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SureSelect Target Enrichment System for Illumina Paired

1. 8 While keeping the snap cap on insert a pipette tip through the pre split septa then slowly remove the sheared DNA 9 Transfer each sheared DNA sample approximately 50 uL to a separate well of a 96 well plate or strip tube Instructions in this manual are for sample processing in 96 well PCR plates When processing a small number of samples you can instead use strip tubes or individual tubes that are compatible with the thermal cycler and magnetic separation device used in the protocol 46 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Sample Preparation 200 ng DNASamples 3 Step 2 Assess quality optional This step is optional Quality assessment can be done with the 2100 Bioanalyzer instrument For analysis of 200 ng sheared DNA samples Use a High Sensitivity DNA chip and reagent kit See the High Sensitivity DNA Kit Guide at www genomics agilent com for more information on doing this step 1 Figure 6 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Check that the 2100 Bioanalyzer electrodes have been cleaned as instructed in the reagent kit guide Open the 2100 Expert Software version B 02 02 or higher turn on the 2100 Bioanalyzer and check communication Prepare the chip samples and ladder as instructed in the reagent kit guide using 1 uL of each sample for the analysis Load the prepared chip into the 2100 Bioanalyzer and start the run withi
2. Downstream Sequencing Modifications For all FFPE sample derived libraries set up the sequencing run to include adapter trimming To do this step use the IEM Sample Sheet Wizard When prompted by the wizard select the Use Adapter Trimming and Use Adapter Trimming Read 2 options This enables the MiSeq Reporter software to identify the adaptor sequence and trim the adaptor from reads 94 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Appendix Using FFPE derived DNA Samples 6 Modifications for samples assessed using the Agilent NGS FFPE QC Kit Before applying protocol modifications in this section use the Agilent NGS FFPE QC Kit to determine the AACq DNA integrity score and the quantity of amplifiable DNA for each FFPE DNA sample For the complete Agilent NGS FFPE QC Kit protocol go to genomics agilent com and search for document part number G9700 90000 T SureSelect Protocol Modifications Protocol modifications that should be applied to FFPE samples based on the AACq score determined for each sample are detailed in Table 50 Table 50 SureSelect protocol modifications based on AACq DNA integrity score Protocol Step and non FFPE FFPE Samples Parameter Samples 5 AACq lt 1 AACq between 1 and 4 AACq gt 4 DNA input for Library 200 ng based 200 ng based on 100 to 200 ng of amplifiable 100 to 200 ng of amplifiable Preparation page 44 on Qubit Assay Qubit Assay DNA based on qPCR DNA based on
3. 2100 Bioanalyzer and check communication Prepare the chip samples and ladder as instructed in the reagent kit guide using 1 uL of each sample for the analysis Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation Within the instrument context choose the DNA 1000 assay from the drop down list Start the run Enter sample names and comments in the Data and Assay context Verify that the electropherogram shows a distribution with a DNA fragment size peak of approximately 225 to 275 bp Determine the concentration of the library DNA by integrating under the peak A sample electropherogram is shown in Figure 4 Fu 1004 04 F NE tok T T T T T T itt T 15 50 100 150 200 300 400 500 700 1500 bp Figure 4 Analysis of amplified library DNA using a DNA 1000 Bioanalyzer assay SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Sample Preparation 3 pg DNA Samples 2 Option 2 Analysis using the 2200 TapeStation and D1000 ScreenTape For more information to do this step see the Agilent 2200 TapeStation User Manual at www genomics agilent com 1 Prepare the TapeStation samples as instructed in the Agilent 2200 TapeStation User Manual Use 1 uL of each DNA sample diluted with 3 uL of D1000 sample buffer for the analysis CAUTION Make sure that you thoroughly mix the combined DNA and sample buffer on a vortex mixer for 5 seconds for a
4. F01 GACTAGTA G01 ATTGGCTC H01 GATGAATC A02 AGCAGGAA GAGCTGAA c02 AAACATCG D02 GAGTTAGC CGAACTTA F02 GATAGACA G02 AAGGACAC GACAGTGC A03 ATCATTCC B03 GCCACATA ACCACTGT D03 CTGGCATA E03 ACCTCCAA GCGAGTAA ACTATGCA CGGATTGC 104 G04 H04 A05 B05 co5 D05 E05 F05 G05 H05 A06 B06 C06 D06 E06 F06 AACTCACC GCTAACGA CAGATCTG ATCCTGTA CTGTAGCC GCTCGGTA ACACGACC AGTCACTA AACGCTTA GGAGAACA CATCAAGT AAGGTACA CGCTGATC GGTGCGAA CCTAATCC CTGAGCCA AGCCATGC GTACGCAA AGTACAAG ACATTGGC ATTGAGGA GTCGTAGA AGAGTCAA CCGACAAC ACGTATCA GTCTGTCA CTAAGGTC CGACACAC CCGTGAGA GTGTTCTA CAATGGAA AGCACCTC CAGCGTTA TAGGATGA AGTGGTCA ACAGCAGA CATACCAA TATCAGCA ATAGCGAC ACGCTCGA CTCAATGA TCCGTCTA AGGCTAAC CCATCCTC AGATGTAC TCTTCACA CCGAAGTA CGCATACA C10 D10 E10 F10 G10 H10 All B11 C11 D11 E11 F11 G11 H11 A12 B12 C12 D12 E12 F12 G12 AATGTTGC TGAAGAGA AGATCGCA AAGAGATC CAACCACA TGGAACAA CCTCTATC ACAGATTC CCAGTTCA TGGCTTCA CGACTGGA CAAGACTA CCTCCTGA TGGTGGTA AACAACCA AATCCGTC CAAGGAGC TTCACGCA CACCTTAC AAGACGGA ACACAGAA GAACAGGC AACCGAGA ACAAGCTA SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Reference 7 Reference Information for Kits with Original Index Configuration indexing primers in clear capped tubes Use the reference information in
5. includes excess Nuclease free water 15 5 uL 255 75 pL 5x T4 DNA Ligase Buffer green cap 10 uL 165 pL Diluted SureSelect Adaptor Oligo Mix from step1 10 pL 165 pL T4 DNA Ligase red cap 1 5 uL 24 75 uL Total 37 pL 610 5 pL 3 Add 37 uL of the Ligation master mix to each dA tailed purified DNA sample 13 uL in the PCR plate wells 4 Mix well by pipetting up and down SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 53 54 Sample Preparation 200 ng DNA Samples 5 Incubate the plate in the thermal cycler and run the program in Table 26 Do not use a heated lid Table 26 Ligation Thermal Cycler Program Step Temperature Time Step 1 20 C 15 minutes Step 2 4 C Hold Stopping Point If you do not continue to the next step seal the plate and store at 20 C SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Sample Preparation 200 ng DNA Samples 3 Step 8 Purify the sample using AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes Do not freeze the beads at any time 2 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color 3 Add 90 uL of homogeneous AMPure XP beads to each adaptor ligated DNA sample in the PCR plate 50 uL Pipette up and down to mix 4 Incubate samples for 5 minutes at room temperature Put the plate into a magnetic separation device Wait for the soluti
6. 2 minutes or until the residual ethanol completely evaporates 12 Add 15 uL nuclease free water to each sample well 13 Seal the wells with strip caps then mix well on a vortex mixer and briefly spin the plate to collect the liquid 14 Incubate for 2 minutes at room temperature 15 Put the plate in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 16 Remove 13 uL of the cleared supernatant to a fresh PCR plate well You can discard the beads at this time 17 Proceed immediately to the next step Step 7 Ligate the paired end adaptor SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Sample Preparation 200 ng DNA Samples 3 Step 7 Ligate the paired end adaptor Use the SureSelect XT Library Prep Kit ILM for this step Hold samples on ice while setting up this step 1 Dilute the SureSelect Adaptor Oligo Mix brown cap 1 10 in nuclease free water immediately before use Use the diluted oligo mix when preparing the Ligation master mix in the next step For FFPE derived DNA samples skip this dilution step and use the undiluted SureSelect Adaptor Oligo Mix in step 2 below See Chapter 6 for a complete list of modifications recommended for FFPE samples 2 Prepare the appropriate volume of Ligation master mix as described in Table 25 on ice Mix well on a vortex mixer Table 25 Preparation of Ligation master mix Reagent Volume for Volume for 16 reactions 1 reaction
7. 50 Cycle Kit v3 HiSeq 2500 High Output 2 x 100 bp 250 Cycle Kit v4 HiSeq 2000 All Runs 2 x 100 bp 4 x 50 Cycle Kit v3 MiSeq All Runs 2 x 100 bp 300 Cycle Kit v2 MiSeq All Runs 2 x 76 bp 150 Cycle Kit v3 NextSeq 500 All Runs 2 x 100 bp 300 Cycle Kit vi Sequencing run setup guidelines for 8 bp indexes For libraries prepared using kits with 8 bp indexes sequencing runs must be set up to perform an 8 bp index read See the Reference chapter for complete index sequence information SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Indexing and Sample Processing for Multiplexed Sequencing 5 For the HiSeq 2500 and NextSeq 500 v1 platforms use the Cycles settings shown in Table 47 Cycle number settings can be specified on the Run Configuration screen of the instrument control software interface after choosing Custom from the index type selection buttons Table 47 Cycle Number settings for HiSeq and NextSeq platforms Run Segment Cycle Number Read 1 100 Index 1 i7 9 Index 2 i5 0 Read 2 100 For the MiSeq platform use the Illumina Experiment Manager IEM software to generate a Sample Sheet that includes the run parameters specified in Table 48 Table 48 Run parameters for MiSeq platform Sample Sheet Parameter Entry Workflow GenerateFASTO Cycles for Read 1 100 for v2 chemistry 75 for v3 chemistry Cycles for Read 2 100 for v2 chemistry 75 for v3 chemistry Index 1 i7 Sequence Type the
8. 56 Sample Preparation 200 ng DNA Samples Step 9 Amplify the adaptor ligated library This step uses the components listed in Table 27 Thaw the reagents listed below and keep on ice Table 27 Reagents for pre capture PCR amplification Component Storage Location SureSelect Primer brown cap SureSelect XT Library Prep Kit ILM 20 C SureSelect ILM Indexing Pre Capture PCR SureSelect Target Enrichment Kit ILM Indexing Reverse Primer clear cap Hyb Module Box 2 20 C Herculase II Fusion DNA Polymerase red cap Herculase II Fusion DNA Polymerase kit 20 C 5x Herculase II Reaction Buffer clear cap Herculase II Fusion DNA Polymerase kit 20 C 100 mM dNTP Mix green cap Herculase II Fusion DNA Polymerase kit 20 C Do not use the PCR Reaction Buffer or dNTP mix from any other kit SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Sample Preparation 200 ng DNASamples 3 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 Prepare the appropriate volume of pre capture PCR reaction mix as described in Table 28 on ice Mix well on a vortex mixer Table 28 Preparation of SureSelect Pre Capture PCR Reaction Mix Reagent Volume for 1 Volume for 16 reactions reaction includes excess Nuclease free water 6 uL 99 0 uL SureSelect Pr
9. Downstream Sequencing Modifications After determining the amount of sequencing output required for intact DNA samples to meet the goals of your project use the guidelines in Table 53 below to determine the amount of extra sequencing output required for FFPE DNA samples based on the AACq DNA integrity score SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 97 Appendix Using FFPE derived DNA Samples For example if your workflow demands 100 Mb output for intact DNA samples to achieve the required coverage an FFPE sample with DIN score of 4 requires approximately 200 Mb of sequencing output to achieve the same coverage Table 53 Recommended sequencing augmentation for FFPE derived DNA samples DIN value Recommended fold increase for FFPE derived sample gt 8 No extra sequencing output between 5 and 8 Increase sequencing allocation by 1 5x between 3 and 5 Increase sequencing allocation by 2x between 1 5 and 3 Increase sequencing allocation by 3x to 5x lt 1 5 Increase sequencing allocation by 6x to 10x SureSelect Target Enrichment System for Illumina Multiplexed Sequencing SureSelect Target Enrichment System for Illumina Paired End Sequencing Library Protocol 7 Reference Reference Information for Kits with Revised Index Configuration indexing primers in white capped tubes or blue plate 100 Reference Information for Kits with Original Index Configuration indexing primers in clear
10. Level 1 BL1 safety rules e Possible stopping points where samples may be stored at 20 C are marked in the protocol Do not subject the samples to multiple freeze thaw cycles Safety Notes CAUTION e Wear appropriate personal protective equipment PPE when working in the laboratory 1 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 15 1 16 Before You Begin Required Reagents Table 2 Required Reagents for SureSelect Target Enrichment Description Vendor and part number SureSelect ClearSeq or OneSeq Capture Library SureSelect Reagent Kit Illumina ILM platforms HiSeq platform 16 reactions HiSeq platform 96 reactions HiSeq platform 480 reactions MiSeq platform 16 reaction MiSeq platform 96 reactions MiSeq platform 480 reactions Agencourt AMPure XP Kit 5 mL 60 mL 450 mL Herculase Il Fusion DNA Polymerase includes dNTPs and 5x Buffer 200 Reactions processes 100 XT libraries 400 Reactions Dynabeads MyOne Streptavidin T1 2mL 10 mL 100 mL 1X Low TE Buffer 10 mM Tris HCl pH 8 0 0 1 mM EDTA 100 Ethanol molecular biology grade Qubit dsDNA HS Assay Kit or Qubit BR dsDNA Assay Kit 100 assays 500 assays Nuclease free Water not DEPC treated Select the appropriate library from Table 3 or Table 4 Agilent p n G9611A p n G9611B p n G9611C p n G9612A p n G9612B p n G9612C Beckman Coulter Genomics p n A63880 p n
11. Spe T T T T T T 1 T 15 50 100 150 200 300 400 500 700 1500 bp Figure 7 Analysis of amplified library DNA using a DNA 1000 Bioanalyzer assay SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Sample Preparation 200 ng DNA Samples 3 Option 2 Analysis using the 2200 TapeStation and D1000 ScreenTape For more information to do this step see the Agilent 2200 TapeStation User Manual at www genomics agilent com 1 Prepare the TapeStation samples as instructed in the Agilent 2200 TapeStation User Manual Use 1 uL of each DNA sample diluted with 3 uL of D1000 sample buffer for the analysis CAUTION Make sure that you thoroughly mix the combined DNA and sample buffer on a vortex mixer for 5 seconds for accurate quantitation 2 Load the sample plate or tube strips from step 1 the D1000 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 a distribution with a DNA fragment size peak of approximately 225 to 275 bp Determine the concentration of the library DNA by integrating under the peak A sample electropherogram is shown in Figure 8 s a 3 E Sample Intensity FU 5 100 300 500 700 Figure 8 Analysis of amplified library DNA using a D1000 ScreenTape Stopping Point If you do not continue to the next step seal the plate and store at 20 C SureSelect Target Enr
12. captured library Refer to the protocol that is included with the Agilent QPCR NGS Library Quantification Kit p n G4880A for more details to do this step 1 Prepare a standard curve using the quantification standard included in the kit according to the instructions provided in the user guide 2 Dilute each index 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 3 Prepare the QPCR master mix with Illumina adaptor specific PCR primers according to instructions provided in the kit 4 Add an aliquot of the master mix to PCR tubes and add template 5 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 6 Use the standard curve to determine the concentration of each unknown index tagged library in nM SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 5 87 5 Indexing and Sample Processing for Multiplexed Sequencing Step 5 Pool samples for multiplexed sequencing The number of indexed libraries that may be multiplexed in a single sequencing lane is determined by the output specifications of the platform used together with the amount of sequencing data required for your research design Calculate the number of indexes that can be combined per lane according to th
13. equivalent Life Technologies p n 12331D or equivalent Pipetman or equivalent Pipetman P10 P20 P200 P1000 or equivalent Savant SpeedVac model DNA120 or equivalent Select a magnetic separator configured to collect magnetic particles on one side of each well Do not use a magnetic separator configured to collect the particles in a ring formation SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Before You Begin 1 Optional Reagents and Equipment Table 6 Optional materials for processing of all samples Description Vendor and part number Ethylene Glycol American Bioanalytical p n AB00455 Tween 20 Sigma Aldrich p n P9416 50ML Tube strip capping tool Agilent p n 410099 PlateLoc Thermal Microplate Sealer with Small Hotplate Agilent p n 65402A Peelable Aluminum Seal for PlateLoc Sealer Agilent p n 24210 001 MicroAmp Clear Adhesive Film Life Technologies p n L12 20 Agilent QPCR NGS Library Quantification Kit Illumina GA Agilent p n G4880A Mx3005P Real Time PCR System Agilent p n 401449 or equivalent Mx3000P Mx3005P 96 well tube plates Agilent p n 410088 or equivalent Mx3000P Mx3005P optical strip caps Agilent p n 401425 or equivalent Table 7 Optional materials for processing of FFPE samples Description Vendor and part number Agilent NGS FFPE QC Kit Agilent 16 reactions p n G9700A 96 reactions p n G9700B TapeStation Genomic DNA Analysis Consumables Genomic DNA ScreenTape Agilent p
14. gDNA samples Sample Preparation 200 ng DNA Samples This chapter describes the steps to prepare libraries for target enrichment from 200 ng gDNA samples Hybridization and Capture This chapter describes the steps to hybridize and capture the prepared library DNA Indexing and Sample Processing for Multiplexed Sequencing This chapter describes the steps to amplify purify and assess quality and quantity of the sample libraries Samples are pooled by mass prior to sequencing Appendix Using FFPE derived DNA Samples This chapter contains recommended protocol modifications for FFPE derived DNA samples Reference This chapter contains reference information including component kit contents and index sequences SureSelect Target Enrichment System for Illumina Multiplexed Sequencing What s New in Version B 3 Support for FFPE derived DNA samples Use the Low Input Library Preparation protocol in Chapter 3 Sample Preparation 200 ng DNA Samples with protocol modifications detailed in Chapter 6 Appendix Using FFPE derived DNA Samples See Table 7 on page 21 for recommended FFPE sample DNA integrity analysis reagents Support for All Exon v6 Capture Libraries see Table 3 on page 17 Updated 96 well plate mixer recommendation see page 20 Updates to information on volume requirements for sample plates or tube strips see page 14 page 19 and page 26 Updates to sequencing guidelines including support f
15. gt 1 5 Mb 12 cycles All Exon and Exome libraries 10 to 12 cycles OneSeq libraries all designs 10 cycles 7 When the PCR amplification program is complete spin the plate or strip tube or strip tube briefly Proceed to Step 2 Purify the amplified captured libraries using AMPure XP beads on page 81 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Indexing and Sample Processing for Multiplexed Sequencing 5 Step 2 Purify the amplified captured libraries using AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes Do not freeze the beads at any time 2 Prepare 400 uL of fresh 70 ethanol per sample plus excess for use in step 9 3 Mix the AMPure XP bead suspension well so that the suspension appears homogeneous and consistent in color 4 Add 90 uL of the homogeneous AMPure XP bead suspension to each 50 uL amplified DNA sample bead suspension in the PCR plate or strip tube 5 Mix thoroughly by pipetting up and down Check that the beads are in a homogeneous suspension in the sample wells Each well should have a uniform color with no layers of beads or clear liquid present 6 Incubate samples for 5 minutes at room temperature 7 Put the plate or strip tube on the magnetic stand at room temperature Wait for the solution to clear approximately 3 to 5 minutes 8 While keeping the plate or tubes in the magnetic stand carefully remove and disca
16. kb up to 0 5 Mb 16 cycles 0 5 Mb up to 1 49 Mb 14 cycles gt 1 5 Mb 12 cycles All Exon and Exome libraries 10 to 12 cycles OneSeq libraries all designs 10 cycles 7 When the PCR amplification program is complete spin the plate or strip tube briefly Proceed to Step 2 Purify the amplified captured libraries using AMPure XP beads on page 81 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 71 5 Indexing and Sample Processing for Multiplexed Sequencing CAUTION Step 1B Amplify the captured libraries with indexing primers containing 6 bp indexes 1 16 In this step the SureSelect enriched DNA libraries are PCR amplified in PCR reactions that include the appropriate indexing primer for each sample This step uses the components listed in Table 41 Thaw then vortex to mix the reagents listed below and keep on ice Table 41 Reagents for post capture indexing by PCR amplification Kit Component Storage Location 5x Herculase II Reaction Buffer Herculase II Fusion DNA Polymerase kit 20 C 100 mM dNTP Mix 25 mM each dNTP Herculase II Fusion DNA Polymerase kit 20 C Herculase II Fusion DNA Polymerase Herculase II Fusion DNA Polymerase kit 20 C SureSelect ILM Indexing Post Capture Forward SureSelect Target Enrichment Kit ILM Indexing PCR Primer Hyb Module Box 2 20 C SureSelect Index reverse primers SureSelect XT Library Prep Kit ILM 20 C Do not use the PC
17. magnetic separation device Wait for the solution to clear approximately 3 to 5 minutes 6 Keep the plate in the magnetic stand Carefully remove and discard the cleared solution from each well Do not touch the beads while removing the solution 7 Continue to keep the plate in the magnetic stand while you dispense 200 uL of freshly prepared 70 ethanol in each sample well 8 Wait for 1 minute to allow any disturbed beads to settle then remove the ethanol 9 Repeat step 7 to step 8 step once 10 Seal the wells with strip caps then briefly spin the plate to collect the residual ethanol Return the plate to the magnetic stand for 30 seconds Remove the residual ethanol with a P20 pipette 11 Dry the samples by placing the unsealed plate on the thermal cycler set to hold samples at 37 C for 1 to 2 minutes or until the residual ethanol completely evaporates 12 Add 15 uL nuclease free water to each sample well 13 Seal the wells with strip caps then mix well on a vortex mixer and briefly spin the plate to collect the liquid 14 Incubate for 2 minutes at room temperature 15 Put the plate in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 16 Remove 13 uL of the cleared supernatant to a fresh PCR plate well You can discard the beads at this time 17 Proceed immediately to the next step Step 8 Ligate the paired end adaptor SureSelect Target Enrichment System for Illumina Multiplexed
18. or PCR hood with UV sterilization and positive air flow 1 Prepare the appropriate volume of pre capture PCR reaction mix as described in Table 17 on ice Mix well on a vortex mixer Table 17 Preparation of SureSelect Pre Capture PCR Reaction Mix Reagent Volume for 1 Volume for 16 reactions reaction includes excess Nuclease free water 21 uL 346 5 uL SureSelect Primer brown cap 1 25 uL 20 6 uL SureSelect ILM Indexing Pre Capture PCR 1 25 uL 20 6 uL Reverse Primer clear cap 5x Herculase II Reaction Buffer clear cap 10 uL 165 pL 100 mM dNTP Mix green cap 0 5 uL 8 25 uL Herculase II Fusion DNA Polymerase red cap Tul 16 5 pL Total 35 pL 577 5 pL 2 Combine 35 uL of the PCR reaction mixture prepared in Table 17 and 15 uL of each purified DNA library sample from step 16 on page 35 Add a single DNA library sample to each well of the plate or strip tube Mix by pipetting SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 37 2 38 Sample Preparation 3 pg DNA Samples 3 Run the program in Table 18 in a thermal cycler Table 18 Pre Capture PCR Thermal Cycler Program Segment Number of Temperature Time Cycles 1 1 98 C 2 minutes 2 4 6 98 C 30 seconds 65 C 30 seconds 72 C 1 minute 3 1 72 C 10 minutes 4 1 4 C Hold Different library preparations can produce slightly different results based on varying DNA quality In most cases five cycles will produce an adeq
19. qPCR quantification quantification Pre capture PCR 10 cycles 10 cycles 10 cycles 13 cycles cycle number page 58 FFPE samples with AACq scores lt 1 should be treated like non FFPE samples at these steps For samples of this type make sure to use the DNA concentration determined by the Qubit Assay instead of the concentration determined by gPCR to cal culate the volume required for 200 ng DNA Downstream Sequencing Modifications After determining the amount of sequencing output required for intact DNA samples to meet the goals of your project use the guidelines in Table 51 to determine the amount of extra sequencing output required for FFPE DNA samples based on the AACq DNA integrity score SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 95 Appendix Using FFPE derived DNA Samples For example if your workflow demands 100 Mb output for intact DNA samples to achieve the required coverage an FFPE sample with AACq score of 1 requires 150 200 Mb of sequencing output to achieve the same coverage Table 51 Recommended sequencing augmentation for FFPE derived DNA samples AACq value Recommended fold increase for FFPE derived sample lt 0 5 between 0 5 and 2 between 2 and 3 5 between 3 5 and 5 gt 5 No extra sequencing output Increase sequencing allocation by 1 5x to 2x Increase sequencing allocation by 3x Increase sequencing allocation by 4x to 5x Increase sequencing alloc
20. step see the Agilent 2200 TapeStation User Manual at www genomics agilent com 1 Prepare the TapeStation samples as instructed in the Agilent 2200 TapeStation User Manual Use 1 uL of each DNA sample diluted with 3 uL of D1000 sample buffer for the analysis CAUTION Make sure that you thoroughly mix the combined DNA and sample buffer on a vortex mixer for 5 seconds for accurate quantitation 2 Load the sample plate or tube strips from step 1 the D1000 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 a DNA fragment size peak between 150 200 bp A sample electropherogram is shown in Figure 3 Sample Intensity FU Figure 3 Analysis of sheared DNA using a D1000 ScreenTape SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 29 2 30 Sample Preparation 3 pg DNA Samples Step 4 Repair the ends Use the SureSelect XT Library Prep Kit ILM for this step To process multiple samples prepare master mixes with overage at each step without the DNA sample Master mixes for preparation of 16 samples including excess are shown in each table as an example Hold samples on ice while setting up this step 1 Prepare the appropriate volume of End Repair master mix as described in Table 10 on ice Mix well on a vortex mixer Table 10 Preparation of End Repair master mix Re
21. the volume of hybridization solution that remains after the 24 hour incubation 2 Maintain the hybridization plate or strip tube at 65 C while you use a multichannel pipette to transfer the entire volume approximately 25 to 29 uL of each hybridization mixture to the plate or strip tube wells containing 200 uL of washed streptavidin beads Mix well by slowly pipetting up and down until beads are fully resuspended Excessive evaporation such as when less than 20 uL remains after hybridization can indicate suboptimal capture performance SeeTable 65 on page 109 for tips to minimize evaporation 3 Cap the wells then incubate the capture plate or strip tube ona 96 well plate mixer mixing vigorously 1400 1800 rpm for 30 minutes at room temperature Make sure the samples are properly mixing in the wells 4 Briefly spin the plate or strip tube in a centrifuge or mini plate spinner 5 Put the plate or strip tube in a magnetic separator to collect the beads Wait until the solution is clear then remove and discard the supernatant 6 Resuspend the beads in 200 uL of SureSelect Wash Buffer 1 Mix by pipetting up and down until beads are fully resuspended 7 Incubate the samples for 15 minutes at room temperature 8 Briefly spin in a centrifuge or mini plate spinner 9 Put the plate or strip tube in the magnetic separator Wait for the solution to clear then remove and discard the supernatant SureSelect Target Enrichment S
22. 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 Target Enrichment System for Illumina Multiplexed Sequencing 89 5 Indexing and Sample Processing for Multiplexed Sequencing Step 6 Prepare sequencing samples The sequencing workflow for FFPE derived DNA libraries should employ the modifications discussed in the Appendix starting on page 93 Modifications include the requirement for adapter trimming and increased sequencing depth for lower integrity DNA samples The optimal seeding concentration for SureSelect target enriched libraries is 6 to 8 pM on HiSeq or MiSeq instruments and 1 2 to 1 3 pM on the NextSeq platform Seeding concentration and cluster density may also need to be optimized based on the DNA fragment size range for the library and on the desired output and data quality Follow Illumina s recommendation for a PhiX control in a low concentration spike in for improved sequencing quality control Proceed to cluster amplification using the appropriate Illumina Paired End Cluster Generation Kit See Table 46 for kit configurations compatible with the recommended read length Table 46 Illumina Kit Configuration Selection Guidelines Platform Run Type Read Length SBS Kit Configuration Chemistry HiSeq 2500 Rapid Run 2 x 100 bp 200 Cycle Kit vi HiSeq 2500 Rapid Run 2 x 100 bp 200 Cycle Kit v2 HiSeg 2500 High Output 2 x 100 bp 4 x
23. 221 ng uL prepare 3 4 uL of a 221 ng uL dilution of each library 2 For prepped libraries with DNA concentrations below 221 ng uL use a vacuum concentrator to concentrate the samples at lt 45 C a Add the entire 30 uL volume 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 Dehydrate using a vacuum concentrator on low heat less than 45 C c Reconstitute with nuclease free water to a final concentration of 221 ng uL Pipette up and down along the sides of the tube for optimal recovery d Mix well on a vortex mixer and spin in a centrifuge for 1 minute 3 Transfer each 3 4 uL gDNA library sample 750 ng to a separate well of a 96 well plate or strip tube Seal the wells and keep on ice You must avoid evaporation from the small volumes of the capture during the 16 or 24 hour 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 27 uL of water at 65 C for 24 hours as a test Include water 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 4 pL For a partial list of tested options showing minimal evaporation refer to Alternative Captu
24. 59 Step 11 Assess quality and quantity 60 This section contains instructions for the preparation of gDNA libraries from 200 ng DNA samples For higher input 3 ug DNA samples see the library preparation protocol on page 23 The sample preparation protocol is used to prepare DNA libraries for sequencing using the Illumina paired read platform For each sample to be sequenced an individual indexed library is prepared For an overview of the SureSelect target enrichment workflow see Figure 1 on page 12 The steps in this section differ from the Illumina protocol in the use of the Covaris system for gDNA shearing smaller target shear size elimination of size selection by gel purification implementation of AMPure XP beads for all purification steps and primers used for PCR Refer to the Illumina protocol Preparing Samples for Multiplexed Paired End Sequencing p n1005361 or the appropriate Illumina protocol for more information git Agilent Technologies 43 3 44 Sample Preparation 200 ng DNA Samples Step 1 Shear the DNA Make sure genomic DNA samples are of high quality with an OD 260 280 ratio ranging from 1 8 to 2 0 Use the Qubit system to quantify genomic DNA before library preparation For FFPE derived DNA samples review the protocol modifications detailed in the Appendix on page 93 before starting the library preparation protocol For each DNA sample to be sequenced prepare 1 library 1 Set up the Covaris E
25. 8 nt index sequence for each individual sample see Table 37 enter in Data Section for on page 65 each sample SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 91 5 Indexing and Sample Processing for Multiplexed Sequencing Step 6 Prepare sequencing samples 92 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing SureSelect Target Enrichment System for Illumina Paired End Sequencing Library Protocol ee 6 7 e Appendix Using FFPE derived DNA Samples Modifications for all FFPE DNA samples 94 Modifications for samples assessed using the Agilent NGS FFPE QC Kit 95 Modifications for samples assessed using Agilent s Genomic DNA ScreenTape 97 FFPE derived DNA samples may be used in the Low Input 200 ng DNA Library Preparation protocol and subsequent Target Enrichment protocol after making the minor protocol modifications detailed in this chapter Protocol modifications that should be applied to all FFPE samples are detailed on page 94 Additional protocol modifications may be appropriate depending on the integrity of the FFPE sample DNA DNA integrity may be assessed using the Agilent NGS FFPE QC Kit or using the Agilent 2200 TapeStation system and Genomic DNA ScreenTape The Agilent NGS FFPE QC Kit provides a qPCR based assay for DNA sample integrity determination Results include the precise quantity of amplifiable DNA in the sample and a AACq
26. A63881 p n A63882 Agilent p n 600677 p n 600679 Life Technologies p n 65601 p n 65602 p n 65603 Life Technologies p n 12090 015 or equivalent Sigma Aldrich p n E7023 Life Technologies p n 032851 Life Technologies p n 032850 p n 032853 Ambion Cat AM9930 SureSelect ClearSeq and OneSeq reagents must be used within one year of receipt t HiSeq and MiSeq Reagent Kits are also compatible with the NextSeq 500 platform SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Before You Begin 1 Table 3 SureSelect Capture Libraries SureSelect Human AllExonv 5190 8863 5190 88684 tt SureSelect Human All Exon v6 UTRs 5190 8881 5190 8882 z SureSelect Human All Exon v6 COSMIC 5190 9307 5190 9308 SureSelect Human All Exon v6 Plus 1 5190 8866 5190 8867 SureSelect Human All Exon v6 Plus 2 5190 8869 5190 8870 SureSelect Clinical Research Exome 5190 7338 5190 7339 SureSelect Focused Exome 5190 7787 5190 7788 SureSelect Focused Exome Plus 1 5190 7790 5190 7791 SureSelect Focused Exome Plus 2 5190 7793 5190 7795 SureSelect Human All Exon v5 5190 6208 5190 6209 SureSelect Human All Exon v5 UTRs 5190 6213 5190 6214 SureSelect Human All Exon v5 IncRNA 5190 6446 5190 6447 SureSelect Human All Exon v5 Plus 5190 6211 5190 6212 SureSelect Human All Exon v4 5190 4631 5190 4632 5190 4634 SureSelect Human All E
27. Backbone Custom 0 5 2 9 Mb 5190 8889 5190 8890 OneSegq Hi Res CNV Backbone Custom 3 5 9 Mb 5190 8892 5190 8893 OneSegq Hi Res CNV Backbone Custom 6 11 9 Mb 5190 8895 5190 8896 18 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing CAUTION Required Equipment Before You Begin 1 Sample volumes exceed 0 2 ml in certain steps of this protocol If you plan to use plates or strip tubes besides the SureCycler 8800 compatible plasticware listed in the table below first make sure that the plasticware holds 20 31 ml per well when processing 3 ug DNA samples or gt 0 28 ml per well when processing 200 ng DNA samples Samples may be transferred to 1 5 ml tubes for processing where required with possible impacts on sample throughput and yield Table 5 Required Equipment for SureSelect Target Enrichment Description SureCycler 8800 Thermal Cycler or equivalent 96 well plate module for SureCycler 8800 Thermal Cycler SureCycler 8800 compatible plasticware 96 well plates OR 8 well strip tubes Tube cap strips domed DNA Analysis Platform and Consumables Agilent 2100 Bioanalyzer Laptop Bundle Agilent 2100 Bioanalyzer Electrophoresis Set Agilent DNA 1000 Kit Agilent High Sensitivity DNA Kit OR Agilent 2200 TapeStation D1000 ScreenTape D1000 Reagents High Sensitivity D1000 ScreenTape High Sensitivity D1000 Reagents Qubit Fluorometer Qubit Assay Tubes Vendor and part number Ag
28. Before you begin view hands on videos of SureSelect procedures at http www agilent com genomics protocolvideos alee m SureSelect Target Enrichment System for Illumina Paired End Multiplexed Sequencing Library Version B 3 June 2 SureSelect platform manufactur with Agilent SurePrint Technology For Research Use Only Not for use in Diagnostic Procedures oe Agilent Technologies Notices Agilent Technologies Inc 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 G7530 90000 Edition Version B 3 June 2015 Printed in USA Agilent Technologies Inc 5301 Stevens Creek Blvd Santa Clara CA 95051 USA Acknowledgment Oligonucleotide sequences 2006 2008 and 2011 Illumina Inc All rights reserved Only for use with the Illumina sequencer systems 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 Notice to Purchaser SureS
29. DNA integrity score Protocol modifications based on AACgq scores for individual samples are detailed on page 95 The Agilent 2200 TapeStation system combined with the Genomic DNA ScreenTape assay provides a microfluidics based method for determination of a DNA Integrity Number DIN score Protocol modifications based on DIN scores for individual samples are detailed on page 97 wit Agilent Technologies 93 6 Appendix Using FFPE derived DNA Samples Modifications for all FFPE DNA samples SureSelect Protocol Modifications Protocol modifications that should be applied to all FFPE samples are detailed in Table 49 Table 49 SureSelect protocol modifications for all FFPE samples Workflow Step and page Parameter Condition for non FFPE Condition for FFPE Samples Samples Library Preparation using Duration of DNA Shearing 6 minutes 4 minutes Low Input DNA page 45 Library Preparation using Dilution of SureSelect Adaptor Use 1 10 dilution of Use undiluted SureSelect Adaptor Low Input DNA page 53 Oligo Mix for Ligation reaction SureSelect Adaptor Oligo Mix Oligo Mix Hybridization page 65 Amount of prepared library 750 ng 500 750 ng use maximum added to Hybridization available in range Post capture PCR Amount of captured DNA 14 ul 30 ul decrease the amount of page 75 to page 76 OR bead suspension added to water added to post capture PCR by page 78 to page 79 PCR 16 ul to compensate for greater volume of captured DNA
30. ILM Indexing Pre Capture PCR Reverse Primer SureSelect ILM Indexing Post Capture Forward PCR Primer tube with green cap tube with blue cap tube with brown cap tube with purple cap tube with clear cap tube with orange cap tube with yellow cap tube with green cap tube with blue cap tube with brown cap tube with purple cap tube with clear cap tube with orange cap tube with green cap tube with blue cap tube with brown cap tube with purple cap tube with clear cap tube with orange cap SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 107 7 108 Reference Nucleotide Sequences of SureSelect 6 bp Indexes Original Kit Configuration Refer to the sequence information in Table 64 only if your kit includes Library Prep kit p n 5500 0105 or p n 5500 0075 with 6 bp indexing primers provided in 16 clear capped tubes original kit configuration Table 64 SureSelect Indexes 1 16 Index Number Sequence 1 ATCACG 2 CGATGT 3 TTAGGC 4 TGACCA 5 ACAGTG 6 GCCAAT 7 CAGATC 8 ACTTGA 9 GATCAG 10 TAGCTT 11 GGCTAC 12 CTTGTA 13 AAACAT 14 CAAAAG 15 GAAACC 16 AAAGCA SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Alternative Capture Equipment Combinations Reference 7 Table 65 below lists combinations of thermal cyclers lid temperatures plates or strip tubes and sealing methods that have shown minimal evaporation when used for the Hybridizatio
31. PCR plate well You can discard the beads at this time Stopping Point If you do not continue to the next step seal the plate and store at 20 C SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 59 3 Sample Preparation 200 ng DNA Samples Step 11 Assess quality and quantity Sample analysis can be done with either the 2100 Bioanalyzer instrument or the 2200 TapeStation instrument Option 1 Analysis using the 2100 Bioanalyzer and DNA 1000 Assay See the DNA 1000 Kit Guide at www genomics agilent com for more information on doing this step 1 Check that the 2100 Bioanalyzer electrodes have been cleaned as instructed in the reagent kit guide Open the 2100 Expert Software version B 02 02 or higher turn on the 2100 Bioanalyzer and check communication Prepare the chip samples and ladder as instructed in the reagent kit guide using 1 uL of each sample for the analysis Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation Within the instrument context choose the DNA 1000 assay from the drop down list Start the run Enter sample names and comments in the Data and Assay context Verify that the electropherogram shows a distribution with a DNA fragment size peak of approximately 225 to 275 bp Determine the concentration of the library DNA by integrating under the peak A sample electropherogram is shown in Figure 7 Fu
32. R Reaction Buffer or dNTP mix from any other kit Prepare one indexing amplification reaction for each DNA library When processing FFPE derived DNA samples some details of this step should be modified See Table 49 on page 94 for more information To avoid cross contaminating libraries set up PCR mixes in a dedicated clean area or PCR hood with UV sterilization and positive air flow 78 1 Determine the appropriate index assignments for each sample See Table 64 in the Reference chapter for sequences of the index portion of the SureSelect PCR Primers Index 1 through Index 16 used to amplify the DNA libraries in this step Use a different indexing primer for each sample to be sequenced in the same lane SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Indexing and Sample Processing for Multiplexed Sequencing 5 2 Prepare the appropriate volume of PCR reaction mix as described in Table 42 on ice Mix well on a vortex mixer Table 42 Preparation of post capture PCR Reaction mix Reagent Volume for 1 Volume for 16 reactions reaction includes excess Nuclease free water 22 5 uL 382 5 uL 5x Herculase II Reaction Buffer clear cap 10 uL 170 pL Herculase II Fusion DNA Polymerase red cap Tul 17 uL 100 mM dNTP Mix green cap 0 5 pL 8 5 uL SureSelect ILM Indexing Post Capture Forward 1 uL 17 uL PCR Primer orange cap Total 35 pL 595 pL 3 Add 35 uL of the PCR reaction mix prepared
33. Remove the cleared supernatant approximately 32 uL to a fresh PCR plate well You can discard the beads at this time Stopping Point If you do not continue to the next step seal the plate and store at 20 C SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 35 2 36 Sample Preparation 3 pg DNA Samples Step 10 Amplify the adaptor ligated library This step uses the components listed in Table 16 Thaw the reagents listed below and keep on ice Table 16 Reagents for pre capture PCR amplification Component Storage Location SureSelect Primer SureSelect XT Library Prep Kit ILM 20 C SureSelect ILM Indexing Pre Capture PCR SureSelect Target Enrichment Kit ILM Indexing Reverse Primer Hyb Module Box 2 20 C Herculase II Fusion DNA Polymerase Herculase II Fusion DNA Polymerase kit 20 C 5x Herculase II Reaction Buffer Herculase II Fusion DNA Polymerase kit 20 C 100 mM dNTP Mix Herculase II Fusion DNA Polymerase kit 20 C Do not use the PCR Reaction Buffer or dNTP mix from any other kit This protocol uses half of the adaptor ligated library for amplification The remainder can be saved at 20 C for future use if needed SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Sample Preparation 3 pg DNA Samples 2 CAUTION To avoid cross contaminating libraries set up PCR reactions all components except the library DNA in a dedicated clean area
34. Sequencing 33 2 34 Sample Preparation 3 pg DNA Samples Stopping Point Step 8 Ligate the paired end adaptor Use the SureSelect XT Library Prep Kit ILM for this step Hold samples on ice while setting up this step 1 Prepare the appropriate volume of Ligation master mix as described in Table 14 on ice Mix well on a vortex mixer Table 14 Preparation of Ligation master mix Reagent Volume for 1 reaction Volume for 16 reactions includes excess Nuclease free water 15 5 uL 255 75 uL 5x T4 DNA Ligase Buffer green cap 10 uL 165 uL SureSelect Adaptor Oligo Mix brown cap 10 pL 165 pL T4 DNA Ligase red cap 1 5 uL 24 75 uL Total 37 pL 610 5 pL 2 Add 37 uL of the Ligation master mix to each dA tailed purified DNA sample 13 uL in the PCR plate wells 3 Mix well by pipetting up and down 4 Incubate the plate in the thermal cycler and run the program in Table 15 Do not use a heated lid Table 15 Ligation Thermal Cycler Program Step Temperature Time Step 1 20 C 15 minutes Step 2 4 C Hold If you do not continue to the next step seal the plate and store at 20 C SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Sample Preparation 3 pg DNA Samples 2 Step 9 Purify the sample using AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes Do not freeze the beads at any time 2 Mix the bead suspension well so that the reagent a
35. a and 12 212 Computer Software and for the Department of Defense DFARS 252 227 7015 Technical Data Commercial 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 2 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing In this Guide This guide provides an optimized protocol for Illumina paired end multiplexed library preparation using the SureSelect Library Prep and Capture System 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 Sample Preparation 3 pg DNA Samples This chapter describes the steps to prepare libraries for target enrichment from 3 ug
36. ading tips into the 2200 TapeStation as instructed in the Agilent 2200 TapeStation User Manual Start the run 3 Verify that the electropherogram shows the peak of DNA fragment size positioned between 250 and 350 bp A sample electropherogram is shown in Figure 10 4 Measure the concentration of each library by integrating under the entire peak If you wish to more precisely quantify the target enriched samples prior to pooling proceed to Step 4 Quantify each index tagged library by QPCR optional on page 87 Otherwise proceed to Step 5 Pool samples for multiplexed sequencing on page 88 If you do not continue to the next step store the libraries at 4 C overnight or at 20 C for up to one month SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 85 5 Indexing and Sample Processing for Multiplexed Sequencing Step 3 Assess indexed library DNA quantity and quality Sample Intensity FU doa 2 a g Figure 10 Post capture analysis of amplified indexed library DNA using the 2200 TapeStation with a High Sensitivity D1000 ScreenTape 86 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Indexing and Sample Processing for Multiplexed Sequencing Step 4 Quantify each index tagged library by QPCR optional You can use the Agilent QPCR NGS Library Quantification Kit for Illumina to accurately determine the concentration of each index tagged
37. ads remain suspended in solution after 5 minutes carefully remove and discard 100 ul of cleared solution from near the bottom of the wells and continue incubating the plate in the magnetic stand for an additional 3 minutes After the remaining suspension has cleared remove and discard the remaining cleared solution approximately 180 ul from the wells 8 Continue to keep the plate in the magnetic stand while you dispense 200 uL of 70 ethanol in each sample well Use fresh 70 ethanol for optimal results 9 Wait for 1 minute to allow any disturbed beads to settle then remove the ethanol 10 Repeat step 8 to step 9 once SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 49 3 Sample Preparation 200 ng DNA Samples 11 Seal the wells with strip caps then briefly spin the plate to collect the residual ethanol Return the plate to the magnetic stand for 30 seconds Remove the residual ethanol with a P20 pipette 12 Dry the samples by placing the unsealed plate on the thermal cycler set to hold samples at 37 C for 3 to 5 minutes or until the residual ethanol completely evaporates Do not dry the bead pellet to the point that the pellet appears cracked during any of the bead drying steps in the protocol Elution efficiency is significantly decreased when the bead pellet is excessively dried 13 Add 32 uL nuclease free water to each sample well 14 Seal the wells with strip caps then mix well on a vort
38. agent Volume for 1 reaction Volume for 16 reactions includes excess Nuclease free water 35 2 uL 580 8 uL 10x End Repair Buffer clear cap 10 pL 165 pL dNTP Mix green cap 1 6 uL 26 4 uL T4 DNA Polymerase purple cap Tul 16 5 pL Klenow DNA Polymerase yellow cap 2 uL 33 uL T4 Polynucleotide Kinase orange cap 2 2 uL 36 3 uL Total 52 pl 858 pL 2 Add 52 uL of the master mix to each PCR plate sample well containing purified sheared DNA Mix by pipetting up and down 3 Incubate the plate in the thermal cycler and run the program in Table 11 Do not use a heated lid Table 11 End Repair Thermal Cycler Program Step Temperature Time Step 1 20 C 30 minutes Step 2 4 C Hold SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Sample Preparation 3 pg DNA Samples 2 Step 5 Purify the sample using AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes Do not freeze the beads at any time 2 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color 3 Add 180 uL of homogeneous AMPure XP beads to each 100 uL end repaired DNA sample in the PCR plate Pipette up and down 10 times to mix 4 Incubate samples for 5 minutes at room temperature Put the plate into a magnetic separation device Wait for the solution to clear approximately 3 to 5 minutes 6 Keep the plate in the magnetic stand Carefully remove and discard the cl
39. ation by 6x to 10x SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Appendix Using FFPE derived DNA Samples 6 Modifications for samples assessed using Agilent s Genomic DNA ScreenTape Before applying protocol modifications in this section use the Agilent 2200 TapeStation system and Genomic DNA ScreenTape to determine the DNA Integrity Number DIN score for each sample For more information on how to obtain DIN numbers using the TapeStation system go to genomics agilent com and search for document part number G5991 5442 Use the DIN score to determine whether additional SureSelect protocol or downstream sequencing modifications are appropriate for each sample T SureSelect Protocol Modifications Protocol modifications that should be applied to FFPE samples based on DIN score are detailed in Table 52 Table52 SureSelect protocol modifications based on DIN score Protocol Step and non FFPE FFPE Samples Parameter Samples DIN gt 5 DIN between 2 and 5 DIN lt 2 DNA input for Library 200 ng based 200 ng based on 100 to 200 ng of amplifiable 100 to 200 ng of amplifiable Preparation page 44 on Qubit Assay Qubit Assay DNA based on qPCR DNA based on qPCR quantification quantification Pre capture PCR 10 cycles 10 cycles 10 cycles 13 cycles cycle number page 58 Use the Agilent NGS FFPE QC Kit for qPCR based sample quantification See Table 7 on page 21 for ordering information
40. ble 2 on page 16 1 Prewarm SureSelect Wash Buffer 2 at 65 C in a circulating water bath or heat block for use in Step 3 Capture the hybridized DNA using streptavidin coated beads on page 71 2 Vigorously resuspend the Dynabeads MyOne Streptavidin T1 magnetic beads on a vortex mixer The magnetic beads settle during storage 3 For each hybridization sample add 50 uL of the resuspended beads to wells of a fresh PCR plate or strip tube 4 Wash the beads a b c d Add 200 uL of SureSelect Binding Buffer Mix by pipetting up and down until beads are fully resuspended Put the plate or strip tube into a magnetic separator device Wait until the solution is clear then remove and discard the supernatant Repeat step a through step d two more times for a total of 3 washes 5 Resuspend the beads in 200 uL of SureSelect Binding Buffer If you are equipped for higher volume magnetic bead captures the streptavidin beads may be batch washed in an Eppendorf tube or conical vial Start the batch washing procedure using excess bead solution After resuspending the washed beads in the appropriate volume of SureSelect Binding Buffer aliquot 200 ul of the washed beads to plate or strip tube wells to be used for hybridization capture 70 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Hybridization and Capture 4 Step 3 Capture the hybridized DNA using streptavidin coated beads 1 Estimate and record
41. capped tubes 105 Alternative Capture Equipment Combinations 109 This chapter contains reference information including component kit contents and index sequences ait Agilent Technologies 99 7 Reference CAUTION This chapter contains two sets of index sequence and kit content information The first section covers kits with 8 bp indexes supplied in Library Prep Kit p n 5500 0132 or 5500 0133 typically received December 2014 or later The second section covers kits with 6 bp indexes supplied in Library Prep Kit p n 5500 0105 or 5500 0075 typically received before December 2014 Verify that you are referencing the information appropriate for your kit version before you proceed Reference Information for Kits with Revised Index Configuration indexing primers in white capped tubes or blue plate Use the reference information in this section if your kit includes Library Prep Kit p n 5500 0132 or 5500 0133 If your kit does not include one of these component kits see page 105 for kit content and indexing primer information Kit Contents XT Each SureSelect Reagent Kit contains the following component kits Table 54 SureSelect Reagent Kit Contents Revised Index Configuration Product Storage Condition 16 Reactions 96 Reactions 480 Reactions SureSelect XT Library Prep Kit ILM 20 C 5500 0132 5500 0133 5 x 5500 0133 SureSelect Target Enrichment Box 1 Room Temperature 5190 8645 5190 8646 5 x 5190 8646 SureSelect Ta
42. ccurate quantitation 2 Load the sample plate or tube strips from step 1 the D1000 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 a distribution with a DNA fragment size peak of approximately 225 to 275 bp Determine the concentration of the library DNA by integrating under the peak A sample electropherogram is shown in Figure 5 s a 3 E Sample Intensity FU 5 100 300 500 700 Figure 5 Analysis of amplified library DNA using a D1000 ScreenTape SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 41 2 Sample Preparation 3 pg DNA Samples Step 12 Assess quality and quantity 42 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing SureSelect Target Enrichment System for Illumina Paired End Sequencing Library Protocol ee 3 CAUTION 7 e Sample Preparation 200 ng DNA Samples Step 1 Shear the DNA 44 Step 2 Assess quality optional 47 Step 3 Repair the ends 48 Step 4 Purify the sample using AMPure XP beads 49 Step 5 Adenylate the 3 end of the DNA fragments 51 Step 6 Purify the sample using AMPure XP beads 52 Step 7 Ligate the paired end adaptor 53 Step 8 Purify the sample using AMPure XP beads 55 Step 9 Amplify the adaptor ligated library 56 Step 10 Purify the amplified library with AMPure XP beads
43. ch area In particular never use materials designated to post PCR work areas for pre PCR segments of the workflow 2 Maintain clean work areas Clean pre PCR surfaces that pose the highest risk of contamination daily using a 10 bleach solution 3 Always use dedicated pre PCR pipettors with nuclease free aerosol resistant tips to pipette dedicated pre PCR solutions 4 Wear powder free gloves Use good laboratory hygiene including changing gloves after contact with any potentially contaminated surfaces Do not mix stock solutions of gDNA on a vortex mixer Instead gently tap the tube with your finger to mix the sample e 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 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Before You Begin e For each protocol step that requires removal of tube cap strips reseal the tubes with a fresh strip of domed caps Cap deformation may result from exposure of the cap strips to the heated lid of the thermal cycler and from other procedural steps Reuse of strip caps can cause sample loss sample contamination or imprecision in sample temperatures during thermal cycler incubation steps e In general follow Biosafety
44. ded in white capped tubes or A01 through H12 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 75 5 Indexing and Sample Processing for Multiplexed Sequencing provided in blue plate to each well Add only one of the 16 or 96 possible indexing primers to each reaction well 5 Add the DNA library samples to the PCR reactions a Obtain the PCR plate or strip tube containing 30 uL of bead bound target enriched DNA samples from ice prepared on page 72 b Pipette each DNA sample up and down until the bead suspension is homogeneous then transfer 14 uL of the sample to the appropriate well of the PCR plate or strip tube containing PCR reaction mix and indexing primer c Mix the PCR reactions well by pipetting d Store the remaining library bound beads at 20 C for future use if needed 6 Transfer the PCR plate or strip tube to a thermal cycler and run the PCR amplification program shown in Table 39 Table 39 Post Capture PCR cycling program Segment Number of Cycles Temperature Time 1 1 98 C 2 minutes 2 10 to 16 Cycles 98 C 30 seconds See Table 40 for recommendations based 57 C 30 seconds on Capture Library size 72 C 1 minute 3 1 72 C 10 minutes 4 1 4 C Hold 76 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Indexing and Sample Processing for Multiplexed Sequencing 5 Table 40 Post capture PCR cycle number recommendations Capture Size Cycles 1
45. e capacity of your platform and the amount of sequencing data required per sample 1 Combine the libraries such that each index tagged sample is present in equimolar amounts in the pool For each library use the formula below to determine the amount of indexed sample to use Volume of Index VXC x C i where V f is the final desired volume of the pool C f is the desired final concentration of all the DNA in the pool is the number of indexes and C i is the initial concentration of each indexed sample Table 45 shows an example of the amount of 4 index tagged samples of different concentrations and Low TE needed for a final volume of 20 uL at 10 nM Table 45 Example of indexed sample volume calculation for total volume of 20 uL Component V f C i C f Volume to use pL Sample 1 20 uL 20 nM 10 nM 4 2 5 Sample 2 20 uL 10 nM 10 nM 4 5 Sample 3 20 uL 17 nM 10 nM 4 2 9 Sample 4 20 uL 25 nM 10 nM 4 2 Low TE 76 2 Adjust the final volume of the pooled library to the desired final concentration SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Indexing and Sample Processing for Multiplexed Sequencing 5 If the final volume of the combined index 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 index tagged samples is greater than the final desired volume V f lyophilize and reconstitute
46. e the most current protocol Go to genomics agilent com and search for G7530 90000 Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an experiment This protocol differs from the Illumina Multiplexed Paired End sequencing manual and other SureSelect protocols at several steps Pay close attention to the primers used for each amplification step and the blocking agents used during hybridization Agilent cannot guarantee the SureSelect Target Enrichment kits and cannot provide technical support for the use of non Agilent protocols or instruments to process samples for enrichment os Agilent Technologies 11 1 Before You Begin Overview of the Workflow Overview of the Workflow The SureSelect target enrichment workflow is summarized in Figure 1 The estimated time requirements for each step are summarized in Table 1 SureSelect XT i Genomic DNA samples NGS Target Enrichment Fig UNSUNG DNA canara Workflow Sail DNA fragments of 150 200 bp Prepare samples using SureSelect Library Prep Kit Design target Adaptor tagged DNA libraries sequences in SureDesign PCR amplify E ea ame Prepared DNA libraries Hybridize using SureSelect reagents and protocol l Capture hybrids on magnetic beads PCR amplify using indexing primers lt _ 1 Pool captured samples Optional v Figure 1 Overall target enriched sequencing sample p
47. eared solution from each well Do not touch the beads while removing the solution 7 Continue to keep the plate in the magnetic stand while you dispense 200 uL of freshly prepared 70 ethanol in each sample well 8 Wait for 1 minute to allow any disturbed beads to settle then remove the ethanol 9 Repeat step 7 to step 8 step once 10 Seal the wells with strip caps then briefly spin the plate to collect the residual ethanol Return the plate to the magnetic stand for 30 seconds Remove the residual ethanol with a P20 pipette 11 Dry the samples by placing the unsealed plate on the thermal cycler set to hold samples at 37 C for 3 to 5 minutes or until the residual ethanol completely evaporates 12 Add 32 uL nuclease free water to each sample well 13 Seal the wells with strip caps then mix well on a vortex mixer and briefly spin the plate to collect the liquid 14 Incubate for 2 minutes at room temperature 15 Put the plate in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 16 Remove the cleared supernatant approximately 30 uL to a fresh PCR plate well You can discard the beads at this time Stopping Point If you do not continue to the next step seal the plate and store at 20 C SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 31 2 Sample Preparation 3 pg DNA Samples Step 6 Adenylate the 3 end of the DNA fragments Use the SureSelect XT Library Pre
48. elect capture libraries and reagents must be used within one year of receipt Warranty The material contained in this document is provided as is and is subject to being changed with out notice in future editions Fur ther to the maximum extent permitted by applicable law Agi lent 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 merchant ability and fitness for a particular purpose Agilent shall not be lia ble for errors or for incidental or consequential damages in con nection with the furnishing use or performance 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 doc ument that conflict with these terms the warranty terms in the separate 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 Dat
49. eturn the plate to the magnetic stand for 30 seconds Remove the residual ethanol with a P20 pipette 11 Dry the samples by placing the unsealed plate on the thermal cycler set to hold samples at 37 C for 1 to 2 minutes or until the residual ethanol completely evaporates 12 Add 30 uL nuclease free water to each sample well 13 Seal the wells then mix well on a vortex mixer and briefly spin the plate in a centrifuge or mini plate spinner to collect the liquid 14 Incubate for 2 minutes at room temperature 15 Put the plate in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 16 Remove the cleared supernatant approximately 30 uL to a fresh PCR plate well You can discard the beads at this time Stopping Point If you do not continue to the next step seal the plate and store at 20 C SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 39 2 40 Sample Preparation 3 pg DNA Samples Step 12 Assess quality and quantity Quality assessment can be done with either the 2100 Bioanalyzer instrument or the 2200 TapeStation instrument Option 1 Analysis using the 2100 Bioanalyzer and DNA 1000 Assay See the DNA 1000 Kit Guide at www genomics agilent com for more information on doing this step 1 Check that the 2100 Bioanalyzer electrodes have been cleaned as instructed in the reagent kit guide Open the 2100 Expert Software version B 02 02 or higher turn on the
50. ex mixer and briefly spin the plate to collect the liquid 15 Incubate for 2 minutes at room temperature 16 Put the plate in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 17 Remove the cleared supernatant approximately 30 uL to a fresh PCR plate well You can discard the beads at this time Stopping Point If you do not continue to the next step seal the plate and store at 20 C 50 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Sample Preparation 200 ng DNA Samples 3 Step 5 Adenylate the 3 end of the DNA fragments Use the SureSelect XT Library Prep Kit ILM for this step Hold samples on ice while setting up this step 1 Prepare the appropriate volume of Adenylation master mix as described in Table 23 on ice Mix well on a vortex mixer Table 23 Preparation of Adenylation master mix Reagent Volume for 1 reaction Volume for 16 reactions includes excess Nuclease free water 11 uL 181 5 pL 10x Klenow Polymerase Buffer blue cap 5 uL 82 5 uL dATP green cap Tul 16 5 pL Exo Klenow red cap 3 pL 49 5 uL Total 20 pL 330 pL 2 Add 20 uL of the Adenylation master mix to each end repaired purified DNA sample approximately 30 uL 3 Mix well by pipetting up and down 4 Incubate the plate in the thermal cycler and run the program in Table 24 Do not use a heated lid Table 24 dA Tailing Thermal Cycler Program Step Temperature Time S
51. from step 8 5 uL 85 uL Capture Library lt 3 Mb 2 uL 34 uL Total 20 pL 340 uL SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Hybridization and Capture 4 10 Maintain the gDNA library Block Mix plate or strip tube at 65 C while you add 20 uL of the Capture Library Hybridization Mix from step 9 to each sample well Mix well by pipetting up and down 8 to 10 times The hybridization reaction wells now contain approximately 27 to 29 uL depending on the degree of evaporation during the thermal cycler incubation 11 Seal the wells with strip caps or using the PlateLoc Thermal Microplate Sealer Make sure that all wells are completely sealed CAUTION Wells must be adequately sealed to minimize evaporation or your results can be negatively impacted When using the SureCycler 8800 thermal cycler and sealing with strip caps make sure to use domed strip caps and to place a compression mat over the PCR plate or strip tubes in the thermal cycler 12 Incubate the hybridization mixture for 16 or 24 hours at 65 C witha heated lid at 105 C SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 69 4 Hybridization and Capture Step 2 Prepare streptavidin coated magnetic beads The hybrid capture protocol uses reagents provided in SureSelect Target Enrichment Box 1 stored at room temperature in addition to the streptavidin coated magnetic beads obtained from another supplier see Ta
52. g Conditions Where Used SureSelect Hyb 1 SureSelect Target Enrichment Box 1 RT page 66 SureSelect Hyb 2 SureSelect Target Enrichment Box 1 RT page 66 SureSelect Hyb 3 SureSelect Target Enrichment Kit ILM Warm to Room page 66 Indexing Hyb Module Box 2 20 C Temperature RT SureSelect Hyb 4 SureSelect Target Enrichment Box 1 RT page 66 SureSelect Indexing Block 1 SureSelect Target Enrichment Kit ILM Thaw on ice page 66 Indexing Hyb Module Box 2 20 C SureSelect Block 2 SureSelect Target Enrichment Kit ILM Thaw on ice page 66 Indexing Hyb Module Box 2 20 C SureSelect ILM Indexing Block 3 SureSelect Target Enrichment Kit ILM Thaw on ice page 66 Indexing Hyb Module Box 2 20 C SureSelect RNase Block SureSelect Target Enrichment Kit ILM Thaw on ice page 67 Indexing Hyb Module Box 2 20 C Capture Library 80 C Thaw on ice page 68 64 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing CAUTION Hybridization and Capture 4 For each sample library prepared do one hybridization and capture Do not pool samples at this stage The hybridization reaction requires 750 ng of prepared DNA in a volume of 3 4 uL initial concentration of 221 ng uL For FFPE derived DNA samples add the maximum amount of DNA available in range of 500 750 ng DNA See Chapter 6 for a complete list of modifications recommended for FFPE samples 1 For prepped libraries with DNA concentrations above
53. ge 88 Stopping Point If you do not continue to the next step store the libraries at 4 C for up to one week or at 20 C for longer periods SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 83 84 5 Indexing and Sample Processing for Multiplexed Sequencing Step 3 Assess indexed library DNA quantity and quality 35 100 150 200 300 400 500 600 1000 2000 10380 bp Figure 9 Post capture analysis of amplified indexed library DNA using the 2100 Bioanalyzer and a High Sensitivity DNA Assay SureSelect Target Enrichment System for Illumina Multiplexed Sequencing CAUTION Indexing and Sample Processing for Multiplexed Sequencing 5 Option 2 Analysis using the Agilent 2200 TapeStation and High Sensitivity D1000 ScreenTape Use a High Sensitivity D1000 ScreenTape p n 5067 5584 and reagent kit p n 5067 5585 to analyze the amplified indexed DNA For more information to do this step see the Agilent 2200 TapeStation User Manual at www genomics agilent com 1 Prepare the TapeStation samples as instructed in the Agilent 2200 TapeStation User Manual Use 2 uL of each indexed DNA sample diluted with 2 uL of High Sensitivity D1000 sample buffer for the analysis Make sure that you thoroughly mix the combined DNA and sample buffer on a vortex mixer for 5 seconds for accurate quantitation Stopping Point 2 Load the sample plate or tube strips from step 1 the High Sensitivity D1000 ScreenTape and lo
54. h protocol step that requires removal of tube cap strips make sure to reseal the tubes with a fresh strip of caps Reuse of strip caps can cause sample loss sample contamination or imprecision in sample temperatures during incubations CAUTION 6 To each gDNA library sample well prepared in step 3 on page 65 add 5 6 uL of the SureSelect Block Mix prepared in Table 32 Pipette up and down to mix 7 Cap the wells then transfer the sealed plate or strip tube to the thermal cycler and run the following program shown in Table 33 Use a heated lid set at 105 C to hold the temperature at 65 C Make sure that the DNA Block Mix samples are held at 65 C for at least 5 minutes before adding the remaining hybridization reaction components in step 10 below Table 33 Thermal cycler program for DNA Block Mix prior to hybridization Step Temperature Time Step 1 95 C 5 minutes Step 2 65 C Hold at least 5 minutes The lid of the thermal cycler is hot and can cause burns Use caution when working near the lid 8 Prepare the appropriate dilution of SureSelect RNase Block based on the size of your Capture Library according to Table 34 Prepare the amount required for the number of hybridization reactions in the run plus excess Keep the mixture on ice until it is used in step 9 Table 34 Preparation of RNase Block dilution Capture Library Size RNase Block dilution Volume of dilute RNase Block parts RNase Block parts
55. he captured libraries with indexing primers containing 8 bp indexes AQI H12 74 Step 1B Amplify the captured libraries with indexing primers containing 6 bp indexes 1 16 78 Step 2 Purify the amplified captured libraries using AMPure XP beads 81 Step 3 Assess indexed library DNA quantity and quality 83 Step 4 Quantify each index tagged library by QPCR optional 87 Step 5 Pool samples for multiplexed sequencing 88 Step 6 Prepare sequencing samples 90 This chapter describes the steps to add index tags by amplification and to purify and assess quality and quantity of the captured libraries Sample pooling instructions are provided to prepare the indexed samples for multiplexed sequencing Note that Step 1A and Step 1B are alternative protocols Follow the protocol appropriate for the set of indexing primers provided with your kit Use Step 1A if your kit includes the reconfigured 8 bp indexing primers A01 to H12 provided in white capped tubes 16 reaction kits or a blue plate 96 reaction kits Use Step 1B if your kit includes the original 6 bp indexing primers 1 to 16 provided in clear capped tubes it Agilent Technologies 73 5 Indexing and Sample Processing for Multiplexed Sequencing CAUTION This chapter includes instructions for kits containing two different sets of indexing primers Verify that you are referencing the information appropriate for your kit version before you proceed Step 1A covers kits with 8 bp inde
56. ichment System for Illumina Multiplexed Sequencing 61 3 Sample Preparation 200 ng DNA Samples Step 11 Assess quality and quantity 62 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing SureSelect Target Enrichment System for Illumina Paired End Sequencing Library Protocol ee 4 CAUTION 7 e Hybridization and Capture J e Step 1 Hybridize DNA samples to the Capture Library 64 Step 2 Prepare streptavidin coated magnetic beads 70 Step 3 Capture the hybridized DNA using streptavidin coated beads 71 This chapter describes the steps to hybridize the prepared gDNA libraries with a target specific Capture Library After hybridization the targeted molecules are captured on streptavidin beads Each DNA library sample must be hybridized and captured individually prior to addition of the indexing tag by PCR The ratio of Capture Library to gDNA library is critical for successful capture ait Agilent Technologies 63 4 Hybridization and Capture Step 1 Hybridize DNA samples to the Capture Library In this step the prepared gDNA libraries are hybridized to a target specific Capture Library This step uses the SureSelect Reagent Kit components listed in Table 30 Thaw each component under the conditions indicated in the table Vortex each reagent to mix then spin tubes briefly to collect the liquid Table 30 Reagents for Hybridization Kit Component Storage Location Thawin
57. ilent p n G8800A Agilent p n G8810A Agilent p n 410088 Agilent p n 410092 Agilent p n 410096 Agilent p n G2943CA Agilent p n G2947CA Agilent p n 5067 1504 Agilent p n 5067 4626 Agilent p n G2964AA or G2965AA Agilent p n 5067 5582 Agilent p n 5067 5583 Agilent p n 5067 5584 Agilent p n 5067 5585 Life Technologies p n 032857 Life Technologies p n 032856 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 19 Before You Begin Table 5 Required Equipment for SureSelect Target Enrichment Description Vendor and part number Covaris Sample Preparation System S series or E series model Covaris sample holders 96 microTUBE plate E series only microTUBE for individual sample processing DNA LoBind Tubes 1 5 mL PCR clean 250 pieces Microcentrifuge Plate or strip tube centrifuge 96 well plate mixer Magnetic separator Multichannel pipette P10 P20 P200 and P1000 pipettes Vortex mixer Vacuum concentrator Ice bucket Powder free gloves Sterile nuclease free aerosol barrier pipette tips Covaris Covaris p n 520078 Covaris p n 520045 Eppendorf p n 022431021 or equivalent Eppendorf microcentrifuge model 5417C or equivalent Labnet International MPS1000 Mini Plate Spinner p n C1000 requires adapter p n C1000 ADAPT for use with strip tubes or equivalent Eppendorf ThermoMixer C p n 5382 000 015 and Eppendorf SmartBlock 96 PCR p n 5306 000 006 or
58. imer brown cap 1 25 uL 20 6 pL SureSelect ILM Indexing Pre Capture PCR 1 25 uL 20 6 uL Reverse Primer clear cap 5x Herculase II Reaction Buffer clear cap 10 uL 165 pL 100 mM dNTP Mix green cap 0 5 pL 8 3 uL Herculase II Fusion DNA Polymerase red cap 1 uL 16 5 uL Total 20 uL 330 pL 2 Add 20 uL of the PCR reaction mixture prepared in Table 28 to each purified DNA library sample 80 uL in the PCR plate wells Mix by pipetting SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 57 3 Sample Preparation 200 ng DNA Samples 3 Run the program in Table 29 in a thermal cycler Table 29 Pre Capture PCR Thermal Cycler Program Segment Number of Temperature Time Cycles 1 1 98 C 2 minutes 2 10 98 C 30 seconds 65 C 30 seconds 72 C 1 minute 3 1 72 C 10 minutes 4 1 4 C Hold FFPE derived DNA samples may require a different cycle number for amplification depending on DNA integrity See page 95 or page 97 for DNA integrity score based cycle number recommendations 58 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Sample Preparation 200 ng DNA Samples 3 Step 10 Purify the amplified library with AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes Do not freeze the beads at any time 2 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color 3 Add 90 uL of homogeneous AMP
59. in Table 42 to each sample well of a fresh PCR plate or strip tube 4 Add 1 uL of the appropriate SureSelect PCR Primer Index 1 through Index 16 provided in clear capped tubes Add only one of the 16 possible indexing primers to each reaction well 5 Add the DNA library samples to the PCR reactions a Obtain the PCR plate or strip tube containing 30 uL of bead bound target enriched DNA samples from ice prepared on page 72 b Pipette each DNA sample up and down until the bead suspension is homogeneous then transfer 14 uL of the sample to the appropriate well of the PCR plate or strip tube containing PCR reaction mix and indexing primer c Mix the PCR reactions well by pipetting d Store the remaining library bound beads at 20 C for future use if needed SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 79 5 Indexing and Sample Processing for Multiplexed Sequencing 6 Transfer the PCR plate or strip tube to a thermal cycler and run the PCR amplification program shown in Table 39 Table 43 Post Capture PCR cycling program Segment Number of Cycles Temperature Time 1 1 98 C 2 minutes 2 10 to 16 Cycles 98 C 30 seconds See Table 44 for recommendations based 57 C 30 seconds on Capture Library size 72 C 1 minute 3 1 72 C 10 minutes 4 1 4 C Hold Table 44 Post capture PCR cycle number recommendations Capture Size Cycles 1 kb up to 0 5 Mb 16 cycles 0 5 Mb up to 1 49 Mb 14 cycles
60. l plate Tt See Table 58 on page 103 for a plate map See Table 59 on page 104 for index sequences SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 101 7 Reference Table 56 SureSelect Target Enrichment Box 1 Content Kit Component 16 Reactions 96 or 480 Reactions SureSelect Hyb 1 SureSelect Hyb 2 SureSelect Hyb 4 SureSelect Binding Buffer SureSelect Wash Buffer 1 SureSelect Wash Buffer 2 tube with orange cap tube with red cap tube with black cap bottle bottle bottle tube with orange cap tube with red cap tube with black cap bottle bottle bottle Table 57 SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2 Content Kit Component 16 Reactions 96 Reactions 480 Reactions SureSelect Hyb 3 SureSelect Indexing Block 1 SureSelect Block 2 SureSelect ILM Indexing Block 3 SureSelect RNase Block SureSelect ILM Indexing Pre Capture PCR Reverse Primer SureSelect ILM Indexing Post Capture Forward PCR Primer tube with yellow cap tube with green cap tube with blue cap tube with brown cap tube with purple cap tube with clear cap tube with orange cap tube with yellow cap tube with green cap tube with blue cap tube with brown cap tube with purple cap tube with clear cap tube with orange cap bottle tube with green cap tube with blue cap tube with brown cap tube with purple cap tube with clear cap tube wi
61. n 5067 5365 Genomic DNA Reagents Agilent p n 5067 5366 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 21 1 22 Before You Begin Optional Reagents and Equipment SureSelect Target Enrichment System for Illumina Multiplexed Sequencing SureSelect Target Enrichment System for Illumina Paired End Sequencing Library Protocol ee 2 CAUTION 70 Sample Preparation 3 pg DNA Samples Step 1 Shear the DNA 24 Step 2 Purify the sample using AMPure XP beads 26 Step 3 Assess quality optional 28 Step 4 Repair the ends 30 Step 5 Purify the sample using AMPure XP beads 31 Step 6 Adenylate the 3 end of the DNA fragments 32 Step 7 Purify the sample using AMPure XP beads 33 Step 8 Ligate the paired end adaptor 34 Step 9 Purify the sample using AMPure XP beads 35 Step 10 Amplify the adaptor ligated library 36 Step 11 Purify the amplified library with AMPure XP beads 39 Step 12 Assess quality and quantity 40 This section contains instructions for the preparation of gDNA libraries from 3 ug DNA samples For lower input 200 ng DNA samples and FFPE derived DNA samples see the library preparation protocol on page 43 The sample preparation protocol is used to prepare DNA libraries for sequencing using the Illumina paired read platform For each sample to be sequenced an individual indexed library is prepared For an overview of the SureSelect T target enrichment workflo
62. n 7 Setting Value Duty Cycle 10 Intensity 5 Cycles per Burst 200 Time 6 cycles of 60 seconds each Set Mode Frequency sweeping Temperature 4 to7 C 7 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 each sheared DNA sample approximately 130 uL toa separate well of a 96 well plate or strip tube SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 25 2 26 Sample Preparation 3 pg DNA Samples Step 2 Purify the sample using AMPure XP beads Instructions in this manual are for sample processing in 96 well PCR plates When processing a small number of samples you can instead use strip tubes or individual tubes that are compatible with the thermal cycler and magnetic separation device used in the protocol The total liquid volume is 0 31 ml in step 4 through step 7 of the protocol below If you are using plates or strip tubes besides the recommended SureCycler 8800 compatible plasticware see Table 5 on page 19 first make sure that wells hold this volume Samples may be transferred to 1 5 ml tubes for this step if needed 1 Let the AMPure XP beads come to room temperature for at least 30 minutes Do not freeze the beads at any time 2 Prepare 400 uL of 70 ethanol per sample plus excess for use in step 8 The freshly prepared 70 ethanol ma
63. n five minutes after preparation Within the instrument context choose the High Sensitivity DNA assay from the drop down list Start the run Enter sample names and comments in the Data and Assay context Check that the electropherogram shows a DNA fragment size peak between 120 150 bp A sample electropherogram is shown in Figure 6 ru mr T T T T T t T 35 100 150 200 300 400 10380 bp Analysis of sheared DNA using a High Sensitivity DNA Bioanalyzer assay 47 3 48 Sample Preparation 200 ng DNA Samples Step 3 Repair the ends Use the SureSelect XT Library Prep Kit ILM for this step To process multiple samples prepare master mixes with overage at each step without the DNA sample Master mixes for preparation of 16 samples including excess are shown in each table as an example Hold samples on ice while setting up this step 1 Prepare the appropriate volume of End Repair master mix as described in Table 21 on ice Mix well on a vortex mixer Table 21 Preparation of End Repair master mix Reagent Volume for 1 reaction Volume for 16 reactions includes excess Nuclease free water 35 2 uL 580 8 uL 10x End Repair Buffer clear cap 10 pL 165 pL dNTP Mix green cap 1 6 uL 26 4 uL T4 DNA Polymerase purple cap Tul 16 5 pL Klenow DNA Polymerase yellow cap 2 uL 33 uL T4 Polynucleotide Kinase orange cap 2 2 uL 36 3 uL Total 52 pl 858 pL 2 Add 52 uL of the master mix to each PCR plate well co
64. n in wash solution volumes to 200 uL including ethanol washes for example see step 8 on page 27 and capture washes for example see step 6 on page 71 e Revision of hybridization setup instructions see page 64 through page 69 Revisions do not affect the final composition of the hybridization reaction but format and order of certain preparation steps have been modified e Removal of SureSelect Elution Buffer and Neutralization Buffer from SureSelect Target Enrichment Box 1 p n 5190 8645 or 5190 8646 see Table 56 on page 102 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Content 1 2 Before You Begin 11 Overview of the Workflow 12 Procedural Notes 14 Safety Notes 15 Required Reagents 16 Required Equipment 19 Optional Reagents and Equipment 21 Sample Preparation 3 pg DNA Samples 23 Step 1 Shear the DNA 24 Step 2 Purify the sample using AMPure XP beads 26 Step 3 Assess quality optional 28 Step 4 Repair the ends 30 Step 5 Purify the sample using AMPure XP beads 31 Step 6 Adenylate the 3 end of the DNA fragments 32 Step 7 Purify the sample using AMPure XP beads 33 Step 8 Ligate the paired end adaptor 34 Step 9 Purify the sample using AMPure XP beads 35 Step 10 Amplify the adaptor ligated library 36 Step 11 Purify the amplified library with AMPure XP beads 39 Step 12 Assess quality and
65. n protocol on page 64 Refer to this list for additional equipment and plasticware combination options for hybridization beyond the combinations used for protocol optimization and supported by Agilent Note that minimal evaporation is required to ensure optimal capture results Table 65 Tested options that show minimal evaporation PCR Machine Agilent Mx3005P Real Time PCR System Agilent Mx3005P Real Time PCR System ABI GeneAmp 9700 ABI Veriti p n 4375786 Eppendorf Mastercycler BioRad MJ Research PTC 200 BioRad MJ Research PTC 200 BioRad MJ Research PTC 200 Plate Strips Mx3005P Strip Tubes Agilent p n 401428 Life Technologies ABI MicroAmp Optical 96 well plates p n N8010560 Life Technologies ABI MicroAmp Optical 96 well plates p n N8010560 Life Technologies ABI MicroAmp Optical 96 well plates p n N8010560 Eppendorf 8 Tube PCR Tubes Mx4000 Strip Tubes Agilent p n 410022 Mx4000 Strip Tubes Agilent p n 410022 Mx3005P 96 well plate Agilent p n 410088 Cover Mx3005P Optical Strip Caps Agilent p n 401425 MicroAmp clear adhesive film p n 4306311 MicroAmp caps p n N8010535 MicroAmp clear adhesive film p n 4306311 Attached caps Mx4000 Optical Caps Agilent p n 401024 Mx3005P Optical Strip Caps Agilent p n 401425 Mx3005P Optical Strip Caps Agilent p n 401425 Comments Heated lid Heated lid use ABI compression pad 4312639 use t
66. nol 10 Repeat step 8 to step 9 once 11 Seal the wells with strip caps then briefly spin the plate to collect the residual ethanol Return the plate to the magnetic stand for 30 seconds Remove the residual ethanol with a P20 pipette 12 Dry the samples by placing the unsealed plate on the thermal cycler set to hold samples at 37 C for 3 to 5 minutes or until the residual ethanol completely evaporates Do not dry the bead pellet to the point that the pellet appears cracked during any of the bead drying steps in the protocol Elution efficiency is significantly decreased when the bead pellet is excessively dried 13 Add 50 uL nuclease free water to each sample well 14 Seal the wells with strip caps then mix well on a vortex mixer and briefly spin the plate to collect the liquid 15 Incubate for 2 minutes at room temperature 16 Put the plate in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 17 Remove the cleared supernatant approximately 48 uL to a fresh PCR plate well You can discard the beads at this time Stopping Point If you do not continue to the next step seal the plate and store at 20 C SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 27 2 Sample Preparation 3 pg DNA Samples Step 3 Assess quality optional This step is optional Quality assessment can be done with either the 2100 Bioanalyzer instrument or the 2200 TapeStation instrument O
67. ntaining the sheared DNA samples approximately 48 50 uL Mix by pipetting up and down 3 Incubate the plate in the thermal cycler and run the program in Table 22 Do not use a heated lid Table 22 End Repair Thermal Cycler Program Step Temperature Time Step 1 20 C 30 minutes Step 2 4 C Hold SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Sample Preparation 200 ng DNASamples 3 Step 4 Purify the sample using AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes Do not freeze the beads at any time 2 Prepare 400 uL of 70 ethanol per sample plus excess for use in step 8 The freshly prepared 70 ethanol may be used for subsequent purification steps run on the same day The complete Library Preparation protocol requires 1 6 mL of fresh 70 ethanol per sample 3 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color 4 Add 180 uL of homogeneous AMPure XP beads to each end repaired DNA sample approximately 100 uL in the PCR plate Pipette up and down 10 times to mix 5 Incubate samples for 5 minutes at room temperature Put the plate into a magnetic separation device Wait for the solution to clear approximately 3 to 5 minutes 7 Keep the plate in the magnetic stand Carefully remove and discard the cleared solution from each well Do not touch the beads while removing the solution If some magnetic be
68. on to clear approximately 3 to 5 minutes 6 Keep the plate in the magnetic stand Carefully remove and discard the cleared solution from each well Do not touch the beads while removing the solution 7 Continue to keep the plate in the magnetic stand while you dispense 200 uL of freshly prepared 70 ethanol in each sample well 8 Wait for 1 minute to allow any disturbed beads to settle then remove the ethanol 9 Repeat step 7 and step 8 step once 10 Seal the wells with strip caps then briefly spin the plate to collect the residual ethanol Return the plate to the magnetic stand for 30 seconds Remove the residual ethanol with a P20 pipette 11 Dry the samples by placing the unsealed plate on the thermal cycler set to hold samples at 37 C for 1 to 2 minutes or until the residual ethanol completely evaporates 12 Add 32 uL nuclease free water to each sample well 13 Seal the wells with strip caps then mix well on a vortex mixer and briefly spin the plate to collect the liquid 14 Incubate for 2 minutes at room temperature 15 Put the plate in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 16 Remove the cleared supernatant approximately 30 uL to a fresh PCR plate well You can discard the beads at this time Stopping Point If you do not continue to the next step seal the plate and store at 20 C SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 55 3
69. or the NextSeq platform see page 90 to page 91 Updates to estimated time requirements for the protocol see Table 1 on page 13 What s New in Version B 2 Support for the OneSeq Capture Libraries see Table 4 on page 18 and Table 40 on page 77 Support for ClearSeq Capture Libraries including ClearSeq Comprehensive Cancer XT Libraries see Table 4 on page 18 What s New in Version B 1 Support for kits with either 8 bp indexes A01 to H12 revised index configuration typically received December 2014 or later or 6 bp indexes 1 to 16 original index configuration typically received before December 2014 Kits with 8 bp indexes include indexing primers provided in white capped tubes 16 Reactions or blue plate 96 Reactions For indexing protocol details see page 74 4 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing For kit content details see page 100 For nucleotide sequences of the 8 bp indexes see page 104 Kits with 6 bp indexes include indexing primers provided in clear capped tubes For indexing protocol details see page 78 For kit contents see page 105 For nucleotide sequences of the 6 bp indexes see page 108 e Support for sample processing in 96 well plates or 8 well strip tubes See Table 5 on page 19 for recommended thermal cycler and plasticware Protocol modifications for plate strip tube sample processing are located throughout this guide Key modifications include reductio
70. p Kit ILM for this step Hold samples on ice while setting up this step 1 Prepare the appropriate volume of Adenylation master mix as described in Table 12 on ice Mix well on a vortex mixer Table 12 Preparation of Adenylation master mix Reagent Volume for 1 reaction Volume for 16 reactions includes excess Nuclease free water 11 uL 181 5 pL 10x Klenow Polymerase Buffer blue cap 5 uL 82 5 uL dATP green cap Tul 16 5 pL Exo Klenow red cap 3 pL 49 5 uL Total 20 pL 330 pL 2 Add 20 uL of the Adenylation master mix to each end repaired purified DNA sample approximately 30 uL 3 Mix well by pipetting up and down 4 Incubate the plate in the thermal cycler and run the program in Table 13 Do not use a heated lid Table 13 dA Tailing Thermal Cycler Program Step Temperature Time Step 1 37 C 30 minutes Step 2 4 C Hold 32 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Sample Preparation 3 pg DNA Samples 2 Step 7 Purify the sample using AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes Do not freeze the beads at any time 2 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color 3 Add 90 uL of homogeneous AMPure XP beads to each 50 uL dA tailed DNA sample in the PCR plate Pipette up and down 10 times to mix 4 Incubate samples for 5 minutes at room temperature Put the plate into a
71. ppears homogeneous and consistent in color 3 Add 90 uL of homogeneous AMPure XP beads to each adaptor ligated DNA sample in the PCR plate 50 uL Pipette up and down to mix 4 Incubate samples for 5 minutes at room temperature Put the plate into a magnetic separation device Wait for the solution to clear approximately 3 to 5 minutes 6 Keep the plate in the magnetic stand Carefully remove and discard the cleared solution from each well Do not touch the beads while removing the solution 7 Continue to keep the plate in the magnetic stand while you dispense 200 uL of freshly prepared 70 ethanol in each sample well 8 Wait for 1 minute to allow any disturbed beads to settle then remove the ethanol 9 Repeat step 7 and step 8 step once 10 Seal the wells with strip caps then briefly spin the plate to collect the residual ethanol Return the plate to the magnetic stand for 30 seconds Remove the residual ethanol with a P20 pipette 11 Dry the samples by placing the unsealed plate on the thermal cycler set to hold samples at 37 C for 1 to 2 minutes or until the residual ethanol completely evaporates 12 Add 32 uL nuclease free water to each sample well 13 Seal the wells with strip caps then mix well on a vortex mixer and briefly spin the plate to collect the liquid 14 Incubate for 2 minutes at room temperature 15 Put the plate in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 16
72. pplement the circulated water chiller with ethylene glycol to 20 volume to prevent freezing Refer to the Covaris instrument user guide for more details 2 Put a Covaris microTube into the loading and unloading station Keep the cap on the tube You can use the 96 microTube plate see Table 5 on page 19 for the DNA shearing step when preparing multiple gDNA samples in the same experiment 3 Use the Qubit dsDNA Assay to determine the concentration of your gDNA sample Follow the instructions for the instrument 4 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 5 Use a tapered 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 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Sample Preparation 3 pg DNA Samples 2 6 Secure the microTube in the tube holder and shear the DNA with the settings in Table 8 or Table 9 depending on the Covaris instrument SonoLab software version used The target DNA fragment size is 150 to 200 bp Table8 Shear settings for Covaris instruments using SonoLab software version 7 or newer Setting Value Duty Factor 10 Peak Incident Power PIP 175 Cycles per Burst 200 Treatment Time 360 seconds Bath Temperature 4 to 8 C Table9 Shear settings for Covaris instruments using SonoLab software prior to versio
73. ption 1 Analysis using the 2100 Bioanalyzer and DNA 1000 Assay Use a DNA 1000 chip and reagent kit for analysis of the 3 ug sheared DNA samples using the 2100 Bioanalyzer See the DNA 1000 Kit Guide at www genomics agilent com for more information on doing this step 1 Check that the 2100 Bioanalyzer electrodes have been cleaned as instructed in the reagent kit guide Open the 2100 Expert Software version B 02 02 or higher turn on the 2100 Bioanalyzer and check communication Prepare the chip samples and ladder as instructed in the reagent kit guide using 1 uL of each sample for the analysis Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation Within the instrument context choose the DNA 1000 assay from the drop down list Start the run Enter sample names and comments in the Data and Assay context Check that the electropherogram shows a DNA fragment size peak between 150 200 bp A sample electropherogram is shown in Figure 2 eg a 100 20 w xo 32500 fe Figure 2 Analysis of sheared DNA using a DNA 1000 Bioanalyzer assay 28 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Sample Preparation 3 pg DNA Samples Option 2 Analysis using the 2200 TapeStation and D1000 ScreenTape Use a D1000 ScreenTape and D1000 Reagents for analysis of the 3 ug sheared DNA samples using the 2200 TapeStation For more information to do this
74. quantity 40 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Contents 3 Sample Preparation 200 ng DNA Samples 43 Step 1 Shear the DNA 44 Step 2 Assess quality optional 47 Step 3 Repair the ends 48 Step 4 Purify the sample using AMPure XP beads 49 Step 5 Adenylate the 3 end of the DNA fragments 51 Step 6 Purify the sample using AMPure XP beads 52 Step 7 Ligate the paired end adaptor 53 Step 8 Purify the sample using AMPure XP beads 55 Step 9 Amplify the adaptor ligated library 56 Step 10 Purify the amplified library with AMPure XP beads 59 Step 11 Assess quality and quantity 60 4 Hybridization and Capture 63 Step 1 Hybridize DNA samples to the Capture Library 64 Step 2 Prepare streptavidin coated magnetic beads 70 Step 3 Capture the hybridized DNA using streptavidin coated beads 71 5 Indexing and Sample Processing for Multiplexed Sequencing 73 Step 1A Amplify the captured libraries with indexing primers containing 8 bp indexes AQI H12 74 Step 1B Amplify the captured libraries with indexing primers containing 6 bp indexes 1 16 78 Step 2 Purify the amplified captured libraries using AMPure XP beads 81 Step 3 Assess indexed library DNA quantity and quality 83 Step 4 Quantify each index tagged library by QPCR optional 87 Step 5 Pool samples for multiplexed sequencing 88 Step 6 Prepare sequencing samples 90 8 SureSelect Target Enrichment System for Illumina Multiplexed Seq
75. quencing 5 Prepare one indexing amplification reaction for each DNA library When processing FFPE derived DNA samples some details of this step should be modified See Table 49 on page 94 for more information CAUTION To avoid cross contaminating libraries set up PCR mixes in a dedicated clean area or PCR hood with UV sterilization and positive air flow 1 Determine the appropriate index assignments for each sample See Table 59 in the Reference chapter for sequences of the index portion of the SureSelect 8 bp Indexes A01 through H12 indexing primers used to amplify the DNA libraries in this step Use a different indexing primer for each sample to be sequenced in the same lane 2 Prepare the appropriate volume of PCR reaction mix as described in Table 38 on ice Mix well on a vortex mixer Table 38 Preparation of post capture PCR Reaction mix Reagent Volume for 1 Volume for 16 reactions reaction includes excess Nuclease free water 18 5 uL 314 5 uL 5x Herculase II Reaction Buffer clear cap 10 uL 170 pL Herculase II Fusion DNA Polymerase red cap Tul 17 yL 100 mM dNTP Mix green cap 0 5 pL 8 5 uL SureSelect ILM Indexing Post Capture Forward Tul 17 uL PCR Primer orange cap Total 31 pL 527 pL 3 Add 31 uL of the PCR reaction mix prepared in Table 38 to each sample well of a fresh PCR plate or strip tube 4 Add 5 uL of the appropriate indexing primer SureSelect 8 bp Indexes AO1 through H02 provi
76. rd the cleared solution from each well Do not disturb the beads while removing the solution 9 Continue to keep the plate or tubes in the magnetic stand while you dispense 200 uL of freshly prepared 70 ethanol in each sample well 10 Wait for 1 minute to allow any disturbed beads to settle then remove the ethanol 11 Repeat step 9 and step 10 once for a total of two washes Make sure to remove all of the ethanol at each wash step 12 Seal the wells with strip caps then briefly spin to collect the residual ethanol Return the plate or strip tube to the magnetic stand for 30 seconds Remove the residual ethanol with a P20 pipette 13 Dry the samples by placing the unsealed plate or strip tube on the thermal cycler set to hold samples at 37 C for 1 to 2 minutes or until the residual ethanol completely evaporates 14 Add 30 uL of nuclease free water to each sample well SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 81 5 Indexing and Sample Processing for Multiplexed Sequencing 15 Seal the sample wells then mix well on a vortex mixer and briefly spin to collect the liquid 16 Incubate for 2 minutes at room temperature 17 Put the plate or strip tube in the magnetic stand and leave for 2 minutes or until the solution is clear 18 Remove the cleared supernatant approximately 30 uL to a fresh well You can discard the beads at this time Stopping Point If you do not continue to the next step
77. re Equipment Combinations on page 109 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 65 4 66 Hybridization and Capture 4 Prepare the Hybridization Buffer by mixing the components in Table 31 at room temperature If a precipitate forms warm the Hybridization Buffer at 65 C for 5 minutes Keep the prepared Hybridization Buffer at room temperature until it is used in step 9 Table 31 Preparation of Hybridization Buffer Reagent Volume for 1 reaction Volume for 16 reactions includes excess SureSelect Hyb 1 orange cap or bottle 6 63 pL 116 pL SureSelect Hyb 2 red cap 0 27 pL 4 7 uL SureSelect Hyb 3 yellow cap or bottle 2 65 pL 46 4 uL SureSelect Hyb 4 black cap or bottle 3 45 uL 60 4 uL Total 13 pL 227 5 Prepare Hybridization Buffer for at least 5 reaction equivalents per run to allow accurate pipetting volumes 5 Prepare the SureSelect Block Mix by mixing the components in Table 32 Keep the mixture on ice until it is used in step 6 Table 32 Preparation of SureSelect Block Mix Reagent Volume for 1 reaction Volume for 16 reactions includes excess SureSelect Indexing Block 1 green cap 2 5 uL 42 5 uL SureSelect Block 2 blue cap 2 5 uL 42 5 uL SureSelect ILM Indexing Block 3 brown cap 0 6 pL 10 2 uL Total 5 6 pL 95 2 pL SureSelect Target Enrichment System for Illumina Multiplexed Sequencing CAUTION Hybridization and Capture 4 For eac
78. reparation workflow 12 SureSelect Target Enrichment System for Ilumina Multiplexed Sequencing Before You Begin 1 Table 1 Estimated time requirements up to 16 sample run size Step Time Library Preparation 5 hours Hybridization and Capture 16 or 24 hours Post capture amplification 1 hour QC using Bioanalyzer or TapeStation and sample pooling 1 5 hours SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 13 1 14 Before You Begin Procedural Notes e This protocol supports sample processing in the plates or strip tubes specified in Table 5 on page 19 Certain protocol steps include liquid volumes exceeding 0 2 ml which can be accommodated by the recommended plasticware If using different plasticware you must first verify that wells can accommodate at least 0 31 ml for processing of 3 ug DNA samples or can accommodate at least 0 28 ml for processing of 200 ng DNA samples If needed samples may be transferred to 1 5 ml tubes for the high volume protocol steps with possible impacts on sample throughput and yield 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 Use best practices to prevent PCR product contamination of samples throughout the workflow 1 Assign separate pre PCR and post PCR work areas and use dedicated equipment supplies and reagents in ea
79. rget Enrichment Kit ILM 20 C 5190 4455 5190 4456 5190 4457 Indexing Hyb Module Box 2 SureSelect capture libraries and reagents must be used within one year of receipt 100 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Reference 7 The contents of each of the component kits listed in Table 54 are described in the tables below Table 55 SureSelect XT Library Prep Kit ILM Content Revised Index Configuration Kit Component 16 Reactions 96 or 480 Reactions 10X End Repair Buffer 10X Klenow Polymerase Buffer 5X T4 DNA Ligase Buffer T4 DNA Ligase Exo Klenow T4 DNA Polymerase Klenow DNA Polymerase T4 Polynucleotide Kinase dATP dNTP Mix SureSelect Adaptor Oligo Mix SureSelect Primer forward primer SureSelect 8 bp Index reverse primers tube with clear cap tube with blue cap tube with green cap tube with red cap tube with red cap tube with purple cap tube with yellow cap tube with orange cap tube with green cap tube with green cap tube with brown cap tube with brown cap SureSelect 8bp Indexes A01 through H02 provided in 16 white capped tubes tube with clear cap tube with blue cap tube with green cap tube with red cap tube with red cap tube with purple cap tube with yellow cap tube with orange cap tube with green cap tube with green cap tube with brown cap tube with brown cap SureSelect 8bp Indexes A01 through H12 provided in blue 96 wel
80. series or S series instrument a Check that the water in the Covaris tank is filled with fresh deionized water to the appropriate fill line level according to the manufacturer s recommendations for the specific instrument model and sample tube or plate in use b Check that the water covers the visible glass part of the tube c On the instrument control panel push the Degas button Degas the instrument for least 30 minutes before use or according to the manufacturer s recommendations d Set the chiller temperature to between 2 C to 5 C to ensure that the temperature reading in the water bath displays 5 C e Optional Supplement the circulated water chiller with ethylene glycol to 20 volume to prevent freezing Refer to the Covaris instrument user guide for more details 2 Put a Covaris microTube into the loading and unloading station Keep the cap on the tube You can use the 96 microTube plate see Table 5 on page 19 for the DNA shearing step when preparing multiple gDNA samples in the same experiment 3 Use the Qubit dsDNA Assay to determine the concentration of your gDNA sample Follow the instructions for the instrument For FFPE derived DNA samples with significantly degraded DNA use the concentration of amplifiable DNA as determined by qPCR and use the maximum amount of DNA available in the range of 100 200 ng See Chapter 6 for more information on when protocol modifications are appropriate for FFPE samples S
81. store the libraries at 4 C for up to one week or at 20 C for longer periods 82 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Indexing and Sample Processing for Multiplexed Sequencing 5 Step 3 Assess indexed library DNA quantity and quality Option 1 Analysis using the Agilent 2100 Bioanalyzer and High Sensitivity DNA Assay Use the Bioanalyzer High Sensitivity DNA Assay to analyze the amplified indexed DNA See the High Sensitivity DNA Kit Guide at www genomics agilent com for more information on doing this step 1 Set up the 2100 Bioanalyzer as instructed in the reagent kit guide 2 Prepare the chip samples and ladder as instructed in the reagent kit guide using 1 uL of each sample for the analysis 3 Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation 4 Verify that the electropherogram shows the peak of DNA fragment size positioned between 250 and 350 bp A sample electropherogram is shown in Figure 9 5 Measure the concentration of each library by integrating under the entire peak For accurate quantification make sure that the concentration falls within the linear range of the assay If you wish to more precisely quantify the target enriched samples prior to pooling proceed to Step 4 Quantify each index tagged library by QPCR optional on page 87 Otherwise proceed to Step 5 Pool samples for multiplexed sequencing on pa
82. tep 1 37 C 30 minutes Step 2 4 C Hold SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 51 3 52 Sample Preparation 200 ng DNA Samples Step 6 Purify the sample using AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes Do not freeze the beads at any time 2 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color 3 Add 90 uL of homogeneous AMPure XP beads to each 50 uL dA tailed DNA sample in the PCR plate Pipette up and down 10 times to mix 4 Incubate samples for 5 minutes at room temperature Put the plate into a magnetic separation device Wait for the solution to clear approximately 3 to 5 minutes 6 Keep the plate in the magnetic stand Carefully remove and discard the cleared solution from each well Do not touch the beads while removing the solution 7 Continue to keep the plate in the magnetic stand while you dispense 200 uL of freshly prepared 70 ethanol in each sample well 8 Wait for 1 minute to allow any disturbed beads to settle then remove the ethanol 9 Repeat step 7 to step 8 step once 10 Seal the wells with strip caps then briefly spin the plate to collect the residual ethanol Return the plate to the magnetic stand for 30 seconds Remove the residual ethanol with a P20 pipette 11 Dry the samples by placing the unsealed plate on the thermal cycler set to hold samples at 37 C for 1 to
83. th orange cap 102 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Table 58 1 A A01 B B01 c c01 D D01 E E01 F F01 G G01 H H01 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Reference 7 Plate map for SureSelect 8bp Indexes A01 through H12 provided in blue plate in Library Prep kit p n 5500 0133 2 A02 B02 c02 D02 E02 F02 G02 H02 3 A03 B03 c03 D03 E03 F03 G03 H03 4 A04 B04 C04 D04 E04 F04 G04 H04 A05 B05 C05 D05 E05 F05 G05 H05 A06 B06 c06 D06 E06 F06 G06 H06 7 A07 B07 C07 D07 E07 F07 G07 H07 A08 B08 c08 D08 E08 F08 G08 H08 A09 B09 c09 D09 E09 F09 G09 H09 10 A10 B10 C10 D10 E10 F10 G10 H10 11 All B11 C11 D11 E11 F11 G11 H11 12 A12 B12 C12 D12 E12 F12 G12 H12 103 7 Reference Nucleotide Sequences of SureSelect Indexes A01 to H12 Each index is 8 nt in length See page 90 for sequencing run setup requirements for sequencing libraries using 8 bp indexes Table 59 SureSelect Indexes for indexing primers provided in blue 96 well plate or white capped tubes Sequence Sequence Sequence Sequence ATGCCTAA GAATCTGA C01 AACGTGAT 201 CACTTCGA E GCCAAGAC
84. this section if your kit includes Library Prep Kit p n 5500 0105 or 5500 0075 If your kit does not include this component kit see page 100 for kit content and indexing primer information Kit Contents Each SureSelect Reagent Kit contains the following component kits Table 60 SureSelect Reagent Kit Contents Original Index Configuration Product Storage Condition 16 Reactions 96 Reactions 480 Reactions SureSelect XT Library Prep Kit ILM 20 C 5500 0105 5500 0075 5 x 5500 0075 SureSelect Target Enrichment Box 1 Room Temperature 5190 4393 5190 4394 5190 4395 SureSelect Target Enrichment Kit ILM 20 C 5190 4455 5190 4456 5190 4457 Indexing Hyb Module Box 2 SureSelect capture libraries and reagents must be used within one year of receipt SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 105 7 106 Reference The contents of each of the component kits listed in Table 60 are described in the tables below Table 61 SureSelect XT Library Prep Kit ILM Content Original Index Configuration Kit Component Format 10X End Repair Buffer 10X Klenow Polymerase Buffer 5X T4 DNA Ligase Buffer T4 DNA Ligase Exo Klenow T4 DNA Polymerase Klenow DNA Polymerase T4 Polynucleotide Kinase dATP dNTP Mix SureSelect Adaptor Oligo Mix SureSelect Primer forward primer PCR Primer Index 1 through Index 16 reverse primers containing 6 bp index sequences tube with clear cap tube
85. uate yield for subsequent capture without introducing bias or non specific products If yield is too low or too high where non specific high molecular weight products are observed adjust the number of cycles accordingly to amplify the remaining library template SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Sample Preparation 3 pg DNA Samples 2 Step 11 Purify the amplified library with AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes Do not freeze the beads at any time 2 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color 3 Add 90 uL of homogeneous AMPure XP beads to each 50 uL amplified DNA sample in the PCR plate Pipette up and down to mix 4 Incubate samples for 5 minutes at room temperature Put the plate into a magnetic separation device Wait for the solution to clear approximately 3 to 5 minutes 6 Keep the plate in the magnetic stand Carefully remove and discard the cleared solution from each well Do not touch the beads while removing the solution 7 Continue to keep the plate in the magnetic stand while you dispense 200 uL of freshly prepared 70 ethanol in each sample well 8 Wait for 1 minute to allow any disturbed beads to settle then remove the ethanol 9 Repeat step 7 and step 8 step once 10 Seal the wells with strip caps then briefly spin the plate to collect the residual ethanol R
86. uencing Contents 6 Appendix Using FFPE derived DNASamples 93 Modifications for all FFPE DNA samples 94 SureSelect Protocol Modifications 94 Downstream Sequencing Modifications 94 Modifications for samples assessed using the Agilent NGS FFPEQC Kit 95 SureSelect Protocol Modifications 95 Downstream Sequencing Modifications 95 Modifications for samples assessed using Agilent s Genomic DNA Screenlape 97 SureSelect Protocol Modifications 97 Downstream Sequencing Modifications 97 7 Reference 99 Reference Information for Kits with Revised Index Configuration indexing primers in white capped tubes or blue plate 100 Kit Contents 100 Nucleotide Sequences of SureSelect Indexes A01 to H12 104 Reference Information for Kits with Original Index Configuration indexing primers in clear capped tubes 105 Kit Contents 105 Nucleotide Sequences of SureSelect 6 bp Indexes Original Kit Configuration 108 Alternative Capture Equipment Combinations 109 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 9 Contents 10 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing SureSelect Target Enrichment System for Illumina Paired End Sequencing Library Protocol ee 1 070 Before You Begin 2 Overview of the Workflow 12 Procedural Notes 14 Safety Notes 15 Required Reagents 16 Required Equipment 19 Optional Reagents and Equipment 21 Make sure you hav
87. ure XP beads to each 50 uL amplified DNA sample in the PCR plate Pipette up and down to mix 4 Incubate samples for 5 minutes at room temperature Put the plate into a magnetic separation device Wait for the solution to clear approximately 3 to 5 minutes 6 Keep the plate in the magnetic stand Carefully remove and discard the cleared solution from each well Do not touch the beads while removing the solution 7 Continue to keep the plate in the magnetic stand while you dispense 200 uL of freshly prepared 70 ethanol in each sample well 8 Wait for 1 minute to allow any disturbed beads to settle then remove the ethanol 9 Repeat step 7 and step 8 step once 10 Seal the wells with strip caps then briefly spin the plate to collect the residual ethanol Return the plate to the magnetic stand for 30 seconds Remove the residual ethanol with a P20 pipette 11 Dry the samples by placing the unsealed plate on the thermal cycler set to hold samples at 37 C for 1 to 2 minutes or until the residual ethanol completely evaporates 12 Add 30 uL nuclease free water to each sample well 13 Seal the wells then mix well on a vortex mixer and briefly spin the plate in a centrifuge or mini plate spinner to collect the liquid 14 Incubate for 2 minutes at room temperature 15 Put the plate in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 16 Remove the cleared supernatant approximately 30 uL to a fresh
88. ureSelect Target Enrichment System for Illumina Multiplexed Sequencing Sample Preparation 200 ng DNASamples 3 4 Dilute 200 ng of high quality gDNA with 1X Low TE Buffer in a 1 5 mL LoBind tube to a total volume of 50 uL 5 Use a tapered pipette tip to slowly transfer the 50 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 19 or Table 20 depending on the Covaris instrument SonoLab software version used The target DNA fragment size is 150 to 200 bp For FFPE derived DNA samples reduce the duration of shearing from 6 minutes to 4 minutes See Chapter 6 for a complete list of modifications recommended for FFPE samples Table 19 Settings for Covaris instruments using SonoLab software version 7 or newer Setting Value Duty Factor 10 Peak Incident Power PIP 175 Cycles per Burst 200 Treatment Time 360 seconds Bath Temperature 4 to 8 C Table 20 Settings for Covaris instruments using SonoLab software prior to version 7 Setting Value Duty Cycle 10 Intensity 5 Cycles per Burst 200 Time 6 cycles of 60 seconds each Set Mode Frequency sweeping Temperature 4 to7 C SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 45 3 Sample Preparation 200 ng DNA Samples 7 Put the Covaris microTube back into the loading and unloading station
89. w see Figure 1 on page 12 The steps in this section differ from the Illumina protocol in the use of the Covaris system for gDNA shearing smaller target shear size elimination of size selection by gel purification implementation of AMPure XP beads for all purification steps and primers used for PCR Refer to the Illumina protocol Preparing Samples for Multiplexed Paired End Sequencing p n1005361 or the appropriate Illumina protocol for more information ait Agilent Technologies 23 2 24 Sample Preparation 3 pg DNA Samples Step 1 Shear the DNA Make sure genomic DNA samples are of high quality with an OD 260 280 ratio ranging from 1 8 to 2 0 Use the Qubit system to quantify genomic DNA before library preparation For each DNA sample to be sequenced prepare 1 library 1 Set up the Covaris E series or S series instrument a Check that the water in the Covaris tank is filled with fresh deionized water to the appropriate fill line level according to the manufacturer s recommendations for the specific instrument model and sample tube or plate in use b Check that the water covers the visible glass part of the tube c On the instrument control panel push the Degas button Degas the instrument for least 30 minutes before use or according to the manufacturer s recommendations d Set the chiller temperature to between 2 C to 5 C to ensure that the temperature reading in the water bath displays 5 C e Optional Su
90. water Required per hybridization reaction gt 3 0 Mb 25 1 3 2 uL lt 3 0 Mb 10 1 9 5 ul SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 67 4 68 Hybridization and Capture Prepare the Capture Library mixture described in step 9 below near the end of the 65 C hold step of gt 5 minute duration described in Table 33 Keep the mixture at room temperature briefly until adding the mixture to sample wells in step 10 Do not keep solutions containing the Capture Library at room temperature for extended periods 9 Prepare the Capture Library Hybridization Mix appropriate for your Capture Library size according to Table 35 Capture Libraries gt 3 Mb or Table 36 Capture Libraries lt 3 Mb Mix well by vortexing at high speed for 5 seconds then spin down briefly Keep the mixture at room temperature briefly until use in step 10 Table 35 Preparation of Capture Library Hybridization Mix for gt 3 Mb Capture Libraries Reagent Volume for 1 reaction Volume for 16 reactions includes excess Hybridization Buffer mixture from step 4 13 pL 221 uL 25 RNase Block solution from step 8 2 uL 34 uL Capture Library gt 3 Mb 5 uL 85 uL Total 20 pL 340 uL Table 36 Preparation of Capture Library Hybridization Mix for lt 3 Mb Capture Libraries Reagent Volume for 1 reaction Volume for 16 reactions includes excess Hybridization Buffer mixture from step 4 13 uL 221 uL 10 RNase Block solution
91. with blue cap tube with green cap tube with red cap tube with red cap tube with purple cap tube with yellow cap tube with orange cap tube with green cap tube with green cap tube with brown cap tube with brown cap 16 tubes with clear caps See Table 64 on page 108 for index sequences SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Table 62 SureSelect Target Enrichment Box 1 Content Kit Component 16 Reactions 96 Reactions Reference 7 480 Reactions SureSelect Hyb 1 SureSelect Hyb 2 SureSelect Hyb 4 SureSelect Binding Buffer SureSelect Wash Buffer 1 SureSelect Wash Buffer 2 SureSelect Elution Buffer SureSelect Neutralization Buffer tube with orange cap tube with red cap tube with black cap bottle bottle bottle tube with blue cap bottle tube with orange cap tube with red cap tube with black cap bottle bottle bottle bottle bottle bottle tube with red cap bottle bottle bottle bottle bottle bottle Table 63 SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2 Content The provided SureSelect Elution Buffer and Neutralization Buffer are not used in the workflow described in this manual Kit Component SureSelect Hyb 3 16 Reactions 96 Reactions 480 Reactions bottle tube with yellow cap SureSelect Indexing Block 1 SureSelect Block 2 SureSelect ILM Indexing Block 3 SureSelect RNase Block SureSelect
92. wo layers of film Heated lid Heated lid use ABI compression pad 4312639 use two layers of film Lid heating set to 75 C Heated lid Heated lid Heated lid SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 109 www agilent com In This Book This guide contains information to run the SureSelect target enrichment protocol Agilent Technologies Inc 2015 Version B 3 June 2015 p n G7530 90000 oe Agilent Technologies
93. xes supplied in Library Prep Kit p n 5500 0132 or 5500 0133 in format of white capped tubes or blue plate typically received December 2014 or later Step 1B covers kits with 6 bp indexes supplied in Library Prep Kit p n 5500 0105 or 5500 0075 in format of clear capped tubes typically received before December 2014 Step 1A Amplify the captured libraries with indexing primers containing 8 bp indexes A01 H12 74 In this step the SureSelect enriched DNA libraries are PCR amplified in PCR reactions that include the appropriate indexing primer for each sample This step uses the components listed in Table 37 Thaw then vortex to mix the reagents listed below and keep on ice Table 37 Reagents for post capture indexing by PCR amplification Kit Component Storage Location 5x Herculase II Reaction Buffer Herculase II Fusion DNA Polymerase kit 20 C 100 mM dNTP Mix 25 mM each dNTP Herculase II Fusion DNA Polymerase kit 20 C Herculase II Fusion DNA Polymerase Herculase II Fusion DNA Polymerase kit 20 C SureSelect ILM Indexing Post Capture Forward SureSelect Target Enrichment Kit ILM Indexing PCR Primer Hyb Module Box 2 20 C SureSelect 8 bp Indexes reverse primers SureSelect XT Library Prep Kit ILM 20 C Do not use the PCR Reaction Buffer or dNTP mix from any other kit SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Indexing and Sample Processing for Multiplexed Se
94. xon v4 UTRs 5190 4636 5190 4637 5190 4639 SureSelect Mouse All Exon 5190 4641 5190 4642 5190 4644 SureSelect Human X Chromosome 5190 4651 5190 4652 5190 4653 SureSelect Custom 1 kb up to 499 kb 5190 4806 5190 4807 5190 4809 reorder 5190 4811 5190 4812 5190 4814 SureSelect Custom 0 5 Mb upto2 9Mb 5190 4816 5190 4817 5190 4819 reorder 5190 4821 5190 4822 5190 4824 SureSelect Custom 3 Mb up to 5 9 Mb 5190 4826 5190 4827 5190 4829 reorder 5190 4831 5190 4832 5190 4834 SureSelect Custom 6 Mb up 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 one year of receipt SureSelect Target Enrichment System for Illumina Multiplexed Sequencing 17 1 Before You Begin Table4 Compatible ClearSeq and OneSeq Capture Libraries Capture Library 16 96 480 Reactions Reactions Reactions ClearSeq Comprehensive Cancer XT 5190 8011 5190 8012 ClearSeq Comprehensive Cancer Plus XT 5190 8014 5190 8015 ClearSeq Inherited Disease XT 5190 7518 5190 7519 ClearSeq Inherited Disease Plus XT 5190 7521 5190 7522 ClearSeq DNA Kinome XT 5190 4646 5190 4647 5190 4649 OneSeg Constitutional Research Panel 5190 8702 5190 8703 OneSegq Hi Res CNV Backbone Custom 1 499 kb 5190 8705 5190 8887 OneSeg Hi Res CNV
95. y be used for subsequent purification steps run on the same day The complete Library Preparation protocol requires 2 mL of fresh 70 ethanol per sample 3 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color 4 Add 180 uL of homogeneous AMPure XP beads to each sheared DNA sample approximately 130 uL in the PCR plate Pipette up and down 10 times to mix 5 Incubate samples for 5 minutes at room temperature 6 Put the plate into a magnetic separation device Wait for the solution to clear approximately 3 to 5 minutes 7 Keep the plate in the magnetic stand Carefully remove and discard the cleared solution from each well Do not touch the beads while removing the solution If some magnetic beads remain suspended in solution after 5 minutes carefully remove and discard 100 ul of cleared solution from near the bottom of the wells and continue incubating the plate in the magnetic stand for an additional 3 minutes After the remaining suspension has cleared remove and discard the remaining cleared solution approximately 210 ul from the wells SureSelect Target Enrichment System for Illumina Multiplexed Sequencing Sample Preparation 3 pg DNA Samples 2 8 Continue to keep the plate in the magnetic stand while you dispense 200 uL of 70 ethanol in each sample well Use fresh 70 ethanol for optimal results 9 Wait for 1 minute to allow any disturbed beads to settle then remove the etha
96. ystem for Illumina Multiplexed Sequencing 71 4 Hybridization and Capture CAUTION It is important to maintain bead suspensions at 65 C during the washing procedure below to ensure specificity of capture Make sure that the SureSelect Wash Buffer 2 is pre warmed to 65 C before use Do not use a tissue incubator or other devices with significant temperature fluctuations for the incubation steps 10 Wash the beads with SureSelect Wash Buffer 2 a Resuspend the beads in 200 uL of 65 C prewarmed Wash Buffer 2 Pipette up and down until beads are fully resuspended b Cap the wells then incubate the sample plate or strip tube for 10 minutes at 65 C on the thermal cycler c Put the plate or strip tube in the magnetic separator Wait for the solution to clear then remove and discard the supernatant d Repeat step a through step c for a total of 3 washes Make sure all of the wash buffer has been removed during the final wash 11 Add 30 uL of nuclease free water to each sample well Pipette up and down to resuspend the beads Keep the samples on ice until they are used on page 75 Captured DNA is retained on the streptavidin beads during the post capture amplification step 72 SureSelect Target Enrichment System for Illumina Multiplexed Sequencing SureSelect Target Enrichment System for Illumina Paired End Sequencing Library Protocol 5 Indexing and Sample Processing for Multiplexed Sequencing Step 1A Amplify t

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