Agilent XT HS2 DNA User Manual

Download

SureSelect XT HS2 DNA System

DNA Library Preparation and Target Enrichment for the Illumina Platform

Protocol

Version D0, April 2021

SureSelect platform manufactured with Agilent SurePrint Technology

For Research Use Only. Not for use in diagnostic procedures.

Agilent Technologies

Notices

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.

Manual Part Number

G9983-90000

Edition

Version D0, April 2021

Printed in USA

Agilent Technologies, Inc. 5301 Stevens Creek Blvd Santa Clara, CA 95051 USA

Acknowledgment

Technical Support

For US and Canada

Call (800) 227-9770 (option 3,4,4) Or send an e-mail to:

ngs.support@agilent.com

For all other regions

Agilent’s world-wide Sales and Support Center contact details for your location can be obtained at

www.agilent.com/en/contact-us/page.

Warranty

The material contained in this document is provided “as is,” and is subject to being changed, without notice, in future editions. Further, to the maximum extent permitted by applicable law, Agilent disclaims all warranties, either express or implied, with regard to this manual and any information contained herein, including but not limited to the implied warranties of merchantability and fitness for a particular purpose. Agilent shall not be liable for errors or for incidental or consequential damages in connection 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 document 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 accordance with the terms of such license.

Restricted Rights Legend

U.S. Government Restricted Rights. Software and technical data rights granted to the federal government include only those rights customarily provided to end user customers. Agilent provides this customary commercial license in Software and technical data pursuant to FAR 12.211 (Technical Data) and 12.212 (Computer Software) and, for the Department of Defense, DFARS

252.227-7015 (Technical Data - Commercial Items) and DFARS 227.7202-3 (Rights in Commercial Computer Software or Computer Software Documentation).

Notice to Purchaser

This product is provided under an agreement between Bio-Rad Laboratories and Agilent Technologies Inc., and the manufacture, use, sale or import of this product is subject to U.S. Pat. No. 6,627,424 and EP Pat. No.1 283 875 81, owned by Bio-Rad Laboratories, Inc. Purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in PCR (but not including real-time PCR) in the Research Field (including all Applied Research Fields, including but not limited to forensics, animal testing, and food testing) and in real-time PCR in the Diagnostics and Prognostics Fields. No rights are granted for use of this product for real-time PCR in the Research Field, including all Applied Research Fields (including but not limited to forensics, animal testing and food testing).

2 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Safety Notices

CAUTION

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.

WARNING

A WARNING 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 personal injury or death. Do not proceed beyond a WARNING notice until the indicated conditions are fully understood and met.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 3

In this Guide...

This guide provides an optimized protocol for preparation of target- enriched Illumina paired- end multiplexed sequencing libraries using the SureSelect XT HS2 DNA system.

1 Before You Begin

This chapter contains information that you should read and understand before you start an experiment.

2 Preparation and Fragmentation of Input DNA

This chapter describes the steps to prepare and fragment gDNA samples, using either mechanical shearing or enzymatic fragmentation, prior to library preparation.

3 Library Preparation

This chapter describes the steps to prepare dual- indexed, molecular- barcoded gDNA sequencing libraries for target enrichment.

4 Hybridization and Capture

This chapter describes the steps to hybridize and capture the prepared DNA library using a SureSelect or ClearSeq Probe Capture Library.

5 Post-Capture Sample Processing for Multiplexed Sequencing

This chapter describes the steps for post- capture amplification and guidelines for sequencing sample preparation.

6 Appendix: Using FFPE-derived DNA Samples

This chapter describes the protocol modifications for gDNA isolated from FFPE samples.

7 Reference

This chapter contains reference information, including component kit contents and index sequences.

4 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

What’s New in Version D0

• Support for SureSelect XT HS Human All Exon V8 Probe.

• Updates to instructions in the “Hybridization and

• New footnotes to Table 11 on page 25 and Table 14 on

• Update to Figure 4 on page 46 and associated text on

• Update to downstream sequencing support information in

• Update to description of flat strip caps in Table 8 on

See Table 3 on page 14 for ordering information and see

Table 28 on page 51 for the hybridization thermal cycling

program recommended for this probe with the SureSelect XT HS2 DNA system. Also see troubleshooting information on page 101 and updates to the Quick Reference Protocol on page 103.

Capture” chapter on page 49 through page 56. Updates

include provision of two separate hybridization thermal cycler programs (Table 28 and Table 29 on page 51) and related changes throughout the chapter.

page 28 on FFPE sample initial fragment size impacts on library fragment size distribution.

page 47.

Table 41 on page 71.

page 19.

What’s New in Version C1

• Updates to index pair sequence tables (page 87 through

page 94) including updates to P5 index platform

descriptions and correction of well position typographical errors

• Updates to downstream sequencing support information (see Table 41 on page 71 and Note on page 86)

• Updates to molecular barcode and associated dark base information (see Figure 8 on page 70) and instructions for processing using the Agilent Genomics NextGen Toolkit (see page 72)

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 5

• Addition of hybridization temperature considerations for

What’s New in Version C0

• Support for revised SureSelect custom probe products,

• Updates to DNA library quantitation/qualification

• Updates to thermal cycler and plasticware

• Updates to Materials Required including updated

• Updates to Optional Materials in Table 8 on page 19,

• Updates to “SureSelect XT HS2 Index Primer Pair

probes designed for use with the SureSelect XT system (see Table 28 on page 51)

produced using an updated manufacturing process beginning August, 2020 (see Table 3 on page 14). Custom probes produced using the legacy manufacturing process are also fully supported by the protocols in this document. Probe information was reorganized (see

Table 3 on page 14), and probe nomenclature throughout

document was updated.

guidelines including increased support for the Agilent’s Fragment Analyzer platform and streamlined sample analysis guidelines (see Table 6 on page 17 and see

page 44 through page 46 and page 65 through page 67).

recommendations and usage instructions (see Caution and Table 5 on page 16 and see Note on page 34).

ordering information for Dynabeads MyOne Streptavidin T1 beads (Table 4 on page 15) and for Eppendorf ThermoMixer C and Qubit Fluorometer (Table 5 on page 16).

including removal of ethylene glycol supplier information (see page 25 for related update to DNA shearing set up instructions).

Information” section (page 86 through page 97) including

addition of P5 primer sequences oriented for the NextSeq, HiSeq 4000 and HiSeq 3000 platforms and reorganization of section content.

6 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Content

1 Before You Begin 9

Overview of the Workflow 10

Procedural Notes 12

Safety Notes 12

Materials Required 13

Optional Materials 19

2 Preparation and Fragmentation of Input DNA 21

Step 1. Prepare and analyze quality of genomic DNA samples 22

Preparation of high-quality gDNA from fresh biological samples 22 Preparation and qualification of gDNA from FFPE samples 22

Step 2. Fragment the DNA 25

Method 1: Mechanical DNA Shearing using Covaris 25 Method 2: Enzymatic DNA Fragmentation 28

3 Library Preparation 31

Step 1. Prepare the Ligation master mix 33

Step 2. Repair and dA-Tail the DNA ends 34

Step 3. Ligate the molecular-barcoded adaptor 36

Step 4. Purify the sample using AMPure XP beads 37

Step 5. Amplify the adaptor-ligated library 39

Step 6. Purify the amplified library with AMPure XP beads 42

Step 7. Assess quality and quantity 44

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 7

Contents

4 Hybridization and Capture 49

Step 1. Hybridize DNA libraries to the probe 50

Step 2. Prepare streptavidin-coated magnetic beads 55

Step 3. Capture the hybridized DNA using streptavidin-coated beads 56

5 Post-Capture Sample Processing for Multiplexed Sequencing 59

Step 1. Amplify the captured libraries 60

Step 2. Purify the amplified captured libraries using AMPure XP beads 63

Step 3. Assess sequencing library DNA quantity and quality 65

Step 4. Pool samples for multiplexed sequencing 68

Step 5. Prepare sequencing samples 70

Step 6. Do the sequencing run and analyze the data 72

Sequence analysis resources 77

6 Appendix: Using FFPE-derived DNA Samples 79

Protocol modifications for FFPE Samples 80

Methods for FFPE Sample Qualification 80

Sequencing Output Recommendations for FFPE Samples 81

7 Reference 83

Kit Contents 84

SureSelect XT HS2 Index Primer Pair Information 86

Troubleshooting Guide 98

Quick Reference Protocol 102

8 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

SureSelect XT HS2 DNA System Protocol

1 Before You Begin

Overview of the Workflow 10 Procedural Notes 12 Safety Notes 12 Materials Required 13 Optional Materials 19

Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an experiment.

NOTE

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 guarantees performance and provides technical support for the SureSelect reagents required for this workflow only when used as directed in this Protocol.

Agilent Technologies

1 Before You Begin

Overview of the Workflow

The SureSelect XT HS2 DNA workflow is summarized in Figure 1. The estimated time requirements for each step are summarized in Table 1.

Figure 1 Overall target-enriched sequencing sample preparation workflow.

10 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Overview of the Workflow

Table 1 Estimated time requirements (up to 16 sample run size)

Step Time

Library Preparation 3.5 hours

Hybridization and Capture 3.5 hours

Post-capture amplification 1 hour

Before You Begin 1

QC using Bioanalyzer or TapeStation platform and sample pooling

1.5 hours

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 11

1 Before You Begin

Procedural Notes

• To prevent contamination of reagents by nucleases, always wear

powder- free laboratory gloves and use dedicated solutions and pipettors with nuclease- free aerosol- resistant tips.

• 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. 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, or equivalent.

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.

• For each protocol step that requires removal of tube cap strips, reseal

the tubes with a fresh strip of 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.

• In general, follow Biosafety Level 1 (BSL1) safety rules.

• 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

12 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

• Wear appropriate personal protective equipment (PPE) when working in the

laboratory.

Before You Begin 1

Materials Required

Materials required to complete the SureSelect XT HS2 protocol will vary based on the following considerations:

• SureSelect XT HS2 DNA Reagent Kit format preference, where some

options include ancillary reagent modules

• DNA sample type: high- quality gDNA derived from fresh/fresh- frozen

samples vs. FFPE- derived gDNA samples

• DNA fragmentation method used in workflow: mechanical

(Covaris- mediated) shearing vs. enzymatic fragmentation

To determine the materials required for your unique needs, first select the preferred kit format from Table 2 below and a compatible target enrichment probe from Table 3. Then refer to Table 4 through Table 7 for additional materials needed to complete the protocols using the selected kit format/DNA sample type/fragmentation method.

Table 2 SureSelect XT HS2 DNA Reagent Kit Varieties

Description Kit Part Number

16 Reaction Kit

SureSelect XT HS2 DNA Reagent Kit G9981A (with Index Pairs 1–16) G9983A (with Index Pairs 1–96)

Reagent Kits with additional component modules

SureSelect XT HS2 DNA Starter Kit

Includes the following modules:

SureSelect XT HS2 DNA Reagent Kit

SureSelect Enzymatic Fragmentation Kit

SureSelect DNA AMPure

SureSelect Streptavidin Beads

SureSelect XT HS2 DNA Reagent Kit with

AMPure

XP/Streptavidin Beads

XP Beads

‡

G9982A (with Index Pairs 1–16) Not applicable

Not applicable G9984A (with Index Pairs 1–96)

96 Reaction Kit

G9983B (with Index Pairs 97–192) G9983C (with Index Pairs 193–288) G9983D (with Index Pairs 289–384)

G9984B (with Index Pairs 97–192) G9984C (with Index Pairs 193–288) G9984D (with Index Pairs 289–384)

†

* 16-reaction kits contain enough reagents for 2 runs containing 8 samples per run.

† 96-reaction kits contain enough reagents for 4 runs containing 24 samples per run.

‡ AMPure, Beckman, and Beckman Coulter are trademarks or registered trademarks of Beckman Coulter, Inc.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 13

1 Before You Begin

Materials Required

Table 3 Compatible Probes

Probe Capture Library 16 Reactions 96 Reactions

Pre-designed Probes

SureSelect XT HS Human All Exon V8 5191-6873 5191-6874

SSel XT HS and XT Low Input Human All Exon V7 5191-4028 5191-4029

SureSelect XT Clinical Research Exome V2 5190-9491 5190-9492

SureSelect XT Mouse All Exon 5190-4641 5190-4642

ClearSeq Comprehensive Cancer XT 5190-8011 5190-8012

ClearSeq Inherited Disease XT 5190-7518 5190-7519

Custom Probes

SureSelect Custom Tier1 1–499 kb Please visit the SureDesign website to design

SureSelect Custom Tier2 0.5 –2.9 Mb

SureSelect Custom Tier3 3 –5.9 Mb

SureSelect Custom Tier4 6 –11.9 Mb

SureSelect Custom Tier5 12–24 Mb

Pre-designed Probes customized with additional Plus custom content

SSel XT HS and XT Low Input Human All Exon V7 Plus 1

SSel XT HS and XT Low Input Human All Exon V7 Plus 2

SureSelect XT Clinical Research Exome V2 Plus 1

SureSelect XT Clinical Research Exome V2 Plus 2

ClearSeq Comprehensive Cancer Plus XT

ClearSeq Inherited Disease Plus XT

Custom SureSelect probes and obtain ordering information. Contact the SureSelect support team (see page 2) or your local representative if you need assistance. Custom probes are also available in a 480 Reaction package.

Please visit the SureDesign website to design the customized Plus content and obtain ordering information. Contact the SureSelect support team (see page 2) or your local representative if you need assistance.

* Custom Probes designed August 2020 or later are produced using an updated manufacturing process; design-size

Tier is shown on labeling for these products. Custom Probes designed and ordered prior to August 2020 may be reordered, with these probes produced using the legacy manufacturing process; design-size Tier is not shown on labeling for the legacy-process products. Custom Probes of both categories use the same optimized target enrichment protocols detailed in this publication.

14 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Table 4 Required Reagents--All Sample Types/Fragmentation Methods

Description Vendor and Part Number Notes

Before You Begin 1

Materials Required

1X Low TE Buffer Thermo Fisher Scientific p/n

12090-015, or equivalent

100% Ethanol (Ethyl Alcohol, 200 proof) Millipore p/n EX0276 —

Qubit BR dsDNA Assay Kit 100 assays 500 assays

Nuclease-free Water Thermo Fisher Scientific p/n

AMPure 5 ml 60 ml 450 ml

Dynabeads MyOne Streptavidin T1 2 ml 10 ml 50 ml

XP Kit

Thermo Fisher Scientific p/n Q32850 p/n Q32853

AM9930

Beckman Coulter Genomics

p/n A63880 p/n A63881 p/n A63882

Thermo Fisher Scientific p/n 65601 p/n 65602 p/n 65604D

10 mM Tris-HCl, pH 7.5-8.0, 0.1 mM EDTA

—

Water should not be DEPC-treated

Separate purchase not required for

use with SureSelect XT HS2 DNA Reagent Kits that include SureSelect DNA AMPure Beads and SureSelect Streptavidin Beads (Agilent p/n G9982A, G9984A, G9984B, G9984C, or G9984D)

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 15

1 Before You Begin

Materials Required

CAUTION

Sample volumes exceed 0.2 ml in certain steps of this protocol. Make sure that the plasticware

used with the selected thermal cycler holds 0.25 ml per well.

Table 5 Required Equipment--All Sample Types/Fragmentation Methods

Description Vendor and Part Number

Thermal Cycler with 96-well, 0.2 ml block Various suppliers

Plasticware compatible with the selected thermal cycler:

96-well plates or 8-well strip tubes Tube cap strips, domed

Nucleic acid analysis system (instrument and consumables) Select one system from Table 6 on page 17

Qubit Fluorometer Thermo Fisher Scientific p/n Q33238

Qubit Assay Tubes Thermo Fisher Scientific p/n Q32856

DNA LoBind Tubes, 1.5-ml PCR clean, 250 pieces Eppendorf p/n 022431021 or equivalent

Microcentrifuge Eppendorf microcentrifuge, model 5417C or equivalent

Plate or strip tube centrifuge Labnet International MPS1000 Mini Plate Spinner, p/n

96-well plate mixer Eppendorf ThermoMixer C, p/n 5382000023 and Eppendorf

Magnetic separator Thermo Fisher Scientific p/n 12331D or equivalent

Multichannel pipette Rainin Pipet-Lite Multi Pipette or equivalent

Single channel pipettes (10-, 20-, 200-, and 1000-µl capacity) Rainin Pipet-Lite Pipettes or equivalent

Sterile, nuclease-free aerosol barrier pipette tips general laboratory supplier

Vortex mixer general laboratory supplier

Ice bucket general laboratory supplier

Powder-free gloves general laboratory supplier

Consult the thermal cycler manufacturer’s

recommendations

C1000 (requires adapter, p/n C1000-ADAPT, for use with strip tubes) or equivalent

SmartBlock 96 PCR, p/n 5306000006, or equivalent

* Select a magnetic separator configured to collect magnetic particles on one side of each well. Do not use a magnetic sep-

arator configured to collect the particles in a ring formation.

16 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Materials Required

Table 6 Nucleic Acid Analysis Platform Options--Select One

Analysis System Vendor and Part Number Information

Agilent 4200/4150 TapeStation Instrument

Consumables:

96-well sample plates

96-well plate foil seals

8-well tube strips

8-well tube strip caps

D1000 ScreenTape

D1000 Reagents

High Sensitivity D1000 ScreenTape

High Sensitivity D1000 Reagents

Agilent 2100 Bioanalyzer Instrument

Agilent 2100 Expert SW Laptop Bundle (optional)

Consumables:

DNA 1000 Kit

High Sensitivity DNA Kit

Agilent 5200/5300/5400 Fragment Analyzer Instrument

Consumables:

NGS Fragment Kit (1-6000 bp)

HS NGS Fragment Kit (1-6000 bp)

Agilent p/n G2991AA/G2992AA

p/n 5042-8502

p/n 5067-5154

p/n 401428

p/n 401425

p/n 5067-5582

p/n 5067-5583

p/n 5067-5584

p/n 5067-5585

p/n G2939BA

p/n G2953CA

p/n 5067-1504

p/n 5067-4626

Agilent p/n M5310AA/M5311AA/M5312AA

p/n DNF-473-0500

p/n DNF-474-0500

Before You Begin 1

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 17

1 Before You Begin

Materials Required

Table 7 Additional Required Materials based on DNA Sample Type/Fragmentation Method

Description Vendor and Part Number Notes

Required for preparation of high-quality DNA samples (not required for FFPE DNA sample preparation)

High-quality gDNA purification system, for example:

QIAamp DNA Mini Kit 50 Samples 250 Samples

Required for preparation of FFPE DNA samples (not required for high-quality DNA sample preparation)

QIAamp DNA FFPE Tissue Kit, 50 Samples Qiagen p/n 56404 —

Deparaffinization Solution Qiagen p/n 19093 —

FFPE DNA integrity assessment system:

Agilent NGS FFPE QC Kit 16 reactions 96 reactions

TapeStation Genomic DNA Analysis Consumables: Genomic DNA ScreenTape Genomic DNA Reagents

Required for mechanical shearing of DNA samples (not required for workflows with enzymatic fragmentation)

Covaris Sample Preparation System Covaris model E220

Covaris microTUBE sample holders Covaris p/n 520045

Required for enzymatic fragmentation of DNA samples (not required for workflows with mechanical shearing)

SureSelect Enzymatic Fragmentation Kit Agilent

Qiagen p/n 51304 p/n 51306

Agilent p/n G9700A p/n G9700B

Agilent p/n 5067-5365 p/n 5067-5366

p/n 5191-4079 (16 reactions) p/n 5191-4080 (96 reactions)

—

Not required for use with SureSelect XT HS2 DNA Starter Kit (Agilent p/n G9982A)

18 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Before You Begin 1

Optional Materials

Table 8 Supplier Information for Optional Materials

Description Vendor and Part Number Purpose

Tween 20 Sigma-Aldrich p/n P9416-50ML Sequencing library

storage (see page 69)

8× flat strip caps Consult the thermal cycler

manufacturer’s recommendations

MicroAmp Clear Adhesive Film Thermo Fisher Scientific p/n 4311971 Improved sealing for flat

PlateLoc Thermal Microplate Sealer with Small Hotplate and Peelable Aluminum Seal for PlateLoc Sealer

* Flat strip caps may be used instead of domed strip caps for protocol steps performed outside of the hybridization/capture

segment of the protocol. Adhesive film may be applied over the flat strip caps for improved sealing properties.

Please contact the SureSelect support team (see page 2) or your local representative for ordering information

Sealing wells for protocol steps outside of hybridization/capture

strip caps*

Sealing wells for protocol steps performed inside or outside of the thermal cycler

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 19

1 Before You Begin

Optional Materials

20 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

SureSelect XT HS2 DNA System Protocol

2 Preparation and Fragmentation of Input DNA

Step 1. Prepare and analyze quality of genomic DNA samples 22

Preparation of high-quality gDNA from fresh biological samples 22 Preparation and qualification of gDNA from FFPE samples 22

Step 2. Fragment the DNA 25

Method 1: Mechanical DNA Shearing using Covaris 25 Method 2: Enzymatic DNA Fragmentation 28

This chapter describes the steps to prepare, quantify, qualify, and fragment input DNA samples prior to SureSelect XT HS2 library preparation and target enrichment. Protocols are provided for two alternative methods of DNA fragmentation–mechanical shearing or enzymatic DNA fragmentation.

The library preparation protocol is compatible with both high- quality gDNA prepared from fresh or fresh- frozen samples and lower- quality DNA prepared from FFPE samples. Modifications required for FFPE samples are included throughout the protocol steps. For a summary of modifications for FFPE samples see Chapter 6, “Appendix: Using FFPE- derived DNA Samples” on page 79.

The library preparation protocol requires 10 ng to 200 ng of input DNA, with adjustments to DNA input amount or quantification method required for some FFPE samples. For optimal sequencing results, use the maximum amount of input DNA available within the recommended range.

Agilent Technologies

2 Preparation and Fragmentation of Input DNA

Step 1. Prepare and analyze quality of genomic DNA samples

Preparation of high-quality gDNA from fresh biological samples

1 Prepare high- quality gDNA using a suitable purification system, such as

Qiagen’s QIAamp DNA Mini Kit, following the manufacturer’s protocol.

NOTE

Make sure genomic DNA samples are of high quality with an OD 260/280 ratio ranging from 1.8 to 2.0.

2 Use the Qubit BR dsDNA Assay Kit to determine the concentration of

each gDNA sample. Follow the manufacturer’s instructions for the instrument and assay kit.

Additional qualification of DNA samples is not required for DNA derived from fresh biological samples. Proceed to “Step 2. Fragment the DNA” on page 25.

Preparation and qualification of gDNA from FFPE samples

1 Prepare gDNA from FFPE tissue sections using Qiagen’s QIAamp DNA

FFPE Tissue Kit and Qiagen’s Deparaffinization Solution, following the manufacturer’s protocol. Elute the final gDNA samples from the MinElute column in two rounds, using 30 µl Buffer ATE in each round, for a final elution volume of approximately 60 µl.

If tissue lysis appears incomplete after one hour of digestion with Proteinase K, add an additional 10 µl of Proteinase K and continue incubating at 56°C, with periodic mixing, for up to three hours.

Store the gDNA samples on ice for same- day library preparation, or at –20°C for later processing.

2 Assess the quality (DNA integrity) for each FFPE DNA sample using one

of the methods below.

22 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Preparation and Fragmentation of Input DNA 2

Preparation and qualification of gDNA from FFPE samples

Option 1: Qualification using the Agilent NGS FFPE QC Kit (Recommended Method)

The Agilent NGS FFPE QC Kit provides a qPCR- based assay for DNA sample integrity determination. Results include a Cq DNA integrity score and the precise quantity of amplifiable DNA in the sample, allowing direct normalization of DNA input for each sample. DNA input recommendations based on Cq scores for individual samples are summarized in Table 9.

a Use the Qubit BR dsDNA Assay Kit to determine the concentration of

each gDNA sample. Follow the manufacturer’s instructions for the instrument and assay kit.

b Remove a 1 µl aliquot of the FFPE gDNA sample for analysis using

the Agilent NGS FFPE QC Kit to determine the Cq DNA integrity score. See the kit user manual at www.agilent.com for more information.

c For all samples with Cq DNA integrity score

Qubit- based gDNA concentration determined in step a, above, to determine volume of input DNA needed for the protocol.

d For all samples with Cq DNA integrity score >1, use the

qPCR- based concentration of amplifiable gDNA, reported by the Agilent NGS FFPE QC Kit results, to determine amounts of input DNA for the protocol.

≤1, use the

Table 9 SureSelect XT HS2 DNA input modifications based on Cq DNA integrity score

Protocol Parameter non-FFPE Samples FFPE Samples

ΔΔCq≤1

DNA input for Library Preparation

* FFPE samples with Cq scores 1 should be treated like non-FFPE samples for DNA input amount determinations. For sam-

ples of this type, make sure to use the DNA concentration determined by the Qubit Assay, instead of the concentration determined by qPCR, to calculate the volume required for 10–200 ng DNA.

10 ng to 200 ng DNA, based on Qubit Assay

ΔΔCq >1

10 ng to 200 ng of amplifiable DNA, based on qPCR quantification

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 23

2 Preparation and Fragmentation of Input DNA

Preparation and qualification of gDNA from FFPE samples

Option 2: Qualification using Agilent’s Genomic DNA ScreenTape assay DIN score

Agilent’s Genomic DNA ScreenTape assay, used in conjunction with Agilent’s TapeStation, provides a quantitative electrophoretic assay for DNA sample integrity determination. This assay reports a DNA Integrity Number (DIN) score for each sample which is used to estimate the appropriate normalization of DNA input required for low- integrity DNA samples.

a Use the Qubit BR dsDNA Assay Kit to determine the concentration of

each gDNA sample. Follow the manufacturer’s instructions for the instrument and assay kit.

b Remove a 1 µl aliquot of the FFPE gDNA sample and analyze using

the Genomic DNA ScreenTape assay. See the user manual at

www.agilent.com for more information.

c Using the DIN score reported for each sample in the Genomic DNA

ScreenTape assay, consult Table 10 to determine the recommended amount of input DNA for the sample.

Table 10 SureSelect XT HS2 DNA input modifications based on DNA Integrity Number (DIN) score

Protocol Parameter

DNA input for Library Preparation

* FFPE samples with DIN>8 should be treated like non-FFPE samples for DNA input amount determinations.

non-FFPE Samples

10 ng to 200 ng DNA, quantified by Qubit Assay

DIN > 8

10 ng to 200 ng DNA, quantified by Qubit Assay

FFPE Samples

DIN 3–8 DIN<3

Use at least 15 ng for more intact samples and at least 40 ng for less intact samples. Use the maximum amount of DNA available, up to 200 ng, for all samples. Quantify by Qubit Assay.

Use at least 50 ng for more intact samples and at least 100 ng for the least intact samples. Use the maximum amount of DNA available, up to 200 ng, for all samples. Quantify by Qubit Assay.

24 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Step 2. Fragment the DNA

Method 1: Mechanical DNA Shearing using Covaris

In this step, gDNA samples are sheared using conditions optimized for either high- quality or FFPE DNA in a 50- µl shearing volume.

The target fragment size and corresponding shearing conditions may vary for workflows using different NGS read lengths. See Table 11 for guidelines. Complete shearing instructions are provided on page 26.

Table 11 Covaris shearing duration based on NGS read length

Preparation and Fragmentation of Input DNA 2

Step 2. Fragment the DNA

NOTE

NGS read length requirement

2 ×100 reads 150 to 200 bp 2 × 120 seconds 240 seconds

2 ×150 reads 180 to 250 bp 2 × 60 seconds 240 seconds

* For FFPE DNA samples, initial DNA fragment size may impact the post-shear fragment size

distribution, resulting in fragment sizes shorter than the target ranges listed in this table. All FFPE samples should be sheared for 240 seconds to generate fragment ends suitable for library construction. Libraries prepared from FFPE samples should be analyzed using an NGS read length suitable for the final library fragment size distribution.

Target fragment size

Shearing duration for high-quality DNA samples

Shearing duration for FFPE DNA samples

Shearing protocols have been optimized using a Covaris model E220 instrument and the 130-l Covaris microTUBE. Consult the manufacturer’s recommendations for use of other Covaris instruments or sample holders to achieve the desired target DNA fragment size.

1 Set up the Covaris E220 instrument. Refer to the instrument user guide.

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.

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 according to the manufacturer’s recommendations, typically 30–60 minutes.

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. Consult the manufacturer’s recommendations for addition of coolant fluids to prevent freezing.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 25

2 Preparation and Fragmentation of Input DNA

Method 1: Mechanical DNA Shearing using Covaris

2 Prepare the DNA samples for the run by diluting 10–200 ng of each

gDNA sample with 1X Low TE Buffer (10 mM Tris-

HCl, pH 7.5- 8.0,

0.1 mM EDTA) to a final volume of 50 µl. Vortex well to mix, then spin briefly to collect the liquid. Keep the samples on ice.

NOTE

Do not dilute samples to be sheared using water. Shearing samples in water reduces the

overall library preparation yield and complexity.

3 Complete the DNA shearing steps below for each of the gDNA samples.

a Transfer the 50-

µl DNA sample into a Covaris microTUBE, using a tapered pipette tip to slowly transfer the sample through the pre- split septum of the cap.

b Spin the microTUBE for 30 seconds to collect the liquid and to

remove any bubbles from the bottom of the tube.

c Secure the microTUBE in the tube holder and shear the DNA with

the settings in Table 12.

Table 12 Shear settings for Covaris E-series instrument (SonoLab software v7 or later)

Setting High-quality DNA

for 2 × 100 read NGS

Duty Factor 10% 10% 10%

Peak Incident Power (PIP) 175 175 175

Cycles per Burst 200 200 200

Treatment Time 2 × 120 seconds 2 × 60 seconds 240 seconds

Bath Temperature 2° to 8° C 2° to 8° C 2° to 8° C

High-quality DNA for 2 × 150 read NGS

FFPE DNA (2 × 100 or 2 × 150 read NGS)

d Use the steps below for two- round shearing of high- quality DNA

samples only:

•Shear for 120 or 60 seconds (see Table 12)

•Spin the microTUBE for 10 seconds

•Vortex the microTUBE at high speed for 5 seconds

•Spin the microTUBE for 10 seconds

•Shear for additional 120 or 60 seconds

•Spin the microTUBE for 10 seconds

•Vortex the microTUBE at high speed for 5 seconds

•Spin the microTUBE for 10 seconds

26 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

NOTE

Preparation and Fragmentation of Input DNA 2

Method 1: Mechanical DNA Shearing using Covaris

e After completing the shearing step(s), put the Covaris microTUBE

back into the loading and unloading station.

f While keeping the snap- cap on, insert a pipette tip through the

pre- split septum, then slowly remove the sheared DNA.

g Transfer the sheared DNA sample (approximately 50 µl) to a 96-

plate or strip tube sample well. Keep the samples on ice.

h After transferring the DNA sample, spin the microTUBE briefly to

collect any residual sample volume. Transfer any additional collected liquid to the sample well used in step g.

It is important to avoid loss of input DNA at this step, especially for low-abundance DNA samples. Visually inspect the microTUBE to ensure that all of the sample has been transferred. If droplets remain in the microTUBE, repeat step h.

The 50- µl sheared DNA samples are now ready for NGS sequencing library preparation, beginning with end repair/dA-

Preparation” on page 31.

tailing. Proceed to “Library

well

NOTE

This is not a stopping point in the workflow, and analysis of the sheared samples is not required before they are used for library preparation. Proceed directly to end-repair and dA-tailing.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 27

2 Preparation and Fragmentation of Input DNA

Method 2: Enzymatic DNA Fragmentation

In this step, gDNA samples are fragmented using Agilent’s SureSelect Enzymatic Fragmentation Kit for ILM (Pre PCR).

1 In wells of a thermal cycler- compatible strip tube or PCR plate, dilute

10 ng to 200 ng of each gDNA sample with nuclease- free water to a final volume of 7 µl.

2 Thaw the vial of 5X SureSelect Fragmentation Buffer, vortex, then place

on ice.

3 Preprogram a thermal cycler with the program in Table 13. Immediately

pause the program, and keep paused until samples are loaded in step 7.

Table 13 Thermal cycler program for enzymatic fragmentation

Step Temperature Time

Step 1 37°C Varies–see Table 14

Step 2 65°C 5 minutes

Step 3 4°C Hold

* Use a reaction volume setting of 10 l, if required for thermal cycler set up.

Optimal fragmentation conditions may vary based on the NGS read length to be used in the workflow. Refer to Table 14 below for the duration at 37°C appropriate for your sample type and required NGS read length.

Table 14 Fragmentation duration based on sample type and NGS read length

NGS read length requirement

2 ×100 reads 150 to 200 bp 15 minutes 15 minutes

2 ×150 reads 180 to 250 bp 10 minutes 15 minutes

* For FFPE DNA samples, initial DNA fragment size may impact the post-fragmentation size distribu-

tion, resulting in fragment sizes shorter than the target ranges listed in this table. All FFPE samples should be incubated at 37°C for 15 minutes to generate fragment ends suitable for library construction. Libraries prepared from FFPE samples should be analyzed using an NGS read length suitable for the final library fragment size distribution.

Target fragment size

Duration of 37°C incubation step (Table 13)

High-quality DNA samples FFPE DNA samples

28 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

4 Prepare the appropriate volume of Fragmentation master mix by

combining the reagents in Table 15.

Mix well by pipetting up and down 20 times or seal the tube and vortex at high speed for 5–10 seconds. Spin briefly to remove any bubbles and keep on ice.

Table 15 Preparation of Fragmentation master mix

Preparation and Fragmentation of Input DNA 2

Method 2: Enzymatic DNA Fragmentation

Reagent Volume for 1 reaction Volume for 8 reactions

(includes excess)

5X SureSelect Fragmentation Buffer 2 µl 18 µl 50 µl

SureSelect Fragmentation Enzyme 1 µl 9 µl 25 µl

Total 3 µl 27 µl 75 µl

Volume for 24 reactions (includes excess)

5 Add 3 µl of the Fragmentation master mix to each sample well

containing 7 µl of input DNA.

6 Mix well by pipetting up and down 20 times or cap the wells and

vortex at high speed for 5–10 seconds. Spin the samples briefly.

7 Immediately place the plate or strip tube in the thermal cycler and

resume the thermal cycling program in Table 13.

8 When the program reaches the 4°C Hold step, remove the samples from

the thermal cycler, add 40 µl of nuclease- free water to each sample, and place the samples on ice.

The 50- µl reactions are now ready for NGS sequencing library preparation, beginning with end repair/dA- tailing. Proceed to “Library Preparation” on page 31.

NOTE

This is not a stopping point in the workflow, and analysis of the enzymatically-fragmented samples is not required before they are used for library preparation. Proceed directly to end-repair and dA-tailing.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 29

2 Preparation and Fragmentation of Input DNA

Method 2: Enzymatic DNA Fragmentation

30 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

SureSelect XT HS2 DNA System Protocol

3 Library Preparation

Step 1. Prepare the Ligation master mix 33 Step 2. Repair and dA-Tail the DNA ends 34 Step 3. Ligate the molecular-barcoded adaptor 36 Step 4. Purify the sample using AMPure XP beads 37 Step 5. Amplify the adaptor-ligated library 39 Step 6. Purify the amplified library with AMPure XP beads 42 Step 7. Assess quality and quantity 44

This chapter describes the steps to prepare DNA libraries for sequencing using the Illumina paired- read platform. For each sample to be sequenced, an individual dual- indexed and molecular- barcoded library is prepared. For an overview of the SureSelect XT HS2 NGS sample preparation workflow, see Figure 1 on page 10.

The NGS library preparation protocol that begins here is used for fragmented DNA samples produced by mechanical shearing (as detailed on

page 25 to page 27) or produced by enzymatic fragmentation (as detailed

on page 28 to page 29). Samples produced by either method should contain 10–200 ng of DNA fragments in a volume of 50 µl.

Protocol steps in this section use the components listed in Table 16. Thaw and mix each component as directed in Table 16 before use (refer to the Where Used column). Remove the AMPure XP beads from cold storage and equilibrate to room temperature for at least 30 minutes in preparation for use on page 37. Do not freeze the beads at any time.

To process multiple samples, prepare reagent mixtures with overage at each step, without the cDNA library sample. Mixtures for preparation of 8 or 24 samples (including excess) are shown in tables as examples.

Agilent Technologies

3 Library Preparation

Table 16 Reagents thawed before use in protocol

Kit Component Storage Location Thawing Conditions Mixing Method Where Used

End Repair-A Tailing Buffer (yellow cap or bottle)

Ligation Buffer (purple cap or bottle)

End Repair-A Tailing Enzyme Mix (orange cap)

T4 DNA Ligase (blue cap) SureSelect XT HS2 Library

SureSelect XT HS2 Adaptor Oligo Mix (clear cap)

SureSelect XT HS2 Library Preparation Kit for ILM (Pre PCR), –20°C

Preparation Kit for ILM (Pre PCR), –20°C

SureSelect XT HS2 Library Preparation Kit for ILM (Pre PCR), –20°C

Thaw on ice (may require >20 minutes) then keep on ice

Place on ice just before use

Thaw on ice then keep on ice

Vortexing page 35

Vortexing page 33

Inversion page 35

Inversion page 33

Vortexing page 36

32 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Library Preparation 3

Step 1. Prepare the Ligation master mix

Prepare the Ligation master mix to allow equilibration to room temperature before use on page 36. Initiate this step before starting the End Repair/dA- tailing protocol; leave samples on ice while completing this step.

1 Vortex the thawed vial of Ligation Buffer for 15 seconds at high speed

to ensure homogeneity.

CAUTION

The Ligation Buffer used in this step is viscous. Mix thoroughly by vortexing at high speed for 15 seconds before removing an aliquot for use. When combining with other reagents, mix well by pipetting up and down 15–20 times using a pipette set to at least 80% of the mixture volume or by vortexing at high speed for 10–20 seconds.

Use flat top vortex mixers when vortexing strip tubes or plates throughout the protocol. If reagents are mixed by vortexing, visually verify that adequate mixing is occurring.

2 Prepare the appropriate volume of Ligation master mix by combining

the reagents in Table 17.

Slowly pipette the Ligation Buffer into a 1.5- ml Eppendorf tube, ensuring that the full volume is dispensed. Slowly add the T4 DNA Ligase, rinsing the enzyme tip with buffer solution after addition. Mix well by slowly pipetting up and down 15–20 times or seal the tube and vortex at high speed for 10–20 seconds. Spin briefly to collect the liquid.

Keep at room temperature for 30–45 minutes before use on page 36.

Table 17 Preparation of Ligation master mix

Reagent Volume for 1 reaction Volume for 8 reactions

(includes excess)

Ligation Buffer (purple cap or bottle) 23 µl 207 µl 575 µl

Volume for 24 reactions (includes excess)

†

T4 DNA Ligase (blue cap) 2 µl 18 µl 50 µl

Total 25 µl 225 µl 625 µl

* The minimum supported run size for 16-reaction kits is 8 samples per run, with kits containing enough reagents for 2 runs of

8 samples each.

† The minimum supported run size for 96-reaction kits is 24 samples per run, with kits containing enough reagents for 4 runs

of 24 samples each.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 33

3 Library Preparation

Step 2. Repair and dA-Tail the DNA ends

1 Preprogram a thermal cycler with the program in Table 18 for the End

Repair and dA- Tailing steps. Immediately pause the program, and keep paused until samples are loaded in step 5.

NOTE

CAUTION

Table 18 Thermal cycler program for End Repair/dA-Tailing

Step Temperature Time

Step 1 20°C 15 minutes

Step 2 72°C 15 minutes

Step 3 4°C Hold

* Use a reaction volume setting of 70 l, if required for thermal cycler set up.

When using Agilent’s SureCycler 8800 thermal cycler, the heated lid may be left on (default setting) throughout the library preparation incubation steps. The heated lid must be on during the hybridization and amplification steps on page 40, page 51 and page 61.

2 Vortex the thawed vial of End Repair- A Tailing Buffer for 15 seconds at

high speed to ensure homogeneity. Visually inspect the solution; if any solids are observed, continue vortexing until all solids are dissolved.

The End Repair-A Tailing Buffer used in this step must be mixed thoroughly by vortexing at high speed for 15 seconds before removing an aliquot for use. When combining with other reagents, mix well either by pipetting up and down 15–20 times using a pipette set to at least 80% of the mixture volume or by vortexing at high speed for 5–10 seconds.

3 Prepare the appropriate volume of End Repair/dA- Tailing master mix,

by combining the reagents in Table 19.

Slowly pipette the End Repair- A Tailing Buffer into a 1.5- ml Eppendorf tube, ensuring that the full volume is dispensed. Slowly add the End Repair- A Tailing Enzyme Mix, rinsing the enzyme tip with buffer solution after addition. Mix well by pipetting up and down 15–20 times or seal the tube and vortex at high speed for 5–10 seconds. Spin briefly to collect the liquid and keep on ice.

34 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Table 19 Preparation of End Repair/dA-Tailing master mix

Library Preparation 3

Step 2. Repair and dA-Tail the DNA ends

Reagent Volume for 1 reaction Volume for 8 reactions

(includes excess)

End Repair-A Tailing Buffer (yellow cap or bottle) 16 µl 144 µl 400 µl

End Repair-A Tailing Enzyme Mix (orange cap) 4 µl 36 µl 100 µl

Total 20 µl 180 µl 500 µl

Volume for 24 reactions (includes excess)

4 Add 20 µl of the End Repair/dA- Tailing master mix to each sample well

containing approximately 50 µl of fragmented DNA. Mix by pipetting up and down 15–20 times using a pipette set to 50 µl or cap the wells and vortex at high speed for 5–10 seconds.

5 Briefly spin the samples, then immediately place the plate or strip tube

in the thermal cycler and resume the thermal cycling program in

Table 18.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 35

3 Library Preparation

Step 3. Ligate the molecular-barcoded adaptor

1 Once the thermal cycler reaches the 4°C Hold step, transfer the samples

to ice while setting up this step.

2 Preprogram a thermal cycler with the program in Table 20 for the

Ligation step. Immediately pause the program, and keep paused until samples are loaded in step 5.

NOTE

Table 20 Thermal cycler program for Ligation

Step Temperature Time

Step 1 20°C 30 minutes

Step 2 4°C Hold

* Use a reaction volume setting of 100 l, if required for thermal cycler set up.

3 To each end- repaired/dA- tailed DNA sample (approximately 70 µl), add

25 µl of the Ligation master mix that was prepared on page 33 and kept at room temperature. Mix by pipetting up and down at least 10 times using a pipette set to 70 µl or cap the wells and vortex at high speed for 5–10 seconds. Briefly spin the samples.

4 Add 5 µl of SureSelect XT HS2 Adaptor Oligo Mix (clear- capped tube)

to each sample. Mix by pipetting up and down 15–20 times using a pipette set to 70 µl or cap the wells and vortex at high speed for 5–10 seconds.

Make sure to add the Ligation master mix and the Adaptor Oligo Mix to the samples in separate addition steps as directed above, mixing after each addition.

5 Briefly spin the samples, then immediately place the plate or strip tube

in the thermal cycler and resume the thermal cycling program in

Table 20.

NOTE

Stopping Point If you do not continue to the next step, seal the sample wells and store

36 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Unique molecular barcode sequences are incorporated into both ends of each library DNA fragment at this step.

overnight at either 4°C or –20°C.

Library Preparation 3

Step 4. Purify the sample using AMPure XP beads

1 Verify that the AMPure XP beads were held at room temperature for at

least 30 minutes before use.

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 0.8 ml of fresh 70% ethanol per sample.

3 Mix the AMPure XP bead suspension well so that the reagent appears

homogeneous and consistent in color.

4 Add 80 µl of homogeneous AMPure XP beads to each DNA sample

(approximately 100 µl) in the PCR plate or strip tube. Pipette up and down 15–20 times or cap the wells and vortex at high speed for 5–10 seconds to mix.

5 Incubate samples for 5 minutes at room temperature.

6 Put the plate or strip tube into a magnetic separation device. Wait for

the solution to clear (approximately 5 to 10 minutes).

7 Keep the plate or strip tube in the magnetic stand. Carefully remove

and discard the cleared solution from each well. Do not touch the beads while removing the solution.

8 Continue to keep the plate or strip tube in the magnetic stand while

you dispense 200 µl of freshly- prepared 70% ethanol in each sample well.

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 samples 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.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 37

3 Library Preparation

Step 4. Purify the sample using AMPure XP beads

12 Dry the samples by placing the unsealed plate or strip tube on the

thermal cycler, set to hold samples at 37°C, until the residual ethanol has just evaporated (typically 1–2 minutes).

NOTE

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 35 µl 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 or strip tube to collect the liquid.

15 Incubate for 2 minutes at room temperature.

16 Put the plate or strip tube in the magnetic stand and leave for

approximately 5 minutes, until the solution is clear.

17 Remove the cleared supernatant (approximately 34 µl) to a fresh PCR

plate or strip tube sample well and keep on ice. You can discard the beads at this time.

It may not be possible to recover the entire 34-µl supernatant volume at this step; transfer the maximum possible amount of supernatant for further processing. To maximize recovery, transfer the cleared supernatant to a fresh well in two rounds of pipetting, using a P20 pipette set at 17 µl.

38 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Library Preparation 3

Step 5. Amplify the adaptor-ligated library

This step uses the components listed in Table 21. Before you begin, thaw the reagents listed below and keep on ice.

Table 21 Reagents for pre-capture PCR amplification

Component Storage Location Mixing Method Where Used

Herculase II Fusion DNA Polymerase (red cap)

5× Herculase II Buffer with dNTPs (clear cap)

SureSelect XT HS2 Index Primer Pairs

* Indexing primer pairs are provided in individual wells of strip tubes (16 reaction kits) or plates (96 reaction kits).

SureSelect XT HS2 Library Preparation Kit for ILM (Pre PCR), –20°C

SureSelect XT HS2 Index Primer Pairs for ILM (Pre PCR),

–20°C

Pipette up and down 15–20 times

Vortexing page 41

page 41

1 Determine the appropriate index pair assignment for each sample. See

Table 51 through Table 58 in the “Reference” chapter for sequences of

the 8 bp index portion of the primers used to amplify the DNA libraries in this step.

Use a different indexing primer pair for each sample to be sequenced in the same lane.

CAUTION

The SureSelect XT HS2 Index Primer Pairs are provided in single-use aliquots. To avoid cross-contamination of libraries, do not retain and re-use any residual volume in wells for subsequent experiments.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 39

3 Library Preparation

Step 5. Amplify the adaptor-ligated library

2 Preprogram a thermal cycler with the program in Table 22. Immediately

pause the program, and keep paused until samples are loaded in step 6.

Table 22 Pre-Capture PCR Thermal Cycler Program

Segment Number of Cycles Temperature Time

1 1 98°C 2 minutes

3 1 72°C 5 minutes

4 1 4°C Hold

* Use a reaction volume setting of 50 l, if required for thermal cycler set up.

See Table 23 for cycle number

98°C 30 seconds

60°C 30 seconds

72°C 1 minute

Table 23 Pre-capture PCR cycle number recommendations

Quality of Input DNA Quantity of Input DNA Cycles

Intact DNA from fresh sample 100 to 200 ng 8 cycles

50 ng 9 cycles

10 ng 11 cycles

FFPE sample DNA 100 to 200 ng

50 ng* 12 cycles

10 ng* 14 cycles

* qPCR-determined quantity of amplifiable DNA or DIN value-adjusted amount of input DNA

11 cycles

40 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Library Preparation 3

Step 5. Amplify the adaptor-ligated library

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.

3 Prepare the appropriate volume of pre- capture PCR reaction mix, as

described in Table 24, on ice. Mix well on a vortex mixer.

Table 24 Preparation of Pre-Capture PCR Reaction Mix

Reagent Volume for 1 reaction Volume for 8 reactions

(includes excess)

5× Herculase II Buffer with dNTPs (clear cap) 10 µl 90 µl 250 µl

Herculase II Fusion DNA Polymerase (red cap) 1 µl 9 µl 25 µl

Total 11 µl 99 µl 275 µl

Volume for 24 reactions (includes excess)

4 Add 11 µl of the PCR reaction mixture prepared in Table 24 to each

purified DNA library sample (34 µl) in the PCR plate wells.

5 Add 5 µl of the appropriate SureSelect XT HS2 Index Primer Pair to

each reaction.

Cap the wells then vortex at high speed for 5 seconds. Spin the plate or strip tube briefly to collect the liquid and release any bubbles.

6 Before adding the samples to the thermal cycler, resume the thermal

cycling program in Table 22 to bring the temperature of the thermal block to 98°C. Once the cycler has reached 98°C, immediately place the sample plate or strip tube in the thermal block and close the lid.

CAUTION

The lid of the thermal cycler is hot and can cause burns. Use caution when working near the lid.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 41

3 Library Preparation

Step 6. Purify the amplified library with AMPure XP beads

1 Verify that the AMPure XP beads were held at room temperature for at

least 30 minutes before use.

2 Prepare 400 µl of 70% ethanol per sample, plus excess, for use in

step 8.

3 Mix the AMPure XP bead suspension well so that the reagent appears

homogeneous and consistent in color.

4 Add 50 µl of homogeneous AMPure XP beads to each 50- µl

amplification reaction in the PCR plate or strip tube. Pipette up and down 15–20 times or cap the wells and vortex at high speed for 5–10 seconds to mix.

5 Incubate samples for 5 minutes at room temperature.

6 Put the plate or strip tube into a magnetic separation device. Wait for

the solution to clear (approximately 5 minutes).

7 Keep the plate or strip tube in the magnetic stand. Carefully remove

and discard the cleared solution from each well. Do not touch the beads while removing the solution.

8 Continue to keep the plate or strip tube in the magnetic stand while

you dispense 200 µl of freshly- prepared 70% ethanol into each sample well.

9 Wait for 1 minute to allow any disturbed beads to settle, then remove

the ethanol.

10 Repeat step 8 and step 9 step once.

11 Seal the wells with strip caps, then briefly spin the samples 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.

12 Dry the samples by placing the unsealed plate or strip tube on the

thermal cycler, set to hold samples at 37°C, until the residual ethanol has just evaporated (typically 1–2 minutes).

13 Add 15 µl 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 or strip tube to collect the liquid.

15 Incubate for 2 minutes at room temperature.

16 Put the plate or strip tube in the magnetic stand and leave for 2 to

3 minutes, until the solution is clear.

42 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Library Preparation 3

Step 6. Purify the amplified library with AMPure XP beads

17 Remove the cleared supernatant (approximately 15 µl) to a fresh PCR

plate or strip tube sample well and keep on ice. You can discard the beads at this time.

NOTE

It may not be possible to recover the entire 15-µl supernatant volume at this step; transfer the maximum possible amount of supernatant for further processing.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 43

3 Library Preparation

Step 7. Assess quality and quantity

Analyze a five- fold dilution of each sample using one of the platforms listed in Table 25. Follow the instructions in the linked user guide provided for each assay in Table 25, after reviewing the SureSelect library qualification steps on page 45. Each analysis method provides an electropherogram showing the size distribution of fragments in the sample and tools for determining the concentration of DNA in the sample. See

Table 26 for fragment size distribution guidelines for various sample types.

Representative electropherograms generated using the TapeStation system are provided to illustrate typical results for libraries prepared from several types of input DNA.

Table 25 Pre-capture library analysis options

Analysis platform Assay used at this step Link to assay instructions Amount of library

sample to analyze

Agilent 4200 or 4150 TapeStation system

Agilent 2100 Bioanalyzer system DNA 1000 Kit Agilent DNA 1000 Kit Guide 1 µl of five-fold

Agilent 5200, 5300, or 5400 Fragment Analyzer system

D1000 ScreenTape Agilent D1000 Assay Quick

Guide

NGS Fragment Kit (1-6000 bp) Agilent NGS Fragment Kit

(1-6000 bp) Kit Guide

1 µl of five-fold dilution

dilution

2 µl of five-fold dilution

Table 26 Pre-capture library qualification guidelines

NGS read length for fragmentation protocol selection

2 ×100 reads Mechanical shearing Intact DNA 300 to 400 bp

2 ×150 reads Mechanical shearing Intact DNA 330 to 450 bp

Fragmentation method Input DNA type Expected library DNA

fragment size peak position

FFPE DNA 200 to 400 bp

Enzymatic fragmentation Intact DNA 300 to 400 bp

FFPE DNA 200 to 400 bp

FFPE DNA 200 to 450 bp

Enzymatic fragmentation Intact DNA 330 to 450 bp

FFPE DNA 200 to 450 bp

44 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Library Preparation 3

Step 7. Assess quality and quantity

1 Set up the instrument as instructed in the appropriate user guide (links

provided in Table 25).

2 Prepare samples for analysis by diluting 1 µl of each prepared library

sample in 4 µl of nuclease- free water.

3 Prepare the diluted samples for analysis and set up the assay as

instructed in the appropriate user guide. Load the analysis assay into the instrument and complete the run.

4 Verify that the electropherogram shows the expected DNA fragment size

peak position (see Table 26 for guidelines). Sample TapeStation system electropherograms are shown for libraries prepared from sheared DNA designed for 2 ×100 bp reads in Figure 2 (high- quality DNA), Figure 3 (medium- quality FFPE DNA), and Figure 4 (low- quality FFPE DNA).

Electropherograms obtained using the other analysis platform options listed in Table 25 are expected to show similar fragment size profiles.

5 Determine the concentration of the library DNA by integrating under

the peak.

Figure 2 Pre-capture library prepared from a sheared high-quality gDNA sample ana-

lyzed using a D1000 ScreenTape assay.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 45

3 Library Preparation

Step 7. Assess quality and quantity

Figure 3 Pre-capture library prepared from a typical FFPE gDNA sample (fragmented by

shearing) analyzed using a D1000 ScreenTape assay.

Figure 4 Pre-capture library prepared from a low-quality FFPE gDNA sample (fragment-

ed by shearing) analyzed using a D1000 ScreenTape assay.

46 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Library Preparation 3

Step 7. Assess quality and quantity

Observation of a low molecular weight peak, in addition to the expected library fragment peak, indicates the presence of adaptor- dimers in the library. It is acceptable to proceed to target enrichment with library samples for which adaptor- dimers are observed in the electropherogram at low abundance, similar to that seen in the example electropherogram in

Figure 4. See Troubleshooting on page 100 for additional considerations.

NOTE

Stopping Point If you do not continue to the next step, seal the sample wells and store at

For libraries being prepared for whole-genome sequencing (not specifically supported by this user guide), samples with an adaptor-dimer peak must be subjected to an additional round of SPRI-purification. To complete, first dilute the sample to 50 µl with nuclease free water, then follow the SPRI purification procedure on page 42.

4°C overnight or at –20°C for prolonged storage.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 47

3 Library Preparation

Step 7. Assess quality and quantity

48 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

SureSelect XT HS2 DNA System Protocol

4 Hybridization and Capture

Step 1. Hybridize DNA libraries to the probe 50 Step 2. Prepare streptavidin-coated magnetic beads 55 Step 3. Capture the hybridized DNA using streptavidin-coated beads 56

This chapter describes the steps to hybridize the prepared gDNA libraries with a target- specific probe. After hybridization, the targeted molecules are captured on streptavidin beads. Each DNA library sample is hybridized and captured individually.

The standard single- day SureSelect XT HS2 protocol includes the hybridization step immediately followed by capture and amplification steps. If required, the hybridized samples may be held overnight with capture and amplification steps completed the following day by using the simple protocol modifications noted on page 52.

CAUTION

The ratio of probe to prepared gDNA library is critical for successful capture.

Agilent Technologies

4 Hybridization and Capture

Step 1. Hybridize DNA libraries to the probe

In this step, the prepared gDNA libraries are hybridized to a target- specific probe using probe- specific hybridization conditions. For each sample library prepared, do one hybridization and capture. Do not pool samples at this stage.

The hybridization reaction requires 500- 1000 ng of prepared DNA in a volume of 12 µl. Use the maximum amount of prepared DNA available in this range.

This step uses the components listed in Table 27. Thaw each component under the conditions indicated in the table. Vortex each reagent to mix, then spin tubes briefly to collect the liquid.

Table 27 Reagents for Hybridization

Kit Component Storage Location Thawing Conditions Where Used

SureSelect XT HS2 Blocker Mix (blue cap)

SureSelect RNase Block (purple cap)

SureSelect Fast Hybridization Buffer (bottle)

Probe –80°C Thaw on ice page 53

SureSelect XT HS2 Target Enrichment Kit ILM Hyb Module, Box 2 (Post PCR), –20°C

Thaw on ice page 52

Thaw on ice page 53

Thaw and keep at Room Temperature

page 53

50 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Hybridization and Capture 4

Step 1. Hybridize DNA libraries to the probe

1 Preprogram a thermal cycler (with heated lid ON) with the program in

Table 28 for the SureSelect XT HS Human All Exon V8 Probe or in Table 29 for all other probes. Immediately pause the program, and keep

paused until samples are loaded in step 4 on page 52.

Table 28 Hybridization program for SureSelect XT HS Human All Exon V8 Probe

Segment # Number of Cycles Temperature Time

1 1 95°C 5 minutes

2 1 65°C 10 minutes

3 1 65°C 1 minute (Pause cycler here for reagent addition; see step 7 on page 54)

4 60 65°C 1 minute

37°C 3 seconds

5 1 65°C 60 minutes

6 1 65°C Hold briefly until ready to begin capture steps on page 56

* Use a reaction volume setting of 30 l (final volume of hybridization reactions in Segment 4).

Table 29 Hybridization program for all other probes

Segment # Number of Cycles Temperature Time

1 1 95°C 5 minutes

2 1 65°C 10 minutes

3 1 65°C 1 minute (Pause cycler here for reagent addition; see step 7 on page 54)

4 60 65°C

†

37°C 3 seconds

†

65°C

1 minute

Hold briefly until ready to begin capture steps on page 56

* Use a reaction volume setting of 30 l (final volume of hybridization reactions during cycling in Segment 4).

† Hybridization at 65°C is optimal for probes designed for the SureSelect XT HS2/XT HS/XL Low Input platforms. Reducing the

hybridization temperature (Segments 4 and 5) may improve performance for probes designed for the SureSelect XT platform, including SureSelect XT Human All Exon V6 (62.5°C), SureSelect XT Clinical Research Exome V2 (62.5°C) and custom probes originally designed for use with SureSelect XT system (60°C–65°C).

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 51

4 Hybridization and Capture

Step 1. Hybridize DNA libraries to the probe

NOTE

CAUTION

The Hybridization reaction may be run overnight with the following protocol modifications:

• In the final segment of the thermal cycler program (Table 28 or Table 29), replace the 65°C Hold step with a 21°C Hold step.

• The hybridized samples may be held at 21°C for up to 16 hours. Begin the capture preparation steps on page 55 on day 2, after the overnight hold.

2 Place 1000 ng of each prepared gDNA library sample into the

hybridization plate or strip tube wells and then bring the final volume in each well to 12 µl using nuclease- free water. If 1000 ng DNA is not available for any of the samples, use the maximum amount available, within the 500–1000 ng range.

3 To each DNA library sample well, add 5 µl of SureSelect XT HS2

Blocker Mix (blue cap). Seal the wells then vortex at high speed for 5 seconds. Spin briefly to collect the liquid and release any bubbles.

The lid of the thermal cycler is hot and can cause burns. Use caution when working near the lid.

4 Transfer the sealed sample plate or strip to the thermal cycler and

resume the thermal cycling program (Table 28 or Table 29 on page 51), allowing the cycler to complete Segments 1 and 2 of the program.

Important: The thermal cycler must be paused during Segment 3 to allow additional reagents to be added to the Hybridization wells in

step 7 on page 54.

During Segments 1 and 2 of the thermal cycling program, begin preparing the additional hybridization reagents as described in step 5 and step 6 below. If needed, you can finish these preparation steps after pausing the thermal cycler in Segment 3.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment

5 Prepare a 25% solution of SureSelect RNase Block (1 part RNase Block

to 3 parts water) according to Table 30. Prepare the amount required for the number of hybridization reactions in the run, plus excess. Mix well and keep on ice.

Table 30 Preparation of RNase Block solution

Hybridization and Capture 4

Step 1. Hybridize DNA libraries to the probe

Reagent Volume for 1 reaction Volume for 8 reactions

(includes excess)

SureSelect RNase Block 0.5 µl 4.5 µl 12.5 µl

Nuclease-free water 1.5 µl 13.5 µl 37.5 µl

Total 2 µl 18 µl 50 µl

NOTE

Prepare the mixture described in step 6, below, just before pausing the thermal cycler in Segment 3. Keep the mixture at room temperature briefly until the mixture is added to the

Volume for 24 reactions (includes excess)

DNA samples in step 7 on page 54. Do not keep solutions containing the probe at room temperature for extended periods.

6 Prepare the Probe Hybridization Mix appropriate for your probe design

size. Use Table 31 for probes 3 Mb or Table 32 for probes <3 Mb.

Combine the listed reagents at room temperature. Mix well by vortexing at high speed for 5 seconds then spin down briefly. Proceed immediately to step 7.

Table 31 Preparation of Probe Hybridization Mix for probes≥3 Mb

Reagent Volume for 1 reaction Volume for 8 reactions

(includes excess)

Volume for 24 reactions (includes excess)

25% RNase Block solution (from step 5) 2 µl 18 µl 50 µl

Probe (with design 3 Mb) 5 µl 45 µl 125 µl

SureSelect Fast Hybridization Buffer 6 µl 54 µl 150 µl

Total 13 µl 117 µl 325 µl

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 53

4 Hybridization and Capture

Step 1. Hybridize DNA libraries to the probe

Table 32 Preparation of Probe Hybridization Mix for probes<3 Mb

Reagent Volume for 1 reaction Volume for 8 reactions

(includes excess)

25% RNase Block solution (from step 5) 2 µl 18 µl 50 µl

Probe (with design<3 Mb) 2 µl 18 µl 50 µl

SureSelect Fast Hybridization Buffer 6 µl 54 µl 150 µl

Nuclease-free water 3 µl 27 µl 75 µl

Total 13 µl 117 µl 325 µl

Volume for 24 reactions (includes excess)

7 Once the thermal cycler starts Segment 3 (1 minute at 65°C), pause the

program. With the cycler paused, and while keeping the DNA + Blocker samples in the cycler, transfer 13 µl of the room- temperature Probe Hybridization Mix from step 6 to each sample well.

Mix well by pipetting up and down slowly 8 to 10 times.

The hybridization reaction wells now contain approximately 30 µl.

8 Seal the wells with fresh domed strip caps. Make sure that all wells are

completely sealed. Vortex briefly, then spin the plate or strip tube briefly to remove any bubbles from the bottom of the wells. Immediately return the plate or strip tube to the thermal cycler.

9 Resume the thermal cycling program to allow hybridization of the

prepared DNA samples to the probe.

CAUTION

Wells must be adequately sealed to minimize evaporation, or your results can be negatively impacted.

Before you do the first experiment, make sure the plasticware and capping method are appropriate for the thermal cycler. Check that no more than 4 µl is lost to evaporation under the conditions used for hybridization.

54 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Hybridization and Capture 4

Step 2. Prepare streptavidin-coated magnetic beads

The remaining hybridization capture steps use the reagents in Table 33.

NOTE

Table 33 Reagents for Capture

Kit Component Storage Location Where Used

SureSelect Binding Buffer SureSelect Target Enrichment Kit, ILM Hyb Module, Box 1

SureSelect Wash Buffer 1 SureSelect Target Enrichment Kit, ILM Hyb Module, Box 1

SureSelect Wash Buffer 2 SureSelect Target Enrichment Kit, ILM Hyb Module, Box 1

SureSelect Streptavidin Beads OR Dynabeads MyOne Streptavidin T1 Beads

If performing same-day hybridization and capture, begin the bead preparation steps below approximately one hour after starting hybridization in step 9 on page 54. If performing next-day capture after an overnight hold at 21°C, begin the bead preparation steps below on day 2, just before you are ready to start the capture steps on page 56.

page 55

(Post PCR), RT

page 56

(Post PCR), RT

page 56

(Post PCR), RT

4°C page 55

1 Vigorously resuspend the vial of streptavidin beads on a vortex mixer.

The magnetic beads settle during storage.

2 For each hybridization sample, add 50 µl of the resuspended beads to

wells of a fresh PCR plate or strip tube.

3 Wash the beads:

a Add 200 µl of SureSelect Binding Buffer.

b Mix by pipetting up and down 20 times or cap the wells and vortex

at high speed for 5–10 seconds then spin down briefly.

c Put the plate or strip tube into a magnetic separator device.

d Wait 5 minutes or until the solution is clear, then remove and

discard the supernatant.

e Repeat step a through step d two more times for a total of 3 washes.

4 Resuspend the beads in 200 µl of SureSelect Binding Buffer.

NOTE

If you are equipped for higher-volume magnetic bead captures, the streptavidin beads may instead be batch-washed in an Eppendorf tube or conical vial.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 55

4 Hybridization and Capture

Step 3. Capture the hybridized DNA using streptavidin-coated beads

1 After all streptavidin bead preparation steps are complete and with the

hybridization thermal cycling program in the final hold segment (see

Table 28 or Table 29 on page 51), transfer the samples to room

temperature.

2 Immediately transfer the entire volume (approximately 30 µl) of each

hybridization mixture to wells containing 200 µl of washed streptavidin beads using a multichannel pipette.

Pipette up and down 5–8 times to mix then seal the wells with fresh caps.

3 Incubate the capture plate or strip tube on a 96- well plate mixer,

mixing vigorously (at 1400–1900 rpm), for 30 minutes at room temperature.

Make sure the samples are properly mixing in the wells.

4 During the 30- minute incubation for capture, prewarm SureSelect Wash

Buffer 2 at 70°C as described below.

a Place 200- µl aliquots of Wash Buffer 2 in wells of a fresh 96- well

plate or strip tubes. Aliquot 6 wells of buffer for each DNA sample in the run.

b Cap the wells and then incubate in the thermal cycler held at 70°C

until used in step 9.

5 When the 30- minute capture incubation period initiated in step 3 is

complete, spin the samples briefly to collect the liquid.

6 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.

7 Resuspend the beads in 200 µl of SureSelect Wash Buffer 1. Mix by

pipetting up and down 15–20 times, until beads are fully resuspended.

8 Put the plate or strip tube in the magnetic separator. Wait for the

solution to clear (approximately 1 minute), then remove and discard the supernatant.

56 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Hybridization and Capture 4

Step 3. Capture the hybridized DNA using streptavidin-coated beads

CAUTION

It is important to maintain bead suspensions at 70°C during the washing procedure below to ensure specificity of capture.

Make sure that the SureSelect Wash Buffer 2 is pre-warmed to 70°C before use.

Do not use a tissue incubator, or other devices with significant temperature fluctuations, for the incubation steps.

9 Remove the plate or strip tubes from the magnetic separator and

transfer to a rack at room temperature. Wash the beads with Wash

Buffer 2, using the protocol steps below.

a Resuspend the beads in 200 µl of 70°C prewarmed Wash Buffer 2.

Pipette up and down 15–20 times, until beads are fully resuspended.

b Seal the wells with fresh caps and then vortex at high speed for

8 seconds. Spin the plate or strip tube briefly to collect the liquid without pelleting the beads.

Make sure the beads are in suspension before proceeding.

c Incubate the samples for 5 minutes at 70°C on the thermal cycler

with the heated lid on.

d Put the plate or strip tube in the magnetic separator at room

temperature.

e Wait 1 minute for the solution to clear, then remove and discard the

supernatant.

f Repeat step a through step e five more times for a total of 6 washes.

10 After verifying that all wash buffer has been removed, add 25 µl of

nuclease- free water to each sample well. Pipette up and down 8 times to resuspend the beads.

Keep the samples on ice until they are used on page 62.

NOTE

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 57

Captured DNA is retained on the streptavidin beads during the post-capture amplification step.

4 Hybridization and Capture

Step 3. Capture the hybridized DNA using streptavidin-coated beads

58 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

SureSelect XT HS2 DNA System Protocol

5 Post-Capture Sample Processing for Multiplexed Sequencing

Step 1. Amplify the captured libraries 60 Step 2. Purify the amplified captured libraries using AMPure XP beads 63 Step 3. Assess sequencing library DNA quantity and quality 65 Step 4. Pool samples for multiplexed sequencing 68 Step 5. Prepare sequencing samples 70 Step 6. Do the sequencing run and analyze the data 72

This chapter describes the steps to amplify, purify, and assess quality and quantity of the captured libraries. Sample pooling instructions are provided to prepare the indexed, molecular barcoded samples for multiplexed sequencing. Sequencing run setup and sequencing data analysis steps will vary according to your NGS platform and data analysis pipeline; guidelines for these downstream steps are also provided in this chapter.

Agilent Technologies

5 Post-Capture Sample Processing for Multiplexed Sequencing

Step 1. Amplify the captured libraries

In this step, the SureSelect- enriched DNA libraries are PCR amplified.

This step uses the components listed in Table 34. Before you begin, thaw the reagents listed below and keep on ice. Remove the AMPure XP beads from cold storage and equilibrate to room temperature for at least 30 minutes in preparation for use on page 63. Do not freeze the beads at any time.

Table 34 Reagents for post-capture PCR amplification

Component Storage Location Mixing Method Where Used

Herculase II Fusion DNA Polymerase (red cap)

5× Herculase II Buffer with dNTPs (clear cap)

SureSelect Post-Capture Primer Mix (clear cap)

Prepare one amplification reaction for each DNA library.

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.

SureSelect XT HS2 Target Enrichment Kit ILM Hyb Module, Box 2 (Post PCR), –20°C

Pipette up and down 15–20 times

Vortexing page 62

page 62

60 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Post-Capture Sample Processing for Multiplexed Sequencing 5

Step 1. Amplify the captured libraries

1 Preprogram a thermal cycler with the program in Table 35. Immediately

pause the program, and keep paused until samples are loaded in step 5.

Table 35 Post-capture PCR Thermal Cycler Program

Segment Number of Cycles Temperature Time

1 1 98°C 2 minutes

2 10–16

(See Table 36 for hybridization probe design size-based cycle number recommendations)

3 1 72°C 5 minutes

4 1 4°C Hold

* Use a reaction volume setting of 50 l, if required for thermal cycler set up.

98°C 30 seconds

60°C 30 seconds

72°C 1 minute

Table 36 Post-capture PCR cycle number recommendations

Probe Capture Library Size Cycles

Probes <0.2 Mb 16 cycles

Probes 0.2–3 Mb 12–16 cycles

Probes 3–5 Mb 11–12 cycles

Probes >5 Mb (including Human All Exon probes) 10–11 cycles

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 61

5 Post-Capture Sample Processing for Multiplexed Sequencing

Step 1. Amplify the captured libraries

2 Prepare the appropriate volume of PCR reaction mix, as described in

Table 37, on ice. Mix well on a vortex mixer.

Table 37 Preparation of post-capture PCR reaction mix

Reagent Volume for 1 reaction Volume for 8 reactions

(includes excess)

Nuclease-free water 13 µl 117 µl 325 µl

5× Herculase II Buffer with dNTPs (clear cap) 10 µl 90 µl 250 µl

Herculase II Fusion DNA Polymerase (red cap) 1 µl 9 µl 25 µl

SureSelect Post-Capture Primer Mix (clear cap) 1 µl 9 µl 25 µl

Total 25 µl 225 µl 625 µl

Volume for 24 reactions (includes excess)

3 Add 25 µl of the PCR reaction mix prepared in Table 37 to each sample

well containing 25 µl of bead- bound target- enriched DNA (prepared on

page 57 and held on ice).

4 Mix the PCR reactions well by pipetting up and down until the bead

suspension is homogeneous. Avoid splashing samples onto well walls; do not spin the samples at this step.

5 Place the plate or strip tube in the thermal cycler and resume the

thermal cycling program in Table 35.

6 When the PCR amplification program is complete, spin the plate or

strip tube briefly. Remove the streptavidin- coated beads by placing the plate or strip tube on the magnetic stand at room temperature. Wait 2 minutes for the solution to clear, then remove each supernatant (approximately 50 µl) to wells of a fresh plate or strip tube.

The beads can be discarded at this time.

62 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Post-Capture Sample Processing for Multiplexed Sequencing 5

Step 2. Purify the amplified captured libraries using AMPure XP beads

1 Verify that the AMPure XP beads were held at room temperature for at

least 30

minutes before use.

2 Prepare 400 µl of fresh 70% ethanol per sample, plus excess, for use in

step 8.

3 Mix the AMPure XP bead suspension well so that the suspension

appears homogeneous and consistent in color.

4 Add 50 µl of the homogeneous AMPure XP bead suspension to each

amplified DNA sample (approximately 50 µl) in the PCR plate or strip tube. Mix well by pipetting up and down 15–20 times or cap the wells and vortex at high speed for 5–10 seconds.

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.

5 Incubate samples for 5 minutes at room temperature.

6 Put the plate or strip tube on the magnetic stand at room temperature.

Wait for the solution to clear (approximately 3 to 5 minutes).

7 Keep the plate or tubes in the magnetic stand. Carefully remove and

discard the cleared solution from each well. Do not disturb the beads while removing the solution.

8 Continue to keep the plate or tubes in the magnetic stand while you

dispense 200 µl of freshly- prepared 70% ethanol in each sample well.

9 Wait for 1 minute to allow any disturbed beads to settle, then remove

the ethanol.

10 Repeat step 8 and step 9 once for a total of two washes. Make sure to

remove all of the ethanol at each wash step.

11 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.

12 Dry the samples by placing the unsealed plate or strip tube on the

thermal cycler, set to hold samples at 37°C, until the residual ethanol has just evaporated (typically 1–2 minutes).

13 Add 25 µl of Low TE to each sample well.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 63

5 Post-Capture Sample Processing for Multiplexed Sequencing

Step 2. Purify the amplified captured libraries using AMPure XP beads

14 Seal the sample wells with strip caps, then mix well on a vortex mixer

and briefly spin to collect the liquid without pelleting the beads.

15 Incubate for 2 minutes at room temperature.

16 Put the plate or strip tube in the magnetic stand and leave for

2 minutes or until the solution is clear.

17 Remove the cleared supernatant (approximately 25 µl) to a fresh well.

You can discard the beads at this time.

64 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Post-Capture Sample Processing for Multiplexed Sequencing 5

Step 3. Assess sequencing library DNA quantity and quality

Analyze each library using one of the platforms listed in Table 38. Follow the instructions in the linked user guide provided for each assay in

Table 38, after reviewing the post- capture library qualification steps on page 66. See Table 39 for fragment size distribution guidelines for various

sample types. Representative electropherograms generated using the TapeStation system are provided to illustrate typical results for post- capture libraries prepared from selected sample types.

Table 38 Post-capture library analysis options

Analysis platform Assay used at this step Link to assay instructions Amount of library

sample to analyze

Agilent 4200 or 4150 TapeStation system

Agilent 2100 Bioanalyzer system

Agilent 5200, 5300, or 5400 Fragment Analyzer system

High Sensitivity D1000 ScreenTape

High Sensitivity DNA Kit Agilent High Sensitivity DNA Kit

HS NGS Fragment Kit (1-6000 bp)

Agilent High Sensitivity D1000 Assay Quick Guide

Guide

Agilent HS NGS Fragment Kit (1-6000 bp) Kit Guide

2 µl

1 µl

2 µl

Table 39 Post-capture library qualification guidelines

NGS read length for fragmentation protocol selection

2 ×100 reads Intact DNA 200 to 400 bp (see Figure 5 for sample electropherogram)

2 ×150 reads Intact DNA 230 to 450 bp

Input DNA type Expected DNA fragment size peak position

FFPE DNA 200 to 400 bp (see Figure 6 and Figure 7 for sample

electropherograms)

FFPE DNA 200 to 450 bp

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 65

5 Post-Capture Sample Processing for Multiplexed Sequencing

Step 3. Assess sequencing library DNA quantity and quality

1 Set up the instrument as instructed in the appropriate user guide (links

provided in Table 38).

2 Prepare the samples for analysis and set up the assay as instructed in

the appropriate user guide. Load the analysis assay into the instrument and complete the run.

3 Verify that the electropherogram shows the expected DNA fragment size

peak position (see Table 39 for guidelines). Sample TapeStation system electropherograms are shown for libraries prepared from sheared DNA designed for 2 ×100 bp reads in Figure 5 (high- quality input DNA),

Figure 6 (medium- quality FFPE input DNA), and Figure 7 (low- quality

FFPE input DNA).

Electropherograms obtained using the other analysis platform options listed in Table 38 are expected to show similar fragment size profiles.

4 Determine the concentration of the library DNA by integrating under

the entire peak.

Figure 5 Post-capture library prepared from a high-quality gDNA sample analyzed using

a High Sensitivity D1000 ScreenTape assay.

66 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Post-Capture Sample Processing for Multiplexed Sequencing 5

Step 3. Assess sequencing library DNA quantity and quality

Figure 6 Post-capture library prepared from a typical FFPE gDNA sample analyzed us-

ing a High Sensitivity D1000 ScreenTape assay.

Figure 7 Post-capture library prepared from a low-quality FFPE gDNA sample analyzed

using a High Sensitivity D1000 ScreenTape assay.

Stopping Point If you do not continue to the next step, seal the plate and store at 4°C

overnight or at –20°C for prolonged storage.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 67

5 Post-Capture Sample Processing for Multiplexed Sequencing

Step 4. 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 the capacity of your platform and the amount of sequencing data required per sample.

Combine the libraries such that each index- tagged sample is present in equimolar amounts in the pool using one of the following methods:

Method 1: Dilute each sample to be pooled to the same final

concentration (typically 4 nM–15 nM, or the concentration of the most dilute sample) using Low TE, then combine equal volumes of all samples to create the final pool.

Method 2: Starting with samples at different concentrations, add the

appropriate volume of each sample to achieve equimolar concentration in the pool, then adjust the pool to the desired final volume using Low TE. The formula below is provided for determination of the amount of each indexed sample to add to the pool.

Volume of Index

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

(typically 4 nM–15 nM or the concentration of the most dilute sample)

# is the number of indexes, and

C(i) is the initial concentration of each indexed sample

Table 40 shows an example of the amount of 4 index- tagged samples

(of different concentrations) and Low TE needed for a final volume of 20 µl at 10 nM DNA.

68 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Vf Cf

---------------------------------=

# Ci

Post-Capture Sample Processing for Multiplexed Sequencing 5

Step 4. Pool samples for multiplexed sequencing

Table 40 Example of volume calculation for total volume of 20 µl at 10 nM concentration

Component V(f) C(i) C(f) # Volume to use (µl)

Sample 1 20 µl 20 nM 10 nM 4 2.5

Sample 2 20 µl 10 nM 10 nM 4 5

Sample 3 20 µl 17 nM 10 nM 4 2.9

Sample 4 20 µl 25 nM 10 nM 4 2

Low TE 7.6

If you store the library before sequencing, add Tween 20 to 0.1% v/v and store at - 20°C short term.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 69

5 Post-Capture Sample Processing for Multiplexed Sequencing

Step 5. Prepare sequencing samples

The final SureSelect XT HS2 library pool is ready for direct sequencing using standard Illumina paired- end primers and chemistry. Each fragment in the prepared library contains one target insert surrounded by sequence motifs required for multiplexed sequencing using the Illumina platform, as shown in Figure 8.

Figure 8 Content of SureSelect XT HS2 sequencing library. Each fragment contains one

target insert (blue) surrounded by the Illumina paired-end sequencing elements (black), unique dual sample indexes (red and green), duplex molecular barcodes (brown) and the library PCR primers (yellow).

Libraries can be sequenced on the Illumina HiSeq, MiSeq, NextSeq or NovaSeq platform using the run type and chemistry combinations shown in Table 41.

Proceed to cluster amplification using the appropriate Illumina Paired- End Cluster Generation Kit. See Table 41 for kit configurations compatible with the recommended read length.

The optimal seeding concentration for SureSelect XT HS2 target- enriched libraries varies according to sequencing platform, run type, and Illumina kit version. See Table 41 for guidelines. 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. Begin optimization using a seeding concentration in the middle of the range listed in Table 41.

Follow Illumina’s recommendation for a PhiX control in a low- concentration spike- in for improved sequencing quality control.

70 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Post-Capture Sample Processing for Multiplexed Sequencing 5

Step 5. Prepare sequencing samples

Table 41 Illumina Kit Configuration Selection Guidelines

Platform Run Type Read Length SBS Kit Configuration Chemistry Seeding

Concentration

HiSeq 2500 Rapid Run 2 × 100 bp 200 Cycle Kit v2 9–10 pM

HiSeq 2500 High Output 2 × 100 bp 250 Cycle Kit v4 12–14 pM

MiSeq All Runs 2 × 100 bp or

2 × 150 bp

MiSeq All Runs 2 × 75 bp 150 Cycle Kit v3 12–16 pM

NextSeq 500/550 All Runs 2 × 100 bp or

2 × 150 bp

HiSeq 3000/4000 All Runs 2 × 100 bp or

2 × 150 bp

NovaSeq 6000 Standard

Workflow Runs

NovaSeq 6000 Xp Workflow

Runs

2 × 100 bp or

2 × 150 bp

2 × 100 bp or

2 × 150 bp

300 Cycle Kit v2 9–10 pM

300 Cycle Kit v2.5 1.2–1.5 pM

300 Cycle Kit v1 230–240 pM

300 Cycle Kit v1.0 or v1.5 300–600 pM

300 Cycle Kit v1.0 or v1.5 200–400 pM

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 71

5 Post-Capture Sample Processing for Multiplexed Sequencing

Step 6. Do the sequencing run and analyze the data

The guidelines below provide an overview of SureSelect XT HS2 library sequencing run setup and analysis considerations. Links are provided for additional details for various NGS platforms and analysis pipeline options.

• Each of the sample- level indexes requires an 8- bp index read. For complete index sequence information, see Table 51 on page 87 through

Table 58 on page 94.

• For the HiSeq, NextSeq, and NovaSeq platforms, set up the run using the instrument’s user interface, following the guidelines on page 73.

• For the MiSeq platform, set up the run using Illumina Experiment Manager (IEM) using the steps detailed on page 73 to page 76 to generate a custom sample sheet.

• Demultiplex using Illumina’s bcl2fastq software to generate paired end reads based on the dual indexes and remove sequences with incorrectly paired P5 and P7 indexes.

• The MBC sequence and dark bases are located at the 5’ end of both Read 1 and Read 2. Use the Agilent Genomics NextGen Toolkit (AGeNT) for molecular barcode extraction and trimming (see page 77 for more information). If your sequence analysis pipeline excludes MBCs and is incompatible with AGeNT, you can trim or mask the first five bases from each read before alignment as described in the Note on page 77.

• Before aligning reads to the reference genome, Illumina adaptor sequences should be trimmed from the reads using Agilent’s AGeNT trimmer module, which properly accounts for the degenerate MBCs in the adaptor sequence. See page 77 for more information. Do not use the adaptor trimming options in Illumina Experiment Manager (IEM). Make sure any IEM adaptor trimming option checkboxes are cleared (deselected) when setting up the sequencing run.

72 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Post-Capture Sample Processing for Multiplexed Sequencing 5

Step 6. Do the sequencing run and analyze the data

HiSeq/NextSeq/NovaSeq platform sequencing run setup guidelines

Set up sequencing runs using the instrument control software interface, using the settings shown in Table 42. For HiSeq runs, select Dual Index on the Run Configuration screen of the instrument control software interface and enter the Cycles settings in Table 42.

For the NextSeq or NovaSeq platform, open the Run Setup screen of the instrument control software interface and enter the Read Length settings in Table 42. In the Custom Primers section, clear (do not select) the checkboxes for all primers (Read 1, Read 2, Index 1 and Index 2).

Table 42 Run settings

Run Segment Cycles/Read Length

Read 1 100 or 150

Index 1 (i7) 8

Index 2 (i5) 8

Read 2 100 or 150

MiSeq platform sequencing run setup guidelines

Use the Illumina Experiment Manager (IEM) software to generate a custom Sample Sheet according to the guidelines below. Once a Sample Sheet has been generated, index sequences need to be manually changed to the SureSelect XT HS2 indexes used for each sample. See page 87 through

page 94 for nucleotide sequences of the SureSelect XT HS2 index pairs.

Set up a custom Sample Sheet:

1 In the IEM software, create a Sample Sheet for the MiSeq platform

using the following Workflow selections.

• Under Category, select Other.

• Under Application, select FASTQ Only.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 73

5 Post-Capture Sample Processing for Multiplexed Sequencing

Step 6. Do the sequencing run and analyze the data

2 On the Workflow Parameters screen, enter the run information, making

sure to specify the key parameters highlighted below. In the Library Prep Workflow field, select TruSeq Nano DNA. In the Index Adapters

field, select TruSeq DNA CD Indexes (96 Indexes). Clear both adaptorSettings, since adaptor trimming must be performed using Agilent’s AGeNT software (see page 77).

If TruSeq Nano DNA is not available in the Sample Prep Kit field, instead select TruSeq HT.

trimming checkboxes under FASTQ Only Workflow- Specific

3 Using the Sample Sheet Wizard, set up a New Plate, entering the

required information for each sample to be sequenced. In the I7 Sequence column, assign each sample to any of the Illumina i7 indexes. The index will be corrected to the i7 sequence from the SureSelect XT HS2 index pair at a later stage.

74 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Post-Capture Sample Processing for Multiplexed Sequencing 5

Step 6. Do the sequencing run and analyze the data

Likewise, in the I5 Sequence column, assign any of the Illumina i5 indexes, to be corrected to the i5 sequence from the SureSelect XT HS2 index pair at a later stage.

4 Finish the sample sheet setup tasks and save the sample sheet file.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 75

5 Post-Capture Sample Processing for Multiplexed Sequencing

Step 6. Do the sequencing run and analyze the data

Editing the Sample Sheet to include SureSelect XT HS2 dual indexes

• Open the Sample Sheet file in a text editor and edit the i7 and i5 index information for each sample in columns 5–8 (highlighted below). See

page 87 through page 94 for nucleotide sequences of the SureSelect XT

HS2 indexes.

• In column 5 under I7_Index_ID, enter the SureSelect XT HS2 index pair number assigned to the sample. In column 6 under index, enter the corresponding P7 index sequence.

• In column 7 under I5_Index_ID, enter the SureSelect XT HS2 index pair number assigned to the sample. In column 8 under index2, enter the corresponding P5 index sequence.

• If the run includes more than 96 samples, the sample sheet may be edited to include additional sample rows containing the assigned SureSelect XT HS2 index pair sequences in column 6 (P7 index) and column 8 (P5 index).

Figure 9 Sample sheet for SureSelect XT HS2 library sequencing

5 Save the edited Sample Sheet in an appropriate file location for use in

the run.

76 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

NOTE

Post-Capture Sample Processing for Multiplexed Sequencing 5

Sequence analysis resources

Guidelines are provided below for typical NGS analysis pipeline steps appropriate for SureSelect XT HS2 library data analysis. Your NGS analysis pipeline may vary.

Use the Illumina bcl2fastq software to generate paired end reads by demultiplexing sequences based on the dual indexes and to remove sequences with incorrectly paired P5 and P7 indexes.

The demultiplexed FASTQ data needs to be pre- processed to remove sequencing adaptors and extract the molecular barcode (MBC) sequences using the Agilent Genomics NextGen Toolkit (AGeNT). AGeNT is a set of Java- based software modules that provide MBC pre- processing adaptor trimming and duplicate read identification. This toolkit is designed to enable building, integrating, maintaining, and troubleshooting internal analysis pipelines for users with bioinformatics expertise. For additional information and to download this toolkit, visit the AGeNT page at

www.agilent.com.

If your sequence analysis pipeline excludes MBCs, you can remove the first 5 bases from Read 1 and Read 2 by either masking or trimming before proceeding to further analysis. To remove during demultiplexing via masking, include the base mask N5Y*,I8,I8,N5Y* (where * may be replaced with the actual read length, matching the read length value in the RunInfo.xml file). Alternatively, the first 5 bases may be trimmed from the demultiplexed fastq files using a suitable processing tool of your choice, such as seqtk. Alternatively, the AGeNT trimmer module can be used to remove the MBCs and properly remove adaptor sequences as well. Standard adaptor trimmers will fail to remove the MBC sequences from the opposite adaptor (refer to Figure 8).

The trimmed reads should be aligned, and MBC tags added to the aligned BAM file using a suitable tool such as the BWA- MEM. Once alignment and tagging are complete, the AGeNT LocatIt module may be used to generate consensus reads and mark or remove duplicates. The resulting BAM files are ready for downstream analysis including variant discovery.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 77

5 Post-Capture Sample Processing for Multiplexed Sequencing

Sequence analysis resources

78 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

SureSelect XT HS2 DNA System Protocol

6 Appendix: Using FFPE-derived DNA Samples

Protocol modifications for FFPE Samples 80 Methods for FFPE Sample Qualification 80 Sequencing Output Recommendations for FFPE Samples 81

This chapter summarizes the protocol modifications to apply to FFPE samples based on the integrity of the FFPE sample DNA.

Agilent Technologies

6 Appendix: Using FFPE-derived DNA Samples

Protocol modifications for FFPE Samples

Protocol modifications that should be applied to FFPE samples are summarized in Table 43.

Table 43 Summary of protocol modifications for FFPE samples

Workflow Step and page Parameter Condition for non-FFPE Samples Condition for FFPE Samples

gDNA Sample Preparation page 22

DNA input for Library Preparation page 22

DNA Shearing page 26 Mode of DNA

Pre-capture PCR page 40 Cycle number 8–11 11–14

Sequencing page 81 Output augmentation Per project requirements 1× to 10× based on determined

Qualification of DNA Integrity

Input amount and means of quantification

Shearing

Not required Required

10 ng to 200 ng, quantified by Qubit assay

2 × 120 seconds (for 2 × 100 reads)

2 × 60 seconds (for 2 × 150 reads)

Based on determined DNA integrity (see Table 9 on page 23 and Table 10 on page 24)

240 seconds (continuous, for all read lengths)

DNA integrity (see Table 44 and

Table 45 on page 81)

Methods for FFPE Sample Qualification

DNA integrity may be assessed using the Agilent NGS FFPE QC Kit or using the Agilent TapeStation system with the 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 to allow direct normalization of input DNA amount and a Cq DNA integrity score used to design other protocol modifications.

The Agilent TapeStation instrument, combined with the Genomic DNA ScreenTape assay, provides an automated electrophoresis method for determination of a DNA Integrity Number (DIN) score used to estimate amount of input DNA required for sample normalization and to design other protocol modifications.

80 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Appendix: Using FFPE-derived DNA Samples 6

Sequencing Output Recommendations for FFPE Samples

After determining the amount of sequencing output required for intact DNA samples to meet the goals of your project, use the guidelines below to determine the amount of extra sequencing output required for FFPE DNA samples.

Samples qualified using ΔΔCq: For samples qualified based on the Cq DNA

integrity score, use the guidelines in Table 44. For example, if your workflow demands 100 Mb output for intact DNA samples to achieve the required coverage, an FFPE sample with Cq score of 1 requires 200– 400 Mb of sequencing output to achieve the same coverage.

Table 44 Recommended sequencing augmentation for FFPE-derived DNA samples

ΔΔCq value Recommended fold increase for FFPE-derived sample

<0.5 No extra sequencing output

between 0.5 and 2 Increase sequencing allocation by 2× to 4×

>2 Increase sequencing allocation by 5× to 10× or more

Samples qualified using DIN: For samples qualified based on the Genomic

DNA ScreenTape assay DIN integrity score, use the guidelines in Table 45. 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 200–400 Mb of sequencing output to achieve the same coverage.

Table 45 Recommended sequencing augmentation for FFPE-derived DNA samples

DIN value Recommended fold increase for FFPE-derived sample

8 No extra sequencing output

between 3 and 8 Increase sequencing allocation by 2× to 4×

<3 Increase sequencing allocation by 5× to 10× or more

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 81

6 Appendix: Using FFPE-derived DNA Samples

Sequencing Output Recommendations for FFPE Samples

82 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

SureSelect XT HS2 DNA System Protocol

7 Reference

Kit Contents 84 SureSelect XT HS2 Index Primer Pair Information 86 Troubleshooting Guide 98 Quick Reference Protocol 102

This chapter contains reference information, including component kit contents, index sequences, troubleshooting information, and a quick- reference protocol for experienced users.

Agilent Technologies

7 Reference

Kit Contents

SureSelect XT HS2 Target Enrichment System Reagent Kits include the component kits listed in Table 46. Detailed contents of each of the multi- part component kits listed in Table 46 are shown in Table 47 through Table 50 on the following pages.

Table 46 Component Kits

Component Kit Name Storage

Condition

Standard Component Modules

SureSelect XT HS2 Library Preparation Kit for ILM (Pre PCR)

SureSelect XT HS2 Index Primer Pairs for ILM (Pre PCR)

SureSelect Target Enrichment Kit, ILM Hyb Module, Box 1 (Post PCR)

SureSelect XT HS2 Target Enrichment Kit ILM Hyb Module, Box 2 (Post PCR)

Optional Component Modules

SureSelect DNA AMPure® XP Beads +4°C 5191-5739*

SureSelect Streptavidin Beads +4°C 5191-5741* 5191-5742

SureSelect Enzymatic Fragmentation Kit

–20°C 5500-0146 5500-0147

–20°C 5191-5687 (Index Pairs 1–16) 5191-5688 (Index Pairs 1–96),

Room Temperature

–20°C 5191-6686 5191-6688

–20°C 5191-4079* 5191-4080

Component Kit Part Number

16 Reaction Kits 96 Reaction Kits

5191-5689 (Index Pairs 97–192),

5191-5690 (Index Pairs 193–288), OR

5191-5691 (Index Pairs 289–384)

5190-9685 5190-9687

5191-5740

†

‡

* Provided with the 16-Reaction SureSelect XT HS2 DNA Starter Kit, p/n G9982A.

† Provided with 96-Reaction Reagent Kit part numbers G9984A, G9984B, G9984C, G9984D.

‡ Purchased separately; not included with SureSelect XT HS2 DNA Reagent Kits, but use is supported by the protocols in this

publication.

84 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Reference 7

Kit Contents

Table 47 SureSelect XT HS2 Library Preparation Kit for ILM (Pre PCR) Content

Kit Component 16 Reaction Kit Format 96 Reaction Kit Format

End Repair-A Tailing Enzyme Mix tube with orange cap tube with orange cap

End Repair-A Tailing Buffer tube with yellow cap bottle

T4 DNA Ligase tube with blue cap tube with blue cap

Ligation Buffer tube with purple cap bottle

SureSelect XT HS2 Adaptor Oligo Mix tube with white cap tube with white cap

Herculase II Fusion DNA Polymerase tube with red cap tube with red cap

5× Herculase II Reaction Buffer with dNTPs tube with clear cap tube with clear cap

Table 48 SureSelect XT HS2 Index Primer Pairs for ILM (Pre PCR) Content

Kit Component 16 Reaction Kit Format 96 Reaction Kit Format

SureSelect XT HS2 Index Primer Pairs for ILM (Pre PCR)

Blue 8-well strip tube (index pairs 1-8), AND

White 8-well strip tube (index pairs 9-16)

Orange 96-well plate (index pairs 1–96), OR

Blue 96-well plate (index pairs 97–192), OR

Green 96-well plate (index pairs 193–288), OR

Red 96-well plate (index pairs 289–384)

Table 49 SureSelect Target Enrichment Kit, ILM Hyb Module Box 1 (Post PCR) Content

Kit Component 16 Reaction Kit Format 96 Reaction Kit Format

SureSelect Binding Buffer bottle bottle

SureSelect Wash Buffer 1 bottle bottle

SureSelect Wash Buffer 2 bottle bottle

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 85

7 Reference

SureSelect XT HS2 Index Primer Pair Information

Table 50 SureSelect XT HS2 Target Enrichment Kit, ILM Hyb Module Box 2 (Post PCR) Content

Kit Component 16 Reaction Kit Format 96 Reaction Kit Format

SureSelect Fast Hybridization Buffer bottle bottle

SureSelect XT HS2 Blocker Mix tube with blue cap tube with blue cap

SureSelect RNase Block tube with purple cap tube with purple cap

SureSelect Post-Capture Primer Mix tube with clear cap tube with clear cap

Herculase II Fusion DNA Polymerase tube with red cap tube with red cap

5× Herculase II Reaction Buffer with dNTPs tube with clear cap tube with clear cap

SureSelect XT HS2 Index Primer Pair Information

The SureSelect XT HS2 Index Primer Pairs are provided pre- combined. Each member of the primer pair contains a unique 8- bp P5 or P7 index, resulting in dual- indexed NGS libraries. The nucleotide sequence of the index portion of each primer is provided in Table 51 through Table 58. See page 72 for sequencing run setup requirements for sequencing libraries using 8- bp indexes.

NOTE

P7 indexes are shown in a single orientation, applicable to any of the supported Illumina platforms. P5 indexes are shown in two orientations for different platforms; check the table column headings carefully before selecting the P5 sequences.

The first P5 index orientation is applicable to the supported platforms NovaSeq 6000 with v1.0 chemistry, MiSeq, and HiSeq 2500. This orientation is also applicable to the HiSeq 2000 platform that is not specifically supported in this user manual.

The second P5 index orientation is applicable to the supported platforms NovaSeq 6000 with v1.5 chemistry, NextSeq 500/550, HiSeq 4000 and HiSeq 3000. This orientation is also applicable to the iSeq 100, MiniSeq, and HiSeq X platforms that are not specifically supported in this user manual.

One primer pair is provided in each well of 8- well strip tubes (16 reaction kits; see Figure 10 for a map) or of 96- well plates (96 reaction kits; see

page 96 through page 97 for plate maps). Each well contains a single- use

aliquot of a specific pair of forward plus reverse primers.

86 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Reference 7

SureSelect XT HS2 Index Primer Pair Information

Table 51 SureSelect XT HS2 Index Primer Pairs 1–48, provided in orange 96-well plate or in strip tubes

Primer

Well P7 Index P5 Index

Pair #

NovaSeq (v1.0 chemistry), MiSeq, HiSeq 2500

1 A01 CAAGGTGA ATGGTTAG CTAACCAT 25 A04 AGATGGAT TGGCACCA TGGTGCCA

2 B01 TAGACCAA CAAGGTGA TCACCTTG 26 B04 GAATTGTG AGATGGAT ATCCATCT

3 C01 AGTCGCGA TAGACCAA TTGGTCTA 27 C04 GAGCACTG GAATTGTG CACAATTC

4 D01 CGGTAGAG AGTCGCGA TCGCGACT 28 D04 GTTGCGGA GAGCACTG CAGTGCTC

5 E01 TCAGCATC AAGGAGCG CGCTCCTT 29 E04 AATGGAAC GTTGCGGA TCCGCAAC

6 F01 AGAAGCAA TCAGCATC GATGCTGA 30 F04 TCAGAGGT AATGGAAC GTTCCATT

7 G01 GCAGGTTC AGAAGCAA TTGCTTCT 31 G04 GCAACAAT TCAGAGGT ACCTCTGA

8 H01 AAGTGTCT GCAGGTTC GAACCTGC 32 H04 GTCGATCG GCAACAAT ATTGTTGC

9 A02 CTACCGAA AAGTGTCT AGACACTT 33 A05 ATGGTAGC GTCGATCG CGATCGAC

10 B02 TAGAGCTC CTACCGAA TTCGGTAG 34 B05 CGCCAATT ATGGTAGC GCTACCAT

11 C02 ATGTCAAG TAGAGCTC GAGCTCTA 35 C05 GACAATTG CGCCAATT AATTGGCG

12 D02 GCATCATA ATGTCAAG CTTGACAT 36 D05 ATATTCCG GACAATTG CAATTGTC

13 E02 GACTTGAC GCATCATA TATGATGC 37 E05 TCTACCTC ATATTCCG CGGAATAT

14 F02 CTACAATG GACTTGAC GTCAAGTC 38 F05 TCGTCGTG TCTACCTC GAGGTAGA

15 G02 TCTCAGCA CTACAATG CATTGTAG 39 G05 ATGAGAAC TCGTCGTG CACGACGA

16 H02 AGACACAC TCTCAGCA TGCTGAGA 40 H05 GTCCTATA ATGAGAAC GTTCTCAT

17 A03 CAGGTCTG AGACACAC GTGTGTCT 41 A06 AATGACCA GTCCTATA TATAGGAC

18 B03 AATACGCG CAGGTCTG CAGACCTG 42 B06 CAGACGCT AATGACCA TGGTCATT

19 C03 GCACACAT AATACGCG CGCGTATT 43 C06 TCGAACTG CAGACGCT AGCGTCTG

20 D03 CTTGCATA GCACACAT ATGTGTGC 44 D06 CGCTTCCA TCGAACTG CAGTTCGA

21 E03 ATCCTCTT CTTGCATA TATGCAAG 45 E06 TATTCCTG CGCTTCCA TGGAAGCG

22 F03 GCACCTAA ATCCTCTT AAGAGGAT 46 F06 CAAGTTAC TATTCCTG CAGGAATA

23 G03 TGCTGCTC GCACCTAA TTAGGTGC 47 G06 CAGAGCAG CAAGTTAC GTAACTTG

24 H03 TGGCACCA TGCTGCTC GAGCAGCA 48 H06 CGCGCAAT CAGAGCAG CTGCTCTG

P5 Index

NovaSeq (v1.5 chemistry), NextSeq, HiSeq 4000, HiSeq 3000

Primer Pair #

Well P7 Index P5 Index

NovaSeq (v1.0 chemistry), MiSeq, HiSeq 2500

P5 Index

NovaSeq (v1.5 chemistry), NextSeq, HiSeq 4000, HiSeq 3000

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 87

7 Reference

SureSelect XT HS2 Index Primer Pair Information

Table 52 SureSelect XT HS2 Index Primer Pairs 49–96, provided in orange 96-well plate

Primer Pair #

Well P7 Index P5 Index

NovaSeq (v1.0 chemistry), MiSeq, HiSeq 2500

P5 Index

NovaSeq (v1.5 chemistry), NextSeq, HiSeq 4000, HiSeq 3000

Primer Pair #

Well P7 Index P5 Index

NovaSeq (v1.0 chemistry), MiSeq, HiSeq 2500

P5 Index

NovaSeq (v1.5 chemistry), NextSeq, HiSeq 4000, HiSeq 3000

49 A07 TGAGGAGT CGCGCAAT ATTGCGCG 73 A10 AACGCATT ATAGTGAC GTCACTAT

50 B07 ATGACGAA TGAGGAGT ACTCCTCA 74 B10 CAGTTGCG AACGCATT AATGCGTT

51 C07 TACGGCGA ATGACGAA TTCGTCAT 75 C10 TGCCTCGA CAGTTGCG CGCAACTG

52 D07 AGCGAGTT TACGGCGA TCGCCGTA 76 D10 AAGGCTTA TGCCTCGA TCGAGGCA

53 E07 TGTATCAC AGCGAGTT AACTCGCT 77 E10 GCAATGAA AAGGCTTA TAAGCCTT

54 F07 GATCGCCT TGTATCAC GTGATACA 78 F10 AAGAACCT GCAATGAA TTCATTGC

55 G07 GACTCAAT GATCGCCT AGGCGATC 79 G10 CTGTGCCT AAGAACCT AGGTTCTT

56 H07 CAGCTTGC GACTCAAT ATTGAGTC 80 H10 TACGTAGC CTGTGCCT AGGCACAG

57 A08 AGCTGAAG CAGCTTGC GCAAGCTG 81 A11 AAGTGGAC TACGTAGC GCTACGTA

58 B08 ATTCCGTG AGCTGAAG CTTCAGCT 82 B11 CAACCGTG AAGTGGAC GTCCACTT

59 C08 TATGCCGC ATTCCGTG CACGGAAT 83 C11 CTGTTGTT CAACCGTG CACGGTTG

60 D08 TCAGCTCA TATGCCGC GCGGCATA 84 D11 GCACGATG CTGTTGTT AACAACAG

61 E08 AACTGCAA TCAGCTCA TGAGCTGA 85 E11 GTACGGAC GCACGATG CATCGTGC

62 F08 ATTAGGAG AACTGCAA TTGCAGTT 86 F11 CTCCAAGC GTACGGAC GTCCGTAC

63 G08 CAGCAATA ATTAGGAG CTCCTAAT 87 G11 TAGTCTGA CTCCAAGC GCTTGGAG

64 H08 GCCAAGCT CAGCAATA TATTGCTG 88 H11 TTCGCCGT TAGTCTGA TCAGACTA

65 A09 TCCGTTAA GCCAAGCT AGCTTGGC 89 A12 GAACTAAG ATACGAAG CTTCGTAT

66 B09 GTGCAACG TCCGTTAA TTAACGGA 90 B12 AAGCCATC GAGATTCA TGAATCTC

67 C09 AGTAACGC GTGCAACG CGTTGCAC 91 C12 AACTCTTG AAGCCATC GATGGCTT

68 D09 CATAGCCA AGTAACGC GCGTTACT 92 D12 GTAGTCAT AACTCTTG CAAGAGTT

69 E09 CACTAGTA CATAGCCA TGGCTATG 93 E12 CTCGCTAG GTAGTCAT ATGACTAC

70 F09 TTAGTGCG CACTAGTA TACTAGTG 94 F12 AGTCTTCA CAGTATCA TGATACTG

71 G09 TCGATACA TTAGTGCG CGCACTAA 95 G12 TCAAGCTA CTTCGTAC GTACGAAG

72 H09 ATAGTGAC TCGATACA TGTATCGA 96 H12 CTTATCCT TCAAGCTA TAGCTTGA

88 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

SureSelect XT HS2 Index Primer Pair Information

Table 53 SureSelect XT HS2 Index Primer Pairs 97–144, provided in blue 96-well plate

Reference 7

Primer Pair #

Well P7 Index P5 Index

NovaSeq (v1.0 chemistry), MiSeq, HiSeq 2500

P5 Index

NovaSeq (v1.5 chemistry), NextSeq, HiSeq 4000, HiSeq 3000

Primer Pair #

Well P7 Index P5 Index

NovaSeq (v1.0 chemistry), MiSeq, HiSeq 2500

P5 Index

NovaSeq (v1.5 chemistry), NextSeq, HiSeq 4000, HiSeq 3000

97 A01 TCATCCTT CTTATCCT AGGATAAG 121 A04 CAGGCAGA AGACGCCT AGGCGTCT

B01 AACACTCT TCATCCTT AAGGATGA 122 B04 TCCGCGAT CAGGCAGA TCTGCCTG

C01 CACCTAGA AACACTCT AGAGTGTT 123 C04 CTCGTACG TCCGCGAT ATCGCGGA

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

D01 AGTTCATG CACCTAGA TCTAGGTG 124 D04 CACACATA CTCGTACG CGTACGAG

E01 GTTGGTGT AGTTCATG CATGAACT 125 E04 CGTCAAGA CACACATA TATGTGTG

F01 GCTACGCA GTTGGTGT ACACCAAC 126 F04 TTCGCGCA CGTCAAGA TCTTGACG

G01 TCAACTGC GCTACGCA TGCGTAGC 127 G04 CGACTACG TTCGCGCA TGCGCGAA

H01 AAGCGAAT TCAACTGC GCAGTTGA 128 H04 GAAGGTAT CGACTACG CGTAGTCG

A02 GTGTTACA AAGCGAAT ATTCGCTT 129 A05 TTGGCATG GAAGGTAT ATACCTTC

B02 CAAGCCAT GTGTTACA TGTAACAC 130 B05 CGAATTCA TTGGCATG CATGCCAA

C02 CTCTCGTG CAAGCCAT ATGGCTTG 131 C05 TTAGTTGC CGAATTCA TGAATTCG

D02 TCGACAAC CTCTCGTG CACGAGAG 132 D05 GATGCCAA TTAGTTGC GCAACTAA

E02 TCGATGTT TCGACAAC GTTGTCGA 133 E05 AGTTGCCG GATGCCAA TTGGCATC

F02 CAAGGAAG TCGATGTT AACATCGA 134 F05 GTCCACCT AGTTGCCG CGGCAACT

G02 ATTGATGC AGAGAATC GATTCTCT 135 G05 ATCAAGGT GTCCACCT AGGTGGAC

H02 TCGCAGAT TTGATGGC GCCATCAA 136 H05 GAACCAGA ATCAAGGT ACCTTGAT

A03 GCAGAGAC TCGCAGAT ATCTGCGA 137 A06 CATGTTCT GAACCAGA TCTGGTTC

B03 CTGCGAGA GCAGAGAC GTCTCTGC 138 B06 TCACTGTG CATGTTCT AGAACATG

C03 CAACCAAC CTGCGAGA TCTCGCAG 139 C06 ATTGAGCT TCACTGTG CACAGTGA

D03 ATCATGCG CAACCAAC GTTGGTTG 140 D06 GATAGAGA ATTGAGCT AGCTCAAT

E03 TCTGAGTC ATCATGCG CGCATGAT 141 E06 TCTAGAGC GATAGAGA TCTCTATC

F03 TCGCCTGT TCTGAGTC GACTCAGA 142 F06 GAATCGCA TCTAGAGC GCTCTAGA

G03 GCGCAATT TCGCCTGT ACAGGCGA 143 G06 CTTCACGT GAATCGCA TGCGATTC

H03 AGACGCCT GCGCAATT AATTGCGC 144 H06 CTCCGGTT CTTCACGT ACGTGAAG

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 89

7 Reference

SureSelect XT HS2 Index Primer Pair Information

Table 54 SureSelect XT HS2 Index Primer Pairs 145–192, provided in blue 96-well plate

Primer Pair #

Well P7 Index P5 Index

NovaSeq (v1.0 chemistry), MiSeq, HiSeq 2500

P5 Index

NovaSeq (v1.5 chemistry), NextSeq, HiSeq 4000, HiSeq 3000

Primer Pair #

Well P7 Index P5 Index

NovaSeq (v1.0 chemistry), MiSeq, HiSeq 2500

P5 Index

NovaSeq (v1.5 chemistry), NextSeq, HiSeq 4000, HiSeq 3000

145 A07 TGTGACTA CTCCGGTT AACCGGAG 169 A10 CGCTCAGA CTAACAAG CTTGTTAG

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

B07 GCTTCCAG TGTGACTA TAGTCACA 170 B10 TAACGACA CGCTCAGA TCTGAGCG

C07 CATCCTGT GCTTCCAG CTGGAAGC 171 C10 CATACTTG TAACGACA TGTCGTTA

D07 GTAATACG CATCCTGT ACAGGATG 172 D10 AGATACGA CATACTTG CAAGTATG

E07 GCCAACAA GTAATACG CGTATTAC 173 E10 AATCCGAC AGATACGA TCGTATCT

F07 CATGACAC GCCAACAA TTGTTGGC 174 F10 TGAAGTAC AATCCGAC GTCGGATT

G07 TGCAATGC CATGACAC GTGTCATG 175 G10 CGAATCAT TGAAGTAC GTACTTCA

H07 CACATTCG TGCAATGC GCATTGCA 176 H10 TGATTGGC CGAATCAT ATGATTCG

A08 CAATCCGA CACATTCG CGAATGTG 177 A11 TCGAAGGA TGATTGGC GCCAATCA

B08 CATCGACG CAATCCGA TCGGATTG 178 B11 CAGTCATT TCGAAGGA TCCTTCGA

C08 GTGCGCTT CATCGACG CGTCGATG 179 C11 CGCGAACA CAGTCATT AATGACTG

D08 ATAGCGTT GTGCGCTT AAGCGCAC 180 D11 TACGGTTG CGCGAACA TGTTCGCG

E08 GAGTAAGA ATAGCGTT AACGCTAT 181 E11 AGAACCGT TACGGTTG CAACCGTA

F08 CTGACACA GAGTAAGA TCTTACTC 182 F11 AGGTGCTT AGAACCGT ACGGTTCT

G08 ATACGTGT CTGACACA TGTGTCAG 183 G11 ATCGCAAC AGGTGCTT AAGCACCT

H08 GACCGAGT ATACGTGT ACACGTAT 184 H11 GCCTCTCA ATCGCAAC GTTGCGAT

A09 GCAGTTAG GACCGAGT ACTCGGTC 185 A12 TCGCGTCA GCCTCTCA TGAGAGGC

B09 CGTTCGTC GCAGTTAG CTAACTGC 186 B12 GAGTGCGT TCGCGTCA TGACGCGA

C09 CGTTAACG CGTTCGTC GACGAACG 187 C12 CGAACACT GCATAAGT ACTTATGC

D09 TCGAGCAT CGTTAACG CGTTAACG 188 D12 TAAGAGTG AGAAGACG CGTCTTCT

E09 GCCGTAAC TCGAGCAT ATGCTCGA 189 E12 TGGATTGA TAAGAGTG CACTCTTA

F09 GAGCTGTA GCCGTAAC GTTACGGC 190 F12 AGGACATA TGGATTGA TCAATCCA

G09 AGGAAGAT GAGCTGTA TACAGCTC 191 G12 GACATCCT AGGACATA TATGTCCT

H09 CTAACAAG AGGAAGAT ATCTTCCT 192 H12 GAAGCCTC GACATCCT AGGATGTC

90 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

SureSelect XT HS2 Index Primer Pair Information

Table 55 SureSelect XT HS2 Index Primer Pairs 193–240, provided in green 96-well plate

Reference 7

Primer Pair #

Well P7 Index P5 Index

NovaSeq (v1.0 chemistry), MiSeq, HiSeq 2500

P5 Index

NovaSeq (v1.5 chemistry), NextSeq, HiSeq 4000, HiSeq 3000

Primer Pair #

Well P7 Index P5 Index

NovaSeq (v1.0 chemistry), MiSeq, HiSeq 2500

P5 Index

NovaSeq (v1.5 chemistry), NextSeq, HiSeq 4000, HiSeq 3000

193 A01 GTCTCTTC GAAGCCTC GAGGCTTC 217 A04 GCGGTATG CACGAGCT AGCTCGTG

194 B01 AGTCACTT GTCTCTTC GAAGAGAC 218 B04 TCTATGCG GCGGTATG CATACCGC

195 C01 AGCATACA AGTCACTT AAGTGACT 219 C04 AGGTGAGA TCTATGCG CGCATAGA

196 D01 TCAGACAA AGCATACA TGTATGCT 220 D04 CACAACTT AGGTGAGA TCTCACCT

197 E01 TTGGAGAA TCAGACAA TTGTCTGA 221 E04 TTGTGTAC CACAACTT AAGTTGTG

198 F01 TTAACGTG TTGGAGAA TTCTCCAA 222 F04 TCACAAGA TTGTGTAC GTACACAA

199 G01 CGTCTGTG TTAACGTG CACGTTAA 223 G04 GAAGACCT TCACAAGA TCTTGTGA

200 H01 AACCTAAC CGTCTGTG CACAGACG 224 H04 AGTTCTGT GAAGACCT AGGTCTTC

201 A02 AGAGTGCT AACCTAAC GTTAGGTT 225 A05 GCAGTGTT AGTTCTGT ACAGAACT

202 B02 TTATCTCG AGAGTGCT AGCACTCT 226 B05 AGGCATGC GCAGTGTT AACACTGC

203 C02 CATCAGTC TTATCTCG CGAGATAA 227 C05 AAGGTACT AGGCATGC GCATGCCT

204 D02 AAGCACAA CATCAGTC GACTGATG 228 D05 CACTAAGT AAGGTACT AGTACCTT

205 E02 CAGTGAGC AAGCACAA TTGTGCTT 229 E05 GAGTCCTA CACTAAGT ACTTAGTG

206 F02 GTCGAAGT CAGTGAGC GCTCACTG 230 F05 AGTCCTTC GAGTCCTA TAGGACTC

207 G02 TCTCATGC GTCGAAGT ACTTCGAC 231 G05 TTAGGAAC AGTCCTTC GAAGGACT

208 H02 CAGAAGAA TCTCATGC GCATGAGA 232 H05 AAGTCCAT TTAGGAAC GTTCCTAA

209 A03 CGGATAGT CAGAAGAA TTCTTCTG 233 A06 GAATACGC AAGTCCAT ATGGACTT

210 B03 CACGTGAG CGGATAGT ACTATCCG 234 B06 TCCAATCA GAATACGC GCGTATTC

211 C03 TACGATAC CACGTGAG CTCACGTG 235 C06 CGACGGTA TCCAATCA TGATTGGA

212 D03 CGCATGCT TACGATAC GTATCGTA 236 D06 CATTGCAT CGACGGTA TACCGTCG

213 E03 GCTTGCTA CGCATGCT AGCATGCG 237 E06 ATCTGCGT CATTGCAT ATGCAATG

214 F03 GAACGCAA GCTTGCTA TAGCAAGC 238 F06 GTACCTTG ATCTGCGT ACGCAGAT

215 G03 ATCTACCA GAACGCAA TTGCGTTC 239 G06 GAGCATAC GTACCTTG CAAGGTAC

216 H03 CACGAGCT ATCTACCA TGGTAGAT 240 H06 TGCTTACG GAGCATAC GTATGCTC

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 91

7 Reference

SureSelect XT HS2 Index Primer Pair Information

Table 56 SureSelect XT HS2 Index Primer Pairs 241–288, provided in green 96-well plate

Primer Pair #

Well P7 Index P5 Index

NovaSeq (v1.0 chemistry), MiSeq, HiSeq 2500

P5 Index

NovaSeq (v1.5 chemistry), NextSeq, HiSeq 4000, HiSeq 3000

Primer Pair #

Well P7 Index P5 Index

NovaSeq (v1.0 chemistry), MiSeq, HiSeq 2500

P5 Index

NovaSeq (v1.5 chemistry), NextSeq, HiSeq 4000, HiSeq 3000

241 A07 AAGAGACA TGCTTACG CGTAAGCA 265 A10 CAATGCTG CATGAATG CATTCATG

242 B07 TAGCTATG AAGAGACA TGTCTCTT 266 B10 CTTGATCA CAATGCTG CAGCATTG

243 C07 TCTGCTAC TAGCTATG CATAGCTA 267 C10 GCGAATTA CTTGATCA TGATCAAG

244 D07 GTCACAGA TCTGCTAC GTAGCAGA 268 D10 GTTCGAGC GCGAATTA TAATTCGC

245 E07 CGATTGAA GTCACAGA TCTGTGAC 269 E10 GCCAGTAG GTTCGAGC GCTCGAAC

246 F07 GAGAGATT CGATTGAA TTCAATCG 270 F10 AAGGTCGA GCCAGTAG CTACTGGC

247 G07 TCATACCG GAGAGATT AATCTCTC 271 G10 AGTGAAGT CACTTATG CATAAGTG

248 H07 TCCGAACT TCATACCG CGGTATGA 272 H10 GTTGCAAG ATAACGGC GCCGTTAT

249 A08 AGAGAGAA TCCGAACT AGTTCGGA 273 A11 AGCCGGAA GTTGCAAG CTTGCAAC

250 B08 GATCGTTA AGAGAGAA TTCTCTCT 274 B11 AACAGCCG AGCCGGAA TTCCGGCT

251 C08 GCGCTAGA GATCGTTA TAACGATC 275 C11 CTAGTGTA AACAGCCG CGGCTGTT

252 D08 ATGACTCG GCGCTAGA TCTAGCGC 276 D11 GAGGCTCT CTAGTGTA TACACTAG

253 E08 CAATAGAC ATGACTCG CGAGTCAT 277 E11 CTCCGCAA GAGGCTCT AGAGCCTC

254 F08 CGATATGC CAATAGAC GTCTATTG 278 F11 CGCTATTG CTCCGCAA TTGCGGAG

255 G08 GTCAGAAT CGATATGC GCATATCG 279 G11 GTGTTGAG CGCTATTG CAATAGCG

256 H08 CATAAGGT GCACTACT AGTAGTGC 280 H11 TCACCGAC GTGTTGAG CTCAACAC

257 A09 TGTTGGTT GATTCGGC GCCGAATC 281 A12 CGGTAATC TCACCGAC GTCGGTGA

258 B09 ATACTCGC TGTTGGTT AACCAACA 282 B12 GTGACTGC CGGTAATC GATTACCG

259 C09 AATGCTAG ATACTCGC GCGAGTAT 283 C12 CGACTTGT GTGACTGC GCAGTCAC

260 D09 GCCTAGGA AATGCTAG CTAGCATT 284 D12 GATAGGAC CGACTTGT ACAAGTCG

261 E09 GCAACCGA GCCTAGGA TCCTAGGC 285 E12 AAGTACTC GATAGGAC GTCCTATC

262 F09 ATACTGCA GCAACCGA TCGGTTGC 286 F12 GCTCTCTC AAGTACTC GAGTACTT

263 G09 TCTCCTTG ATACTGCA TGCAGTAT 287 G12 CTACCAGT GCTCTCTC GAGAGAGC

264 H09 CATGAATG TCTCCTTG CAAGGAGA 288 H12 GATGAGAT CTACCAGT ACTGGTAG

92 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

SureSelect XT HS2 Index Primer Pair Information

Table 57 SureSelect XT HS2 Index Primer Pairs 289–336, provided in red 96-well plate

Reference 7

Primer Pair #

Well P7 Index P5 Index

NovaSeq (v1.0 chemistry), MiSeq, HiSeq 2500

P5 Index

NovaSeq (v1.5 chemistry), NextSeq, HiSeq 4000, HiSeq 3000

Primer Pair #

Well P7 Index P5 Index

NovaSeq (v1.0 chemistry), MiSeq, HiSeq 2500

P5 Index

NovaSeq (v1.5 chemistry), NextSeq, HiSeq 4000, HiSeq 3000

289 A01 AGATAGTG GATGAGAT ATCTCATC 313 A04 AGCTACAT GATCCATG CATGGATC

290 B01 AGAGGTTA AGATAGTG CACTATCT 314 B04 CGCTGTAA AGCTACAT ATGTAGCT

291 C01 CTGACCGT AGAGGTTA TAACCTCT 315 C04 CACTACCG CGCTGTAA TTACAGCG

292 D01 GCATGGAG CTGACCGT ACGGTCAG 316 D04 GCTCACGA CACTACCG CGGTAGTG

293 E01 CTGCCTTA GCATGGAG CTCCATGC 317 E04 TGGCTTAG GCTCACGA TCGTGAGC

294 F01 GCGTCACT CTGCCTTA TAAGGCAG 318 F04 TCCAGACG TGGCTTAG CTAAGCCA

295 G01 GCGATTAC GCGTCACT AGTGACGC 319 G04 AGTGGCAT TCCAGACG CGTCTGGA

296 H01 TCACCACG GCGATTAC GTAATCGC 320 H04 TGTACCGA AGTGGCAT ATGCCACT

297 A02 AGACCTGA TCACCACG CGTGGTGA 321 A05 AAGACTAC TGTACCGA TCGGTACA

298 B02 GCCGATAT AGACCTGA TCAGGTCT 322 B05 TGCCGTTA AAGACTAC GTAGTCTT

299 C02 CTTATTGC GCCGATAT ATATCGGC 323 C05 TTGGATCT TGCCGTTA TAACGGCA

300 D02 CGATACCT CTTATTGC GCAATAAG 324 D05 TCCTCCAA TTGGATCT AGATCCAA

301 E02 CTCGACAT CGATACCT AGGTATCG 325 E05 CGAGTCGA TCCTCCAA TTGGAGGA

302 F02 GAGATCGC CTCGACAT ATGTCGAG 326 F05 AGGCTCAT CGAGTCGA TCGACTCG

303 G02 CGGTCTCT GAGATCGC GCGATCTC 327 G05 GACGTGCA AGGCTCAT ATGAGCCT

304 H02 TAACTCAC CGGTCTCT AGAGACCG 328 H05 GAACATGT GACGTGCA TGCACGTC

305 A03 CACAATGA TAACTCAC GTGAGTTA 329 A06 AATTGGCA GAACATGT ACATGTTC

306 B03 GACTGACG CACAATGA TCATTGTG 330 B06 TGGAGACT AATTGGCA TGCCAATT

307 C03 CTTAAGAC GACTGACG CGTCAGTC 331 C06 AACTCACA TGGAGACT AGTCTCCA

308 D03 GAGTGTAG CTTAAGAC GTCTTAAG 332 D06 GTAGACTG AACTCACA TGTGAGTT

309 E03 TGCACATC GAGTGTAG CTACACTC 333 E06 CGTAGTTA GTAGACTG CAGTCTAC

310 F03 CGATGTCG TGCACATC GATGTGCA 334 F06 CGTCAGAT CGTAGTTA TAACTACG

311 G03 AACACCGA CGATGTCG CGACATCG 335 G06 AACGGTCA CGTCAGAT ATCTGACG

312 H03 GATCCATG AACACCGA TCGGTGTT 336 H06 GCCTTCAT AACGGTCA TGACCGTT

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 93

7 Reference

SureSelect XT HS2 Index Primer Pair Information

Table 58 SureSelect XT HS2 Index Primer Pairs 337–384, provided in red 96-well plate

Primer Pair #

Well P7 Index P5 Index

NovaSeq (v1.0 chemistry), MiSeq, HiSeq 2500

P5 Index

NovaSeq (v1.5 chemistry), NextSeq, HiSeq 4000, HiSeq 3000

Primer Pair #

Well P7 Index P5 Index

NovaSeq (v1.0 chemistry), MiSeq, HiSeq 2500

P5 Index

NovaSeq (v1.5 chemistry), NextSeq, HiSeq 4000, HiSeq 3000

337 A07 TGAGACGC GCCTTCAT ATGAAGGC 361 A10 CTGAGCTA GCACAGTA TACTGTGC

338 B07 CATCGGAA TGAGACGC GCGTCTCA 362 B10 CTTGCGAT CTGAGCTA TAGCTCAG

339 C07 TAGGACAT CATCGGAA TTCCGATG 363 C10 GAAGTAGT CTTGCGAT ATCGCAAG

340 D07 AACACAAG TAGGACAT ATGTCCTA 364 D10 GTTATCGA GAAGTAGT ACTACTTC

341 E07 TTCGACTC AACACAAG CTTGTGTT 365 E10 TGTCGTCG GTTATCGA TCGATAAC

342 F07 GTCGGTAA TTCGACTC GAGTCGAA 366 F10 CGTAACTG TGTCGTCG CGACGACA

343 G07 GTTCATTC GTCGGTAA TTACCGAC 367 G10 GCATGCCT CGTAACTG CAGTTACG

344 H07 AAGCAGTT GTTCATTC GAATGAAC 368 H10 TCGTACAC GCATGCCT AGGCATGC

345 A08 ATAAGCTG AAGCAGTT AACTGCTT 369 A11 CACAGGTG TCGTACAC GTGTACGA

346 B08 GCTTAGCG ATAAGCTG CAGCTTAT 370 B11 AGCAGTGA CACAGGTG CACCTGTG

347 C08 TTCCAACA GCTTAGCG CGCTAAGC 371 C11 ATTCCAGA AGCAGTGA TCACTGCT

348 D08 TACCGCAT TTCCAACA TGTTGGAA 372 D11 TCCTTGAG ATTCCAGA TCTGGAAT

349 E08 AGGCAATG TACCGCAT ATGCGGTA 373 E11 ATACCTAC TCCTTGAG CTCAAGGA

350 F08 GCCTCGTT AGGCAATG CATTGCCT 374 F11 AGACCATT ATACCTAC GTAGGTAT

351 G08 CACGGATC GCCTCGTT AACGAGGC 375 G11 CGTAAGCA AGACCATT AATGGTCT

352 H08 GAGACACG CACGGATC GATCCGTG 376 H11 TCTGTCAG CGTAAGCA TGCTTACG

353 A09 AGAGTAAG GAGACACG CGTGTCTC 377 A12 CACAGACT TCTGTCAG CTGACAGA

354 B09 AGTACGTT AGAGTAAG CTTACTCT 378 B12 GTCGCCTA CACAGACT AGTCTGTG

355 C09 AACGCTGC AGTACGTT AACGTACT 379 C12 TGCGCTCT GTCGCCTA TAGGCGAC

356 D09 GTAGAGCA AACGCTGC GCAGCGTT 380 D12 GCTATAAG TGCGCTCT AGAGCGCA

357 E09 TCCTGAGA GTAGAGCA TGCTCTAC 381 E12 CAACAACT GCTATAAG CTTATAGC

358 F09 CTGAATAG TCCTGAGA TCTCAGGA 382 F12 AGAGAATC CTCTCACT AGTGAGAG

359 G09 CAAGACTA CTGAATAG CTATTCAG 383 G12 TAATGGTC AGACGAGC GCTCGTCT

360 H09 GCACAGTA CAAGACTA TAGTCTTG 384 H12 GTTGTATC TAATGGTC GACCATTA

94 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Index Primer Pair Strip Tube and Plate Maps

SureSelect XT HS2 Index Primer Pairs 1- 16 (provided with 16 reaction kits) are supplied in a set of two 8- well strip tubes as detailed below.

Figure 10 Map of the SureSelect XT HS2 Index Primer Pairs for ILM (Pre PCR) strip tubes

provided with 16 reaction kits

The blue strip contains Index Primer Pairs 1- 8, with pair #1 supplied in the well proximal to the numeral 1 etched on the strip’s plastic end tab.

The white strip contains Index Primer Pairs 9- 16, with pair #9 supplied in the well proximal to the numeral 9 etched on the strip’s plastic end tab.

When using the strip tube- supplied index primer pairs in the library preparation protocol, re- seal any unused wells using the fresh foil seal strips provided with the index strip tubes.

Reference 7

See Table 59 on page 96 through Table 62 on page 97 for plate maps showing positions of the SureSelect XT HS2 Index Primer Pairs provided with 96 reaction kits.

CAUTION

The SureSelect XT HS2 Index Primer Pairs are provided in single-use aliquots. To avoid cross-contamination of libraries, use each well in only one library preparation reaction. Do not retain and re-use any residual volume for subsequent experiments.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 95

7 Reference

Index Primer Pair Strip Tube and Plate Maps

Table 59 Plate map for SureSelect XT HS2 Index Primer Pairs 1-96, provided in orange plate

1 2 3 4 5 6 7 8 9 10 11 12

A 1 9 17 25 33 41 49 57 65 73 81 89

B 2 10182634 42 50 58 66 74 82 90

C 3 11192735 43 51 59 67 75 83 91

D 4 12202836 44 52 60 68 76 84 92

E 5 13212937 45 53 61 69 77 85 93

F 6 14223038 46 54 62 70 78 86 94

G 7 15233139 47 55 63 71 79 87 95

H 8 16243240 48 56 64 72 80 88 96

Table 60 Plate map for SureSelect XT HS2 Index Primer Pairs 97-192, provided in blue plate

1 2 3 4 5 6 7 8 9 10 11 12

A 97 105 113 121 129 137 145 153 161 169 177 185

B 98 106 114 122 130 138 146 154 162 170 178 186

C 99 107 115 123 131 139 147 155 163 171 179 187

D 100 108 116 124 132 140 148 156 164 172 180 188

E 101 109 117 125 133 141 149 157 165 173 181 189

F 102 1110 118 126 134 142 150 158 166 174 182 190

G 103 111 119 127 135 143 151 159 167 175 183 191

H 104 112 120 128 136 144 152 160 168 176 184 192

96 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Reference 7

Index Primer Pair Strip Tube and Plate Maps

Table 61 Plate map for SureSelect XT HS2 Index Primer Pairs 193-288, provided in green plate

1 2 3 4 5 6 7 8 9 10 11 12

A 193 201 209 217 225 233 241 249 257 265 273 281

B 194 202 210 218 226 234 242 250 258 266 274 282

C 195 203 211 219 227 235 243 251 259 267 275 283

D 196 204 212 220 228 236 244 252 260 268 276 284

E 197 205 213 221 229 237 245 253 261 269 277 285

F 198 206 214 222 230 238 246 254 262 270 278 286

G 199 207 215 223 231 239 247 255 263 271 279 287

H 200 208 216 224 232 240 248 256 264 272 280 288

Table 62 Plate map for SureSelect XT HS2 Index Primer Pairs 289-384, provided in red plate

1 2 3 4 5 6 7 8 9 10 11 12

A 289 297 305 313 321 329 337 345 353 361 369 377

B 290 298 306 314 322 330 338 346 354 362 370 378

C 291 299 307 315 323 331 339 347 355 363 371 379

D 292 300 308 316 324 332 340 348 356 364 372 380

E 293 301 309 317 325 333 341 349 357 365 373 381

F 294 302 310 318 326 334 342 350 358 366 374 382

G 295 303 311 319 327 335 343 351 359 367 375 383

H 296 304 312 320 328 336 344 352 360 368 376 384

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 97

7 Reference

Troubleshooting Guide

If recovery of gDNA from samples is low

✔ Using excess tissue for gDNA isolation can reduce yield. Use only the

amount of each specific tissue type recommended by the gDNA isolation protocol.

✔ Tissue sample lysis may not have been optimal during gDNA isolation.

Monitor the extent of sample lysis during the Proteinase K digestion at 56°C by gently pipetting the digestion reaction every 20–30 minutes, visually inspecting the solution for the presence of tissue clumps. If clumps are still present after the 1- hour incubation at 56°C, add another 10 µl of Proteinase K and continue incubating at 56°C, with periodic mixing and visual inspections, for up to two additional hours. When the sample no longer contains clumps of tissue, move the sample to room temperature until lysis is complete for the remaining samples. Do not over- digest. Individual samples may be kept at room temperature for up to 2 hours before resuming the protocol. Do not exceed 3 hours incubation at 56°C for any sample.

If yield of pre-capture libraries is low

✔ The library preparation protocol includes specific thawing, temperature

control, pipetting, and mixing instructions which are required for optimal performance of the highly viscous buffer and enzyme solutions used in the protocol. Be sure to adhere to all instructions when setting up the reactions.

✔ Ensure that the ligation master mix (see page 33) is kept at room

temperature for 30–45 minutes before use.

✔ PCR cycle number may require optimization. Repeat library preparation

for the sample, increasing the pre- capture PCR cycle number by 1 to 2 cycles. If a high molecular weight peak (>500 bp) is observed in the electropherogram for a sample with low yield, the DNA may be overamplified. Repeat library preparation for the sample, decreasing the pre- capture PCR cycle number by 1 to 3 cycles.

✔ DNA isolated from degraded samples, including FFPE tissue samples,

may be over- fragmented or have modifications that adversely affect library preparation processes. Use the Agilent NGS FFPE QC Kit to determine the precise quantity of amplifiable DNA in the sample and allow direct normalization of input DNA amount.

98 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Reference 7

Troubleshooting Guide

✔ Performance of the solid- phase reversible immobilization (SPRI)

purification step may be poor. Verify the expiration date for the vial of AMPure XP beads used for purification. Adhere to all bead storage and handling conditions recommended by the manufacturer. Ensure that the beads are kept at room temperature for at least 30 minutes before use. Use freshly- prepared 70% ethanol for each SPRI procedure.

✔ DNA elution during SPRI purification steps may be incomplete. Ensure

that the AMPure XP beads are not overdried just prior to sample elution.

If solids observed in the End Repair-A Tailing Buffer

✔ Vortex the solution at high speed until the solids are dissolved. The

observation of solids when first thawed does not impact performance, but it is important to mix the buffer until all solutes are dissolved.

If pre-capture library fragment size is larger than expected in electropherograms

✔ Shearing may not be optimal. For intact, high- quality DNA samples,

ensure that shearing is completed using the two- round shearing protocol provided, including all spinning and vortexing steps.

✔ Any bubbles present on the microTUBE filament may disrupt complete

shearing. Spin the microTUBE for 30 seconds before the first round of shearing to ensure that any bubbles are released.

If pre-capture library fragment size is different than expected in electropherograms

✔ FFPE DNA pre- capture libraries may have a smaller fragment size

distribution due to the presence of DNA fragments in the input DNA that are smaller than the target DNA shear size.

✔ DNA fragment size selection during SPRI purification depends upon

using the correct ratio of sample to AMPure XP beads. Before removing an aliquot of beads for the purification step, mix the beads until the suspension appears homogeneous and consistent in color and verify that you are using the bead volume recommended for pre- capture purification on page 42.

SureSelect XT HS2 DNA Library Preparation and Target Enrichment 99

7 Reference

Troubleshooting Guide

If low molecular weight adaptor-dimer peak is present in pre-capture library electropherograms

✔ The presence of a low molecular weight peak, in addition to the

expected peak, indicates the presence of adaptor- dimers in the library. It is acceptable to proceed to target enrichment with library samples for which adaptor- dimers are observed in the electropherogram at low abundance, similar to the example shown in Figure 4 on page 46. The presence of excessive adaptor- dimers in the samples may be associated with reduced yield of pre- capture libraries. If excessive adaptor- dimers are observed, verify that the adaptor ligation protocol is being performed as directed on page 36. In particular, ensure that the Ligation master mix is mixed with the sample prior to adding the Adaptor Oligo Mix to the mixture. Do not add the Ligation master mix and the Adaptor Oligo Mix to the sample in a single step.

✔ For whole- genome sequencing (not specifically supported by this

protocol), samples with an adaptor- dimer peak must be subjected to an additional round of SPRI- purification. To complete, first dilute the sample to 50 µl with nuclease free water, then follow the SPRI purification procedure on page 42.

If yield of post-capture libraries is low

✔ PCR cycle number may require optimization. Repeat library preparation

and target enrichment for the sample, increasing the post- capture PCR cycle number by 1 to 2 cycles.

✔ The RNA Probe used for hybridization may have been compromised.

Verify the expiration date on the Probe vial or Certificate of Analysis. Adhere to the recommended storage and handling conditions. Ensure that the Capture Library Hybridization Mix is prepared immediately before use, as directed on page 53, and that solutions containing the Probe are not held at room temperature for extended periods.

If post-capture library fragment size is different than expected in electropherograms

✔ DNA fragment size selection during SPRI purification depends upon

100 SureSelect XT HS2 DNA Library Preparation and Target Enrichment

Agilent XT HS2 DNA User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

Overview of the Workflow

Procedural Notes

Safety Notes

Materials Required

Optional Materials

Step 1. Prepare and analyze quality of genomic DNA samples

Preparation of high-quality gDNA from fresh biological samples

Preparation and qualification of gDNA from FFPE samples

Step 2. Fragment the DNA

Method 1: Mechanical DNA Shearing using Covaris

Method 2: Enzymatic DNA Fragmentation

Step 1. Prepare the Ligation master mix

Step 2. Repair and dA-Tail the DNA ends

Step 3. Ligate the molecular-barcoded adaptor

Step 4. Purify the sample using AMPure XP beads

Step 5. Amplify the adaptor-ligated library

Step 6. Purify the amplified library with AMPure XP beads

Step 7. Assess quality and quantity

Step 1. Hybridize DNA libraries to the probe

Step 2. Prepare streptavidin-coated magnetic beads

Step 3. Capture the hybridized DNA using streptavidin-coated beads

Step 1. Amplify the captured libraries

Step 2. Purify the amplified captured libraries using AMPure XP beads

Step 3. Assess sequencing library DNA quantity and quality

Step 4. Pool samples for multiplexed sequencing

Step 5. Prepare sequencing samples

Step 6. Do the sequencing run and analyze the data

Sequence analysis resources

Protocol modifications for FFPE Samples

Methods for FFPE Sample Qualification

Sequencing Output Recommendations for FFPE Samples

Kit Contents

SureSelect XT HS2 Index Primer Pair Information

Index Primer Pair Strip Tube and Plate Maps

Troubleshooting Guide