Agilent Technologies 6217A Service Manual

Agilent TechnologiesSureSelectXT2 Target Enrichment System for Illumina Multiplexed SequencingFeaturing Pre-Capture Indexing Reagents and ProtocolsPro

10 SureSelectXT2 Target Enrichment System for IlluminaContents

11SureSelectXT2 Target Enrichment System for Illumina Multiplexed Sequencing ProtocolAgilent Technologies1Before You BeginOverview of the Workflow

1 Before You Begin Overview of the Workflow12 SureSelectXT2 Target Enrichment System for IlluminaOverview of the WorkflowThe SureSelectXT2 target enr

Before You Begin 1 Procedural NotesSureSelectXT2 Target Enrichment System for Illumina 13Procedural Notes• This User Guide includes protocols for libr

1 Before You Begin Required Reagents14 SureSelectXT2 Target Enrichment System for IlluminaRequired ReagentsTable 1 Required Reagents for SureSelectXT

Before You Begin 1 Required ReagentsSureSelectXT2 Target Enrichment System for Illumina 15Table 2 SureSelectXT2 Capture LibrariesCapture Library 16 S

1 Before You Begin Required Equipment16 SureSelectXT2 Target Enrichment System for IlluminaRequired EquipmentTable 3 Required Equipment for SureSelec

Before You Begin 1 Optional Reagents and EquipmentSureSelectXT2 Target Enrichment System for Illumina 17Optional Reagents and EquipmentVacuum concentr

1 Before You Begin Optional Reagents and Equipment18 SureSelectXT2 Target Enrichment System for Illumina

19SureSelectXT2 Target Enrichment System for Illumina Multiplexed Sequencing ProtocolAgilent Technologies2Sample Preparation (1 µg DNA Samples)Step 1.

2 SureSelectXT2 Target Enrichment System for IlluminaNotices© Agilent Technologies, Inc. 2015No part of this manual may be reproduced in any form or b

2 Sample Preparation (1 µg DNA Samples) Step 1. Shear DNA20 SureSelectXT2 Target Enrichment System for IlluminaStep 1. Shear DNAMake sure genomic DNA

Sample Preparation (1 µg DNA Samples) 2 Step 1. Shear DNASureSelectXT2 Target Enrichment System for Illumina 216 Secure the microTube in the tube hold

2 Sample Preparation (1 µg DNA Samples) Step 1. Shear DNA22 SureSelectXT2 Target Enrichment System for Illumina9 Transfer each 50-µL sheared DNA samp

Sample Preparation (1 µg DNA Samples) 2 Step 2. Assess quality (optional)SureSelectXT2 Target Enrichment System for Illumina 23Step 2. Assess quality

Figure 2 Analysis of sheared DNA using a DNA 1000 Bioanalyzer assay. 2 Sample Preparation (1 µg DNA Samples) Step 2. Assess quality (optional)24 Sure

Sample Preparation (1 µg DNA Samples) 2 Step 3. Repair the endsSureSelectXT2 Target Enrichment System for Illumina 25Step 3. Repair the endsUse the Su

2 Sample Preparation (1 µg DNA Samples) Step 3. Repair the ends26 SureSelectXT2 Target Enrichment System for Illumina4 Incubate the plate in the Sure

Sample Preparation (1 µg DNA Samples) 2 Step 4. Purify the sample using AMPure XP beadsSureSelectXT2 Target Enrichment System for Illumina 27Step 4. P

2 Sample Preparation (1 µg DNA Samples) Step 4. Purify the sample using AMPure XP beads28 SureSelectXT2 Target Enrichment System for Illumina11 Seal

Sample Preparation (1 µg DNA Samples) 2 Step 5. Adenylate the 3' end of the DNA fragmentsSureSelectXT2 Target Enrichment System for Illumina 29St

SureSelectXT2 Target Enrichment System for Illumina 3Safety NoticesCAUTIONA CAUTION notice denotes a hazard. It calls attention to an operating proced

2 Sample Preparation (1 µg DNA Samples) Step 6. Ligate the pre-capture indexing adaptor30 SureSelectXT2 Target Enrichment System for IlluminaStep 6.

Sample Preparation (1 µg DNA Samples) 2 Step 7. Purify the indexed DNA using AMPure XP beadsSureSelectXT2 Target Enrichment System for Illumina 31Step

2 Sample Preparation (1 µg DNA Samples) Step 8. Amplify the indexed library32 SureSelectXT2 Target Enrichment System for IlluminaStep 8. Amplify the

Sample Preparation (1 µg DNA Samples) 2 Step 8. Amplify the indexed librarySureSelectXT2 Target Enrichment System for Illumina 333 Run the program in

2 Sample Preparation (1 µg DNA Samples) Step 9. Purify the amplified library with AMPure XP beads34 SureSelectXT2 Target Enrichment System for Illumi

Sample Preparation (1 µg DNA Samples) 2 Step 10. Assess quality with the 2100 Bioanalyzer DNA 1000 AssaySureSelectXT2 Target Enrichment System for Ill

2 Sample Preparation (1 µg DNA Samples) Step 10. Assess quality with the 2100 Bioanalyzer DNA 1000 Assay36 SureSelectXT2 Target Enrichment System for

37SureSelectXT2 Target Enrichment System for Illumina Multiplexed Sequencing ProtocolAgilent Technologies3Sample Preparation (100 ng DNA Samples)Step

3 Sample Preparation (100 ng DNA Samples) Step 1. Shear DNA38 SureSelectXT2 Target Enrichment System for IlluminaStep 1. Shear DNAMake sure genomic D

Sample Preparation (100 ng DNA Samples) 3 Step 1. Shear DNASureSelectXT2 Target Enrichment System for Illumina 396 Secure the microTube in the tube ho

4 SureSelectXT2 Target Enrichment System for IlluminaIn this Guide...This guide describes an optimized protocol for Illumina paired-end multiplexed li

3 Sample Preparation (100 ng DNA Samples) Step 1. Shear DNA40 SureSelectXT2 Target Enrichment System for Illumina9 Transfer each 50-µL sheared DNA sa

Sample Preparation (100 ng DNA Samples) 3 Step 2. Assess quality (optional)SureSelectXT2 Target Enrichment System for Illumina 41Step 2. Assess qualit

Figure 4 Analysis of 100 ng sheared DNA sample using a High-Sensitivity DNA Bioanalyzer assay. 3 Sample Preparation (100 ng DNA Samples) Step 2. Ass

Sample Preparation (100 ng DNA Samples) 3 Step 3. Repair the endsSureSelectXT2 Target Enrichment System for Illumina 43Step 3. Repair the endsUse the

3 Sample Preparation (100 ng DNA Samples) Step 3. Repair the ends44 SureSelectXT2 Target Enrichment System for Illumina4 Incubate the plate in the Su

Sample Preparation (100 ng DNA Samples) 3 Step 4. Purify the sample using AMPure XP beadsSureSelectXT2 Target Enrichment System for Illumina 45Step 4.

3 Sample Preparation (100 ng DNA Samples) Step 4. Purify the sample using AMPure XP beads46 SureSelectXT2 Target Enrichment System for Illumina11 Sea

Sample Preparation (100 ng DNA Samples) 3 Step 5. Adenylate the 3' end of the DNA fragmentsSureSelectXT2 Target Enrichment System for Illumina 47

3 Sample Preparation (100 ng DNA Samples) Step 6. Ligate the pre-capture indexing adaptor48 SureSelectXT2 Target Enrichment System for IlluminaStep 6

Sample Preparation (100 ng DNA Samples) 3 Step 7. Purify the indexed DNA using AMPure XP beadsSureSelectXT2 Target Enrichment System for Illumina 49St

SureSelectXT2 Target Enrichment System for Illumina 5What’s New in Version E.0• Support for kits supplied with either of two indexing primer configura

3 Sample Preparation (100 ng DNA Samples) Step 8. Amplify the indexed library50 SureSelectXT2 Target Enrichment System for IlluminaStep 8. Amplify th

Sample Preparation (100 ng DNA Samples) 3 Step 9. Purify the amplified library with AMPure XP beadsSureSelectXT2 Target Enrichment System for Illumina

3 Sample Preparation (100 ng DNA Samples) Step 10. Assess quality with the 2100 Bioanalyzer DNA 1000 Assay52 SureSelectXT2 Target Enrichment System f

Sample Preparation (100 ng DNA Samples) 3 Step 10. Assess quality with the 2100 Bioanalyzer DNA 1000 AssaySureSelectXT2 Target Enrichment System for I

3 Sample Preparation (100 ng DNA Samples) Step 10. Assess quality with the 2100 Bioanalyzer DNA 1000 Assay54 SureSelectXT2 Target Enrichment System f

55SureSelectXT2 Target Enrichment System for Illumina Multiplexed Sequencing ProtocolAgilent Technologies4HybridizationStep 1. Pool indexed DNA sample

4 Hybridization Step 1. Pool indexed DNA samples for hybridization56 SureSelectXT2 Target Enrichment System for IlluminaStep 1. Pool indexed DNA samp

Hybridization 4 Step 2. Hybridize gDNA library pools to the SureSelect Capture LibrarySureSelectXT2 Target Enrichment System for Illumina 57Step 2. Hy

4 Hybridization Step 2. Hybridize gDNA library pools to the SureSelect Capture Library58 SureSelectXT2 Target Enrichment System for Illumina4 In a PC

Hybridization 4 Step 3. Prepare streptavidin-coated magnetic beadsSureSelectXT2 Target Enrichment System for Illumina 59Step 3. Prepare streptavidin-c

6 SureSelectXT2 Target Enrichment System for Illumina

4 Hybridization Step 4. Capture the hybridized DNA using streptavidin beads60 SureSelectXT2 Target Enrichment System for IlluminaStep 4. Capture the

Hybridization 4 Step 4. Capture the hybridized DNA using streptavidin beadsSureSelectXT2 Target Enrichment System for Illumina 6110 Wash the beads wit

4 Hybridization Step 4. Capture the hybridized DNA using streptavidin beads62 SureSelectXT2 Target Enrichment System for Illumina

63SureSelectXT2 Target Enrichment System for Illumina Multiplexed Sequencing ProtocolAgilent Technologies5Post-Capture Sample Processing for Multiplex

5 Post-Capture Sample Processing for Multiplexed Sequencing Step 1. Amplify the captured libraries64 SureSelectXT2 Target Enrichment System for Illum

Post-Capture Sample Processing for Multiplexed Sequencing 5 Step 1. Amplify the captured librariesSureSelectXT2 Target Enrichment System for Illumina

5 Post-Capture Sample Processing for Multiplexed Sequencing Step 2. Purify the amplified captured libraries using AMPure XP beads66 SureSelectXT2 Tar

Post-Capture Sample Processing for Multiplexed Sequencing 5 Step 2. Purify the amplified captured libraries using AMPure XP beadsSureSelectXT2 Target

5 Post-Capture Sample Processing for Multiplexed Sequencing Step 3. Assess quality with the 2100 Bioanalyzer High Sensitivity DNA assay68 SureSelectX

Post-Capture Sample Processing for Multiplexed Sequencing 5 Step 3. Assess quality with the 2100 Bioanalyzer High Sensitivity DNA assaySureSelectXT2 T

SureSelectXT2 Target Enrichment System for Illumina 7Content1 Before You Begin 11Overview of the Workflow 12Procedural Notes 13Safet

5 Post-Capture Sample Processing for Multiplexed Sequencing Step 4. Prepare samples for multiplexed sequencing70 SureSelectXT2 Target Enrichment Syst

Post-Capture Sample Processing for Multiplexed Sequencing 5 Step 4. Prepare samples for multiplexed sequencingSureSelectXT2 Target Enrichment System f

5 Post-Capture Sample Processing for Multiplexed Sequencing Step 5. Optional: Quantify captured library pools by QPCR72 SureSelectXT2 Target Enrichme

Post-Capture Sample Processing for Multiplexed Sequencing 5 Step 6. Optional: Pool captured libraries for sequencingSureSelectXT2 Target Enrichment Sy

5 Post-Capture Sample Processing for Multiplexed Sequencing Step 6. Optional: Pool captured libraries for sequencing74 SureSelectXT2 Target Enrichmen

75SureSelectXT2 Target Enrichment System for Illumina Multiplexed Sequencing ProtocolAgilent Technologies6ReferenceReference Information for Kits with

6 Reference Reference Information for Kits with Revised Index Configuration (indexing primers in white-capped tubes or blue plate)76 SureSelectXT2 Ta

Reference 6 Kit ContentsSureSelectXT2 Target Enrichment System for Illumina 77The contents of each of the component kits listed in Table 33 are descri

6 Reference Kit Contents78 SureSelectXT2 Target Enrichment System for IlluminaTable 37 Plate map for Indexes A01 through H12 provided in blue plate i

Reference 6 Nucleotide Sequences of SureSelectXT2 Indexes A01 to H12SureSelectXT2 Target Enrichment System for Illumina 79Nucleotide Sequences of Sure

8 SureSelectXT2 Target Enrichment System for IlluminaContents 3 Sample Preparation (100 ng DNA Samples) 37Step 1. Shear DNA 38Step 2. Asse

6 Reference Reference Information for Kits with Original Index Configuration (indexing primers in clear-capped tubes)80 SureSelectXT2 Target Enrichme

Reference 6 Kit ContentsSureSelectXT2 Target Enrichment System for Illumina 81The contents of each of the component kits listed in Table 39 are descri

6 Reference Nucleotide Sequences of SureSelect Pre-Capture Indexes-Original Index Configuration82 SureSelectXT2 Target Enrichment System for Illumina

Reference 6 Nucleotide Sequences of SureSelect Pre-Capture Indexes-Original Index ConfigurationSureSelectXT2 Target Enrichment System for Illumina 83T

6 Reference Nucleotide Sequences of SureSelect Pre-Capture Indexes-Original Index Configuration84 SureSelectXT2 Target Enrichment System for Illumina

Reference 6 Nucleotide Sequences of SureSelect Pre-Capture Indexes-Original Index ConfigurationSureSelectXT2 Target Enrichment System for Illumina 85T

6 Reference Nucleotide Sequences of SureSelect Pre-Capture Indexes-Original Index Configuration86 SureSelectXT2 Target Enrichment System for Illumina

Reference 6 Nucleotide Sequences of SureSelect Pre-Capture Indexes-Original Index ConfigurationSureSelectXT2 Target Enrichment System for Illumina 87T

www.agilent.comAgilent TechnologiesIn This BookThis guide contains information to run the SureSelectXT2 Target Enrichment System for Illumina Multiple

Contents SureSelectXT2 Target Enrichment System for Illumina 9Reference Information for Kits with Original Index Configuration (indexing primers in cl