Agilent XT HS2 DNA User Manual

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.

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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

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