SureSelectXT2 Target Enrichment System for Illumina Multiplexed Sequencing Featuring Pre-Capture Indexing Reagents and Protocols Protocol Version E0, January 2015 Before you begin, view hands-on videos of SureSelect procedures at SureSelect platform manufactured with Agilent SurePrint Technology http://www.agilent.com/genomics/protocolvideos. Research Use Only. Not for use in Diagnostic Procedures.
Notices © Agilent Technologies, Inc. 2015 Warranty Notice to Purchaser 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.
Safety Notices CA U T I O N A CAUTION notice denotes a hazard. It calls attention to an operating 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. WARN I NG A WARNING notice denotes a hazard.
In this Guide... This guide describes an optimized protocol for Illumina paired-end multiplexed library preparation using the SureSelectXT2 Library Prep and Capture System. This protocol is specifically optimized 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. The SureSelectXT2 Library Prep and Capture System features pre-capture indexing reagents and protocols.
What’s New in Version E.0 • Support for kits supplied with either of two indexing primer configurations. Kits with revised index configuration (typically received February 2015 or later) include indexing primers A01 through H02 provided in white-capped tubes (16 Reaction kits) or indexing primers A01 through H12 provided in a blue plate (96 Reaction kits). For kit content details see page 76. For nucleotide sequences of the 8-bp indexes in this revised configuration, see Table 38 on page 79.
SureSelectXT2 Target Enrichment System for Illumina
Content 1 Before You Begin 11 Overview of the Workflow 12 Procedural Notes 13 Safety Notes 13 Required Reagents 14 Required Equipment 16 Optional Reagents and Equipment 2 17 Sample Preparation (1 µg DNA Samples) 19 Step 1. Shear DNA 20 Step 2. Assess quality (optional) 23 Step 3. Repair the ends 25 Step 4. Purify the sample using AMPure XP beads 27 Step 5. Adenylate the 3' end of the DNA fragments 29 Step 6. Ligate the pre-capture indexing adaptor 30 Step 7.
Contents 3 Sample Preparation (100 ng DNA Samples) 37 Step 1. Shear DNA 38 Step 2. Assess quality (optional) 41 Step 3. Repair the ends 43 Step 4. Purify the sample using AMPure XP beads 45 Step 5. Adenylate the 3' end of the DNA fragments 47 Step 6. Ligate the pre-capture indexing adaptor 48 Step 7. Purify the indexed DNA using AMPure XP beads 49 Step 8. Amplify the indexed library 50 Step 9. Purify the amplified library with AMPure XP beads 51 Step 10.
Contents Reference Information for Kits with Original Index Configuration (indexing primers in clear-capped tubes) 80 Kit Contents 80 Nucleotide Sequences of SureSelect Pre-Capture Indexes-Original Index Configuration 82 SureSelectXT2 Target Enrichment System for Illumina 9
Contents 10 SureSelectXT2 Target Enrichment System for Illumina
SureSelectXT2 Target Enrichment System for Illumina Multiplexed Sequencing Protocol 1 Before You Begin Overview of the Workflow 12 Safety Notes 13 Required Reagents 14 Required Equipment 16 Optional Reagents and Equipment 17 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 SureSelectXT2 target enrichment workflow is summarized in Figure 1. Figure 1 12 Overall target-enriched sequencing sample preparation workflow.
Before You Begin Procedural Notes 1 Procedural Notes • This User Guide includes protocols for library preparation using either 1 g DNA samples (see Chapter 2 on page 19) or 100 ng DNA samples (see Chapter 3 on page 37). Make sure that you are following the appropriate protocol for your DNA input amount. After the prepared libraries are amplified, both DNA input options use the same protocol for hybridization and post-capture processing.
1 Before You Begin Required Reagents Required Reagents Required Reagents for SureSelectXT2 Target Enrichment Table 1 Description Vendor and part number SureSelectXT2 Capture Library* Select one library from Table 2 SureSelect XT2 Agilent Reagent Kit* HiSeq platform (HSQ), 16 Samples HiSeq platform (HSQ), 96 Samples HiSeq platform (HSQ), 480 Samples p/n G9621A p/n G9621B p/n G9621C MiSeq platform (MSQ), 16 Samples MiSeq platform (MSQ), 96 Samples MiSeq platform (MSQ), 480 Samples p/n G9622A p/n
Before You Begin Required Reagents Table 2 SureSelectXT2 Capture Libraries Capture Library 16 Samples 96 Samples 480 Samples SureSelectXT2 Clinical Research Exome* 5190-7345 5190-7346 5 × 5190-7346 5190-7797 5190-7798 5 × 5190-7798 5190-7800 5190-7806 5 × 5190-7806 5190-6216 5190-6217 5 × 5190-6217 5190-6221 5190-6222 5 × 5190-6222 SureSelect XT2 Focused Exome* SureSelect XT2 Focused Exome Plus 1* SureSelect XT2 Human All Exon v5 * SureSelectXT2 Human All Exon v5 + UTRs* Sur
1 Before You Begin Required Equipment Required Equipment Table 3 Required Equipment for SureSelectXT2 Target Enrichment Description Vendor and part number SureCycler 8800 Thermal Cycler Agilent p/n G8800A 96 well plate module for SureCycler 8800 Thermal Cycler Agilent p/n G8810A SureCycler 8800-compatible 96-well plates Agilent p/n 410088 Optical strip caps Agilent p/n 401425 Tube cap strips, domed Agilent p/n 410096 Compression mats Agilent p/n 410187 2100 Bioanalyzer Laptop Bundle Agile
Before You Begin Optional Reagents and Equipment Table 3 1 Required Equipment for SureSelectXT2 Target Enrichment Description Vendor and part number Vacuum concentrator Savant SpeedVac, model DNA120, with 96-well plate rotor, model RD2MP, or equivalent Ice bucket Powder-free gloves Sterile, nuclease-free aerosol barrier pipette tips Timer Water bath or heat block suitable for incubation temperatures up to 65°C Optional Reagents and Equipment Table 4 Optional Reagents and Equipment Description Ven
1 18 Before You Begin Optional Reagents and Equipment SureSelectXT2 Target Enrichment System for Illumina
SureSelectXT2 Target Enrichment System for Illumina Multiplexed Sequencing Protocol 2 Sample Preparation (1 µg DNA Samples) Step 1. Shear DNA 20 Step 2. Assess quality (optional) 23 Step 3. Repair the ends 25 Instructions for Library Prep Kit p/n 5500-0102 or 5500-0103 26 Step 5. Adenylate the 3' end of the DNA fragments 29 Step 6. Ligate the pre-capture indexing adaptor 30 Step 7. Purify the indexed DNA using AMPure XP beads 31 Step 8. Amplify the indexed library 32 Step 9.
2 Sample Preparation (1 µg DNA Samples) Step 1. Shear DNA Step 1. Shear DNA NOTE Make sure genomic DNA samples are of high quality with an OD 260/280 ratio ranging from 1.8 to 2.0. For each DNA sample to be sequenced, prepare 1 library. 1 Use the Qubit dsDNA BR Assay to determine the concentration of your gDNA sample. Follow the instructions for the instrument. 2 Dilute 1 µg of high-quality gDNA with 1X Low TE Buffer in a 1.5-mL LoBind tube to a total volume of 50 µL.
Sample Preparation (1 µg DNA Samples) Step 1. Shear DNA 2 6 Secure the microTube in the tube holder and shear the DNA with the settings in Table 5 or Table 6, depending on the Covaris instrument SonoLab software version used. The target DNA fragment size is 150 to 200 bp.
2 Sample Preparation (1 µg DNA Samples) Step 1. Shear DNA 9 Transfer each 50-µL sheared DNA sample to a separate well of a SureCycler 8800 96-well plate. CA U T I O N Use SureCycler 8800-compatible 96-well plates (see Table 3 on page 16) for all SureSelectXT2 protocol steps done in 96-well plates. Protocols were optimized using the specified plates; use of other plasticware may negatively impact your results and is not supported by Agilent.
Sample Preparation (1 µg DNA Samples) Step 2. Assess quality (optional) 2 Step 2. Assess quality (optional) NOTE You can use Agilent’s 2200 TapeStation for rapid analysis of multiple samples at this step. For analysis of 1 g sheared DNA samples, use the D1000 ScreenTape (p/n 5067-5582) and associated reagents. See the 2200 TapeStation and D1000 ScreenTape protocols for information on sample preparation and data analysis. This step is optional.
2 Sample Preparation (1 µg DNA Samples) Step 2. Assess quality (optional) Figure 2 24 Analysis of sheared DNA using a DNA 1000 Bioanalyzer assay.
Sample Preparation (1 µg DNA Samples) Step 3. Repair the ends 2 Step 3. Repair the ends Use the SureSelectXT2 Library Prep Kit. CA U T I O N This section contains two versions of instructions for the end repair reaction. The first section covers kits supplied with Library Prep Kit p/n 5500-0130 or 5500-0131, with two separate reagent vials containing the End Repair Enzyme Mix and End Repair Oligo Mix (typically received February, 2015 or later).
2 Sample Preparation (1 µg DNA Samples) Step 3. Repair the ends 4 Incubate the plate in the SureCycler thermal cycler and run the program in Table 8. Do not use a heated lid. Table 8 End-Repair Thermal Cycler Program Step Temperature Time Step 1 20°C 30 minutes Step 2 4°C Hold Instructions for Library Prep Kit p/n 5500-0102 or 5500-0103 1 Add 50 µL of SureSelect End Repair Master Mix to each sheared DNA sample. 2 Mix well by pipetting up and down or by gentle vortexing.
Sample Preparation (1 µg DNA Samples) Step 4. Purify the sample using AMPure XP beads 2 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.
2 Sample Preparation (1 µg 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 on the SureCycler thermal cycler, set to hold samples at 37°C, for 5 to 10 minutes or until the residual ethanol completely evaporates. 13 Add 22 µL nuclease-free water to each sample well.
Sample Preparation (1 µg DNA Samples) Step 5. Adenylate the 3' end of the DNA fragments 2 Step 5. Adenylate the 3' end of the DNA fragments 1 Add 20 µL of SureSelect dA-Tailing Master Mix to each end-repaired, purified DNA sample (approximately 20 µL). 2 Mix well by pipetting up and down or by gentle vortexing. CA U T I O N SureSelectXT2 master mixes are viscous and thorough mixing is required to combine these mixtures with other solutions.
2 Sample Preparation (1 µg DNA Samples) Step 6. Ligate the pre-capture indexing adaptor Step 6. Ligate the pre-capture indexing adaptor See the Reference section for sequences of the index portion of the indexing adaptors that are ligated to gDNA libraries in this section. Be sure to keep the sample plate at 4°C or on ice while doing step 1 and step 2 sequentially as outlined below. 1 Add 5 µL of SureSelect Ligation Master Mix to each A-tailed DNA sample.
Sample Preparation (1 µg DNA Samples) Step 7. Purify the indexed DNA using AMPure XP beads 2 Step 7. Purify the indexed DNA 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 60 µL of homogeneous AMPure XP beads to each 50-µL indexing adaptor-ligated DNA sample in the PCR plate. Pipette up and down to mix.
2 Sample Preparation (1 µg DNA Samples) Step 8. Amplify the indexed library Step 8. Amplify the indexed library This protocol uses half of the indexing adaptor-ligated library for amplification. The remainder can be saved at –20°C for future use, if needed. 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.
Sample Preparation (1 µg DNA Samples) Step 8. Amplify the indexed library 2 3 Run the program in Table 13 in a SureCycler thermal cycler. . Table 13 NOTE Pre-Capture PCR Thermal Cycler Program Segment Number of Cycles Temperature Time 1 1 98°C 2 minutes 2 5 98°C 30 seconds 60°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.
2 Sample Preparation (1 µg DNA Samples) Step 9. Purify the amplified library with AMPure XP beads Step 9. 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 60 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 (1 µg DNA Samples) Step 10. Assess quality with the 2100 Bioanalyzer DNA 1000 Assay 2 Step 10. Assess quality with the 2100 Bioanalyzer DNA 1000 Assay NOTE You can use Agilent’s 2200 TapeStation for rapid analysis of multiple samples at this step. For analysis of indexed DNA amplicons prepared from 1-g gDNA samples, use the D1000 ScreenTape (p/n 5067-5582) and associated reagents.
2 Sample Preparation (1 µg DNA Samples) Step 10. Assess quality with the 2100 Bioanalyzer DNA 1000 Assay Figure 3 36 DNA 1000 Assay analysis of amplified library DNA prepared using the 1-g DNA input Sample Preparation protocol.
SureSelectXT2 Target Enrichment System for Illumina Multiplexed Sequencing Protocol 3 Sample Preparation (100 ng DNA Samples) Step 1. Shear DNA 38 Step 2. Assess quality (optional) 41 Step 3. Repair the ends 43 Step 4. Purify the sample using AMPure XP beads 45 Step 5. Adenylate the 3' end of the DNA fragments 47 Step 6. Ligate the pre-capture indexing adaptor 48 Step 7. Purify the indexed DNA using AMPure XP beads 49 Step 8. Amplify the indexed library 50 Step 9.
3 Sample Preparation (100 ng DNA Samples) Step 1. Shear DNA Step 1. Shear DNA NOTE Make sure genomic DNA samples are of high quality with an OD 260/280 ratio ranging from 1.8 to 2.0. For each DNA sample to be sequenced, prepare 1 library. 1 Use the Qubit dsDNA BR Assay to determine the concentration of your gDNA sample. Follow the instructions for the instrument. 2 Dilute 100 ng of high-quality gDNA with 1X Low TE Buffer in a 1.5-mL LoBind tube to a total volume of 50 µL.
Sample Preparation (100 ng DNA Samples) Step 1. Shear DNA 3 6 Secure the microTube in the tube holder and shear the DNA with the settings in Table 14 or Table 15, depending on the Covaris instrument SonoLab software version used. The target DNA fragment size is 150 to 200 bp.
3 Sample Preparation (100 ng DNA Samples) Step 1. Shear DNA 9 Transfer each 50-µL sheared DNA sample to a separate well of a SureCycler 8800 96-well plate. CA U T I O N Use SureCycler 8800-compatible 96-well plates (see Table 3 on page 16) for all SureSelectXT2 protocol steps done in 96-well plates. Protocols were optimized using the specified plates; use of other plasticware may negatively impact your results and is not supported by Agilent.
Sample Preparation (100 ng DNA Samples) Step 2. Assess quality (optional) 3 Step 2. Assess quality (optional) NOTE You can use Agilent’s 2200 TapeStation for rapid analysis of multiple samples at this step. For analysis of 100 ng sheared DNA samples, use the High-Sensitivity D1000 ScreenTape (p/n 5067-5584) and associated reagents. See the 2200 TapeStation and High-Sensitivity D1000 ScreenTape protocols for information on sample preparation and data analysis. This step is optional.
3 Sample Preparation (100 ng DNA Samples) Step 2. Assess quality (optional) Figure 4 42 Analysis of 100 ng sheared DNA sample using a High-Sensitivity DNA Bioanalyzer assay.
Sample Preparation (100 ng DNA Samples) Step 3. Repair the ends 3 Step 3. Repair the ends Use the SureSelectXT2 Library Prep Kit. CA U T I O N This section contains two versions of instructions for the end repair reaction. The first section covers kits supplied with Library Prep Kit p/n 5500-0130 or 5500-0131, with two separate reagent vials containing the End Repair Enzyme Mix and End Repair Oligo Mix (typically received February, 2015 or later).
3 Sample Preparation (100 ng DNA Samples) Step 3. Repair the ends 4 Incubate the plate in the SureCycler thermal cycler and run the program in Table 17. Do not use a heated lid. Table 17 End-Repair Thermal Cycler Program Step Temperature Time Step 1 20°C 30 minutes Step 2 4°C Hold Instructions for Library Prep Kit p/n 5500-0102 or 5500-0103 1 Add 50 µL of SureSelect End Repair Master Mix to each sheared DNA sample. 2 Mix well by pipetting up and down or by gentle vortexing.
Sample Preparation (100 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 (100 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 on the SureCycler thermal cycler, set to hold samples at 37°C, for 5 to 10 minutes or until the residual ethanol completely evaporates. 13 Add 22 µL nuclease-free water to each sample well.
Sample Preparation (100 ng DNA Samples) Step 5. Adenylate the 3' end of the DNA fragments 3 Step 5. Adenylate the 3' end of the DNA fragments 1 Add 20 µL of SureSelect dA-Tailing Master Mix to each end-repaired, purified DNA sample (approximately 20 µL). 2 Mix well by pipetting up and down or by gentle vortexing. CA U T I O N SureSelectXT2 master mixes are viscous and thorough mixing is required to combine these mixtures with other solutions.
3 Sample Preparation (100 ng DNA Samples) Step 6. Ligate the pre-capture indexing adaptor Step 6. Ligate the pre-capture indexing adaptor See the Reference section for sequences of the index portion of the indexing adaptors that are ligated to gDNA libraries in this section. Be sure to keep the sample plate at 4°C or on ice while doing step 1 through step 3 sequentially as outlined below.
Sample Preparation (100 ng DNA Samples) Step 7. Purify the indexed DNA using AMPure XP beads 3 Step 7. Purify the indexed DNA 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 50 µL of homogeneous AMPure XP beads to each 50-µL indexing adaptor-ligated DNA sample in the PCR plate. Pipette up and down to mix.
3 Sample Preparation (100 ng DNA Samples) Step 8. Amplify the indexed library Step 8. Amplify the indexed 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 21, on ice. Mix well on a vortex mixer.
Sample Preparation (100 ng DNA Samples) Step 9. Purify the amplified library with AMPure XP beads 3 Step 9. 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 50 µL of homogeneous AMPure XP beads to each 50-µL amplified DNA sample in the PCR plate. Pipette up and down to mix.
3 Sample Preparation (100 ng DNA Samples) Step 10. Assess quality with the 2100 Bioanalyzer DNA 1000 Assay Step 10. Assess quality with the 2100 Bioanalyzer DNA 1000 Assay NOTE You can use Agilent’s 2200 TapeStation for rapid analysis of multiple samples at this step. For analysis of indexed DNA amplicons prepared from 100-ng gDNA samples, use the D1000 ScreenTape (p/n 5067-5582) and associated reagents.
Sample Preparation (100 ng DNA Samples) Step 10. Assess quality with the 2100 Bioanalyzer DNA 1000 Assay Figure 5 3 DNA 1000 Assay analysis of amplified library DNA prepared using the 100-ng DNA input Sample Preparation protocol.
3 54 Sample Preparation (100 ng DNA Samples) Step 10.
SureSelectXT2 Target Enrichment System for Illumina Multiplexed Sequencing Protocol 4 Hybridization Step 1. Pool indexed DNA samples for hybridization 56 Step 2. Hybridize gDNA library pools to the SureSelect Capture Library 57 Step 3. Prepare streptavidin-coated magnetic beads 59 Step 4. Capture the hybridized DNA using streptavidin beads 60 This chapter describes the steps to pool indexed gDNA libraries and then hybridize the pooled gDNA libraries with a SureSelectXT2 Capture Library.
4 Hybridization Step 1. Pool indexed DNA samples for hybridization Step 1. Pool indexed DNA samples for hybridization In this step, you pool the indexed gDNA samples, before hybridization to the SureSelectXT2 Capture Library. Each hybridization reaction requires a total of 1500 ng indexed gDNA, made up of equal amounts of 8 or 16 individual libraries. See Table 23 for the recommended pool composition for your SureSelectXT2 Capture Library.
Hybridization Step 2. Hybridize gDNA library pools to the SureSelect Capture Library 4 Step 2. Hybridize gDNA library pools to the SureSelect Capture Library 1 To each 7-µL indexed gDNA pool, add 9 µL of SureSelect XT2 Blocking Mix. Pipette up and down to mix. 2 Cap the wells, then transfer the sealed plate to the thermal cycler and run the following program shown in Table 24. Use a heated lid, set at 105°C, to hold the temperature at 65°C.
4 Hybridization Step 2. Hybridize gDNA library pools to the SureSelect Capture Library 4 In a PCR plate (kept on ice), for each hybridization reaction well, combine the indicated volumes of SureSelectXT2 Capture Library and dilute RNase Block, according to Table 26. Mix well by pipetting. Table 26 Preparation of Capture Library/RNase Block mixture Capture Library Size Volume of Capture Library per hybridization reaction Volume of dilute RNase Block per hybridization reaction <3.
Hybridization Step 3. Prepare streptavidin-coated magnetic beads 4 Step 3. Prepare streptavidin-coated magnetic beads 1 Prewarm SureSelect XT2 Wash 2 at 65°C in a water bath or heat block for use in “Step 4. Capture the hybridized DNA using streptavidin beads”. 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 µL of the resuspended beads to wells of a SureCycler 8800 PCR plate.
4 Hybridization Step 4. Capture the hybridized DNA using streptavidin beads Step 4. Capture the hybridized DNA using streptavidin beads 1 Estimate and record the volume of hybridization solution that remains after the 24 hour incubation. CA U T I O N Excessive evaporation, such as when less than 52 µL remains after hybridization, can indicate suboptimal capture performance.
Hybridization Step 4. Capture the hybridized DNA using streptavidin beads 4 10 Wash the beads with SureSelect XT2 Wash 2: a Resuspend the beads in 200 µL of 65°C prewarmed SureSelect XT2 Wash 2. Pipette up and down until the beads are fully resuspended. b Incubate the sample plate for 5 minutes at 65°C on the SureCycler thermal cycler. c Briefly spin the plate in a centrifuge or mini-plate spinner. d Put the plate in the magnetic separator.
4 62 Hybridization Step 4.
SureSelectXT2 Target Enrichment System for Illumina Multiplexed Sequencing Protocol 5 Post-Capture Sample Processing for Multiplexed Sequencing Step 1. Amplify the captured libraries 64 Step 2. Purify the amplified captured libraries using AMPure XP beads 66 Step 3. Assess quality with the 2100 Bioanalyzer High Sensitivity DNA assay 68 Step 4. Prepare samples for multiplexed sequencing 70 Step 5. Optional: Quantify captured library pools by QPCR 72 Step 6.
5 Post-Capture Sample Processing for Multiplexed Sequencing Step 1. Amplify the captured libraries Step 1. Amplify the captured libraries In this step, the SureSelect-enriched indexed library DNA pools are PCR amplified. The protocol uses half of the bead-bound captured library pool for amplification. The remainder can be saved at –20°C for future use, if needed.
Post-Capture Sample Processing for Multiplexed Sequencing Step 1. Amplify the captured libraries 5 5 Place the plate in a thermal cycler and run the PCR amplification program shown in Table 28 using the cycle number specified in Table 29.
5 Post-Capture Sample Processing for Multiplexed Sequencing Step 2. Purify the amplified captured libraries using AMPure XP beads 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 70% ethanol per sample, plus excess, for use in step 9. 3 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color.
Post-Capture Sample Processing for Multiplexed Sequencing Step 2. Purify the amplified captured libraries using AMPure XP beads 5 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 µL) to a fresh tube or plate well. You can discard the beads at this time. 18 Remove 1 µL of the purified captured library pool from the sample and combine with 9 µL of 1 X Low TE Buffer for Bioanalyzer analysis.
5 Post-Capture Sample Processing for Multiplexed Sequencing Step 3. Assess quality with the 2100 Bioanalyzer High Sensitivity DNA assay Step 3. Assess quality with the 2100 Bioanalyzer High Sensitivity DNA assay NOTE You can use Agilent’s 2200 TapeStation for rapid analysis of multiple samples at this step. For analysis of captured DNA, use the High-Sensitivity D1000 ScreenTape (p/n 5067-5584) and associated reagents.
Post-Capture Sample Processing for Multiplexed Sequencing Step 3. Assess quality with the 2100 Bioanalyzer High Sensitivity DNA assay 5 If the yield is too low or non-specific peaks are observed in the electropherogram, repeat the PCR with more or fewer cycles. The goal is to minimize cycles, while you produce enough library for application to the flow cell. Figure 6 Analysis of amplified captured DNA using the High Sensitivity DNA Assay.
5 Post-Capture Sample Processing for Multiplexed Sequencing Step 4. Prepare samples for multiplexed sequencing Step 4. Prepare samples for multiplexed sequencing The final SureSelectXT2-enriched samples contain pools of either 8 or 16 indexed libraries, based on the Capture Library size and resulting pre-capture pooling strategy.
Post-Capture Sample Processing for Multiplexed Sequencing Step 4. Prepare samples for multiplexed sequencing 5 concentration of each sample may be accurately determined as described in “Step 5. Optional: Quantify captured library pools by QPCR” on page 72. Sequencing run setup guidelines If samples will not be further combined in post-capture pools, proceed to cluster amplification using the Illumina Paired-End Cluster Generation Kit; refer to the manufacturer’s instructions for this step.
5 Post-Capture Sample Processing for Multiplexed Sequencing Step 5. Optional: Quantify captured library pools by QPCR Step 5. Optional: Quantify captured library pools by QPCR For accurate determination of the DNA concentration in each captured library pool, use the QPCR NGS Library Quantification Kit (for Illumina). Refer to the protocol that is included with the QPCR NGS Library Quantification Kit (p/n G4880A) for more details to do this step.
Post-Capture Sample Processing for Multiplexed Sequencing Step 6. Optional: Pool captured libraries for sequencing 5 Step 6. Optional: Pool captured libraries for sequencing See Table 32 on page 74 for post-capture pooling guidelines, based on your SureSelectXT2 Capture Library size and sequencing design. Pooling instructions are provided below. 1 Combine the capture pools such that each index-tagged sample is present in equimolar amounts in the final sequencing sample pool.
5 Post-Capture Sample Processing for Multiplexed Sequencing Step 6. Optional: Pool captured libraries for sequencing Table 32 Example of capture pool volume calculations for a 20-µL final sequencing sample pool containing 10 nM DNA Component V(f) C(i) C(f) # Volume to use (µL) Sample 1 20 µL 20 nM 10 nM 6 5.0 Sample 2 20 µL 15 nM 10 nM 6 6.7 Low TE 8.3 3 If you store the library before sequencing, add Tween 20 to 0.1% v/v and store at –20°C short term.
SureSelectXT2 Target Enrichment System for Illumina Multiplexed Sequencing Protocol 6 Reference Reference Information for Kits with Revised Index Configuration (indexing primers in white-capped tubes or blue plate) 76 Reference Information for Kits with Original Index Configuration (indexing primers in clear-capped tubes) 80 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) 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 5500-0130 or 5500-0131. If your kit does not include one of these component kits, see page 80 for kit content and indexing primer information.
Reference Kit Contents 6 The contents of each of the component kits listed in Table 33 are described in the tables below.
6 Reference Kit Contents Table 37 Plate map for Indexes A01 through H12 provided in blue plate in Library Prep kit p/n 5500-0131 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 F02 F03 F04 F0
Reference Nucleotide Sequences of SureSelectXT2 Indexes A01 to H12 6 Nucleotide Sequences of SureSelectXT2 Indexes A01 to H12 Each index is 8 nt in length. Sequencing protocols must be run using the third-read protocol that is also used for 9-bp indexes.
6 Reference 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 5500-0102 or 5500-0103 and Pre-Capture Index Kit p/n 5190-3933, 5190-3936 or 5190-3937.
Reference Kit Contents 6 The contents of each of the component kits listed in Table 39 are described in the tables below.
6 Reference Nucleotide Sequences of SureSelect Pre-Capture Indexes-Original Index Configuration Nucleotide Sequences of SureSelect Pre-Capture Indexes-Original Index Configuration The nucleotide sequence of each SureSelectXT2 Pre-Capture Index provided with the original kit configuration is provided in the tables below. Refer to the sequence information below only if your kit includes p/n 5190-3933 or p/n 5190-3936 and p/n 5190-3937, with indexing primers provided in individual clear-capped tubes.
Reference Nucleotide Sequences of SureSelect Pre-Capture Indexes-Original Index Configuration Table 45 6 SureSelectXT2 Pre-capture Indexes 17-32 Index Number Sequence 17 AAGGTACA 18 ACACAGAA 19 ACAGCAGA 20 ACCTCCAA 21 ACGCTCGA 22 ACGTATCA 23 ACTATGCA 24 AGAGTCAA 25 AGATCGCA 26 AGCAGGAA 27 AGTCACTA 28 ATCCTGTA 29 ATTGAGGA 30 CAACCACA 31 CAAGACTA 32 CAATGGAA SureSelectXT2 Target Enrichment System for Illumina 83
6 Reference Nucleotide Sequences of SureSelect Pre-Capture Indexes-Original Index Configuration Table 46 84 SureSelectXT2 Pre-capture Indexes 33-48 Index Number Sequence 33 CACTTCGA 34 CAGCGTTA 35 CATACCAA 36 CCAGTTCA 37 CCGAAGTA 38 CCGTGAGA 39 CCTCCTGA 40 CGAACTTA 41 CGACTGGA 42 CGCATACA 43 CTCAATGA 44 CTGAGCCA 45 CTGGCATA 46 GAATCTGA 47 GACTAGTA 48 GAGCTGAA SureSelectXT2 Target Enrichment System for Illumina
Reference Nucleotide Sequences of SureSelect Pre-Capture Indexes-Original Index Configuration Table 47 6 SureSelectXT2 Pre-capture Indexes 49-64 Index Number Sequence 49 GATAGACA 50 GCCACATA 51 GCGAGTAA 52 GCTAACGA 53 GCTCGGTA 54 GGAGAACA 55 GGTGCGAA 56 GTACGCAA 57 GTCGTAGA 58 GTCTGTCA 59 GTGTTCTA 60 TAGGATGA 61 TATCAGCA 62 TCCGTCTA 63 TCTTCACA 64 TGAAGAGA SureSelectXT2 Target Enrichment System for Illumina 85
6 Reference Nucleotide Sequences of SureSelect Pre-Capture Indexes-Original Index Configuration Table 48 86 SureSelectXT2 Pre-capture Indexes 65-80 Index Number Sequence 65 TGGAACAA 66 TGGCTTCA 67 TGGTGGTA 68 TTCACGCA 69 AACTCACC 70 AAGAGATC 71 AAGGACAC 72 AATCCGTC 73 AATGTTGC 74 ACACGACC 75 ACAGATTC 76 AGATGTAC 77 AGCACCTC 78 AGCCATGC 79 AGGCTAAC 80 ATAGCGAC SureSelectXT2 Target Enrichment System for Illumina
Reference Nucleotide Sequences of SureSelect Pre-Capture Indexes-Original Index Configuration Table 49 6 SureSelectXT2 Pre-capture Indexes 81-96 Index Number Sequence 81 ATCATTCC 82 ATTGGCTC 83 CAAGGAGC 84 CACCTTAC 85 CCATCCTC 86 CCGACAAC 87 CCTAATCC 88 CCTCTATC 89 CGACACAC 90 CGGATTGC 91 CTAAGGTC 92 GAACAGGC 93 GACAGTGC 94 GAGTTAGC 95 GATGAATC 96 GCCAAGAC SureSelectXT2 Target Enrichment System for Illumina 87
www.agilent.com In This Book This guide contains information to run the SureSelectXT2 Target Enrichment System for Illumina Multiplexed Sequencing protocol featuring pre-capture indexing. Agilent Technologies, Inc.
