Instrument Control Software Registration Hybridization Fluidics o Imaging The GeneAtlas Imaging Station Figure 6 2 1s used to image the array strip 64 GeneAtlas System User s Guide Figure 6 2 The GeneAtlas Imaging Station To run the Imaging Step in the workflow 1 Make sure the Imager tab is displayed Figure 6 3 It will be automatically displayed if you have come to this step directly from the Fluidics step otherw
53. ion User s Guide P N 08 0311 provides information about the proper placement of the units on a laboratory bench and connecting the components Fluidics Station Imaging Station Monitor Keyboard Mouse and Barcode Reader to the computer workstation The guide also provides information for performing the Instrument Verification IV to ensure the system is fully operational after power up 4 GeneAtlas System User s Guide User Documentation The operation of the GeneAtlas Instrument requires familiarity with other user documentation Those manuals that are relevant for you will depend on your system configuration a Site Preparation Guide GeneAtlas System and Assays P N 08 0307 a GeneChip 3 IVT Express Manual for use with GeneAtlas System User s Guide P N 702833 a GeneAtlas System User s Guide P N 08 0306 a GeneAtlas System Setup and Verification User s Guide P N 08 0311 a Provided Hybridization Oven Manual Safety Information and Warnings This section deals with safety issues and hazards concerning the Imaging Station present during regular operation To ensure safe operation of the GeneAtlas Instrument read this section completely before operating the instruments CAUTION The power supply cord is used as the main disconnect device Ensure that the socket outlet 1s located and installed near the equipment and is easily accessible ATTENTION Le cordon d alimentation est utilis comme interrupteur general
54. ise click the Imager tab to display the controls Figure 6 3 Imager window Utility Actions v Open Imaging Station Start No Strips Added 2 Click the Strip button ELM The Add Strip Window appears Figure 6 4 Chapter 6 Imaging 65 Figure 6 4 Add Strip dialog box for Imager Add Strip Scan or enter the bar code Bar Code Strip Name 3 Scan or enter the Bar Code required of an Array Strip you have registered The Strip Name field is automatically populated Figure 6 5 Figure 6 5 Add Strip dialog box for Imager Add Strip Scan or enter the bar code Bar Code 5600030000000121212000 Strip Name Test 1 Instrument Imaging 1 l Sample 1 Sample 2 l Sample 3 l Sample 4 4 From the Instrument drop down box select the correct Imaging station if more than one is available Select the arrays that you wish to image by default all are selected Click Add The Array Strip information is displayed on the left side of the screen and the Open Imaging Station button is activated Figure 6 6 66 GeneAtlas System User s Guide NOTE If you are re imaging a previously imaged array the following notice appears Array Sample 1 5ample 7 Sample 3 has been imaged previously Do you wish to re image ezana i Click Yes to re image the array New data files will be created and named with the following format Array Name Re Image Number For example if the
55. lace any liquids or containers holding liquids on or near electrical systems Ergonomic Hazards The workstation has a user interface that may pose ergonomic issues To avoid fatigue or muscle pain follow basic precautions including the following a Read understand and follow your workplace ergonomic recommendations Move computer monitor keyboard and mouse user interface so that it can be used comfortably Take short regular breaks away from the instruments Make sure the area is well lit and you are able to see the information on the screen clearly Table 1 2 summarizes possible hazards Table 1 2 GeneAtlas Instrument Hazards Powa presene pesen ST TT TT Instrument weight heavy instrument 6 GeneAtlas System User s Guide Table 1 2 GeneAtlas Instrument Hazards Continued paa presen pet ES IMPORTANT If you use the GeneAtlas Instrument in a manner not specified in this user s guide you may impair the protection provided by the equipment Electromagnetic Compatibility EMC A good EMC environment is critical to the instrument since large noises may lead to unpredictable results Please consider the following cautions Keep the instrument away from high dischargeable equipment such as pacemakers electric welding equipment etc Keep the instrument away from frequently starting up high power consuming equipment such as refrigerators centrifuges etc Keep the instrument away from a
56. les the product of the initial imaging They are used to generate the cell intensity data file after the grid has been aligned The DAT file must be opened to perform manual gridding or to run the grid alignment algorithm in the GeneAtlas Viewer If the cell intensity data CEL file has been generated you can click the Cell Intensity button 3 view the cell intensity data in the DAT Image window 92 GeneAtlas System User s Guide GeneAtlas DAT File Exposures Each array is imaged twice with different exposure times The image data from both exposures are in the GeneAtlas Array DAT file You can switch between the different exposures for checking the gridding etc by using the Exposure button see Displaying Different Exposures GeneAtlas DAT Files Only on page 100 CEL Files CEL files are cell intensity data files produced using the DAT file data after gridding and feature extraction You cannot perform grid alignment or cell generation on a CEL file JPG Files JPG files are a copy of the DAT image in a standard image file format they provide an image file with a reduced file size for QC inspection archiving and publication Moving the Components Out of the Viewer You can move the following components to a different location on your screen by clicking in the title bar and dragging the box to the new location Figure A 7 a Properties box u Grid box Figure A 7 Dragging boxes outside of the GeneAtlas Viewer Pr
57. list of the arrays on the strip being processed AY e Affymetr x 14 4 FLUIDIC 5 qc Utility Actions Pause Sample File Mame Gender Probe Array Type Probe Array Position Sample 1 HT HG U133 Plus PM A01 Fluidics 1 01 13 12 HT HG U133 Plus PM COL HT HG U133 Plus PM E01 HT HG U133 Plus PM GOL When the Wash and Stain process is finished you are notified on the user interface Figure 5 29 Chapter 5 Fluidics Wash and Stain 59 A AK a a Affymetr x FLUIDIC 5 n 17 Click Stop The Stop Notice opens Figure 5 30 A Stop are you sure 18 Click Yes to stop Fluidics run The Go to Imager button appears Figure 5 31 60 GeneAtlas System User s Guide Affymetrix FLUIDICS e Utility Actions Go to Imager Wash B Temperature 38 C 38 C 19 Click Go To Imager This automatically removes the Strip from the Fluidics tab If you do not click the Go to Imager button you will need to remove the strip from the tab manually 20 Open the Fluidics Station 21 Unlatch the Hyb Tray and Imaging Tray Array Strip 22 Remove the trays disposing of the wash and hyb trays properly and placing the Imaging Tray and Array Strip in the Imaging Station IMPORTANT Affymetrix recommends imaging the arrays as soon as the fluidics operation is finished Removing Strip Manually from the Fluidics Tab This step is necessary if you do not click the Go To Imager button when you finish the Flu
58. lues Cell intensity values are generated automatically after Running any grid alignment algorithm a Saving a DAT file after manual gridding You can also regenerate the intensity values without performing one of these other steps To regenerate the intensity values 1 In the Grids toolbar click the Image Processing button Image Processing 2 Select Regenerate CEL Intensity from the list New CEL file data is generated Exporting Images in Other Formats You have two options for exporting a copy of the image Copying Images to the Computer Clipboard a Creating a JPG File on page 110 Copying Images to the Computer Clipboard To copy an image to the computer clipboard 1 Display the image you wish to copy in the Image window El TIP You can zoom in on a specific region of the image if you desire before copying 2 Click the Copy button in the image window toolbar Figure A 43 or Press CTRL C Figure A 43 Copy button 78 Cul Date 2 ga dat ima Q JEl 9 Copy button The image in the Image window is copied to the clipboard You can then paste the image into a graphics program such as Paint and save it as a graphics file Creating a JPG File You can create a JPG copy of a DAT file for archive purposes To create a JPG copy of a DAT file 1 From the File menu select Create JPG from DAT The Open dialog box opens Figure A 44 Appendix A Using the Viewer 111 Figure A 44 Open di
59. mplete hybridization The Go to Fluidics button is active Figure 4 12 Chapter 4 Hybridization 41 KY Affymetrix HYBRIDIZATION Utility Actions v Go to Fluidics Hyb 1 4 Set temperature to 45 C Hybridization process is complete Please remove the strip from the hybridization station 15 Click Go To Fluidics or click the Fluidics tab to proceed to the next step IMPORTANT Going over the recommended hyb time may cause changes in data quality Affymetrix recommends processing the arrays through fluidics and imaging as soon as hybridization is finished 42 GeneAtlas System User s Guide Fluidics Wash and Stain Fluidics is performed using the GeneAtlas Fluidics Station and the GeneAtlas Instrument Control software Figure 5 1 Figure 5 1 Array Processing Workflow GeneAtlas Array Steps performed using GeneAtlas Processing Workflow Instrument Control Software Arrays in Sample ARR files l Hybridize Array Strip Hybridization and Sample i Perform wash and stain steps Fluidics on array strip i Image arrays on array strip and Autosave data in DAT files Imaging Autogenerate intensity data Register Sample s and Assign s Ss E Registration Import CEL files into downstream data analysis software programs It requires two sets of steps a Preparing Reagents and Filling Trays on page 45 a Loading Fluidics Station and Setting Up Run on page 49 a Make sure to follow instructions in
60. ng outside of the selected thresholds are tagged as Exclude In general Affymetrix highly encourages users to create a running log of these parameters to monitor quality and potentially flag outlier samples The QC functionality built into the GeneAtlas Software can help with this Evaluation of particular samples should be based on the examination of all sample and array performance metrics in light of the history of the metrics performance in an individual tissue and array type Prior to excluding samples from downstream analysis users should consult the Affymetrix troubleshooting website for potential reasons the controls may have failed and possible resolutions for the issue For example if hybridization controls are not spiked into the cocktail then obviously the controls will fail while the rest of the array data may be perfectly fine The QC Report displays information on a Signal Value u Hybridization Controls u Labeling Controls a Sample Quality Housekeeping Genes The features of the QC Report are described in the following sections a Opening a QC Report a Summary View on page 76 a Detail View on page 77 The QC Parameters on page 77 Printing the OC Report on page 78 Saving the OC Report on page 79 Opening a QC Report To open a QC Report 1 From the Utility Actions menu select View QC Report The Open dialog box appears Figure 7 2 Figure 7 2 Open dialog box Open File Seles Current Directory C Comman
61. ning the Fluidics Station door The trays need to be placed on the Fluidics Station deck Figure 5 13 Figure 5 13 Fluidics Station Tray Deck Hyb Tray Array Wash B Strip Tray slot Tray slot Imaging Tray Wash A Stain slot Tray slot A np 5 N 2 Retention Lever Em 7 Load the Wash B tray Figure 5 14 Make sure it 1s seated properly 52 GeneAtlas System User s Guide Figure 5 14 Load Wash B tray Notched Corner Wash B Tra 8 Load the Wash A tray Figure 5 15 Gently push the tray to the notched upper left corner after seating Figure 5 15 Load Wash A tray a e p p FI Notched Corner J O 0 Og A B C D E zn EB c D lt c D 06808008 O900000908 6 0 00 8 0 8 080 D 6 0 000 6 0 00 0 6 00 000 D 6 0 06 000 e 8 80 80 8 0 8 0 80 s80 B 00 060000800 p T 9 Open the retaining lever in front of the Hybridization Tray and Imaging Tray locations on the Fluidics Station Tray Deck by rotating the left hand side of the lever in the counter clockwise direction Figure 5 16 Chapter 5 Fluidics Wash and Stain 53 Figure 5 16 Opening the retaining lever IMPORTANT The positioning label shapes on the Tray Deck and on the plastic trays are slightly different 10 Load the mated Hyb tray and array strip on the deck of the Fluidics Station in the Position l
62. nterference and 2 This device must accept any interference received including interference that may cause undesired operation This Class A digital apparatus meets all requirements of the Canadian Interference Causing Equipment Regulation Cet appareil num rique de la classe A respecte toutes les exigences du R glement sur le mat riel broullier du Canada Chapter 1 The GeneAtlas Instrument Overview 11 Regulatory Approval This device has been approved by the following regulatory agencies Table 1 8 Table 1 8 Regulatory Approval Regulatory Agency Certification EU EMC Directive 2008 108 EC EU Low Voltage Directive 73 23 EEC IEC 61010 1 CSA C22 1010 1 1992 Canada UL 61010A 1 2002 USA EN 61010 1 2001 EU Mechanical Safety EN 1050 1996 IEC 60825 1 1993 A1 1997 A2 2001 EN60825 1 1994 A1 2002 A2 2001 for a Class 1 LED product Product Safety for Electrical Equipment for Measurement Control and Laboratory Use Pollution Degree 2 Over voltage Category II North American standards harmonized to IEC 61010 1 CAN CSA C22 2 No 61010 1 2004 Canada UL 61010 1 USA Low Voltage Directive 73 23 EEC EU EN 61010 1 2001 General requirements EN 61010 2 010 1994 A1 Requirements for laboratory equipment for the heating of materials as set forth in the Design Input Requirements document Electromagnetic Conformity for Industrial Scientific and Medical ISM equipment Group I Class A industrial loca
63. ntroduction The GeneAtlas Instrument is a modular microarray system for processing four arrays in parallel Each of the four arrays are attached to pegs and arranged on a single strip Figure 1 1 The GeneAtlas System can run about two strips per day Refer to the Affymetrix website for more information Figure 1 1 The GeneAtlas System Four Array Strip Affymetrix designed the GeneAtlas Imaging Station and Fluidics Station Figure 1 2 to assist processing samples After completing target preparation you then perform the hybridization washing and staining and imaging processes of the workflow on separate modular components 1 e the Fluidics Station and Imaging Station which are connected to a single computer workstation as illustrated in Figure 1 3 Figure 1 2 The GeneAtlas Imaging Station and Fluidics Station 2 GeneAtlas System User s Guide Figure 1 3 The GeneAtlas Instrument Connections Imaging Station Fluidics Station Barcode Reader Hybridization Station The workflow for processing an array strip on the GeneAtlas System is simple and requires limited hands on time Table 1 1 illustrates the workflow used in the GeneAtlas System and the components used to perform that workflow A computer workstation monitors and controls all of the workflow steps Table 1 1 GeneAtlas Instrument Workflow Step in Workflow Register Samples Hybridize Array Strip Wash and Stain Array Strip Image Array
64. ny strong magnetic field Do not connect many power cables to the junction box to which the instrument is connected Do not plug in or pull out any other equipment to the same junction box while the instrument is running GeneAtlas Instrument Specifications Table 1 3 Table 1 4 and Table 1 5 list the important instrument specifications Table 1 3 The Specifications of the Combined GeneAtlas Instrument Parameter Working Environment Temperature Range 59 F to 86 F 15 C to 30 C indoor use only Relative Humidity Range 10 90 Pollution Degree 2 environment Installation Category lI Altitude lt 2000m Shipping and Storage Conditions Temperature Range 40 F to 1 40 F 40 C to 60 C Relative Humidity Range 10 95 Electrical Supply Provide voltage frequency or power rating per unit label Circuit breaker Main Supply Voltage Mains supply voltage fluctuations up 10 of the Fluctuations nominal supply voltage Transient overvoltages typically present on the mains supply Chapter 1 The GeneAtlas Instrument Overview Table 1 4 Specifications of the GeneAtlas Imaging Station Specifications Supported Array Formats Excitation Wavelengths nm Autofocus Wavelengths nm Resolution Sensitivity Imaging Time for a strip of 4 arrays Digital Resolution File Format Operating System Dimensions Noise dB Weight Kg Power Supply Description Array strip mated with the imaging tray speci
65. o the interior of the GeneAtlas Instrument through any other openings except that is needed to perform operations related with consumables Removing the housing may damage the instrument components and result in hazardous exposure to LED light hazardous voltage or moving parts If the protective housing is damaged users are not allowed to operate the instrument any more Hazards Chapter 1 The GeneAtlas Instrument Overview 5 a WARNING Do not open the instrument mechanical cabinet or stick fingers into the instrument Moving the unit s axes can cause a risk of pinch or crush hazards Be aware of the placement of all assemblies before starting a run Make sure the instrument s enclosure is secure before beginning a run if it is not make sure no one is working inside the system Read understand and follow the safety information contained in this manual prior to operating or using this equipment Pay close attention to all safety labels Mechanical Hazards Do not open the instrument mechanical cabinet or stick fingers into the instruments Moving the unit s axes can cause a risk of pinch or crush hazards Be aware of the placement of all assemblies before starting a run Make sure the instrument s enclosure 1s secure before beginning a run if it is not make sure no one is working inside the system Electrical Hazards Do not use the instruments if you see damaged or frayed electrical cords Tag and report them as unsafe Do not p
66. olvents such as alcohol or acetone to clean the system because they may damage the exterior surfaces a Use soft cloth to clean the surface of the instruments Do not use liquid cleaner Do not power on and off the instruments frequently The power off time should be longer than 30 seconds a Do not place the instruments on a bench that is not level a Do not shake the instruments while moving them from one location to another Monthly Wipe down the outer surface of the instrument with a dry cloth Maintenance Occasionally you must replace the rear fuses and clean the fan window The procedures are simple see and Replacing the Fuses Fuses may be blown because the public power supply network sometimes has problem The GeneAtlas Imaging Station contains two fuses the rating of which is 5mm diameter x20mm height AC 250V T2 5A for the Imaging Station and AC 250V T3 15A fuse for the Fluidic Station There is a marking beside the fuse box which is located next to the power cord plug You can open the fuse box remove the fuse holder to replace the fuses with the same rating 1 Open the rear fuse box by gently prying open with a standard flat head screwdriver Using the screwdriver gently pry out the fuse holder Replace the fuses Return the fuse holder to the fuse box Close the box Ur uw d 114 GeneAtlas System User s Guide 1 Fuse Box 2 Power Plug 3 USB Connection for Imaging Station 4 Fan Wind
67. on array strip and Autosave data in DAT files e Imaging Autogenerate intensity data Import CEL files into downstream data analysis software programs The GeneAtlas Hybridization tab allows you to track the array strips that are in hybridization A timer notifies you when hybridization is finished and tracks the time since completion before the array strip was removed x NOTE The computer workstation does not control the hybridization station The hybridization station does not shut down when hybridization is complete To perform hybridization 1 Prepare samples for hybridization see the Assay manual for details 2 Register array strips and proceed to the Hybridization tab 3 Click the Hybridization tab 36 GeneAtlas System User s Guide NL Affymetrix A HYBRIDIZATION 4 Click the Strip button BES The Add Strip Window appears Figure 4 3 Add Strip Scan or enter the bar code Bar Code Strip Name s CS Time Temperature 5 Scan or enter the Bar Code required of an Array Strip you have registered The Strip Name field is automatically populated 6 From the Instrument drop down box select the correct hybridization station if multiple stations are available The Time and Temperature settings are automatically populated from the installed library files Add Strip Scan or enter the bar code Bar Code 5600030000000121212001 Strip Name Strip 02 Time 16 00 Temper
68. operties 2 A al El Array Information File View Help 2Z Open File d Help Ctrl_Date_2 ga cel gt 1000 El Fluidics Genelh ie HT Hum can Geru El Grid Alignment este Pixel X 426 Pixel Y 1002 Intensity 16 You can move the Image window outside of the Viewer by clicking on the file name tab and dragging the window out of the viewer Appendix A Using the Viewer 93 To dock the boxes back in the GeneAtlas Viewer a Double click on the box title bar or the file name tab in the Image window To choose a new location for the box 1 Click on the title bar and drag the box back into the GeneAtlas Viewer The docking hints buttons appear in the Viewer Figure A 8 Figure A 8 Using the Docking Hints buttons SAME El Array Information File View Help EF Open File J Help Ctrl Date 2 ga dat 3 Ct Date 2 ga cel BEELI OORTE FEE Genelhe HT Hurt El Grid Alignment Indicator DOX 2 Move the Cursor to the docking hint button A gray box appears to show where the box will dock 3 Release the mouse button The box is docked in the selected location The Box title bar contains some controls for the Properties and Grid boxes Figure A 9 Figure A 9 Box title bars To close a box u Click the Close E34 button To use the Autohide feature a Click the AutoHide button J in the box The box 1s closed and a tab is displayed on the left side o
69. ows 5 FireWire Connection for Imaging Station 1 USB Connection to Outlet 2 Fuse Box 3 Power Cord to Outlet 4 Fan Window Appendix B Instrument Care 115 Cleaning the Fan Window The air filter prevents dust or other fine contaminants from entering the Fluidics Station and Imaging Station You must clean the air filter when you can see a build up of contaminants on the air filter surface 1 Using a standard flat head screwdriver gently pry off the fan window Remove the air filter out from the air filter supporter and Wipe the filter clean using a lint free cloth Return the air filter to the supporter U c Re install the fan window 116 GeneAtlas System User s Guide Index Symbols CEL files see CEL files DAT files see DAT files JPG files see JPG files A adjusting colors 99 adjusting contrast 99 AGCC LibFilelmporter log 17 Aligning Subgrids on Arrays on Array Plates 104 alignment subgrid 103 ARR files see Sample ARR files array information 16 array name 16 array processing workflow 14 hybridization 15 35 imaging 15 63 performing fluidics wash and stain 15 registering samples and arrays 15 21 tracking gridding and CEL file generation 15 87 Array s on Array Plates align subgrids 104 attributes sample 15 template 15 user 15 Audit file 80 Audit files introduction 17 Autoscale function 99 C Care 113 CEL files generation 15 in View
70. piking directly into eukaryotic total RNA samples labeled aRNA targets are prepared and hybridized onto GeneChip expression arrays The resultant signal intensities for the poly A RNA controls serve as sensitive indicators of the efficiency of the labeling reaction and are independent of input sample RNA quality Sample Quality Housekeeping Genes Housekeeping genes are gene transcripts that are constitutively expressed on most samples These transcripts serve as internal controls are useful for monitoring the quality of the starting sample and are subject to any variability in the labeling of the sample and hybridization for the array For Human Mouse and Rat 3 expression arrays GAPDH is used to assess RNA sample and assay quality The signal values for the 3 probe sets are compared to the signal values for the corresponding 5 probe sets If the ratio is greater than 3 the sample is failed Printing the QC Report NOTE The Print function only prints the content displayed on the QC Report screen at the time Summary or Detail To print out the QC Report 1 Click the Print button The Print dialog box opens Figure 7 6 Figure 7 6 Print dialog box Printer M ame j sse z PSU VwWaenderwaman Properties Status Ready Type HP LaserJet 4250 P5 Where 3420 Central 4112 SoftDev Comment HP BA Laserlet 4250tr F Print to file r Print range r Copies Al Humber of copies 1 Pages from mmi to a
71. processed by the GeneAtlas System An Audit file is produced for each physical array and tracks all the processing steps that were performed on the array including multiple imagings and regridding The audit file has the same root name as the physical array Log Files Log files are produced by different GeneAtlas components The logs provide a record of the tasks performed by different components such as the migration tools and the installer These log files may provide useful information for troubleshooting problems The different log files include Systemlog XML XML file with system information UserSettings XML XML file with software configuration settings fluidics log Text file with info on Fluidics Station use Imager log Text file with info on Imaging Station Control use GeneAtlas LibFileImporter Text file with info on use of the Library File Importer log with date and time code FS log with date and time Text file with information on the Fluidics Script code installation The log files can be found in the C Command Console WLogs folder and can be viewed with a text editor or web browser Log files for the GeneAtlas Instrument control processes are placed in subdirectories of the C Command Console Logs folder You can place relevant log files into a zip folder that you can then send to Affymetrix customer support for troubleshooting see Collect Logs on page 84 18 GeneAtlas System User s Guide Data Organiza
72. r the selected array Figure A 4 Viewer menu Command Console Viewer File View Help Viewer tool ba Pr Aen e d Saye A TEM B um Information Array ID Array Name Barcode Design Type Properties box Ex pression Probe Array Type HT HG U133 Sample File Name Fluidics Fluidics Actual Time Fluidics Serial Numb Fluidics Wash B Ma Fluidics Wash B Min Fluidics Wash B Set Grid Alignment Grid Algorithm Versic 3 0 0 167 Grid Corners Grid Status Auto aligned Grid box cursor position in _ Status bar Pixel X 544 Pixel Y 0 Intensity 106 Plus Plv 150 79 1149 83 1 54beldd3 5a7a 482b a P N Gene Chi i P HT Huma Ger Led x i mage Window MES The viewer has the following components when it first opens s Viewer menu a Viewer tool bar Status bar displays cursor position and intensity of selected pixel cell Additional components are visible when a DAT or CEL file is displayed To learn about the Image window tool bar see Changing the Display of the Image on page 96 89 90 GeneAtlas System User s Guide To learn about the Properties box see Learning about the Image File on page 102 To learn about the Grid box see Checking the Grid Alignment on page 103 After opening the Viewer you can use the Open dialog box to find and open other DAT and CEL files see Opening the Image Viewer in the GeneAtlas System on page 88 Array and Grid
73. re 5 6 or Figure 5 7 6 Add 350 uL of Stain Cocktail 1 to the corresponding positions in the Wash A Stain Tray Figure 5 6 or Figure 5 7 Stain Cocktail 1 1s pinkish in color due to presence of fluorescent dye 7 Add 350 uL of Stain Cocktail 2 to the corresponding positions in the Wash A Stain Tray Figure 5 6 or Figure 5 7 48 GeneAtlas System User s Guide Figure 5 6 IVT WT Tray maps eJ g use eJ g useA eJ uie1S v useA eJ Uule1S v USeAA O Note The Wash B Tray deck position is labeled with a circle Keay q AH d ns Bed gt im S e lt Un ct Z Es CD gt a I x og Z Q N ct 9 5 e lt Aes Bu Beu Aes Buibe wy Volume Well wash A 850 uL stain 1 350 pL stain 2 350 pL wash B 850 uL _ Array Holding Buffer 200 uL Volume Well m Wash A 850 uL _ Stain 1 350 uL stain 2 350 pL C wash B 850 pL Array Holding Buffer 200 uL Chapter 5 Fluidics Wash and Stain 49 Loading Fluidics Station and Setting Up Run After preparing the consumable trays for the fluidics run you need to a Display the Fluidics tab Add the strip Load the trays u Start the run To run the Fluidics step in the workflow 1 Make sure the Fluidics tab is displayed It will be automatically displayed if you have come to this step directly from the Hybridization step otherwise click the Fluidics tab to display the controls
74. rent status of instruments Help button Click to display help Utility Actions menu Used to select utilities see Chapter 7 GeneAtlas Utilities on page 73 20 GeneAtlas System User s Guide Registering Samples Each sample on an array strip must be registered in the GeneAtlas software prior to processing in the GeneAtlas System The registration process creates a sample file ARR for each sample Figure 3 1 Figure 3 1 Array Processing Workflow GeneAtlas Array Steps performed using GeneAtlas Processing Workflow Instrument Control Software Arrays in Sample ARR files Hybridize Array Strip Hybridization and Sample Perform wash and stain steps A Fluidics on array strip i Image arrays on array strip and Autosave data in DAT files e Imaging Autogenerate intensity data Register Sample s and Assign tS Registration Import CEL files into downstream data analysis software programs This chapter describes Sample Registration a Using Templates on page 28 a Importing Sample Data on page 29 a Editing Sample Files on page 31 22 GeneAtlas System User s Guide Sample Registration 1 Click Start Programs gt Affymetrix GeneAtlas or click on the GeneAtlas shortcut on the desktop to launch the GeneAtlas software The GeneAtlas Home window appears Figure 3 2 XY Affymetr x HOME I Hybridization 2 Click the Registration tab Figure 3 3 e AC Affymetr x REGISTRA
75. rs via phone or E mail Contact information is listed below Affymetrix Inc 3420 Central Expressway Santa Clara CA 95051 USA E mail support affymetrix com Tel 1 888 362 2447 1 888 DNA CHIP Fax 1 408 731 5441 Affymetrix UK Ltd Voyager Mercury Park Wycombe Lane Wooburn Green High Wycombe HP10 OHH United Kingdom E mail saleseurope affymetrix com E mail supporteurope affymetrix com UK and Others Tel 44 0 1628 552550 France Tel 0800919505 Germany Tel 01803001334 Fax 44 0 1628 552585 9 if after service or calibration the instrument does not perform to the specifications stated in Table 1 3 10 GeneAtlas System User s Guide Affymetrix Japan K K ORIX Hamamatsucho Bldg 7F 1 24 8 Hamamatsucho Minato ku Tokyo 105 0013 Japan E mail salesjapan affymetrix com E mail supportjapan affymetrix com Tel 81 3 6430 4020 Fax 81 3 6430 4021 Regulatory and Conformity GeneAtlas Instrument Compliance We Affymetrix Inc 3420 Central Expressway Santa Clara California 95051 USA www affymetrix com declare under sole responsibility that the Affymetrix GeneAtlas Instrument and associated Workstation with software is manufactured in conformity with the regulations and certifications stated in this section using U S and Non U S components This device complies with Part 15 of FCC Rules Operation is subject to the following two conditions 1 This device may not cause harmful i
76. s Select the library files to download AutoFocus_Array Fluidics est L HG3 u213 W HT HG L133 Plus PM AY HT MG 430 PM HT Rat230 PM CJ HuGene 1_1 st v1 HT_MG 430_PM Click the Abort button to halt the download When the download is halted or finished a list of the downloaded library files is displayed Figure 7 18 Figure 7 18 Download complete list The following files have been E downloaded 550070 protocoal 550112 96MC HT MG 430 PM cdf HT MG 430 PM 4 MEDIA HT MG 430 PM S5CAN PARAMS HT Rat ll DM ner laz 6 Click OK to close the list Collect Logs Log files are produced by the different GeneAtlas components The logs provide a record of the tasks performed by the different components such as the Fluidics and Imaging Stations and the installer These log files may provide useful information for troubleshooting problems Chapter 7 GeneAtlas Utilities 85 The Collect Logs utility creates a zip package of all the log files for the GeneAtlas System including log files for the Fluidics Station and the Imaging Station Upon request you can send the zip package to Affymetrix Support To create a zip package of the log files 1 From the Utility Actions menu select Collect Logs A notice that the zip file 1s being created appears Figure 7 19 KX 5 Affymetrix HOME i n Utility Actions Hybridization Fluidics Zip file is being created please wait When the file
77. s Figure 3 24 NOTE If you plan to import a template select all of the sample files of an Array Strip Figure 3 24 Edit Files window Affymetrix Edit Files Import Template Import Data Add Column Barcode Lot Number Expiration Date Gender Sample File Name text date text Sample 1 556000390000000121212000 12 12 2012 Mate Sample 2 55000300000003 21212000 12 12 2012 Female Sample 3 S3 500030000000121212000 12 12 2012 Female 5ample 4 S36000300000001 21212000 12 12 2012 Male Chapter 3 Registering Samples 33 3 If you want to a Add a sample attribute to the data grid See Step 9 on page 25 Import a template See Step 2 to Step 3 on page 28 Import data See Step 4 to Step on page 30 Edit an attribute value Select the item in the data grid and enter a new value NOTE In the Edit Files window items that are shaded in gray cannot be modified 34 GeneAtlas System User s Guide Hybridization Hybridization is the second step in the array processing workflow Figure 4 1 after registering the sample and array It is done using the Hybridization Station Figure 4 1 Array Processing Workflow GeneAtlas Array Steps performed using GeneAtlas Processing Workflow Instrument Control Software Register Sample s and Assign m d P Registration Arrays in Sample ARR files e Hybridize Array Strip Hybridization and Sample j Perform wash and stain steps E Fluidics on array strip Image arrays
78. s to manage the attributes used for a particular experiment see Template and User Attributes on page 15 for more information Array Information Information about the array used with the sample Each array is assigned an array name during registration The array name is used to identify the DAT CEL and CHP data files that are generated during analysis Figure 2 2 Sample File with associated arrays and data files Sample File Sample ARR With Sample Attributes Array name FirstArray J used for DAT CEL and CHP files Ed DAT File FirstArray DAT E Ei CEL File FirstArray CEL A set of data files is produced for each array in the Sample file The data files include a Image DAT file below a Intensity CEL Data Files on page 17 Chapter 2 Getting Started With the GeneAtlas System 17 Image DAT file The DAT file contains pixel intensity values collected from an Affymetrix Imaging Station along with the gridding information used during feature extraction When a DAT file is regridded the existing CEL file is over written New DAT files and CEL files are created only when an array is re imaged Intensity CEL Data Files The CEL file stores the results of the intensity calculations on the pixel values of the DAT file Other File Types Audit and Log files track the tasks performed by different software components Audit Files An Audit file is an XML file that tracks the processing of each physical array
79. some of the grids are misaligned or if you have manually aligned the main grid To run the alignment algorithm again on an array that uses sub grids 1 In the Grids toolbar click on the Image Processing button and select Realign All Grids from the menu Figure A 30 Figure A 30 Grids tool bar for array with grids Lr Image Processing Realign All Grids Regenerate CEL Intensity Progress bars display the progress of the alignment and cell generation Figure A 31 Appendix A Using the Viewer 105 Figure A 31 Progress bars 10 complete Command Console Viewer Convert Cr When the process is finished new gridding information and a new CEL file are generated A notice of completion appears Figure A 32 Figure A 32 Notice Command Console Viewer E Algorithm was executed successfully 3 Command Console Viewer File View Help EF Open File Help Properties je lA l Z El Array Information El Fluidics Grids Hit Active Grid 7 EU BRE E LE Ll d E Image Processing Pixel X 708 Pixel Y 528 Intensity 3 NOTE This will align all the subgrids on the array Manually Aligning the Subgrids If the algorithm alignment fails an error message appears Figure A 34 106 GeneAtlas System User s Guide Figure A 34 Notice of grid misalignment Command Console Viewer Failed to execute the Grid Alignment algorithm The error is
80. sted coordinates to all channels and exposures Saving Image display options 2 Select a new color for the grids from the Grid line color drop down box 3 Click OK to close the dialog box and enable the changes Learning about the Image File The Properties box displays information about the image file displayed in the window The information can be displayed in alphabetical order or ordered by different categories depending upon the type of file displayed For a DAT file a Array Information a Fluidics a Grid Alignment Hyb Image Imager For a CEL file a Array Information a Cel a Fluidics a Imager Appendix A Using the Viewer 103 Figure A 28 Properties box esL S __ Properties tool bar El Array Information Expression HT_HG U133 Plus Pb Sample File Hame El Fluidics Fluidics Actual Time Fluidics Serial Numb Fluidics Wash B Ma Fluidics Wash B Min Fluidics fash B Set El Grd Alignment Grid Algorithm erse 3 0 0 167 Grid Comer 150 73 1143 83 1 Auto aligned Hyb Max Temperatu Hub Min Temperatur Hub Set Temperatur Hub Start Time To expand or collapse a component a Click on the button to the left of the component To sort the data in a different way u Click the Category Sort or the Alphabetical Sort l l button The Grid information category displays information about the grids a Grids when a
81. ted Completed Completed Stopped Download Library Files The Download Library Files function lets you download the latest library files from the NetAffx website You will need to create a NetAffx account if you have not already done so To download libraries 1 From the Utility Actions menu select Download Library Files Figure 7 14 Chapter 7 GeneAtlas Utilities KS Affymetrix HOME Utility Actions Updates available Download Library Files j Hybridization Fluidics The Net ffx Account Information dialog box opens Figure 7 15 Email Password Reqister How 2 If you have not created a NetAffx account click the Register Now link to do so 3 If you have created a NetAffx account enter your email and password and click the OK button The NetAffx Library Files dialog box opens Figure 7 16 s NetAffx Library Files Select the library Files to download AutoFocus_Array L FluidicsT est DIHG U219 L HT HG 1133 Plus PM L HT MG 430 PM L HT Rai230 PM CJ HuGene 1_1 st v1 Caneel The list includes u Files for Instrument Verification AutoFocus Array 83 84 GeneAtlas System User s Guide FluidicsTest a Files for different array types a HT_HG_U219_PM a HT MG 430 PM a HT Rat230 pm 4 Select the checkbox next to the libraries you wish to download 5 Click the Download button The dialog box displays the progress of the download s NetAffx Library File
82. tes that can be assigned to a Sample file An Express template is provided with the GeneAtlas application This template contains a set of attributes that might be useful while analyzing CEL files using the Partek Express Software You can assign this template to sample files during registration This automatically adds all the attributes in the template to the Sample file You can then enter values for each attribute This allows you to standardize the attributes that are assigned to samples User Attributes User attributes are created on the fly during the registration of a sample and array This allows you to create a quick note for a particular sample file User attributes are not listed in a template they have usually been added to a specific sample file They can be used in filtering and search operations just like the template attributes Hybridizing Arrays and Samples For Array strips the hybridization is performed in the hybridization station The GeneAtlas Instrument Control software provides a timer that informs you when the array strip should be removed See Chapter 4 Hybridization on page 35 Performing Fluidics The arrays on the Array strip are washed and stained using the GeneAtlas Fluidics Station controlled using the GeneAtlas Instrument Control software The software and its use are described in Chapter 5 Fluidics Wash and Stain on page 43 Running Imager The arrays on the Array strip are imaged using the
83. tion Start Imaging 1 wd Cleese remove strip w Sample 1 w Sample 2 Sample 3 Sample 4 Imaging process is complete Please remove the strip from the imaging station 15 Click Open Imaging Station and remove the array strip from the imager 16 Click Close Imaging Station The Imaging Station door closes 17 Click on the dropdown arrow in the Strip box on the left of the screen and select Remove Strip from the menu 72 GeneAtlas System User s Guide Testi 7 c Remove Strip 5000300000004 21212000 e Please remove strip w Sample 1 Sample 2 The Remove Strip Notice appears Figure 6 19 Are you sure you want to remove the strip 5600030000000121212000 7 18 Click Yes The array strip is removed from the system GeneAtlas Utilities The GeneAtlas Utilities provide the functions shown in Table 7 1 Table 7 1 The GeneAtlas utilities Utility Function See Updates Available Notifies you of updates for GeneAtlas software that Links to the Affymetrix can be downloaded at affymetrix com Update Page for the software Edit Files Enables you to select and edit sample files ARR Editing Sample Files on page 31 View QC Report Shows the QC Summary Report for one or more user Viewing a QC Report on selected samples page 74 View Audit Files Shows the audit file for a user selected sample Viewing Audit Files on page 80 View Image Shows the intensity data CEL for a user selected Appendix
84. tion in the GeneAtlas System To use the GeneAtlas System you need to understand the structures the software provides for organizing your data during and after generation GeneAtlas data is placed in the C GeneAtlas Data folder You can create subfolders in the C GeneAtlas Data folder during sample registration using the Create New Folder feature in the Save dialog box Figure 2 3 Save dialog box C GeneAtlas Data Folder Date Introduction to the Software Interface This section describes basic features of the GeneAtlas Instrument Control Software including How to start the software Parts of the screen Basic navigation features To start the software a Click the GeneAtlas Instrument Control software icon on the desktop The software opens to the home tab described below Chapter 2 Getting Started With the GeneAtlas System 19 The Home Page The Home tab Figure 2 4 provides an overview of the status of the different instruments Affymetrix elp butto OE Kun Utility Actions menu adde l Hybridization Instrument Status The Home tab has the following components Workflow tabs Allows you to switch between Registration tab see Chapter 3 Registering Samples on page 21 Hybridization tab see Chapter 4 Hybridization on page 35 Fluidics tab see Chapter 5 Fluidics Wash and Stain on page 43 maging tab see Chapter 6 maging on page 63 Instrument Status Displays cur
85. tions ICES 003 Industry Canada Interference Causing Equipment Standard Digital Apparatus Class A Canada FCC Part 15 Radio Frequency Emissions for Class A Equipment USA EMC Directive 89 336 EEC EU EN 61326 1997 A2 2001 General EMC Requirements EN 55011 1998 Radio Frequency Emissions EN 61000 3 2 2000 Harmonic Current Emissions EN 61000 3 3 1995 Voltage Fluctuations and Flicker Compliant with directive 2002 96 EC WEEE 371123740 WEEE German Registration WEEE Registration France Complies with requirements of Radio communications Electromagnetic Compatibility Standard 2008 12 GeneAtlas System User s Guide CE Mark Declaration of Conformity The Affymetrix GeneAtlas Instrument conforms with the relevant provisions of the following standard s and or other normative document s GeneAtlas Imaging Station EMC Directive 89 336 EEC Low Voltage Directive 73 23 EEC ICES 003 Industry Canada Interference Causing Equipment Standard Digital Apparatus Class A Canada FCC Part 15 Radio Frequency Emissions for Class A Equipment USA CAN CSA C22 2 No 61010 1 04 Canada as harmonized to IEC 61010 1 UL 61010 1 USA as harmonized to IEC 61010 1 EMC Directive 89 336 EEC GeneAtlas Fluidics Station Low Voltage Directive 73 23 EEC ICES 003 Industry Canada Interference Causing Equipment Standard Digital Apparatus Class A Canada FCC Part 15 Radio Frequency Emissions for Class A Equipment USA
86. u can switch between the different exposures for checking the gridding etc by using the Exposure button The Exposure button displays the currently displayed exposure time To switch between views of the different exposures a Click the Exposure button Figure A 24 Figure A 24 Exposure Button IT 1800ms The other DAT Exposure is displayed Displaying the Grid You can display or hide the grid using the Show Grid Corners button To display or hide the grid Click the Show Grid Corners button Appendix A Using the Viewer 101 Figure A 25 Outside edges of grid displayed m Ctil Date 2 ga dat E GeneCh ir HT Human Ger BENEI TEC HET El Hh Yt Teta BT Bi SRR BEE fet Ta E Se SEPAR BSS Ra ra La RU ee HB dR EBE EERE aa H Ha HHHH BEREHCERE El See Checking the Grid Alignment on page 103 of this manual for more information about manual gridding Changing Settings for the Grid Display To change settings for the grid display 1 From the View menu select Options 102 GeneAtlas System User s Guide The Options dialog box opens Figure A 27 Figure A 27 Options dialog box Options JPEG Creation Compression Ratio 2 Sampling Ratio 4 Review Window Enable Review window on startup Show only items with grid alignment errors Remove item upon close Polling Interval sec Grid Lines Grid line color Manual Grid Adjustment C Apply adju
87. vailable Displays the pixel coordinates for the corners of each grid Checking the Grid Alignment This chapter describes the use of the GeneAtlas Viewer for aligning failed grids u Aligning Grids on page 103 a Regenerating Intensity Values on page 110 For general information about grid alignment see Array and Grid Types on page 90 Aligning Grids Sometimes one or more grids may require alignment The failed grids are marked with an X in the Grids box Figure A 29 104 GeneAtlas System User s Guide Figure A 29 Array with misaligned grids 3 Command Console Viewer File View Help 7 Open File Help x r Failed Grid HT HG U133 Plus PM A04 DAT gt lt gt 1000 Misaligned grids are marked with an X Pixel X 463 Pixel Y 977 Intensity 14 If the grid alignment fails you can a Run the grid alignment algorithm see below Perform a manual alignment on the failed grids see page 105 You use the same controls and steps to align subgrids for GeneAtlas arrays as you do for cartridge arrays with the following exceptions a You may have to check the grid alignment for both exposures see Displaying Different Exposures GeneAtlas DAT Files Only on page 100 You can select an option to apply adjusted coordinates to all channels and exposures see Changing the Manual Grid Adjustment Setting on page 109 Running the Grid Alignment Algorithm You can run the grid alignment algorithm if
88. ximum of four strips For details on adding a strip see Step 3 to Step 4 on page 23 Enter the sample file names NOTE If a sample file name in the data grid is not found in the data file xls selected for import no data will be imported for the sample Click the Import Data button LL XY Affymetr x REGISTRATION Utility Actions Import Template Import Data Save and Proceed gt gt text text Sample 9 HT HG U133 Plus PM Sample 10 HT HG U133 Plus PM Sample 11 HT HG U133 Plus PM Sample 12 HT HG U133 Plus PM The Open dialog box appears Figure 3 21 Chapter 3 Registering Samples 31 Look in Data E ck Eg My Recent J Test 3 Data xls Documents My Network File name Places Files of type Excel Worksheet xls xlsx Y 5 Select the data file xls and click Open The sample information from the data file appears in the data grid Figure 3 22 GA Affymetrix REGISTRATION A J Utility Actions v Import Data 1 Save and Proceed gt gt Sample File Name Probe Array Type v Probe Array Position Age v Gender v Sample Type v Tissue v Date of sample prep text text text text v text v text v text v text Sample 9 HT HG U133 Plus PM 25 Male RNA Blood Sample 10 HT HG U133 Plus PM 14 Maie RNA Kidney 12 8 2009 Lot 0000000 Sample 11 HT HG U133 Plus PM 54 Maie RNA Lung Test 3 v 5600030000000121212002 Dp DNE Tu Sample 12 HT HG U133 Plus PM 36 Female RNA
89. ype Y Probe Array Position text v text Sample 1 HT HG U133 Plus PM Test 1 Y Sample 2 HT HG U133 Plus PM 5500030000000121212000 Lot 0000000 Sample 3 HT_HG U133 Plus PM as Sample 4 HT HG U133 Plus PM E In the data grid enter the sample attribute values NOTE You can import sample data to populate the data grid see page 29 Some attribute values can be edited in the sample file ARR see page 30 10 When sample registration is complete click the Save and Proceed button LE The Save dialog box appears Figure 3 12 CAGeneAtlas Data Create New Folder 11 You can save the data 1n the default folder or create a new folder for the data A Click Create New Folder The Create New Folder dialog box opens Figure 3 13 Create New Folder New Folder Name Test 1 Chapter 3 Registering Samples 27 B Enter a name for the new folder C Click Create D Select a folder in which to save your data Figure 3 14 C GeneAtlas Data Create New Folder 12 Click OK in the Save dialog box Your sample files ARR are saved to the selected folder and a confirmation message appears Figure 3 15 Sample Files Created e Go to Hybridization 13 Click OK or Click Go to Hybridization to proceed to the Hybridization tab Figure 3 16 NOTE You may enter up to four array strips simultaneously during the registration process armia Affymetrix i HYBRIDIZATION

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