SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library Illumina HiSeq and MiSeq Multiplexed Sequencing Platforms Protocol Version B.2, April 2015 Before you begin, view hands-on videos of SureSelect procedures at http://www.agilent.com/genomics/protocolvideos. SureSelect platform manufactured with Agilent SurePrint Technology For Research Use Only. Not for use in Diagnostic Procedures.
Notices © Agilent Technologies, Inc. 2015 Warranty No part of this manual may be reproduced in any form or by any means (including electronic storage and retrieval or translation into a foreign language) without prior agreement and written consent from Agilent Technologies, Inc. as governed by United States and international copyright laws. The material contained in this document is provided “as is,” and is subject to being changed, without notice, in future editions.
In this Guide... This guide provides an optimized protocol for Illumina paired-end multiplexed library preparation using the SureSelectXT Library Prep and Capture System. This guide describes the recommended operational procedures to capture the genomic regions of interest using biotinylated RNA oligomer libraries to enrich targeted regions of the genome from repetitive sequences and sequences unrelated to the research focus prior to sample sequencing using the Illumina platform.
What’s New in Version B.2 • Support for the OneSeq Capture Libraries (see Table 4 on page 16 and Table 40 on page 73). • Support for ClearSeq Capture Libraries, including ClearSeq Comprehensive Cancer XT Libraries (see Table 4 on page 16). 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).
Content 1 Before You Begin 9 Overview of the Workflow Procedural Notes 12 Safety Notes 13 Required Reagents 14 Optional Reagents 16 Required Equipment 17 Optional Equipment 19 2 10 Sample Preparation (3 µg DNA Samples) 21 Step 1. Shear the DNA 22 Step 2. Purify the sample using AMPure XP beads 24 Step 3. Assess quality (optional) 26 Step 4. Repair the ends 28 Step 5. Purify the sample using AMPure XP beads 29 Step 6. Adenylate the 3' end of the DNA fragments 30 Step 7.
Contents 3 Sample Preparation (200 ng DNA Samples) 41 Step 1. Shear the DNA 42 Step 2. Assess quality (optional) 45 Step 3. Repair the ends 46 Step 4. Purify the sample using AMPure XP beads 47 Step 5. Adenylate the 3' end of the DNA fragments 49 Step 6. Purify the sample using AMPure XP beads 50 Step 7. Ligate the paired-end adaptor 51 Step 8. Purify the sample using AMPure XP beads 52 Step 9. Amplify the adaptor-ligated library 53 Step 10. Purify the amplified library with AMPure XP beads Step 11.
Contents Kit Contents 88 Nucleotide Sequences of SureSelectXT Indexes A01 to H12 92 Reference Information for Kits with Original Index Configuration (indexing primers in clear-capped tubes) 93 Kit Contents 93 Nucleotide Sequences of SureSelectXT 6-bp Indexes-Original Kit Configuration 96 Alternative Capture Equipment Combinations 97 SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing 7
Contents 8 SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library Protocol 1 Before You Begin Overview of the Workflow 10 Procedural Notes 12 Safety Notes 13 Required Reagents 14 Optional Reagents 16 Required Equipment 17 Optional Equipment 19 Make sure you have 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.
1 Before You Begin Overview of the Workflow Overview of the Workflow The SureSelectXT target enrichment workflow is summarized in Figure 1. The estimated time requirements for each step are summarized in Table 1. Figure 1 10 Overall target-enriched sequencing sample preparation workflow.
Before You Begin Overview of the Workflow Table 1 1 Estimated time requirements Step Time Prepped Library Production 1 day Library Hybridization 16 or 24 hours Streptavidin bead preparation 30 minutes Hybrid capture and washing 2 hours DNA purification 30 minutes Post-capture indexing and amplification 1 hour PCR product purification 30 minutes QC using Bioanalyzer or TapeStation 2 to 3 hours Pooling of indexed samples by mass < 1 hour SureSelectXT Target Enrichment System for Illumi
1 Before You Begin Procedural Notes Procedural Notes • To prevent contamination of reagents by nucleases, always wear powder-free laboratory gloves and use dedicated solutions and pipettors with nuclease-free aerosol-resistant tips. • 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 each area.
Before You Begin Safety Notes 1 Safety Notes CA U T I O N • Wear appropriate personal protective equipment (PPE) when working in the laboratory.
1 Before You Begin Required Reagents Required Reagents Table 2 Required Reagents for SureSelectXT Target Enrichment Description Vendor and part number SureSelect, ClearSeq or OneSeq Capture Library * SureSelectXT 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 Select the appropriate library from Table 3 or Table 4 Agilent p/n G9611A p/
Before You Begin Required Reagents Table 3 SureSelectXT Capture Libraries* Capture Library 16 Reactions 96 Reactions SureSelect XT Clinical Research Exome 5190-7338 5190-7339 – SureSelect XT 5190-7787 5190-7788 – 5190-7790 5190-7791 – Focused Exome SureSelectXT Focused Exome Plus 1 480 Reactions SureSelect XT Focused Exome Plus 2 5190-7793 5190-7795 – SureSelect XT Human All Exon v5 5190-6208 5190-6209 – SureSelect XT Human All Exon v5 + UTRs 5190-6213 5190-6214 – 5190
1 Before You Begin Optional Reagents Table 4 Compatible ClearSeq and OneSeq Capture Libraries Capture Library 16 Reactions 96 Reactions 480 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 OneSeq Constitutional Research Panel 5190-8702 5190-8703 – OneSeq
Before You Begin Required Equipment 1 Required Equipment Table 6 Required Equipment for SureSelectXT Target Enrichment Description Vendor and part number SureCycler 8800 Thermal Cycler, or equivalent Agilent p/n G8800A 96 well plate module for SureCycler 8800 Thermal Cycler Agilent p/n G8810A SureCycler 8800-compatible plasticware: 96-well plates Agilent p/n 410088 8-well strip tubes Agilent p/n 410092 OR Tube cap strips, domed Agilent p/n 410096 DNA Analysis Platform and Consumables Agilen
1 Before You Begin Required Equipment Table 6 Required Equipment for SureSelectXT Target Enrichment Description Vendor and part number Microcentrifuge Eppendorf microcentrifuge model 5417C or equivalent Plate or strip tube centrifuge Labnet International MPS1000 Mini Plate Spinner p/n C1000 (requires adapter, p/n C1000-ADAPT, for use with strip tubes) or equivalent Nutator plate mixer BD Diagnostics p/n 421105, or equivalent Magnetic separator Life Technologies p/n 12331D or equivalent* Multich
Before You Begin Optional Equipment 1 Optional Equipment Table 7 Optional Equipment for SureSelectXT Target Enrichment Description Vendor and part number Tube-strip capping tool Agilent p/n 410099 PlateLoc Thermal Microplate Sealer with Small Hotplate Agilent p/n G5402A Peelable Aluminum Seal for PlateLoc Sealer Agilent p/n 24210-001 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
1 20 Before You Begin Optional Equipment SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library Protocol 2 Sample Preparation (3 µg DNA Samples) Step 1. Shear the DNA 22 Step 2. Purify the sample using AMPure XP beads 24 Step 3. Assess quality (optional) 26 Step 4. Repair the ends 28 Step 5. Purify the sample using AMPure XP beads 29 Step 6. Adenylate the 3' end of the DNA fragments 30 Step 7. Purify the sample using AMPure XP beads 31 Step 8. Ligate the paired-end adaptor 32 Step 9.
2 Sample Preparation (3 µg DNA Samples) Step 1. Shear the DNA Step 1. Shear the DNA NOTE 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.
Sample Preparation (3 µg DNA Samples) Step 1. Shear the DNA 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.
2 Sample Preparation (3 µg DNA Samples) Step 2. Purify the sample using AMPure XP beads Step 2. Purify the sample using AMPure XP beads NOTE Instructions in this protocol 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. 1 Let the AMPure XP beads come to room temperature for at least 30 minutes.
Sample Preparation (3 µg DNA Samples) Step 2. Purify the sample using AMPure XP beads 2 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. 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.
2 Sample Preparation (3 µg DNA Samples) Step 3. Assess quality (optional) 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. Option 1: Analysis using the 2100 Bioanalyzer and DNA 1000 Assay Use a DNA 1000 chip and reagent kit for analysis of the 3 g sheared DNA samples using the 2100 Bioanalyzer. See the DNA 1000 Kit Guide at www.genomics.agilent.
Sample Preparation (3 µg DNA Samples) Step 3. Assess quality (optional) 2 Option 2: Analysis using the 2200 TapeStation and D1000 ScreenTape Use a D1000 ScreenTape and D1000 Reagents for analysis of the 3 g sheared DNA samples using the 2200 TapeStation. 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.
2 Sample Preparation (3 µg DNA Samples) Step 4. Repair the ends 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.
Sample Preparation (3 µg DNA Samples) Step 5. Purify the sample using AMPure XP beads 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 µL of homogeneous AMPure XP beads to each 100-µL end-repaired DNA sample in the PCR plate. Pipette up and down 10 times to mix.
2 Sample Preparation (3 µg DNA Samples) Step 6. Adenylate the 3' end of the DNA fragments Step 6. 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 12, on ice. Mix well on a vortex mixer.
Sample Preparation (3 µg DNA Samples) Step 7. Purify the sample using AMPure XP beads 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 µL of homogeneous AMPure XP beads to each 50-µL dA-tailed DNA sample in the PCR plate. Pipette up and down 10 times to mix.
2 Sample Preparation (3 µg DNA Samples) Step 8. Ligate the paired-end adaptor 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 µL 255.
Sample Preparation (3 µg DNA Samples) Step 9. Purify the sample using AMPure XP beads 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 appears homogeneous and consistent in color. 3 Add 90 µL of homogeneous AMPure XP beads to each adaptor-ligated DNA sample in the PCR plate (50 µL). Pipette up and down to mix.
2 Sample Preparation (3 µg DNA Samples) Step 10. Amplify the adaptor-ligated library 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.
Sample Preparation (3 µg DNA Samples) Step 10. Amplify the adaptor-ligated library CA U T I O N 2 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 17, on ice. Mix well on a vortex mixer.
2 Sample Preparation (3 µg DNA Samples) Step 10. Amplify the adaptor-ligated library 3 Run the program in Table 18 in a thermal cycler. . Table 18 NOTE 36 Pre-Capture PCR Thermal Cycler Program Segment Number of Cycles Temperature Time 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.
Sample Preparation (3 µg DNA Samples) Step 11. Purify the amplified library with AMPure XP beads 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 µL of homogeneous AMPure XP beads to each 50-µL amplified DNA sample in the PCR plate. Pipette up and down to mix.
2 Sample Preparation (3 µg DNA Samples) Step 12. Assess quality and quantity 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.
Sample Preparation (3 µg DNA Samples) Step 12. Assess quality and quantity 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 µL of each DNA sample diluted with 3 µL of D1000 sample buffer for the analysis.
2 40 Sample Preparation (3 µg DNA Samples) Step 12.
SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library Protocol 3 Sample Preparation (200 ng DNA Samples) Step 1. Shear the DNA 42 Step 2. Assess quality (optional) 45 Step 3. Repair the ends 46 Step 4. Purify the sample using AMPure XP beads 47 Step 5. Adenylate the 3' end of the DNA fragments 49 Step 6. Purify the sample using AMPure XP beads 50 Step 7. Ligate the paired-end adaptor 51 Step 8. Purify the sample using AMPure XP beads 52 Step 9.
3 Sample Preparation (200 ng DNA Samples) Step 1. Shear the DNA Step 1. Shear the DNA NOTE 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.
Sample Preparation (200 ng DNA Samples) Step 1. Shear the DNA 3 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.
3 Sample Preparation (200 ng DNA Samples) Step 1. Shear the DNA 9 Transfer each sheared DNA sample (approximately 50 µL) to a separate well of a 96-well plate or strip tube. NOTE 44 Instructions in this protocol 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.
Sample Preparation (200 ng DNA Samples) Step 2. Assess quality (optional) 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 Check that the 2100 Bioanalyzer electrodes have been cleaned as instructed in the reagent kit guide.
3 Sample Preparation (200 ng DNA Samples) Step 3. Repair the ends 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.
Sample Preparation (200 ng DNA Samples) Step 4. Purify the sample using AMPure XP beads 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 µL of 70% ethanol per sample, plus excess, for use in step 8. NOTE The freshly-prepared 70% ethanol may be used for subsequent purification steps run on the same day. The complete Library Preparation protocol requires 1.
3 Sample Preparation (200 ng DNA Samples) Step 4. Purify the sample using AMPure XP beads 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.
Sample Preparation (200 ng DNA Samples) Step 5. Adenylate the 3' end of the DNA fragments 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.
3 Sample Preparation (200 ng DNA Samples) Step 6. Purify the sample using AMPure XP beads 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 µL of homogeneous AMPure XP beads to each 50-µL dA-tailed DNA sample in the PCR plate. Pipette up and down 10 times to mix.
Sample Preparation (200 ng DNA Samples) Step 7. Ligate the paired-end adaptor 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. 2 Prepare the appropriate volume of Ligation master mix, as described in Table 25, on ice.
3 Sample Preparation (200 ng DNA Samples) Step 8. Purify the sample using AMPure XP beads 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 µL of homogeneous AMPure XP beads to each adaptor-ligated DNA sample in the PCR plate (50 µL). Pipette up and down to mix.
Sample Preparation (200 ng DNA Samples) Step 9. Amplify the adaptor-ligated library 3 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.
3 Sample Preparation (200 ng DNA Samples) Step 9. Amplify the adaptor-ligated library CA U T I O N 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.
Sample Preparation (200 ng DNA Samples) Step 9. Amplify the adaptor-ligated library 3 3 Run the program in Table 29 in a thermal cycler. .
3 Sample Preparation (200 ng DNA Samples) Step 10. Purify the amplified library with AMPure XP beads 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 µL of homogeneous AMPure XP beads to each 50-µL amplified DNA sample in the PCR plate. Pipette up and down to mix.
Sample Preparation (200 ng DNA Samples) Step 11. Assess quality and quantity 3 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.
3 Sample Preparation (200 ng DNA Samples) Step 11. Assess quality and quantity 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 µL of each DNA sample diluted with 3 µL of D1000 sample buffer for the analysis.
SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library Protocol 4 Hybridization and Capture Step 1. Hybridize DNA samples to the Capture Library 60 Step 2. Prepare streptavidin-coated magnetic beads 66 Step 3. Capture the hybridized DNA using streptavidin-coated beads 67 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.
4 Hybridization and Capture Step 1. Hybridize DNA samples to the Capture Library 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 SureSelectXT 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.
Hybridization and Capture Step 1. Hybridize DNA samples to the Capture Library 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 µL (initial concentration of 221 ng/µL). 1 For prepped libraries with DNA concentrations above 221 ng/µL, prepare 3.4 µL of a 221 ng/µL dilution of each library.
4 Hybridization and Capture Step 1. Hybridize DNA samples to the Capture Library 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.
Hybridization and Capture Step 1. Hybridize DNA samples to the Capture Library CA U T I O N 4 For each 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. 6 To each gDNA library sample well prepared in step 3 on page 61, add 5.6 µL of the SureSelect Block Mix prepared in Table 32. Pipette up and down to mix.
4 Hybridization and Capture Step 1. Hybridize DNA samples to the Capture Library NOTE Prepare the Capture Library mixture described in step 9, below, near the end of the 65°C hold step of >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.
Hybridization and Capture Step 1. Hybridize DNA samples to the Capture Library 4 10 Maintain the gDNA library + Block Mix plate or strip tube at 65°C while you add 20 µL 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 µL, depending on the degree of evaporation during the thermal cycler incubation.
4 Hybridization and Capture Step 2. Prepare streptavidin-coated magnetic beads 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 Table 2 on page 14). 1 Prewarm SureSelect Wash Buffer 2 at 65°C in a circulating water bath or heat block for use in “Step 3.
Hybridization and Capture Step 3. Capture the hybridized DNA using streptavidin-coated beads 4 Step 3. Capture the hybridized DNA using streptavidin-coated beads 1 Estimate and record the volume of hybridization solution that remains after the 24 hour incubation.
4 Hybridization and Capture Step 3. Capture the hybridized DNA using streptavidin-coated beads CA U T I O N 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.
SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library Protocol 5 Indexing and Sample Processing for Multiplexed Sequencing Step 1A. Amplify the captured libraries with indexing primers containing 8-bp indexes A01–H12 70 Step 1B. Amplify the captured libraries with indexing primers containing 6-bp indexes 1–16 74 Step 2. Purify the amplified captured libraries using AMPure XP beads 77 Step 3. Assess indexed library DNA quantity and quality 79 Step 4.
5 Indexing and Sample Processing for Multiplexed Sequencing Step 1A. Amplify the captured libraries with indexing primers containing 8-bp indexes A01–H12 CA U T I O N 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.
Indexing and Sample Processing for Multiplexed Sequencing Step 1A. Amplify the captured libraries with indexing primers containing 8-bp indexes A01–H12 5 Prepare one indexing amplification reaction for each DNA library. CA U T I O N 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.
5 Indexing and Sample Processing for Multiplexed Sequencing Step 1A. Amplify the captured libraries with indexing primers containing 8-bp indexes A01–H12 5 Add the DNA library samples to the PCR reactions: a Obtain the PCR plate or strip tube containing 30 µL of bead-bound target-enriched DNA samples from ice (prepared on page 68).
Indexing and Sample Processing for Multiplexed Sequencing Step 1A. Amplify the captured libraries with indexing primers containing 8-bp indexes A01–H12 Table 40 5 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 >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.
5 Indexing and Sample Processing for Multiplexed Sequencing Step 1B. Amplify the captured libraries with indexing primers containing 6-bp indexes 1–16 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.
Indexing and Sample Processing for Multiplexed Sequencing Step 1B. Amplify the captured libraries with indexing primers containing 6-bp indexes 1–16 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 reaction Volume for 16 reactions (includes excess) Nuclease-free water 22.5 µL 382.
5 Indexing and Sample Processing for Multiplexed Sequencing Step 1B. Amplify the captured libraries with indexing primers containing 6-bp indexes 1–16 6 Transfer the PCR plate or strip tube to a thermal cycler and run the PCR amplification program shown in Table 39.
Indexing and Sample Processing for Multiplexed Sequencing Step 2. Purify the amplified captured libraries using AMPure XP beads 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 µL 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.
5 Indexing and Sample Processing for Multiplexed Sequencing Step 2. Purify the amplified captured libraries using AMPure XP beads 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 µL) to a fresh well. You can discard the beads at this time.
Indexing and Sample Processing for Multiplexed Sequencing Step 3. Assess indexed library DNA quantity and quality 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.
5 Indexing and Sample Processing for Multiplexed Sequencing Step 3. Assess indexed library DNA quantity and quality Figure 9 80 Post-capture analysis of amplified indexed library DNA using the 2100 Bioanalyzer and a High Sensitivity DNA Assay.
Indexing and Sample Processing for Multiplexed Sequencing Step 3. Assess indexed library DNA quantity and quality 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.
5 Indexing and Sample Processing for Multiplexed Sequencing Step 3. Assess indexed library DNA quantity and quality Figure 10 82 Post-capture analysis of amplified indexed library DNA using the 2200 TapeStation with a High Sensitivity D1000 ScreenTape.
Indexing and Sample Processing for Multiplexed Sequencing Step 4. Quantify each index-tagged library by QPCR (optional) 5 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 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.
5 Indexing and Sample Processing for Multiplexed Sequencing Step 5. Pool samples 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.
Indexing and Sample Processing for Multiplexed Sequencing Step 5. Pool samples 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 to the desired volume. 3 If you store the library before sequencing, add Tween 20 to 0.
5 Indexing and Sample Processing for Multiplexed Sequencing Guidelines for sequencing sample preparation and run setup Guidelines for sequencing sample preparation and run setup Use the appropriate Illumina Paired-End Cluster Generation Kit to do cluster amplification. Refer to the instructions that are included with the Illumina Paired-End Cluster Generation Kit. The optimal seeding concentration for SureSelectXT libraries is 6 to 8 pM, depending on the desired output and data quality.
SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library Protocol 6 Reference Reference Information for Kits with Revised Index Configuration (indexing primers in white-capped tubes or blue plate) 88 Reference Information for Kits with Original Index Configuration (indexing primers in clear-capped tubes) 93 Alternative Capture Equipment Combinations 97 This chapter contains reference information, including component kit contents and index sequences.
6 Reference Reference Information for Kits with Revised Index Configuration (indexing primers in white-capped tubes or blue plate) CA U T I O N 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).
Reference Kit Contents 6 The contents of each of the component kits listed in Table 47 are described in the tables below.
6 Reference Kit Contents Table 49 SureSelect Target Enrichment-Box 1 Content Kit Component 16 Reactions 96 or 480 Reactions SureSelect Hyb 1 tube with orange cap tube with orange cap SureSelect Hyb 2 tube with red cap tube with red cap SureSelect Hyb 4 tube with black cap tube with black cap SureSelect Binding Buffer bottle bottle SureSelect Wash Buffer 1 bottle bottle SureSelect Wash Buffer 2 bottle bottle Table 50 SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2 Con
Reference Kit Contents Table 51 6 Plate map for SureSelect 8bp Indexes A01 through H12, provided in blue plate in Library Prep kit p/n 5500-0133 1 2 3 4 5 6 7 8 9 10 11 12 A A01 A02 A03 A04 A05 A06 A07 A08 A09 A10 A11 A12 B B01 B02 B03 B04 B05 B06 B07 B08 B09 B10 B11 B12 C C01 C02 C03 C04 C05 C06 C07 C08 C09 C10 C11 C12 D D01 D02 D03 D04 D05 D06 D07 D08 D09 D10 D11 D12 E E01 E02 E03 E04 E05 E06 E07 E08 E09 E10 E11 E12 F F01 F0
6 Reference Nucleotide Sequences of SureSelectXT Indexes A01 to H12 Nucleotide Sequences of SureSelectXT Indexes A01 to H12 Each index is 8 nt in length. See page 86 for sequencing run setup requirements for sequencing libraries using 8-bp indexes.
Reference 6 Reference Information for Kits with Original Index Configuration (indexing primers in clear-capped tubes) Reference Information for Kits with Original Index Configuration (indexing primers in clear-capped tubes) Use the reference information in 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 88 for kit content and indexing primer information.
6 Reference Kit Contents The contents of each of the component kits listed in Table 53 are described in the tables below.
Reference Kit Contents Table 55 6 SureSelect Target Enrichment-Box 1 Content Kit Component 16 Reactions 96 Reactions 480 Reactions SureSelect Hyb 1 tube with orange cap tube with orange cap bottle SureSelect Hyb 2 tube with red cap tube with red cap tube with red cap SureSelect Hyb 4 tube with black cap tube with black cap bottle SureSelect Binding Buffer bottle bottle bottle SureSelect Wash Buffer 1 bottle bottle bottle SureSelect Wash Buffer 2 bottle bottle bottle SureSelect
6 Reference Nucleotide Sequences of SureSelectXT 6-bp Indexes-Original Kit Configuration Nucleotide Sequences of SureSelectXT 6-bp Indexes-Original Kit Configuration Refer to the sequence information in Table 57 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).
Reference Alternative Capture Equipment Combinations 6 Alternative Capture Equipment Combinations Table 58 below lists combinations of thermal cyclers, lid temperatures, plates or strip tubes and sealing methods that have shown minimal evaporation when used for the Hybridization protocol on page 60. Refer to this list for additional equipment and plasticware combination options for hybridization, beyond the combinations used for protocol optimization and supported by Agilent.
www.agilent.com In This Book This guide contains information to run the SureSelectXT target enrichment protocol. Agilent Technologies, Inc. 2015 Version B.
