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CMS_082581 - Applied Biosystems

1. 35 bp 50 bp 1 Flowcell 2 Flowcells 1 Flowcell 2 Flowcells Instrument Buffer 2157 4125 2949 5710 Storage Buffer 343 587 423 748 Cleave 1 Solution 124 224 169 316 Cleave 2 1 Solution 124 224 169 316 Reset Buffer 91 164 91 164 Ligase Buffer 42 74 56 101 Universal Buffer 26 41 33 55 Imaging Buffer 82 142 107 191 Table 32 Fill volumes for paired end sequencing 35 bp Volume mL F5 P2 Tag F5 BC Tag 1 Flowcell 2 Flowcells 1 Flowcell 2 Flowcells Instrument Buffer 2477 4954 2477 4954 Storage Buffer 448 896 448 896 Cleave 1 Solution 78 156 78 156 Cleave 2 1 Solution 78 156 78 156 Reset Buffer 72 145 72 145 Ligase Buffer SY 75 37 75 Universal Buffer 36 73 36 73 Imaging Buffer 68 135 68 135 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix C On Instrument Reagent Volumes and Reagent Strip Layouts Recommended fill volumes for on instrument reagents Mate pair sequencing F3 R3 Tags Table 33 Fill volumes for mate pair sequencing 25 35 or 50 bp Volume per tag mL 25 bp 35 bp 50 bp 1 Flowcell 2 Flowcells 1 Flowcell 2 Flowcells 1 Flowcell 2 Flowcells Instrument Buffer 1629 3069 2157 4125 2949 5710 Storage Buffer 289 480 343 587 423 748 Cleave 1 Solution 93 163 124 224 169 316 Cleave 2 1 Solution 93 163 124 224 169 316 Reset Buffer 91 164 91 164 91 164 Ligase Buffer 33 55 42 74 56 101 Universal Buffer 21 32 26 41 33 55 Imaging Buff
2. Note If you do not select the correct read length for the number of barcodes specified in your run definition file the SOLIDO System software will display an error If at least 1 library on the slide uses Barcode 17 or higher ten bases of barcode tag should be read g Select Primer Set 2 and Primer Set 3 e If sequencing only one end of the barcoded fragment library select F3 for Primer Set 2 If sequencing both ends of the barcoded fragment library select F5 BC for Primer Set 2 and F3 for Primer Set 3 h Enter read lengths for Primer Set 2 and if used Primer Set 3 A typical read length for the F3 tag is 35 50 bases A typical read length for the F5 BC tag is 25 bases Note For barcoded fragment libraries of short inserts such as small RNA samples consult your Field Applications Specialist for the recommended sequencing reagents and read length i Select the appropriate mask to use j Click Next Applied Biosystems SOLiD9 4 System Instrument Operation Guide 63 Chapter 3 Set Up Control and Monitor the Run Create a multiplex sequencing run record Figure 42 Complete the information in the Select Run Type and Mask pane solid0054 SOLID 4 0 User lab user SETUP gt Create Edit Run gt Select Run Type and Mask The following pages will guide you through to set up a run Required fields are noted with an asterisk Select type of run to create Create New Run Fragment O Mate Pair Pa
3. Multiplex fragment 2 F3 Tag 1 BC Tag sequencing 35 Part No Part No 4449352 4449308 Part No Part No 4449388 4449316 c Y 38 Multiplex paired end 3 F3 Tag 2 F5 BC Tag 1 BC Tag sequencing Part No 4449308 Part No Part No a gt 4449388 4463232 o 423 EB gt Part No 4449316 r N od N 3 Place the reagent strip s in an ABgene 96 well square well storage plate and centrifuge at 160 x g for 2 minutes 4 Verify that the reagent strip blocks are oriented and seated properly in the chiller block The block must engage the orientation key to fit properly The orientation key cut corner is in the upper left position when the block is placed on the instrument see Figure 22 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Chapter 2 Prepare and Install Slides and Reagents Install reagent stripls on the instrument Figure 22 The Reagent Strip Block IMPORTANT Ensure that the chiller block temperature is below 10 C before proceeding with reagent strip installation Under the System Status menu select Cooling from the Chiller drop down menu see Figure 9 on page 26 5 When the temperature is less than 10 C place the reagents in the appropriate location in the chiller block see Table 6 and Figure 26 on page 41 See Figures 23 to 25 for the appropriate location of reagent strips on the chiller block For information about the contents of the reagent strip tubes see Appen
4. Cycles Cyde3 Cyce 1 BC Tag Primer B Cyce1 1 TCyae2 des 500 1_ Bridge Probe Primer C BC Tag Primer C Bridge Probe C Cyde L 76yde2 es 00 Ges 00 Oye CHEE BETETPURE D Bridge Probe o S Cycles Cyce3 Cycle 1 Bridge Probe BC Tag Primer E Bridge Probe Cycle1 7 Cyde2 A ar gt Sequence up to 50 bp Sequence up to 35 bp jua Sequence 5 or 10 bp DNA Fer EET Ta 0 12 3 4 5 6 7 8 9 1011 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 DOCOCCCOOOCOCCOCCOCOCOOCCCCCCCOCOOCCCCCCCCCOCOCCCOCCCCCOCCCCOCCCOCCOCCOCCOCCCOCOOO g PrimerA Cycle 1 yde2 C Cycle 3 ycle4 O O Cycle 5 ycle6 Cycle 7 ycle8 Cycle 9 r B O O Cycle 1 cle 2 Cycle 3 ycde4 Cycle 5 dle 6 O Cycle 7 sycle 8 Cycle 9 3 Tag Primer C Bridge Probe Cycle O O Cycle 3 jde4 Cycle 5 RT O Cycle 7 8 O Cycle 9 a Bridge Probe Cycle 1 C Cycle 3 C Cycle 5 C Cycle 7 E Cycle 9 Primer E Bridge Probe Cycle 1 RST cycles eT Cycle 5 EE Cyde 7 JER Cycles The workflow for each type of sequencing run is shown in Figure 4 14 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Chapter 1 Introduction 1 SOLiD 4 System run types Figure 4 Sequencing run workflows Fragment sequencing Paired end sequencing Mate pair
5. Focusing 3 Ensure that Do auto exposure with auto focus is checked Enter a value 5000 less than the nominal Z distance for Z Min Enter a value 5000 greater than the nominal Z distance for Z Max Enter 30 for the Number of Steps and choose Histogram Range from the drop down menu in the Focusing Method Selection pane see Figure 86 IMPORTANT Leave the Auto Focus Options window open on the Desktop Do not click the Save to File button Values entered in the Focusing box are used by the Imager software even if the values are not saved Applied Biosystems SOLIDO 4 System Instrument Operation Guide 115 B Appendix B Supplemental Procedures Manually find the focus range Figure 86 Enter auto focus options Uw Auto Focus Options m Focus Settings Flowcell 1 Flowcell 2 Outside Flowcells Z Min 653103 648830 652503 Z Max 663103 658830 662503 Revert to Revert to Revert to Number of Steps 30 MV Do auto exposure with auto focus Focusing Method Selection Histogram Range m 4 Use the mouse to move the green box to the upper left corner of the slide 5 In theImager window click the Autofocus button then wait about 20 seconds for the Imager to focus on the slide The Camera 1 indicator displays BUSY while the autofocus is running When the Camera 1 indicator displays READY click AutoExpose and wait for the Camera 1 indicator to display READY Click AutoFocus so that the Ima
6. P2 P1 ratio P2 positive beads pulse spin R3 tag remove the supernatant resuspend the beads Satay plot sonicate the beads tag templated bead preparation templated beads titration 166 Plot indicating noise to signal for each dye The frequency of template positive beads that meet a defined threshold of spectral purity and signal intensity after a single ligation step Library template concentration that gives the best sequencing results Double stranded oligonucleotide ligated at the 5 end of the library Double stranded oligonucleotide ligated at the 3 end of the library The frequency of template positive beads P2 relative to total beads P1 deposited on the slide this metric is also referred to as 96 P2 Positive value SOLiD P1 DNA beads with fully extended and amplified template Place the tube in a picofuge and spin for a few seconds to bring down any beads or liquid stuck on the walls of the tube Tag to be sequenced using primers specific to the Internal Adaptor sequence Use a pipette to carefully remove the liquid from the tube without disturbing any beads The beads can be resuspended in one of two ways Gently pipet the solution up and down until the beads are suspended Using a slower speed to aspirate and expel the solution minimizes the amount of beads that stick to the inside of the pipette tip Vortex the solution until all of the beads are suspended Place the beads in a
7. Default CJ End quote sal Default LJ Cere tilo L_neset_ 6 Finish entering the sample library and if applicable barcode information using the fill down function You can access this function by selecting Edit Fill Down If you assign multiple barcodes to a single library enter the barcodes in quotes separated by commas see Figure 75 Figure 75 Complete the sample library and barcode information E solid0062 20091217 PE BC txt OpenOffice org Calc File Edit View Insert Format Tools Data Window Help B Bado ERG Vis X56 9D 0N 4 by MOBEQA OY dx MT EE DAA y y tU Arial x 10 v B7 UI e F G H version userld tunType isMultiplexing runName runDesc mask protocol 2 v1 2 lab user PAIRED END TRUE solid 062 20091217 PE BC 4 spot mask sf SOLID PE Multiplex primerSet baseLength 5 5_ F5 BC 25 50 sampleName sampleDesc spotAssignments primarySetting library application multiplexinaSeries barcodes bcSample1 1Barcode 1MM Lib1 BC Kit Module 1 16 1 2 bcSample1 1Barcode 1MM Lib2 BC Kit Module 1 16 3 4 bcSample1 1Barcode 1MM Lib3 BC Kit Module 1 16 5 5 bcSample2 2Barcode 1MM Lib1 BC Kit Module 1 16 1 bcSample2 2Barcode 1MM Lib2 BC Kit Module 1 16 2 bcSample2 2Barcode 1MM Lib3 BC Kit Module 1 16 3 bcSample2 2Barcode 1MM Lib4 BC Kit Module 1 16 4 bcSample2 2Barcode 1MM Lib5 BC Kit Module 1 16 5 bcSample2 2Barcode 1MM Lib6 BC Kit Module 1 16 6 bcSample2 2Barcode 1MM Lib BC K
8. Detectthe focus NIE Ls GEB E S tide den uode d 49 Startthe WEA TUI T ce obtu Lebe tantu mtt rte TAA ade od rne aeta acd 50 Monitor the WFA run io ereinen eee ee n Rn hn 52 View therurmlog s minean pese p ERES mea eden sedula Ge De dte 52 Viewithe heat itriaps eso Leo Le Ri eo UE 2 nde SE IAM S 52 View cycle SCalis tet ce ed dos tdo tene da d e e cid teta ee 53 View the WEA report srra orc sus Glee s ber Rs LEER IR EENER DRAN bel ER MEE 53 Generate the WFA report 00 ccc eect eee e ee eaee 53 Determine the optimal titration pOint 02 000 54 Determine the bead deposition density for a sequencing FUN L eee 55 Section 3 2 Set up and perform a sequencing Un e e e e e K eee eee 56 Materials and equipment required see 56 WOFKIloW eese ssi essa RR RE RU Re dare aie ee ua e Ro a Met 56 Workflow overview 0 0 cece m en 56 Create a sequencing non multiplex run record e 58 Create a multiplex sequencing run record c ooocccocccc eese 61 Detect the focus range ern 69 Startihe sequenclng4 uris s boo LI e eL EE RES 70 Control tlie r n 2x Ta A R e ERR X E met dtd 73 Pause Resunme RU ia 73 Dee The Run Control menu coke tne oben a a da ER Rec PE Ra 74 Use imaging and analysis controls for specific spots 0 0 cece eee eee eee 76 Monitor the UN ae RIS A eb PE Na E PNG URL EP UU d 77 View thie rum log 2 A aevi edem s dee epe es 77 View the heat maps escinsel ni dc XE g R R R a N mH mre 77 View cycle SM
9. In the Text Importer window empty the Text Delimiter box by clicking in the Text Delimiter box and pressing the Backspace key to delete the quotation marks see Figure 73 Then click OK Figure 73 Empty the Text Delimiter box Text Import solid0062_20091217_PE_BC txt Import Character set Western Europe Windows 1252 WinLatin 1 From row 1 Separator options O Fixed width Separated by Tab CI comma C Semicolon O Space C Merge delimiters Text delimiter Fields Column type E Standard Standard Standard Standard Standard ersion userId runType isMultiplexing runName L 2 lab_user PAIRED END TRUE solid0062 rimerSet baseLength c 5 BC l dl ael cal co ro 5 Select Tools Autocorrect Options In the Custom Quotes tab uncheck the Replace box in the Double Quotes section see Figure 74 If this box is checked the file will not be imported correctly into ICS because directional quotation marks will be used instead of non directional straight quotation marks 106 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix B Supplemental Procedures B Set up a run by importing a Run Definition file Figure 74 Uncheck Replace in the Double quotes section AutoCorrect A n N Replace Exceptions Options Custom Quotes Simgle quotes C Replace Start quote CJ Default C End quote Default Default Double quotes OR Start quote
10. Lib BC Tag MM10 Reset Buffer Multiplex paired end sequencing up to 50 bases for the F3 tag of barcoded fragment libraries Barcodes 1 96 90 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix A Applied Biosystems SOLIDO 4 System Instrument Operation Guide Required Materials Set up and perform a sequencing run Item part GT Kit componentls number used in SOLIDO ToP Reset Buffer Sequencing and or Instrument Buffer WFA Gl L Kit 4452688 dd 10X Instrument Buffer Cleave Solution 1 Cleave 2 1 Kit Cleave 2 1 Parts 1 and 2 Storage Buffer Imaging Buffer Kit Imaging Buffer Parts 1 and 2 1X T4 Ligase Buffer Kit Ligase Buffer Parts 1 and 2 Universal Buffer Kit Universal Buffer Parts 1 and 2 SOLIDO Flowcell O rings 4398217 10 pack of flowcell O rings Sequencing and or WFA SOLIDO XD Slide amp Deposition Kit v2 4456997 4 x 2 Sequencing Slides Slide Deposition Buffer v2 Slide Storage Buffer Bead deposition Other components SOLIDO Mixing Strip Tube with Zinc 4449506 SOLID Mixing Strip Tube 4406595 Required equipment Table 18 Required equipment Sequencing run Item Source SOLIDO 4 System Applied Biosystems PN 4452773 110 V Applied Biosystems PN 4452774 220 V SOLIDO 4 Analyzer Applied Biosystems PN 4444317 SOLIDO 3 Plus to SOLiD94 Upgrade Kit SOLIDO 3 to SOLiD94 Upgrade Kit Applied
11. Phosphatase 7 cycles or 35 bp Image 29 34 Cleave 10 cycles or 50 bp Ligation cycle without 2 5 3 Ligate priming F3 R3 ase lease A Phosphatase Image Cleave Applied Biosystems SOLID 4 System Instrument Operation Guide 129 Appendix D Instrument Process Times Times for entire processes Process Estimated Time h Steps poseen Ber Sequencing Primers 4 5 Reset 24 30 7 cycles or 35 bp ee Prime Ligate Dark Ligate Capper Enzyme Image Cleave RevPhosphatase Sequencing Primers 5 6 Reset 25 30 7 cycles or 35 bp Wu im Bridge Probe Ligate Dark Ligate Capper Enzyme Image Cleave RevPhosphatase Ligation cycle without 3 5 4 5 Ligate priming F5 P2 F5 BC Dare Ligate z Capper Enzyme Image Cleave RevPhosphatase Times for entire processes 130 Table 37 Times for entire processes Process Total Runtimet Workflow Analysis WFA 4 5 hours Fragment 35 bp 4 4 5 days Fragment 50 bp 6 7 days Mate paired 25 25 bp 7 8 days Mate paired 35 35 bp 8 9 days Mate paired 50 50 bp 13 14 days Paired end 50 35 bp 12 14 days Multiplex fragment 35 5 bp 5 5 5 days Multiplex fragment 35 10 bp 6 6 5 days Applied Biosystems SOLID 4 System Instrument Operation Guide Appendix D Instrument Process Times Times for entire processes Process Total Runtime Multiplex fragment 50 5 bp 7 8 days Multiplex fragment 50 10 bp 8 9 da
12. Primary Analysis Application Library defaultLibrary Sample default primary v Optional Type or Select gt w Select settings Secondary Analysis Description To assign samples to spots select a sample in the list and then click on spots to assign that sample to them Sample Mask 1 spot mask sf Spots Sample 1 Sample 4 Assign Samples to spots in the mask on the Specify Samples and Analysis pane A blue or white circle on the mask indicates that the numbered sample has been assigned to a spot Clicking on a white circle selects that spot and clicking on a blue circle un assigns that spot To assign a sample select it from the Sample list then click on a spot with no sample assigned to it 5 Click Finish to return to the Setup Task pane 6 Choose either to assign run to a flowcell for immediate use or to an instrument database to store for later use then click OK 7 Toassign a run previously saved to the database a Click on Manage Runs in the task pane b Click the run then select Assign to Flowcell 60 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Section 3 2 Set up and perform a sequencing run Create a multiplex sequencing run record c Choose a flowcell then click OK Create a multiplex sequencing run record Note For instructions to set up a sequencing non multiplex run record go to the previous section Create a
13. Reassemble after the chamber after drying is complete 7 Repeat steps 2 4 twice for a total of 3 volume measurements 8 Take the average volume from the three measurements 9 Cleanthe deposition chamber thoroughly after the calibration is complete 10 Measure the exact volume of each chamber using this technique Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix B Supplemental Procedures B Clean the Instrument Buffer bottle 11 Mark each chamber with exact volume as to best support correct loading procedures Bubbles during e Significant bubbles during slide loading or during sequencing can cause large deposition or regions of bead loss across the slide sequencing e Ensure that the instrument buffer lines are properly primed prior to slide loading Clean the Instrument Buffer bottle Regular cleaning of the Instrument Buffer bottle is required for every run Failure to clean the Instrument Buffer bottle regularly may allow microbial contaminants to proliferate in the system If the level of Instrument Buffer in the Instrument Buffer bottle falls below the recommended fill volume do not add new Instrument Buffer to top off the Buffer that has been standing in the Instrument Buffer bottle Topping off can lead to contamination Required Table 20 Required equipment Clean the Instrument Buffer bottle equipment Item Source Beaker or graduated cylinder Major Laboratory Supplier MLS Bottle bru
14. WARRANTY OR UNDER ANY STATUTE INCLUDING WITHOUT LIMITATION ANY TRADE PRACTICE UNFAIR COMPETITION OR OTHER STATUTE OF SIMILAR IMPORT OR ON ANY OTHER BASIS FOR DIRECT INDIRECT PUNITIVE INCIDENTAL MULTIPLE CONSEQUENTIAL OR SPECIAL DAMAGES SUSTAINED BY THE BUYER OR ANY OTHER PERSON OR ENTITY WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES INCLUDING WITHOUT LIMITATION DAMAGES ARISING FROM OR RELATED TO LOSS OF USE LOSS OF DATA FAILURE OR INTERRUPTION IN THE OPERATION OF ANY EQUIPMENT OR SOFTWARE DELAY IN REPAIR OR REPLACEMENT OR FOR LOSS OF REVENUE OR PROFITS LOSS OF GOOD WILL LOSS OF BUSINESS OR OTHER FINANCIAL LOSS OR PERSONAL INJURY OR PROPERTY DAMAGE NO AGENT EMPLOYEE OR REPRESENTATIVE OF APPLIED BIOSYSTEMS HAS ANY AUTHORITY TO MODIFY THE TERMS OF THIS LIMITED WARRANTY STATEMENT OR TO BIND APPLIED BIOSYSTEMS TO ANY AFFIRMATION REPRESENTATION OR WARRANTY CONCERNING THE PRODUCT THAT IS NOT CONTAINED IN THIS LIMITED WARRANTY STATEMENT AND ANY SUCH Applied Biosystems SOLiD9 4 System Instrument Operation Guide 145 Appendix H Instrument Warranty Information Damages claims and returns MODIFICATION AFFIRMATION REPRESENTATION OR WARRANTY MADE BY ANY AGENT EMPLOYEE OR REPRESENTATIVE OF APPLIED BIOSYSTEMS WILL NOT BE BINDING ON APPLIED BIOSYSTEMS UNLESS IN A WRITING SIGNED BY AN EXECUTIVE OFFICER OF APPLIED BIOSYSTEMS THIS WARRANTY IS LIMITED TO T
15. by implication or by estoppel This product is for internal research purposes only and is not for use in commercial applications of any kind including without limitation quality control and commercial services such as reporting the results of purchaser s activities for a fee or other form of consideration For information on obtaining additional rights please contact outlicensing dlifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners Covaris is a trademark of Covaris Inc HydroShear is a registered trademark of Genomic Solutions Inc ABgene is a trademark of ABgene LTD Limited NanoDrop is a trademark of NanoDrop Technologies Kimwipes is a trademark of Kimberly Clark Corporation IKA and ULTRA TURRAXX are trademarks of IKA Werke GMBH Eppendorf LoBind is a registered trademark of Eppendorf International O 2012 Life Technologies Corporation All rights reserved Contents About This e Hs LTTE 9 Userattentlom Words una A AAA AE 9 B CHAPTER Introduction ss ss eee e e x e o 11 SOLIDO 4 System run types a ei ne rah RR rn ln 11 Workflow analysis WEA run ssssssesssssssess IRR 12 Sequencing iecoris eb mere Pet espe een ese nrc edb dens 12 Software operation and data analysis eee 16 S CHAPTER 2 Prepare and Install Slides and Reagents 17 Mate
16. ipmelit c a bb Nee etd eS 103 Required consumables sssssssssssllellllllls ls e 103 Replace the SOLID Light Source RR rne 104 Required equipment ad R KE Rd 0 RL L K vase ace RKR RAR KKK eee ie UE 104 Set up a run by importing a Run Definition file 0000s 105 Manually find the focus range s e 108 Select the flowcell then find the beads on the slide lessen 109 Find the focal range oli el de p e ia e Sa dw pees 110 Calculate then set the focal range esses 114 Shut down the SOLID 4 Analyzer ssssssssssssssssss n 119 Required equipment esses mn 119 Reset theirobot position 1i sien geen bbsR Roa DUEMERA Santee bee H RAE Me es 119 Store the slide in a flowcell raen eaa eee ER eee ZR T da 119 Modify the Barcode Error Correction Level 120 APPENDIX C On Instrument Reagent Volumes and Reagent Strip LAVOULS sos ep dina ND des cuu Eu dtd s 121 Recommended fill volumes for on instrument reagents cece e eee eee ees 121 Volumes of on instrument reagents coco 121 Fragment sequencing F3 Tag e 122 Paired end sequencing F5 BC F5 P2 Tad 122 Mate pair sequencing F3 R3 Tags 123 Barcode sequencing BC Tag re 123 Workflow analysis WEA ccoo coins pee nce ep ee EIN aid fa d 123 Applied Biosystems SOLID 4 System Instrument Operation Guide 5 Contents Reagent strip layouts sers 124 Fragment paired end sequencing F3Tag esses 124 Mat
17. picofuge and pulse spin for a few seconds to bring down any beads stuck on the walls of the tube Do not over spin the beads or the beads aggregate into a pellet Indicator of spectral purity and signal intensity of the beads Place the tube containing the beads in the appropriate tube holder then place in the Covaris S2 System afterwards run the appropriate program A length of DNA to be sequenced Process of adding library template to beads by emulsion PCR enriching the beads to remove beads without template then modifying the 3 end of the template on the beads to prepare for bead deposition and sequencing SOLiD P1 DNA Beads with amplified library template attached Library template concentration used to prepare an emulsion Applied Biosystems SOLiD9 4 System Instrument Operation Guide Glossary titration metric Product of P2 positive beads and the On Axis beads the titration that generates the highest titration metric value is the optimal titration point for a given library usable beads Number of beads that are called during color calling workflow analysis Type of run on the SOLiD system in which a small portion of templated beads are WFA run deposited and analyzed to test for templated bead quality Applied Biosystems SOLiD9 4 System Instrument Operation Guide 167 Glossary 168 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Barcode Error Correction Level and multiplex sequencing run record 61 modify 1
18. see Figure 50 If you do not see bead images or if you see out of focus bead images set the focus range manually see Manually find the focus range on page 108 Figure 50 Beads in focus Start the sequencing run Note Before starting the run make sure that the air filter at the back of the instrument is not clogged with dust see Clean the air filter on page 98 1 Ensure that there is adequate disk space for images and results of the sequence run for the minimum needed disk space see Table 9 To know the amount of disk space that is available click Manage Runs on the task pane 70 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Section 3 2 Set up and perform a sequencing run Start the sequencing run Table 9 Minimum required disk space to store images and results Minimum space KT needed for Primary Minimum space needed for Run Type Sp Analysis results Secondary Analysis results needed for images NE spch csfasta mapping pairing QV qual Fragment 35 bp 2 7 TB 1 3 TB 200 GB Fragment 50 bp 3 8 TB 1 8 TB 200 GB Mate paired 25 25 bp 3 8 TB 1 8TB lt 200 GB Mate paired 35 35 bp 5 3 TB 2 4 TB lt 200 GB Mate paired 50 50 bp 7 6 TB 3 3 TB lt 200 GB Paired end 50 35 bp 6 5 TB 3 1 TB 200 GB Multiplex fragment 35 5 bp 3 1 TB 1 5 TB 200 GB Multiplex fragment 35 10 bp 3 4 TB 1 7 TB 200 GB Multiplex fragment 50 5 bp 4 2 TB 2 1 TB 200
19. then find the beads on the slide on page 109 Shut down the SOLiD 4 Analyzer The instrument can be shut down using the Instrument Shutdown wizard in the ICS For instruments that have UPS in the event of power failure an uninterrupted power supply UPS is activated UPS systematically shuts down the instrument Analysis jobs are stopped and Linux is shut down Slides are preserved in Storage Buffer Required Table 29 Required equipment Shut down the instrument equipment Item Source UPS Applied Biosystems 4397781 North America 4393695 International 1 Open the Instrument Shutdown wizard by choosing Wizards Instrument Shutdown 2 Follow the instructions in the wizard Reset the robot position The Robot standby script sends the robot back to home position 1 Open the Utility Scripts menu by choosing Tools Utility Scripts 2 Select Robot standby 3 Select Run Script Store the slide in a flowcell The Store flowcell script fills the flowcell with Storage Buffer 1 Open the Utility Scripts menu by choosing Tools Utility Scripts 2 Select Store flowcell Applied Biosystems SOLiD9 4 System Instrument Operation Guide 119 B Appendix B Supplemental Procedures Modify the Barcode Error Correction Level 3 Select Run Script Modify the Barcode Error Correction Level A Barcode Error Correction Level is set at 0 mismatches by default A Barcode Error Correction Level of 1 mismatch increases
20. 000 c cece cence nee 141 Covaris S2 Progralrisu 2 otn eR Entra S ee auta OI LIN PK tutes 142 Covalent Declump T orfane ge nis eR nied vane a LE neue 142 Covalent De cl trpidrt d e tex Atria eet te edo dt EE lel TAS o heheh dy Dia ect ie re ADR 142 APPENDIXH Instrument Warranty Information 143 Computer configuration asas netda ee cece eee Z e Rd Z Z aR R E eA RE S a RET Eu 143 Limited product Warranty nnn 143 Warranty period effective date e 144 MWartantyelaliTis e n dota co dades daa de Ment ni ET TE 144 Warranty exceptions esee mre ln 145 WarrantillmitatonS secs bEPL ada 145 6 Applied Biosystems SOLiD 4 System Instrument Operation Guide Contents Damages claims and returnS ss e 146 Sa IP ERE H UR a 146 E Ia ski emet a ds vinea rue ee cei del 146 Ret urfe c A A rei cm AET PI Re uteris aes 146 APPENDIX Life Technologies End User Software License Agreement e e e e e eee 147 About this agreement o 147 TIUS a ste L ERR REDE EE ERES A VELA ed E e tp 148 CODY TIGNES yo tener tec lentas ep cas is is de MEE de o TEE P 148 AAA A A E AA 148 Useotthe software 0004 dd ia 148 Dora 148 Wiel Lo esce A te Lib ete patte ad aee p NEA de eds 149 TAIANA esca ute AA a aee cock da 149 US governmerit end Users 2 sait a CeM bee biet ded eee es 149 European community end users sssusussseelllllllsl sse 149 Regulated uses ai eg nena A NN A R Rg NIRE DITS 149 Limited warranty and
21. 3 Using an appropriate pipettor and pipette tip press down the plunger button then place the tip into one of the portholes of the well Slowly release the pipettor plunger button then aspirate the SOLiD XD Slide Deposition Buffer v2 Fresh SOLiD XD Slide Deposition Buffer v2 is drawn into the well to replace the old Deposition Buffer Repeat this procedure for the other wells 4 Gently loosen the SOLiD Deposition Chamber screws As the screws are loosened more fresh SOLiD XD Slide Deposition Buffer v2 is drawn into the Deposition Chamber 5 Open the SOLiD Deposition Chamber lid then carefully remove the SOLiD 4 or Opti Slide Carrier assembly from the Deposition Chamber 6 Immediately pour Slide Storage Buffer over the slide to completely cover the beads Allow the Slide Storage Buffer to flow off onto a paper towel or into a waste container 7 Repeat step 6 Applied Biosystems SOLIDO 4 System Instrument Operation Guide 31 Install the slide 32 Chapter 2 Prepare and Install Slides and Reagents Install slide s on the instrument 8 Immediately place the SOLiD 4 or Opti Slide Carrier assembly onto the instrument or into the SOLiD Slide Storage Chamber IMPORTANT The Deposition Chamber should not have any contact with Slide Storage Buffer or Overlay Buffer from previous versions of SOLIDO instruments STOPPING POINT If you are storing the slide place the SOLiD 4 or Opti Slide Carrier assembly into t
22. 4398217 Procedure Insert the O ring into the groove on the flowcell so that the smooth side is on top see Figure 68 Run your finger around the O ring to flatten any high spots 102 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix B Supplemental Procedures B Clean the reagent strip cover Figure 68 Install the SOLiD9 O Ring on a flowcell Clean the reagent strip cover Inspect the top and bottom of the reagent strip covers after each run for splattered wet or dry reagents If you see reagent on a cover clean the cover see Table 27 Required Table 25 Required equipment Clean the reagent strip cover equipment Item Source Pipette or graduated cylinder Major Laboratory Supplier MLS Scrub brush MLS T For the SDS of any chemical not distributed by Applied Biosystems contact the chemical manufacturer Before handling any chemicals refer to the SDS provided by the manufacturer and observe all relevant precautions Required Table 26 Required consumables Clean the reagent strip cover consumables iR CHER Extran 300 VWR EM EX0996 2 Kimwipes wipers Major Laboratory Supplier MLS Deionized water MLS Cotton swabs MLS Table 27 Clean the reagent strip cover according to location or amount of reagent splatter If you see Then Minor splashes Wipe the reagent strip cover with a Kimwipes wiper If the reagents are dried first wet the Kimwipes wip
23. BC Tag 0 eee eee eee eee 126 Multiplex fragment paired end sequencing BC Tag 00 0 eee 127 Workflow analysis WFA o s 0 Ie 128 Recommended fill volumes for on instrument reagents Volumes of IMPORTANT Verify that there is sufficient volume of Reset Buffer for each tag on instrument sequenced The SOLiD ToP Instrument Buffer contains sufficient Reset Buffer to reset reagents two tags see On Instrument Reagent Volumes and Reagent Strip Layouts on page 121 For multiplex sequencing a bottle of Reset Buffer is included with multiplex sequencing kits to account for sequencing the BC tag Table 30 Volumes of on instrument reagents provided in the SOLIDO ToP Instrument Buffer Kit Volume mL 1X Instrument Buffer 8000 Cleave 1 Solution 350 Cleave 2 1 Solution 350 Reset Buffer 170 Imaging Buffer 250 Ligase Buffer 120 Universal Buffer 75 Storage Buffer 825 Applied Biosystems SOLiD9 4 System Instrument Operation Guide 121 Appendix C On Instrument Reagent Volumes and Reagent Strip Layouts Recommended fill volumes for on instrument reagents Fragment sequencing F3 Tag Paired end sequencing F5 BC F5 P2 Tag 122 Volumes shown are after preparation according to the instructions in Chapter 2 Install on instrument reagents on page 25 Table 31 Fill volumes for fragment sequencing 35 or 50 bp Volume mL
24. Buffer Multiplex sequencing up to 50 bases for the F3 tag of barcoded fragment libraries Barcodes 1 16 SOLIDO ToP Fragment BC Sequencing Kit BC Frag Lib MM50 10 4452699 SOLiD9 ToP Sequencing Kit Frag Lib F3 Tag MM50 SOLIDO ToP Frag Lib Seq F3 Tag Primer A Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer B Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer C Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer D Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer E Master Mix 50 SOLIDO Mixing Strip Tube with Zinc SOLiD9 ToP Sequencing Kit BC Frag Lib BC Tag MM10 Reset Buffer Multiplex sequencing up to 50 bases for the F3 tag of barcoded fragment libraries Barcodes 1 96 Applied Biosystems SOLiD9 4 System Instrument Operation Guide 89 A Appendix A Applied Biosystems SOLiD9 4 System Instrument Operation Guide Required Materials Set up and perform a sequencing run Item part number Components Kit components used in SOLIDO ToP Paired End Sequencing Kit BC Frag Lib MM50 35 5 4459181 SOLIDO ToP Sequencing Kit Frag Lib F3 Tag MM50 SOLIDO ToP Frag Lib Seq F3 Tag Primer A Master Mix 50 SOLiD ToP Frag Lib Seq F3 Tag Primer B Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer C Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer D Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer E Master Mix 50 SOLiD Mixing Strip T
25. Corporation or its suppliers and is protected by international laws of copyright The law provides for civil and criminal penalties for anyone in violation of the laws of copyright License Use of the software 1 1 Subject to the terms and conditions of this Agreement Life Technologies LLC grants you a non exclusive license only to install and use the Software to operate the single instrument or workstation in connection with which this License was purchased and to display analyze and otherwise manipulate data generated by the use of such product There is no limit to the number of computers on which you may install and use the Software to display analyze and otherwise manipulate such data 2 If the Software uses registration codes access to the number of licensed copies of Software is controlled by a registration code For example if you have a registration code that enables you to use five copies of Software simultaneously you cannot install the Software on more than five separate computers 3 You may make one copy of the Software in machine readable form solely for backup or archival purposes You must reproduce on any such copy all copyright notices and any other proprietary legends found on the original You may not make any other copies of the Software except as permitted under Section 1 above Restrictions 1 You agree that you will not copy or use the Software or the associated documentation in whole or in part except as exp
26. Description Abort Run Run aborts and analysis jobs are cancelled Set run as completed Run is set as completed Secondary analysis jobs start Change Primer Schedule The Change Primer Schedule command allows you to choose a first and second primer in any order Depending on how you set up the run only primers from a selected primer set are available in the sub menu Set Early Pause Point The Set Early Pause Point command allows you to define when the instrument pauses This command can be used to replenish reagents at a more convenient time than the time defined by the software Note that if the Change Run Progress Point command see below is selected any changes to the Early Pause Point resets to the default Change Run Progress Point The Change Run Progress Point command allows you to back up or skip to any specific point in the run Ensure that you select points that are consistent with the progress of the run Repeating primers can be performed by using the Change Run Progress Point command selecting a primer to repeat resuming the run and using the Set Early Pause Point command to pause the run after the repeated primer has completed The Instrument Control Software ICS allows you to control imaging for individual spots on a slide With the controls you can deposit fragment and mate paired library samples onto the same slide and turn off imaging of a spot To access the imaging and analysis controls in the Sample Slid
27. GB Multiplex fragment 50 10 bp 4 6 TB 2 3 TB 200 GB Multiplex paired end 50 35 5 bp 6 9 TB 3 3 TB 200 GB Multiplex paired end 50 35 10 bp 7 3 TB 3 5 TB 200 GB 2 Click Start Run 3 If there is not enough room to store the data for the run then the Start Run dialog box appears see Figure 51 Choose the appropriate option see Table 51 Figure 51 This Start Run dialog appears if there is not enough room to store data for the run A There is not enough free disk space to complete this run and the currently active runs An additional 3 748GB is required Delete Images Table 10 Choose one of the three options to manage disk space Option Description Start Anyway Initiates the run The instrument pauses Itself when it runs out of free disk space Delete Imagest Launches Historical Runs page in SETS Images and or results can be deleted through SETS Cancel Aborts the run t For more information on creating more available disk space see the Applied Biosystems SOLIDO 4 System SETS Software User Guide Part no 4448411 IMPORTANT Before deleting an image ensure that data analysis from the previous run is satisfactory and complete For more information refer to the Applied Biosystems SOLiD 4 System SETS Software User Guide Part no 4448411 Applied Biosystems SOLIDO 4 System Instrument Operation Guide 71 Chapter 3 Set Up Control and Monitor the Run Start t
28. Guide 117 Appendix B Supplemental Procedures Manually find the focus range Figure 88 Record the average focal value in Notepad E Untitled Notepad ES File Edit Format View Help highest 653930 lowest 648790 average of highest and lowest 651360 set range 5 inis 640560 9556360 10 Subtract 5000 counts from the calculated average then enter that value in the Z Min box Add 5000 counts to the average then enter that value into the Z Max box Confirm that the Number of Steps is 30 Click Save to File see Figure 89 Figure 89 Enter the focus settings UM Auto Focus Options m x Focus Settings Flowcell 1 Flowcel 2 Dutside Flowcells Z Min 6531 03 646360 652503 Z Max 6631 03 65636 662503 Number of Steps 30 v Do auto exposure with auto focus Focusing Method Selection Histogram Range m 11 Click Yes when the warning displays see Figure 90 118 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix B Supplemental Procedures Shut down the SOLiD9 4 Analyzer Figure 90 Click Yes when the Save Focus Range Warning displays UM Confirm Save Focus Range dd Save Z min and max for flowcell 2 to file 12 When the Flowcell Selection window appears choose the number of the flowcell 1 or 2 that has just been measured and click OK The focal range is saved 13 Optional Repeat all previous steps starting from Select the flowcell
29. RETURNED WITHIN 90 DAYS OF THE DELIVERY DATE THESE ARE YOUR SOLE AND EXCLUSIVE REMEDIES FOR ANY BREACH OF WARRANTY WARRANTY CLAIMS MUST BE MADE WITHIN THE APPLICABLE WARRANTY PERIOD Third party products This Software uses third party software components from several sources Portions of these software components are copyrighted and licensed by their respective owners Various components require distribution of source code or if a URL is used to point the end user to a source code repository and the source code is not available at such site the distributor must for a time determined by the license offer to provide the source code In such cases please contact your Life Technologies Corporation representative As well various licenses require that the end user receive a copy of the license Such licenses may be found on the distribution media in a folder called Licenses In order to use this Software the end user must abide by the terms and conditions of these third party licenses After installation the licenses may also be found in a folder named Licenses located in the Software installation s root directory Limitation of liability IN NO EVENT SHALL APPLIED BIOSYSTEMS OR ITS SUPPLIERS BE RESPONSIBLE OR LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE INCLUDING WITHOUT LIMITATION ANY TRADE PRACTICE UNFAIR COMPETITION OR OTHER STATUTE OF SIMILAR IMPORT OR ON ANY OTHER BASIS FOR SPECIAL INDIRECT INCIDENTA
30. Refit and screw down the access cover on the housing 6 In the System Status menu on the ICS click Reset to reset the lamp timer Figure 69 Orientation of the SOLID Light Source 104 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix B Supplemental Procedures Set up a run by importing a Run Definition file Set up a run by importing a Run Definition file A sequencing run can be set up by importing a Run Definition file that has been created offline Setting up a run by importing a run definition file saves time re entering information of a repeated run 1 Click Manage Runs in the task pane on the left menu pane of the ICS see Figure 70 Figure 70 Manage Runs hsolid0062 SO User lab_user File Tools Wizards Window Help Welcome lab_user Run Name Run Name Created by 9 Created by mi Assign Run 7 9 ng os B ex Os ll Assign Run BEAL pur d Sample Slide Sample Slide esee gt 2 Click Export Run see Figure 71 Figure 71 Export Run solid0062 SOLID 4 File Wizards Welcome lab user User lab_user Tools Window Help Setup gt Manage Runs The SOLID Run Manager provides a sumrnary view of recent run activity and allows operators to load masks import and export s selected in the grid it can be addressed by the buttons in the toolbar below lr Images Data Free Space 3754G A T neue Data Free Space ooo RN gr Setup E3 Import Run Espera Edit Selected Ru
31. SOFTWARE IF YOU DO NOT AGREE TO THE TERMS AND CONDITIONS OF THIS AGREEMENT YOU SHOULD PROMPTLY RETURN THIS SOFTWARE TOGETHER WITH ALL PACKAGING TO APPLIED BIOSYSTEMS AND YOUR PURCHASE PRICE WILL BE REFUNDED This Agreement accompanies the Life Technologies SOLiD System Software Suite Software and related explanatory materials Documentation The term Software also includes any upgrades modified versions updates additions and copies of the Software licensed to you by Life Technologies The term Life Technologies as used in this License means Life Technologies LLC The term License or Agreement means this End User Software License Agreement The term you or Licensee means the purchaser from Life Technologies of this license to use the Software Applied Biosystems SOLiD9 4 System Instrument Operation Guide 147 EN Appendix Life Technologies End User Software License Agreement Title Title Title ownership rights and intellectual property rights in and to the Software and Documentation shall at all times remain with Life Technologies LLC and its subsidiaries and their suppliers All rights not specifically granted by this License including Federal and international copyrights are reserved by Life Technologies Corporation or their respective owners Copyright The Software including its structure organization code user interface and associated Documentation is a proprietary product of Life Technologies
32. Supplemental Procedures This appendix covers Clean the deposition chamber ro 95 Calibrate the deposition chamber volume e oo 9 Clean the Instrument Buffer bottle oooooooooocccoocorroromorrr nooo 97 Install the SOLiD System Flowcell O ring sees 102 Clean the reagent Strip cover initan a a e 103 Replace the SOLIDO Light Source uis poo Lex ws certos TREE ERRAT 104 Set up a run by importing a Run Definition file ooooooooo 105 Manually find the focus range e 108 Shut down the SOLD 4 Analyzer eot Geis ett bade bobs eases 119 Reset the robot position 6 6 6 cece reren 119 Store the slideinaflowcell lees 119 Modify the Barcode Error Correction Level 0 eee eee eee eens 120 Clean the deposition chamber The enhanced surface chemistry on the SOLiD XD Slides is extremely robust and serves to efficiently bind templated beads but it is also extremely sensitive to contaminants Contaminants will result in a decrease in bead binding if the proper protocol is not followed In addition use only supported consumables and reagents for bead preparation and deposition Common sources of contamination include e Improper cleaning of deposition chambers e Incomplete drying of the deposition chamber e Inadequate buffer exchanges and washes in bead samples It is critical that the user follows the proper procedure for cleaning and preparation of the deposition chamber Fol
33. accessories Depending on the weight moving or lifting may require two or more persons If you decide to lift or move the instrument after it has been installed do not attempt to do so without the assistance of others the use of appropriate moving equipment and proper lifting techniques Ensure you have a secure comfortable grip on the instrument or accessory Make sure that the path from where the object is to where it is being moved is clear of obstructions Do not lift an object and twist your torso at the same time Keep your spine in a good neutral position while lifting with your legs Participants should coordinate lift and move intentions with each other before lifting and carrying For smaller packages rather than lifting the object from the packing box carefully tilt the box on its side and hold it stationary while someone else slides the contents out of the box Applied Biosystems SOLiD9 4 System Instrument Operation Guide 155 4 Appendix J Safety Safety and electromagnetic compatibility EMC standards A CAUTION Moving Parts Moving parts can crush pinch and cut Keep hands clear of moving parts while operating the instrument Disconnect power before servicing Electrical WARNING Fuse Installation Before installing the instrument verify that the fuses are properly installed and the fuse voltage matches the supply voltage Replace fuses only with the type and rating specified for the unit Improper fuses
34. coms Go Tol 2 t Gotof1 G Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix B Supplemental Procedures B Manually find the focus range 2 In the Imager window click the AutoFocus button then wait about 20 seconds for the Imager to focus on the slide see Figure 80 The Camera 1 indicator displays BUSY while autofocus is running When the Camera 1 indicator displays READY click AutoExpose then wait for the Camera 1 indicator to show READY Click AutoFocus so that the Imager can focus again this time with correct exposure settings Note where the focus peak falls in the focus range by looking at the value that displays where the peak is located Figure 80 Click AutoFocus and AutoExpose so that the focus peak falls in the focus range Qu SOLID Imager 1 9 10 f e View Run Options Devices Took Windows Heb lum Acque ME eos SA Genua SH 266055 IP 127001 Pot BOI 2 Stage Status tete H eel pag Pot Saba Motion Poson Speed Acceleration B Depth fo Z Saaste Ree 00 Golive Dais View Clear Ad Save s Fi Fa Cow READY x 233000 100 100 Shite Status ocur Stats e Status 1869 00 100 100 EE m B cows B cow 5e Nudge Size o a Re Caldeate Stage GoToX nm AJo p soror hen vbe T N Enable Joystick v TES Go To Fiducial Mak EHE o Status Stage Cort Re Enable Jog Td Nix Go e s 2 j Go To 1 2 fV ShowFlonCel 3 When the Imager displays READY click the Acquire button then examin
35. discontinuing use of the Software removing all copies from your computers and storage media and returning the Software and Documentation and all copies thereof to Life Technologies Corporation Life Technologies Corporation may terminate this Agreement if you fail to comply with all of its terms in which case you agree to discontinue using the Software remove all copies from your computers and storage media and return the Software and Documentation and all copies thereof to Life Technologies Corporation The Software is a commercial item as that term is defined in 48 C F R 2 101 Oct 1995 consisting of commercial computer software and commercial computer software documentation as such terms are used in 48 C F R 12 212 Sept 1995 Consistent with 48 C F R 12 212 and 48 C F R 227 7202 1 through 227 7202 4 June 1995 all U S Government End Users acquire the Software with only those rights set forth herein If this Software is used within a country of the European Community nothing in this Agreement shall be construed as restricting any rights available under the European Community Software Directive O J Eur Comm No L 122 42 1991 You acknowledge that the Software has not been cleared approved registered or otherwise qualified collectively Approval by Life Technologies LLC with any regulatory agency for use in diagnostic or therapeutic procedures or for any other use requiring compliance with any fe
36. mate pair sequencing Tag 1 125 Workflow Analysis WFA run 128 related documentation 161 replace the SOLiD Light Source 104 required kits and equipment sequencing run 86 Workflow Analysis WEA run 83 robot position reset 119 run Change Primer Schedule command 76 Change Run Progress Point command 76 fill volumes for on instrument reagents 121 log view for a sequencing run 77 log view for a Workflow Analysis WFA run 52 multiplex record create for sequencing run 61 number of beads per well 11 Pause Run Resume Run 73 pause resume 73 purpose of different types 11 record create for Workflow Analysis WFA run 46 Run Control menu 74 Set Early Pause Point command 76 standard record create for sequencing run 58 Stop Run command 76 summary about 11 time estimated 11 types overview 11 view cycle scans in a sequencing run 78 view heat maps in a sequencing run 77 Workflow Analysis WFA 44 Run Control Menu commands 74 Run Definition file import to set up a run 105 S safety alerts on instrument 154 biohazard 158 chemical 158 cleaning and decontamination 156 Applied Biosystems SOLiD 4 System Instrument Operation Guide electrical 156 instrument 155 physical injury 155 standards 156 symbols on instrument 153 Safety Data Sheets SDSs obtaining 162 Scan Slide and pausing resuming a run 73 sequencing run about 12 Change Primer Schedule command 76 Change Run Progress Point command 76 create a multiplex run reco
37. method is to use the Run wizard see below The second method is to import a txt file that contains the run definition see Set up a run by importing a Run Definition file on page 105 It saves time re entering information of a repeated run This file can be generated on an off instrument computer 1 Click Create Runs in the Setup task pane on the left menu pane of the ICS see Figure 38 Figure 38 Use the run wizard to create a sequencing run solid0062 SOLID 4 0 User lab user File Tool Wizards Window Help Welcome lab_user Run Name e Run N Created by Create i Assign Run 7 e ecc n X NIL ES Sample Slide San X Create Runs Hide Samples lt lt m BD Flow cell 1 2 Complete the information in the Select Run Type and Mask pane see Figure 39 a Select the type of run If performing a fragment sequencing run select Fragment f performing a mate pair sequencing run select Mate Pair If performing a paired end sequencing run select Paired End o Optional Type a new run name o Optional Enter a description Ensure that the Run Protocol is set to SOLiD4 a Select Primer Set 1 and Primer Set 2 m If performing a fragment sequencing run leave F3 as Primer Set 1 e If performing a mate pair sequencing run select R3 as Primer Set 1 and F3 for Primer Set 2 IMPORTANT The R3 tag must be run first in a mate pair sequencing run The reagent strips fo
38. number in a prominent location on the outside of the shipping container and return the material to the address designated by the Applied Biosystems representative Applied Biosystems SOLiD9 4 System Instrument Operation Guide Life Technologies End User Software License Agreement This appendix covers About this agreement TTT 147 iE a AEE E E E E E et E eed onec euet EN EA 148 Copyrights onire she bls tbe hed ae OEE A A uk 148 hui PP v ES 148 Limited warranty and limitation of remedies 0 cee e ee eee ee 150 Third party products ich cesses a 151 Limitation of liability 9 TN 9 RR cee eee ee 151 Generali bites 152 About this agreement This appendix is the Life Technologies End User Software License Agreement for Instrument Operating and Associated Bundled Software and Limited Product Warranty NOTICE TO USER PLEASE READ THIS APPLIED BIOSYSTEMS SOLiD 4 SYSTEM SOFTWARE SUITE END USER SOFTWARE LICENSE AGREEMENT CAREFULLY THIS IS THE CONTRACT BETWEEN YOU AND APPLIED BIOSYSTEMS LLC REGARDING THE OPERATING SOFTWARE FOR YOUR APPLIED BIOSYSTEMS WORKSTATION OR OTHER INSTRUMENT AND BUNDLED SOFWARE INSTALLED WITH YOUR OPERATING SOFTWARE THIS AGREEMENT CONTAINS WARRANTY AND LIABILITY DISCLAIMERS AND LIMITATIONS YOUR INSTALLATION AND USE OF THE APPLIED BIOSYSTEMS SOFTWARE IS SUBJECT TO THE TERMS AND CONDITIONS CONTAINED IN THIS AGREEMENT AND YOU WILL BE BOUND BY THESE TERMS AND CONDITIONS IF YOU INSTALL OR USE THIS
39. right corner of the flowcell panel to open a dialog box describing a series of instrument events 2 After you finish viewing the Run Log click Close located at the bottom of the Run Log window 1 Toview the heat map showing bead densities found in the focal map images click Heat Map located at the top right corner of the flowcell panel see Figure 58 2 Look for Uniform deposition of beads on the slide e The actual average bead deposition density panel value being similar in value to the targeted average bead deposition density for example 300 000 beads panel A large number of missing panels could indicate a deposition problem Note The heat map is not available until after the completion of the first sequencing cycle The software refines the focal map using images collected during the first sequencing cycle and does not display the heat map until after all of the images collected during both the Prescan and cycle 1 have been processed This may take up to 30 minutes depending on the number of panels that were imaged 3 After viewing the Heat Map click Close located at the bottom of the Heat Map window Applied Biosystems SOLiD9 4 System Instrument Operation Guide 77 Chapter 3 Set Up Control and Monitor the Run Monitor the run Figure 58 Bead Count left Bead Signal center and Image Signal right heat maps Bead Count Bead Signal Bead Count Bead Signal Image Signal L Over Limit MN under Lim
40. sequencing Run F3 Tag Primers A to E Run F5 P2 Tag Primers A to E Run R3 Tag Primers A to E up to 10 cycles per primer up to 7 cycles per primer up to 10 cycles per primer Multiplex fragment sequencing Multiplex paired end sequencing Run F3 Tag Primers A to E up to 10 cycles per primer Applied Biosystems SOLiD9 4 System Instrument Operation Guide 15 Chapter 1 Introduction Software operation and data analysis Software operation and data analysis 16 The SOLiD 4 System comprises multiple complementary analysis software components that complete primary analysis image acquisition signal processing color calling and quality control and secondary analysis alignment to a reference genome SNP identification and base calling of fragment mate pair and paired end sequencing experiments For information describing the relationship between ICS SOLiD Instrument Control Software SETS SOLiD Experimental Tracking System and BioScope Software refer to the Applied Biosystems SOLiD 4 Software Operation and Data Analysis Quick Reference Guide Part no 4448432 For additional secondary and tertiary analysis tools visit the SOLiD Software Development Community website http solidsoftwaretools com You can integrate standalone tools from the SOLiD Software Development Community with BioScope to perform more automated analysis For details refer to the BioScope Software for Scientists Guide Part n
41. sequencing non multiplex run record on page 58 There are two ways to create a multiplex sequencing run The first method is to use the Run wizard see below The second method is to import a Run Definition file created offline see Set up a run by importing a Run Definition file on page 105 IMPORTANT For all barcodes gt Barcode 16 ten bases of barcode tag must be read While creating a multiplex sequencing run file on the System the barcode definition file for the 1 96 series already specifies ten bases of sequence If at least 1 library on the slide uses barcode 17 or higher ten bases of barcode tag will be read for all other libraries on the slide even if they are on separate spots 1 Optional If desired modify the Barcode Error Correction Level A Barcode Error Correction Level is set at 0 mismatches by default A Barcode Error Correction Level of 1 mismatch increases the number of reads by about 10 but lower quality sequencing reads are included in the data For information about how to modify the Barcode Error Correction Level see Modify the Barcode Error Correction Level on page 120 2 Click Create Runs in the Setup task pane on the left menu pane of the ICS see Figure 41 Applied Biosystems SOLiD9 4 System Instrument Operation Guide 61 5 Chapter 3 Set Up Control and Monitor the Run Create a multiplex sequencing run record Figure 41 Use the run wizard to create a multiplex sequencing run f
42. sequencing run record Figure 44 Enter the information for the libraries in the sample Create New Library For each library present in a multiplexed sample enter a Library Name select the Multiplexing Series used and specify which Barcode s have been added during that library s creation Library Name Control_1 1 Select Multiplexing Series 2 Select Barcodes 3 Review selected barcodes for this library EC Kit Module 1 16 1 EC Kit Module 1 16 1 Dz os 14 Os o O Da 19 Dio Du O12 More about the Selected Multiplexing Series 0 3 Barcodes on DNA and RNA libraries made with O14 SOLID Barcoding Kits Module 1 16 PN 4444836 Dis and 4427046 Use 5 bp sequencing reagents to sequence these barcodes 116 IMPORTANT Be sure to select the correct Multiplexing Series for your run You can obtain additional information by selecting the Multiplexing Series and looking at the lower left panel More about the Selected Multiplexing Series If at least 1 library uses Barcode 17 or higher on the slide you should select the BC Kit Modules 1 96 option for each library on the slide Table8 Contents of the Multiplexing Series Multiplexing Series Contents BC Kit Module 1 16 16 barcodes in the SOLIDO Fragment Library Barcoding Kit Module 1 16 Part no 4444636 and SOLIDO Transcriptome Multiplexing Kit Part no 4427046 BC Kit Modules 1 96 96 barcodes in the SOLiD9 Fragment Library Barcoding K
43. terminal main lG ceoeo Terminal that can receive or supply alternating current or voltage Terminal that can receive or supply alternating or direct current or voltage Do not dispose of this product in Xl unsorted municipal waste CAUTION To minimize negative mmm disposal options environmental impact from disposal of electronic waste do not dispose of electronic waste in unsorted municipal waste Follow local municipal waste ordinances for proper disposal provision and contact customer service for information about responsible Safety alerts on this instrument Additional text may be used with one of the symbols described above when more specific information is needed to avoid exposure to a hazard See the following table for safety alerts found on the instrument Safety label Location on Instrument CAUTION Hot Surfaces Flow cell cover and inside arc lamp box CAUTION Replace only with CERMAX tx300f 300 w lamp Arc lamp box cover WARNING HOT Do not remove lamp until 15 minutes after disconnecting power supply Arc lamp box cover CAUTION Crush pinch hazard Syringe pump 154 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix J Safety J Safety information for instruments not manufactured by Life Technologies Safety information for instruments not manufactured by Life Technologies Some of the accessories provided as part of the ins
44. the SOLiD Opti Slide Carrier Using a different slide carrier may lead to run failure a Move the retainers out so that the slide fits into the SOLiD 4 or Opti Slide Carrier To do this push down on the two spring knobs in the SOLiD 4 or Opti Slide Carrier and slide the knobs towards the outside edges of the carrier see Figure 7 A Applied Biosystems SOLiD9 4 System Instrument Operation Guide Chapter 2 Prepare and Install Slides and Reagents 7 Deposit the beads on the slides b Place the slide against the alignment nubs in the SOLiD 4 or Opti Slide Carrier c Ensure that the slide is precisely positioned in the SOLiD 4 or Opti Slide Carrier and then slide the retainers inward until they hold the slide in position see Figure 7 B Note To move the retainers over the slide do not push down the knobs Figure 7 A The SOLiD 4 Slide Carrier with spring knobs and retainers pushed out and B with spring knobs and retainers pushed in the slide is held under the retainers Spring knob Spring retainer Orientation key Blue on the side Spring knob Spring retainer Orientation key Deposition Chamber base then place the appropriate SOLiD Deposition Chamber lid on top see Figure 8 IMPORTANT After washing do not let the slide sit for more than 10 minutes before depositing beads Note The SOLiD Deposition Chamber top must engage with the orientation key to fit properly T
45. the number of reads by about 1076 but lower quality sequencing reads are included in the data To modify the Barcode Error Correction Level 1 In SETS log in as the Administrator 2 Select Analysis Primary Analysis Settings New 3 In the Barcode Error Correction Level drop down list select O or 1 see Figure 91 4 Save the primary analysis setting then apply the setting to the multiplex sequencing run when the run is created Figure 91 In Barcode Error Correction Level select 0 or 1 Primary Analysis Setting Owner Administrator Name Barcode 1MM Description Minimum Calls Barcode Error Correction leve Low and high thresholds for traffic light parameters 120 Applied Biosystems SOLiD9 4 System Instrument Operation Guide On Instrument Reagent Volumes and Reagent Strip Layouts This appendix covers Recommended fill volumes for on instrument reagents ooooommmmoo 121 Paired end sequencing F5 BC F5 P2 Tag oo 122 Mate pair sequencing F3 R3 Tags 0 eee eee ee 123 Barcode sequencing BC Tag 6 6 6 e 123 Workflow analysis WFA cress reared vinse ern NE TT TR TE ENT E 123 Reagent strip layouts erea R eke ee men eh e ht hh ere RR REFER E 124 Fragment paired end sequencing F3 Tag 0 eee ee eee ee 124 Mate Pair sequencing F3 Iag eee R E KRE R eens 125 Mate Pair sequencing R3 Tag 125 Paired end sequencing F5 P2 Tag 126 Multiplex paired end sequencing F5
46. 20 barcode sequencing fill volumes for reagents 123 barcode creating a library for a barcoded sample 65 beads concentration chart 20 depositing 20 deposition density determining 21 45 55 number per well according to run type 11 number to use according to run 21 biohazard safety 158 bottles position of buffer bottles in chiller block 29 position of buffers and waste in the instrument 27 position of reagents in the instrument 28 C Change Primer Schedule command 76 Change Run Progress Point command 76 checklists and workflow tracking forms 135 chemical safety 158 clean flush the fluidiclines 99 the reagent strip cover 103 cleaning safety 156 Covaris S2 System operation and maintenance 141 cycle scans viewing for WFA run 53 viewing in sequencing run 78 D data analysis about 16 decontamination safety 156 density bead deposition determining 21 45 55 documentation related 161 Applied Biosystems SOLiD 4 System Instrument Operation Guide Index E electrical safety 156 electromagnetic compatibility EMC standards 156 end user software license agreement 147 Eppendorf Lo Bind tubes use of 19 F fill volumes for on instrument reagents 121 flowcell O ring install 102 storing the slide in 119 flush the fluidic lines 99 focus range detecting 49 finding manually 108 fragment sequencing fill volumes for on instrument reagents 121 reagent strip layouts 124 G glossary 165 H heat maps viewi
47. 61 on page 82 4 After you finish viewing the Cycle Scans close the Cycle Scans window Note If the WFA run appears problematic you can 1 Allow the run to continue and troubleshoot after the run or 2 Pause the run and troubleshoot Consult an Applied Biosystems SOLiD Field Applications Specialist View the WFA report Generate the WFA The WFA report is automatically generated and available in SETS when the WEA run report finishes see Figure 37 Refer to the Applied Biosystems SOLiD 4 System SETS Software User Guide Part no 4448411 Applied Biosystems SOLiD9 4 System Instrument Operation Guide 53 Chapter 3 Set Up Control and Monitor the Run View the WEA report Determine the optimal titration point 54 Figure 37 One WFA report for a single 4 well slide displayed in SETS Template Bead Titration and Selection Report for solid0054_20090827_WFA_JPB215_217 Windows Internet Explorer GO gi notti a isalresutisckdonssl noss 20090627 ra e amp zis 2t7 acreportidefau hm File Edit View Favorites Tools Help Sis de e Template Bead Teration and Selection Report for sola M E eh Ep rage gt ons Report Summary Name Template Bead Titration and Selection Report for solid0054 20090827 WFA JPB215 217 Created By corona Date Created Fri Aug 28 08 56 10 GMT 07 00 2009 Report Results wfaSample 1 JPB215 wfaSample 2 JPB216 wfaSampl
48. 62 1 WEA Created by lab_user Created by lab_user SD asson fue o Edit Run 2 ded Ll RunLogs Heat Map E A Edit Run 7 kar DI Run Logs f Heat Map Sample Slide Sample Slide Show Samples gt gt 3 Samples in 4_spot_WFA_mask_sf Show Samples gt gt 1 Sample in 4 spot V ate Runs age Runs v Cell Details 1Cell2 Protocol Protocol b SOLID WFA F3 1 bases Y SOLID WFA F3 Pre Scan MEME Pre Sean L a C C BE LE IMPORTANT Do not disturb the SOLiD 4 Analyzer while in operation and do not open the flowcell during a pause in the run Significant perturbations for example opening system parts vibrations during the run is detrimental to the results Monitor the WFA run Note To monitor the run remotely use SETS from any computer to connect to the networked instrument refer to the Applied Biosystems SOLiD 4 System SETS Software User Guide Part no 4448411 If desired set up e mail notification regarding the instrument run and system information using SETS refer to the Applied Biosystems SOLiD 4 System SETS Software User Guide View the run log 1 Click Run Log located at the top right corner of the flowcell panel A dialog box opens describing a series of instrument events 2 After you finish viewing the Run Log click Close located at the bottom of the Run Log window View the heat maps 1 Toview the heat map showing bead densities found in the focal map images click Heat Map
49. 7 Required Table 15 Required equipment WFA run equipment Itemt Source SOLIDO 4 System Applied Biosystems PN 4452773 110 V Applied Biosystems PN 4452774 220 V SOLIDO 4 Analyzer Applied Biosystems PN 4452775 SOLIDO 3 Plus to SOLiD94 Upgrade Kit Applied Biosystems PN 4452784 SOLIDO 3 to SOLiD94 Upgrade Kit Applied Biosystems PN 4452785 SOLIDO Light Source Applied Biosystems PN 4383441 SOLIDO Slide Storage Chamber Applied Biosystems PN 4406354 SOLiD9 Deposition Chambers 1 Wellt Applied Biosystems PN 4406352 SOLiD Deposition Chambers 4 Well8 Applied Biosystems PN 4406358 SOLiD Deposition Chambers 8 Well8 Applied Biosystems PN 4406359 SOLiD Uninterruptible Power Supply UPS Applied Biosystems PN 4397781 SOLIDO UPS North America Applied Biosystems PN 4393695 220 V SOLiD UPS International SOLIDO Accessory Disk Drive Applied Biosystems PN 4426101 SOLiD9 Bead Concentration Chart Applied Biosystems PN 4415131 84 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix A Applied Biosystems SOLIDO 4 System Instrument Operation Guide Required Materials Set up and perform a workflow analysis WFA run Item Source Covaris S2 System 110 V for U S customers 220 V for international customers The system includes e Covaris S2 sonicator Latitude laptop from Dell e MultiTemp Ill Thermostatic Circulator e Covaris 2 series Machine Holder for one 1 5 mL microcentrifuge tube e Covar
50. 90 days Applied Biosystems warrants that chemicals and other consumable products will be free of defects in materials and workmanship when received by the buyer but not thereafter unless otherwise specified in documentation accompanying the product Applied Biosystems warrants that for a period of ninety 90 days from the date the warranty period begins the tapes diskettes or other media bearing the operating software of the product if any will be free of defects in materials and workmanship under normal use If there is a defect in the media covered by the above warranty and the media is returned to Applied Biosystems within the ninety 90 day warranty period Applied Biosystems will replace the defective media Unless indicated herein Applied Biosystems makes no warranty whatsoever in regard to products or parts furnished by third parties including but not limited to the non APC branded UPS or APC UPS Covaris S2 Genomic Solutions HydroShear DNA Shearing Device Recirculating Chiller and IKA ULTRA TURRAX purchased or obtained from a third party Such products or parts will be subject to the warranties if any of their respective manufacturers to the extent they are transferable or otherwise available to Applied Biosystems buyer Applied Biosystems at its sole discretion may refuse to provide buyer with support or service for buyer s use of Covaris S2 in a method not described in a SOLiD System protocol Applied Biosyst
51. A run 3 Start the WFA run Figure 35 This Start Run dialog appears if there is not enough room to store data for the run A There is not enough free disk space to complete this run and the currently active runs An additional 3 748G8 is required Start Anyway Table 7 Options for managing disk space Option Description Start Anyway Initiates the run The instrument pauses itself when it runs out of free disk space Delete Images Launches Historical Runs page in SETS Images and or results can be deleted through SETS Cancel Aborts the run t For more information on creating more available disk space refer to the Applied Biosystems SOLiD9 4 System SETS Software User Guide Part no 4448411 IMPORTANT Before deleting any images ensure that data analysis from the previous run is satisfactory and complete For more information refer to the Applied Biosystems SOLiD 4 System SETS Software User Guide Part no 4448411 3 After the run has been initiated you can click the Run Log Cycle Scans and Heat Map buttons located at the top of the appropriate flowcell panel to learn more information about the current run see Figure 36 Applied Biosystems SOLiD9 4 System Instrument Operation Guide 51 3 Chapter 3 Set Up Control and Monitor the Run Monitor the WFA run Figure 36 How to learn more about the current run d0062 SOLID 4 0 User lab_user Run Name E i 2 1 Wi e Run Name by
52. Biosystems PN 4452784 Applied Biosystems PN 4452785 SOLIDO Light Source Applied Biosystems PN 4388441 SOLiD Slide Storage Chamber Applied Biosystems PN 4406354 SOLIDO Deposition Chambers 1 Well Applied Biosystems PN 4406352 SOLIDO Deposition Chambers 4 Well8 Applied Biosystems PN 4406358 SOLIDO Deposition Chambers 8 Well Applied Biosystems PN 4406359 SOLiD 4 Slide Carriers8 Applied Biosystems PN 4453027 Applied Biosystems SOLiD9 4 System Instrument Operation Guide 91 Appendix A Applied Biosystems SOLiD9 4 System Instrument Operation Guide Required Materials Set up and perform a sequencing run Item Source SOLIDO Uninterruptible Power Supply UPS Applied Biosystems PN 4397781 SOLIDO UPS North America Applied Biosystems PN 4393695 220 V SOLIDO UPS International SOLiD9 Accessory Disk Drive Applied Biosystems PN 4426101 SOLiD9 Bead Concentration Chart Applied Biosystems PN 4415131 Covaris9 S2 System 110 V for U S customers 220 V for international customers The system includes e Covaris S2 sonicator Latitude laptop from Dell e MultiTemp Ill Thermostatic Circulator e Covaris 2 series Machine Holder for one 1 5 mL microcentrifuge tube e Covaris 2 series Machine Holder for one 0 65 mL microcentrifuge tube e Covaris 2 series Machine Holder for one 13 mm x 65 mm tube e Covaris 2 Series Machine Holder for one m
53. C JEL Multiplex paired end sequencing gt F3 F5 BC a BC ELT The type of sequencing run depends on the type of library to be sequenced see Table 2 Table 2 Types of sequencing runs possible for each library type Library type Possible sequencing run type s Fragment library Fragment sequencing Paired end sequencing Mate paired library Mate pair sequencing Barcoded fragment library Multiplex fragment sequencing Multiplex paired end sequencing During a SOLiD sequencing run two probe sets are used to maximize the fraction of mappable beads read length and sequencing throughput Mappable beads are beads amplified with template that map to the reference genome Applied Biosystems SOLiD9 4 System Instrument Operation Guide 1 Chapter 1 Introduction SOLIDO 4 System run types Compared to terminator based sequencing chemistry with SOLID System sequencing base information is not collected instead five rounds of primers Primers A B C D and E are used to sequence template by ligation of di base labeled probes for each tag for examples see Figure 3 Figure 3 Di base sequencing in a mate pair sequencing run top and in a multiplex paired end sequencing run bottom 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 0123456 7 8 9 10 Cycles Cyde3 Oyde C BG Tag PrmerA Cyde 77 6yde2
54. F3 tag of barcoded fragment libraries Barcodes 1 16 88 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix A Applied Biosystems SOLIDO 4 System Instrument Operation Guide Required Materials A Set up and perform a sequencing run Item part e Kit componentls number used in SOLIDO ToP SOLIDO ToP Sequencing Kit Frag Lib F3 Tag MM35 Multiplex Fragment BC Sequencing Kit BC Frag Lib MM35 10 4452698 SOLIDO ToP Frag Lib Seq F3 Tag Primer A Master Mix 35 SOLIDO ToP Frag Lib Seq F3 Tag Primer B Master Mix 35 SOLIDO ToP Frag Lib Seq F3 Tag Primer C Master Mix 35 SOLIDO ToP Frag Lib Seq F3 Tag Primer D Master Mix 35 SOLIDO ToP Frag Lib Seq F3 Tag Primer E Master Mix 35 SOLiD Mixing Strip Tube with Zinc SOLiD9 ToP Sequencing Kit BC Frag Lib BC Tag MM10 Reset Buffer sequencing up to 35 bases for the F3 tag of barcoded fragment libraries Barcodes 1 96 SOLIDO ToP Fragment BC Sequencing Kit BC Frag Lib MM50 5 4452697 SOLiD9 ToP Sequencing Kit Frag Lib F3 Tag MM50 SOLiD ToP Frag Lib Seq F3 Tag Primer A Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer B Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer C Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer D Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer E Master Mix 50 SOLIDO Mixing Strip Tube with Zinc SOLiD9 ToP Sequencing Kit BC Frag Lib BC Tag MM5 Reset
55. HE BUYER OF THE PRODUCT FROM APPLIED BIOSYSTEMS AND IS NOT TRANSFERABLE Some countries or jurisdictions limit the scope of or preclude limitations or exclusion of warranties of liability such as liability for gross negligence or willful misconduct or of remedies or damages as or to the extent set forth above In such countries and jurisdictions the limitation or exclusion of warranties liability remedies or damages set forth above shall apply to the fullest extent permitted by law and shall not apply to the extent prohibited by law Damages claims and returns Damages Claims Returns 146 If shipping damage to the product is discovered contact the shipping carrier and request inspection by a local agent Secure a written report of the findings to support any claim Do not return damaged goods to Applied Biosystems without first securing an inspection report and contacting Applied Biosystems Technical Support for a Return Authorization RA number After a damage inspection report is received by Applied Biosystems Applied Biosystems will process the claim unless other instructions are provided Do not return any material without prior notification and authorization If for any reason it becomes necessary to return material to Applied Biosystems contact Applied Biosystems Technical Support or your nearest Applied Biosystems subsidiary or distributor for a return authorization RA number and forwarding address Place the RA
56. L MULTIPLE PUNITIVE OR CONSEQUENTIAL DAMAGES ARISING OUT OF THE POSSESSION OR USE OF OR THE INABILITY TO USE THE SOFTWARE OR DOCUMENTATION EVEN IF APPLIED BIOSYSTEMS IS ADVISED IN ADVANCE OF THE POSSIBILITY OF SUCH DAMAGES INCLUDING WITHOUT LIMITATION DAMAGES ARISING FROM OR RELATED TO LOSS OF USE LOSS OF DATA DOWNTIME OR FOR LOSS OF REVENUE PROFITS GOODWILL OR BUSINESS OR OTHER FINANCIAL LOSS IN ANY CASE THE ENTIRE LIABILITY OF APPLIED BIOSYSTEMS AND ITS SUPPLIERS UNDER THIS LICENSE OR ARISING OUT OF THE USE OF THE SOFTWARE SHALL NOT EXCEED IN THE AGGREGATE THE PURCHASE PRICE OF THE PRODUCT SOME STATES COUNTRIES OR JURISDICTIONS LIMIT THE SCOPE OF OR PRECLUDE LIMITATIONS OR EXCLUSION OF REMEDIES OR DAMAGES OR OF LIABILITY SUCH AS LIABILITY FOR GROSS NEGLIGENCE OR WILLFUL MISCONDUCT AS OR TO THE EXTENT SET FORTH ABOVE OR DO NOT ALLOW IMPLIED WARRANTIES TO BE EXCLUDED IN SUCH STATES COUNTRIES OR JURISDICTIONS THE LIMITATION OR EXCLUSION OF WARRANTIES REMEDIES DAMAGES OR LIABILITY SET FORTH ABOVE MAY NOT APPLY TO YOU HOWEVER ALTHOUGH THEY SHALL NOT APPLY TO THE EXTENT PROHIBITED BY LAW THEY SHALL APPLY TO THE FULLEST EXTENT PERMITTED BY LAW YOU MAY ALSO HAVE OTHER RIGHTS THAT VARY BY STATE COUNTRY OR OTHER JURISDICTION Applied Biosystems SOLiD9 4 System Instrument Operation Guide 151 General ER Appendix Life Technologies End User Software License Agreement General 152 This Agreement shall be governe
57. NESS FOR A PARTICULAR PURPOSE OR MERCHANTABILITY OR THAT THE SOFTWARE OR DOCUMENTATION IS NON INFRINGING ALL OTHER WARRANTIES ARE EXPRESSLY DISCLAIMED WITHOUT LIMITING THE GENERALITY OF THE FOREGOING APPLIED BIOSYSTEMS MAKES NO WARRANTIES THAT THE SOFTWARE WILL MEET YOUR REQUIREMENTS THAT OPERATION OF THE LICENSED SOFTWARE WILL BE UNINTERRUPTED OR ERROR FREE OR WILL CONFORM EXACTLY TO THE DOCUMENTATION OR THAT APPLIED BIOSYSTEMS WILL CORRECT ALL PROGRAM ERRORS APPLIED BIOSYSTEMS SOLE LIABILITY AND RESPONSIBILITY FOR BREACH OF WARRANTY RELATING TO THE SOFTWARE OR DOCUMENTATION SHALL BE LIMITED AT APPLIED BIOSYSTEMS SOLE OPTION TO 1 CORRECTION OF ANY ERROR IDENTIFIED TO APPLIED BIOSYSTEMS IN A WRITING FROM YOU IN A SUBSEQUENT RELEASE OF THE SOFTWARE WHICH SHALL BE SUPPLIED TO YOU FREE OF CHARGE 2 ACCEPTING A RETURN OF THE PRODUCT AND REFUNDING THE PURCHASE PRICE UPON RETURN OF THE PRODUCT AND REMOVAL OF ALL COPIES OF THE SOFTWARE FROM YOUR COMPUTERS AND STORAGE DEVICES 3 REPLACEMENT OF THE DEFECTIVE SOFTWARE WITH A FUNCTIONALLY EQUIVALENT PROGRAM AT NO CHARGE TO YOU OR 4 PROVIDING A REASONABLE WORK AROUND WITHIN A REASONABLE TIME APPLIED BIOSYSTEMS SOLE LIABILITY AND RESPONSIBILITY UNDER THIS AGREEMENT FOR BREACH OF WARRANTY RELATING TO MEDIA IS THE Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix Life Technologies End User Software License Agreement Third party products REPLACEMENT OF DEFECTIVE MEDIA
58. No Run Defined Run Control jj No Run Defined A g 3 Load Flowcell Door Closed Locked E g Clear Flowcell Unlock Doors Applied Biosystems SOLiD9 4 System Instrument Operation Guide Chapter 2 Prepare and Install Slides and Reagents Install on instrument reagents 4 After the chiller temperature is lt 10 C install the prepared 1X Instrument Buffer and Storage Buffer into the appropriate positions in the cabinet see Figure 10 CAUTION POTENTIAL OVERHEAD HAZARD Use caution when working inside the cabinet Figure 10 Positions of buffers and waste in the cabinet Waste 1x Instrument Buffer Storage Buffer Applied Biosystems SOLID 4 System Instrument Operation Guide 27 Chapter 2 Prepare and Install Slides and Reagents Install on instrument reagents 5 Install Cleave Solution 1 prepared Cleave Solution 2 1 and Reset Buffer in the appropriate positions on the side of the instrument see Figure 11 Make sure that the tubes on the caps of the reagent bottles are fully screwed on e CAUTION POTENTIAL OVERHEAD HAZARD Use caution when working inside the cabinet Keep the instrument side door closed over the Cleave Solution and Reset Buffer bottles Figure 11 Positions of reagent bottles on the side of the instrument SOLID Instrument Buffer Kit Cleave 2 1 Part2 Byte 2445676 z Cleave Solution 1 Reset Buffer Cleave Solution 2 1 28 Applied Biosystems SOLiD9 4 System Instrument Operati
59. RRRRRRRRRRRRNNN Executing Scan Slide Filter 3 of 4 Panel 1503 of 2358 Run Name Created by f Assign Run L3 Eo Run dif clear ME Runko Sample Slide Hide Samples Protocol Pre Scan Primers A B C D and E of F5 BC Stop Run b Running Run Control commands u Load Flowcell UL co e No Run Defined z coT M G m gA Clear Flowcell IMPORTANT Do not disturb the SOLiD 4 Analyzer while in operation and do not open the flowcell during a pause in the run Significant perturbations for example opening system parts vibrations during the run are detrimental to the results Table 11 Run Control commands Command Command is available while the instrument is Stop Run Running or paused Reset Current Primer Paused within Primer Cycle Change Primer Schedule Paused Set Early Pause Point Running or paused Change Run Progress Point Paused t Certain commands have sub menus that allow you to control every step in a run protocol Applied Biosystems SOLiD9 4 System Instrument Operation Guide 3 Chapter 3 Set Up Control and Monitor the Run Control the run Use imaging and analysis controls for specific spots 76 Stop Run The Stop Run command launches a Stop Run dialog box Choose the appropriate option in the dialog according to Table 12 Table 12 Options available in the Stop Run dialog Option
60. S A A A A NAT ees Aino 78 APPENDIX A Applied Biosystems SOLiD 4 System Instrument Operation Guide Required Materials eee 83 Set up and perform a workflow analysis WFA pun I 83 Required Applied Biosystems reagent kits cece eee eae 83 Required equipment isa A DE ND EE ERA E 84 Required consumables sse e teenies 86 Set up and perform a sequencing run 0 0c cece eee tee en 86 Required Applied Biosystems reagent kits 0c cee eee eee eens 86 Applied Biosystems SOLiD 4 System Instrument Operation Guide Contents Required equipment seen 91 Required consumables ie ortos tete WP HR DO e bU emus 93 APPENDIX B Supplemental Procedures 95 Clean the deposition chamber sss 95 Deposition chambers previously used with the SOLID 3 or SOLiD 3 Plus System 96 Calibrate the deposition chamber volume sse 96 Bubbles during deposition or sequencing nc 97 Clean the Instrument Buffer bottle IH 97 Required equipment sees rns 97 Clean the air INE ni i een poete obe em oer obse ttai eode 98 Required consumables 98 Flush the fluidic lines ocio 2 ae aee a ARR E Re Rd 99 Required consumables eese per ee Lp at SIR RE 99 Install the SOLID System Flowcell O ring II 102 Required equipments tan oes td 102 Procedure erecta a pole a gaye ees el e re LOS 102 Clean the reagent strip cover 2 0c cc cece ese rn 103 Regq ired eg
61. Storage Chambers are provided for use with all chambers 8 Or equivalent but validation of the equipment for library preparation is required Required consumables Table 19 Required consumables Sequencing run Item Source Nuclease free Water Applied Biosystems AM9932 ABgene 96 1 2 mL square well storage plates ABgene AB 1127 3 mm adhesive disks Grace Bio Labs ST200 Ethylene glycol American Bioanalytical AB00455 01000 CF 1 Calibration Fluid Kit Thermo Scientific CF 1 PR 1 Conditioning Kit Thermo Scientific PR 1 1 5 mL LoBind Tubes Eppendorf 022431021 Kimwipes Major Laboratory Supplier MLS Filtered pipettor tips MLS t Applied Biosystems has validated this protocol using this specific material Substitution may adversely affect system performance T The NanoDrop Conditioning Kit is useful for reconditioning the sample measurement pedestals to a hydrophobic state if they become unconditioned refer to the Nanodrop user s manual for more information The PR 1 kit consists of a container of specially formulated polishing compound and a supply of convenient applicators Applied Biosystems SOLiD9 4 System Instrument Operation Guide 93 A Appendix A Applied Biosystems SOLiD9 4 System Instrument Operation Guide Required Materials Set up and perform a sequencing run 94 Applied Biosystems SOLiD9 4 System Instrument Operation Guide
62. Tit 3 After clearing each flowcell open the appropriate flowcell chamber 4 Remove the SOLiD 4 or Opti Slide Carrier assembly from the previous run If the slide will be reused place the SOLiD 4 or Opti Slide Carrier assembly into a SOLiD Slide Storage Chamber then fill the chamber with Slide Storage Buffer Store the SOLiD Slide Storage Chamber at 4 C 5 Clean the flowcell block with 70 ethanol and Kimwipes wipers to remove residue Applied Biosystems SOLIDO 4 System Instrument Operation Guide 33 34 Chapter 2 Prepare and Install Slides and Reagents Install slide s on the instrument 6 Inspect the O ring and reseat it if necessary see Figure 17 for details see Appendix B Install the SOLIDO System Flowcell O ring on page 102 Check the O ring for cuts and abrasions If any abnormalities are observed replace it Inspect the O ring grooves for debris or contamination and clean with water as needed Figure 17 O ring installed on flowcell O ring Ensure that the instrument and storage buffer lines are primed before loading a slide or the slide will dry out Insert the SOLiD 4 or Opti Slide Carrier onto the instrument Work quickly to prevent the slide from drying out a Remove the SOLiD 4 or Opti Slide Carrier assembly from the SOLIDO Deposition Chamber or from the SOLiD Slide Storage Chamber b Place the SOLiD 4 or Opti Slide Carrier assembly into the open flowcell en
63. USER GUIDE applied biosystems by Kafe technologies Applied Biosystems SOLiD 4 System INSTRUMENT OPERATION GUIDE Publication Part Number 4448379 Rev C Revision Date May 2012 Library Templated Bead Preparation Preparation 1 o For Research Use Only Not intended for any animal or human therapeutic or diagnostic use technologies For Research Use Only Not intended for any animal or human therapeutic or diagnostic use The information in this guide is subject to change without notice DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF NOTICE TO PURCHASER LIMITED USE LABEL LICENSE Research Use Only The purchase of this product conveys to the purchaser the limited non transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser No right to resell this product or any of its components is conveyed expressly
64. ages see Figure 84 Applied Biosystems SOLIDO 4 System Instrument Operation Guide 113 B Appendix B Supplemental Procedures Manually find the focus range Calculate then set the focal range 114 Figure 84 Examples of in focus images m I tee e 145 H tte t et te a Beads in focus Back of slide in focus x CY UF beh A pon tis a A ft U 1 559 L 3 A a Back of heater block in focus Reflection of beads in focus 9 Uncheck the Go Live check box in the Imager window to close the live image window 1 When the image is in focus record the value of the Z distance in the Focus Status pane This is the nominal Z distance for this flowcell Be sure to record the correct Z distance value not the value in the GoTo window see Figure 85 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix B Supplemental Procedures Manually find the focus range Figure 85 Record the nominal Z distance Ue SOLID Imager 1 9 10 Stage Status Pot Su Motion Posion cow ion 26812 00 a Nudge Sue 10 Z GoToX ES Go To Y ELENA G vj S ShowFiowCel 2 Zoom In Flowcel Go To Fiducial Mak Cama SM 208255 IP 127001 Poe 9 Expone Time 2 3 enfe Rrrr 1 Ba Dei a X Sasae Bae 001 GoUve Shutter Status EE o Delad View Den A Sacr Aa Focus Status Fiet Status Nosepiece Status TEC come ME cows y Stop v tania GoTof al Ss teb S Jog Senstivy 5 Z 2 Choose Options
65. al Buffer Partt S O Universal Buffer Part2 OOOO Imaging Buffer Part 1 Imaging Buffer Part 2 Cleave Solution i OOOO Cleave Solution 2 1 Part S O Cleave Solution 2 1 Part2 OOOO Reset Buffer S O Slide Storage Buffer F3 Tag Sequencing Kit S F3 Primera i S A O E A OO OOOO AO O n gnu L L L Td Ligase Buffer Part 2 F3 Mixing Strip Tube R3 F5 P2 F5 BC Tag Sequencing Kit R3 F5 P2 F5 BC Primer C R3 F5 P2 F5 BC Primer D Strip Tube Applied Biosystems SOLiD9 4 System Instrument Operation Guide 139 Appendix E Checklists and Workflow Tracking Forms Workflow tracking set up a sequencing run 8 well Workflow tracking set up a sequencing run 8 well Date Run Date Samples 1 to 4 Sampename J 600 E eee Concentration pap _ Deposition Volume il Volare Is aed Beads Left pru p E Samples 5 to 8 Sample Name Sample information A600 Concentration beads pL Deposition Volume uL Volume Left EA E 3 Beads Left EL G O Deposition Buffer v2 PT Slide Prep Reagent ooo Instrument Buffer PT CS H T4 Ligase Buffer Universal Butter Part Universal Buffer Part2 SSS Imaging Buffer Pati PT Imaging Buffer Pat2 PT Cleave Solution o O Cleave Solution 2 1 Parti PT Cleave Solution 2 1 Part2 OOOO Reset Bu
66. and Storage Buffer lines Install slide s on instrument Install reagent strip s on instrument Set up control and monitor the run see Chapter 3 Deposit the beads on the slide s For a WFA run the beads are quantitated using the Applied Biosystems SOLiDO Bead Concentration Chart Part no 4415131 and 15 million beads are deposited in one well of a 4 Well SOLiD Deposition Chamber For a sequencing run the choice of SOLiD Deposition Chamber depends on factors such as the requirements of the experiment number of libraries being assessed the size of the genome and the sequencing coverage required Three SOLiD Deposition Chamber designs are available for use see Table 3 Table3 Three deposition chamber designs Deposition chamber Number of image panels 1 Well 2357 4 Well 426 per well 8 Well 186 per well Applied Biosystems SOLiD9 4 System Instrument Operation Guide Chapter 2 Prepare and Install Slides and Reagents Tips Install on instrument reagents Prepare 1X Instrument Buffer using glycerol and 10X Instrument Buffer provided in the SOLiD Instrument Buffer Kit You may formulate 1X Instrument Buffer in 8 L batches as needed or prepare it in larger volumes and store at 4 C until ready for use Prepare T4 Ligase Buffer Imaging Buffer Universal Buffer and Cleave Solution 2 1 by combining the two parts provided in the SOLiD ToP Instrument Buffer Kit Cool the chiller block prior to installa
67. apter 3 Set Up Monitor the run solid0045 20100119 PE BC WTA 2 File View Options Help LO BRA AH a lt Images Layout Control and Monitor the Run Figure 61 View the focal map and the image for each fluorescent dye signal BC_P3_01_V1 0152 S OLiD Panel Browser a aja a Focamap v ram ha Apply TXR A L FOCALMAP FAM cv3 solid0045 20100119 PE solid0045 20100119 PE solid0045 20100119 PE 01 19 10 13 46 54 01 20 10 14 46 43 01 20 10 15 05 08 Min 496 Max 16383 Min 0 Max 255 Min 1 Max 255 A XR solid0045_20100119_PE 01 20 10 15 20 42 Min 2 Max 255 cy solid0045_20100119_PE_ 01 20 10 15 34 55 Min 4 Max 255 4 After you finish viewing the Cycle Scans close the Cycle Scans window Note If the sequencing run appears problematic you can 1 Allow the run to continue and troubleshoot after the run or 2 Pause the run and troubleshoot Consult a SOLiD System Field Applications Specialist Applied Biosystems SOLiD9 4 System Instrument Operation Guide A Applied Biosystems SOLiD 4 System Instrument Operation Guide Required Materials This appendix covers Set up and perform a workflow analysis WFA run Required Applied Biosystems reagent kits o oooooooommmmmmmmoomoo oo Required equipment ss cos0 eR RR ds ee eek ee ERE SERT RR Pe RR e X Une Required COS aleg c KEZ Kg R R
68. are labeled and their positions on the slide are recorded to derive a focal map followed by a single ligation cycle The run can be monitored using the Run Log Heat Map and Cycle Scans through the Instrument Control Software ICS or from the SETS browser Each flowcell generates a separate Run Log that records high level events such as fluidic modules and slide scanning The start and stop times of these events and any pauses or errors that occur during the run are also recorded The Run Log is particularly useful in helping you anticipate and schedule reagent refills or troubleshoot instrument errors Heat maps are generated from the analysis of the focal map images and analysis of each ligation cycle A heat map is a colorized display of a particular metric bead count bead signal or image signal across all the panels for a run for definitions of the metrics see the Glossary on page 165 For each flowcell the corresponding Cycle Scans window provides nearly real time feedback on initial data quality on a per cycle basis Applied Biosystems SOLiD9 4 System Instrument Operation Guide 57 Chapter 3 Set Up Control and Monitor the Run Create a sequencing non multiplex run record Create a sequencing non multiplex run record Note For instructions to set up a multiplex sequencing run record go to the next section Create a multiplex sequencing run record on page 61 There are two ways to create a sequencing run The first
69. are recorded to derive a focal map and then they undergo a single ligation cycle The run can be monitored through the ICS or the SETS browser by using the Run Log Heat Map and Cycle Scans Each flowcell generates a separate Run Log that records high level events such as fluidic modules and slide scanning The start and stop times of these events as well as any pauses or errors during the run are also recorded The Run Log is particularly useful in helping you anticipate and schedule reagent refills or troubleshoot instrument errors Heat maps are generated from the analysis of the focal map images and analysis of each ligation cycle A heat map is a colorized display of a particular metric bead count bead signal or image signal across all the panels for a run for definitions of the metrics see the Glossary on page 165 For each flowcell the corresponding Cycle Scans window provides nearly real time feedback on initial data quality on a per cycle basis View the WFA report You can view a WFA report in the SOLiD Experiment Tracking System SETS after the run is complete Three important metrics are generated in the WEA report P2 P1 ratio On Axis beads and Titration Metric These metrics guide the selection of the best performing bead population based on different titration points used in ePCR for definitions of the metrics see the Glossary on page 165 In general the closer the image data points are on axis the highe
70. c alignment during color increase in any subsequent ligation calling cycle for each sequencing primer 2 The fraction of Best The fraction can vary depending on the A significant drop in the fraction of good beads usable beads quality of the library the efficiency of the good beads in the initial ligation PCR and the enrichment process cycles would indicate a reason to abate TE ause the run and to troubleshoot the As a guideline the fraction is around 0 5 p M Me performance 0 6 in the first ligation cycle of each primer and drops to 0 2 0 3 in the last cycle Applied Biosystems SOLIDO 4 System Instrument Operation Guide 79 Chapter 3 Set Up Control and Monitor the Run Monitor the run Parameter Normal run Problematic run 3a 3b Effective exposure Gradual increase from ligation cycles 1 5 250 ms or greater in ligation cycle 1 or times or higher for each sequence primer when instrument times out when the Performance varies from slide to slide and effective exposure time exceeds as a function of the age of the SOLID Light 500 ms Long exposure times may Source t indicate replacement of the SOLIDO As a guideline the effective exposure time Light Sourcet is typically 20 40 ms in the first ligation cycle and increases to 100 300 ms in the tenth cycle 4 Satay plots The first cycle of any primer should showa An abnormal fuzzy Satay plot in the relatively clean Satay plot with most fi
71. can damage the instrument wiring system and cause a fire WARNING Ensure appropriate electrical supply For safe operation of the instrument Plug the system into a properly grounded receptacle with adequate current capacity Ensure the electrical supply is of suitable voltage Never operate the instrument with the ground disconnected Grounding continuity is required for safe operation of the instrument WARNING Power Supply Line Cords Use properly configured and approved line cords for the power supply in your facility Cleaning and CAUTION CLEANING AND DECONTAMINATION decontamination Before using a cleaning or decontamination method other than those recommended by the manufacturer verify with the manufacturer that the proposed method will not damage the equipment Safety and electromagnetic compatibility EMC standards The instrument design and manufacture complies with the standards and requirements for safety and electromagnetic compatibility as noted in the following table Safety Reference Description EU Directive 2006 95 EC European Union Low Voltage Directive IEC 61010 1 Safety requirements for electrical equipment for EN 61010 1 measurement control and laboratory use Part 1 General requirements UL 61010 1 CSA C22 2 No 61010 1 IEC 61010 2 010 Safety requirements for electrical equipment for EN 61010 2 010 measurement control and laboratory use Part 2 010 Particular requirements fo
72. d 0 TRER d ene Set up and perform a sequencing TUN 6 cee Required Applied Biosystems reagent kits 0 Required equipment Rn ER RE EMI RR pe EE Required consumables oooooococorooconcccrrrn eee Set up and perform a workflow analysis WFA run Required Applied Biosystems reagent kits Table 14 Required Applied Biosystems reagent kits WFA run Item part number Components Kit component s used in SOLIDO ToP Instrument Buffer Kit 4452688 Reset Buffer Glycerol 10X Instrument Buffer Cleave Solution 1 Cleave 2 1 Kit Cleave 2 1 Parts 1 and 2 Storage Buffer Imaging Buffer Kit Imaging Buffer Parts 1 and 2 1X T4 Ligase Buffer Kit 1X Ligase Buffer Parts 1 and 2 Universal Buffer Kit Universal Buffer Parts 1 and 2 Sequencing and or WFA SOLiD9 XD Slide amp Deposition Kit v2 4456997 4 x 2 Sequencing Slides Slide Deposition Buffer v2 Slide Storage Buffer Bead deposition SOLiD9 ToP Workflow Analysis Reagents 4453237 SOLIDO ToP Workflow Analysis Reagents WFA Applied Biosystems SOLiD9 4 System Instrument Operation Guide 83 A Appendix A Applied Biosystems SOLiD9 4 System Instrument Operation Guide Required Materials Set up and perform a workflow analysis WFA run Item part number Components Ait ARES SOLiD9 Flowcell O rings 10 pack of flowcell O rings Sequencing and or WFA 439821
73. d by laws of the State of California exclusive of its conflict of laws provisions This Agreement shall not be governed by the United Nations Convention on Contracts for the International Sale of Goods This Agreement contains the complete agreement between the parties with respect to the subject matter hereof and supersedes all prior or contemporaneous agreements or understandings whether oral or written If any provision of this Agreement is held by a court of competent jurisdiction to be contrary to law that provision will be enforced to the maximum extent permissible and the remaining provisions of this Agreement will remain in full force and effect The controlling language of this Agreement and any proceedings relating to this Agreement shall be English You agree to bear any and all costs of translation if necessary The headings to the sections of this Agreement are used for convenience only and shall have no substantive meaning All questions concerning this Agreement shall be directed to Life Technologies 850 Lincoln Centre Drive Foster City CA 94404 1128 Attention Legal Department Applied Biosystems SOLiD9 4 System Instrument Operation Guide Safety WARNING GENERAL SAFETY Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device Ensure that anyone using this product has received instructions in general safety practices for laboratories an
74. d the safety information provided in this document Before using an instrument or device read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device Before handling chemicals read and understand all applicable Safety Data Sheets SDSs and use appropriate personal protective equipment gloves gowns eye protection etc To obtain SDSs see the Documentation and Support section in this document Symbols on this instrument Symbols may be found on the instrument to warn against potential hazards or convey important safety information In this document the symbol is used along with user attention words described in the About This Guide section to highlight important safety information The following table lists the meaning of these symbols Symbol English Caution risk of danger gt Consult the manual for further safety information Caution hazardous waste Refer to SDS s and local regulations for handling and disposal Caution hot surface Caution risk of electrical shock Moving parts Potential slipping hazard Potential overhead hazard gt gt ee Applied Biosystems SOLIDO 4 System Instrument Operation Guide 153 J Appendix J Safety Safety alerts on this instrument Symbol English On Off On Off Standby Earth ground terminal ground Protective conductor
75. deral or state law regulating diagnostic or therapeutic products blood products medical devices or any similar product hereafter collectively referred to as federal or state drug laws or the Software has been validated for any such regulated uses The Software must not be used for any purpose that would require any such Approval or validation unless proper Approval is obtained or validation is completed You agree that if you elect to use the Software for a purpose that would subject you or the Software to the jurisdiction of any federal or state drug laws you will be solely responsible for obtaining any required Approvals and otherwise ensuring that your use of the Software complies with such laws or any required validation Applied Biosystems SOLiD9 4 System Instrument Operation Guide 149 Appendix Life Technologies End User Software License Agreement Limited warranty and limitation of remedies Limited warranty and limitation of remedies 150 Limited Warranty Life Technologies warrants that during the same period as of the SOLiD 4 Analyzer for which this Software is an instrument operating software the Software will function substantially in accordance with the functions and features described in the Documentation delivered with the Software when properly installed and that for a period of ninety days from the beginning of the applicable warranty period as described below the tapes CDs diskettes or other media bearing the Soft
76. dix C Reagent strip layouts on page 124 Table 6 Where to place the strip tubes If using flowcelL Then place the strip tube s in the 1 Front block 2 Rear block Applied Biosystems SOLiD9 4 System Instrument Operation Guide 39 Chapter 2 Prepare and Install Slides and Reagents Install reagent strip s on the instrument Figure 23 Reagent strip block layout for WFA Primer E Primer D Primer C E E Primer B po H Primer A SOLIDO ToP Sequencing Kit Frag Lib F3 Tag MM50 SOLIDO ToP Sequencing Kit Frag Lib F3 Tag MM35 SOLiD ToP Sequencing Kit MP Lib Seq F3 Tag MM 50 SOLiD ToP Sequencing Kit MP Lib Seq R3 Tag MM 50 SOLIDO ToP Sequencing Kit MP Lib Seq R3 Tag MM 35 40 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Chapter 2 Prepare and Install Slides and Reagents Install reagent stripls on the instrument Figure 25 Reagent strip block layouts for sequencing F5 P2 and F5 BC tags SOLIDO Mixing Strip Tube Primer E Primer D am rimer C am Primer B Primer A Cae SOLiD ToP Paired End Seq Kit Frag Lib F5 P2 Tag MM35 SOLiD ToP Paired End Seq Kit BC Frag Lib F5 BC Tag MM35 Figure 26 Position of the sequencing reagent blocks for flowcells 1 and 2 Z Applied Biosystems SOLID 4 System Instrument Operation Guide 41 Chapter 2 Prepare and Install Slides and Reagents Install rea
77. ducts not supplied or authorized by Applied Biosystems modification or repair of the product not authorized by Applied Biosystems relocation or movement of the instrument by buyer or by any third party not acting on behalf of Applied Biosystems or intrusive activity including without limitation computer viruses hackers or other unauthorized interactions with instrument or software that detrimentally affects normal operations Parts in contact with any liquid are considered wetted and may be deemed user replaceable and not be covered by the above warranties including but not limited to seals filters gaskets shearing assemblies valves syringes syringe adapters syringe shields and output tubing Warranty limitations THE FOREGOING PROVISIONS SET FORTH APPLIED BIOSYSTEMS SOLE AND EXCLUSIVE REPRESENTATIONS WARRANTIES AND OBLIGATIONS WITH RESPECT TO THE PRODUCTS WARRANTIED HEREIN AND APPLIED BIOSYSTEMS MAKES NO OTHER WARRANTY OF ANY KIND WHATSOEVER EXPRESSED OR IMPLIED INCLUDING WITHOUT LIMITATION WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE WHETHER ARISING FROM A STATUTE OR OTHERWISE IN LAW OR FROM A COURSE OF DEALING OR USAGE OF TRADE ALL OF WHICH ARE EXPRESSLY DISCLAIMED THE REMEDIES PROVIDED HEREIN ARE THE BUYER S SOLE AND EXCLUSIVE REMEDIES WITHOUT LIMITING THE GENERALITY OF THE FOREGOING TO THE FULL EXTENT ALLOWED BY LAW IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT
78. dule a dialog displays see Figure 53 Figure 53 Pause Run dialog for pre mixing module Pause Run This module contains pre mixing Select the type of pause to perform How do you want to pause the run Pause after current module completes Recommended pause Pauses the run after the current module completes On resume ofthe run execution will begin with the next scheduled module 5 Con Applied Biosystems SOLiD9 4 System Instrument Operation Guide 73 Chapter 3 Set Up Control and Monitor the Run Control the run Use the Run Control menu 74 The default setting is to pause after the current module completes so that pre mixing is not interrupted If you want to pause the run within a pre mixing module select Pause anyway see Figure 54 Figure 54 Pause Run dialog options for pre mixing module i Pause Run This module contains pre mixing Selectthe type of pause to perform How do you wantto pause the run Pause anyway Pause anyway Pause after current module completes auses the run after aborting the current module On resume ofthe run resume or remix script will be executed prior to the execution of the current module When you resume a run to use the pre mixed reagents in the strip tube a dialog box displays see Figure 55 While it is recommended to simply resume the run the system provides an option to remix the reagents by clicking Remix Resume In this case the pre
79. e 3 JP C IO ET l Anabel Bees E NT P2 Rfu 191013 P2_Rfu 154872 P2_Rfu 146822 P2_Exp 35 P2_Exp 147 P2 Exp 51 P2 Gain 32 P2 Gain 32 P2_Gain 32 P T8 mesian per panel 44799 P1 median per pane 44113 P41 mesian per panel 38490 PZ median per panel 35831 I median per panen 34282 P23 mecian per panei 31807 P2 P1 ratio 0 P2 P 1 ratio 78 P2 P1 ratio 83 N2S 9 N2s 1196 N2s 6 la On Axis 68 On Axis 65 On Axis 80 Titration Metric 54 Titration Metric 150 Titration Metric 66 a Determine which titration has the highest titration metric This titration is the optimal titration point Applied Biosystems SOLID 4 System Instrument Operation Guide Section 3 1 Set up and perform a workflow analysis WFA run 3 View the WFA report Determine the 1 Calculate the concentration of P2 positive beads using the formula below where bead deposition X is the volume of templated beads used for the WEA sample equivalent to 15 density for a million beads and P2 is given by the WFA report sequencing run xe 15 x 10 beads H beads pL according to NanoDrop P2 beads panel x 426 panels X uL Y P2 positive beads uL Example For a sample with a concentration of 500 000 beads uL measured by NanoDrop ND 1000 where the WFA report indicates a P2 value of 20 000 beads panel 15 x 10 beads 500 000 beads pL 20 000 beads panel x 426
80. e 30 Complete the information in the Specify Samples and Analysis pane User lab_user SETUP gt Create Edit Run gt Specify Samples and Analysis Select how many samples you need for this run For Application Primary and Secondary Analysis columns in a sequencing or multiplexing run you can click the column F remaining rows How many samples will you need for your mask 3 v Sample Name Primary Analysis Secondary Analysis Description wfaSample 1 default primary none wfaSample 2 default primary none wfaSample 3 default primary none To assign samples to spots select a sample in the list and then click on spots to assign that sample to them Sample Mask 4 spot WFA mask sf Spots v 3 wfaSample 3 4 Choose either to assign a run to a flowcell for immediate use or to store the run in the instrument database for later use then click OK 5 Toassign a run previously saved to the database a Click on Manage Runs in the task pane b Click the run then select Assign to Flowcell 48 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Section 3 1 Set up and perform a workflow analysis WFA run Detect the focus range c Choose a flowcell then click OK 6 Optional Repeat the above steps for the other flowcell if performing a WFA run on a second slide Detect the focus range Note If automatic focus range detection fails determine t
81. e Pair sequencing F3 Tag sn 125 Mate Pair sequencing R3 Tag esee 125 Paired end sequencing F5b P2T g A sees ous e ele IteieA hry ewido ve RR TUERI E TUS 126 Multiplex paired end sequencing F5 BC Tag rr 126 Multiplex fragment paired end sequencing BC Tag n n 0 cece eee ee eee eee 127 Workflow analysis WFA 3 ocd neti blica 128 APPENDIX D Instrument Process Times ssuueuee 129 Times for individual processes 0 000 cece eee eee eee eee e 129 Times for entire processes sss er lue eed ed UU oe ATE ee pe A ees 130 APPENDIX E SOLiD 4 Analyzer Plumbing System Schematic 133 APPENDIX F Checklists and Workflow Tracking Forms 135 Workflow checklists set up a workflow analysis or sequencing run n 136 Workflow tracking set up and perform a workflow analysis WFA pun 000s 137 Workflow tracking set up a sequencing run 1 well 0 ccc cece eee ences 138 Workflow tracking set up a sequencing run 4 well 0 ccc ccc cece nrnna 139 Workflow tracking set up a sequencing run 8 well 0 ccc ccc een eas 140 APPENDIX G The Covaris S2 System uuu 141 Operation notes scie e Pr ee ge Eleg A RN eS 141 Fillthetank istoc A eR C EMETIVURiN a MUERTE CREER 141 Degas thiewaler oz sc er e aite deg idee eon 141 Set tlie chlller 3i Sateen aes Laelia pa vene ever eu 141 Perform required maintenance of the Covaris S2 System
82. e display of the ICS right click a sample name to display options menu These options are not available until the focal map prescan is complete The options menu includes Enabled e Imaging Turned Off Spot discarded Enabled Enabled is the default setting and allows for both imaging and analysis of the spot Applied Biosystems SOLiD9 4 System Instrument Operation Guide Monitor the run View the run log View the heat maps Section 3 2 Set up and perform a sequencing run Monitor the run Imaging Turned Off Imaging Turned Off turns off imaging but allows analysis to complete Spot Discarded Spot Discarded turns off both imaging and analysis Spot Discarded can be used to remove problematic samples from the software workflow Use of Spot Discarded updates the Sample Slide display according to the selection in Spot Discarded Use of Spot Discarded also affects subsequent cycles see Figure 57 Figure 57 The Sample Slide display updates after using Imaging Turned Off or Spot Discarded Imaging Spot Turned Of Discarded Note To monitor the run remotely use SETS from any computer to connect to the networked instrument Refer to the Applied Biosystems SOLiDO 4 System SETS Software User Guide Part no 4448411 Set up optional email notification regarding instrument run and system information using SETS Refer to the Applied Biosystems SOLiD 4 System SETS Software User Guide 1 Click Run Log located at the top
83. e during the warranty period The following parts of the HydroShear DNA Shearing Device are use replaceable and not covered by the warranty on the HydroShear DNA Shearing Device shearing assembly syringes syringe adapters syringe shields and output tubing Applied Biosystems reserves the right to use new repaired or refurbished instruments or components for warranty and post warranty service agreement replacements Repair or replacement of products or components that are under warranty does not extend the original warranty period Applied Biosystems SOLiD9 4 System Instrument Operation Guide 143 Appendix H Instrument Warranty Information Warranty period effective date Applied Biosystems warrants that all optional accessories supplied with its SOLiD 4 Analyzer such as peripherals printers and special monitors will be free of defects in materials and workmanship for a period of ninety 90 days from the date the warranty begins Applied Biosystems will repair or replace at its discretion defective accessories during this warranty period After this warranty period Applied Biosystems will pass on to the buyer to the extent that it is permitted to do so the warranty of the original manufacturer for such accessories With the exception of consumable and maintenance items replaceable products or components used on or in the instrument are themselves warranted to be free of defects in materials and workmanship for a period of ninety
84. e the image on the screen see Figure 81 Applied Biosystems SOLiD9 4 System Instrument Operation Guide 111 B Appendix B Supplemental Procedures Manually find the focus range Figure 81 Examine the image on the screen Image in focus Image out of focus Note To zoom on the image double left click the mouse To un zoom double right click To drag the image hold down the left mouse button 4 If the image is in focus proceed to Calculate then set the focal range on page 114 If the image is out of focus proceed to step 5 5 Check the Go Live check box in the Imager window By checking the Go Live check box a live image of the flowcell displays You can use this image to manually find the focal range see Figure 82 Figure 82 Check Go Live to manually find the focal range Cu SOLID Imager 1 9 10 Fie View Run Options Devices Tools Windows Help CAMERA lelo Status ALE pote Camera SN 266255 IP 127001 Pot 8081 Stage Status Expone Time 32 ME um Pot Status Hoon Pomin Speed Acceleration AR DexaVew Dew M Saveds Fes Status Nosepece Status f x 23x00 100 mm o NS uu 100 m HH oe LET BH oc B ow Stage Contest aj rr m er ia SL Enable F Show Depa 2 Go To Fiducial Mark Jog Jog gei El gates Z geih Z 112 Applied Biosystems SOLIDO 4 System Instrument Operation Guide Appendix B Supplemental Procedures Manually find the focus range 6 Click the focus slider bar in the Imager window A black box app
85. eader and use ctrl D for column fill down which will c remaining rows How many samples will you need for your mask He vi Create New Run 1 Select Run Type Sample Name Primary Analysis Description Re and Mask L bcsamplel default primary v Please select a sample in the above table to define its libraries Create Barcode Library Library Name Application Secondary Analysis 3tBarcodes Multiplexing Series Options BC sangle Optional Type or Select v Select settings vii BC Kit Module 1 96 Ex BC sample2 Optional Type or Select v Select settings vit BC Kit Module 1 96 Bw x BC_sample3 lt Optional Type or Select gt v Select settings vit BC Kit Module 1 96 wx BC sample4 Optional Type or Select v Select settings vit BC Kit Module 1 96 wx BC_samples Optional Type or Select v Select settings vit BC Kit Module 1 96 wx BC_sample6 Optional Type or Select gt v Select settings gt vit BC Kit Module 1 96 wx BC sample Optional Type or Select v Select settings vit BC Kit Module 1 96 Bx BC sample8 1 BC Kit Module 1 96 E X BC_sample9 Optional Type or Select v Select settings vit BC Kit Module 1 96 wx Control 1 Optional Type or Select v Select settings vit BC Kit Module 1 96 wx To assign samples to spot
86. ears see Figure 83 Figure 83 Use the black box to change the focal distance Qu SOLID Imager 1 9 10 File View Pun Options Devices Took Windows Help Stage Status Pot Suh Mobon Pomon Speed Acceleston 2955100 100 100 2681200 100 100 Nudge Sze 10 Z Go Tox 26652 seo Go To Y 26808 v Pe Lait ie Stage Zoom In Fiowcel Go To Fiducial Mark fV Enable Joystick f ShowFlowCel 2 v Comes SM 266255 IP 127001 Pot 8081 Expone Time 23 9 Belg Berna S BaDepih 8 SauseRae o01 GoUve Shuster Status 7 When the black box is visible hold down the Ctrl key and hold down either the right or left arrow key on the keyboard Holding down Ctrl and the arrow key right or left arrow simultaneously changes the focal distance in 300 count intervals 8 While watching the live image on the screen scan the focal distance Use the Ctrl right arrow keys to scan the focal distance upward When the live image is in focus release the keys then record the value shown in the black box of the Imager window e Ifan in focus image cannot be found then scan downward using the Ctrl left arrow keys past the starting point while watching the live image on the screen When the live image is in focus release the keys then record the value shown in the black box of the Imager window The value in the black box of the imager window when the image is in focus is the nominal Z distance For examples of in focus im
87. eath or serious injury Except for IMPORTANTS the user attention words in user documentation appear with an open triangle figure that contains a hazard symbol These hazard symbols are identical to the hazard symbols that are affixed to the instrument See the Safety appendix for descriptions of the symbols Applied Biosystems SOLiD9 4 System Instrument Operation Guide 9 About This Guide User attention words 10 Applied Biosystems SOLiD9 4 System Instrument Operation Guide SOLiD 4 System run types Introduction On the Applied Biosystems SOLiD 4 System you can perform a workflow analysis WFA run and a sequencing run see Table 1 Table 1 Run types on the SOLiD9 4 System WFA Sequencing Purpose Assess various preparations of templated beads to determine the potential quality of sequence data Evaluate the fraction of P2 positive beads Useas a tool to determine the deposition density for sequencing slides Generate sequencing data for fragment or mate paired libraries Run summary e P1 and P2 bead counting Single ligation cycle Report generation Multiple ligation cycles for each of 5 primers resulting in up to 50 bases of sequence information per tag Estimated run time 4 5 hours e 4 14 days Deposition chamber 4 well e 1 well e 4 well 8 well Number of beads 15 million beads per well 708 million beads per well 1 well 128 million beads per wel
88. ected in the grid it can be addressed by the buttons in the toolbar below Images Data Free Space 3754G A Results Data Free Space ooo MERE IES Import Run R Export Run B Edit Selected Run vl Assion to Flowcell gt Import Mask Run Name Run Creator Status Type Mask Protocol Sam solid0062_20091217_PE_BC lab user Not Started Paired End BC 4 spot mask sf SOLID PE Mult solid0062 20091217 FRAG BC lab user Not Started Fragment BC 1 spot mask sf SOLID PE Mult 11 Please select a file to import the run definition Look in B My Documents My Recent Documents My Documents My Computer My Network Places d c E LabVIEW Data a My Music B My Pictures le Updater5 E selido062 20091217 PE BC txt File name Files of type Tab delimited data To assign a run previously saved to the database a Click on Manage Runs in the task pane b Click the run then select Assign to Flowcell c Choose a flowcell then click OK Manually find the focus range 108 You should first attempt automatic range detection see Chapter 3 Detect the focus range on page 49 If automatic detection fails then use the manual mode Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix B Supplemental Procedures B Manually find the focus range Select the flo
89. ee eee DRE NEED 169 Applied Biosystems SOLID 4 System Instrument Operation Guide About This Guide Note For general safety information see the Safety appendix in this document For important safety information related to Corvaris S2 system please refer to the user documentation of the product When a hazard symbol and hazard type appear by a chemical name or instrument hazard see the Safety appendix for the complete alert on the chemical or instrument CAUTION ABBREVIATED SAFETY ALERTS Hazard symbols and hazard types specified in procedures may be abbreviated in this document For the complete safety information see the Safety appendix in this document User attention words Five user attention words may appear in this document Each word implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product IMPORTANT Provides information that is necessary for proper instrument operation or accurate chemistry kit use CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury DANGER Indicates an imminently hazardous situation that if not avoided will result in d
90. el Figure 67 Do not shut down the power to the Linux head node and compute nodes e SOLiD 4 Analyzer idle for more than two weeks If the SOLiD 4 Analyzer will not be used for more than two weeks click ShutDown Figure 67 Close down the SOLiD Instrument Control Software and power off the instrument Applied Biosystems SOLiD9 4 System Instrument Operation Guide 101 Appendix B Supplemental Procedures Install the SOLIDO System Flowcell O ring Figure 67 Click Cancel if performing regular maintenance or ShutDown to power off the instrument gt Instrument Shutdown Wizard Shutdown Linux System Shutdown Linux System y To power off Linux head node and compute nodes press the Vindovs x ShutDown button Linux head node Wait 30 sec for the system to shutdown Verify whether the system is shutdown successfully by inspecting the light indicator on the front panel C npute node If the light indicator is still on please contact AB technical support Compute node Compute node Install the SOLID System Flowcell O ring After each run check the SOLiD System Flowcell O ring for cuts and abrasions If you see any abnormalities replace the O ring Inspect the O ring groove for debris or contamination Clean the O ring groove with water as needed Required Table 24 Required equipment Install the SOLiD9 Flowcell O ring equipment Item Source SOLiD9 System Flowcell O ring 10 pack Applied Biosystems
91. ems does not warrant that the operation of the instrument or its operating software will be uninterrupted or be error free Warranty period effective date Any applicable warranty period under these sections begins on the earlier of the date of installation or ninety 90 days from the date of shipment for hardware and software installed by Applied Biosystems personnel For all hardware and software installed by the buyer or anyone other than Applied Biosystems and for all other products the applicable warranty period begins the date the product is delivered to the buyer Warranty claims 144 Warranty claims must be made within the applicable warranty period or for chemicals or other consumable products within thirty 30 days after receipt by the buyer unless otherwise specified in the documentation accompanying the product Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix H Instrument Warranty Information Warranty exceptions Warranty exceptions The above warranties do not apply to defects resulting from misuse neglect or accident including without limitation operation with incompatible solvents or samples in the system operation outside of the environmental or use specifications or not in conformance with the instructions for the instrument system software or accessories improper or inadequate maintenance by the user installation of software or interfacing or use in combination with software or pro
92. er moistened with deionized water or a dilution of Extran 300 wash detergent in deionized water Applied Biosystems SOLIDO 4 System Instrument Operation Guide 103 Appendix B Supplemental Procedures Replace the SOLiD9 Light Source If you see Then Reagents in the holes of the of the reagent strip cover Wipe the inside surfaces of the holes with a cotton swab If the reagents are dried first wet the cotton swab as necessary with deionized water or a dilution of Extran 300 wash detergent in deionized water Large areas of dried reagents Wash the covers in a dilution of Extran wash detergent 10 mL Extran 300 in 1 L of deionized water If needed use a scrub brush Replace the SOLiD Light Source CAUTION PHYSICAL INJURY HAZARD Hot Surface Surface of the SOLID Light Source may be hot Use care when working around the SOLiD Light Source to avoid being burned Replace the SOLiD Light Source in the SOLiD 4 Analyzer every 1500 hours Required Table 28 Replace the SOLIDO Light Source equipment Item Source SOLIDO Light Source Applied Biosystems 4388441 1 Unscrew the 4 screws retaining the light box access cover on the top of the instrument 2 Remove the cover to the light source from the housing 3 Pull the light source straight up and out of the unit 4 Slide anew SOLiD Light Source into place Ensure that the light source is oriented in the correct direction see Figure 69 5
93. er 66 109 82 142 107 191 Barcode Table 34 Fill volumes for barcode sequencing 5 or 10 bp BC Tag Volume mL 5 bp 10 bp 1 Flowcell 2 Flowcells 1 Flowcell 2 Flowcells Instrument Buffer 638 1088 883 1577 Storage Buffer 189 279 227 356 Cleave 1 Solution 32 41 47 72 Cleave 2 1 Solution 32 41 47 72 Reset Buffer 91 164 91 164 Ligase Buffer 15 19 18 25 Universal Buffer 13 14 15 19 Imaging Buffer 33 43 41 59 Workflow analysis WFA Table 35 Fill volumes for workflow analysis WFA Volume mL 1 Flowcell 2 Flowcells Instrument Buffer 294 400 Storage Buffer 116 134 Cleave 1 Solution 0 0 Cleave 2 1 Solution 0 0 Applied Biosystems SOLID 4 System Instrument Operation Guide 123 E Appendix C On Instrument Reagent Volumes and Reagent Strip Layouts Reagent strip layouts Volume mL 1 Flowcell 2 Flowcells Reset Buffer 43 67 Ligase Buffer 12 12 Universal Buffer 0 0 Imaging Buffer 26 30 Reagent strip layouts Fragment paired end sequencing F3 Tag m SOLIDO Mixing Strip Tube with Zinc Ligase l ProbeA Probe B G ee Ligase Phosphate PA vo J ProbeA Probe B x d Tag x lt I Lib F3 Tag Ligase Probe A Probe B y C d 4 Lib F3 Tag Ligase Probe B z B ME a Lib F3 Tag Ligase Phosph Probe A Probe B Primer A 124 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Ap
94. everse Reverse BC Frag Lib F5 BC Enzyme Primer B Probe A Probe B A Tag Primer B Capper F5 BC Reverse Reverse Enzyme Primer A Probe A Probe B a do E Multiplex fragment paired end sequencing BC Tag BC Frag Lib BC Tag Bridge Probes BC Primer E BC Primer C BC Primer D BC Frag Lib BC Tag Primers BC Frag Lib BC Tag Probes BC BC Primer A Primer B O Probe A a BS S Applied Biosystems SOLiD9 4 System Instrument Operation Guide 127 C Appendix C On Instrument Reagent Volumes and Reagent Strip Layouts Reagent strip layouts Workflow analysis WFA a B E Ligase P L A Probe A noon afro Fida rimer y ap nalysis Reagents 7 I EA 128 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Instrument Process Times This appendix covers Times for individual processes rr 129 Times for entire processes cles ee 130 Times for individual processes Table 36 Times for individual processes Process Estimated Time h Steps rcs m Focal Map 2 3 Reset P2 Label Image Sequencing Primers 3 4 Reset 3 4 1 amp 2 and Ligation F3 R3 Prime Leste tal Ligate 15 17 Dark Ligate 5 cycles or 25 bp Phosphatase 18 22 7 cycles or 35 bp Image 28 33 lese 10 cycles or 50 bp Sequencing Primers 4 5 Reset 4 5 ae and Ligation Prime 1 cycle or 5 bp Bridge Probe 16 18 Ligate 5 cycles or 25 bp Dark Ligate 19 23
95. everse Reverse Frag Lib F5 P2 Tag 9 Enzyme Primer D Probe A Probe B l Primer D Ligase Capper Reverse Reverse Frag Lib F5 P2 Tag 9 Enzyme Probe A L Probe B y Primer C Ligase Capper F5 P2 Reverse Reverse Frag Lib F5 P2 Tag 9 Enzyme J PrimerB Probe A Probe B Primer B Ligase Capper F5 P2 Reverse Reverse 9 Enzyme Primer A Probe A Probe B a an A Multiplex paired end sequencing F5 BC Tag Ligase Capper F5 BC Reverse Reverse Reverse s Enzyme Primer E Probe A Probe B Phosphatase Ligase Capper F5 BC Reverse Reverse Reverse BC Frag Lib F5 BC 9 Enzyme PrimerD Probe A Probe B Phosphatase Tag Primer D Lise Capper F5 BC Reverse Reverse Reverse BC Frag Lib F5 BC ges Enzyme Primer C Probe A Probe B Phosphatase Tag Primer C Liaase Capper F5 BC Reverse Reverse Reverse BC Frag Lib F5 BC 9 Enzyme Primer B Probe A Probe B Phosphatase Tag Primer B anza Capper F5 BC Reverse Reverse Reverse BC Frag Lib F5 BC 9 Enzyme Primer A Probe A Probe B Phosphatase Tag Primer A 126 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix C On Instrument Reagent Volumes and Reagent Strip Layouts C Reagent strip layouts Capper F5 BC Reverse Reverse Enzyme Primer E Probe A Probe B A i Capper F5 BC Reverse Reverse BC Frag Lib F5 BC Enzyme Primer D Probe A Probe B jT Tag Primer D Capper F5 BC Reverse Reverse BC Frag Lib F5 BC Enzyme Primer C Probe A Probe B V ii Tag Primer C Capper F5 BC R
96. ffer T Slide Storage Buffer PT F3 Tag SegueneimgKit PT CET ooo F8 PimerB OO LLL LL L E L T L L A AAN AAN AAA i OOOO A pT R3 F5 P2 F5 BC Tag Sequencing Kit R3 F5 P2 F5 BC Mixing Strip Tube 140 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Operation notes Fill the tank Degas the water Set the chiller Perform required maintenance of the Covaris S2 System Applied Biosystems SOLIDO 4 The Covaris S2 System This appendix covers Operation Motes este tale eet did 141 A teet eve eM cenae tue UE 141 Degas the Waters isses are ree dime heri vaccinia e ues Rhe ding ie dia 141 Set the chiller oe LEE DU ELO PU EPA T IET ELAREELAPRELATE 141 Perform required maintenance of the Covaris S2 System 5 141 Covaris S2 Programs e Ld vendu i pa paella 142 Covalent Declump 1 0 0 0 e 142 Covalent Declump Fisica cid pe e bid EP ass 142 Fill the tank with fresh deionized water to the proper fill line The water should cover the visible part of the tube Degas the water for 30 minutes To maintain degassed water keep the pump continuously on during operation and sample processing Set the chiller temperature to between 2 5 C to ensure that the temperature reading in the water bath displays 5 C The circulated water chiller should be supplemented with 20 ethylene glycol The Covaris S2 System requires regular maintenance to work properly Perform the tasks i
97. fully pipet a sample of templated beads into the well through the porthole As the area of the well fills lower the top of the SOLiD Deposition Chamber so that it becomes level Proceed to step 9 With the SOLiD Deposition Chamber flat carefully pipet a sample of templated beads into one of the 4 or 8 wells through the porthole Repeat step 7 to fill each of the remaining wells with each sample of templated beads Note each sample s well position relative to the slide orientation Place 3 mm adhesive disks over all the portholes in the SOLiD Deposition Chamber Centrifuge the slide in the SOLiD Deposition Chamber at 167 x g for 10 minutes in a plate centrifuge Applied Biosystems SOLiD9 4 System Instrument Operation Guide 11 12 13 Chapter 2 Prepare and Install Slides and Reagents Install on instrument reagents Incubate the SOLiD Deposition Chamber at 37 C for 1 hour After incubation centrifuge the slide in the SOLIDO Deposition Chamber at 167 x g for 10 minutes in a plate centrifuge For high density depositions wash the slides with SOLiD XD Slide Deposition Buffer v2 or SOLiD XD Slide Storage Buffer to gently remove unbound beads prior to instrument loading Install on instrument reagents Note For information about recommended fill volumes for on instrument reagents see Appendix C Recommended fill volumes for on instrument reagents on page 121 Pre pare Note Prepare buffers just prior t
98. g Lib Seq F5 BC Tag Primer B Master Mix 35 SOLiD ToP Frag Lib Seq F5 BC Tag Primer C Master Mix 35 SOLIDO ToP Frag Lib Seq F5 BC Tag Primer D Master Mix 35 SOLIDO ToP Frag Lib Seq F5 BC Tag Primer E Master Mix 35 SOLIDO Mixing Strip Tube Paired end sequencing of RNA libraries up to 50 bases for the forward read and 35 bases for the reverse read Applied Biosystems SOLiD9 4 System Instrument Operation Guide 87 A Appendix A Applied Biosystems SOLiD9 4 System Instrument Operation Guide Required Materials Set up and perform a sequencing run Item part Component Kit component s number used in SOLIDO ToP Mate SOLIDO ToP Sequencing Kit M P Lib F3 Tag MM35 Mate pair Paired Sequencing Kit M P Lib MM35 35 4452684 SOLiD9 ToP MP Lib Seq F3 Tag Primer A Master Mix 35 SOLiD ToP MP Lib Seq F3 Tag Primer B Master Mix 35 SOLiD9 ToP MP Lib Seq F3 Tag Primer C Master Mix 35 SOLiD9 ToP MP Lib Seq F3 Tag Primer D Master Mix 35 SOLiD9 ToP MP Lib Seq F3 Tag Primer E Master Mix 35 SOLiD Mixing Strip Tube with Zinc SOLIDO ToP Sequencing Kit M P Lib R3 Tag MM35 SOLIDO ToP MP Lib Seq R3 Tag Primer A Master Mix 35 SOLiD9 ToP MP Lib Seq R3 Tag Primer B Master Mix 35 SOLiD ToP MP Lib Seq R3 Tag Primer C Master Mix 35 SOLiD9 ToP MP Lib Seq R3 Tag Primer D Master Mix 35 SOLIDO ToP MP Lib Seq R3 Tag Primer E Master Mix 35 SOLIDO Mixing Strip Tube with Zinc sequencing up
99. gaging the alignment key on the carrier with the corresponding part on the flowcell c Slide the two slide carrier lock down tabs on the flowcell inward until they are positioned over and flush with the carrier see Figure 18 IMPORTANT Ensure that the tabs are flush with the carrier If necessary loosen the Allen screws further then slide the tabs over the slide carrier d To properly seat the carrier on the flowcell gradually tighten the 2 Allen screws on both lock down tabs in an alternating fashion to 20 inch pounds e Rotate the flowcell up and lock it into the scan position Applied Biosystems SOLiD9 4 System Instrument Operation Guide 9 Close the instrument do 10 Click the Load Flowcell Figure 18 Slide carrier lockdown tabs Install slide s on the instrument Chapter2 Prepare and Install Slides and Reagents Slide carrier lock down tab Slide carrier lock down tab OrS s button located at the bottom of the flowcell panel Each flowcell has its own Load Flowcells button see Figure 19 Figure 19 Click Load Flowcells to load the flowcell with buffer ocol un Defined Run Control E Load Flowcell y 14 Clear Flowcell Protocol No Run Defined Y Run Control E s Load Flowcell g Clear Flowcell Applied Biosystems SOLiD9 4 System Instrument Operation Guide 35 Chapter 2 Prepare and Install Slides and Reagents Install reagent strip s on the in
100. gent strip s on the instrument 6 Place the cover over the reagent strips and secure them using the cover fasteners see Figure 27 Note Ensure that the top and bottom of the cover is free of splattered wet or dry reagents see Appendix B Clean the reagent strip cover on page 103 Figure 27 Securely fastened reagent strip covers m Read SDS For Research Use Only M Cover fastener Cover fastener 42 Applied Biosystems SOLID 4 System Instrument Operation Guide Set Up Control and Monitor the Run This chapter covers Section 3 1 Set up and perform a workflow analysis WFA run 44 m Materials and equipment required 6 c cece ee eee 44 B WOrktlOW ess ica a THAT aT dt es ee Lo eg re Ris 44 B Create a WFA run record escorias A is Da 46 B Detect the focus range nia la a 49 B Start the WEA DADA Ra R ei SEA eb acte arte ee rat N 50 B Monitor the WEA TOi rr mre aae EEEo E E hh hh hr 52 B View th WEA Teport sese o Ue pee PERSE 53 Section 3 2 Set up and perform a sequencing run o ooooooooococcmmmoo o 56 m Materials and equipment required 0 ees 56 W WO rk flO Wiest Ta RTT Z TN A AS aad etie 56 m Create a sequencing non multiplex run record o 58 m Create a multiplex sequencing run record 6 eee 61 mM Detect the focus range clle 69 B Start the sequencing run 6 eee eens 70 B Control the tun ss eese eee wea ack wea eve trace eee eos 73 B Monitor t
101. ger can focus again this time with the correct exposure settings 6 When the Imager displays READY click the Acquire button then confirm that the image is in focus 7 Record the Z distance value from the Focus Status pane see Figure 87 116 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix B Supplemental Procedures B Manually find the focus range Figure 87 Record the Z distance value Qu SOLID Imager 1 9 10 F e View Run Options Devices Took Windows Help 0 CAMERA info e v BENE ren ba pp P Stage Status Exposue Tee 123 2 Gan 78 gt perea D gt Pot Sims Motion Postion Speed Acceleestion BL Deri 8 27 Spsg fate 001 Golwe 2655100 100 100 Shuster Status Focus Status 2681200 0 100 Nudge Sie 10 Z Re Calbvate Stage GoToX EN asi Go Toy Zoom beat Y aa E E Go To Fiducial Mak 8 Open Notepad then record the measured focal value for the spot on the slide that has been focussed 9 Repeat steps 4 8 for the top right bottom left bottom right and center of the slide The range of these values should not exceed 5000 units Calculate the average value of the highest and lowest spots and record this value in Notepad see Figure 88 IMPORTANT If the range of these values exceeds 5000 units the flowcell is out of alignment Call your Applied Biosystems Field Service Engineer to align the flowcell Applied Biosystems SOLiD9 4 System Instrument Operation
102. he SOLiD Slide Storage Chamber then fill with 5 mL Slide Storage Buffer Store the slide at 4 C until the slide is ready for use see Figure 15 Figure 15 SOLiD Slide Storage Chamber IMPORTANT Before removing a slide from a previous run ensure that the run images and data collected from the previous run are satisfactory For more information refer to the Applied Biosystems SOLiD 4 System SETS Software User Guide PN 4448411 1 Check to see if a SOLiD 4 or Opti Slide Carrier assembly from a previous run is present in the flowcell chamber If a SOLiD 4 or Opti Slide Carrier assembly is present in the flowcell chamber proceed with steps 2 to 4 If a SOLiD 4 or Opti Slide Carrier assembly is not present in the flowcell chamber skip to step 5 For each flowcell to be used click the Clear Flowcell button at the bottom of the flowcell panel see Figure 16 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Hide samples lt lt Protocol Chapter 2 Prepare and Install Slides and Reagents Install slide s on the instrument Figure 16 Click Clear Flowcell to flush the contents from the flowcell Hide samples Protocol No Run Defined Ls Run control Dj No Run Defined y Run Control 8 K Start Re K Start Rur IT ocked E C 4 Clear Flowicell y 14 Clear Flowcell AA Doors inline Fluidics is Online SoM of 297m
103. he TU ia Se dhs a Saha reed 77 Applied Biosystems SOLiD9 4 System Instrument Operation Guide 43 Chapter 3 Set Up Control and Monitor the Run Materials and equipment required Section 3 1 Set up and perform a workflow analysis WFA run Materials and equipment required See Appendix A on page 83 for a list of equipment kits and consumables necessary to setup a workflow analysis WFA run Workflow Monitor the run View the run log View the heat maps View cycle scans View the WFA report Generate the WFA report Determine the optimal titration point Determine the bead deposition density for a sequencing run Workflow overview Create a WFA run record A WEA run record is created using the SOLiD Instrument Control Software ICS Detect the focus range Two methods exist for determining the focus range automatic and manual You should first attempt automatic range detection If automatic detection fails use the manual mode 44 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Section 3 1 Set up and perform a workflow analysis WFA run 3 Workflow Start the WFA run Start the run by using the SOLiD Instrument Control Software Before starting a run you may need to remove previous data may need to ensure maximum disk space for new results Monitor the run While executing a WFA run the instrument performs a Prescan In a Prescan all of the beads are labeled and their positions on the slide
104. he focus range manually see Manually find the focus range on page 108 1 Close the front doors of the SOLiD 4 Analyzer Open the Imager window by choosing Windows Imaging System 2 Select Tools Detect Focusing Range 3 In the focusing range dialog specify the stage template file by entering the name of the stage template file directly or by clicking the Browse button to navigate to a suitable stage template file see Figure 31 The stage template must match the slide in the target flowcell s If you created a run from the ICS then select the file imagingMap STG from the subdirectory in C Runs whose name matches the name of the run for example select C Runs Solid0327_20081209_2_Oct_Test imagingMap STG Figure 31 Detect Focusing Range dialog UM Detect Focusing Range Select Stage Template Browse Please select flowcelt 2 lt Go Cancel P 4 Select the flowcell using the drop down menu then press Go The Imager works for several minutes while it determines the range The blue progress bar indicates how close it is to completion see Figure 32 You can also click Cancel so that the Imager aborts the ranging operation Figure 32 Detect Focusing Range Dialog while detection in progress UM Detect Focusing Range Select Stage Template C Runs solid0327 20081204 1x15 S3Quad 1 imagingMap STG So aed Searching focusing range at 22218 24504 Please select flowcell
105. he information in the run record using a separate spreadsheet program see Set up a run by importing a Run Definition file on page 105 Choose to assign the run to a flowcell for immediate use or to an instrument database to store for later use then click OK To assign a run previously saved to the database a Click on Manage Runs in the task pane b Click the run then select Assign to Flowcell c Choose a flowcell then click OK Applied Biosystems SOLID 4 System Instrument Operation Guide Section 3 2 Set up and perform a sequencing run Detect the focus range Detect the focus range Note If automatic focus range detection fails determine the focus range manually see Manually find the focus range on page 108 1 Close the front doors of the SOLiD 4 Analyzer Open the Imager window by choosing Windows Imaging System Select Tools Detect Focusing Range In the focusing range dialog specify the stage template file by entering the name directly or by clicking the Browse button to navigate to a suitable one see Figure 47 The stage template must match the slide in the target flowcell s If you created a run from the ICS then select the file imagingMap STG from the subdirectory in C Y Runs whose name matches the name of the run for example in Figure 48 C Runs VSolid0327 20081209 2 Oct TestVimagingMap STG is selected Figure 47 Detect Focusing Range dialog UM Detect Focusing Range Se
106. he manufacturer s cleanup procedures as recommended in the SDS Handle chemical wastes in a fume hood Ensure use of primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage After emptying a waste container seal it with the cap provided Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply WARNING HAZARDOUS WASTE from instruments Waste produced by the instrument is potentially hazardous Follow the guidelines noted in the preceding General Chemical Handling warning WARNING 4L Reagent and Waste Bottle Safety Four liter reagent and waste bottles can crack and leak Each 4 liter bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Biological hazard safety A WARNING Potential Biohazard Depending on the samples used on this instrument the surface ma
107. he orientation key is in the lower left position when the slide carrier assembly is placed on the instrument This orientation must be maintained in order to preserve the correct sample order when the 4 Well or 8 Well SOLiD Deposition Chamber is used Applied Biosystems SOLiD9 4 System Instrument Operation Guide 23 2 Chapter 2 Prepare and Install Slides and Reagents Deposit the beads on the slides Deposit the beads 1 24 Figure 8 SOLIDO Deposition Chambers 1 Well 4 Well and 8 Well Screw tab Tighten the four screw tabs on the SOLiD Deposition Chamber in a crisscross pattern until the lid is securely attached Twist the tabs flat Sonicate the beads using the Covalent Declump 3 program on the Covaris S2 System for program conditions see Appendix G Covalent Declump 3 on page 142 Afterward pulse spin but do not pellet the beads Repeat step 1 Use a pipettor with an appropriate tip to pipet the bead solution up and down a few times then withdraw the sample of templated beads from the microcentrifuge tube IMPORTANT Samples must be deposited onto the slide immediately after sonication to minimize clumping and maximize monolayering Perform one of the following sequences If the SOLiD9 Deposition Chamber has Then perform steps 1 well 5 6 and 9 to 11 4 or 8 wells 7 to 11 Elevate and tilt the SOLiD Deposition Chamber with the entry porthole of the well at the lowest point Care
108. he sequencing run 4 After the run has been initiated you can click the Run Log and Heat Map buttons located at the top of the appropriate flowcell panel to learn more information about the current run see Figure 52 You can also select the appropriate flowcell in the Cycle Scans menu on the task bar on the left Figure 52 How to learn more about the current run solid0419 iD 4 0 User lab_user F cols wW Welcome lab_user Flow Cell 1 Running E Run Name A solid0419 1 F7wt duds Created by lab user Created by Al isign Run fa Edit Run Z ciek aul i M f Assign Run Edit R ne Clear B Run Logs A Hee Sample Slide Sample Slide Create Runs 1 Sample in 1 spot mask sf El Flow Cell Details E Details A Ig Cycle Scan lia EET Ex Description primaryAnalysisSetting default primary E Libraries Secondary ia none Numbers 1 E E Sample Loading Protocol b SOLiD4 F5 BC F3 25 50 bases Protocol MEM ccce F560 mmmmm NEHEHH HENHEHNH NEHH 335 F3 F5 BC PrimerD Ligation 5 Prime Buffer Line Chiller 4 0 C Cooling Y Lamp 689 hrs On 5 Executing Scan Slide Filter 3 of 4 Panel 1503 of 2358 y RunControl_f E Pre Scan Primers A B C D and E of F5 BC No Run Defined IMPORTANT Do not disturb the SOLiD 4 Analyzer while in operation and do not open the flowcell during a pause in the run Significant
109. icroTube e Covaris microTube Prep Station e Covaris Water Tank Label Kit e Covaris microTubes 1 pack of 25 For system materials summary refer to Covaris S2 System Materials Summary Applied Biosystems SOLIDO 4 System Site Preparation Guide Applied Biosystems PN 4387833 110 V Applied Biosystems PN 4392718 220 V or Covaris 6 Tube Magnetic Stand Applied Biosystems AM10055 Microcentrifuge 5417R refrigerated without rotor Eppendorf 022621807 120 V 60 Hz Eppendorf 022621840 230 V 50 Hz FA 45 24 11 fixed angle rotor 24 x 1 5 2 mL including aluminum lid aerosol tight Eppendorf 022636006 NanoDrop ND 1000 Spectrophotometer computer required Thermo Scientific ND 1000 Tabletop Centrifuge for 96 well plate Major Laboratory Supplier MLS Vortexer MLS Picofuge MLS Magnetic stirrer MLS Refrigerator 4 C MLS Freezer 20 C MLS Pipettors 20 uL MLS Pipettors 200 uL MLS Pipettors 1000 uL MLS 92 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix A Applied Biosystems SOLIDO 4 System Instrument Operation Guide Required Materials Set up and perform a sequencing run t Applied Biosystems has validated this protocol using this specific material Substitution may adversely affect system performance i The SOLIDO 4 Analyzer is shipped with 2 of each size SOLiD Deposition Chambers and SOLIDO 4 Slide Carriers Two SOLIDO Slide
110. ides brief step by step procedures for loading SOLIDO 4 System and running the SOLiD9 4 System Instrument Operation Quick Reference Card Applied Biosystems 4448639 Provides all the information that you need to set up SOLIDO 4 System Site the SOLiD9 4 System Preparation Guide Applied Biosystems 4448411 Provides an alternate platform to monitor runs SOLIDO 4 System SETS modify settings and reanalyze previous runs that Software User Guide are performed on the SOLIDO 4 System Applied Biosystems Describes the software and provides procedures SOLIDO 4 System ICS for common tasks see the Instrument Control Software Help Software BioScope Software for 4448431 Provides a bioinformatics analysis framework for Scientists Guide flexible application analysis data generated mapping SNPs count reads from sequencing runs Applied Biosystems SOLiD9 4 System Instrument Operation Guide 161 Documentation and Support Obtaining SDSs Obtaining SDSs 162 Document poe Description number Working with 4452359 Provides an online suite of software tools for Next SOLiDBioScope com Generation Sequencing NGS analysis Quick Reference Card SOLIDBioScope com leverages the scalable resources of cloud computing to perform computation intensive NGS data processing Applied Biosystems 4448432 Describes the relationship between the software SOLIDO 4 System comprising the SOLID 4 platform and provides Softwa
111. ides for this step Click Lock Doors Click Run Script to begin instrument flush Note The script takes approximately 13 minutes to complete After the script has completed click Next In theSystem Wash screen click Unlock Doors and remove all of the bottles from the side of the instrument the chiller block and the cabinet You will now perform a dry flush of the system Figure 66 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix B Supplemental Procedures Flush the fluidic lines Figure 66 Complete the actions in the second System Wash screen Instrument Shutdown Wizard System wash Follow the steps to run the systematic wash scripts System Wash The following steps guide the user in preparing the SOLID instrument for long term shutdown by systematic wash steps Run shutdown script with air 1 Unlock and open the doors Doors UnLocked Successfully Remove all the bottles from the side of the instrument the chiller block and the cabinet Lock the doors Doors Locked Successfully Re run the script to run air through the lines 10 To begin the dry instrument flush click Run Script Note This script takes approximately 13 minutes to complete 11 Perform one of the following SOLID 4 Analyzer in continuous use If the SOLiD 4 Analyzer is in continuous use and you are performing this flush every three months as required for preventive maintenance click Canc
112. ims and returns e eee eee 146 Computer configuration Applied Biosystems supplies or recommends certain configurations of computer hardware software and peripherals for use with its instrumentation Applied Biosystems reserves the right to decline support for or impose extra charges for supporting nonstandard computer configurations or components that have not been supplied or recommended by Applied Biosystems Applied Biosystems also reserves the right to require that computer hardware and software be restored to the standard configuration prior to providing service or technical support For systems that have built in computers or processing units installing unauthorized hardware or software may void the Warranty or Service Plan Limited product warranty Applied Biosystems warrants that all standard components of the SOLiD 4 Analyzer IKA9 ULTRA TURRAX Tube Drive the Covaris S2 System APC UPS and the recirculating chiller will be free of defects in materials and workmanship for a period of one 1 year from the date the warranty period begins Applied Biosystems will repair or replace at its discretion all defective components during this warranty period Applied Biosystems warrants the Genomic Solutions HydroShear DNA Shearing Device will be free of defects in materials and workmanship for a period of one 1 year from the date the warranty period begins Applied Biosystems will replace a defective HydroShear DNA Shearing Devic
113. ired End O WFA 2 Ix Multiplexing Identify the run and enter a description Run Name solid0054_20100203_PE_BC Description Assign a Primer Set and of bases Primers run in sequential order Select Run Protocol SOLID Y Barcode and Sequencing with 5 primer wal Primer Set 1 10 Bases wv Primer Set 2 25 Bases v Primer Set 3 v 4 Complete the information in the Specify Samples and Analysis pane see Figure 43 on page 65 a Select the number of samples that will be used in the run A sample consists of a bead sample that may contain many barcoded libraries A single sample that will run in multiple spots on the same slide counts as only one sample otherwise the number of samples typically matches the number of spots on the mask b Click the first sample name to select it Change the sample name if desired After you select the sample you are able to enter the names primary analysis and description of libraries that are in the sample 64 Applied Biosystems SOLID 4 System Instrument Operation Guide Section 3 2 Set up and perform a sequencing run Create a multiplex sequencing run record Figure 43 Complete the information in the Specify Samples and Analysis pane SETUP gt Create Edit Run gt Specify Samples and Analysis Select how many samples you need for this run For Application Primary and Secondary Analysis columns in a sequencing or multiplexi
114. is 2 series Machine Holder for one 0 65 mL microcentrifuge tube e Covaris 2 series Machine Holder for one 13 mm x 65 mm tube e Covaris 2 Series Machine Holder for one microTUBE e Covaris microTUBE Prep Station e Covaris Water Tank Label Kit e Covaris microTubes 1 pack of 25 For system materials summary refer to Covaris S2 System Materials Summary Applied Biosystems SOLIDO 4 System Site Preparation Guide Applied Biosystems PN 4387833 110 V Applied Biosystems PN 4392718 220 V or Covaris 6 Tube Magnetic Stand Applied Biosystems AM10055 Microcentrifuge 5417R refrigerated without Eppendorf Ino 022621807 120 V 60 Hz Eppendorf 022621840 230 V 50 Hz FA 45 24 11 fixed angle rotor Eppendorf 24 x 1 5 2 mL including aluminum lid 022636006 aerosol tight NanoDrop ND 1000 Spectrophotometer computer required Thermo Scientific ND 1000 Tabletop Centrifuge for 6 well plate Major Laboratory Supplier MLS Vortexer MLS Picofuge MLS Magnetic stirrer MLS Refrigerator 4 C MLS Freezer 20 C MLS Pipettors 20 uL MLS Pipettors 200 uL MLS Pipettors 1000 uL MLS Applied Biosystems SOLiD9 4 System Instrument Operation Guide 85 A Appendix A Applied Biosystems SOLiD9 4 System Instrument Operation Guide Required Materials Set up and perform a sequencing run Required consumables system performance Applied Biosystems ha
115. it over Limit under Limit Close Close View cycle scans 1 Select the appropriate flowcell Flow Cell 1 or Flow Cell 2 in the Cycle Scan menu on the task bar on the left The top section of the Cycle Scans window lists all the scans per ligation cycle for the slide and the bottom section shows scan information sorted by sample for the scan selected in the top section Use the parameters shown in Figure 59 to assess the progress of the sequencing run see Table 13 78 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Section 3 2 Set up and perform a sequencing run Monitor the run Figure 59 Parameters available in the Cycle Scans window to assess the progress of the sequencing run 1 Failed panels 2 Best Good Beads usable beads 3a 3b Effective exposure times 4 Satay plots lab_user lizar YN ada Cycle Scans gt Flow Cell 1 solido045_20100119 PE BC108 10 The Cycle Scan Reporting Panel presents scan data from the current run in tabular format Click on any scan to display sample details in the bottom panel To view used scans exclusively checl control labeled Show Used Scans Only below The format of bead counts and value bar scaling can be adjusted via the controls labeled Display Bead Counts and Display Value Bar Scaling i ang used ShowUsed Scans Only Display Bead Counts as Average Beads Per Panel x Display Value Bar Scaling as Fixed v ES Expo
116. it 1 96 Part no 4449637 or 48 barcodes in the SOLID RNA Barcode Modules Multiplexing Series A 10 barcodes that are in the SOLIDO Small RNA Expression Kit 1 10 t For the latest information regarding Multiplexing Series and barcode kits contact your Field Applications Specialist 66 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Section 3 2 Set up and perform a sequencing run Create a multiplex sequencing run record Multiplexing Series Contents Multiplexing Series B 20 barcodes that are supplied with the SOLIDO 3 System or 1 20 16 barcodes supplied with the SOLIDO 3 Plus System Use the Multiplexing Series B 1 20 option with the SOLIDO Transcriptome Multiplex Kit which contains up to 16 barcodes 7 Click the selection box for the barcode or barcodes assigned to the library 8 Click Save when you have completed the information for the first library 9 Optional Repeat step 5 to step 7 for all remaining libraries The Libraries panel of the Specify Samples and Analysis screen now shows information for all libraries for a particular sample see Figure 45 Figure 45 The Libraries panel shows information for all libraries File Welcome lab_user SETUP gt Create Edit Run gt Specify Samples and Analysis Select how many samples you need for this run For Application Primary and Secondary Analysis columns in a sequencing or multiplexing run you can click the column h
117. it Module 1 16 7 bcSample2 2Barcode 1MM Lib8 BC Kit Module 1 16 8 bcSample2 2Barcode 1MM Lib9 BC Kit Module 1 16 9 bcSample2 2Barcode 1MM Lib10 BC Kit Module 1 16 10 bcSample2 2Barcode 1MM Lib11 BC Kit Module 1 16 11 bcSample2 2Barcode 1MM Lib12 BC Kit Module 1 16 12 bcSample2 2Barcode 1MM Lib13 BC Kit Module 1 16 13 bcSample2 2Barcode 1MM Lib14 BC Kit Module 1 16 14 bcSample2 2Barcode 1MM Lib15 BC Kit Module 1 16 15 bcSample2 2Barcode 1MM Lib16 BC Kit Module 1 16 16 bcSample3 3 Barcode 1MM Lib1 BC Kit Module 1 16 1 IMPORTANT Be sure to review each sample s library and barcode assignment before starting a run After a run starts you cannot change the library or barcode assignment Applied Biosystems SOLIDO 4 System Instrument Operation Guide 107 B Appendix B Supplemental Procedures Manually find the focus range amp Create Runs Manage Runs El Flow Cell Details imr Cycle Scan System Status Prime Buffer Line Save the file by selecting File Save Do not select Save As because scalc does not allow files to be saved as txt files Click Keep Current Format In ICS click Manage Runs Click Import Run and select the run definition file you modified with scalc see Figure 76 Figure 76 Import the run definition file Setup gt Manage Runs The SOLID Run Manager provides a summary view of recent run activity and allows operators to load masks import and export sample sheets as well sel
118. itional information about biohazard guidelines is available at www cdc gov In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO Laboratory Biosafety Manual third edition found at www who int csr resources publications biosafety WHO_CDS_CSR_LYO_2004_11 en Applied Biosystems SOLiD9 4 System Instrument Operation Guide 159 J Appendix J Safety Biological hazard safety 160 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Documentation and Support Related documentation The following related documents are shipped with the system Part Document Description number Applied Biosystems 4445673 Provides procedures for preparing libraries SOLIDO 4 System Library Preparation Guide Applied Biosystems 4445674 Provides brief step by step procedures for SOLIDO 4 System Library preparing libraries Preparation Quick Reference Card Applied Biosystems 4448378 Describes how to prepare templated beads by SOLIDO 4 System emulsion PCR ePCR required before sequencing Templated Bead on the SOLIDO 4 System Preparation Guide Applied Biosystems 4448329 Provides brief step by step procedures for SOLIDO 4 System preparing templated beads by emulsion PCR Templated Bead ePCR required before sequencing on the SOLIDO Preparation Quick 4 System Reference Card Applied Biosystems 4448380 Prov
119. its The following table lists the required Applied Biosystems reagent kits for sequencing run For part numbers of individual boxes see Figure 23 on page 40 Table 17 Required Applied Biosystems reagent kits Sequencing run Item part Gampanent Kit componentls number used in SOLiD9 ToP SOLiD ToP Frag Lib Seq F3 Tag Primer A Master Mix 35 Fragment Sequencing Kit ne l sequencing up to SOLID ToP Frag Lib Seq F3 Tag P B Master Mix 35 Frag Lib F3 Tag L a oles 35 bases MM35 4449352 SOLIDO ToP Frag Lib Seq F3 Tag Primer C Master Mix 35 SOLiD ToP Frag Lib Seq F3 Tag Primer D Master Mix 35 SOLiD ToP Frag Lib Seq F3 Tag Primer E Master Mix 35 SOLIDO Mixing Strip Tube with Zinc 86 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix A Applied Biosystems SOLIDO 4 System Instrument Operation Guide Required Materials A Set up and perform a sequencing run Item part C Kit componentls number used in SOLiD9 ToP SOLIDO ToP Frag Lib Seq F3 Tag Primer A Master Mix 50 Fragment xd E SOLIDO ToP Frag Lib Seq F3 Tag Primer B Master Mix 50 P e TIE MM50 4449388 SOLiD ToP Frag Lib Seq F3 Tag Primer C Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer D Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer E Master Mix 50 SOLIDO Mixing Strip Tube with Zinc SOLiD ToP Paired SOLID ToP Sequencing Kit Frag Lib F3 Tag MM50 Paired end End Seque
120. izard by choosing Wizards gt Instrument Shutdown Figure 64 Figure 64 Open Instrument Shutdown Wizard 2 In the Wizard s introduction screen click Next 3 In the System Wash screen click Unlock Doors Figure 65 Applied Biosystems SOLiD9 4 System Instrument Operation Guide 99 100 Appendix B Supplemental Procedures Flush the fluidic lines Figure 65 Complete the actions in the first System Wash screen Instrument Shutdown Wizard System Wash Ir System Wash The following steps guide the user in preparing the SOLID instrument for long term shutdown by systematic wash steps Run shutdown script with DI water 1 Unlock and open the doors Fil the Cleave1 Cleave2 Reset bottles with at least 30mL of DI H20 Fill the Ligase Buffer Phosphatase Buffer Imaging Buffer bottles with at least 30mL of DI H20 Fill the Instrument Buffer and the Storage Buffer bottles with 250mL of DI H20 Load both flowcells with blank slides and carriers Lock the doors Run shutdown script Replace all of the bottles from the side of the instrument the chiller block and the cabinet with rinsed out bottles All bottles except the Instrument Buffer and Storage Buffer bottles should contain at least 30 mL of deionized water The Instrument Buffer and Storage Buffer bottles These two bottles require 250 mL Loada slide onto each of the flowcells You can load used slides or 1x 3 inch standard microscope sl
121. l 4 well 56 million beads per well 8 well t One tag for fragment sequencing and two tags for mate pair sequencing and paired end sequencing The reverse read tag in paired end sequencing is currently limited to 25 bases Only 5 or 10 bases of the barcode tag need to be sequenced t Total run time for dual slide run Run time varies with read length and type of sequencing run Times may deviate depending on imaging time Applied Biosystems SOLiD9 4 System Instrument Operation Guide 11 1 Chapter 1 Introduction SOLIDO 4 System run types Use Figure 1 to choose the run type that most closely meets your sequencing needs Figure 1 Relationship between the workflow analysis WFA and sequencing runs on the SOLIDO System Yes Templated bead preparation at cr Templated bead preparation at two library concentrations Quantitative PCR qPCR optimal library concentration Workflow analysis WFA run Sequencing run on on SOLIDO Analyzer SOLIDO Analyzer Workflow analysis You can optimize sequencing results by performing workflow analysis WFA runs A WFA run WFA run analyzes a quadrant of a slide that undergoes a single ligation cycle The quadrant contains beads deposited at a lower density than the density of beads deposited for a sequencing run A WEA run determines the e Optimal library concentration the library concentration for optimal preparation of templated beads using the library You use this library concent
122. l Buffer Part 2 bottle Applied Biosystems SOLIDO 4 System Instrument Operation Guide 25 Chapter 2 Prepare and Install Slides and Reagents Install on instrument reagents 2 Gently mix the contents by slowly inverting the bottle 3 to 5 times to ensure thorough mixing and to minimize bubbles 3 After mixing apply the Universal Buffer label supplied with the bottle Prepare Cleave 1 Transfer the contents of the Cleave Solution 2 1 Part 1 bottle to the contents of the Solution 2 1 Cleave Solution 2 1 Part 2 bottle 2 Gently mix the contents by slowly inverting the bottle 3 5 times to ensure thorough mixing and to minimize bubbles Install reagents 1 If needed flush the tubing of the SOLiD 4 Analyzer fluidics system see 26 Appendix B Flush the fluidic lines on page 99 Note If the SOLiD 4 Analyzer is in continuous use the fluidics system should be flushed every three months If the SOLiD 4 Analyzer will sit idle for more than two weeks the fluidics system should be flushed and the instrument powered down with the fluidics lines empty 2 Double click the SOLiD Instrument Control Software icon to launch the SOLiD Instrument Control Software if it is not already open 3 Under the System Status menu select Cooling from the Chiller drop down menu see Figure 9 Figure 9 Select Cooling to cool the flowcell Protocol Protocol System Status Prime Buffer Line biler 0 C 0 h On Y SHE Ore n
123. lect Stage Template v Browse Please select flowcell 2 Go Cancel Select the flowcell using the drop down menu then press Go The Imager works for several minutes while it determines the range The blue progress bar indicates how close it is to completion see Figure 48 You can also click Cancel so that the Imager aborts the ranging operation Figure 48 Detect Focusing Range Dialog while detection is in progress CN Detect Focusing Range Select Stage Template C Runs solid0327_20081204_1x15_S3Quad_1 imagingMap STG v Browse co cme Searching focusing range at 22218 24504 B Please select flowcel v 5 When the Imager is done a dialog appears see Figure 49 Click Yes if you want to replace the values in the local settings file Click No if you want the Imager to discard the newly calculated focus range Applied Biosystems SOLiD9 4 System Instrument Operation Guide 69 Chapter 3 Set Up Control and Monitor the Run Start the sequencing run Figure 49 Confirm that you want to replace the local settings file UM Focusing Range Detection B The focus range for flowcell 2 is 660621 670621 Do you want to save these for this flowcell 6 Verify the validity of the newly calculated focus range by taking images at random locations IMPORTANT You should see images of beads ensuring that the algorithm was able to focus on the beads and not on other artifacts
124. limitation of remedies lesse 150 Thirdparty produele ii earnan e RD ea ERE Io ees te inst 151 Limitation of liability i e nen m edet hed Pee Pulp EE dd 151 General xc ciu tr beu tbt tete bred nb eate b pep eb RIEN 152 APPENDIX J Safety uuueeleseeeseeeee eene 153 Symbols on this instr rment oe bbb RING UP EIN eve eM DN Bo e 153 Safety alerts on this Instrument en 154 Safety information for instruments not manufactured by Life Technologies 155 Instr rrentiately A eR bb RT eer bite d bep ex b tS 155 ej E 155 Physical injury a uiu ec e ie mae doe Vra eee aa ee pic 155 Electrical sx does ged md te x MEC LEE exa eg eI Mei Red 156 Cleaning and decontamination sss eys g K r R C cece KR AL e e 156 Safety and electromagnetic compatibility EMC standards 2 0 ccc cece eee eee 156 Y vein on wus eer REA ERR ees ee MA i ee 156 EME Se ee ee ee ee ee et 157 Environmental design 0 Kd aR occ 157 Chemical safely icccuet atic Lol id de e cba LM ous ee ei 158 Biological hazard safety cece eect ete tenet tenet 0 158 Documentation and Support 0c ccc eee eee eee eee 161 Related documentation 0 0 c cece c cnc 161 Obtaining SDSS i hte O ha de Joust 162 Applied Biosystems SOLiD 4 System Instrument Operation Guide 7 Contents Obtaining support tit Sees A URP A Pee S UE 163 Limited product Warranty see n 163 Glossary asas As 165 far CPU C DO ies ween Mon
125. located at the top right hand corner of the flowcell panel see Figure 36 on page 52 52 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Section 3 1 Set up and perform a workflow analysis WFA run 3 View the WFA report 2 Look for Uniform deposition of beads on the slide The actual average bead deposition density panel value being similar in value to the targeted average bead deposition density panel of 25 000 beads panel for WFA run A large number of missing panels could indicate a deposition problem Note The heat map may not immediately be available after the completion of the Prescan The software must process all the images collected during the Prescan before the heat map is available This process may take up to 30 minutes depending on the number of panels imaged 3 After you finish viewing the Heat Map click Close located at the bottom of the Heat Map window View cycle scans 1 Select the appropriate flowcell Flow Cell 1 or Flow Cell 2 in the Cycle Scan menu on the task bar on the left Note Details regarding which parameters to monitor are described in the sequencing run section see Monitor the run on page 77 2 Click the heat map link to view the heat map for that cycle see Figure 60 on page 81 3 Left click any square panel on the heat map to open the panel browser window The panel browser allows you to view the focal map and the image for each fluorescent dye signal see Figure
126. low the proceeding instructions 1 Clean the deposition chamber by washing thoroughly in hot water for at least 1 minute 2 Then thoroughly rinse with DI water for 2 minutes 3 Dry chambers completely using lint free wipes i e Kimwipes or air dry overnight at room temperature 4 Do not put the chambers in 37 C to dry as this allows any residual buffer contaminants from the gasket to seep out and will adversely affect your deposition Applied Biosystems SOLiD9 4 System Instrument Operation Guide 95 B Appendix B Supplemental Procedures Calibrate the deposition chamber volume Deposition chambers previously used with the SOLiD9 3 or SOLiD 3 Plus System 5 Do not dry with compressed air because any contaminants in the air system will affect the deposition For users who have upgraded from the SOLiD 3 or SOLiD 3 Plus System to the SOLID 4 System and who are thus still using the same deposition chambers it is recommended that they stop using detergents to clean the deposition chamber since the SOLiD XD Slides are highly sensitive to any detergent contamination For deposition chambers used before with the SOLiD 3 or SOLiD 3 Plus System an additional cleaning protocol outlined below should be conducted 1 Rinse the deposition chamber top bottom and carrier thoroughly with hot water for at least 1 minute 2 Fill a sonicator capable of holding the deposition chamber top with DI water to the a
127. lt 5 When the Imager is done a dialog appears see Figure 33 Click Yes if you want to replace the values in the local settings file Click No if you want the Imager to discard the newly calculated focus range Applied Biosystems SOLiD9 4 System Instrument Operation Guide 49 3 Chapter 3 Set Up Control and Monitor the Run Start the WFA run Figure 33 Confirm that you want to replace the local settings file ov Focusing Range Detection B The focus range for flowcell 2 is 660621 670621 Do you want to save these for this flowcell e JU jJ 6 Verify the validity of the newly calculated focus range by taking images at random locations IMPORTANT You should see images of beads ensuring that the algorithm was able to focus on the beads and not on other artifacts see Figure 34 If you do not see bead images or if you see out of focus bead images set the focus range manually see Manually find the focus range on page 108 Figure 34 Beads in focus Start the WFA run Note Before starting the run make sure the air filter at the back of the instrument is not clogged with dust see Clean the air filter on page 98 1 Click Start Run 2 If there is not enough room to store the data the Start Run dialog appears see Figure 35 Choose the appropriate option see Table 7 50 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Section 3 1 Set up and perform a workflow analysis WF
128. lumbing System Schematic 134 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Checklists and Workflow Tracking Forms This appendix covers Workflow checklists set up a workflow analysis or sequencing run 136 Workflow tracking set up and perform a workflow analysis WFA run 137 Workflow tracking set up a sequencing run l Well o oooooommmoo o 138 Workflow tracking set up a sequencing run 4 well ooooooooo o 139 Workflow tracking set up a sequencing run LR welD 0 00 000s 140 Applied Biosystems SOLiD9 4 System Instrument Operation Guide 135 Appendix F Checklists and Workflow Tracking Forms Workflow checklists set up a workflow analysis or sequencing run Workflow checklists set up a workflow analysis or sequencing run Equipment Reagents Preparation Steps Covaris S2 System 1X TEX Buffer SOLID Bead Deposition Buffer v2 Concentration Chart Double distilled filtered SOLID Deposition water Chamber 1 5 mL LoBind Tubes SOLID Slide Carrier 3 mm adhesive disks NanoDrop ND 1000 SOLID System 6 Tube magnetic rack Sequencing Slide Vortexer Slide Prep Reagent Picofuge Filtered pipettor tips Pipettors SOLID 4 Analyzer 8 L reagent bottle Graduated cylinder Magnetic stir bar Pipettors Deposit the beads 10X Instrument Buffer Storage Buffer 1X T4 DNA Ligase Buffer Parts 1 and 2 Universal Buffer Parts 1 and 2 Imaging Buffer Parts 1 and 2 Cleave Sol
129. mber of beads that meet a stringent set of criteria based on spectral purity and intensity Tag to be sequenced using primers specific to the P1 Adaptor sequence Tag to be sequenced using reverse ligation chemistry and using primers specific to the P2 Adaptor sequence for paired end sequencing of non barcoded fragment libraries Tag to be sequenced using reverse ligation chemistry and using primers specific to the Internal Adaptor sequence for paired end sequencing of barcoded fragment libraries Library consisting of a sheared DNA fragment with P1 and P2 Adaptors ligated to the 5 end and 3 end respectively The number of beads that meet criteria less stringent than best beads criteria based on spectral purity and intensity The average signal intensity of every pixel whether or not it is associated with a bead or not Double stranded oligonucleotide located between two tags to be sequenced Set of DNA tags prepared from the same biological sample to be sequenced on the SOLiD System Beads with template that map back to the reference genome Library consisting of two DNA tags a known distance apart linked by an internal adaptor with P1 and P2 Adaptors ligated to the 5 end and 3 end respectively Method to analyze multiple biological samples in a single spot using barcodes Applied Biosystems SOLiD9 4 System Instrument Operation Guide 165 Glossary N25 plot on axis beads optimal titration point P1 Adaptor P2 Adaptor
130. n Assign to Flowcell F Import Mask Create Runs Run Name Run Creator Status Type Mask E solid0062_20091217_FRAG_BC lab_user Not Started Fragment BC 1_spot_mask_sf Manage Runs ems pe 8 L L E i solid 062 20091217 PE BC lab user Not Started Paired End BC 4 spot mask sf Elf Flow Cell Details 3 Open the Run Definition file that you exported using scalc see Figure 72 You can use another spreadsheet program Applied Biosystems SOLiD9 4 System Instrument Operation Guide 105 B Appendix B Supplemental Procedures Set up a run by importing a Run Definition file Figure 72 Open the Run Definition file File Edit View Favorites Tools Help E Q Back gt 2 JO search Folders E a My Documents Address v SE Name Size Type Date Modified File and Folder Tasks LabVIEW Data File Folder 5 6 2008 11 35 AM aj Renae VG Any Music File Folder 4 18 2008 6 19 AM Emy Pictures File Folder 2 8 2009 9 32 AM iy Move this file Updaters File Folder 8 10 2007 8 19 AM P Copy this file Enp e 1KB Text Document 12 16 2009 4 09 PM 9 Publish this file to the Web ied e E mail this file Edit a Print this File 7 Zip K XK Delete this file Open With E Notepad Send To B scale B swriter no a e Internet Explorer a ree SORS A WordPad O Shared Documents Create Shortcut j R TE Mj My Computer Delete Choose Program X My Network Places e A Properties 4
131. n the table below see Table 38 Table 38 Required maintenance of the Covaris S2 System Required maintenance task Frequency to perform task Degas water for 30 minutes prior to use Before every use Change water Daily Clean with bleach Every two weeks System Instrument Operation Guide 141 Covaris S2 Programs Appendix G The Covaris S2 System Covaris S2 Programs Covalent Declump 1 Covalent Declump 3 142 Table 39 Covalent Declump 1 1 cycle Treatment 1 followed by 1 cycle Treatment 2 Treatment 1 Treatment 2 Duty Cycle 2 5 Intensity 6 9 Cycles Burst 100 100 Time 5 sec 30 sec Target wattage power 4 15 performance estimate WIT t Not programmed Table 40 Covalent Declump 3 3 cycles Treatment 1 followed by 1 cycle Treatment 2 Treatment 1 Treatment 2 Duty Cycle 2 5 Intensity 6 9 Cycles Burst 100 100 Time 5 sec 30 sec Target wattage power 4 15 performance estimate WIT t Not programmed Applied Biosystems SOLiD9 4 System Instrument Operation Guide Instrument Warranty Information This appendix covers Computer configuration iiisssssseeeeeeeeee eene 143 Limited product warranty een 143 Warranty period effective dat rr 144 Warranty claims ccv d ves encodes em ee Cito E 144 Warranty exceptions excede e RS LI etta 145 Warranty limitations ses gs eR N e 0 N a d E E N Ne eee hn 145 Damages cla
132. ncing Kit DNA Frag Lib MM50 35 4459179 SOLIDO ToP Frag Lib Seq F3 Tag Primer A Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer B Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer C Master Mix 50 SOLiD ToP Frag Lib Seq F3 Tag Primer D Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer E Master Mix 50 SOLIDO Mixing Strip Tube with Zinc SOLIDO ToP Sequencing Kit Frag Lib F5 P2 Tag MM35 SOLi SOLi SOLi SOLi SOLi DO ToP Frag Lib Seq F5 P2 Tag Primer A Master Mix 35 DO ToP Frag Lib Seq F5 P2 Tag Primer B Master Mix 35 DO ToP Frag Lib Seq F5 P2 Tag Primer C Master Mix 35 DO ToP Frag Lib Seq F5 P2 Tag Primer D Master Mix 35 DO ToP Frag Lib Seq F5 P2 Tag Primer E Master Mix 35 SOLIDO Mixing Strip Tube sequencing of fragment libraries up to 50 bases for the forward read and 35 bases for the reverse read SOLIDO ToP Paired End Sequencing Kit RNA Frag Lib MM50 35 4459180 SOLiD9 ToP Sequencing Kit Frag Lib F3 Tag MM50 SOLIDO ToP Frag Lib Seq F3 Tag Primer A Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer B Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer C Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer D Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer E Master Mix 50 SOLIDO Mixing Strip Tube with Zinc SOLIDO ToP Sequencing Kit Frag Lib F5 BC Tag MM35 SOLIDO ToP Frag Lib Seq F5 BC Tag Primer A Master Mix 35 SOLIDO ToP Fra
133. nd the STG file superimposes the slide layout on the Imager screen 4 Usethe mouse to drag the green box cursor to a position on the upper left side of the slide Dragging the green box cursor moves the flowcell stage to that corner of the slide see Figure 78 on page 110 Applied Biosystems SOLiD9 4 System Instrument Operation Guide 109 Appendix B Supplemental Procedures Manually find the focus range Figure 78 Use the mouse to drag the green box cursor and to move the flowcell stage the same way Find the focal 1 range Set the Filter Status to WL white light see Figure 79 Figure 79 Click WL white light to set the Filter Status Qu SOLID Imager 1 9 10 Fle View Run Options Devices Tools Windows Help Stage Status Pot Status Motion Position Speed Acceleration x 2339000 100 100 M1 os EN uus 100 100 NudgeSue 10 7 Go ToX 2138 Suo Go To Y hess U S Enable Joystick S Show Herd 2 gt A A L le L adv ae Stage Zoom in Flowceli 110 Update Stage Statut Go To Fiducial Mark Applied Bibsystems SOLID CAMERA Info Dd NC NE Acquire Aspe Camea SN 266255 IP 127001 Pot 8081 Expone Tee 100 Gan 32 v Senna 1 y Ba Depth TEJ Sasae Rate foo FT GoLwe Shutter Status BE o Focus Status B cows Sin Wi J Sr 3 Stop Auto Focus f Enable Jog Jog Serativy 5 Z Focus Curve Docs Side Shape teen sut View ML Ss ec loser Nos SO Nosepece TEES VEN
134. ng for Workflow Analysis WFA run 52 viewing in sequencing run 77 Image Prep and pausing resuming a run 73 install on instrument reagents 25 O ring 34 reagent strips 36 slides on the instrument 31 the SOLiD Flowcell O ring 102 instrument process times 129 instrument safety 155 169 Index introduction 11 L library creating for barcoded samples 65 Light Source replacing 104 Lo Bind tubes Eppendorf use of 19 log view run log for a Workflow Analysis WFA run 52 view run log in a sequencing run 77 M maintenance Covaris TM 52 System 141 manually find the focus range 108 mate pair sequencing fill volumes for on instrument reagents 121 Tag 1 reagent strip layouts 125 Material Data Safety Sheets MSDSs See Safety Data Sheets SDSs mismatches and Barcode Error Correction Level 120 multiplex sequencing run Barcode Error Correction Level 61 create a run record 61 N needle and needle holder 36 0 on Instrument reagent volumes and reagent strip layouts 121 Opti Slide Carrier SOLID 23 O ring install 34 picture 34 P pause 76 Pause Run Resume Run 73 physical injury safety 155 plumbing system schematic 133 pre mixing 73 prepare and install slides and reagents 17 process times entire 130 individual 129 170 R reagent fill volumes on instrument 121 install on instrument reagents 25 strip cover clean 103 strips install 36 reagent strip layouts fragment sequencing 124
135. ng run you can click the remaining rows il E How many samples will you need For your mask 1 A Create New Run 1 Select Run Type Sample Name Primary Analysis Description and Mask bcSample1 default primary v Please select a sample in the above table to define its librari Create Barcode Library Library Name Application Secondary Analysis Barcodes Mult BC sample lt Optional Type or Select w 1 Select settinas gt 1 BCH 5 If performing a paired end sequencing run click Create New p to begin entering library information for that sample see Figure 43 For a barcode sample in a fragment sequencing run select Barcoded Sample and click Create New Library to begin entering library information for that sample see Figure 43 IMPORTANT Each barcoded sample requires at least one library If there are any non barcoded fragment library samples to be sequenced on the same slide as barcoded samples select Non barcoded Sample for that sample Note Non barcoded samples cannot be sequenced on the same slide as barcoded samples for paired end sequencing runs 6 Enter information for the first library present in the sample see Figure 44 a Enter the library name b Select a Multiplexing Series Applied Biosystems SOLiD9 4 System Instrument Operation Guide 65 Chapter 3 Set Up Control and Monitor the Run Create a multiplex
136. ntific and Medical ISM Radio Frequency Generators Reference Description Directive 2002 96 EC European Union WEEE Directive Waste electrical and electronic equipment Directive 2002 95 EC European Union RoHS Directive Restriction of hazardous substances in electrical and electronic equipment Directive 2006 66 EC European Union Battery Directive MII Order 39 PRC Management Methods for Controlling Pollution by Electronic Information Products 157 4 Appendix J Safety Chemical safety Chemical safety WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials To obtain SDSs see the Documentation and Support section in this document Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood Check regularly for chemical leaks or spills If a leak or spill occurs follow t
137. o 4448431 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Prepare and Install Slides and Reagents This chapter covers m Materials and equipment required 6 6 c cece eee eens 17 B WOrKELOW G56 eno HERPES IM a aaa REPRE 18 EU m 19 m Deposit the beads on the slides 0 16 20 B Install on instrument reagents 0 0 coe eee een nee 25 S Install slide s on the instrument 0 0 0 eee eee eens 31 m Install reagent strip s on the instrument e rrn rerna rnrn 36 Materials and equipment required See Appendix A Applied Biosystems SOLiD 4 System Instrument Operation Guide Required Materials on page 83 for a list of equipment kits and consumables necessary to set up a workflow analysis WFA run See Appendix A Applied Biosystems SOLiD 4 System Instrument Operation Guide Required Materials on page 83 for a list of equipment kits and consumables necessary to set up a sequencing run Applied Biosystems SOLiD9 4 System Instrument Operation Guide 17 7 Chapter 2 Prepare and Install Slides and Reagents Workflow Workflow Workflow overview 18 Deposit the beads on the slide s Wash the beads Prepare the slide s Deposit the beads Install on instrument reagents Prepare 1x Instrument Buffer Prepare 1x T4 Ligase Buffer Prepare Imaging Buffer Prepare Universal Buffer Prepare Cleave Solution 2 1 Install reagents Check the waste level Prime instrument
138. o use on the SOLiD 4 Analyzer 1X Instrument 1 Add 800 mL of 10X Instrument Buffer to an empty 8 L reagent bottle Buffer IMPORTANT Regular cleaning of the 8 L Instrument Buffer bottle is required for every run see Appendix B Clean the Instrument Buffer bottle on page 97 Failure to clean the Instrument Buffer bottle regularly may allow microbial contaminants to proliferate in the system Never top off the Instrument Buffer bottle 2 Add 1600 mL of glycerol to the reagent bottle Use a graduated cylinder to measure the glycerol 3 Add 5600 mL of double distilled water rinsing residual glycerol from the graduated cylinder 4 Using a clean magnetic stir bar mix the solution for 10 minutes to ensure homogeneity 5 Remove the stir bar and install the prepared buffer on the SOLiD 4 Analyzer or store at 4 C until ready for use Prepa re 1X T4 1 Transfer the contents of the 1X T4 Ligase Buffer Part 1 tube to the 1X T4 Ligase Ligase Buffer Prepare Imaging 1 Buffer 2 Prepare Universal 1 Buffer Buffer Part 2 bottle Gently mix the contents by slowly inverting the bottle 3 to 5 times to ensure thorough mixing and to minimize bubbles Transfer the contents of the Imaging Buffer Part 1 bottle to the Imaging Buffer Part 2 bottle Gently mix the contents by slowly inverting the bottle 3 to 5 times to ensure thorough mixing and to minimize bubbles Transfer the contents of the Universal Buffer Part 1 bottle to the Universa
139. ods exist for determining the focus range automatic and manual You should first attempt automatic range detection If automatic detection fails use the manual mode Start the sequencing run The run is started using the SOLiD Instrument Control Software Before starting a run previous data should be removed to ensure maximum disk space for new results The instrument is limited to run 5 primers per flowcell If you are performing a sequencing run with multiple tags the instrument automatically pauses and stays paused until the reagents are replaced and the run is resumed Control the run You can control how the SOLiD 4 System collects sequencing data with the SOLiDO Instrument Control Software ICS With the ICS you can repeat a primer to improve the real time primary analysis results or set an early pause point to change reagents on your schedule You can choose to turn off imaging of specific samples to collect only the best sequencing data To control the ICS use the Run Control drop down menu Use the Run Control menu only if you understand clearly the series of fluidic and imaging steps in a run Skipping or repeating certain steps could lead to errors in the resulting data For example with the ICS menus you can repeat a ligation however you must first cleave the fluorescent label from the ligation product Monitor the run While executing a sequencing run the instrument performs a Prescan In a Prescan all of the beads
140. on Guide Chapter 2 Prepare and Install Slides and Reagents Install on instrument reagents 6 Install the prepared Imaging Buffer prepared 1X T4 Ligase Buffer and prepared Universal Buffer into the appropriate positions in the chiller block see Figure 12 Make sure that the tubes on the caps of the reagent bottles are fully screwed on Figure 12 Positions of buffer bottles in the chiller block 1X T4 Ligase Buffer Universal Buffer Imaging Buffer Check the waste 1 Check the level of waste in the 10 L carboy level 2 If the carboy is more than Y4 full properly dispose of the waste according to your institution s environmental health and safety guidelines Applied Biosystems SOLiD9 4 System Instrument Operation Guide 29 20 Chapter 2 Prepare and Install Slides and Reagents Install on instrument reagents Prime Instrument IMPORTANT The priming of the lines must be performed prior to each run whether and Storage Buffer the Instrument and or Storage Buffers were changed or not lines 1 Under the System Status menu click Prime see Figure 13 Figure 13 Click Prime to prime the instrument and storage buffer lines r lab_user Run Name Run Name Created by 9 Created by o E mf Assign Run C co o Besse B neat Map d M B assign Run Bi esc Run df clear MY Run Loos Be sl Mw Y E Setup Sample Slide Sample Slide X Create Runs ES Manage Runs Hide Samples lt lt Hide Samples lt lt Wi C
141. on in the Select Run Type and Mask pane see Figure 29 on page 47 a Select the WFA option b Optional Type a new run name c Optional Enter a description d Ensure that the mask 4_spot_WFA_mask_sf is selected e Click Next Applied Biosystems SOLiD9 4 System Instrument Operation Guide Section 3 1 Set up and perform a workflow analysis WFA run Create a WFA run record Figure 29 Complete the information in the Select Run Type and Mask pane File Welcome lab user SETUP gt Create Edit Run gt Select Run Type and Mask e The following pages will guide you through to set up a run Required fields are noted with an asterisk Select type of run to create Fragment Mate Pair Paired End Create New Run Multiplexing Identify the run and enter a description Run Name solid0D62_20091222_WFA Description Assign a Primer Set and of bases Primers run in sequential order Select Run Protocol SOLD WEAR Sequencing For Workflow Analys Primer Set 1 F 1 Bases Select mask to use 4 kant lt Bad Next gt Finish Imaging is Online Fluidics is Online 45m of 297m 3 Complete the information in the Specify Samples and Analysis pane see Figure 30 a Assign samples to spots b Click Finish Applied Biosystems SOLiD9 4 System Instrument Operation Guide 47 Create a WFA run record Chapter3 Set Up Control and Monitor the Run Figur
142. ophotometer Refer to Quantitate the beads using the NanoDrop ND 1000 Spectrophotometer in the Applied Biosystems SOLiD 4 System Templated Bead Preparation Guide Part no 4448378 5 Transfer the appropriate number of beads to a 1 5 mL Eppendorf LoBind Tube and store the remaining beads at 4 C Table 4 Number of beads to use according to the type of run and deposition chamber SOLiD9 Target number of P2 Maximum threshold Type of run Deposition positive beads per number of beads per Chamber well well WFA 4 Well 15 million 30 million Sequencing 1 Well 708 million 778 million Sequencing 4 Well 128 million 141 million Sequencing 8 Well 56 million 61 million Target number of P2 positive beads per well The targeted bead deposition density is 300 000 P2 positive beads per panel and the maximum threshold bead deposition density for all beads P2 positive or not is 330 000 beads per panel Exceeding the maximum threshold number of beads per well may result in decreased sequence quality Bead deposition densities may deviate from the targeted bead deposition density of 300 000 P2 positive beads per panel due to variability in the bead quantitation process The calculated bead concentration based on the WFA report is the most accurate because it specifically measures the concentration of P2 positive beads see Chapter 3 Determine the bead deposition density for a sequencing run on page 55 If WFA run resul
143. page 142 Pulse spin but do not pellet the beads 2 Ifa WFA run has already been performed use the results from the WFA report to estimate the bead concentration and proceed to step 5 otherwise use the Applied Biosystems SOLiD Bead Concentration Chart Part no 4415131 to estimate the bead concentration see Figure 5 Figure 5 The SOLiD9 Bead Concentration Chart For best results use the Applied Biosystems SOLiD9 Bead Concentration Chart Part no 4415131 supplied separately ds a F FEEBBBEBN 125K 250K 500K 750K 1 25M 1 5M 3 Adjust the volume of beads so that the color of the bead solution matches a color in the optimal range 750 000 1 25 million beads uL see Figure 5 and Figure 6 Figure 6 SOLiD9 Bead Concentration Chart workflow Match beads to color chart 750 000 to 1 25 million beads uL Too light Too dark Color of suspension Place bead suspension in magnetic rack and remove some of the supernatant Match beads to color chart 750 000 to 1 25 million beads uL Dilute with more 1x TEX Buffer Too light Too dark Color of suspension Matched Quantitate beads using the NanoDrop ND 1000 Spectrophotometer 20 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Chapter 2 Prepare and Install Slides and Reagents Deposit the beads on the slides 4 When the bead concentration is within the accurate range quantitate the beads using the NanoDrop ND 1000 Spectr
144. panels 30 uL XyL 30 UL Y P2 positive beads uL 284 000 P2 positive beads pL 2 Use the calculated concentration in place of the value determined by the NanoDrop ND 1000 Spectrophotometer for more accurate deposition densities when preparing slides for sequencing It is recommended that you use this resulting calculated bead concentration to determine the volume of beads for deposition 3 Multiply that volume by 120 to calculate the volume of beads for deposition Note The 2076 overage has been estimated empirically and is attributable to bead loss during washing and bead deposition steps The overage factor may be adjusted by individual operators based on their experiences Desired P2 positive beads to be deposited Z uL bead solution Y P2 positive beads uL Example For a sample with a concentration of 284 000 P2 positive beads pL to be deposited in one well of an 8 well SOLiD Deposition Chamber at a density of 300 000 beads panel 56 million P2 positive beads x 12096 Z UL bead solution 284 000 P2 positive beads uL 237 UL bead solution Applied Biosystems SOLiD9 4 System Instrument Operation Guide 55 Chapter 3 Set Up Control and Monitor the Run Materials and equipment required Section 3 2 Set up and perform a sequencing run Materials and equipment required Workflow Workflow overview 56 See Appendix A on page 83 for a list of equipment kits and consumables necessary to set up a seq
145. pendix C On Instrument Reagent Volumes and Reagent Strip Layouts C Reagent strip layouts Mate Pair sequencing F3 Tag SOLIDO Mixing Strip Tube with Zinc MP Lib F3 Tag Primer E MP Lib F3 Tag Primer D MP Lib F3 Tag Primer C MP Lib F3 Tag Primer B MP Lib F3 Tag Primer A oS oo Phosphatase Phosphatase Mate Pair sequencing R3 Tag gt a y SOLIDO Mixing Strip Tube with Zinc GY Ligase Phosphatase R3 H ProbeA Probes Y Probe Y i iD ag dada Ra y Primer E Primer E ZZ Bridge YY ProbeB j 7 7 P SA lt d Q Q G A p P y A A UY GJ d E MP Lib R3 Tag Primer D A Op lt G PPKG Ul UY M R3 V ES 4 4 MP Lib R3 Tag j ProbeA 1 ProbeB Primer C Y 7 primer c LD lt Ligase Phosphatase Jf Y AY A 4 G 7 MP Lib R3 Tag ProbeB S L y Y Primer B le a g K lt d V m V M Focal C MP Lib R3 Ta Ligase Phosphatase Primer A ProbeA j ProbeB 4 Mop i b y H 7 7 Y Z Applied Biosystems SOLiD9 4 System Instrument Operation Guide 125 E Appendix C On Instrument Reagent Volumes and Reagent Strip Layouts Reagent strip layouts Paired end sequencing F5 P2 Tag 4 4 4 gt p SOLIDO Mixing Strip Tube i Ligas Capper Reverse Reverse Frag Lib F5 P2 Tag 9 Enzyme i Probe A J Probe B Phosphatase Primer E Ligase Capper R
146. perturbations for example opening system parts vibrations during the run are detrimental to the results 72 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Control the run Section 3 2 Set up and perform a sequencing run Control the run Pause Resume Note Special considerations need to be made when using the Pause Run Resume Run Run function in certain modules When the run has been paused in the middle of or at the end of the Scan Slide module and if the Scan Slide module of the same cycle needs to be repeated itis recommended that you resume the run starting from the Image Prep module Pausing the run is generally followed by automatic filling of the flowcell with Storage Buffer Resuming from the Image Prep module fills the flowcell with Imaging Buffer again which is required to perform the functions in the Scan Slide module You can resume from the Image Prep module using the Change Run Progress Point command in the Run Control menu see Use the Run Control menu on page 74 Eleven modules contain a pre mixing step for the next module e Prime Ligate Mix Ligate Dark Ligate Mix Dark Ligate Phosphatase Mix e Cleave Ligate Mix e Prime Bridge Ligate Mix Bridge Ligate Phosphatase Mix e Dark Ligate Capper Enzyme Mix e Cleave RevPhosphatase Mix e RevPhosphatase Ligate Mix s RevPhosphatase Bridge Ligate Mix Bridge Ligate Capper Enzyme Mix When you try to pause a run in the middle of the mo
147. position Chamber with the SOLIDO 4 Slide Carrier or SOLiD Opti Slide Carrier IMPORTANT For upgraded systems with new slide carriers the SOLiD Deposition Chamber should be recalibrated using the SOLiD 4 Slide Carrier to determine the exact deposition volume Table 5 Approximate Deposition Buffer volumes Deposition chamber Volume per well uL 1 Well 550 4 Well 400 8 Well 300 Note Deposition Buffer volume is dependent on the slide carrier as well as the Deposition Chamber and volumes vary from slide carrier to slide carrier In addition residual SOLiD XD Slide Deposition Buffer v2 that remains on the surface of the slide after washing may alter the volume slightly Thoroughly clean rinse and dry the SOLiD Deposition Chamber before deposition a Clean the SOLIDO Deposition Chamber by rinsing it with MilliQ water Note Do not wash the SOLiD Deposition Chamber with ethanol because ethanol damages the adhesive on the O ring b Blot the SOLIDO Deposition Chamber dry on a lab wipe Be particularly careful to dry around the O ring and to remove fluid from the fill ports Insert a new slide into the SOLiD 4 or Opti Slide Carrier see Figure 7 IMPORTANT Do not touch the slide surface IMPORTANT The SOLiD 4 and Opti Slide Carriers are painted blue across the side see Figure 7 If you are using the SOLiD ToP reagent strips for sequencing you must use either the SOLiD 4 Slide Carrier or
148. ppropriate level No additives should be added to DI water used for sonication 3 Place only the deposition chamber top into the sonicator and sonicate at a medium low setting for 10 minutes 4 Remove the sonicated deposition chamber top and thoroughly rinse with DI water for 2 minutes 5 Rinse the deposition chamber bottom and carrier with DI water for 2 minutes 6 Drythe chambers thoroughly with lint free wipes or allow the chambers to air dry at room temperature overnight Calibrate the deposition chamber volume 96 The proper filling of the deposition chamber is crucial for uniform depositions e Underestimation of the chamber volume can cause bubbles to remain in the chamber during centrifugation and result in bead loss and uneven depositions e Overestimation of chamber volume will lead to decreased densities due to beads remaining in the pipette To properly calibrate the volume of the deposition chamber follow the protocol 1 Obtain a new or used SOLiD XD Slide 2 Assemble the deposition chamber with the SOLiD XD Slide 3 Pipeta1mL of DI water into the deposition chamber assembly Once the chamber is full adjust the pipette until the remaining water is at the end of the pipette tip 4 Subtract this measurement from the 1 mL to yield the chamber volume 5 Record the deposition chamber volume measurement 6 Disassemble the deposition chamber and dry the chamber and the slide with a lint free wipe
149. r Set and of bases Primers run in sequential order Select Run Protocol SOLiD4 Y Barcode and Sequencing with 5 primer walkaway Primer Set 1 F5 P2 25 bases Y Primer Set 2 F3 Preview Select mask to use 1 spot mask sf Back Next gt Finish Cancel i Imaging is Online Fluidics is Onlir 3 Complete the information in the Specify Samples and Analysis pane see Figure 40 Applied Biosystems SOLiD9 4 System Instrument Operation Guide 59 Chapter 3 Set Up Control and Monitor the Run Create a sequencing non multiplex run record a Select the number of samples that will be used in the run If a single sample will be run in multiple spots of the same slide it counts as only one sample Otherwise the number of samples typically matches the number of spots on the mask b Optional Edit the Sample Name Library Analysis settings and Description Figure 40 Complete the information in the Specify Samples and Analysis pane solid0010 SOLID 4 0 User lab_user SETUP gt Create Edit Run gt Specify Samples and Analysis remaining rows Select how many samples you need for this run For Application Primary and Secondary Analysis columns in a sequencing or multiplexing run you can click the column header and use ctrl D How many samples will you need for your mask 1 v Create New Run 1 Select Run Type and Mask Sample Name
150. r laboratory equipment for the heating of materials 156 Applied Biosystems SOLiD9 4 System Instrument Operation Guide EMC Environmental design Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix J Safety J Safety and electromagnetic compatibility EMC standards Reference Description IEC 61010 2 081 EN 61010 2 081 Safety requirements for electrical equipment for measurement control and laboratory use Part 2 081 Particular requirements for automatic and semi automatic laboratory equipment for analysis and other purposes IEC 60825 1 EN 60825 1 Safety of laser products Part 1 Equipment classification and requirements 21 CFR 1040 10 and 1040 11 as applicable U S FDA Health and Human Services HHS Radiological health performance standards for laser products and Radiological health performance standards for specific purpose laser products Reference Description Directive 2004 108 EC European Union EMC Directive EN 61326 1 Electrical Equipment for Measurement Control and Laboratory Use EMC Requirements Part 1 General Requirements FCC Part 18 47 CFR U S Standard Industrial Scientific and Medical Equipment AS NZS 2064 Limits and Methods of Measurement of Electromagnetic Disturbance Characteristics of Industrial Scientific and Medical ISM Radiofrequency Equipment ICES 001 Issue 3 Industrial Scie
151. r the F3 tag do not contain Focal Map reagents 58 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Section 3 2 Set up and perform a sequencing run Create a sequencing non multiplex run record If performing a paired end sequencing run select F5 P2 as Primer Set 1 and F3 for Primer Set 2 If performing a paired end sequencing run on a library prepared using the SOLiD Total RNA Seq Kit select F5 BC as Primer Set 1 and F3 for Primer Set 2 f Enter read lengths for Primer Set 1 and if used 2 A typical read length for the F3 and R3 tags is 35 50 bases A typical read length for the F5 P2 tag or F5 BC tag is 35 bases g Select the appropriate mask to use h Click Next Note If a message window appears during these steps then continue setting up run as indicated If a message remains after all selections have been made correct the entries before clicking Next Figure 39 Complete the information in the Select Run Type and Mask pane lid0054 SOLiD 4 er lab_user File Welcome lab user SETUP gt Create Edit Run gt Select Run Type and Mask e The following pages will guide you through to set up a run Required fields are noted with an asterisk Select type of run to create Create New Run Fragment O Mate Pair Paired End O WFA Multiplexing Identify the run and enter a description Run Name solid0054_20100203_PE Description Assign a Prime
152. r the quality of data obtained due to good bead deposition and chemistry From the WFA data you can also estimate the bead deposition density to be used when preparing a slide for sequencing Differences between bead concentration measured on the NanoDrop ND 1000 Spectrophotometer and the concentration actually detected by the bead counting algorithm on the instrument may occur In order to maximize throughput in a SOLiD System sequencing run a bead density of 300 000 P2 positive beads per panel is recommended Applied Biosystems SOLiD9 4 System Instrument Operation Guide 45 Chapter 3 Set Up Control and Monitor the Run Create a WFA run record Create a WFA run record 46 There are two ways to create a WFA run The first method is to use the Run wizard see below The second method is to import a txt file that contains the run definition see Set up a run by importing a Run Definition file on page 105 Importing a txt file saves time re entering information of a repeated run The txt file can be generated on an off instrument computer 1 Click Create Runs in the Setup task pane on the left menu pane of the ICS see Figure 28 on page 46 Figure 28 Use the Run wizard to create a WFA run fi solid0062 SOLID 4 0 User lab user File Tools Wizards Window Help Welcome lab_user wl Assign Run Bi Edic aun d dear BP Run toos Y Sample Slide Hide Samples lt lt 2 Complete the informati
153. ration for any preparation of templated beads for that library as long as the scale of templated bead preparation is the same Bead enrichment efficiency the proportion of beads that have been successfully amplified using emulsion PCR ePCR as a fraction of the total number of beads prepared You use this value to accurately deposit successfully amplified beads for a sequencing run WEA runs require the same materials as those materials needed for sequencing runs If you perform multiple WFA runs routinely you should order additional SOLiD Instrument Buffer Kits To perform a WFA run prepare slide and install reagents according to the procedure in Chapter 2 Prepare and Install Slides and Reagents on page 17 then set up and monitor the run according to the procedure in Chapter 3 Set Up Control and Monitor the Run on page 43 Sequencing run There are five types of sequencing runs that can be performed on the SOLiD 4 System fragment sequencing paired end sequencing mate pair sequencing multiplex fragment sequencing and multiplex paired end sequencing see Figure 2 12 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Chapter 1 Introduction 1 SOLiD9 4 System run types Figure 2 Types of sequencing runs Fragment sequencing p F3 SLM Paired end sequencing gt F3 F5 P2 aa P1 Adaptor Mate pair sequencing P P1 Adaptor Multiplex fragment sequencing gt F3 gt B
154. rd 61 create a standard run record 58 fill volumes for on instrument reagents 121 pause resume for pre mixing 73 required kits and equipment 86 Run Control menu 74 Set Early Pause Point command 76 set up and perform a run 56 Stop Run command 76 view cycle scans 78 view heat maps 77 view run log 77 workflow 56 Set Early Pause Point command 76 set up a run by importing a Run Definition file 105 set up control and monitor the run 43 shut down the SOLiD M 4 Analyzer 119 Slide Carrier SOLID 4 23 slides prepare and install 31 slide carrier lockdown tabs 35 SOLID Slide Storage Chamber 32 SOLID 4 Slide Carrier 23 SOLID Opti Slide Carrier 23 software license agreement 147 operation about 16 SOLID Slide Carrier lockdown tabs 35 SOLID 4 Analyzer plumbing system schematic 133 Stop Run command 76 store the slide in a flowcell 119 strip layouts reagent fragment sequencing 124 mate pair sequencing Tag 1 125 Workflow Analysis WFA run 128 Applied Biosystems SOLiD 4 System Instrument Operation Guide Index supplemental procedures clean the reagent strip cover 103 flush the fluidic lines 99 install the SOLiD Flowcell O ring 102 manually find the focus range 108 replace the SOLiD Light Source 104 set up arun by importing a Run Definition file 105 shut down the SOLiD 4 Analyzer 119 store the slide in a flowcell 119 support obtaining 163 symbols on instrument 153 syringe 31 T times entire processe
155. re Integrated quick step procedures on operating each software Workflow Quick Reference to perform data analysis Guide Applied Biosystems 4452360 Provides a quick guide to the sequencing kits you SOLID 4 System Product need to perform fragment paired end mate pair Selection Guide multiplex fragment and multiplex paired end sequencing Applied Biosystems 4451929 Provides a brief summary of changes made SOLIDO System SOLIDO 3 between the SOLIDO 3 Plus System Plus to SOLIDO 4 System documentation and the SOLiD 4 System User Documentation documentation Changes Applied Biosystems 4449773 Provides a checklist to ensure that all necessary SOLIDO 4 Upgrade preparations are made before upgrading to the Checklist SOLIDO 4 System and provides a list of consumables you can order Note For additional documentation see Limited product warranty on page 163 Safety Data Sheets SDSs are available from www lifetechnologies com support Note For the SDSs of chemicals not distributed by Life Technologies contact the chemical manufacturer Applied Biosystems SOLiD9 4 System Instrument Operation Guide Documentation and Support Obtaining support Obtaining support For the latest services and support information for all locations go to www lifetechnologies com support At the website you can e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities Search through f
156. requently asked questions FAQs Submit a question directly to Technical Support Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents Obtain information about customer training Download software updates and patches Limited product warranty Life Technologies Corporation and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechnologies com support Applied Biosystems SOLiD9 4 System Instrument Operation Guide 163 Documentation and Support Limited product warranty 164 Applied Biosystems SOLiD9 4 System Instrument Operation Guide barcode barcoded fragment library BC tag bead count bead signal best beads F3 tag F5 P2 tag F5 BC tag fragment library good beads image signal internal adaptor library mappable beads mate paired library multiplexing Glossary Unique sequence identifier added to the sample during library construction Fragment library with a barcode sequence appended to the 3 end of the sheared DNA fragments Barcode tag The number of beads detected in a panel The average signal intensity of every pixel associated with a bead Nu
157. ressly permitted in this Agreement 2 You agree that you will not reverse assemble decompile or otherwise reverse engineer the Software 3 You agree that you will not remove any proprietary copyright trade secret or warning legend from the Software or any Documentation 4 You agree to fully comply with all export laws and restrictions and regulations of the United States or applicable foreign agencies or authorities You agree that you will not export or reexport directly or indirectly the Software into any country prohibited by the United States Export Administration Act and the regulations thereunder or other applicable United States law 148 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Trial Termination US government end users European community end users Regulated uses Appendix Life Technologies End User Software License Agreement License 5 You agree that you will not modify merge the Software or any interest therein or create derivative works based on the Software or any part thereof 6 You agree that you will not sell rent transfer or distribute this license or the Software or any part thereof or any interest therein to any other person If this license is granted on a trial basis you are hereby notified that license management software may be included to automatically cause the Software to cease functioning at the end of the trial period You may terminate this Agreement by
158. rials and equipment required nn 17 VORTON themes eot teed oa o tu d tes ada a EU A E H N D Bd Ad e Aout qe 18 Workflow overview seetri 0 c 09T KR RII HR I rn 18 A EE 19 General at T E EEE AEE DEE A A E 19 Covas US TTT 19 Deposit the beads on the slides n nnan see 20 Waslithe beads N g a ni id dd bide 20 Prepare the slidels 5 mid ct rece ees eem ee eru HR 22 Deposit the beads rn 24 Install on instrument reagents lesser 25 Prepare 1X instrument Buffer 2 osse entre saa id Yuan 25 Prepare 1X T4 Ligase Buffer e 25 Prepare Imaging Buffer sse 25 Prepare Universal Buffer is ida a Ue n ee a a e ore dps edd 25 Prepare Cleave Solution 2 1 26 Install feagents bec o onec wt rts 26 Checlcthe waste level 2isresnlniverfes dt A a USE ce iecit 29 Prime Instrument and Storage Buffer Unes 0 0 00 cece eee ee eee eee eee ees 30 Install slide s on the instrument n nanunua arrn nr rnrn rnrn eet n 31 Prepare the slide ca R R r a daddy 31 Installihe slide cocaina Pta ba dais 32 Install reagent stripls on the instrument rr 36 Applied Biosystems SOLiD 4 System Instrument Operation Guide 3 Contents S CHAPTER 3 Set Up Control and Monitor the Run 43 Section 3 1 Set up and perform a workflow analysis WFA run 44 Materials and equipment required see 44 Work PRETI dai pa 44 WorktloW OVeEVIGW 2 dai A ERT T Lbs a 44 Create a WFA run record coococcccccc o 46
159. rst cycle is a reason to pause the run points clustered on the four color axes and and troubleshoot the performance with minimal fraction of the points clustered around the origin The quality of the Satay plot typically degrades gradually with each ligation cycle for a single primer cycle becoming more fuzzy in the last cycle t Exposure time is indicative of the signal intensity of the beads The instrument uses an auto exposure routine on a per sample basis to maximize bead signal with minimal image saturation Shorter exposure times are associated with efficient ligation of the fluorescent probes i You should replace the SOLiD Light Source in the Life Technologies SOLIDO 4 Analyzer every 1500 hours of use See Appendix B for instructions 2 Click the heat map link to view the heat map for that cycle see Figure 60 80 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Section 3 2 Set up and perform a sequencing run Monitor the run Figure 60 Click the heat map link to view the heat map for that cycle BC PC 01 V1 Cycle Heat Map DER Usable Beads Best Beads Best Good Beads Close 3 Left double click any square panel on the heat map to open the panel browser window The panel browser allows you to view the focal map and the image for each fluorescent dye signal see Figure 61 Applied Biosystems SOLiD9 4 System Instrument Operation Guide 81 82 Z Ch
160. rt Scans H Focal Heat Map j Scan Date Complete Used Panels Failed Panels Usable Beads Best Beads Best Good Beads Bead Color Balance Effective Ex CLA re t Map ME EN 149 060 s T 176 719 ma sl 15 X Create Runs BC PA 02 V1 Jan 19 2010 6 03 PM yes amp HeatMap 2358 63 2 237 362 143 850 173 227 18 BC PB 01 V1 Jan 20 2010 12 11 PM yes HeatMap 2358 2 236 962 147 254 172 L n m BC PB 02 V1 Jan20 2010 3 51 PM yes M HeatMap 2358 0 2 237 375 142 330 L 1 BC PC 01 1 Jan 20 2010 9 00 PM yes HeatMap 2358 4 2 236 477 145 119 170 L AT BC PC 02 V1 Jan21 2010 12 40 AM yes xa HeatMap 2358 62 2 236 843 138 528 167 867 16 BC PD 01 V1 Jan 21 2010 4 40 AM yes A HeatMap 2358 68 2 237 058 140 680 164 034 SS 15 BC PD 02 V1 Jan 21 2010 7 09 AM yes xe HeatMap 2358 61 2 237 012 135 303 162 608 19 Sample data for scan BC PA 01 V1 Sample Panels Failed Panels Usable Beads Best Beads Best Good Beads Bead Color Balance Effective Exposure Times ms g bcSample1 2358 59 2 236 556 149 060 176 748 HEN EA 15 15 9 9 Satay N25 P System Status Prime Buffer Line Chiller 3 9 C Cooling Y Lamp O hrs On y Table 13 Cycle Scans window Distinguish normal runs from problematic runs Parameter Normal run Problematic run 1 Failed panels number of In general the number of failed panels Run begins with extremely high panels that failed image should be relatively small and consistent number of failed panels or dramati
161. s select a sample in the list and then click on spots to assign that sample to them Sample Mask 1_spot_mask_sf Spots bcSample1 1 beSample1 lt Back Imaging is Online Fluidics is Online untitled Paint Applied Biosystems SOLIDO 4 System Instrument Operation Guide 67 Chapter 3 Set Up Control and Monitor the Run Create a multiplex sequencing run record 10 11 12 13 14 15 16 68 IMPORTANT Review the Selected Barcodes for each library to ensure that they are correct and be sure that the libraries are correctly assigned to the sample After a run is started you cannot change the barcode or library assignment Repeat step 5 to step 9 for all remaining samples Assign samples to unassigned spots in the mask White circles are unassigned and blue circles are assigned To assign a sample a Select it from the Sample list b Click a white unassigned spot To remove a sample from an assigned blue spot click the spot The sample is removed and the spot turns white Once all of the samples have at least 1 library click Finish and select Save to Database Click Manage Runs in the task pane on the left menu pane of the ICS see Figure 46 Figure 46 Manage Runs P solid0062 SOLID 4 0 User lab user File Tools Wizards Window Help lab_user Run b 9 Create E Assign Run 9 m A ex b SM f Sample Slide San C Optional To complete t
162. s 130 individual processes 129 run estimated 11 tracking forms workflow 135 training information on 163 V volumes fill for on instrument reagents 121 W WEA See Workflow Analysis Workflow Analysis WFA run about 12 creating a run record 46 fill volumes for on instrument reagents 121 reagent strip layouts 128 required kits and equipment 83 set up and perform a run 44 view run log 52 workflow 44 workflow checklists and tracking forms 135 workflow diagrams bead concentration chart 20 preparing slides and reagents 18 relationship between types of runs 12 Workflow Analysis WFA run 44 171 a TRL Headquarters 6 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 For support visit www lifetechnologies com support technologies www lifetechnologies com
163. s validated this protocol using this specific material Substitution may adversely affect The SOLiD9 4 Analyzer is shipped with 2 of each size SOLIDO Deposition Chambers and SOLIDO 4 Slide Carriers Two SOLiDO Slide Storage Chambers are provided for use with all chambers co Table 16 Required consumables WFA run Or equivalent but validation of the equipment for library preparation is required Item Source Nuclease free Water Applied Biosystems AM9932 ABgene 96 1 2 mL square well storage plates ABgene AB 1127 3 mm adhesive disks Grace Bio Labs ST200 Ethylene glycol American Bioanalytical AB00455 01000 CF 1 Calibration Fluid Kit Thermo Scientific CF 1 PR 1 Conditioning Kitt Thermo Scientific PR 1 1 5 mL LoBind Tubes Eppendorf 022431021 Kimwipes9 Major Laboratory Supplier MLS Filtered pipettor tips MLS t Applied Biosystems has validated this protocol using this specific material Substitution may adversely affect system performance i The NanoDrop Conditioning Kit is useful for reconditioning the sample measurement pedestals to a hydrophobic state if they become unconditioned refer to the Nanodrop user s manual for more information The PR 1 kit consists of a container of specially formulated polishing compound and a supply of convenient applicators Set up and perform a sequencing run Required Applied Biosystems reagent k
164. sh MLS Table 21 Required consumables Clean the Instrument Buffer bottle Item Source Deionized water Major Laboratory Supplier MLS Bleach MLS Water MLS t For the SDS of any chemical not distributed by Applied Biosystems contact the chemical manufacturer Before handling any chemicals refer to the SDS provided by the manufacturer and observe all relevant precautions 1 Rinse the Instrument Buffer bottle three times with approximately 1 L of deionized water 2 Inspect the Instrument Buffer bottle for visible signs of microbial contaminants 3 If any contaminants are present clean the Instrument Buffer bottle with bleach a Pour approximately 500 mL of bleach into the Instrument Buffer bottle b Add approximately 500 mL of water to the bleach in the bottle c Scrub the bottle with a bottle brush d Rinse the bottle at least two times with water e Rinse the bottle at least three more times with deionized water Applied Biosystems SOLiD9 4 System Instrument Operation Guide 97 B Appendix B Supplemental Procedures Clean the air filter Clean the air filter Regular cleaning of the air filter is required once a month Failure to clean the air filter can cause the filter to clog with dust which may lead to poor air flow and temperature control for the flowcells You should clean the filter between runs but not during runs Required Table 22 Required consumables Clean the air filter cons
165. sis Reagents Applied Biosystems SOLID 4 System Instrument Operation Guide 137 Appendix F Checklists and Workflow Tracking Forms Workflow tracking set up a sequencing run 1 well Workflow tracking set up a sequencing run 1 well Sample Name Sample information A600 Concentration beads Deposition Volume uL Volume Left Beads Left Side ea O PEOS S LL Deposition Butera OOS Slide Prep Reagent SOO Instrument Buffer SOO Storage Buffer T T4 Ligase Buffer Part PO T4 Ligase Buffer Part2 Universal Buffer Part OOO Universal Buffer Part2 S Imaging Buffer Pati PY AS Cleave Solution 1 Cleave Solution 2 1 Part T Cleave Solution 2 1 Part2 PO ResetBuffer T SideStorageBuffer F3 Tag Sequencing Kit L F3 PrmerA S F3 PrimerB T L E A L L AAA o E AAA E y y l O Ee J ES F3 Primer C F3 Primer D Sequencing Kit R3 F5 P2 F5 BC Primer D R3 F5 P2 F5 BC Primer E Strip Tube BC Primers BC Probes 138 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix F Checklists and Workflow Tracking Forms Workflow tracking set up a sequencing run 4 well Workflow tracking set up a sequencing run 4 well Samples 1 to 4 Sample information C MN MEE NEC EN NEN Concentration beads uL pL Beads Left Lot numbers Slide Deposition Buffer v2 AAA 1 AE MERE SEEN n OOOO Univers
166. strument 11 12 13 Click Yes to start loading the flowcell IMPORTANT The flowcell should be loaded within 1 2 minutes of slide installation Slides should be installed one at a time with flowcells loaded before installing a second slide Check for leaks to ensure that the slide does not dry out If only one flowcell will be used ensure that unused flowcell slide carrier lock down tabs are pushed all the way in and tightened before closing the unused flowcell Install reagent strip s on the instrument 1 36 Clean the needle and bottom of the needle holder with a Kimwipes wiper see Figure 20 If needed first moisten the Kimwipes wiper with deionized water or use a pre moistened alcohol pad Figure 20 Needle and needle holder Needle holder Needle Thaw the appropriate reagent strip s on ice See Figure 21 to determine which reagent strips should be used for a sequencing run Use reagent strips from the boxes that match the arrow symbol for the appropriate library and tag IMPORTANT Verify that there is sufficient volume of Reset Buffer for each tag sequenced The SOLiD ToP Instrument Buffer contains sufficient Reset Buffer to reset two tags see Appendix C On Instrument Reagent Volumes and Reagent Strip Layouts on page 121 For multiplex sequencing a bottle of Reset Buffer is included with multiplex sequencing kits to account for sequencing the BC tag Applied Biosystems SOLiD9 4 System In
167. strument Operation Guide Chapter 2 Prepare and Install Slides and Reagents Install reagent stripls on the instrument The blue circled numbers indicate the order in which the tags should be sequenced for each library type The XX represents read length options in bases 35 and 50 for F3 and R3 tags 5 and 10 for BC tag Note The 5 bp barcode sequencing reagents can be used if only barcodes 1 16 are being used The 10 bp barcode sequencing reagents are needed for any set that includes barcodes 17 96 For example if you are sequencing 32 barcoded fragment libraries using barcodes 1 32 you should use the 10 bp barcode sequencing reagents Only 5 bp or 10 bp barcode reads not both may be sequenced on a single slide Applied Biosystems SOLiD9 4 System Instrument Operation Guide 37 Chapter 2 Prepare and Install Slides and Reagents Install reagent strip s on the instrument Figure 21 Reagent strip box symbols corresponding to fragment sequencing paired end sequencing mate pair sequencing multiplex fragment sequencing and multiplex paired end sequencing Fragment sequencing 1 F3 Tag Part No 35 4449352 Part No 4449388 Paired end sequencing 2 F3 Tag 1 F5 P2 Tag DNA gt 9 n 4449388 4463230 Paired end sequencing F3T F5 BC T cu 2 F3 Tag 1 ag Mate pair sequencing 2 F3 Tag Q R3 Tag Part No 35 Part No 4449364 4449376 Part No Part No Mate pair tag mn
168. tem must be specially adapted to prepare beads for the Life Technologies SOLiD 4 System Do not use the Covaris S1 sonicator or an unadapted Covaris S2 System for bead preparation For more information contact a Life Technologies SOLiD System Applications Specialist Ensure that the Covaris S2 System is degassed that bubbles are not present in the system and that the instrument and tube are properly aligned for appropriate sonication of beads To ensure optimal sonication by the Covaris S2 System use the appropriate adaptor with the Covaris S2 System For sample volumes lt 200 uL use a 0 5 mL Eppendorf LoBind Tube and 0 65 mL tube adaptor For sample volumes between 200 uL and 600 uL use a 1 5 mL Eppendorf LoBind Tube and 1 5 mL Applied Biosystems SOLiD9 4 System Instrument Operation Guide 19 2 Chapter 2 Prepare and Install Slides and Reagents Deposit the beads on the slides tube adaptor For sample volumes between 600 uL and 1 2 mL use a 2 0 mL Eppendorf LoBind Tube and the same adaptor as used for the 1 5 mL tubes Place the tube collar at the indicator line of the adaptor Ensure that the white line on the tube holder is visible Deposit the beads on the slides Wash the beads Note The bead wash procedure is for one WFA or sequencing run sample 1 Sonicate P2 enriched beads using the Covalent Declump 1 program on the Covaris S2 System for program conditions see Appendix G Covalent Declump 1 on
169. tion of buffers Because the tubing from the Instrument and Storage Buffer bottles to the flowcell is long prime the lines before installing the slides in the flowcell Install slide s on the instrument Remove the slide from the Deposition Chamber and prepare it for installation on the instrument Each flowcell can be loaded with the slide independently of each other Install reagent strip s on the instrument Install the workflow analysis or sequencing reagent strips on the reagent strip chiller block Cool the chiller block prior to installation of reagent strips Tips General e Prior to deposition store the slides at 20 C to ensure optimal bead deposition and to minimize loss of P2 enriched beads e Remove the slides from storage at 20 C 5 minutes prior to use If only one slide from the two pack is used place the remaining slide back into the box and place the box back into the resealable pouch The pouch should be resealed and stored at 20 C The stored slide should be used within 1 month Use Eppendorf LoBind Tubes to perform all steps requiring 0 5 mL 1 5 mL and 2 0 mL tubes Eppendorf LoBind Tubes from other vendors may have a chemical coating that can have adverse effects on bead deposition e Adjust microcentrifuge speeds and times according to the g forces specified in the protocols 160 x g 167 x g Covaris S2 The procedures are optimized for the Covaris S2 System The Covaris S2 System Sys
170. to 35 bases for each tag SOLIDO ToP Mate Paired Sequencing Kit M P Lib MM50 50 4452685 SOLIDO ToP Sequencing Kit M P Lib F3 Tag MM50 SOLIDO ToP MP Lib Seq F3 Tag Primer A Master Mix 50 SOLiD9 ToP MP Lib Seq F3 Tag Primer B Master Mix 50 SOLiD9 ToP MP Lib Seq F3 Tag Primer C Master Mix 50 SOLiD9 ToP MP Lib Seq F3 Tag Primer D Master Mix 50 SOLIDO ToP MP Lib Seq F3 Tag Primer E Master Mix 50 SOLIDO Mixing Strip Tube with Zinc SOLIDO ToP Sequencing Kit M P Lib R3 Tag MM50 SOLIDO ToP MP Lib Seq R3 Tag Primer A Master Mix 50 SOLiD9 ToP MP Lib Seq R3 Tag Primer B Master Mix 50 SOLiD9 ToP MP Lib Seq R3 Tag Primer C Master Mix 50 SOLIDO ToP MP Lib Seq R3 Tag Primer D Master Mix 50 SOLIDO ToP MP Lib Seq R3 Tag Primer E Master Mix 50 SOLIDO Mixing Strip Tube with Zinc Mate pair sequencing up to 50 bases for each tag SOLIDO ToP Fragment BC Sequencing Kit BC Frag Lib MM35 5 4452696 SOLiD9 ToP Sequencing Kit Frag Lib F3 Tag MM35 SOLIDO ToP Frag Lib Seq F3 Tag Primer A Master Mix 35 SOLiD ToP Frag Lib Seq F3 Tag Primer B Master Mix 35 SOLIDO ToP Frag Lib Seq F3 Tag Primer C Master Mix 35 SOLIDO ToP Frag Lib Seq F3 Tag Primer D Master Mix 35 SOLIDO ToP Frag Lib Seq F3 Tag Primer E Master Mix 35 SOLIDO Mixing Strip Tube with Zinc SOLIDO ToP Sequencing Kit BC Frag Lib BC Tag MM5 Reset Buffer Multiplex sequencing up to 35 bases for the
171. trument system are not designed or built by Life Technologies Consult the manufacturer s documentation for the information needed for the safe use of these products Instrument safety General CAUTION Do not remove instrument protective covers If you remove the AN protective instrument panels or disable interlock devices you may be exposed to serious hazards including but not limited to severe electrical shock laser exposure crushing or chemical exposure CAUTION Solvents and Pressurized fluids Wear eye protection when working with any pressurized fluids Use caution when working with any polymeric tubing that is under pressure Extinguish any nearby flames if you use flammable solvents Do not use polymeric tubing that has been severely stressed or kinked Do not use polymeric tubing with tetrahydrofuran or nitric and sulfuric acids Be aware that methylene chloride and dimethyl sulfoxide cause polymeric tubing to swell and greatly reduce the rupture pressure of the tubing Be aware that high solvent flow rates 40mL min may cause a static charge to build up on the surface of the tubing and electrical sparks may result Physical injury CAUTION Moving and Lifting Injury The instrument is to be moved and positioned only by the personnel or vendor specified in the applicable site preparation guide Improper lifting can cause painful and permanent back injury Things to consider before lifting or moving the instrument or
172. ts are not available it is possible to estimate the bead concentration using the SOLiD Bead Concentration Chart and NanoDrop spectrometer measurement It is recommended you target an additional overage volume to account for measurement variability especially for off instrument bead quantitation Typical overages can range as high as 20 50 and can vary with the library operator sample type and other factors 6 Place the tube of aliquoted beads in a magnetic rack for at least 1 minute After the solution clears remove and discard the supernatant 7 Remove the SOLiD XD slide s from 20 C storage to equilibrate at room temperature 8 Resuspend the beads in 400 uL of SOLiD XD Slide Deposition Buffer v2 Vortex thoroughly then pulse spin IMPORTANT Do not pellet the beads Applied Biosystems SOLiD9 4 System Instrument Operation Guide 21 2 Chapter 2 Prepare and Install Slides and Reagents Deposit the beads on the slides Prepare the slide s 22 9 10 Place the tube in a magnetic rack for at least 1 minute After the solution clears remove and discard the supernatant Note It may take longer than 1 minute for the solution to clear If necessary slowly pipet the solution up and down in the tube until the beads pellet against the magnet Repeat steps step 8 and step 9 twice Resuspend the beads in the volume of SOLiD XD Slide Deposition Buffer v2 listed in Table 5 calibration of the SOLiD De
173. u solid0062 SOLiD 4 0 User lab user Run Name o Created by e Res Bcc dece Bes ens ec lE min tn cn 0 ce Mis ee ec Sd Setup Sample Slide Sample Slide 9 Manage Runs ij Cycle Scan BD Flow cet 1 EB Flow cen 2 Protocol Protocol i System Status 7 Prime Buffer Line Prime Chiller 4 0 Cooling v Oh o tamp Ohrs 7 No Run Defined Ls Bun control jj No Run Defined Run Control 8 8 a Load Flowcell y Load Flowcell e K Start Ru K Start Run Door Closed Locked rA Clear Flowcell ImagngisOnine Fluidics is Online Salle omof297m 3 Complete the information in the Select Run Type and Mask pane see Figure 42 a Select the type of run If sequencing only one end of the barcoded fragment library select Fragment e If sequencing both ends of the barcoded fragment library select Paired End b Ensure that the Multiplexing option is checked c Optional Type a new run name d Optional Enter a description e Ensure that the Run Protocol is set to SOLiD4 Multiplex f Select BC for Primer Set 1 Enter the read length for the BC Primer according to the barcodes used in the libraries 62 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Section 3 2 Set up and perform a sequencing run Create a multiplex sequencing run record Barcodes used on the slide Set the read length to bases Barcodes 1 16 only 5 Barcodes 17 96 10
174. ube with Zinc SOLIDO ToP Sequencing Kit BC Frag Lib F5 BC Tag MM35 SOLIDO ToP Frag Lib Seq F5 BC Tag Primer A Master Mix 35 SOLiD9 ToP Frag Lib Seq F5 BC Tag Primer B Master Mix 35 SOLiD ToP Frag Lib Seq F5 BC Tag Primer C Master Mix 35 SOLiD9 ToP Frag Lib Seq F5 BC Tag Primer D Master Mix 35 SOLIDO ToP Frag Lib Seq F5 BC Tag Primer E Master Mix 35 SOLIDO Mixing Strip Tube SOLiD9 ToP Sequencing Kit BC Frag Lib BC Tag MM5 Reset Buffer Multiplex paired end sequencing up to 50 bases for the F3 tag of barcoded fragment libraries Barcodes 1 16 SOLiD ToP Paired End Sequencing Kit BC Frag Lib MM50 35 10 4459182 SOLIDO ToP Sequencing Kit Frag Lib F3 Tag MM50 SOLIDO ToP Frag Lib Seq F3 Tag Primer A Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer B Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer C Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer D Master Mix 50 SOLIDO ToP Frag Lib Seq F3 Tag Primer E Master Mix 50 SOLIDO Mixing Strip Tube with Zinc SOLIDO ToP Sequencing Kit BC Frag Lib F5 BC Tag MM35 SOLIDO ToP Frag Lib Seq F5 BC Tag Primer A Master Mix 35 SOLiD9 ToP Frag Lib Seq F5 BC Tag Primer B Master Mix 35 SOLiD ToP Frag Lib Seq F5 BC Tag Primer C Master Mix 35 SOLIDO ToP Frag Lib Seq F5 BC Tag Primer D Master Mix 35 SOLIDO ToP Frag Lib Seq F5 BC Tag Primer E Master Mix 35 SOLIDO Mixing Strip Tube SOLIDO ToP Sequencing Kit BC Frag
175. uencing run Prepare and install slide s and reagents Chapter 2 Create a sequencing run record Create a multiplex sequencing run record Fragment Paired end Multiplex fragment Mate pair Multiplex paired end Detect the focus range Start the sequencing run Control the run Monitor the run Pause resume run View the run log Use the Run Control menu View the heat maps Use image controls View cycle scans Create a sequencing non multiplex run record A sequencing non multiplex run record is created using the SOLiD Instrument Control Software ICS Primary Analysis Settings and Secondary Analysis Settings should be created prior to creating a sequencing run record using SETS refer to the Applied Biosystems SOLiD 4 System SETS Software User Guide Part no 4448411 Create a multiplex sequencing run record A multiplex sequencing run record is created using the SOLiD Instrument Control Software ICS At this step barcodes are matched to libraries The run record file can be created using a spreadsheet program either on or off the instrument Primary Analysis Settings and Secondary Analysis Settings should be created prior to creating a sequencing run record using SETS refer to the Applied Biosystems SOLiD 4 System SETS Software User Guide Part no 4448411 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Section 3 2 Set up and perform a sequencing run 3 Workflow Detect the focus range Two meth
176. umables Item Source KimwipesO wipers Major Laboratory Supplier MLS 1 Atthe back of the instrument remove the plastic filter cover with the filter see Figure 62 Figure 62 Remove the plastic filter cover E WN S ON 2 Separate the plastic cover from the air filter see Figure 64 Figure 63 Separate the plastic cover left from the air filter right 3 Use a Kimwipes wiper to clean the filter If the filter is heavily clogged rinse the filter under running water and dry thoroughly overnight 4 Reinstall the filter and plastic filter cover on the instrument 98 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix B Supplemental Procedures Flush the fluidic lines Flush the fluidic lines If the SOLiD 4 Analyzer is in continuous use the fluidics system in the instrument generally does not need much routine care beyond regular cleaning of the Instrument Buffer bottle and flushing of the lines every three months However if the SOLiD 4 Analyzer will be sitting idle for more than two weeks flush the fluidic system and power off the instrument with the fluidic lines empty Note The instrument flush procedure comprises two scripts Each script takes approximately 13 minutes to complete Required Table 23 Required consumables Flush the fluidic lines consumables Item Source Deionized water Major Laboratory Supplier MLS Slides MLS 1 Open the Instrument Shutdown w
177. ution 1 Cleave Solution 2 1 Parts 1 and 2 Reset Buffer Glycerol Double distilled water Deposition Buffer v2 Filtered pipettor tips Allen wrench 7096 ethanol SOLID Slide Storage Kimwipes Chamber optional Slide Storage Buffer Tabletop centrifuge O 96 well square well O Thaw appropriate reagent storage plate strip s D 00 D DO DD D DD 0 00 3 o g S D 8 da i B s o S 1 S SOLID 4 Analyzer Pipettors D D D D O D U mu 0 uum OO OOO OOO Install reagent strip s 136 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Appendix E Checklists and Workflow Tracking Forms Workflow tracking set up and perform a workflow analysis WFA run Workflow tracking set up and perform a workflow analysis WFA run Sample information pre WFA A600 C NEM mu 1 T 1 beads L uL y y WFAreprt S yO gt A NUES DENEN RN P2 Bp ie A A A E Sample information post WFA e AM TT beads uL Volume Left Beads Left OOO lotes SO ET AS i DepositionBufferv2 Slide Prep Reagent Instrument Bute Storage Butter O TaligaseBuferPati O SSS T4 Ligase Buffer Pat2 S Universal Buffer Pat 1 UniwrsalBuferPatag SOSS COCOCOCOCOCCCCCOCCCCCCC Imaging Buffer Pati fe Imaging BufferPart2 T Cleave Solution 4 T Cleave Solution 2 1 Part CS D E Reset Buffer Slide Storage Buffer Slide Storage Buffer Buffer Workflow Analy
178. vious pre mixed reagent is delivered to the waste and a new reagent is mixed Figure 55 The options to resume or remix resume the run are available Resume Remix Resume Use the pre mixed reagents as is Recommended if pausing time is lt 1 hour Remix Resume Waste the existing pre mixed reagents and make a new mixed reagent Recommeded if pausing time is gt 1 hour Note One reagent strip contains reagents enough for 10 cycles per primer Therefore if the remix option is used the cycle number which can be used with the reagent strip is reduced 1 Click Run Control to display the Run Control menu see Figure 56 Applied Biosystems SOLiD9 4 System Instrument Operation Guide Section 3 2 Set up and perform a sequencing run Control the run Figure 56 Click Run Control to display the Run Control menu for that flowcell SOLID 4 0 User lab_user tails ocked Flow Cell 1 Running Run Name E aoao 20100412 PE MCF7wta 1 Created by lab_user aa Run EA Edt Run H ces U Run Logs Heat Map Sample Slide 1 Sample in 1 spot mask sf El Details MCF Description primaryAnalysisSetting E Libraries Secondary defa none Ly default primary El Sample Loading 1 Protocol b SOLID4 F5 BC F3 25 50 bases Pre Sco ae F560 DERRER Eunn ie ttt i 1 i F3 i F5 BC PrimerD Ligation 5 Scan Slide gt HERRRRERRRRRRRRRRRRRRRRRRRHR
179. ware will be free of defects in materials and workmanship under normal use The above warranties do not apply to defects resulting from misuse neglect or accident including without limitation operation outside of the environmental or use specifications or not in conformance with the instructions for any instrument system software or accessories improper or inadequate maintenance by the user installation of software or interfacing or use in combination with software or products not supplied or authorized by Life Technologies intrusive activity including without limitation computer viruses hackers or other unauthorized interactions with instrument or software that detrimentally affects normal operations and modification or repair of the products not authorized by Life Technologies Warranty Period Commencement Date The applicable warranty period for software begins on the earlier of the date of installation or three 3 months from the date of shipment for software installed by Life Technologies personnel For software installed by the purchaser or anyone other than Life Technologies the warranty period begins on the date the software is delivered to you The applicable warranty period for media begins on the date the media is delivered to the purchaser APPLIED BIOSYSTEMS MAKES NO OTHER WARRANTIES OF ANY KIND WHATSOEVER EXPRESS OR IMPLIED WITH RESPECT TO THE SOFTWARE OR DOCUMENTATION INCLUDING BUT NOT LIMITED TO WARRANTIES OF FIT
180. wcell 1 Close the front doors of the SOLiD 4 Analyzer Open the Imager window by then find the beads choosing Window Imaging System on the slide 2 Select the Show Flowcell box in the bottom left hand corner of the Imager window then choose the number of the flowcell to set Flowcell 1 or Flowcell 2 see Figure 77 Figure 77 Click Show Flowcell to find the beads on the slide ity SOLiD Imager 1 9 8 1 File View Run Options Devices Tools Windows Help Slide Shape CAMERA Info Status BEH Acquire AutoExpose Camera SN 266272 IP 127 0 0 1 Port 8081 Stage Status Exposure Time 216 E Gain 32 Binning 1 v Port Status Motion Position Speed Acceleration Bit Depth 8 y Saturate Rate 0 01 GoLive Default View Clear All Save As COMI READY x 8917 00 100 100 m Shutter Status Focus Status Filter Status Nosepiece Status Y 2669900 100 100 BENE com ENS cov E come L COME Stage Control 658683 a Nudge Size 500 q Re Calibrate Stage I Go ToX en Update Stage Status bo Tol ES 4 Sus gt Cote 256904 Zoom In Flowcell Stop Auto Focus V Enable Joysti Y Go To Fiduciary Mark v Enable Jog 1 ShowFlowCell 1 XY Iu IHE GoTo j GoTo h 3 3 From the View menu choose Stage Template then navigate to the C Runs directory Find the folder in the Runs directory that corresponds to the run just set up then choose the STG file for the run Choosing Stage Template a
181. y be considered a biohazard Use appropriate decontamination methods when working with biohazards 158 Applied Biosystems SOLID 4 System Instrument Operation Guide Appendix J Safety J Biological hazard safety A WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following In the U S U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cdc gov biosafety Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Add
182. ycle Scan ED Flow ce 1 EB Flow cel 2 Protocol Protocol Prime Buffer Line Prime Chiller 4 0 C Cooling Y Lamp 0 hi O ame PS No Run Defined Lov Run control jj No Run Defined v Run Control 8 l l s Load Flowcell y Load Flowcell K Start Ru K Start Rur oec ecos E Unlock Doors Imagingis Online Fluidics is Online I SoM of 297M 9 2 During priming open the middle front door of the SOLiD 4 Analyzer to check that the syringe is filled with buffer when the plunger is at the aspiration stage see Figure 14 Confirm that the buffer lines are filled with buffer If the syringe is not filled with buffer even at the last stroke click Prime again Applied Biosystems SOLiD9 4 System Instrument Operation Guide Chapter 2 Prepare and Install Slides and Reagents Install slide s on the instrument Figure 14 Syringe on the SOLiD 4 Analyzer I Install slide s on the instrument Prepare the slide IMPORTANT Before removing the slide from the SOLiD Deposition Chamber ensure that either the instrument flowcell is ready or a SOLiD Slide Storage Chamber is available 1 Remove the 3 mm adhesive disks 2 Pour enough SOLiD XD Slide Deposition Buffer v2 to cover the top of the SOLiD Deposition Chamber 1 5 mL in each port for the 1 Well SOLiD Deposition Chamber and 6 mL for the 4 Well and 8 Well SOLiD Deposition Chambers
183. ys Multiplex paired end 50 35 5 bp 13 15 days Multiplex paired end 50 35 10 bp 14 16 days T The estimated time is for a dual slide run Estimated times may vary due to differences in imaging time Applied Biosystems SOLiD9 4 System Instrument Operation Guide 131 D Appendix D Instrument Process Times Times for entire processes 132 Applied Biosystems SOLID 4 System Instrument Operation Guide SOLiD9 4 Analyzer Plumbing System Schematic BEES ens SOLID 4 Analyzer PLUMBING SYSTEM SCHEMATIC PIN 4387534 CHEMISTRY CABINET SIDEWALL UPPER LEFT CLEAVE SOLUTION 2 TUBE ASSEMBLY CLEAVE SOLUTION 1 TUBE ASSEMBLY po SNL a5 FLOWCELLS AO wstrumenr 122 UNIVERSAL BUFFER WASTE BOTTLE TUBE ASSEMBLY um D TUBE ASSEMBLY Ew EE ucase Y t t Do k FLOWCELL 1 TUBE ASSEMBLY TUBE ASSEMBLY 5 RESET n FLOWCELL 2 TUBE ASSEMBLY 1 TUBE ASSEMBLY R aum IMAGING BUFFER 2 TUBE ASSEMBLY T TUBE ASSEMBLY d SPILL TRAY 12 1 SWITCHING MANIFOLD VALVE INSTRUMENT BUFFER TUBE ASSEMBLY WASTE MANIFOLD INSTRUMENT WASTE BOTTLE TUBE ASSEMBLY BUFFER SPLITTER WASTE TUBE COUPLER GANTRY N UBE ASSEMBLY STORAGE BUFFER TUBE ASSEMBLY 8 BUFFER COMMON TUBE ASSEMBLY ROBOT GANTRY BUFFER IMAGING CHEMISTRY WASTE SIDECART SPILL TRAY Applied Biosystems SOLiD9 4 System Instrument Operation Guide 133 Appendix E SOLIDO 4 Analyzer P

CMS_082581 - Applied Biosystems

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