SureSelect Target Enrichment System for SOLiD 5500 Multiplexed
Contents
1. Figure 6 Prepped library shown in red Prepped Library 42 SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing Hybridization 3 Table 21 PCR program Step Temperature Time Step 1 95 C 5 minutes Step 2 65 C Hold 8 Use a heated lid on the thermal cycler at 105 C to hold the temperature of the plate on the thermal cycler at 65 C CAUTION The lid of the thermal cycler is hot and can cause burns Use caution when working near the lid 9 While the sample incubates at 95 C combine 13 uL of hybridization buffer with 7 uL of prepared SureSelect Capture Library mix at room temperature 10 When the thermal cycler reaches 65 C maintain the plate at 65 C while you use a multi channel pipette to add 20 uL of the hybridization buffer and SureSelect Capture Library mix to the prepped library Slowly pipette up and down 2 to 3 times to mix The hybridization mixture is now 27 to 29 uL depending on the degree of evaporation during the 95 C step 11 Seal the wells with strip caps or double adhesive film Make sure all wells are completely sealed Use new adhesive seals or strip caps The structural integrity of the seals and caps can be compromised during the 95 C step 12 Incubate the hybridization mixture for 24 hours at 65 C with a heated lid at 105 C Samples can be hybridized for up to 72 hours but when you hybridize at longer periods test the sample to make sure that evaporation2. CAUTION To avoid cross contaminating libraries set up PCR reactions all components except the library DNA in a dedicated clean area or PCR hood with UV sterilization and positive air flow Prepare 1 amplification reaction for each hybrid capture Include a negative no template control To see the nucleotide sequence in each of the barcode included in SureSelect reagent kits see SureSelect Barcodes for SOLID on page 66 1 For 1 library In a PCR tube strip tube or plate prepare the reaction mix in Table 22 on ice Mix well by gently pipetting up and down 2 For multiple libraries a Prepare the reaction mix in Table 22 on ice Mix well on a vortex mixer b Add 34 uL of the reaction mix to each well or tube c Add 2 uL of the appropriate barcode SureSelect LT BC1 through BC16 clear cap from the SureSelect 55500 Indexing Construction Kit or BC1 through BC96 from the SureSelect LTI5500 BC1 BC96 plate to each well and mix by pipetting See Figure 9 SureSelect LTI5500 BC1 BC96 plate barcode orientation on page 70 to locate the barcodes on the index plate Use a different barcode primer for each sample to be sequenced in the same lane Use Table 23 as a guide to determine the number of barcodes to pool per sequencing lane 48 SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing Addition of Barcode Tags by Post Hybridization Amplification 4 Include full sets of 4 barcodes in each l
3. If you do not continue to the next step seal the sheared DNA sample plate and store at 4 C overnight or at 20 C for prolonged storage SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 23 2 Sample Preparation Step 3 Assess quality with the 2100 Bioanalyzer 4000 3000 Sample Intensity FU o o 1000 el MW 13 bp Figure 3 Analysis of sheared DNA using the 2200 TapeStation with a D1K ScreenTape The electropherogram shows an average DNA fragment size of about 150 bp 1000 si 300 1 24 SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing Step 4 Repair the ends Sample Preparation 2 Use reagents from the SureSelect XT Library Prep Kit 55500 1 For 1 library In a 1 5 mL LoBind tube strip tube or plate prepare the reaction mix in Table 13 on ice Mix well by gently pipetting up and down 2 For multiple libraries a Prepare the reaction mix in Table 13 on ice Mix gently on a vortex mixer b Add 52 uL of the reaction mix to each well or tube Use a pipette to add 48 uL of each DNA sample to each well or tube Mix by pipetting Change pipette tips between samples Table 13 End Repair Reagent Volume for 1 Library pL Volume for 12 Libraries pL includes excess Sheared DNA 10x End Repair Buffer clear cap dNTP Mix green cap T4 DNA Polymerase purple cap Klenow DNA Polymerase yellow cap
4. 62 SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing Reference 5 Table 29 SureSelect Target Enrichment Box 2 55500 Kit Component SureSelect Hyb 3 yellow cap SureSelect Indexing Block 1 green cap SureSelect Block 2 blue cap SureSelect LT Indexing Block 3 brown cap SureSelect RNase Block purple cap Table 30 SureSelect XT Library Prep Kit 55200 Kit Component 10x End Repair Buffer clear cap T4 Polynucleotide Kinase orange cap 10x Klenow Polymerase Buffer blue cap T4 DNA Ligase red cap Exo Klenow red cap T4 DNA Polymerase purple cap Klenow DNA Polymerase yellow cap dATP green cap dNTP Mix green cap 5x T4 DNA Ligase Buffer SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 63 5 64 Reference Table31 SureSelect 5500 Indexing Construction Kit Component SureSelect 1715500 P1 purple cap SureSelect 1715500 IA blue cap SureSelect LTI Pre Capture Primer green cap SureSelect LT BC1 through BC16 clear cap or SureSelect 1715500 BC1 BC96 plate SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing Reference 5 Other Reagent Kits Content These reagents are from kits other than the SureSelect Reagent kit Make sure you use only the reagents listed here Table 32 Herculase Il Fusion DNA Polymerase Agilent Component DMSO green cap 5x Herculase Il Rxn B
5. Table 4 Required Reagents for Hybridization Description Vendor and part number Dynabeads MyOne Streptavidin T1 Life Technologies 2mL Cat 65601 10 mL Cat 65602 100 mL Cat 65603 Nuclease free Water not DEPC treated Ambion Cat AM9930 Optional Reagents Table5 Optional Reagents Description Vendor and part number SureSelect gDNA Extraction Kit 50 reaction kit Agilent p n G7505A 250 reaction kit Agilent p n G7505B SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 11 1 12 Before You Begin Required Equipment Table 6 Required Equipment for Library Prep and Post Hybridization Amplification Description Vendor and part number 2100 Bioanalyzer or 2200 TapeStation System Mx3005P Real Time PCR System Thermal cycler Covaris Sample Preparation System S series or E series model Covaris microTUBE with AFA fiber and snap cap Eppendorf Microcentrifuge Model 5417R Eppendorf fixed angle rotor with standard lid DNA LoBind Tubes 1 5 mL PCR clean 250 pieces Qubit Fluorometer E Gel iBase and E Gel Safe Imager Combo Kit or Safe Imager Real Time Transilluminator and E Gel iBase Power System Dynal DynaMag 2 magnetic stand P10 P20 P200 and P1000 pipettes Vacuum concentrator Ice bucket Agilent p n G2938C Agilent p n G2964AA or G2965AA Agilent p n 401449 or equivalent Agilent SureCycler Life Technologies Veriti Thermal Cycler BioRad MJ Resea
6. FU M xls MW 2 e S s bp Figure 5 Analysis of amplified library DNA using the 2200 TapeStation with a DTK ScreenTape The electropherogram shows an average DNA fragment size of about 200 bp SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 37 2 Sample Preparation Step 12 Assess quality and quantity with the 2100 Bioanalyzer 38 SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing CAUTION SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing Protocol 3 e Hybridization Step 1 Hybridize the library 40 Step 2 Prepare magnetic beads 44 Step 3 Select hybrid capture with SureSelect 45 This chapter describes the steps to combine the prepped library with the hybridization reagents blocking agents and the SureSelect capture library The ratio of SureSelect capture library to prepped library is critical for successful capture CAUTION Refer to SureSelect Reagent Kit Content on page 62 for a complete content listing of each SureSelect Target Enrichment kit You must avoid evaporation from the small volumes of the capture during the 24 hour or greater incubation If you want to use a different combination of thermal cycler lid temperature plates or strips and sealing method strip caps or sealing tape first test the conditions Incubate 29 uL of SureSelect Hybridization Buffer without DNA at 65 C for 24 hours or l
7. 5 mL LoBind tube You can discard the beads at this time If you do not continue to the next step store the samples at 4 C for up to a week or at 20 C for longer periods SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing Addition of Barcode Tags by Post Hybridization Amplification 4 Step 3 Remove primer dimers from the sample using Agencourt AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes 2 Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze 3 Add 60 uL of homogenous AMPure beads to a 1 5 mL LoBind tube and add amplified library 750 uL Mix well on a vortex mixer and incubate for 5 minutes 4 Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes 5 Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes 6 Continue to keep the tube in the magnetic stand while you dispense 500 uL of 70 ethanol in each tube Use fresh 70 ethanol for optimal result 7 Let the tube sit for 1 minute to allow any disturbed beads to settle and remove the ethanol 8 Repeat step 6 and step 7 once 9 Dry the samples on the 37 C heat block for 5 minutes or until the residual ethanol is completely evaporated Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency
8. 7 Verify the results Check that the electropherogram shows a distribution with a peak size around 150 bp Stopping Point If you do not continue at the next step store the purified DNA in RNase free water at 4 C 22 SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing CAUTION Sample Preparation 2 Post Shearing Cleanup 1 T T t 00 400 500 1000 1500 be Figure 2 Analysis of sheared DNA using a DNA 1000 Bioanalyzer assay The electro pherogram shows a single peak in the size range of 150 bp 2200 TapeStation and D1K ScreenTape You can use the 2200 TapeStation for rapid analysis of multiple samples Use the D1K ScreenTape and D1K Reagents For more information to do this step see the Agilent 2200 TapeStation User Manual 1 Prepare the TapeStation samples as instructed in the Agilent 2200 TapeStation User Manual Use 1 uL of each sheared DNA sample diluted with 3 uL of D1K sample buffer for the analysis Make sure that you thoroughly mix the combined DNA and D1K sample buffer on a vortex mixer for 5 seconds or more for accurate quantitation Stopping Point 2 Load the sample plate or tube strips from step 1 the DIK ScreenTape and loading tips into the 2200 TapeStation as instructed in the Agilent 2200 TapeStation User Manual Start the run 3 Verify that the electropherogram shows an average DNA fragment size of about 150 bp A sample electropherogram is shown in Figure 3
9. Items and DFARS 227 7202 3 Rights in Commercial Computer Software or Com puter Software Documentation Safety Notices CAUTION A CAUTION notice denotes a haz ard It calls attention to an operat ing procedure practice or the like that if not correctly performed or adhered to could result in damage to the product or loss of important data Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met A WARNING notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly per formed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indicated condi tions are fully understood and met SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing In this Guide This guide describes the recommended operational procedures to capture genomic regions of interest using the Agilent SureSelect Target Enrichment System Kit for SOLiD 5500 Multiplex Sequencing This protocol is specifically developed and optimized to use Biotinylated RNA oligomer libraries or Bait to enrich targeted regions of the genome from repetitive sequences and sequences unrelated to the research focus The SureSelect T Target Enrichment System Kit for SOLiD 5500 Multiplex Sequencing is designed to work on the SOLiD 5500 system For 5011 4 systems refer to the Target Enrichment
10. System and DNA 1000 Assay 1 Check that the 2100 Bioanalyzer electrodes have been cleaned and dried as instructed in the reagent kit guide Open the Agilent 2100 Expert Software version B 02 07 or higher turn on the 2100 Bioanalyzer instrument and check communication Prepare the chip samples and ladder as instructed in the reagent kit guide then mix on the IKA vortex mixer that is included with the 2100 Bioanalyzer instrument Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation Within the instrument context choose the appropriate assay from the drop down list Start the run Enter sample names and comments in the Data and Assay context Verify the results Check that the electropherogram shows a distribution with a peak size around 200 bp SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 35 2 Sample Preparation Pre Capture Amp 6 Cycle 4 200bp Pre Capture Amp H 200bp Pre Capture Amp i 50 100 19 20 00 50 700 1000 1500 te Figure 4 Analysis of amplified prepped library DNA using a DNA 1000 assay The elec tropherogram shows a single peak in the size range of 200 bp 8 Ifthe concentration of your sample is greater than the high end of the dynamic range of the Bioanalyzer DNA 1000 assay gt 50 ng uL use the Qubit Fluorometer to quantitate the library Dilu
11. library template As an alternative you can prepare one PCR master mix as outlined in Table 16 Split the master mix into three small scale 10 pL PCR reactions and run for 4 5 or 6 cycles Clean these PCR reactions using the AMPure XP protocol outlined in Step 11 Purify the sample using the Agencourt AMPure XP beads with these modifications Use 30 pL of AMPure XP beads and elute with 20 uL of nuclease free water Run these cleaned samples on DNA1000 chip on the Bioanalyzer as described in Step 12 Assess quality and quantity with the 2100 Bioanalyzer Use the optimal cycle number to repeat PCR at the 50 reaction scale SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 33 2 Sample Preparation Stopping Point 34 Step 11 Purify the sample using the Agencourt AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze Add 90 uL of homogenous AMPure XP beads to a 1 5 mL LoBind tube and add the ligated library 50 uL Mix well on a vortex mixer and incubate for 5 minutes Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes Continue to keep the tube in the magnetic stand while
12. pL sample 3 Incubate at 22 C for 15 minutes SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 29 2 Sample Preparation Stopping Point 30 Step 9 Purify the sample using the Agencourt AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze Add 90 uL of homogenous AMPure XP beads to a 1 5 mL LoBind tube and add the ligated DNA library 50 uL Mix well on a vortex mixer and incubate for 5 minutes Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes Continue to keep the tube in the magnetic stand while you dispense 0 5 mL of 70 ethanol in each tube Use fresh 70 ethanol for optimal result Let the tube sit for 1 minute to allow any disturbed beads to settle and remove the ethanol Use fresh 70 ethanol for optimal result Repeat step 6 and step 7 once Dry the samples on the 37 C heat block for 5 minutes until the residual ethanol is completely evaporated Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency is significantly decreased when the bead pellet is excessively dried 10 Add 30 uL nuclease free water mix well a
13. reagents are included the SureSelect XT Library Prep Kit 55500 3 Incubate in a thermal cycler for 30 minutes at 37 C If you use a heated lid make sure that the lid temperature does not exceed 50 C SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 27 2 Sample Preparation Step 7 Purify the sample using Agencourt AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze Add 90 uL of homogenous AMPure XP beads to a 1 5 mL LoBind tube and add the A tailed DNA library 50uL Mix well on a vortex mixer and incubate for 5 minutes Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes Continue to keep the tube in the magnetic stand while you dispense 500 uL of 70 ethanol in each tube Use fresh 70 ethanol for optimal result Let the tube sit for 1 minute to allow any disturbed beads to settle and remove the ethanol Repeat step 6 and step 7 step once Dry the samples on the 37 C heat block for 5 minutes or until the residual ethanol completely evaporates Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency is significantly decreased w
14. start the run within five minutes after preparation Within the instrument context choose the appropriate assay from the drop down list Start the run Enter sample names and comments in the Data and Assay context Verify the results SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing CAUTION Addition of Barcode Tags by Post Hybridization Amplification 4 Post Capture Amp Ib Capture 8 Cycles oy U EES Se ee RS Ls ee ee meine T r T T T T T T 100 150 200 300 400 500 1000 2000 10380 te Figure 7 Analysis of Amplified Capture DNA using the High Sensitivity DNA Kit The electropherogram shows a peak in the size range of approximately 260 bp 2200 TapeStation and High Sensitivity DIK ScreenTape Use the 2200 TapeStation to analyze the barcoded DNA Use the High Sensitivity ScreenTape and High Sensitivity D1K Reagents For more information to do this step see the Agilent 2200 TapeStation User Manual 1 Prepare the TapeStation samples as instructed in the Agilent 2200 TapeStation User Mamual Use 2 uL of each amplified library DNA sample diluted with 2 uL of High Sensitivity sample buffer for the analysis Make sure that you thoroughly mix the combined DNA and D1K sample buffer on a vortex mixer for 5 seconds or more for accurate quantitation Stopping Point 2 Load the sample plate or tube strips from step 1 the High Sensi
15. you dispense 500 uL of 70 ethanol in each tube Use fresh 70 ethanol for optimal result Let the tube sit for 1 minute to allow any disturbed beads to settle and remove the ethanol Repeat step 6 and step 7 once Dry the samples on the 37 C heat block for 5 minutes until the residual ethanol completely evaporates Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency is significantly decreased when the bead pellet is excessively dried 10 Add 30 uL nuclease free water mix well on a vortex mixer and incubate for 2 minutes at room temperature 11 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 12 Remove approximately 30 uL of the supernatant to a fresh 1 5 mL LoBind tube You can discard the beads at this time If you do not continue to the next step store the samples at 20 C SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing Sample Preparation 2 Step 12 Assess quality and quantity with the 2100 Bioanalyzer The hybridization protocol in the following section requires 500 ng of each amplified DNA library Measure the concentration of each library using one of the methods detailed below Once DNA concentration for each sample is determined calculate the volume of the library to be used for hybridization using the following formula Volume uL 500 ng concentration ng uL 2100 Bioanalyzer
16. Bind tube and add the sheared DNA library 130 uL Mix well on a vortex mixer and incubate for 5 minutes Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes Add 91 uL of homogenous AMPure XP beads to a new 1 5 mL LoBind tube Keep the original tube in the magnetic stand Do not touch the beads while you carefully move the cleared solution approximately 273 uL from the original tube to the new 1 5 mL LoBind tube Discard the beads from the original tube Mix well on a vortex mixer and incubate for 5 minutes Put the new tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes Keep the tube in the magnetic stand Do not touch the beads while you carefully remove the cleared solution from the tubes 10 Continue to keep the tube in the magnetic stand while you dispense 0 5 mL of 70 ethanol in each tube Use fresh 70 ethanol for optimal result 11 Let the tube sit for 1 minute to allow any disturbed beads to settle and remove the ethanol 12 Repeat step 10 and step 11 once 13 Dry the samples on the 37 C heat block for 5 minutes until the residual ethanol is completely evaporated Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency is significantly decreased when the bead pellet is excessively dried 14 Add 50 uL nuclease free water mix well on a vortex mixer and incubate for 2 minutes at
17. C81 BC89 BC2 BC10 BC18 26 BC34 BC42 BC50 BC58 BC66 BC74 BC82 90 BC3 BC11 BC19 BC27 BC35 BC43 BC51 BC59 BC67 BC75 BC83 BC91 BC12 BC20 BC28 BC36 BC44 BC52 BC60 BC68 BC76 BC84 BC92 BCS BC13 BC21 BC29 BC37 BC45 BC53 BC61 BC69 BC77 BC85 BC93 BC6 BC14 BC22 BC30 BC38 BC46 BC54 62 70 BC78 86 BC94 BC15 BC23 BC31 BC39 BC47 BC55 BC63 BC71 BC79 BC87 BC95 BC8 BC16 BC24 BC32 BC40 BC48 BC56 BC64 BC72 BC80 BC88 96 Figure 9 SureSelect LTI5500 BC1 BC96 plate barcode orientation 70 SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing Alternative Capture Equipment Combinations Reference 5 Table 36 lists combinations of thermal cycler lid temperature plates or strips and sealing method strip caps or sealing tape other than those used in this protocol that have shown minimal evaporation Refer to this list for additional of equipment combination options for hybridization Note that minimal evaporation is needed to ensure good capture results Table 36 Tested options that show minimal evaporation PCR Machine Plate Strips Cover Comments Agilent Mx3005P Mx3000P Strip Tubes MX3000P Optical Strip Heated Lid QPCR 401428 Caps 401425 Agilent Mx3005P MicroAmp Optical MicroAmp
18. Clear Heated Lid QPCR ABI GeneAmp 9700 ABI Veriti 4375786 Eppendorf Mastercycler BioRad MJ Research PTC 200 BioRad MJ Research PTC 200 BioRad MJ Research PTC 200 96 well reaction plate N801 0560 MicroAmp Optical 96 well Reaction Plate N801 0560 MicroAmp Optical 96 well Reaction Plate N801 0560 Eppendorf 8 Tube PCR Tubes Agilent strip tubes 410022 Mx4000 Agilent strip tubes 410022 Mx4000 Agilent 96 well Plate 410088 Mx3000 3005 Adhesive Film 4306311 MicroAmp Caps 8caps strip N801 0535 MicroAmp Clear Adhesive Film 4306311 Attached lids Agilent Optical cap 410024 Mx4000 Agilent Optical cap 401425 Mx3000 3005 Agilent Optical cap 401425 Mx3000 3005 ABI compression pad 4312639 Use two layers of film Heated Lid Heated Lid ABI compression pad 4312639 Use two layers of film Lid heating set to 75 C Heated Lid Heated Lid Heated Lid SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 71 5 Reference Alternative Capture Equipment Combinations 72 SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing www agilent com In This Book This guide contains information to run the SureSelect Target Enrichment System for SOLiD 5500 Multiplexed Sequencing protocol with the SureSelect Target Enrichment Kits for AB SOLiD 5500 Multiplexed Sequencing Agilent Tec
19. SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing Protocol Version A1 August 2015 SureSelect platform manufactured with Agilent SurePrint Technology For Research Use Only Not for use in diagnostic procedures GE Agilent Technologies Notices Agilent Technologies Inc 2012 2015 No part of this manual may be reproduced in any form or by any means including elec tronic storage and retrieval or translation into a foreign language without prior agree ment and written consent from Agilent Technologies Inc as governed by United States and international copyright laws Manual Part Number 67530 90004 Edition Version 1 August 2015 Agilent Technologies Inc 5301 Stevens Creek Rd Santa Clara CA 95051 USA Acknowledgement Oligonucleotide sequences 2006 and 2008 Applied Biosystems a division of Life Technologies Inc All rights reserved Only for use with the Applied Biosystems SOLID System Sequencing and associated assays Technical Support For technical product support contact your local Agilent Support Services representa tive For US and Canada call 800 227 9770 option 3 4 4 For other countries find your support center telephone numbers at www agilent com chem contactus Or send an e mail to SureSelect Support agilent com SureSelect capture libraries and reagents must be used within one year of receipt Warranty The material contained
20. T4 Polynucleotide Kinase orange cap Nuclease free water Total Volume 48 10 1 6 2 2 2 35 2 100 125 20 12 5 25 27 5 440 650 52 pL sample 3 Incubate the mixture at 22 C for 30 minutes SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 25 2 Sample Preparation Stopping Point 26 Step 5 Purify the sample using the Agencourt AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze Add 180 uL of homogenous AMPure XP beads to a 1 5 mL LoBind tube and add the repaired DNA library 100 uL Mix well on a vortex mixer and incubate for 5 minutes Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes Continue to keep the tube in the magnetic stand while you dispense 0 5 mL of 70 ethanol in each tube Use fresh 70 ethanol for optimal result Let the tube sit for 1 minute to allow any disturbed beads to settle and remove the ethanol Repeat step 6 and step 7 once Dry the samples on the 37 C heat block for 5 minutes until the residual ethanol is completely evaporated Do not dry the bead pellet to the point that the bead pellet appears cracked Elution effic
21. TATCGTGAGT 40 AAAAGGGTTA 4 TGTGGGATTG 42 GAATGTACTA 43 CGCTAGGGTT 44 AAGGATGATC SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 67 5 Reference Table 35 SureSelect Barcodes 1 to 96 continued Barcode Number Sequence 45 GTACTTGGCT 46 GGTCGTCGAA 47 GAGGGATGGC 48 GCCGTAAGTG 49 ATGTCATAAG 50 GAAGGCTTGC 51 AAGCAGGAGT 52 GTAATTGTAA 53 GTCATCAAGT 54 AAAAGGCGGA 55 AGCTTAAGCG 56 GCATGTCACC 57 CTAGTAAGAA 58 TAAAGTGGCG 59 AAGTAATGTC 60 GTGCCTCGGT 61 AAGATTATCG 62 AGGTGAGGGT 63 GCGGGTTCGA 64 GTGCTACACC 65 GGGATCAAGC 66 GATGTAATGT 67 GTCCTTAGGG 68 GCATTGACGA SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing Reference Table 35 SureSelect Barcodes 1 to 96 continued Barcode Number Sequence 69 GATATGCTTT 70 GCCCTACAGA 71 ACAGGGAACG 72 AAGTGAATAC 73 GCAATGACGT 74 AGGACGCTGA 75 GTATCTGGGC 76 AAGTTTTAGG 77 ATCTGGTCTT 78 GGCAATCATC 79 AGTAGAATTA 80 GTTTACGGTG 81 GAACGTCATT 82 GTGAAGGGAG 83 GGATGGCGTA 84 GCGGATGAAC 85 GGAAAGCGTT 86 AGTACCAGGA 87 ATAGCAAAGC 88 GTTGATCATG 89 AGGCTGTCTA 90 GTGACCTACT 91 GCGTATTGGG 92 AAGGGATTAC SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 69 5 Reference Table 35 SureSelect Barcodes 1 to 96 continued Barcode Number Sequence 93 GTTACGATGC 94 ATGGGTGTTT 95 GAGTCCGGCA 96 AATCGAAGAG BC1 BC9 BC17 BC25 BC33 BC41 BC49 57 BC65 BC73 B
22. ane A full set of barcodes refers to BC1 through BC4 BC5 through BC8 BC9 through BC12 etc If the number of libraries to be combined in a sequencing lane is not a multiple of 4 based on guides in Table 23 then use multiple barcoding primers in equal ratios to amplify a single library For example for a sequencing sample designed to contain 2 libraries amplify each library using 2 barcoding primers For 3 libraries amplify each library using 4 barcoding primers for a total of 12 barcodes in the sequencing sample d Pipette each DNA sample up and down to make sure that the bead solution is homogenous e Use a pipette to add 14 uL of each DNA sample to each well or tube Mix by pipetting Change pipette tips between samples to avoid cross contamination Table 22 Herculase Master Reagent Volume for 1 Volume for 12 reactions reaction includes excess Captured on bead DNA 14 uL Nuclease free water 22 5 uL 281 25 uL 5x Herculase Il Rxn Buffer clear cap 10 uL 125 uL 100 mM dNTP Mix green cap 0 5 uL 6 25 uL Herculase II Fusion DNA Polymerase red 12 5 pL SureSelect LT BC1 through BC16 clear cap 2 uL Total 50 uL 425 pL 34 pL reaction Included in the Herculase Fusion DNA Polymerase Agilent Do not use the buffer or dNTP mix from any other kit SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 49 4 50 Addition of Barcode Tags by Post H
23. code Tags by Post Hybridization Amplification Step 6 Pool samples for Multiplexed Sequencing 60 SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing Protocol e amp 5 SureSelect Reagent Kit Content 62 Other Reagent Kits Content 65 SureSelect Barcodes for SOLID 66 Alternative Capture Equipment Combinations 71 This chapter contains reference information Agilent Technologies 61 5 Reference SureSelect Reagent Kit Content SureSelect capture libraries and reagents must be used within one year of receipt Each SureSelect Reagent Kit contains one or more of each of these individual kits Table27 SureSelect Reagent Kit Contents Product Storage 16 96 Condition Reactions Reactions SureSelect T Target Enrichment Box 1 5500 Room 5190 5931 5190 5932 Temperature SureSelect Target Enrichment Box 2 5500 20 C 5190 5929 5190 5930 SureSelect XT Library Prep Kit 55500 20 C 5500 0112 5500 0113 SureSelect 5500 Indexing Construction Kit 20 C 5190 5455 5190 5456 The content of each of these kits are described in the next tables Table 28 SureSelect Target Enrichment Box 1 55500 Kit Component SureSelect Hyb 1 orange cap or bottle SureSelect Hyb 2 red cap SureSelect Hyb 4 black cap or bottle SureSelect Binding Buffer SureSelect Wash 1 SureSelect Wash 2
24. e Block dilution for all samples plus excess d Add the amount of diluted SureSelect RNase Block purple cap listed in Table 19 to each capture library and mix by pipetting Table 19 SureSelect Capture Library Capture Size Volume of SureSelect RNase Block Dilution Volume of RNase Library Parts RNase block Block Dilution to Add Parts water lt 3 0 Mb 2 uL 1 9 1096 5yL gt 3 0 Mb 5 uL 1 3 25 2yL SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 41 3 Hybridization 6 Mix the contents in Table 20 to make the correct amount of SureSelect Block mix for the number of samples used Table 20 SureSelect Block Mix Reagent Volume for 1 reaction Volume for 12 reactions includes excess SureSelect Indexing Block 1 green cap 2 5 uL 31 25 uL SureSelect Block 2 blue cap 2 5 uL 31 25 uL SureSelect LT Indexing Block 3 brown cap 0 6 pL 7 5 pL Total 5 6 uL 70 uL 7 Ina separate PCR plate prepare the prepped library for target enrichment a Add 3 4 uL of 147 ng uL prepped library to the C row in the PCR plate Put each sample into a separate well b Add 5 6 uL of the SureSelect Block Mix to each well in row C c Mix by pipetting up and down d Seal the wells of row with caps and put the PCR plate in the thermal cycler Do not heat the Hybridization Buffer or capture library yet only the prepped library with blockers e Start the thermal cycler program in Table 21
25. encing 31 2 Sample Preparation Table 16 Components for PCR mix Reagent Volume for 1 Volume for 12 reactions reaction includes excess Adaptor ligated library 14 uL Nuclease free water 20 5 uL 256 25 pL SureSelect LTI Pre Capture Primer green 4 uL 50 uL 5x Herculase Il Rxn Buffer clear 10 uL 125 uL 100 mM dNTP Mix green 0 5 pL 6 25 uL Herculase Il Fusion DNA Polymerase red 1 uL 12 5 uL Total 50 pL 450 pL 36 pL reaction Included the SureSelect 55500 Indexing Construction Kit t Included in the Herculase II Fusion DNA Polymerase Agilent kit Do not use the buffer or dNTP mix from any other kit 3 Run the program in Table 17 in a thermal cycler Table 17 Step Temperature Time Step 1 98 C 2 minutes Step 2 98 C 30 seconds Step 3 54 C 10 seconds Step 4 72 C 1 minute Step 5 Repeat Step 2 through Step 4 for a total of 4 to 6 times Step 6 72 C 10 minutes Step 7 4 C Hold 32 SureSelect Target Enrichment System for 5011 5500 Multiplexed Sequencing Sample Preparation 2 Different library preparations can produce slightly different results based on varying DNA quality In most cases 5 cycles will produce an adequate yield for subsequent capture without introducing bias or non specific products If yield is too low or non specific high molecular weight products are observed adjust the number of cycles accordingly with the remaining extra
26. he initial concentration of each barcoded sample See Table 25 for the approximate volume of sample to use Table25 Approximate volume of sample to use SOLiD Sequencing Capacity Approximate Sample Final Concentration Volume Needed Needed 1 Lane 50 uL 500 pM Table 26 shows an example of the amount of 4 barcoded samples of different concentrations and Low TE needed for a final volume of 100 uL at 500 pM Table 26 Example of barcode volume calculation for a total volume of 100 uL Component V f C i C f Volume to use pL Sample 1 100 uL 921 pM 500 pM 4 13 6 Sample 2 100 uL 1050 pM 500 pM 4 11 9 Sample 3 100 uL 1352 pM 500 pM 4 9 2 Sample 4 100 uL 684 pM 500 pM 4 18 3 Low TE 47 SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing Addition of Barcode Tags by Post Hybridization Amplification 4 2 Adjust the final volume of the pooled library to the desired final concentration If the final volume of the combined barcode tagged samples is less than the desired final volume V f add Low TE to bring the volume to the desired level If the final volume of the combined barcode tagged samples is greater than the final desired volume V f lyophilize and reconstitute to the desired volume 3 If you store the library before sequencing add Tween 20 to 0 1 v v and store at 20 C short term SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 59 4 Addition of Bar
27. hen the bead pellet is excessively dried 10 Add 15 uL of nuclease free water mix well on a vortex mixer and incubate for 2 minutes at room temperature 11 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 12 Remove the supernatant 15 uL to a fresh 1 5 mL LoBind tube You can discard the beads at this time 13 Proceed immediately to the next step Step 8 Ligate the adaptors 28 SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing Sample Preparation 2 Step 8 Ligate the adaptors Use reagents from the SureSelect XT Library Prep Kit 55500 and the SureSelect 5500 Indexing Construction Kit 1 For 1 library In a 1 5 mL LoBind tube strip tube or plate prepare the reaction mix in Table 15 on ice Mix well by gently pipetting up and down 2 For multiple libraries a Prepare the reaction mix in Table 15 on ice Mix gently on a vortex mixer b Add 35 uL of the reaction mix to each well or tube Use a pipette to add 15 uL of each DNA sample to each well or tube Mix by pipetting Change pipette tips between samples Table 15 Ligation mix Component Volume pL Volume for 12 Libraries pL includes excess A Tailed DNA 15 SureSelect 1715500 P1 purple cap 45 56 25 SureSelect 1715500 IA blue cap 45 56 25 5x T4 DNA Ligase Buffer 10 125 T4 DNA Ligase red cap 1 5 18 75 Nuclease free water 14 5 181 25 50 437 5 35
28. hnologies Inc 2012 2015 Version 0 August 2015 G7530 90004 Revision Al GE Agilent Technologies
29. iency is significantly decreased when the bead pellet is excessively dried 10 Add 30 uL of nuclease free water mix well on a vortex mixer and incubate for 2 minutes at room temperature 11 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 12 Remove the supernatant 30 pL to a fresh 1 5 mL LoBind tube You can discard the beads at this time If you do not continue to the next step store the samples at 20 C SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing Sample Preparation 2 Step 6 Add A Bases to the 3 end of the DNA fragments Use the SureSelect XT Library Prep Kit 55500 1 For 1 library prepare on ice In a PCR tube strip tube or plate prepare the reaction mix in Table 14 Mix well by gently pipetting up and down 2 For multiple libraries prepare on ice a Prepare the reaction mix in Table 14 Mix well on a vortex mixer b Add 20 uL of the reaction mix to each well or tube c Add 30 uL of each DNA sample to each well or tube Mix by pipetting Change pipette tips between samples Table 14 Adding A Bases Reagent Volume for 1 Library Volume for 12 Libraries includes excess End repaired DNA sample 30 uL Nuclease free water 11 uL 137 5 pL 10x Klenow Polymerase Buffer blue cap 5 uL 62 5 uL dATP green cap 12 5 pL Exo Klenow red cap 3 uL 37 5 uL Total Volume 50 pL 250 pL 20 pL sample These
30. in this docu ment is provided as 15 and is sub ject to being changed without notice in future editions Further to the max imum extent permitted by applicable law Agilent disclaims all warranties either express or implied with regard to this manual and any information contained herein including but not limited to the implied warranties of merchantability and fitness for a par ticular purpose Agilent shall not be liable for errors or for incidental or consequential damages in connec tion with the furnishing use or per formance of this document or of any information contained herein Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms the warranty terms in the sep arate agreement shall control Technology Licenses The hardware and or software described in this document are furnished under a license and may be used or copied only in accor dance with the terms of such license Restricted Rights Legend U S Government Restricted Rights Soft ware and technical data rights granted to the federal government include only those rights customarily provided to end user cus tomers Agilent provides this customary commercial license in Software and techni cal data pursuant to FAR 12 211 Technical Data and 12 212 Computer Software and for the Department of Defense DFARS 252 227 7015 Technical Data Commercial
31. iology grade Sigma Aldrich p n E7023 SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 9 1 Before You Begin Table 2 Reagent Reagent Kits 16 Reactions 96 Reactions 480 Reactions SureSelect Reagent Kit 5500 G9615A G9615B N A SureSelect reagents must used within one year of receipt Table3 SureSelect Capture Library select Capture Libraries 16 Reactions 96 Reactions 480 Reactions SureSelect Human All Exon 50Mb 5190 4626 5190 4627 5190 4629 SureSelect Human All Exon V4 5190 4631 5190 4632 5190 4634 SureSelect Human All Exon V4 UTRs 5190 4636 5190 4637 5190 4639 SureSelect Mouse All Exon 5190 4641 5190 4642 5190 4644 SureSelect Custom 1 kb up to 499 Kb 5190 4806 5190 4807 5190 4809 reorder 5190 4811 5190 4812 5190 4814 SureSelect T Custom 0 5 Mb up to 2 9 Mb 5190 4816 5190 4817 5190 4819 reorder 5190 4821 5190 4822 5190 4824 SureSelect T Custom 3 Mb upto 5 9 Mb 5190 4826 5190 4827 5190 4829 reorder 5190 4831 5190 4832 5190 4834 SureSelect Custom 6 Mb to 11 9 Mb 5190 4836 5190 4837 5190 4839 reorder 5190 4841 5190 4842 5190 4844 SureSelect Custom 12 Mb up to 24 Mb 5190 4896 5190 4897 5190 4899 reorder 5190 4901 5190 4902 5190 4904 SureSelect capture libraries must be used within year of receipt 10 SureSelect Target Enrichment System for 5011 5500 Multiplexed Sequencing Before You Begin 1
32. is not extensive SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 43 3 44 Hybridization Step 2 Prepare magnetic beads Use these reagents from the SureSelect Target Enrichment Box 1 5500 SureSelect Binding Buffer SureSelect Wash 2 1 Prewarm SureSelect Wash 2 at 65 C in a circulating water bath for use in Step 3 Select hybrid capture with SureSelect 2 Vigorously resuspend the Dynabeads MyOne Streptavidin T1 on a vortex mixer Magnetic beads settle during storage 3 For each hybridization add 50 uL of Dynabeads MyOne Streptavidin 1 to a 1 5 mL LoBind tube 4 Wash the beads a b c d e Add 200 uL of SureSelect Binding Buffer Mix the beads on a vortex mixer for 5 seconds Put the tubes into a magnetic device such as the Dynal magnetic separator Life Technologies Remove and discard the supernatant Repeat step a through step d for a total of 3 washes 5 Resuspend the beads in 200 uL of SureSelect Binding Buffer SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing Hybridization 3 Step 3 Select hybrid capture with SureSelect Use these reagents from the SureSelect Target Enrichment Box 1 5500 SureSelect Wash 1 SureSelect Wash 2 1 Estimate and record the volume of hybridization that remained after 24 hour incubation 2 Keep the PCR plate or tubes at 65 C in the PCR machine while you add the hybridization mixture directly fr
33. is significantly decreased when the bead pellet is excessively dried 10 Add 30 uL nuclease free water mix well on a vortex mixer and incubate for 2 minutes at room temperature 11 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 12 Remove the supernatant 30 uL to a fresh 1 5 mL LoBind tube You can discard the beads at this time Stopping Point If you do not continue to the next step store the samples at 4 C for up to a week or at 20 C for longer periods SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 53 4 54 Addition of Barcode Tags by Post Hybridization Amplification Step 4 Assess DNA quality Use a Bioanalyzer High Sensitivity DNA Assay or the 2200 TapeStation to assess the quality and size range 2100 Bioanalyzer High Sensitivity DNA Assay You may need to dilute your sample accordingly Refer to the Agilent High Sensitivity DNA Kit Guide at http www chem agilent com en US Search Library _layouts Agilent PublicationSummary aspx whid 59504 Check that the 2100 Bioanalyzer electrodes have been cleaned as instructed in the reagent kit guide Open the Agilent 2100 Expert Software version B 02 07 or higher required to run the High Sensitivity Kit turn on the 2100 Bioanalyzer and check communication 3 Prepare the chip samples and ladder as instructed in the reagent kit guide 4 Load the prepared chip into the 2100 Bioanalyzer and
34. kits and protocols for SOLiD Multiplexed Sequencing 1 Before You Begin This chapter contains information such as procedural notes safety information required reagents and equipment that you should read and understand before you start an experiment 2 Sample Preparation This chapter contains instructions for prepped library production specific to the Life Technologies SOLiD 5500 System 3 Hybridization This chapter describes the steps to combine the prepped library with the blocking agents and the SureSelect capture library 4 Addition of Barcode Tags by Post Hybridization Amplification This chapter describes the steps to amplify purify quantify and pool the barcoded sample libraries after target enrichment hybridization 5 Reference This chapter contains reference information SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 3 SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing Content 1 Before YouBegin 7 Procedural Notes 8 Safety Notes 8 Required Reagents 9 Optional Reagents 11 Required Equipment 12 Optional Equipment 14 2 Sample Preparation 15 Step 1 Quantify and shear DNA 18 Step 2 Size select the sample using the Agencourt AMPure XP beads 20 Step 3 Assess quality with the 2100 Bioanalyzer 22 Step 4 Repairthe ends 25 Step 5 Purify the sample using the Agencourt AMPure XP beads 26 Step 6 Add A Bases to the 3 end of the DNA fragments 27 Ste
35. n the laboratory 8 SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing Before You Begin 1 Required Reagents Table 1 Required Reagents for Library Prep and Post Hybridization Amplification Description Vendor and part number For use with 2100 Bioanalyzer DNA 1000 Kit Agilent p n 5067 1504 High Sensitivity DNA Kit Agilent p n 5067 4626 For use with 2200 TapeStation System D1K ScreenTape Agilent p n 5067 5361 D1K Reagents Agilent p n 5067 5362 High Sensitivity D1K ScreenTape Agilent p n 5067 5363 High Sensitivity D1K Reagents Agilent p n 5067 5364 QPCR NGS Library Quantification Kit SOLID Agilent p n G4881A Herculase II Fusion DNA Polymerase Agilent includes dNTP mix and 5x Buffer 200 reactions p n 600677 400 reactions p n 600679 Each library requires 4 reactions for pre capture amplification and 2 reactions for post capture amplification Nuclease free Water not DEPC treated Ambion Cat AM9930 Agencourt AMPure XP Kit Beckman Coulter Genomics 5 mL p n A63880 60 mL p n A63881 450 mL p n A63882 1X Low TE Buffer 10 mM Tris HCl pH 8 0 0 1 mM EDTA Life Technologies p n 4389764 Qubit dsDNA HS Assay Kit or Life Technologies p n 032851 Qubit dsDNA BR Assay Kit 100 assays 2 1000 ng Life Technologies p n 032850 500 assays 2 1000 ng Life Technologies p n 032853 1000 assays 2 1000 ng Life Technologies p n 033130 Qubit assay tubes Life Technologies p n 032856 100 Ethanol molecular b
36. nal Equipment 14 Make sure you have the most current protocol Go to the Next Gen Sequencing User Manuals page on genomics agilent com and search for manual number G7530 90004 Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an experiment Agilent cannot guarantee the SureSelect Target Enrichment kits and cannot provide technical support for the use of non Agilent protocols to process samples for enrichment E Agilent Technologies 1 Before You Begin Procedural Notes To prevent contamination of reagents by nucleases always wear powder free laboratory gloves and use dedicated solutions and pipettors with nuclease free aerosol resistant tips Maintain a clean work area Do not mix stock solutions and reactions containing gDNA on a vortex mixer Instead gently tap the tube with your finger to mix the sample Avoid repeated freeze thaw cycles of stock and diluted gDNA solutions When preparing frozen reagent stock solutions for use 1 Thaw the aliquot as rapidly as possible without heating above room temperature 2 Mix briefly on a vortex mixer then spin in a centrifuge for 5 to 10 seconds to drive the contents off of walls and lid 3 Store on ice or in a cold block until use In general follow Biosafety Level 1 BL1 safety rules Safety Notes CAUTION Wear appropriate personal protective equipment PPE when working i
37. nding on the Covaris instrument SonoLab software version that is used The target peak for base pair size is 150 bp 18 SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing Sample Preparation 2 Table 11 Shear settings for Covaris instruments that use SonoLab 7 or newer Setting Value Duty Factor 1096 Peak Incident Power PIP 175 Cycles per Burst 100 Treatment Time 360 seconds Bath Temperature 4 to 8 C Table 12 Shear settings for Covaris instruments that use SonoLab software previous to SonoLab 7 Setting Value Duty Cycle 1096 Intensity 5 Cycles per Burst 100 Time 6 cycles of 60 seconds each Set Mode Frequency sweeping Temperature 4 to 7 C Put the Covaris microTube back into the loading and unloading station 8 While keeping the snap cap on insert a pipette tip through the pre split septa then slowly remove the sheared DNA 9 Transfer the sheared DNA into a new 1 5 mL LoBind tube SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 19 2 20 Sample Preparation Step 2 Size select the sample using the Agencourt AMPure XP beads 1 Measure out just enough AMPure XP beads for the number of samples to purify Let the AMPure XP beads come to room temperature for at least 30 minutes Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze Add 143 uL of homogenous AMPure XP beads to a 1 5 mL Lo
38. om the thermal cycler to the bead solution Invert the tube to mix 3 to 5 times Excessive evaporation such as when less than 20 uL remains after hybridization can indicate suboptimal capture performance See Table 36 on page 71 for tips to minimize evaporation 3 Incubate the hybrid capture bead solution on a Nutator or equivalent for 30 minutes at room temperature Make sure the sample is properly mixing in the tube 4 Briefly spin in a centrifuge Separate the beads and buffer on a magnetic separator and remove the supernatant 6 Resuspend the beads 500 uL of SureSelect Wash 1 by mixing on a vortex mixer for 5 seconds 7 Incubate the samples for 15 minutes at room temperature Occasionally mix on a vortex mixer 8 Briefly spin in a centrifuge 9 Separate the beads and buffer on a magnetic separator and remove the supernatant SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 45 3 46 Hybridization 10 Wash the beads Resuspend the beads in 500 uL of 65 C prewarmed SureSelect Wash 2 and mix on a vortex mixer for 5 seconds to resuspend the beads Incubate the samples for 10 minutes at 65 C in a recirculating water bath heat block or equivalent Occasionally mix on a vortex mixer Do not use a tissue incubator It cannot properly maintain temperature Invert the tube to mix The beads may have settled Briefly spin in a centrifuge Separate the beads and buffer on a magnetic sepa
39. onger if applicable as a test Include buffer in each well that you might use including those in the center and those on the edges Check that you do not get extensive evaporation Evaporation should not exceed to 4 pL For a partial list of tested options showing minimal evaporation refer to Alternative Capture Equipment Combinations on page 71 RE Agilent Technologies 39 3 Hybridization Step 1 Hybridize the library For each sample library prepared do one hybridization and capture Do not pool samples at this stage The hybridization reaction requires 500 ng of DNA with a maximum volume of 3 4 uL 1 Ifthe prepped library concentration is below 147 ng uL use a vacuum concentrator to concentrate the sample at lt 45 C a Add the entire 30 uL of prepped library to an Eppendorf tube Poke one or more holes in the lid with a narrow gauge needle You can also break off the cap cover with parafilm and poke a hole in the parafilm b Completely lyophilize Use a vacuum concentrator on low heat less than 45 C to dehydrate Reconstitute with nuclease free water to bring the final concentration to 147 ng uL or greater if sample recovery is of concern Pipette up and down along the sides of the tube for optimal recovery d Mix well on a vortex mixer and spin in a microfuge for 1 minute 2 Optional To test recovery after lyophilization reconstitute the sample to greater than 147 ng uL and check the concent
40. p 7 Purify the sample using Agencourt AMPure XP beads 28 Step 8 Ligate the adaptors 29 Step 9 Purify the sample using the Agencourt AMPure XP beads 30 Step 10 Amplify adaptor ligated library 31 Step 11 Purify the sample using the Agencourt AMPure XP beads 34 Step 12 Assess quality and quantity with the 2100 Bioanalyzer 35 3 Hybridization 39 Step 1 Hybridize the library 40 Step 2 Prepare magnetic beads 44 Step 3 Select hybrid capture with SureSelect 45 SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing Contents 4 Addition of Barcode Tags by Post Hybridization Amplification 47 Step 1 Amplify the captured library on the Streptavidin beads to add barcode tags 48 Step 2 Purify the sample using Agencourt AMPure XP beads 52 Step 3 Remove primer dimers from the sample using Agencourt AMPure XP beads 53 Step 4 Assess DNA quality 54 Step 5 Assess the quantity of each barcode tagged library QPCR 57 Step 6 Pool samples for Multiplexed Sequencing 58 5 Reference 61 SureSelect Reagent Kit Content 62 Other Reagent Kits Content 65 SureSelect Barcodes for SOLID 66 Alternative Capture Equipment Combinations 71 6 SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing Protocol 1 Before You Begin Procedural Notes 8 Safety Notes 8 Required Reagents 9 Optional Reagents 11 Required Equipment 12 Optio
41. pping Point 52 Step 2 Purify the sample using Agencourt AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze Add 60 uL of homogenous AMPure beads to a 1 5 mL LoBind tube and add amplified library 50 uL Mix well on a vortex mixer and incubate for 5 minutes Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes Continue to keep the tube in the magnetic stand while you dispense 500 uL of 70 ethanol in each tube Use fresh 70 ethanol for optimal result Let the tube sit for 1 minute to allow any disturbed beads to settle and remove the ethanol Repeat step 6 and step 7 once 9 Dry the samples on the 37 C heat block for 5 minutes or until the residual ethanol is completely evaporated Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency is significantly decreased when the bead pellet is excessively dried 10 Add 50 uL nuclease free water mix well on a vortex mixer and incubate for 2 minutes at room temperature 11 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 12 Remove the supernatant 50 uL to a fresh 1
42. r QC 1 hour SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 17 2 Sample Preparation Step 1 Quantify and shear DNA 1 Use the Qubit dsDNA Assay to determine the concentration of your gDNA sample Make sure the gDNA is of high quality non degraded Aogo Aggo is 1 8 to 2 0 Follow the instructions for the instrument 2 Set up the Covaris instrument a Check that the water in the Covaris tank is filled with fresh deionized water to fill line level 12 on the graduated fill line label b Check that the water covers the visible glass part of the tube c Set the chiller temperature to between 2 C to 5 C to ensure that the temperature reading in the water bath displays 5 C d Optional Supplement the circulated water chiller with ethylene glycol to 20 volume to prevent freezing e On the instrument control panel push the Degas button Degas the instrument for least 30 minutes before use Refer to the Covaris instrument user guide 3 Dilute 3 ug of high quality gDNA with 1X Low TE Buffer in a 1 5 mL LoBind tube to a total volume of 130 uL 4 Puta Covaris microTube into the loading and unloading station Keep the cap on the tube 5 Useatapered pipette tip to slowly transfer the 130 uL DNA sample through the pre split septa Be careful not to introduce a bubble into the bottom of the tube 6 Secure the microTube in the tube holder and shear the DNA with the settings in Table 11 or Table 12 depe
43. r pipette tips Thermal cycler Agilent SureCycler Life Technologies Veriti Thermal Cycler BioRad MJ Research DNA Engine PTC 200 or equivalent Timer Vortex mixer Water bath or heat block set to 65 C SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 13 1 Before You Begin Optional Equipment Table 8 Optional Equipment for Hybridization Description Vendor and part number Tube strip capping tool Agilent p n 410099 14 SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing Protocol e ee 2 Sample Preparation Step 1 Quantify and shear DNA 18 Step 2 Size select the sample using the Agencourt AMPure XP beads 20 Step 3 Assess quality with the 2100 Bioanalyzer 22 Step 4 Repair the ends 25 Step 5 Purify the sample using the Agencourt AMPure XP beads 26 Step 6 Add A Bases to the 3 end of the DNA fragments 27 Step 7 Purify the sample using Agencourt AMPure XP beads 28 Step 8 Ligate the adaptors 29 Step 9 Purify the sample using the Agencourt AMPure XP beads 30 Step 10 Amplify adaptor ligated library 31 Step 11 Purify the sample using the Agencourt AMPure XP beads 34 Step 12 Assess quality and quantity with the 2100 Bioanalyzer 35 This chapter contains instructions for prepped library production specific to the Life Technologies SOLiD 5500 System Before you begin yo
44. ration on Bioanalyzer DNA 1000 chip See Step 12 Assess quality and quantity with the 2100 Bioanalyzer on page 35147 Alternatively concentrate a 500 ng aliquot at lt 45 C down to 3 4 uL If the sample dries up completely resuspend in 3 4 uL of water and mix ona vortex mixer If processing multiple samples adjust to equivalent volumes before concentrating 3 Mix the components in Table 18 at room temperature to prepare the hybridization buffer 40 SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing Hybridization 3 Table 18 Hybridization Buffer Reagent Volume for 3 Volume for 6 Volume for 12 captures uL captures pL captures pL includesexcess includesexcess includes excess SureSelect Hyb 1 orange cap or 25 50 87 5 bottle SureSelect Hyb 2 red cap 1 2 3 5 SureSelect Hyb 3 yellow cap 10 20 35 SureSelect Hyb 4 black cap or 13 26 45 5 bottle Total 49 98 171 5 13 pL sample 13pL sample 13 pL sample 4 If precipitate forms warm the hybridization buffer at 65 C for 5 minutes 5 Ina PCR plate prepare the SureSelect capture library mix for target enrichment a Keep tubes on ice until step 9 b For each sample add the amount of SureSelect capture library as listed in Table 19 based on the Mb target size of your design c Use nuclease free water to prepare a dilution of the SureSelect RNase Block purple cap as listed in Table 19 Prepare enough RNas
45. rator and remove the supernatant Repeat step a through step e for a total of 3 washes Make sure all of the wash buffer has been removed Mix the beads with 30 uL of nuclease free water on a vortex mixer for 5 seconds to resuspend the beads _ SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing Protocol 4 Addition of Barcode Tags by Post Hybridization Amplification Step 1 Amplify the captured library on the Streptavidin beads to add barcode tags 48 Step 2 Purify the sample using Agencourt AMPure XP beads 52 Step 3 Remove primer dimers from the sample using Agencourt AMPure XP beads 53 Step 4 Assess DNA quality 54 Step 5 Assess the quantity of each barcode tagged library by QPCR 57 Step 6 Pool samples for Multiplexed Sequencing 58 This chapter describes the steps to add barcode tags by amplification purify assess quality and quantity of the libraries and pool barcoded samples for multiplexed sequencing Agilent Technologies 47 4 Addition of Barcode Tags by Post Hybridization Amplification Step 1 Amplify the captured library on the Streptavidin beads to add barcode tags Use reagents from Herculase II Fusion DNA Polymerase Agilent SureSelect 5500 Indexing Construction Kit CAUTION Do not use amplification enzymes other than Herculase Fusion DNA Polymerase Other enzymes have not been validated
46. rch DNA Engine PTC 200 or equivalent Covaris Covaris p n 520045 Eppendorf p n 022621807 120 V 60 Hz Eppendorf p n 022621840 230 V 50 Hz or equivalent Eppendorf p n 022636006 Eppendorf p n 022431021 or equivalent Life Technologies p n 032857 Life Technologies p n G6465 Life Technologies p n G6500 Life Technologies p n G6400 Life Technologies p n 123 21D or equivalent Pipetman P10 P20 P200 P1000 or equivalent Savant SpeedVac or equivalent SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing Before You Begin 1 Table 6 Required Equipment for Library Prep and Post Hybridization Amplification Description Vendor and part number Powder free gloves Sterile nuclease free aerosol barrier pipette tips Timer Vortex mixer Heat block at 37 C Table 7 Required Equipment for Hybridization Description Vendor and part number Mx3000P Mx3005P 96 well tube plates Agilent p n 410088 or equivalent Mx3000P Mx3005P optical strip caps Agilent p n 401425 or equivalent MicroAmp Clear Adhesive Film Life Technologies p n 4306311 or equivalent BD Clay Adams Nutator Mixer BD Diagnostics p n 421105 or equivalent Dynal DynaMag 2 magnetic stand Life Technologies p n 123 21D or equivalent P10 P20 P200 and P1000 pipettes Pipetman P10 P20 P200 P1000 or equivalent Pipet Light Multichannel Pipette 12 channels Rainin p n L12 20 or equivalent Sterile nuclease free aerosol barrie
47. room temperature SureSelect Target Enrichment System for 5011 5500 Multiplexed Sequencing Sample Preparation 2 e sample using the Agencourt AMPure XP beads 15 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 16 Remove the supernatant 50 uL to a fresh 1 5 mL LoBind tube You can discard the beads at this time Stopping Point If you do not continue to the next step store the samples at 20 C SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 21 2 Sample Preparation Step 3 Assess quality with the 2100 Bioanalyzer Quality assessment can be done with either the 2100 Bioanalyzer instrument or the 2200 TapeStation 2100 Bioanalyzer System and DNA 1000 Assay 1 Check that the 2100 Bioanalyzer electrodes have been cleaned and dried as instructed in the reagent kit guide 2 Open the Agilent 2100 Expert Software version B 02 07 or higher turn on the 2100 Bioanalyzer instrument and check communication 3 Prepare the chip samples and ladder as instructed in the reagent kit guide then mix on the IKA vortex mixer that is included with the 2100 Bioanalyzer instrument 4 Load the prepared chip into the 2100 Bioanalyzer instrument and start the run within five minutes after preparation 5 Within the instrument context choose the appropriate assay from the drop down list 6 Start the run Enter sample names and comments in the Data and Assay context
48. rovided in the user guide 3 Dilute each barcode tagged captured library such that it falls within the range of the standard curve Typically this corresponds to approximately a 1 1000 to 1 10 000 dilution of the captured DNA 4 Prepare the QPCR master mix with 5011 adaptor specific PCR primers according to instructions provided in the kit 5 Add an aliquot of the master mix to PCR tubes and add template 6 Ona QPCR system such as the MX3005P run the thermal profile outlined in the QPCR NGS Library Quantification kit user guide Use the SYBR Green instrument setting 7 Use the standard curve to determine the concentration of each unknown barcode tagged library in nM The concentration will be used to accurately pool samples for multiplexed sequencing SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 57 4 58 Addition of Barcode Tags by Post Hybridization Amplification Step 6 Pool samples for Multiplexed Sequencing 1 Combine the libraries such that each barcode tagged sample is present in equimolar amounts in the pool For each library use the formula below to determine the amount of barcoded sample to use V f x C f x C where Volume of Barcoded Sample V f is the final desired volume of the pool C f is the desired final concentration of all the DNA in the pool for example 500 pM for the standard SOLiD protocol is the number of samples to be combined and 1 is t
49. te your prepped library appropriately and use the Bioanalyzer DNA 1000 assay to quantitate again Use the concentration as determined by the Bioanalyzer DNA 1000 assay to calculate the volume of prepped library needed for hybridization 500 ng in Chapter 3 2200 TapeStation and D1K ScreenTape You can use the 2200 TapeStation to analyze the amplified libraries Use the ScreenTape D1K Reagents For more information to do this step see the Agilent 2200 TapeStation User Manual 1 Prepare the TapeStation samples as instructed in the Agilent 2200 TapeStation User Mamual Use 1 uL of each amplified library DNA sample diluted with 3 uL of D1K sample buffer for the analysis CAUTION Make sure that you thoroughly mix the combined DNA and D1K sample buffer on a vortex mixer for 5 seconds or more for accurate quantitation 36 SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing Sample Preparation 2 2 Load the sample plate or tube strips from step 1 the DIK ScreenTape and loading tips into the 2200 TapeStation as instructed in the Agilent 2200 TapeStation User Manual Start the run 3 Verify that the electropherogram shows an average DNA fragment size of about 200 bp A sample electropherogram is shown in Figure 4 Stopping Point If you do not continue to the next step seal the sheared DNA sample plate and store at 4 C overnight at 20 C for prolonged storage 3000 2000 Sample Intensity
50. tivity DIK ScreenTape and loading tips into the 2200 TapeStation as instructed in the Agilent 2200 TapeStation User Manual Start the run 3 Verify that the electropherogram shows an average DNA fragment size of about 260 bp A sample electropherogram is shown in Figure 8 If you do not continue to the next step seal the sheared DNA sample plate and store at 4 C overnight or at 20 C for prolonged storage SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 55 4 56 Addition of Barcode Tags by Post Hybridization Amplification Step 4 Assess DNA quality 1200 1000 800 Sample Intensity FU 600 400 200 0 MW bp Figure 8 Analysis of amplified library DNA using the 2200 TapeStation with a DTK ScreenTape The electropherogram shows a peak size of 260 bp 8 700 100 300 500 SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing Addition of Barcode Tags by Post Hybridization Amplification 4 Step 5 Assess the quantity of each barcode tagged library by QPCR Refer to the protocol that is included with the QPCR NGS Library Quantification Kit SOLiD for more details to do this step 1 Use the QPCR NGS Library Quantification Kit SOLiD to determine the concentration of each barcode tagged captured library 2 Prepare standard curve using the quantification standard included in the kit according to the instructions p
51. u can use the SureSelect gDNA Extraction Kit to extract genomic DNA Refer to the gDNA Extraction Kit Protocol p n 5012 8701 Make sure genomic DNA samples are of high quality with an OD 260 280 ratio ranging from 1 8 to 2 0 E Agilent Technologies 15 2 Sample Preparation Genomic BNA Sam 22 Shear Size Selection ragments with a base pair pea Repair ends nt en if 5 Wi 0 rylate Genomic Locations Bait Design in eArray ey Add Klenow and dATP 3 overhang Ligate LT5500 specific daptors a Ada tor modified ends AMPure XP bead Y purification o unligated adaptors PCR repped Librar ibrary Hybridization 1 per sample rid Capture 24 hours at 65 C election Magnetic bead selection Barcode in PCR and purification Quali Assessment of each barcoded sam le Bioanalyzer and Quantification by QPCR Pool sam m Se uencin Figure 1 16 Overall sequencing sample preparation workflow SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing Sample Preparation 2 Table 10 Overview and time requirements Step Time AB SOLID 5500 Fragment Library Production 8 hours Bioanalyzer QC 1 hour Library Preparation and Hybridization 24 5 hours optional 72 hours Bead Preparation 10 minutes Capture Selection and Washing 2 5 hours Post Hybridization Amplification 1 hour PCR Purification 30 minutes Bioanalyze
52. uffer clear cap 100 mM dNTP Mix green cap Herculase Fusion DNA Polymerase red cap Table 33 D1K Reagents Agilent p n 5067 5362 Components D1K ladder D1K sample buffer Table 34 High Sensitivity D1K Reagents Agilent p n 5067 5364 Components High Sensitivity D1K ladder High Sensitivity D1K sample buffer SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 65 5 66 Reference SureSelect Barcodes for SOLID The nucleotide sequence of each of the SureSelect barcodes is listed in Table 35 Barcode orientation for the SureSelect LTI5500 BC1 BC96 plate is shown in Figure 9 on page 70 Table 35 SureSelect Barcodes 1 to 96 Barcode Number Sequence 1 GTGTAAGAGG 2 AGGGAGTGGT 3 ATAGGTTATA 4 GGATGCGGTC 5 GTGGTGTAAG 6 GCGAGGGACA 7 GGGTTATGCC 8 GAGCGAGGAT 9 AGGTTGCGAC 10 GCGGTAAGCT 11 GTGCGACACG 12 AAGAGGAAAA 13 GCGGTAAGGC 14 GTGCGGCAGA 15 GAGTTGAATG 16 GGGAGACGTT 17 GGCTCACCGC 18 AGGCGGATGA 19 ATGGTAACTG 20 GTCAAGCTTT SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing Reference Table 35 SureSelect Barcodes 1 to 96 continued Barcode Number Sequence 21 GTGCGGTTCC 22 GAGAAGATGA 23 GCGGTGCTTG 24 GGGTCGGTAT 25 AACATGATGA 26 CGGGAGCCCG 27 CAGCAAACTT 28 AGCTTACTAC 29 GAATCTAGGG 30 GTAGCGAAGA 31 GCTGGTGCGT 32 GGTTGGGTGC 33 CGTTGGATAC 34 TCGTTAAAGG 35 AAGCGTAGGA 36 GTTCTCACAT 37 CTGTTATACC 38 GTCGTCTTAG 39
53. vortex mixer and incubate for 2 minutes at room temperature 11 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 12 Remove the supernatant 30 uL to a fresh 1 5 mL LoBind tube You can discard the beads at this time If you do not continue to the next step store the samples at 20 C SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing Sample Preparation 2 Step 10 Amplify adaptor ligated library Use reagents from these kits SureSelect XT Library Prep Kit 55500 SureSelect 5500 Indexing Construction Kit Herculase II Fusion DNA Polymerase Agilent This protocol uses half of the adaptor ligated fragments for amplification The remainder can be saved at 20 C for future use if needed CAUTION To avoid cross contaminating libraries set up PCR reactions all components except the library DNA in a dedicated clean area or PCR hood with UV sterilization and positive air flow 1 For 1 library In a PCR tube strip tube or plate prepare the reaction mix in Table 16 on ice Mix well by gently pipetting up and down 2 For multiple libraries a Prepare the reaction mix in Table 16 on ice Mix well on a vortex mixer b Add 36 uL of the reaction mix to each well or tube c Add 14 uL of each DNA sample to each well or tube Mix by pipetting Change pipette tips between samples SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequ
54. ybridization Amplification Table 23 Sequencing data requirement guidelines Capture size Optimal sequencing output per barcode 1 kb up to 0 5 Mb 0 5 Mb up to 2 9 Mb 3 Mb up to 5 9 Mb 6 Mb up to 11 9 Mb 12 Mb up to 24 Mb Human All Exon v4 Human All Exon v4 UTRs Human All Exon 50 Mb Human DNA Kinome Mouse All Exon 0 1 to 50 Mb 50 to 290 Mb 300 to 590 Mb 600 to 1190 Mb 1 2 to 2 4 Gb 4 Gb 6 Gb 5 Gb 320 Mb 5 Gb For custom libraries Agilent recommends analyzing 100 amount of sequencing data compared to the Capture Library size for each sample Pool samples according to your expected sequencing out put 3 Put the tubes in a thermal cycler and run the program in Table 24 SureSelect Target Enrichment System for SOLID 5500 Multiplexed Sequencing Addition of Barcode Tags by Post Hybridization Amplification 4 Table 24 PCR program Step Temperature Time Step 1 98 C 2 minutes Step 2 98 C 30 seconds Step 3 54 C 10 seconds Step 4 72 C 1 minute Step 5 Repeat Step 2 through Step 4 depending on the size of the capture 0 2 Mb up to 0 49 Mb 12 cycles total 0 5 Mb up to 1 49 Mb 10 cycles total 1 5 Mb up to 2 99 Mb 9 cycles total 3 Mb or more 8 cycles total Step 6 72 C 10 minutes Step 7 4 Hold SureSelect T Target Enrichment System for SOLiD 5500 Multiplexed Sequencing 51 4 Addition of Barcode Tags by Post Hybridization Amplification Sto
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