Contents

Agilent SureSelect XT HS Sequencer Protocol Manual V2 PDF

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Summary of Content for Agilent SureSelect XT HS Sequencer Protocol Manual V2 PDF

SureSelectXT HS Target Enrichment System For Illumina Multiplexed Sequencing Platforms

Protocol Version F0, September 2022

SureSelect platform manufactured with Agilent SurePrint Technology

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

Agilent Technologies

Notices Agilent Technologies, Inc. 2017-2022

No part of this manual may be reproduced in any form or by any means (including elec- tronic storage and retrieval or translation into a foreign language) without prior agree- ment and written consent from Agilent Technologies, Inc. as governed by United States and international copyright laws.

Manual Part Number G9702-90000

Edition Version F0, September 2022

Printed in USA

Agilent Technologies, Inc. 5301 Stevens Creek Blvd

Warranty The material contained in this document is provided as is, and is subject to being changed, with- out notice, in future editions. Fur- ther, to the maximum extent permitted by applicable law, Agi- lent 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 merchant- ability and fitness for a particular purpose. Agilent shall not be lia- ble for errors or for incidental or consequential damages in con- nection 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 doc- ument that conflict with these terms, the warranty terms in the separate agreement shall control.

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2 SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

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.

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing 3

In this Guide...

4 SureS

This guide provides an optimized protocol for preparation of target- enriched Illumina paired- end multiplexed sequencing libraries using SureSelectXT HS Reagent Kits.

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 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.

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.

electXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Whats New in Version F0

SureSelectXT HS Target Enrichment S

Support for SureSelect XT HS Human All Exon V8+UTR and SureSelect XT HS Human All Exon V8+NCV Probes. See Table 3 on page 14 for ordering information.

Design ID information added to Table 3 on page 14 for pre- designed SureSelect probes.

Updates to tube cap strip recommendations from domed caps to flat/domed caps based on specific cycler recommendations (see Table 5 on page 16 and related updates on page 12, page 19, and page 56)

Update to enzymatic fragmentation instructions in step 1 on page 29 for optional use of 1X Low TE Buffer as solvent for DNA samples.

Updates to recommended reagent volumes for 24 reaction runs in Table 15 on page 35 and Table 17 on page 36.

New AmpPure XP bead purification protocol parameter summary tables for experienced users (see Table 19 on page 39, Table 24 on page 44, and Table 35 on page 65).

New Note on cross- platform index equivalence on page 41.

Updates to downstream sequencing support information (see page 75 to page 86). Key updates include guidelines for demultiplexing using Illuminas BCL Convert software (see page 76) and support for Agilents new CReaK tool, replacing the LocatIt tool in AGeNT v3.0 (see page 86).

Support for use of Agilents Alissa Reporter software for SureSelect XT HS DNA library sequence pre- processing and human germline DNA variant analysis (see page 77 and page 85).

Update to Troubleshooting on page 100 on thermal cycler block configuration requirements for efficient heating of SureSelect Wash Buffer 2.

Update to Notice to Purchaser on page 2.

ystem for Illumina Multiplexed Sequencing 5

Whats New in Version E1

6 SureS

Support for SureSelect XT HS Human All Exon V8 Probe (see Table 3 on page 14)

Updates to the Hybridization and Capture chapter on page 51 through page 57, including updates to Table 26 on page 53 and additional minor updates throughout the chapter.

Update to Note on page 26 and new footnote to Table 12 on page 30 on impacts of initial FFPE DNA sample fragment size on final library fragment size distribution.

Whats New in Version E0

Addition of optional Enzymatic DNA Fragmentation

protocol (see page 29 through page 31 for the protocol; also see Table 38 on page 88, Table 41 on page 92, and Troubleshooting on page 97)

New chapter Preparation and Fragmentation of Input DNA starting on page 21 (includes input DNA preparation and mechanical shearing information previously found in Sample Preparation chapter)

Addition of hybridization temperature considerations for probes designed for use with the SureSelect XT system (see footnote to Table 26 on page 53)

Minor updates to instructions in the Hybridization and Capture chapter, including Note on page 53, step 3 on page 53, step 5 on page 54, and step 1 on page 58

Updates to support for optional use of molecular barcodes (see page 21, legend to Figure 14 on page 74, and Note on page 76)

Updates to downstream sequencing platform and kit support information in Table 37 on page 75

Addition of small volume spectrophotometer to Table 5 on page 16

Update to description of flat strip caps in Table 7 on page 19

electXT HS Target Enrichment System for Illumina Multiplexed Sequencing

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 23

Step 2. Fragment the DNA 26

Method 1: Mechanical DNA Shearing using Covaris 26 Method 2: Enzymatic DNA Fragmentation 29

3 Library Preparation 33

Step 1. Repair and dA-Tail the DNA ends 34

Step 2. Ligate the molecular-barcoded adaptor 38

Step 3. Purify the sample using AMPure XP beads 39

Step 4. Amplify the adaptor-ligated library 41

Step 5. Purify the amplified library with AMPure XP beads 44

Step 6. Assess quality and quantity 46

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing 7

Contents

4 Hybridization and Capture 51

Step 1. Hybridize DNA samples to the probe 52

Step 2. Prepare streptavidin-coated magnetic beads 57

Step 3. Capture the hybridized DNA using streptavidin-coated beads 58

5 Post-Capture Sample Processing for Multiplexed Sequencing 61

Step 1. Amplify the captured libraries 62

Step 2. Purify the amplified captured libraries using AMPure XP beads 65

Step 3. Assess sequencing library DNA quantity and quality 67

Step 4. Pool samples for multiplexed sequencing 72

Step 5. Prepare sequencing samples 74

Step 6. Do the sequencing run and analyze the data 76

Sequence analysis resources 84

6 Appendix: Using FFPE-derived DNA Samples 87

Protocol modifications for FFPE Samples 88

Methods for FFPE Sample Qualification 88

Sequencing Output Recommendations for FFPE Samples 89

7 Reference 91

Kit Contents 92

Nucleotide Sequences of SureSelect XT HS Indexes 96

Troubleshooting Guide 97

Quick Reference Protocol 102

8 SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

SureSelectXT HS Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library 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 have the most current protocol. Go to agilent.com and search for G9702- 90000.

To prepare libraries for Agilent SureSelect Cancer All- In- One assays, use the protocols detailed in this publication, while implementing the considerations provided in the SureSelect Cancer All- In- One Target Enrichment Product Overview Guide (publication G9702- 90100).

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

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.

NOTE

NOTE

9Agilent Technologies

1 Before You Begin Overview of the Workflow

Overview of the Workflow

10

The SureSelectXT HS target enrichment 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.

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Before You Begin 1 Overview of the Workflow

SureSelectXT HS Tar

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

QC using Bioanalyzer or TapeStation platform and sample pooling

1.5 hours

get Enrichment System for Illumina Multiplexed Sequencing 11

1 Before You Begin Procedural Notes

Procedural Notes

12

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. 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 20C, are marked in the protocol. Do not subject the samples to multiple freeze/thaw cycles.

Safety Notes

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

CAUTION

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Before You Begin 1 Materials Required

Materials Required

SureSelectXT HS Tar

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

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 workflow, first select the preferred SureSelect XT HS Reagent Kit format from Table 2 below and a compatible target enrichment Probe from Table 3 on page 14. Then refer to Table 4 through Table 6 for additional materials needed to complete the protocols according to your DNA sample type/fragmentation method.

Table 2 SureSelect XT HS Reagent Kit Varieties

Description Kit Part Number

16 Reaction Kit*

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

96 Reaction Kit

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

SureSelect XT HS Reagent Kit for Illumina (ILM) platform

Compatible with HiSeq, MiSeq, NextSeq 500, and NovaSeq 6000 platforms.

G9702A (with Index Primers 116)

G9702B (with Index Primers 1732)

G9702C (with Index Primers 132)**

** Includes 3 single-reaction vials of each index primer 132, for a total of 96 library preparations.

get Enrichment System for Illumina Multiplexed Sequencing 13

1 Before You Begin Materials Required

14

*

Table 3 Compatible Probes

Probe Capture Library Design ID Part Number/Ordering Information

Pre-designed Probes 16 Reactions 96 Reactions

SureSelect XT HS Human All Exon V8 S33266340 5191-6873 5191-6874

SureSelect XT HS Human All Exon V8+UTR S33613271 5191-7401 5191-7402

SureSelect XT HS Human All Exon V8+NCV S33699751 5191-7407 5191-7408

SSel XT HS and XT Low Input Human All Exon V7 S31285117 5191-4028 5191-4029

SureSelect XT Human All Exon V6 S07604514 5190-8863 5190-8864

SureSelect XT Human All Exon V6 + UTRs S07604624 5190-8881 5190-8882

SureSelect XT Clinical Research Exome V2 S30409818 5190-9491 5190-9492

ClearSeq Comprehensive Cancer XT 0425761 5190-8011 5190-8012

Custom Probes

SureSelect Custom Tier1 1499 kb Please visit the SureDesign website to design 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.

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 1224 Mb

Pre-designed Probes customized with additional Plus custom content

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

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.

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

SureSelect XT Human All Exon V6 Plus 1

SureSelect XT Human All Exon V6 Plus 2

SureSelect XT Clinical Research Exome V2 Plus 1

SureSelect XT Clinical Research Exome V2 Plus 2

* Protocols in this document are also compatible with bundled SureSelect XT HS Reagent Kits + Target Enrichment Probes, ordered using p/n G9704A-S, G9705A-S, and G9706A-S. See page 95 for more information.

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-pro- cess products. Custom Probes of both categories use the same optimized target enrichment protocols detailed in this publi- cation.

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Before You Begin 1 Materials Required

SureSelectXT HS Tar

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

Description Vendor and part number

AMPure XP Kit 5 ml 60 ml 450 ml

Beckman Coulter Genomics p/n A63880 p/n A63881 p/n A63882

Dynabeads MyOne Streptavidin T1 2 ml 10 ml 50 ml

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

1X Low TE Buffer (10 mM Tris-HCl, pH 7.5-8.0, 0.1 mM EDTA) 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

Thermo Fisher Scientific p/n Q32850 p/n Q32853

Nuclease-free Water (not DEPC-treated) Thermo Fisher Scientific p/n AM9930

get Enrichment System for Illumina Multiplexed Sequencing 15

1 Before You Begin Materials Required

16

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 (flat or domed, based on cycler/lid requirements)*

Consult the thermal cycler manufacturers recommendations

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

96-well plate mixer Eppendorf ThermoMixer C, p/n 5382000023 and Eppendorf SmartBlock 96 PCR, p/n 5306000006, or equivalent

Small-volume spectrophotometer NanoDrop 2000, Thermo Fisher Scientific p/n ND-2000 or equivalent

Multichannel pipette Rainin Pipet-Lite Multi Pipette or equivalent

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

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

Vortex mixer general laboratory supplier

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Before You Begin 1 Materials Required

DNA Analysis Platform and Consumables

Agilent 2100 Bioanalyzer Instrument

Agilent 2100 Expert SW Laptop Bundle (optional)

DNA 1000 Kit

High Sensitivity DNA Kit

OR

Agilent 4200/4150 TapeStation

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 p/n G2939BA

Agilent p/n G2953CA

Agilent p/n 5067-1504

Agilent p/n 5067-4626

Agilent p/n G2991AA/G2992AA

Agilent p/n 5042-8502

Agilent p/n 5067-5154

Agilent p/n 401428

Agilent p/n 401425

Agilent p/n 5067-5582

Agilent p/n 5067-5583

Agilent p/n 5067-5584

Agilent p/n 5067-5585

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

Ice bucket general laboratory supplier

Powder-free gloves general laboratory supplier

* Consult the thermal cycler manufacturers recommendations for use of either flat or domed strip caps and for any accesso- ries (e.g., compression mats) required for optimal performance with the selected caps. Ensure that the combination of se- lected thermal cycler and plasticware provides complete sealing of sample wells and optimal contact between the instrument heated lid and vial cap for heat transfer.

DNA samples may also be analyzed using the Agilent 5200 Fragment Analyzer, p/n M5310AA, and associated NGS Fragment Kits (DNF-473-0500 and DNF-474-0500). Implement any sample dilution instructions provided in protocols in this document, and then follow the assay instructions provided for each NGS Fragment Kit.

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.

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

Description Vendor and part number

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing 17

1 Before You Begin Materials Required

18

Table 6 Additional Required Materials based on DNA Sample Type/Fragmentation Method

Description Vendor and Part Number

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

Qiagen p/n 51304 p/n 51306

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

OR

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

Agilent p/n G9700A p/n G9700B

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

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

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Before You Begin 1 Optional Materials

Optional Materials

Table 7 Supplier Information for Optional Materials

Description Vendor and Part Number Purpose

Tween 20 Sigma-Aldrich p/n P9416-50ML Sequencing library storage (see page 73)

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

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

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

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

SureSelectXT HS Tar

get Enrichment System for Illumina Multiplexed Sequencing 19

1 Before You Begin Optional Materials

20

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

SureSelectXT HS Target Enrichment 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 23

Step 2. Fragment the DNA 26

Method 1: Mechanical DNA Shearing using Covaris 26

Method 2: Enzymatic DNA Fragmentation 29

This chapter describes the steps to prepare, quantify, qualify, and fragment input DNA samples prior to SureSelect XT HS library preparation and target enrichment. Protocols are provided for two alternative methods of DNA fragmentationmechanical 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 87.

The 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. Analysis using the molecular barcodes is recommended when sample is available in low amounts (1050 ng) or when detecting very low allele frequency variants using small probe designs.

21Agilent Technologies

2 Preparation and Fragmentation of Input DNA Step 1. Prepare and analyze quality of genomic DNA samples

Step 1. Prepare and analyze quality of genomic DNA samples

If you are preparing DNA samples for an Agilent SureSelect Cancer All-In-One assay, use the following modifications to the gDNA sample preparation instructions in this section:

Where required for your experimental design, make sure to prepare reference DNA sample(s) alongside your experimental samples

Use at least 50 ng input gDNA for best results

See publication G9702-90100 for more information.

Preparation of high-quality gDNA from fresh biological samples

NOTE

22

1 Prepare high- quality gDNA using a suitable purification system, such as Qiagens QIAamp DNA Mini Kit, following the manufacturers protocol. The protocol requires 10 ng to 200 ng DNA input.

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

NOTE

2 Use the Qubit BR dsDNA Assay Kit to determine the concentration of each gDNA sample. Follow the manufacturers 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 26.

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Preparation and Fragmentation of Input DNA 2 Preparation and qualification of gDNA from FFPE samples

Preparation and qualification of gDNA from FFPE samples

SureSelectXT HS Tar

1 Prepare gDNA from FFPE tissue sections using Qiagens QIAamp DNA FFPE Tissue Kit and Qiagens Deparaffinization Solution, following the manufacturers protocol. Elute the final gDNA samples from the MinElute column in two rounds, using 30 l Buffer ATE in each round, for a final elution volume of approximately 60 l.

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

NOTE

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

2 Assess the quality (DNA integrity) for each FFPE DNA sample using one of the methods below.

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 8.

a Use the Qubit BR dsDNA Assay Kit to determine the concentration of each gDNA sample. Follow the manufacturers instructions for the instrument and assay kit.

b Remove a 1 l 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 (G9700- 90000) at www.agilent.com for more information.

c For all samples with Cq DNA integrity score 1, use the Qubit- based gDNA concentration determined in step a, above, to determine volume of input DNA needed for the protocol.

get Enrichment System for Illumina Multiplexed Sequencing 23

2 Preparation and Fragmentation of Input DNA Preparation and qualification of gDNA from FFPE samples

24

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.

Table 8 SureSelect XT HS DNA input modifications based on Cq DNA integrity score

Protocol Parameter non-FFPE Samples FFPE Samples

Cq1* Cq >1

DNA input for Library Preparation

10 ng to 200 ng DNA, based on Qubit Assay

10 ng to 200 ng DNA, based on Qubit Assay

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

* 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 de- termined by qPCR, to calculate the volume required for 10200 ng DNA.

Option 2: Qualification using Agilents Genomic DNA ScreenTape assay DIN score

Agilents Genomic DNA ScreenTape assay, used in conjunction with Agilents 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 manufacturers instructions for the instrument and assay kit.

b Remove a 1 l 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 9 to determine the recommended amount of input DNA for the sample.

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Preparation and Fragmentation of Input DNA 2 Preparation and qualification of gDNA from FFPE samples

Table 9 SureSelect XT HS DNA input modifications based on DNA Integrity Number (DIN) score

Protocol Parameter

non-FFPE Samples

FFPE Samples

DIN > 8* DIN 38 DIN<3

DNA input for Library Preparation

10 ng to 200 ng DNA, quantified by Qubit Assay

10 ng to 200 ng DNA, quantified by Qubit Assay

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.

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

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing 25

2 Preparation and Fragmentation of Input DNA Step 2. Fragment the DNA

Step 2. Fragment the DNA

Method 1: Mechanical DNA Shearing using Covaris

26

In this step, 50- l gDNA samples are sheared using conditions optimized for either high- quality or FFPE DNA. The target DNA fragment size is 150 to 200 bp.

NOTE This protocol has been optimized using a Covaris model E220 instrument and the 130-l Covaris microTUBE for a target DNA fragment size of 150 to 200 bp. If you wish to use a different Covaris instrument model/sample holder or if your NGS workflow requires a different DNA fragment size (e.g., for translocation detection with the SureSelect Cancer All-In-One assay), consult the manufacturers recommendations for shearing conditions for the recommended DNA fragment size.

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. All FFPE samples should be sheared for 240 seconds (see Table 10 on page 27) 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.

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

a Check that the water in the Covaris tank is filled with fresh deionized water to the appropriate fill line level according to the manufacturers recommendations for the specific instrument model and sample tube or plate in use.

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 manufacturers recommendations, typically 3060 minutes.

d Set the chiller temperature to between 2C to 5C to ensure that the temperature reading in the water bath displays 5C. Consult the manufacturers recommendations for addition of coolant fluids to prevent freezing.

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Preparation and Fragmentation of Input DNA 2 Method 1: Mechanical DNA Shearing using Covaris

SureSelectXT HS Tar

2 Prepare the DNA samples for the run by diluting 10200 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 l. Vortex well to mix, then spin briefly to collect the liquid. Keep the samples on ice.

Do not dilute samples to be sheared using water. Shearing samples in water reduces the overall library preparation yield and complexity.

NOTE

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

a Transfer the 50- l 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 10.

Table 10 Shear settings for Covaris E-series instrument (SonoLab software v7 or later)

Use the steps below for two- round shearing of high- quality DNA samples only:

Shear for 120 seconds

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 seconds

Spin the microTUBE for 10 seconds

Vortex the microTUBE at high speed for 5 seconds

Spin the microTUBE for 10 seconds

Setting High-quality DNA FFPE DNA

Duty Factor 10% 10%

Peak Incident Power (PIP) 175 175

Cycles per Burst 200 200

Treatment Time 2 120 seconds 240 seconds

Bath Temperature 2 to 8 C 2 to 8 C

get Enrichment System for Illumina Multiplexed Sequencing 27

2 Preparation and Fragmentation of Input DNA Method 1: Mechanical DNA Shearing using Covaris

28

d After completing the shearing step(s), put the Covaris microTUBE back into the loading and unloading station.

e While keeping the snap- cap on, insert a pipette tip through the pre- split septum, then slowly remove the sheared DNA.

f Transfer the sheared DNA sample (approximately 50 l) to a 96- well plate or strip tube sample well. Keep the samples on ice.

g 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 f.

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 g.

NOTE

The 50- l sheared DNA samples are now ready for NGS sequencing library preparation, beginning with end repair/dA- tailing. Proceed to Library Preparation on page 33.

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.

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Preparation and Fragmentation of Input DNA 2 Method 2: Enzymatic DNA Fragmentation

Method 2: Enzymatic DNA Fragmentation

SureSelectXT HS Tar

In this step, gDNA samples are fragmented using Agilents SureSelect Enzymatic Fragmentation Kit.

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 or 1X Low TE Buffer to a final volume of 7 l.

If the DNA concentration is too low to supply the 10200 ng input amount required for your workflow in 7 l, sample volume may be reduced using a suitable concentration method. Alternatively, see Troubleshooting on page 97 for protocol modifications for dilute samples.

2 Thaw the vial of 5X SureSelect Fragmentation Buffer on ice, vortex, then keep on ice.

3 Preprogram a thermal cycler (with the heated lid ON) with the program in Table 11. Immediately pause the program, and keep paused until samples are loaded in step 7.

Table 11 Thermal cycler program for enzymatic fragmentation*

Optimal fragmentation conditions may vary based on the NGS read length to be used in the workflow. Refer to Table 12 for the duration at 37C appropriate for your sample type and required NGS read length.

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

Step Temperature Time

Step 1 37C Variessee Table 12

Step 2 65C 5 minutes

Step 3 4C Hold

get Enrichment System for Illumina Multiplexed Sequencing 29

2 Preparation and Fragmentation of Input DNA Method 2: Enzymatic DNA Fragmentation

30

4 Prepare the appropriate volume of Fragmentation master mix by combining the reagents in Table 13.

Mix well by pipetting up and down 20 times or seal the tube and vortex at high speed for 510 seconds. Spin briefly to remove any bubbles and keep on ice.

on of Fragmentation master mix

Table 12 Fragmentation duration based on sample type and NGS read length

NGS read length requirement

Target fragment size

Duration of 37C incubation step (Table 11)

High-quality DNA samples FFPE DNA samples*

* 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 37C for 15 minutes to generate fragment ends suitable for library construc- tion. Libraries prepared from FFPE samples should be analyzed using an NGS read length suitable for the final library fragment size distribution.

2 100 reads 150 to 200 bp 15 minutes 15 minutes

2 150 reads 180 to 250 bp 10 minutes 15 minutes

Table 13 Preparati

Reagent Volume for 1 reaction

Volume for 8 reactions (includes excess)

Volume for 24 reactions (includes excess)

5X SureSelect Fragmentation Buffer (blue cap) 2 l 18 l 50 l

SureSelect Fragmentation Enzyme (green cap) 1 l 9 l 25 l

Total 3 l 27 l 75 l

5 Add 3 l of the Fragmentation master mix to each sample well containing 7 l of input DNA.

6 Mix well by pipetting up and down 20 times or cap the wells and vortex at high speed for 510 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 11.

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Preparation and Fragmentation of Input DNA 2 Method 2: Enzymatic DNA Fragmentation

SureSelectXT HS Tar

8 When the program reaches the 4C Hold step, remove the samples from the thermal cycler, add 40 l of nuclease- free water to each sample, and place the samples on ice.

The 50- l reactions are now ready for NGS sequencing library preparation, beginning with end repair/dA- tailing. Proceed to Library Preparation on page 33.

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.

get Enrichment System for Illumina Multiplexed Sequencing 31

2 Preparation and Fragmentation of Input DNA Method 2: Enzymatic DNA Fragmentation

32

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

SureSelectXT HS Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library Protocol

3 Library Preparation

Step 1. Repair and dA-Tail the DNA ends 34

Step 2. Ligate the molecular-barcoded adaptor 38

Step 3. Purify the sample using AMPure XP beads 39

Step 4. Amplify the adaptor-ligated library 41

Step 5. Purify the amplified library with AMPure XP beads 44

Step 6. Assess quality and quantity 46

The sample preparation protocol is used to prepare DNA libraries for sequencing using the Illumina paired- read platform. For each sample to be sequenced, an individual indexed and molecular- barcoded library is prepared. For an overview of the SureSelect XT HS target enrichment 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 26 to page 28) or produced by enzymatic fragmentation (as detailed on page 29 to page 31). Samples produced by either method should contain 10200 ng of DNA fragments in a volume of 50 l.

33Agilent Technologies

3 Library Preparation Step 1. Repair and dA-Tail the DNA ends

Step 1. Repair and dA-Tail the DNA ends

34

Protocol steps in this section use the components listed in Table 14. Thaw and mix each component as directed in Table 14 before use.

Remove the AMPure XP beads from cold storage and equilibrate to room temperature in preparation for use on page 39. Do not freeze the beads at any time.

Table 14 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)

SureSelect XT HS Library Preparation Kit for ILM (Pre PCR),* 20C

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

Vortexing page 36

Ligation Buffer (purple cap or bottle)

SureSelect XT HS Library Preparation Kit for ILM (Pre PCR),* 20C

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

Vortexing page 35

End Repair-A Tailing Enzyme Mix (orange cap)

SureSelect XT HS Library Preparation Kit for ILM (Pre PCR),* 20C

Place on ice just before use

Inversion page 36

T4 DNA Ligase (blue cap) SureSelect XT HS Library Preparation Kit for ILM (Pre PCR),* 20C

Place on ice just before use

Inversion page 35

Adaptor Oligo Mix (white cap)

SureSelect XT HS Library Preparation Kit for ILM (Pre PCR),* 20C

Thaw on ice then keep on ice

Vortexing page 38

* May also be labeled as SureSelect XT HS and XT Low Input Library Preparation Kit for ILM (Pre PCR).

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

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Library Preparation 3 Step 1. Repair and dA-Tail the DNA ends

SureSelectXT HS Tar

1 Before starting the end- repair protocol, prepare the Ligation master mix to allow equilibration to room temperature before use.

a 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 1520 times using a pipette set to at least 80% of the mixture volume or by vortexing at high speed for 1020 seconds.

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

b Prepare the appropriate volume of Ligation master mix by combining the reagents in Table 15.

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 1520 times or seal the tube and vortex at high speed for 1020 seconds. Spin briefly to collect the liquid.

Keep at room temperature for 3045 minutes before use on page 38.

Table 15 Preparation of Ligation master mix

Reagent Volume for 1 reaction

Volume for 8 reactions*

(includes excess)

* 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.

Volume for 24 reactions

(includes excess)

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.

Ligation Buffer (purple cap or bottle)

23 l 207 l 598 l

T4 DNA Ligase (blue cap) 2 l 18 l 52 l

Total 25 l 225 l 650 l

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3 Library Preparation Step 1. Repair and dA-Tail the DNA ends

36

2 Preprogram a thermal cycler (with the heated lid ON) with the program in Table 16 for the End Repair and dA- Tailing steps. Immediately pause the program, and keep paused until samples are loaded in step 6.

Table 16 Thermal cycler program for End Repair/dA-Tailing*

3 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.

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

Step Temperature Time

Step 1 20C 15 minutes

Step 2 72C 15 minutes

Step 3 4C Hold

CAUTION 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 1520 times using a pipette set to at least 80% of the mixture volume or by vortexing at high speed for 510 seconds.

4 Prepare the appropriate volume of End Repair/dA- Tailing master mix, by combining the reagents in Table 17.

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 1520 times or seal the tube and vortex at high speed for 510 seconds. Spin briefly to collect the liquid and keep on ice.

Table 17 Preparation of End Repair/dA-Tailing master mix

Reagent Volume for 1 reaction Volume for 8 reactions (includes excess)

Volume for 24 reactions (includes excess)

End Repair-A Tailing Buffer (yellow cap or bottle) 16 l 144 l 416 l

End Repair-A Tailing Enzyme Mix (orange cap) 4 l 36 l 104 l

Total 20 l 180 l 520 l

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Library Preparation 3 Step 1. Repair and dA-Tail the DNA ends

SureSelectXT HS Tar

5 Add 20 l of the End Repair/dA- Tailing master mix to each sample well containing approximately 50 l of fragmented DNA. Mix by pipetting up and down 1520 times using a pipette set to 60 l or cap the wells and vortex at high speed for 510 seconds.

6 Briefly spin the samples, then immediately place the plate or strip tube in the thermal cycler and resume the thermal cycling program in Table 16.

get Enrichment System for Illumina Multiplexed Sequencing 37

3 Library Preparation Step 2. Ligate the molecular-barcoded adaptor

Step 2. Ligate the molecular-barcoded adaptor

38

1 Once the thermal cycler reaches the 4C Hold step, transfer the samples to ice while setting up this step.

2 Preprogram a thermal cycler (with the heated lid ON) for the Ligation step with the program in Table 18. Immediately pause the program, and keep paused until samples are loaded in step 5.

Table 18 Thermal cycler program for Ligation*

3 To each end- repaired/dA- tailed DNA sample (approximately 70 l), add 25 l of the Ligation master mix that was prepared on page 35 and kept at room temperature. Mix by pipetting up and down at least 10 times using a pipette set to 85 l or cap the wells and vortex at high speed for 510 seconds. Briefly spin the samples.

4 Add 5 l of Adaptor Oligo Mix (white capped tube) to each sample. Mix by pipetting up and down 1520 times using a pipette set to 85 l or cap the wells and vortex at high speed for 510 seconds.

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

Step Temperature Time

Step 1 20C 30 minutes

Step 2 4C Hold

Make sure to add the Ligation master mix and the Adaptor Oligo Mix to the samples in separate addition steps as directed in step 3 and step 4 above, mixing after each addition.

NOTE

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.

A unique molecular barcode sequence is incorporated into each library DNA fragment at this step.

NOTE

Stopping Point

If you do not continue to the next step, seal the sample wells and store overnight at either 4C or 20C.

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Library Preparation 3 Step 3. Purify the sample using AMPure XP beads

Step 3. Purify the sample using AMPure XP beads

SureSelectXT HS Tar

In this step, the DNA libraries are purified using AmpPure XP Beads. Critical purification protocol parameters are summarized for experienced users in Table 19.

1 Verify that the AMPure XP beads were held at room temperature for at least 30 minutes before use.

2 Prepare 400 l of 70% ethanol per sample, plus excess, for use in step 8.

Table 19 AMPure XP bead cleanup parameters after adaptor ligation

Parameter Value

Volume of RT AMPure XP bead suspension added to each sample well 80 l

Final elution solvent and volume 35 l nuclease-free water

Amount of eluted sample transferred to fresh well Approximately 34.5 l

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.

NOTE

3 Mix the AMPure XP bead suspension well so that the reagent appears homogeneous and consistent in color.

4 Add 80 l of homogeneous AMPure XP beads to each DNA sample (approximately 100 l) in the PCR plate or strip tube. Pipette up and down 1520 times or cap the wells and vortex at high speed for 510 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.

get Enrichment System for Illumina Multiplexed Sequencing 39

3 Library Preparation Step 3. Purify the sample using AMPure XP beads

40

8 Continue to keep the plate or strip tube in the magnetic stand while you dispense 200 l 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.

12 Dry the samples by placing the unsealed plate or strip tube on the thermal cycler, set to hold samples at 37C, until the residual ethanol has just evaporated (typically 12 minutes).

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.

NOTE

13 Add 35 l 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 34.5 l) 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 34.5-l 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 17.25 l.

NOTE

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Library Preparation 3 Step 4. Amplify the adaptor-ligated library

Step 4. Amplify the adaptor-ligated library

SureSelectXT HS Tar

This step uses the components listed in Table 20. Before you begin, thaw the reagents listed below and keep on ice.

Table 20 Reagents for pre-capture PCR amplification

Component Storage Location Mixing Method Where Used

Herculase II Fusion DNA Polymerase (red cap)

SureSelect XT HS Library Preparation Kit for ILM (Pre PCR)*, 20C

Pipette up and down 1520 times

page 43

5 Herculase II Reaction Buffer (clear cap) SureSelect XT HS Library Preparation Kit for ILM (Pre PCR)*, 20C

Vortexing page 43

100 mM dNTP Mix (green cap) SureSelect XT HS Library Preparation Kit for ILM (Pre PCR)*, 20C

Vortexing page 43

Forward Primer (brown cap) SureSelect XT HS Library Preparation Kit for ILM (Pre PCR)*, 20C

Vortexing page 43

SureSelect XT HS Index Primers A01 through H04 (black-capped tubes)

SureSelect XT HS Index Primers for ILM (Pre PCR), 20C

Vortexing page 43

* May also be labeled as SureSelect XT HS and XT Low Input Library Preparation Kit for ILM (Pre PCR).

Indexing primers are provided in kits containing individual tubes in sets of 116 (A01H02), 1732 (A03H04), or 132 (A01 H04). Thaw only the specific primers assigned to samples in the run.

1 Determine the appropriate index assignments for each sample. See page 96 for more information on the index primers used to amplify the DNA libraries in this step.

Use a different index for each sample to be sequenced in the same lane.

Take care to avoid combining libraries with the same index sequence when multiplexing libraries prepared using different SureSelect kit formats. Indexes A01-H04 in SureSelect XT HS Reagent Kits (provided in black capped-tubes) are equivalent to indexes A01-H04 in Magnis SureSelect XT HS automation kits (provided in black index strips) and to indexes 1-32 in SureSelect XT Low Input Reagent Kits (provided in yellow plate).

NOTE

CAUTION The SureSelect XT HS Index Primers are provided in single-use aliquots. To avoid cross-contamination of libraries, discard each vial after use in one library preparation reaction. Do not retain and re-use any residual volume for subsequent experiments.

get Enrichment System for Illumina Multiplexed Sequencing 41

3 Library Preparation Step 4. Amplify the adaptor-ligated library

42

2 Preprogram a thermal cycler (with the heated lid ON) with the program in Table 21. Immediately pause the program, and keep paused until samples are loaded in step 6.

Table 21 Pre-Capture PCR Thermal Cycler Program*

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

Segment Number of Cycles Temperature Time

1 1 98C 2 minutes

2 8 to 14, based on input DNA quality and quantity (see Table 22)

98C 30 seconds

60C 30 seconds

72C 1 minute

3 1 72C 5 minutes

4 1 4C Hold

Table 22 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*

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

11 cycles

50 ng 12 cycles

10 ng 14 cycles

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Library Preparation 3 Step 4. Amplify the adaptor-ligated library

SureSelectXT HS Tar

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 23, on ice. Mix well on a vortex mixer.

on of Pre-Capture PCR Reaction Mix

Table 23 Preparati

Reagent Volume for 1 reaction Volume for 8 reactions (includes excess)

Volume for 24 reactions (includes excess)

5 Herculase II Reaction Buffer (clear cap) 10 l 90 l 250 l

100 mM dNTP Mix (green cap) 0.5 l 4.5 l 12.5 l

Forward Primer (brown cap) 2 l 18 l 50 l

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

Total 13.5 l 121.5 l 337.5 l

4 Add 13.5 l of the PCR reaction mixture prepared in Table 23 to each purified DNA library sample (34.5 l) in the PCR plate wells.

5 Add 2 l of the appropriate SureSelect XT HS Index Primer 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 release any bubbles.

6 Before adding the samples to the thermal cycler, resume the program in Table 21 to bring the temperature of the thermal block to 98C. Once the cycler has reached 98C, 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.

get Enrichment System for Illumina Multiplexed Sequencing 43

3 Library Preparation Step 5. Purify the amplified library with AMPure XP beads

Step 5. Purify the amplified library with AMPure XP beads

44

In this step, the amplified DNA libraries are purified using AmpPure XP Beads. Critical purification protocol parameters are summarized for experienced users in Table 24.

1 Verify that the AMPure XP beads were held at room temperature for at least 30 minutes before use.

2 Prepare 400 l 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 l of homogeneous AMPure XP beads to each 50- l amplification reaction in the PCR plate or strip tube. Pipette up and down 1520 times or cap the wells and vortex at high speed for 510 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 l 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.

Table 24 AMPure XP bead cleanup parameters after pre-capture PCR

Parameter Value

Volume of RT AMPure XP bead suspension added to each sample well 50 l

Final elution solvent and volume 15 l nuclease-free water

Amount of eluted sample transferred to fresh well Approximately 15 l

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Library Preparation 3 Step 5. Purify the amplified library with AMPure XP beads

SureSelectXT HS Tar

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 37C, until the residual ethanol has just evaporated (typically 12 minutes).

13 Add 15 l 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.

17 Remove the cleared supernatant (approximately 15 l) 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 15-l supernatant volume at this step; transfer the maximum possible amount of supernatant for further processing.

NOTE

get Enrichment System for Illumina Multiplexed Sequencing 45

3 Library Preparation Step 6. Assess quality and quantity

Step 6. Assess quality and quantity

46

Sample analysis can be done with either the 2100 Bioanalyzer instrument or an Agilent TapeStation instrument.

NOTE Using either analysis method, observation of a low molecular weight peak, in addition to the expected library fragment peak, indicates the presence of adaptor-dimers in the library. Adaptor-dimer removal is not required for libraries that will be target-enriched in later steps of the workflow. However, 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 l with nuclease free water, then follow the SPRI purification procedure on page 44.

Option 1: Analysis using the 2100 Bioanalyzer and DNA 1000 Assay

Use a Bioanalyzer DNA 1000 chip and reagent kit. Perform the assay according to the Agilent DNA 1000 Kit Guide.

1 Set up the 2100 Bioanalyzer instrument as instructed in the reagent kit guide.

2 Prepare the chip, samples and ladder as instructed in the reagent kit guide, using 1 l of each sample for the analysis. Load the prepared chip into the instrument and start the run within five minutes after preparation.

3 Verify that the electropherogram shows the peak of DNA fragment size positioned between 300 to 400 bp for high- quality DNA and approximately 200 to 400 bp for FFPE DNA. Sample electropherograms are shown in Figure 2 (library prepared from high- quality DNA), Figure 3 (library prepared from medium- quality FFPE DNA), and Figure 4 (library prepared from low- quality FFPE DNA).

The appearance of an additional low molecular weight 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 sample electropherograms on page 47. See Troubleshooting information on page 99 for additional considerations.

4 Determine the concentration of each library by integrating under the entire peak. For accurate quantification, make sure that the concentration falls within the linear range of the assay.

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Library Preparation 3 Step 6. Assess quality and quantity

SureSelectXT HS Tar

Figure 2 Pre-capture library prepared from a high-quality gDNA sample analyzed using a DNA 1000 Bioanalyzer assay.

Figure 3 Pre-capture library prepared from a typical FFPE gDNA sample analyzed using a DNA 1000 Bioanalyzer assay.

Figure 4 Pre-capture library prepared from a low-quality FFPE gDNA sample analyzed using a DNA 1000 Bioanalyzer assay.

Stopping Point

If you do not continue to the next step, seal the sample wells and store at 4C overnight or at 20C for prolonged storage.

get Enrichment System for Illumina Multiplexed Sequencing 47

3 Library Preparation Step 6. Assess quality and quantity

48

Option 2: Analysis using an Agilent TapeStation and D1000 ScreenTape

Use a D1000 ScreenTape and associated reagent kit. For more information to do this step, see the Agilent D1000 Assay Quick Guide.

1 Prepare the TapeStation samples as instructed in the reagent kit guide. Use 1 l of each DNA sample diluted with 3 l of D1000 sample buffer for the analysis.

CAUTION For accurate quantitation, make sure to thoroughly mix the combined DNA and sample buffer by vortexing the assay plate or tube strip for 1 minute on the IKA MS3 vortex mixer provided with the 4200/4150 TapeStation system before loading the samples.

2 Load the sample plate or tube strips from step 1, the D1000 ScreenTape, and loading tips into the TapeStation as instructed in the reagent kit guide. Start the run.

3 Verify that the electropherogram shows the peak of DNA fragment size positioned between 300 to 400 bp for high- quality DNA and approximately 200 to 400 bp for FFPE DNA. Sample electropherograms are shown in Figure 5 (library prepared from high- quality DNA), Figure 6 (library prepared from medium- quality FFPE DNA), and Figure 7 (library prepared from low- quality FFPE DNA).

The appearance of an additional low molecular weight 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 sample electropherograms on page 49 to page 50. See Troubleshooting information on page 99 for additional considerations.

4 Determine the concentration of the library DNA by integrating under the peak.

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Library Preparation 3 Step 6. Assess quality and quantity

SureSelectXT HS Tar

Figure 5 Pre-capture library prepared from a high-quality gDNA sample analyzed using a D1000 ScreenTape assay.

Figure 6 Pre-capture library prepared from a typical FFPE gDNA sample analyzed using a D1000 ScreenTape assay.

get Enrichment System for Illumina Multiplexed Sequencing 49

3 Library Preparation Step 6. Assess quality and quantity

50

Figure 7 Pre-capture library prepared from a low-quality FFPE gDNA sample analyzed using a D1000 ScreenTape assay.

Stopping Point

If you do not continue to the next step, seal the sample wells and store at 4C overnight or at 20C for prolonged storage.

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

SureSelectXT HS Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library Protocol

4 Hybridization and Capture

Step 1. Hybridize DNA samples to the probe 52

Step 2. Prepare streptavidin-coated magnetic beads 57

Step 3. Capture the hybridized DNA using streptavidin-coated beads 58

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 protocol includes the hybridization step (approximately 90 minutes) 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 53.

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

51Agilent Technologies

4 Hybridization and Capture Step 1. Hybridize DNA samples to the probe

Step 1. Hybridize DNA samples to the probe

52

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

The hybridization reaction requires 5001000 ng of prepared DNA in a volume of 12 l. Use the maximum amount of prepared DNA available within this range.

This step uses the components listed in Table 25. Thaw each component under the conditions indicated in the table. Vortex each reagent to mix, then spin tubes briefly to collect the liquid.

Table 25 Reagents for Hybridization

Kit Component Storage Location Thawing Conditions Where Used

SureSelect XT HS and XT Low Input Blocker Mix (blue cap)

SureSelect XT HS Target Enrichment Kit ILM Hyb Module, Box 2 (Post PCR),* 20C

Thaw on ice page 53

SureSelect RNase Block (purple cap)

SureSelect XT HS Target Enrichment Kit ILM Hyb Module, Box 2 (Post PCR),* 20C

Thaw on ice page 54

SureSelect Fast Hybridization Buffer (bottle)

SureSelect XT HS Target Enrichment Kit ILM Hyb Module, Box 2 (Post PCR),* 20C

Thaw and keep at Room Temperature

page 55

Probe 80C Thaw on ice page 55

* May also be labeled as SureSelect XT HS and XT Low Input Target Enrichment Kit ILM Hyb Module, Box 2 (Post PCR).

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Hybridization and Capture 4 Step 1. Hybridize DNA samples to the probe

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1 Preprogram a thermal cycler (with the heated lid ON) with the program in Table 26. Immediately pause the program, and keep paused until samples are loaded in step 4 on page 54.

*

Table 26 Pre-programmed thermal cycler program for Hybridization

The Hybridization thermal cycling program in Table 26 requires about 90 minutes. The Hybridization reaction may be run overnight with the following protocol modifications:

In segment 5 of the thermal cycler program (Table 26), replace the 65C Hold step with a 21C Hold step.

The hybridized samples may be held at 21C for up to 16 hours. Begin the capture preparation steps on page 57 on day 2, after the overnight hold.

Segment # Number of Cycles Temperature Time

1 1 95C 5 minutes

2 1 65C 10 minutes

3 1 65C 1 minute (Pause cycler here for reagent addition; see step 7 on page 56)

4 60 65C 1 minute

37C 3 seconds

5 1 65C Hold briefly until ready to begin capture steps on page 58

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

Hybridization at 65C is optimal for probes designed for the SureSelect XT HS2/XT HS/XT 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 (62.5C), SureSelect XT Clinical Research Exome V2 (62.5C) and custom probes originally designed for use with SureSelect XT system (60C65C).

NOTE

2 Place 5001000 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 l using nuclease- free water. Use the maximum possible amount of each prepped DNA, within the 5001000 ng range.

3 To each DNA library sample well, add 5 l of SureSelect XT HS and XT Low Input Blocker Mix (blue cap). Seal 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.

get Enrichment System for Illumina Multiplexed Sequencing 53

4 Hybridization and Capture Step 1. Hybridize DNA samples to the probe

54

CAUTION 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 26 on page 53), 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 56.

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.

5 Prepare a 25% solution of SureSelect RNase Block (1 part RNase Block to 3 parts water) according to Table 27. Prepare the amount required for the number of hybridization reactions in the run, plus excess. Mix well and keep on ice.

Table 27 Preparation of RNase Block solution

Reagent Volume for 1 reaction Volume for 8 reactions (includes excess)

Volume for 24 reactions (includes excess)

SureSelect RNase Block (purple cap) 0.5 l 4.5 l 12.5 l

Nuclease-free water 1.5 l 13.5 l 37.5 l

Total 2 l 18 l 50 l

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Hybridization and Capture 4 Step 1. Hybridize DNA samples to the probe

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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 DNA samples in step 7 on page 56. 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 28 for probes 3 Mb or Table 29 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 28 Preparation of Probe Hybridization Mix for probes 3 Mb

Table 29 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 l 18 l 50 l

Probe (with design 3 Mb) 5 l 45 l 125 l

SureSelect Fast Hybridization Buffer 6 l 54 l 150 l

Total 13 l 117 l 325 l

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 l 18 l 50 l

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

SureSelect Fast Hybridization Buffer 6 l 54 l 150 l

Nuclease-free water 3 l 27 l 75 l

Total 13 l 117 l 325 l

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4 Hybridization and Capture Step 1. Hybridize DNA samples to the probe

56

7 Once the thermal cycler starts Segment 3 of the program in Table 26 (1 minute at 65C), pause the program. With the cycler paused, and while keeping the DNA + Blocker samples in the cycler, transfer 13 l 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 l.

8 Seal the wells with fresh 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 l is lost to evaporation under the conditions used for hybridization.

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Hybridization and Capture 4 Step 2. Prepare streptavidin-coated magnetic beads

Step 2. Prepare streptavidin-coated magnetic beads

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The remaining hybridization capture steps use the reagents in Table 30.

NOTE If performing same-day hybridization and capture, begin the bead preparation steps below approximately one hour after starting hybridization in step 9 on page 56. If performing next-day capture after an overnight hold at 21C, begin the bead preparation steps below on day 2, just before you are ready to start the capture steps on page 58.

Table 30 Reagents for Capture

Kit Component Storage Location Where Used

SureSelect Binding Buffer SureSelect Target Enrichment Kit ILM Hyb Module, Box 1 (Post PCR), RT page 57

SureSelect Wash Buffer 1 SureSelect Target Enrichment Kit ILM Hyb Module, Box 1 (Post PCR), RT page 58

SureSelect Wash Buffer 2 SureSelect Target Enrichment Kit ILM Hyb Module, Box 1 (Post PCR), RT page 58

Dynabeads MyOne Streptavidin T1

Follow storage recommendations provided by supplier (see Table 4 on page 15)

page 57

1 Vigorously resuspend the Dynabeads MyOne Streptavidin T1 magnetic beads on a vortex mixer. The magnetic beads settle during storage.

2 For each hybridization sample, add 50 l of the resuspended beads to wells of a fresh PCR plate or a strip tube.

3 Wash the beads:

a Add 200 l of SureSelect Binding Buffer.

b Mix by pipetting up and down 20 times or cap the wells and vortex at high speed for 510 seconds then spin down briefly.

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

d Wait at least 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 l of SureSelect Binding Buffer.

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.

NOTE

get Enrichment System for Illumina Multiplexed Sequencing 57

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

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

58

1 After all streptavidin bead preparation steps are complete, and with the hybridization thermal cycling program in the final hold segment (see Table 26 on page 53), transfer the samples to room temperature.

2 Immediately transfer the entire volume (approximately 30 l) of each hybridization mixture to wells containing 200 l of washed streptavidin beads using a multichannel pipette.

Pipette up and down 58 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 14001800 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 70C as described below.

a Place 200- l 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, with heated lid ON, held at 70C until used in step 9.

5 When the 30- minute 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 all of the supernatant.

7 Resuspend the beads in 200 l of SureSelect Wash Buffer 1. Mix by pipetting up and down 1520 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 all of the supernatant.

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Hybridization and Capture 4 Step 3. Capture the hybridized DNA using streptavidin-coated beads

SureSelectXT HS Tar

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

Make sure that the SureSelect Wash Buffer 2 is pre-warmed to 70C 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 l of 70C prewarmed Wash Buffer 2. Pipette up and down 1520 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 70C in 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 l 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 64.

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

NOTE

get Enrichment System for Illumina Multiplexed Sequencing 59

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

60 SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

SureSelectXT HS Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library Protocol

5 Post-Capture Sample Processing for Multiplexed Sequencing

Step 1. Amplify the captured libraries 62

Step 2. Purify the amplified captured libraries using AMPure XP beads 65

Step 3. Assess sequencing library DNA quantity and quality 67

Step 4. Pool samples for multiplexed sequencing 72

Step 5. Prepare sequencing samples 74

Step 6. Do the sequencing run and analyze the data 76

Sequence analysis resources 84

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.

61Agilent Technologies

5 Post-Capture Sample Processing for Multiplexed Sequencing Step 1. Amplify the captured libraries

Step 1. Amplify the captured libraries

62

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

This step uses the components listed in Table 31. Before you begin, thaw the reagents listed below and keep on ice.

Table 31 Reagents for post-capture PCR amplification

Component Storage Location Mixing Method Where Used

Herculase II Fusion DNA Polymerase (red cap)

SureSelect XT HS Target Enrichment Kit, ILM Hyb Module Box 2 (Post PCR),* 20C

Pipette up and down 1520 times

page 64

5 Herculase II Reaction Buffer (clear cap)

SureSelect XT HS Target Enrichment Kit, ILM Hyb Module Box 2 (Post PCR),* 20C

Vortexing page 64

100 mM dNTP Mix (green cap) SureSelect XT HS Target Enrichment Kit, ILM Hyb Module Box 2 (Post PCR),* 20C

Vortexing page 64

SureSelect Post-Capture Primer Mix (clear cap)

SureSelect XT HS Target Enrichment Kit, ILM Hyb Module Box 2 (Post PCR),* 20C

Vortexing page 64

* May also be labeled as SureSelect XT HS and XT Low Input Target Enrichment Kit ILM Hyb Module Box 2 (Post PCR).

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.

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Post-Capture Sample Processing for Multiplexed Sequencing 5 Step 1. Amplify the captured libraries

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1 Preprogram a thermal cycler (with the heated lid ON) with the program in Table 32. Immediately pause the program, and keep paused until samples are loaded in step 5.

Table 32 Post-capture PCR thermal cycler program

Segment Number of Cycles Temperature Time

1 1 98C 2 minutes

2 9 to 14

See Table 33 for hybridization probe design size-based cycle number recommendations

98C 30 seconds

60C 30 seconds

72C 1 minute

3 1 72C 5 minutes

4 1 4C Hold

Table 33 Post-capture PCR cycle number recommendations

Probe Size/Description Cycles

Probes <0.2 Mb 14 cycles

Probes 0.23 Mb (includes ClearSeq Comp Cancer) 12 cycles

Probes 35 Mb 10 cycles

Probes >5 Mb (includes Human All Exon probes) 9 cycles

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5 Post-Capture Sample Processing for Multiplexed Sequencing Step 1. Amplify the captured libraries

64

2 Prepare the appropriate volume of PCR reaction mix, as described in Table 34, on ice. Mix well on a vortex mixer.

Table 34 Preparation of post-capture PCR Reaction mix

Reagent Volume for 1 reaction Volume for 8 reactions (includes excess)

Volume for 24 reactions (includes excess)

Nuclease-free water 12.5 l 112.5 l 312.5 l

5 Herculase II Reaction Buffer (clear cap) 10 l 90 l 250 l

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

100 mM dNTP Mix (green cap) 0.5 l 4.5 l 12.5 l

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

Total 25 l 225 l 625 l

3 Add 25 l of the PCR reaction mix prepared in Table 34 to each sample well containing 25 l of bead- bound target- enriched DNA (prepared on page 59 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 32.

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 l) to wells of a fresh plate or strip tube.

The beads can be discarded at this time.

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

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

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

SureSelectXT HS Tar

In this step, the amplified enriched DNA libraries are purified using AmpPure XP Beads. Critical purification protocol parameters are summarized for experienced users in Table 35.

1 Let the AMPure XP beads come to room temperature for at least 30 minutes.

2 Prepare 400 l 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 l of the homogeneous AMPure XP bead suspension to each amplified DNA sample (approximately 50 l) in the PCR plate or strip tube. Mix well by pipetting up and down 1520 times or cap the wells and vortex at high speed for 510 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 While keeping 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 l of freshly- prepared 70% ethanol in each sample well.

Table 35 AMPure XP bead cleanup parameters after post-capture PCR

Parameter Value

Volume of RT AMPure XP bead suspension added to each sample well 50 l

Final elution solvent and volume 25 l nuclease-free water

Amount of eluted sample transferred to fresh well Approximately 25 l

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

66

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 37C, until the residual ethanol has just evaporated (typically 12 minutes).

13 Add 25 l of nuclease- free water to each sample well.

14 Seal the sample wells, 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 l) to a fresh well. You can discard the beads at this time.

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Post-Capture Sample Processing for Multiplexed Sequencing 5 Step 3. Assess sequencing library DNA quantity and quality

Step 3. Assess sequencing library DNA quantity and quality

SureSelectXT HS Tar

Option 1: Analysis using the Agilent 2100 Bioanalyzer and High Sensitivity DNA Assay

Use the Bioanalyzer High Sensitivity DNA Assay to analyze the amplified captured DNA. Perform the assay according to the High Sensitivity DNA Kit Guide.

1 Set up the 2100 Bioanalyzer instrument as instructed in the reagent kit guide.

2 Prepare the chip, samples and ladder as instructed in the reagent kit guide, using 1 l of each sample for the analysis.

3 Load the prepared chip into the instrument and start the run within five minutes after preparation.

4 Verify that the electropherogram shows the peak of DNA fragment size positioned between 200 and 400 bp. Sample electropherograms are shown in Figure 8 (library prepared from high- quality DNA), Figure 9 (library prepared from medium- quality FFPE DNA), and Figure 10 (library prepared from low- quality FFPE DNA).

5 Measure the concentration of each library by integrating under the entire peak. For accurate quantification, make sure that the concentration falls within the linear range of the assay.

Figure 8 Post-capture library prepared from a high-quality gDNA sample analyzed using a Bioanalyzer system High Sensitivity DNA assay.

get Enrichment System for Illumina Multiplexed Sequencing 67

5 Post-Capture Sample Processing for Multiplexed Sequencing Step 3. Assess sequencing library DNA quantity and quality

68

Figure 9 Post-capture library prepared from a typical FFPE gDNA sample analyzed us- ing a Bioanalyzer system High Sensitivity DNA assay.

Figure 10 Post-capture library prepared from a low-quality FFPE gDNA sample analyzed using a Bioanalyzer system High Sensitivity DNA assay.

Stopping Point

If you do not continue to the next step, seal the plate and store at 4C overnight or at 20C for prolonged storage.

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Post-Capture Sample Processing for Multiplexed Sequencing 5 Step 3. Assess sequencing library DNA quantity and quality

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Option 2: Analysis using an Agilent TapeStation and High Sensitivity D1000 ScreenTape

Use a High Sensitivity D1000 ScreenTape and associated reagent kit to analyze the amplified captured DNA. For more information to do this step, see the Agilent High Sensitivity D1000 Assay Quick Guide.

1 Prepare the TapeStation samples as instructed in the reagent kit guide. Use 2 l of each indexed DNA sample diluted with 2 l of High Sensitivity D1000 sample buffer for the analysis.

CAUTION For accurate quantitation, make sure to thoroughly mix the combined DNA and sample buffer by vortexing the assay plate or tube strip for 1 minute on the IKA MS3 vortex mixer provided with the 4200/4150 TapeStation system before loading the samples.

2 Load the sample plate or tube strips from step 1, the High Sensitivity D1000 ScreenTape, and loading tips into the TapeStation as instructed in the reagent kit guide. Start the run.

3 Verify that the electropherogram shows the peak of DNA fragment size positioned between 200 and 400 bp. Sample electropherograms are shown in Figure 11 (library prepared from high- quality DNA), Figure 12 (library prepared from medium- quality FFPE DNA), and Figure 13 (library prepared from low- quality FFPE DNA).

4 Determine the concentration of each library by integrating under the entire peak.

get Enrichment System for Illumina Multiplexed Sequencing 69

5 Post-Capture Sample Processing for Multiplexed Sequencing Step 3. Assess sequencing library DNA quantity and quality

70

Figure 11 Post-capture library prepared from a high-quality gDNA sample analyzed using a High Sensitivity D1000 ScreenTape assay.

Figure 12 Post-capture library prepared from a typical FFPE gDNA sample analyzed us- ing a High Sensitivity D1000 ScreenTape assay.

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Post-Capture Sample Processing for Multiplexed Sequencing 5 Step 3. Assess sequencing library DNA quantity and quality

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Figure 13 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 4C overnight or at 20C for prolonged storage.

get Enrichment System for Illumina Multiplexed Sequencing 71

5 Post-Capture Sample Processing for Multiplexed Sequencing Step 4. Pool samples for multiplexed sequencing

Step 4. Pool samples for multiplexed sequencing

72

Volume of Index V f C f # C i

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

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 nM15 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.

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 nM15 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 36 shows an example of the amount of 4 index- tagged samples (of different concentrations) and Low TE needed for a final volume of 20 l at 10 nM DNA.

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Post-Capture Sample Processing for Multiplexed Sequencing 5 Step 4. Pool samples for multiplexed sequencing

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

Table 36 Example of volume calculation for total volume of 20 l at 10 nM concentration

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

Sample 1 20 l 20 nM 10 nM 4 2.5

Sample 2 20 l 10 nM 10 nM 4 5

Sample 3 20 l 17 nM 10 nM 4 2.9

Sample 4 20 l 25 nM 10 nM 4 2

Low TE 7.6

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5 Post-Capture Sample Processing for Multiplexed Sequencing Step 5. Prepare sequencing samples

Step 5. Prepare sequencing samples

74

The final SureSelectXT HS 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 14.

Figure 14 Content of SureSelect XT HS sequencing library. Each fragment contains one target insert (blue) surrounded by the Illumina paired-end sequencing ele- ments (black), the sample index (red), the molecular barcode (MBC; green) and the library bridge PCR primers (yellow). Sequencing of the 10-bp MBC (i5) reads is optional.

Libraries can be sequenced on the Illumina HiSeq, MiSeq, NextSeq, or NovaSeq platform using the run type and chemistry combinations shown in Table 37.

CAUTION Do not use the HiSeq2500 instrument in high-output run mode (v4 chemistry) if your analysis pipeline includes MBC (i5) reads. Poor MBC sequence data quality (lower Q scores, with impacts on coverage and sensitivity of variant calls) has been observed when SureSelectXT HS libraries are sequenced on the HiSeq 2500 instrument in this mode. See Table 37 for alternative run mode/chemistry options for the HiSeq2500 platform. Sequencing performance for Read 1, Read 2, and sample-level index (i7) reads is not affected, and this platform/run mode/chemistry may be used for applications that omit MBC analysis.

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Post-Capture Sample Processing for Multiplexed Sequencing 5 Step 5. Prepare sequencing samples

SureSelectXT HS Tar

Proceed to cluster amplification using the appropriate Illumina Paired- End Cluster Generation Kit. See Table 37 for kit configurations compatible with the recommended read length.

The optimal seeding concentration for SureSelectXT HS target- enriched libraries varies according to sequencing platform, run type, and Illumina kit version. See Table 37 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 37.

Follow Illuminas recommendation for a PhiX control in a low- concentration spike- in for improved sequencing quality control.

Table 37 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 910 pM

HiSeq 2500 High Output 2 100 bp 250 Cycle Kit v4 1214 pM

MiSeq All Runs 2 100 bp or

2 150 bp

300 Cycle Kit v2 910 pM

MiSeq All Runs 2 75 bp 150 Cycle Kit v3 1216 pM

NextSeq 500/550 All Runs 2 100 bp or

2 150 bp

300 Cycle Kit v2.5 1.21.5 pM

HiSeq 3000/4000 All Runs 2 100 bp or

2 150 bp

300 Cycle Kit v1 300400 pM

NovaSeq 6000 Standard Workflow Runs 2 100 bp or

2 150 bp

300 Cycle Kit v1.0 or v1.5 300600 pM

NovaSeq 6000 Xp Workflow Runs 2 100 bp or

2 150 bp

300 Cycle Kit v1.0 or v1.5 200400 pM

* For All-In-One assays that include translocation detection, Agilent strongly recommends using paired-end sequencing read length of at least 2 100 bp and preferably 2 150 bp.

Do not use HiSeq 2500 High Output (v4 chemistry) runs if your analysis pipeline includes MBC (i5) reads. Reduced MBC se- quence quality and lowered Q scores have been observed in sequences obtained from HiSeq 2500 platform runs under these conditions. Sequencing performance for Read 1, Read 2, and sample-level index (i7) reads is not affected.

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Use the guidelines below for SureSelectXT HS library sequencing run setup and analysis.

The sample- level index (i7) requires an 8- bp index read. For complete i7 index sequence information, see Table 48 on page 96.

The 8-bp index sequences in SureSelect XT HS Index Primers A01 through H04 differ from the 8-bp index sequences in index primers A01 through H04 in Agilents SureSelect XT system.

CAUTION

Optional use of the degenerate MBC (i5) requires a 10- bp index read.

If MBC analysis is not needed, you can modify the steps in this section to omit sequencing and analysis of i5 index reads. Use of the MBCs is recommended for detection of very low allele frequency variants and when the DNA sample is present in limited amounts.

Note that if you are using the SureCall (v4.2) All-In-One Analysis workflow for analysis of SureSelect Cancer All-In-One assays, this workflow does not include MBC analysis.

NOTE

For the HiSeq, NextSeq, and NovaSeq platforms, set up the run using the instruments user interface, following the guidelines on page 78.

For the MiSeq platform, set up the run using Illumina Experiment Manager (IEM) using the steps detailed on page 81 to page 84 to generate a custom sample sheet.

Do not use Illuminas IEM adaptor trimming options. Make sure any IEM adaptor trimming option checkboxes are cleared (deselected) when setting up the sequencing run. Adaptors are trimmed in later processing steps using the Agilent software tools described below to ensure proper processing.

Demultiplex using the appropriate Illumina software to generate paired- end reads in FASTQ format. If your workflow excludes use of MBCs, you can demultiplex using Illuminas bcl2fastq, BCL Convert or DRAGEN software.

If your workflow includes use of MBCs, demultiplex using Illuminas bcl2fastq software, using the I2 MBC retrieval steps described below.

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Retrieval of I2 index files containing the MBC (i5) index reads requires offline conversion of .bcl to .fastq files. For information on how to do this step, see page 78 for HiSeq and NextSeq runs and see page 84 for MiSeq runs.

For human germline DNA variant analysis, you can use Agilents Alissa Reporter software for the complete FASTQ file to variant discovery process (see page 85 for more information).

For germline or somatic variant analysis, you can use Agilents AGeNT software modules to process the library read FASTQ files to analysis- ready BAM files (see page 86 for more information).

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HiSeq/NextSeq/NovaSeq platform sequencing run setup guidelines

Set up sequencing runs using the instrument control software interface. A sample run setup for the HiSeq platform using 100 + 100 bp paired- end sequencing with MBC (i5) data collection is shown below.

If using the NextSeq or NovaSeq platform, locate the same parameters on the Run Setup screen, and populate the Read Length fields using the Cycles settings shown in HiSeq platform example above. In the Custom Primers section of the NextSeq or NovaSeq platform Run Setup screen, clear (do not select) the checkboxes for all primers (Read 1, Read 2, Index 1 and Index 2).

BaseSpace currently does not support the sequencing of MBCs as index reads. Set up NextSeq runs using the stand- alone mode if your analysis pipeline includes MBC analysis.

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Retrieve I2 FASTQ files containing MBCs

Retrieval of I2 index files containing the MBC (i5) index reads requires offline conversion of .bcl to .fastq files using one of the two methods below.

Option 1: Use bcl2fastq software with base masking

To generate Index 2 FASTQ files containing the MBCs using the bcl2fastq software, follow Illuminas instructions for use of the software with the following modifications:

1 Use of a sample sheet is mandatory and not optional. Modify the sample sheet to include only the sample index (i7/Index 1) and not the MBC (i5/Index 2) by clearing the contents in the I5_Index_ID and index2 columns.

2 Set mask- short- adapter- reads to value of 0.

3 Use the following base mask: Y*, I8, Y10, Y* (where * should be replaced with the actual read length, with the value entered matching the read length value in the RunInfo.xml file).

CAUTION When generating FASTQ files using Illuminas bcl2fastq software, make sure to clear the contents of the index2 column in the sample sheet as described above. Do not enter an N10 sequence to represent the degenerate MBC; instead, simply leave the column cells cleared. The bcl2fastq software does not treat the N character as a wildcard when found in sample sheet index sequences, and usage in this context will cause a mismatch for any sequence character other than N.

Option 2: Use Broad Institute Picard tools

To generate Index 2 FASTQ files containing the P5 MBCs using the Broad Institute Picard tools, complete the following steps:

1 Use tool ExtractIlluminaBarcodes to find the barcodes. A sample set of commands is shown below (commands used by your facility may vary).

nohup java -jar picard.jar ExtractIlluminaBarcodes BASECALLS_DIR= /Data/Intensities/BaseCalls/ OUTPUT_DIR= LANE=1 READ_STRUCTURE= BARCODE_FILE= METRICS_FILE= NUM_PROCESSORS=

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2 Use tool IlluminaBaseCallsToFastq to generate the FASTQ files based on output of step 1. A sample set of commands is shown below (commands used by your facility may vary).

nohup java -jar picard.jar IlluminaBasecallsToFastq BASECALLS_DIR= /Data/Intensities/BaseCalls/ LANE=1 BARCODES_DIR= READ_STRUCTURE= FLOWCELL_BARCODE= MACHINE_NAME= RUN_BARCODE= ADAPTERS_TO_CHECK=PAIRED_END NUM_PROCESSORS= READ_NAME_FORMAT=CASAVA_1_8 COMPRESS_OUTPUTS=true MULTIPLEX_PARAMS= IGNORE_UNEXPECTED_BARCODES=true TMP_DIR=

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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 HS indexes used for each sample. See Table 48 on page 96 for nucleotide sequences of the SureSelect XT HS system indexes.

If your workflow excludes use of MBCs, modify the steps in this section to collect a Single Index Read (i7) and omit steps for I2 MBC file retrieval.

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.

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). Make sure to clear both adaptor- trimming checkboxes under FASTQ Only Workflow- Specific Settings (circled below), since these are selected by default.

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

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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 a SureSelect XT HS index at a later stage.

Likewise, in the I5 Sequence column, assign any of the Illumina i5 indexes, to be corrected to the degenerate MBC at a later stage.

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

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Edit the Sample Sheet to include SureSelect XT HS indexes and MBCs

1 Open the Sample Sheet file in a text editor and edit the i7 and i5 index information for each sample in columns 58 (highlighted in Figure 15).

In column 5 under I7_Index_ID, enter the name of the SureSelect XT HS index assigned to the sample. In column 6 under index, enter the corresponding SureSelect XT HS Index sequence. See Table 48 on page 96 for nucleotide sequences of the SureSelect XT HS indexes.

In column 7 under I5_Index_ID, enter MBC for all samples. In column 8 under index2, enter text NNNNNNNNNN for all samples to represent the degenerate 10- nucleotide MBC tagging each fragment.

Enter N10 text in the index2 column only when sample sheets are processed using MiSeq Reporter software adjusted to retrieve I2 FASTQ files containing MBCs, as detailed on page 84. Sample sheets processed offline using Illuminas bcl2fastq software must not contain N10 wildcard index sequences. See page 79 for more information.

NOTE

Figure 15 Sample sheet for use with MiSeq platform after MiSeq Reporter reconfiguration

2 Save the edited Sample Sheet in an appropriate file location for use in the MiSeq platform run.

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84

Reconfigure the MiSeq Reporter Software to retrieve I2 FASTQ files

By default, MiSeq Reporter software does not generate FASTQ files for index reads. To generate FASTQ I2 index files containing the MBC reads using MiSeq Reporter, adjust the software settings as described below before the first use of the MiSeq instrument for SureSelect XT HS library sequencing. Once changed, this setting is retained for future runs.

To change this setting, open the file MiSeq Reporter.exe.config. Under the tag, add . You must restart the instrument for this setting change to take effect.

Sequence analysis resources

NOTE If you are using the same instrument for assays other than SureSelect XT HS library sequencing, the configuration file should be edited to value="0"/> and the instrument should be restarted before running the other assay.

If you are using the MiSeqDx platform, run the instrument in research mode to make changes to MiSeq Reporter settings. If research mode is not available on your instrument, you may need to upgrade the system to include the dual boot configuration to allow settings changes in research mode.

The alternative methods for retrieval of I2 FASTQ files described on page 78 for HiSeq and NextSeq platform runs may also be applied to MiSeq platform runs.

Guidelines are provided below for typical NGS analysis pipeline steps appropriate for SureSelect XT HS DNA library data analysis. Your NGS analysis pipeline may vary. For SureSelect Cancer All- In- One assay sequence analysis guidelines, see the assay Product Overview Guide.

Prior to analysis, use the appropriate Illumina demultiplexing software to generate paired- end reads (see page 76 for guidelines). The demultiplexed FASTQ data needs to be pre- processed to remove sequencing adaptors and utilize the i5 MBC reads (if collected) using one of the tools described below.

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Post-Capture Sample Processing for Multiplexed Sequencing 5 Sequence analysis resources

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Using Agilents Alissa Reporter software for germline DNA workflows

Alissa Reporter software provides a complete FASTQ- to- Result solution for Agilents SureSelect assays, processing NGS data from FASTQ format to VCF format, and reporting human germline SNV, InDel and CNV calls.

Alissa Reporter is a cloud- based, multi- tenant software as a service (SaaS) product, delivering integrated pre- processing of SureSelect XT HS DNA library reads (adaptor trimming, MBC extraction and de- duplication) along with secondary data analysis and quality control (QC) analytics using a built- in dashboard. To obtain more information and to purchase access to the software please visit the Alissa Reporter page at www.agilent.com.

Key considerations for SureSelect XT HS DNA assay steps prior to Alissa Reporter software analysis are summarized below:

Alissa Reporter applications are available for germline analysis of human DNA libraries enriched using a pre- designed or custom SureSelect human probe (see page 14). Libraries enriched using SureSelect XT HS Human All Exon V7 or V8 probes are analyzed with the corresponding Human All Exon V7 Germline or Human All Exon V8 Germline application in Alissa Reporter. Libraries enriched using other probes, including additional pre- designed probes, are analyzed using an Alissa Reporter Custom application. The Alissa Reporter console provides tools for importing both pre- designed and custom probe designs from SureDesign and setting up a new Custom application for each imported design.

Human All Exon V8+UTR and Human All Exon V8+NCV designs must be imported into Alissa Reporter for use as Custom-type applications. Use the Catalog-type Alissa Reporter applications, including the Human All Exon V8 Germline application, only for the specific probe indicated for the application without any additional design content.

NOTE

Analysis of FFPE- derived or other DNA samples for detection of somatic variants is not supported at the time of this publication. Please visit the Alissa Reporter page at www.agilent.com for information on the latest Alissa Reporter software version capabilities.

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For CNV calling a co- analysis strategy is used in which unrelated samples from the same Alissa Reporter run are used to determine the reference signal for the target sample (no specific reference sample is required). At least 3 and preferably 8 (or more) unrelated samples need to be analyzed in Alissa Reporter together to obtain a reliable reference signal for CNV calls. For CNV calling on the X and Y chromosomes, unrelated samples of the same gender are required. For best results, process the samples to be used for CNV co- analysis in the same SureSelect run and in the same sequencing run in order to minimize any processing- based variance.

Maximum file size for uploads is 50GB/file (in total 400GB/sample). A maximum of 768 FASTQ files can be uploaded in a run.

File sizes>150M reads are randomly subsampled to 150M reads when using the Human All Exon V7 Germline or Human All Exon V8 Germline application.

Unmerged and merged FASTQ files are supported. Upload of BAM files or other non- FASTQ file formats is not supported.

Using Agilents AGeNT software for germline or somatic DNA workflows

Agilents AGeNT software is a Java- based toolkit used for SureSelect XT HS DNA library read processing steps. The AGeNT tools are 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 and review the AGeNT Best Practices document for processing steps suitable for XT HS DNA libraries summarized below.

Prior to variant discovery, the AGeNT Trimmer module is used to pre- process the demultiplexed SureSelect XT HS library FASTQ data to trim sequencing adaptors and prepare MBC reads for insertion in the aligned BAM file.

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

NOTE CReaK is a deduplication tool introduced in AGeNT version 3.0, replacing the AGeNT LocatIt tool. Please visit the AGeNT page at www.agilent.com and review the FAQs for a detailed comparison of LocatIt and CReaK. LocatIt remains available for backward compatibility but CReaK is the recommended tool.

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6 Appendix: Using FFPE-derived DNA Samples

Protocol modifications for FFPE Samples 88

Methods for FFPE Sample Qualification 88

Sequencing Output Recommendations for FFPE Samples 89

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

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6 Appendix: Using FFPE-derived DNA Samples Protocol modifications for FFPE Samples

Protocol modifications for FFPE Samples

88

Protocol modifications that should be applied to FFPE samples are summarized in Table 38.

Methods for FFPE Sample Qualification

Table 38 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

Qualification of DNA Integrity Not required Required

DNA input for Library Preparation page 23

Input amount and means of quantification

10 ng to 200 ng, quantified by Qubit assay

Based on determined DNA integrity (see Table 8 on page 24 and Table 9 on page 25)

DNA Shearing page 26 Mode of DNA shearing 2 120 seconds 240 seconds (continuous)

Enzymatic Fragmentation of DNA page 29

Duration of 37C incubation 2 100 reads: 15 minutes

2 150 reads: 10 minutes

2 100 reads: 15 minutes

2 150 reads: 15 minutes

Pre-capture PCR page 42 Cycle number 811 1114

Sequencing page 89 Output augmentation Per project requirements 1 to 10 based on determined DNA integrity (see Table 39 and Table 40 on page 89)

DNA integrity may be assessed using the Agilent NGS FFPE QC Kit or using the Agilent TapeStation instrument and 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.

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Sequencing Output Recommendations for FFPE Samples

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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 Cq: For samples qualified based on the Cq DNA integrity score, use the guidelines in Table 39. 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.

Samples qualified using DIN: For samples qualified based on the Genomic DNA ScreenTape assay DIN integrity score, use the guidelines in Table 40. 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 200400 Mb of sequencing output to achieve the same coverage.

Table 39 Recommended sequencing augmentation for FFPE-derived DNA samples

Cq 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

Table 40 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

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SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

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7 Reference

Kit Contents 92

Nucleotide Sequences of SureSelect XT HS Indexes 96

Troubleshooting Guide 97

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.

91Agilent Technologies

7 Reference Kit Contents

Kit Contents

The SureSelect Enzymatic Fragmentation Kit, which may be used for DNA sample fragmentation prior to library preparation using the SureSelect XT HS Reagent Kits, includes the reagents listed in Table 41.

SureSelect XT HS Reagent Kits include the component kits listed in Table 42. Detailed contents of each of the multi- part component kits listed in Table 42 are shown in Table 43 through Table 46.

Table 41 Contents of SureSelect Enzymatic Fragmentation Kit (stored at 20C)

Kit Component 16 Reactions (p/n 5191-4079) 96 Reactions (5191-4080)

SureSelect Fragmentation Enzyme tube with green cap tube with green cap

5 SureSelect Fragmentation Buffer tube with blue cap tube with blue cap

Table 42 Contents of SureSelect XT HS Reagent Kits

Component Kit Name Storage Condition

Component Kit Part Number

16 Reaction Kits (G9702A, G9702B)

96 Reaction Kits (G9702C)

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

20C 5500-0138 5500-0140

SureSelect XT HS Index Primers for ILM (Pre PCR)

20C 5500-0141 (Indexes 116), OR

5500-0142 (Indexes 1732)

5190-9876 (Indexes 1-32)

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

Room Temperature

5190-9685 5190-9687

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

20C 5190-9684 5190-9686

92 SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Reference 7 Kit Contents

Table 43 SureSelect XT HS Library Preparation Kit (Pre PCR) Content

Kit Component 16 Reactions (p/n 5500-0138) 96 Reactions (5500-0140)

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

Adaptor Oligo Mix tube with white cap tube with white cap

Forward Primer tube with brown cap tube with brown cap

100 mM dNTP Mix (25 mM each dNTP) tube with green cap tube with green cap

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

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

Table 44 SureSelect XT HS Index Primers Kit (Pre PCR) Content

Kit Component 16 Reaction Kit, Index Primers 1-16 (p/n 5500-0141)

16 Reaction Kit, Index Primers 17-32 (p/n 5500-0142)

96 Reaction Kit, I Index Primers 1-32 (p/n 5190-9876)

SureSelect XT HS Index Primers (reverse primers containing 8-bp index sequence)*

SureSelect XT HS Index Primers A01 through H02, provided in 16 black-capped tubes

SureSelect XT HS Index Primers A03 through H04, provided in 16 black-capped tubes

SureSelect XT HS Index Primers A01 through H04, provided in 96 black-capped tubes (3 vials of each of 32 different primers)

* See Table 48 on page 96 for index sequences.

CAUTION The SureSelect XT HS Index Primers are provided in single-use aliquots. To avoid cross-contamination of libraries, discard each vial after use in one library preparation reaction. Do not retain and re-use any residual volume for subsequent experiments.

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7 Reference Kit Contents

94

Table 45 SureSelect Target Enrichment Kit ILM Hyb Module, Box 1 (Post PCR) Content

Kit Component 16 Reactions (p/n 5190-9685) 96 Reactions (5190-9687)

SureSelect Binding Buffer bottle bottle

SureSelect Wash Buffer 1 bottle bottle

SureSelect Wash Buffer 2 bottle bottle

Table 46 SureSelect XT HS Target Enrichment Kit ILM Hyb Module, Box 2 (Post PCR) Content

Kit Component 16 Reactions (p/n 5190-9684) 96 Reactions (5190-9686)

SureSelect Fast Hybridization Buffer bottle bottle

SureSelect XT HS and XT Low Input 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

100 mM dNTP Mix (25 mM each dNTP) tube with green cap tube with green cap

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

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

SureSelectXT HS Target Enrichment System for Illumina Multiplexed Sequencing

Reference 7 Kit Contents

SureSelectXT HS Tar

Bundles of SureSelect XT HS Reagent Kits with certain Target Enrichment Probes are available for purchase using the Agilent part numbers listed in Table 47. The SureSelect XT HS Reagent Kits included in these bundles are supplied with the same component kits listed in Table 42 on page 92.

Table 47 Supported SureSelect XT HS Reagent Kit + Probe Bundles

Included SureSelect (SSel) XT HS Probe

Included SureSelect XT HS Reagent Kit

16 Reactions, with Index Primers 116

16 Reactions, with Index Primers 1732

96 Reactions, with Index Primers 132

Custom 1499 kb*

* Kits that include Custom SureSelect Cancer All-In-One panels, designed using Agilents SureDesign application, are ordered using these bundled custom design Agilent part numbers. The Custom SureSelect Cancer All-In-One panels are designated using design IDs beginning with an A charac- ter. (Refer to the probe vial label and the associated Certificate of Analysis to view the design ID.)

G9704A G9705A G9706A

Custom 0.5 2.9 Mb* G9704B G9705B G9706B

Custom 35.9 Mb* G9704C G9705C G9706C

Custom 611.9 Mb* G9704D G9705D G9706D

Custom 1224 Mb* G9704E G9705E G9706E

ClearSeq Comp Cancer G9704G G9705G G9706G

Clinical Research Exome V2 G9704H G9705H G9706H

Clinical Research Exome V2 Plus G9704J G9705J G9706J

Human All Exon V6 G9704K G9705K G9706K

Human All Exon V6 Plus G9704L G9705L G9706L

Human All Exon V6+UTRs G9704M G9705M G9706M

Human All Exon V7 G9704N G9705N G9706N

Human All Exon V7 Plus 1 G9704P G9705P G9706P

Human All Exon V7 Plus 2 G9704Q G9705Q G9706Q

Cancer All-In-One Lung G9704R G9705R G9706R

Cancer All-In-One Solid Tumor G9704S G9705S G9706S

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7 Reference Nucleotide Sequences of SureSelect XT HS Indexes

Nucleotide Sequences of SureSelect XT HS Indexes

96

The SureSelect XT HS Index Primers are provided in individual tubes containing single- use aliquots. Tubes are labeled using well position references indicating the recommended well position for runs of up to 32 samples (see Table 48).

Each index is 8 nt in length. See page 76 for sequencing run setup requirements for sequencing libraries using 8- bp indexes.

Table 48 SureSelect XT HS Indexes 132 (A01H04), provided in black-capped tubes

Index # Well Reference

Sequence Index # Well Reference

Sequence

1 A01 GTCTGTCA 17 A03 AGCAGGAA

2 B01 TGAAGAGA 18 B03 AGCCATGC

3 C01 TTCACGCA 19 C03 TGGCTTCA

4 D01 AACGTGAT 20 D03 CATCAAGT

5 E01 ACCACTGT 21 E03 CTAAGGTC

6 F01 ACCTCCAA 22 F03 AGTGGTCA

7 G01 ATTGAGGA 23 G03 AGATCGCA

8 H01 ACACAGAA 24 H03 ATCCTGTA

9 A02 GCGAGTAA 25 A04 CCGTGAGA

10 B02 GTCGTAGA 26 B04 GACTAGTA

11 C02 GTGTTCTA 27 C04 GATAGACA

12 D02 TATCAGCA 28 D04 GCTCGGTA

13 E02 TGGAACAA 29 E04 GGTGCGAA

14 F02 TGGTGGTA 30 F04 AACAACCA

15 G02 ACTATGCA 31 G04 CGGATTGC

16 H02 CCTAATCC 32 H04 AGTCACTA

CAUTION The 8-bp index sequences in SureSelect XT HS Index Primers A01 through H04 differ from the 8-bp index sequences in index primers A01 through H04 in Agilents SureSelect XT system.

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Reference 7 Troubleshooting Guide

Troubleshooting Guide

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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 56C by gently pipetting the digestion reaction every 2030 minutes, visually inspecting the solution for the presence of tissue clumps. If clumps are still present after the 1- hour incubation at 56C, add another 10 l of Proteinase K and continue incubating at 56C, 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 56C for any sample.

If concentration of FFPE DNA samples is too low for enzymatic fragmentation

The standard enzymatic fragmentation protocol requires 10200 ng input DNA in a volume of 7 l, and uses a final fragmentation reaction volume of 10 l. For dilute FFPE samples, enzymatic fragmentation may be performed using the modified protocol below:

Bring FFPE samples containing 10200 ng DNA to 17 l final volume with 1X Low TE Buffer.

Prepare the Fragmentation master mix as directed in Table 13 on page 30.

Add 3 l of the master mix to each 17- l DNA sample. Mix and spin as directed on page 30.

Run the thermal cycling program in Table 11 on page 29 using the 37C fragmentation duration shown in the table below.

NGS read length High-quality DNA samples FFPE DNA samples

2 100 reads 25 minutes 25 minutes

2 150 reads 15 minutes 25 minutes

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7 Reference Troubleshooting Guide

98

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 35) is kept at room temperature for 3045 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.

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.

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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 44.

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 samples analyzed on page 47 to page 50. 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 38. 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 l with nuclease free water, then follow the SPRI purification procedure on page 44.

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.

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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 Probe Hybridization Mix is prepared immediately before use, as directed on page 55, 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 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 post- capture purification on page 65.

If low % on-target is observed in library sequencing results

Stringency of post- hybridization washes may have been lower than required. Complete the wash steps as directed, paying special attention to the details of SureSelect Wash Buffer 2 washes listed below:

Ensure that SureSelect Wash Buffer 2 is pre- warmed to 70C before use (see page 58). Select a thermal cycler with a block configured for efficient heating of 0.2 ml liquid volumes; ensure that the plasticware containing the wash buffer is fully seated in the thermal cycler block wells, with minimal liquid volume visible above the block during the pre- warming step.

Samples are maintained at 70C during washes (see page 59)

Bead suspensions are mixed thoroughly during washes by pipetting up and down and vortexing (see page 59)

Minimize the amount of time that hybridization reactions are exposed to RT conditions during hybridization setup. Locate a vortex and plate spinner or centrifuge in close proximity to thermal cycler to retain the 65C sample temperature during mixing and transfer steps (step 8 to step 9 on page 56).

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If low uniformity of coverage with high AT-dropout is observed in library sequencing results

High AT- dropout may indicate that hybridization conditions are too stringent to obtain the desired level of coverage for AT- rich targets. Repeat target enrichment at lower stringency using a modified thermal cycler program for hybridization, reducing the hybridization temperature in segments 4 and 5 from 65C to 62.5C or 60C (see Table 26 on page 53).

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An abbreviated summary of the protocol steps is provided below for experienced users. Use the complete protocol on page 22 to page 84 until you are familiar with all of the protocol details such as reagent mixing instructions and instrument settings.

H

Step Summary of Conditions

Library Prep

Prepare, qualify, and fragment DNA samples

Prepare 10200 ng gDNA (in 50 l Low TE for Covaris or in 7 l H20 for enzymatic fragmentation)

For FFPE DNA, qualify integrity and adjust input amount as directed on page 24 and page 25

Mechanically shear DNA using Covaris with shearing conditions on page 27 OR fragment DNA using SureSelect Enzymatic Fragmentation Kit with protocol on page 29 (50 l final volume)

Prepare Ligation master mix

Per reaction: 23 l Ligation Buffer + 2 l T4 DNA Ligase

Keep at room temperature 3045 min before use

Prepare End-Repair/dA-Tailing master mix

Per reaction: 16 l End Repair-A Tailing Buffer + 4 l End Repair-A Tailing Enzyme Mix

Keep on ice

End-Repair and dA-Tail the sheared DNA

50 l fragmented DNA sample + 20 l End Repair/dA-Tailing master mix

Incubate in thermal cycler: 15 min @ 20C, 15 min @ 72C, Hold @ 4C

Ligate adaptor 70 l DNA sample + 25 l Ligation master mix +5 l Adaptor Oligo Mix

Incubate in thermal cycler: 30 min @ 20C, Hold @ 4C

Purify DNA 100 l DNA sample + 80 l AMPure XP bead suspension

Elute DNA in 35 l nuclease-free H2O

Prepare PCR master mix Per reaction: 10 l 5 Herculase II Reaction Buffer + 0.5 l 100 mM dNTP Mix + 2 l Forward Primer + 1 l Herculase II Fusion DNA Polymerase

Keep on ice

Amplify the purified DNA 34.5 l purified DNA + 13.5 l PCR master mix + 2 l assigned SureSelect XT HS Index Primer

Amplify in thermal cycler using program on page 42

Purify amplified DNA 50 l amplified DNA + 50 l AMPure XP bead suspension

Elute DNA in 15 l nuclease-free H2O

Quantify and qualify DNA Analyze 1 l using Agilent 2100 Bioanalyzer or 4200 TapeStation instrument

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Hybridization/Capture

Program thermal cycler Input thermal cycler program on page 53 and pause program

Prep DNA in Hyb plate Adjust 5001000 ng purified prepared library to 12 l volume with nuclease-free H2O

Run pre-hybridization blocking protocol

12 l library DNA + 5 l SureSelect XT HS and XT Low Input Blocker Mix

Run paused thermal cycler program segments 1 through 3; start new pause during segment 3 (1 min @ 65C)

Prepare Probe Hyb Mix Prepare 25% RNase Block dilution, then prepare appropriate mixture below:

Probes 3 Mb: 2 l 25% RNase Block + 5 l Probe + 6 l SureSelect Fast Hybridization Buffer

Probes <3 Mb: 2 l 25% RNase Block + 2 l Probe + 3 l nuclease-free H2O + 6 l SureSelect Fast Hybridization Buffer

Run the hybridization With cycler paused and samples retained in cycler, add 13 l Probe Hyb Mix to wells

Resume the thermal cycler program, completing segments 4 (hybridization) and 5 (65C or 21C hold)

Prepare streptavidin beads Wash 50 l Dynabeads MyOne Streptavidin T1 beads 3 in 200 l SureSelect Binding Buffer

Capture hybridized libraries

Add hybridized samples (~30 l) to washed streptavidin beads (200 l)

Incubate 30 min at RT with vigorous shaking (1400-1800 rpm)

During incubation, pre-warm 6 200 l aliquots per sample of SureSelect Wash Buffer 2 to 70C

Wash captured libraries Collect streptavidin beads with magnetic stand, discard supernatant

Wash beads 1 with 200 l SureSelect Wash Buffer 1 at RT

Wash beads 6 with 200 l pre-warmed SureSelect Wash Buffer 2 (5 minutes at 70C per wash)

Resuspend washed beads in 25 l nuclease-free H2O

Post-capture amplification

Prepare PCR master mix Per reaction: 12.5 l nuclease-free H2O+ 10 l 5 Herculase II Reaction Buffer + 0.5 l 100 mM dNTP Mix + 1 l SureSelect Post-Capture Primer Mix + 1 l Herculase II Fusion DNA Polymerase

Keep on ice

Amplify the bead-bound captured libraries

25 l DNA bead suspension+ 25 l PCR master mix

Amplify in thermal cycler using conditions on page 63

Purify amplified DNA Remove streptavidin beads using magnetic stand; retain supernatant

50 l amplified DNA + 50 l AMPure XP bead suspension

Elute DNA in 25 l nuclease-free H2O

Quantify and qualify DNA Analyze 1 l using Agilent 2100 Bioanalyzer or 4200 TapeStation instrument

Step Summary of Conditions

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In This Book

This guide contains information to run the SureSelectXT HS target enrichment protocol.

Agilent Technologies, Inc. 2017-2022

Version F0, September 2022

*G9702-90000 * p/n G9702-90000

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