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Agilent SureSelect QXT NGS Workstation Option B Sequencer Protocol Manual PDF

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Summary of Content for Agilent SureSelect QXT NGS Workstation Option B Sequencer Protocol Manual PDF

SureSelectQXT Automated Target Enrichment for the Illumina Platform Featuring Transposase-Based Library Prep Technology Automated using Agilent NGS Workstation Option B

Protocol Version F0, November 2021

SureSelect platform manufactured with Agilent SurePrint technology.

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

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Notices Agilent Technologies, Inc. 2014-2016, 2018, 2021

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

Manual Part Number G9681-90010

Edition Version F0, November 2021

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

Warranty The material contained in this document is provided as is, and is subject to being changed, without notice, in future editions. Further, to the maximum extent permitted by applicable law, Agilent disclaims all warranties, either express or implied, with regard to this manual and any information contained herein, including but not limited to the implied warranties of merchantability and fitness for a particular purpose. Agilent shall not be liable for errors or for incidental or consequential damages in connection with the furnishing, use, or performance of this document or of any information contained herein. Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms, the warranty terms in the separate agreement shall control.

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

Acknowledgment Oligonucleotide sequences 2006, 2008, and 2011 Illumina, Inc. All rights reserved. Only for use with the Illumina sequencer systems and associated assays.

Technical Support For US and Canada

Call (800) 227-9770 (option 3,4,4) Or send an e-mail to: ngs.support@agilent.com For all other regions Agilents world-wide Sales and Support Center contact details for your location can be obtained at www.agilent.com/en/contact-us/page.

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.

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 3

In this Guide... This guide describes an optimized protocol for Illumina paired-end multiplexed library preparation using the SureSelectQXT Automated Target Enrichment system.

This protocol is specifically developed and optimized to enrich targeted regions of the genome from repetitive sequences and sequences unrelated to the research focus prior to sample sequencing. Sample processing steps are automated using Agilents NGS Workstation Option B.

1 Before You Begin This chapter contains information that you should read and understand before you start an experiment.

2 Using the Agilent NGS Workstation for SureSelect Target Enrichment This chapter contains an orientation to the Agilent NGS Workstation, an overview of the SureSelect target enrichment protocol, and considerations for designing SureSelect experiments for automated processing.

3 Sample Preparation This chapter describes the steps to prepare gDNA sequencing libraries for target enrichment.

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

5 Indexing and Sample Processing for Multiplexed Sequencing This chapter describes the steps for post-capture amplification and guidelines for sequencing sample preparation.

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

4 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Whats New in Version F0 Support for SureSelect XT HS Human All Exon V8 Probe, SureSelect XT HS Human All Exon

V8+UTR Probe, and SureSelect XT HS Human All Exon V8+NCV Probe (see Table 2 on page 12).

Updates to the NGS Workstation components user guide part numbers (see Table 6 on page 18).

Updated information on the pipette head options and fluid transfer capabilities of the Bravo platform (see page 18).

Updates to downstream sequencing guidelines (see Table 60 on page 84). Updated document look and feel.

Whats New in Version E1 Updates to thermal cycler and plasticware recommendations (see Table 5 on page 14 and see

Caution on page 31) Updates to downstream sequencing support information including sequencing kit selection

and seeding concentration updates (see Table 60 on page 84) and support for the NovaSeq platform (see page 84 through page 89 and see page 94)

Updates to instructions for adaptor trimming using SureCall (see page 88) or AGeNT (see page 89)

Whats New in Version E0 Support for revised SureSelect custom probe products, produced using an updated

manufacturing process beginning August, 2020 (see Table 3 on page 13). Custom probes produced using the legacy manufacturing process are also fully supported by the protocols in this document. Probe information was reorganized (see Table 2 on page 12 and Table 3 on page 13), and probe nomenclature throughout document was updated.

Support for Agilent 4150 TapeStation system and Agilent 5200 Fragment Analyzer system (see page 15).

Minor updates to 2100 Bioanalyzer and 4200/4150 TapeStation use instructions and reference document links (see page 48, page 49, page 80, and page 81.

Updates to ordering information for AMPure XP Kits and 1X Low TE Buffer (see Table 1 on page 12) and for Qubit Fluorometer (see Table 5 on page 14).

Removal reference information for expired SureSelectQXT Reagent Kits p/n G9681A/G9681B, replaced by G9683A/G9683B in 2018 (see Table 1 on page 12, and Table 72 and Table 73 on page 92 for current Reagent Kit information).

Updates to Technical Support contact information (see page 2).

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 5

6 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Content

1 Before You Begin

Procedural Notes 10

Safety Notes 11

Required Reagents 12

Optional Reagents 13

Required Equipment 14

2 Using the Agilent NGS Workstation for SureSelect Target Enrichment

About the Agilent NGS Workstation 18 About the Bravo Platform 18 VWorks Automation Control Software 21

Overview of the SureSelectQXT Target Enrichment Procedure 27

Experimental Setup Considerations for Automated Runs 30 Considerations for Placement of gDNA Samples in 96-well Plates for Automated Processing 30 Considerations for Equipment Setup 31

PCR Plate Type Considerations 31

3 Sample Preparation Step 1. Prepare the genomic DNA samples and Library Prep reagents 34 Step 2. Fragment and adaptor-tag the genomic DNA samples 35 Step 3. Amplify adaptor-ligated libraries 40

Step 4. Purify amplified DNA using AMPure XP beads 45 Step 5. Assess Library DNA quantity and quality 48

4 Hybridization Step 1. Aliquot prepped DNA samples for hybridization 52 Step 2. Hybridize the gDNA library and probe 55 Step 3. Capture the hybridized DNA 64

5 Indexing and Sample Processing for Multiplexed Sequencing Step 1. Amplify the captured libraries to add index tags 72

Step 2. Purify the amplified indexed libraries using AMPure XP beads 78 Step 3. Assess indexed DNA quality 80

Step 4. Quantify each index-tagged library by QPCR (optional) 82 Step 5. Pool samples for Multiplexed Sequencing 82 Step 6. Prepare sequencing samples 83

Step 7. Set up the sequencing run and trim adaptors from the reads 88

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 7

6 Reference

Kit Contents 92

Nucleotide Sequences of SureSelectQXT Dual Indexes 94

Guidelines for Multiplexing with Dual-Indexed Samples 96

8 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

SureSelectQXT Automated Target Enrichment for the Illumina Platform Protocol

1 Before You Begin Procedural Notes 10 Safety Notes 11 Required Reagents 12 Optional Reagents 13 Required Equipment 14

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

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

9

Procedural Notes

The SureSelectQXT system requires high-quality DNA samples for optimal performance. Use best practices for verifying DNA sample quality before initiating the workflow. For best practice, store diluted DNA solutions at 4C to avoid repeated freeze-thaw cycles, which may compromise DNA quality.

Performance of the SureSelectQXT library preparation protocol is very sensitive to variations in amounts of DNA sample and other reaction components. It is important to quantify and dilute DNA samples as described on page 34. Carefully measure volumes for all reaction components, and combine components as described in this instruction manual. Use best-practices for liquid handling, including regular pipette calibration, to ensure precise volume measurement.

Use care in handling the SureSelect QXT Enzyme Mix. After removing the vial from storage at 20C, keep on ice or in a cold block while in use. Return the vial to storage at 20C promptly after use.

Use best-practices to prevent PCR product contamination of samples throughout the workflow: 1 Assign separate pre-PCR and post-PCR pipettors, supplies, and reagents. In particular,

never use materials designated to post-PCR segments for the pre-PCR segments of the workflow. For the pre-PCR workflow steps, always use dedicated pre-PCR pipettors with nuclease-free aerosol-resistant tips to pipette dedicated pre-PCR solutions.

2 Maintain clean work areas. Clean pre-PCR surfaces that pose the highest risk of contamination daily using a 10% bleach solution.

3 Wear powder-free gloves. Use good laboratory hygiene, including changing gloves after contact with any potentially-contaminated surfaces.

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.

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.

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

10 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Safety Notes

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

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 11

Required Reagents

Table 1 Required Reagents for SureSelectQXT Target Enrichment

Description Vendor and part number

SureSelect or ClearSeq Probe Capture Library Select the appropriate probe from Table 2 or Table 3

SureSelectQXT Reagent Kit, 96 Samples (for Illumina HiSeq, MiSeq, and NextSeq platforms)

Agilent p/n G9683B

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, molecular biology grade Sigma-Aldrich p/n E7023

Qubit dsDNA HS Assay Kit or Qubit dsDNA BR Assay Kit 100 assays 500 assays

Thermo Fisher Scientific p/n Q32851 Thermo Fisher Scientific p/n Q32850 p/n Q32853

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

Table 2 Compatible Pre-Designed Probes for Automation

Probe Capture Library 96 Reactions

SureSelect XT HS Human All Exon V8 5191-6875

SureSelect XT HS Human All Exon V8+UTR 5191-7403

SureSelect XT HS Human All Exon V8+NCV 5191-7409

SureSelect XT Human All Exon V7 5191-4006

SureSelect XT Human All Exon V6 5190-8865

SureSelect XT Human All Exon V6 + UTRs 5190-8883

SureSelect XT Human All Exon V6 + COSMIC 5190-9309

SureSelect XT Clinical Research Exome V2 5190-9493

SureSelect XT Focused Exome 5190-7789

SureSelect XT Mouse All Exon 5190-4643

ClearSeq Comprehensive Cancer XT 5190-8013

ClearSeq Inherited Disease XT 5190-7520

12 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Optional Reagents

Pre-designed Probes customized with additional Plus custom content

SureSelect XT 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.

SureSelect XT 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

SureSelect XT Focused Exome Plus 1

SureSelect XT Focused Exome Plus 2

ClearSeq Comprehensive Cancer Plus XT

ClearSeq Inherited Disease Plus XT

Table 3 Compatible Custom Probes for Automation*

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

Probe Capture Library 96 Reactions 480 Reactions

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.

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

Table 2 Compatible Pre-Designed Probes for Automation

Probe Capture Library 96 Reactions

Table 4 Optional Reagents for SureSelectQXT Target Enrichment

Description Vendor and part number

Agilent QPCR NGS Library Quantification Kit (Illumina GA) Agilent p/n G4880A

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 13

Required Equipment Table 5 Required Equipment for SureSelectQXT Target Enrichment

Description Vendor and part number

Agilent NGS Workstation Option B Contact Agilent Automation Solutions for more information: Customerservice.automation@agilent.com

Agilent p/n G5522A (VWorks software version 13.1.0.1366, 13.0.0.1360, or 11.3.0.1195) OR Agilent p/n G5574AA (VWorks software version 13.1.0.1366)

Robotic Pipetting Tips (Sterile, Filtered, 250 L) Agilent p/n 19477-022

Clear Peelable Seal plate seals (for use with the PlateLoc Thermal Plate Sealer)

Agilent p/n 16985-001

Thermal cycler and accessories Various suppliers Important: Not all PCR plate types are supported for use in the VWorks automation protocols for the Agilent NGS Workstation. Select a thermal cycler that is compatible with one of the supported PCR plate types. See supported plate types in the listing below.

PCR plates compatible with the Agilent NGS Workstation and associated VWorks automation protocols

Only the following PCR plates are supported: 96 ABI PCR half-skirted plates (MicroAmp Optical plates),

Thermo Fisher Scientific p/n N8010560 96 Agilent semi-skirted PCR plate, Agilent p/n 401334 96 Eppendorf Twin.tec half-skirted PCR plates, Eppendorf

p/n 951020303 96 Eppendorf Twin.tec PCR plates (full-skirted), Eppendorf

p/n 951020401

Eppendorf twin.tec full-skirted 96-well PCR plates Eppendorf p/n 951020401 or 951020619

Thermo Scientific Reservoirs Thermo Fisher Scientific p/n 1064-15-6

Nunc DeepWell Plates, sterile, 1.3-mL well volume Thermo Fisher Scientific p/n 260251

Axygen 96 Deep Well Plate, 2 mL, Square Well (waste reservoirs; working volume 2.2 mL)

Axygen p/n P-2ML-SQ-C E & K Scientific p/n EK-2440

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

Nucleic acid surface decontamination wipes DNA Away Surface Decontaminant Wipes, Thermo Fisher Scientific p/n 7008, or equivalent

Qubit Fluorometer Thermo Fisher Scientific p/n Q33238

Qubit Assay Tubes Thermo Fisher Scientific p/n Q32856

Vacuum concentrator Savant SpeedVac, model DNA120, with 96-well plate rotor, model RD2MP, or equivalent

Magnetic separator DynaMag-50 magnet, Thermo Fisher Scientific p/n 123-02D or equivalent

14 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

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

Centrifuge Eppendorf Centrifuge model 5804 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

Pipettes (multichannel pipette and P10, P20, P200 and P1000 pipettes)

Rainin Pipet-Lite Pipettes or equivalent

Vortex mixer general laboratory supplier

Ice bucket general laboratory supplier

Powder-free gloves general laboratory supplier

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

* 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.

Table 5 Required Equipment for SureSelectQXT Target Enrichment

Description Vendor and part number

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 15

16 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

SureSelectQXT Automated Target Enrichment for the Illumina Platform Protocol

2 Using the Agilent NGS Workstation for SureSelect Target Enrichment About the Agilent NGS Workstation 18 Overview of the SureSelectQXT Target Enrichment Procedure 27 Experimental Setup Considerations for Automated Runs 30

This chapter contains an orientation to the Agilent NGS Workstation, an overview of the SureSelectQXT target enrichment protocol, and considerations for designing SureSelectQXT experiments for automated processing using the Agilent NGS Workstation.

17

About the Agilent NGS Workstation

Review the user guides listed in Table 6 (available at Agilent.com) to become familiar with the general features and operation of the Agilent NGS Workstation Option B components. Instructions for using the Bravo platform and other workstation components for the SureSelectQXT Target Enrichment workflow are detailed in this user guide.

About the Bravo Platform The Bravo platform is a versatile liquid handler with a nine plate-location platform deck, suitable for handling 96-well, 384-well, and 1536-well plates. The Bravo platform is controlled by the VWorks Automation Control software. Fitted with a choice of three interchangeable disposable-tip pipette heads, it accurately dispenses fluids from 0.3 L to 250 L.

Bravo Platform Deck

The protocols in the following sections include instructions for placing plates and reagent reservoirs on specific Bravo deck locations. Use Figure 1 to familiarize yourself with the location numbering convention on the Bravo platform deck.

CAUTION Before you begin, make sure that you have read and understand operating, maintenance and safety instructions for using the Bravo platform and additional devices included with the workstation. Refer to the user guides listed in Table 6.

Table 6 Agilent NGS Workstation components User Guide reference information

Device User Guide part number

Bravo Platform SD-V1000376 (previously G5562-90000)

VWorks Software G5415-90068 (VWorks versions 13.1.0.1366 and 13.0.0.1360), or G5415-90063 (VWorks version 11.3.0.1195)

BenchCel Microplate Handler G5580-90000

Labware MiniHub G5584-90001

PlateLoc Thermal Microplate Sealer G5585-90010

18 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Figure 1 Bravo platform deck

Setting the Temperature of Bravo Deck Heat Blocks

Bravo deck positions 4 and 6 are equipped with Inheco heat blocks, used to incubate sample plates at defined temperatures during the run. Runs that include high- (85C) or low- (4C) temperature incubation steps may be expedited by pre-setting the temperature of the affected block before starting the run.

Bravo deck heat block temperatures may be changed using the Inheco Multi TEC Control device touchscreen as described in the steps below. See Table 7 for designations of the heat block-containing Bravo deck positions on the Multi TEC control device.

1 Using the arrow buttons, select the appropriate block (CPAC 2 block 1 or CPAC 2 block 2).

2 To set the temperature of the selected block, press the SET button.

Table 7 Inheco Multi TEC Control touchscreen designations

Bravo Deck Position Designation on Inheco Multi TEC Control Screen

4 CPAC 2 1

6 CPAC 2 2

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 19

3 Using the numeral pad, enter the desired temperature. The entered temperature appears in the top, left rectangle. Once the correct temperature is displayed, press the rectangle to enter the temperature.

4 Press the Temp button until the new temperature is displayed on the SET button and until the Temp button is darkened, indicating that the selected heat block is heating or cooling to the new temperature setting. The current temperature of the block is indicated in the center of the display.

Setting the Temperature of Bravo Deck Position 9 Using the ThermoCube Device

Bravo deck position 9 is equipped with a ThermoCube thermoelectric temperature control system, used to incubate components at a defined temperature during the run. During protocols that require temperature control at position 9, you will be instructed to start and set the temperature of the ThermoCube device before starting the run.

ThermoCube temperature settings are modified using the control panel (LCD display screen and four input buttons) on the front panel of the device using the following steps. 1 Turn on the ThermoCube and wait for the LCD screen to display TEMP. 2 Press the UP or DOWN button to change SET TEMP 1 to the required set point. 3 Press the START button.

The ThermoCube then initiates temperature control of Bravo deck position 9 at the displayed set point.

20 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Using the Labware MiniHub

The protocols in the following sections include instructions for placing plates or reservoirs at specific Labware MiniHub positions. Use Figure 2 to familiarize yourself with the required orientations loading plates in the Labware MiniHub for use in SureSelect automation protocols.

For Thermo Scientific reservoirs, place the notched corner facing the center of the hub.

Figure 2 Agilent Labware MiniHub plate orientation.

VWorks Automation Control Software VWorks software, included with your Agilent NGS Workstation, allows you to control the robot and integrated devices using a PC. The Agilent NGS Workstation is preloaded with VWorks software containing all of the necessary SureSelect system liquid handling protocols. General instructions for starting up the VWorks software and the included protocols is provided below. Each time a specific VWorks protocol is used in the SureSelect procedure, any settings required for that protocol are included in the relevant section of this manual.

Logging in to the VWorks software

1 Double-click the VWorks icon or the SureSelectQXT_ILM_v1.0.VWForm shortcut on the Windows desktop to start the VWorks software.

2 If User Authentication dialog is not visible, click Log in on the VWorks window toolbar. 3 In the User Authentication dialog, type your VWorks user name and password, and click OK. (If

no user account is set up, contact the administrator.)

NOTE The instructions in this manual are compatible with VWorks software version 13.1.0.1366, 13.0.0.1360, or 11.3.0.1195, including SureSelectQXT automation protocols version 1.0.

If you have questions about VWorks version compatibility, please contact service.automation@agilent.com.

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 21

VWorks protocol and runset files

VWorks software uses two file types for automation runs, .pro (protocol) files and .rst (runset) files. Runset files are used for automated procedures in which the workstation uses more than one automation protocol during the run.

Using the SureSelectQXT_ILM_v1.0.VWForm to setup and start a run

Use the VWorks form SureSelectQXT_ILM_v1.0.VWForm, shown below, to set up and start each SureSelect automation protocol or runset.

1 Open the form using the SureSelectQXT_ILM_v1.0.VWForm shortcut on your desktop. 2 Use the form drop-down menus to select the appropriate SureSelect workflow step and

number of columns of samples for the run. 3 Once all run parameters have been specified on the form, click Display Initial Workstation

Setup.

22 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

4 The Workstation Setup region of the form will then display the required placement of reaction components and labware on the NGS Workstation for the specified run parameters.

5 After verifying that the NGS Workstation has been set up correctly, click Run Selected Protocol.

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 23

Error messages encountered at start of run

After starting the run, you may see the error messages displayed below. When encountered, make the indicated selections and proceed with the run. Encountering either or both of these error messages is not indicative of a problem with the NGS workstation or your run setup. 1 If you encounter the G-axis error message shown below, select Ignore and Continue, leaving

device in current state.

24 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

2 If you encounter the W-axis error message shown below, select Retry.

Verifying the Simulation setting

VWorks software may be run in simulation mode, during which commands entered on screen are not completed by the NGS workstation. If workstation devices do not respond when you start a run, verify the simulation mode status in VWorks using the following steps. 1 Verify that Simulation is off is displayed on the status indicator (accessible by clicking View >

Control Toolbar).

2 If the indicator displays Simulation is on, click the status indicator button to turn off the simulation mode.

NOTE If you cannot see the toolbar above the SureSelect_XT_Illumina VWorks form, click the Full Screen button to exit full screen mode. If the toolbar is still not visible, right-click on the form and then select Control Toolbar from the menu.

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 25

Finishing a protocol or runset

The window below appears when each run is complete. Click Yes to release the BenchCel racks to allow removal of components used in the current run in preparation for the next .pro or .rst run.

26 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Overview of the SureSelectQXT Target Enrichment Procedure

Figure 3 summarizes the SureSelectQXT target enrichment workflow for samples to be sequenced using the Illumina paired-read sequencing platform. For each sample to be sequenced, individual library preparations, hybridizations, and captures are performed. The samples are then tagged by PCR with an index sequence. Depending on the target size of the SureSelect capture, up to 96 samples can be pooled and sequenced in a single lane using the dual index tags that are provided with SureSelectQXT Library Prep kits.

Table 8 summarizes how the VWorks protocols are integrated into the SureSelectQXT workflow. See Sample Preparation, Hybridization, and Indexing and Sample Processing for Multiplexed Sequencing chapters for complete instructions for use of the VWorks protocols for sample processing.

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 27

Figure 3 Overall sequencing sample preparation workflow.

28 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Table 8 Overview of VWorks protocols and runsets used for SureSelectQXT Target Enrichment

Workflow Step (Protocol Chapter)

Substep VWorks Protocols Used for Agilent NGS Workstation automation

Sample Preparation

Prepare fragmented and adaptor-tagged DNA

LibraryPrep_QXT_ILM_v1.0.rst

Amplify adaptor-tagged DNA Pre-CapturePCR_QXT_ILM_v1.0.pro

Purify DNA using AMPure XP beads AMPureXP_QXT_ILM_v1.0.pro:Pre-Capture PCR

Hybridization

Aliquot prepped libraries for hybridization

Aliquot_Libraries_v1.0.pro

Hybridize prepped DNA to Probe Hybridization_QXT_v1.0.pro

Capture and wash DNA hybrids SureSelectQXT_Capture&Wash_v1.0.rst

Indexing Add index tags by PCR Post-CapturePCR_QXT_ILM_v1.0.pro

Purify DNA using AMPure XP beads AMPureXP_QXT_ILM_v1.0.pro:Post-Capture PCR

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 29

Experimental Setup Considerations for Automated Runs

Agilent SureSelect Automated Library Prep and Capture System runs may include 1, 2, 3, 4, 6, or 12 columns (equivalent to 8, 16, 24, 32, 48, or 96 wells) of gDNA samples to be enriched for sequencing on the Illumina platform. Plan your experiments using complete columns of samples.

The number of columns or samples that may be processed using the supplied reagents will depend on the experimental design. For greatest efficiency of reagent use, plan experiments using at least 3 columns per run. Each 96-reaction kit contains sufficient reagents for 96 reactions configured as 4 runs of 3 columns of samples per run.

Considerations for Placement of gDNA Samples in 96-well Plates for Automated Processing

The Agilent NGS Workstation processes samples column-wise beginning at column 1. gDNA samples should be loaded into 96-well plates column-wise, in well order A1 to H1, then A2 to H2, ending with A12 to H12. When processing partial runs with <12 sample columns, do not leave empty columns between sample columns; always load the plate using the left-most column that is available.

At the hybridization step (see Figure 3), you can add a different Probe Capture Library to each row of the plate. Plan your experiment such that each prepared DNA library corresponds to the appropriate probe row in the sample plate.

For post-capture amplification (see Figure 3), different probes can require different amplification cycle numbers, based on the probe design sizes. It is most efficient to process similar-sized probes on the same plate. See Table 55 on page 78 to determine which probes may be amplified on the same plate.

Post-capture dual index assignments for the DNA samples can affect sample placement decisions at the beginning of the workflow. For example, all samples on the same row of the DNA sample plate must be assigned to the same P5 indexing primer during sample indexing after hybridization (see Figure 3). It is important to review and understand the guidelines for assignment of dual indexing primers on page 74 while planning sample placement for the run to ensure that the indexing design is compatible with the initial DNA sample placement.

Table 9 Columns to Samples Equivalency

Number of Columns Processed Total Number of Samples Processed

1 8

2 16

3 24

4 32

6 48

12 96

30 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Considerations for Equipment Setup Some workflow steps require the rapid transfer of sample plates between the Bravo deck and

a thermal cycler. Locate your thermal cycler in close proximity to the Agilent NGS Workstation to allow rapid and efficient plate transfer.

Several workflow steps require that the sample plate be sealed using the PlateLoc thermal microplate sealer included with the Agilent NGS Workstation, and then centrifuged to collect any dispersed liquid. To maximize efficiency, locate the centrifuge in close proximity to the Agilent NGS Workstation.

PCR Plate Type Considerations Automation protocols include several liquid-handling steps in which reagents are dispensed to PCR plates in preparation for transfer to a thermal cycler. For these steps you must specify the PCR plate type to be used on the SureSelectQXT_ILM_v1.0.VWForm to allow correct configuration of the liquid handling components for the PCR plate type. Before you begin the automation protocol, make sure that you are using a supported PCR plate type. The PCR plate type to be used in the protocol is specified using the menu below. Vendor and part number information is provided for the supported plate types in Table 10 on page 31.

CAUTION The plates listed in Table 10 are compatible with the Agilent NGS Bravo and associated VWorks automation protocols, designed to support use of various thermal cyclers.

Do not use PCR plates that are not listed in Table 10 even if they are compatible with your chosen thermal cycler.

Table 10 Ordering information for supported PCR plates

Description in VWorks menu Vendor and part number

96 ABI PCR half-skirted plates (MicroAmp Optical plates) Thermo Fisher Scientific p/n N8010560

96 Agilent semi-skirted PCR plate Agilent p/n 401334

96 Eppendorf Twin.tec half-skirted PCR plates Eppendorf p/n 951020303

96 Eppendorf Twin.tec PCR plates (full-skirted) Eppendorf p/n 951020401 or 951020619

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 31

32 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

SureSelectQXT Automated Target Enrichment for the Illumina Platform Protocol

3 Sample Preparation Step 1. Prepare the genomic DNA samples and Library Prep reagents 34 Step 2. Fragment and adaptor-tag the genomic DNA samples 35 Step 3. Amplify adaptor-ligated libraries 40 Step 4. Purify amplified DNA using AMPure XP beads 45 Step 5. Assess Library DNA quantity and quality 48

This section contains instructions for gDNA library preparation specific to the Illumina paired-read sequencing platform and to automated processing using the Agilent NGS Workstation.

33

Step 1. Prepare the genomic DNA samples and Library Prep reagents It is important to have all materials prepared in advance of use in the SureSelectQXT automated Library Prep protocol. In this step, the gDNA is carefully quantified and dispensed into the sample plate. Additional reagents that require modification or temperature equilibration before use are also prepared in this step. 1 Remove the DMSO vial from the SureSelect QXT Library Prep Kit Box 2 in 20C storage. Leave

the DMSO vial at room temperature in preparation for use on page 41. 2 Prepare reagents for the purification protocols on page 35 and page 45.

a Transfer the AMPure XP beads to room temperature. The beads should be held at room temperature for at least 30 minutes before use. Do not freeze the beads at any time.

b Prepare 150 mL of fresh 70% ethanol for use in the purification steps. The 70% ethanol may be used for multiple steps done on the same day, when stored in a sealed container.

3 Obtain the bottle of SureSelect QXT Stop Solution from SureSelect QXT Hyb Module Box 1 (stored at room temperature). Verify that the SureSelect QXT Stop Solution contains 25% ethanol, by referring to the container label and the instructions below.

Before the first use of a fresh container, add 1.5 mL of ethanol to the provided bottle containing 4.5 mL of stop solution, for a final ethanol concentration of 25%. Seal the bottle then vortex well to mix. After adding the ethanol, be sure to mark the label for reference by later users.

Keep the prepared 1X SureSelect QXT Stop Solution at room temperature, tightly sealed, until it is used on page 36.

4 Quantify and dilute gDNA samples using two serial fluorometric assays: a Use the Qubit dsDNA BR Assay or Qubit dsDNA HS Assay to determine the initial

concentration of each gDNA sample. Follow the manufacturers instructions for the specific assay kit and the Qubit instrument. This step is critical for successful preparation of input DNA at the required concentration to ensure optimal fragmentation.

b Dilute each gDNA sample with nuclease-free water to a final concentration of 100 ng/L in a LoBind tube.

c Carefully measure the DNA concentration of each of the 100 ng/l dilutions using a second Qubit dsDNA BR or HS Assay.

d Adjust each gDNA sample with nuclease-free water to a final concentration of 10 ng/l in a LoBind tube.

5 Transfer 5 l of the 10 ng/L-DNA samples into the wells of a 96-well Eppendorf plate, column-wise, for processing on the Agilent NGS Workstation, in well order A1 to H1, then A2 to H2, ending with A12 to H12.

6 Seal the plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165C and 1.0 sec.

7 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to remove air bubbles.

Store the sample plate on ice until it is used on page 38.

NOTE SureSelect Automated Library Prep and Capture System runs may include 1, 2, 3, 4, 6, or 12 columns of the plate. See Experimental Setup Considerations for Automated Runs on page 30 for additional sample placement considerations.

34 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Step 2. Fragment and adaptor-tag the genomic DNA samples In this step, automation runset LibraryPrep_QXT_ILM_v1.0.rst is used to enzymatically fragment the gDNA and to add adaptors to ends of the fragments in a single reaction. After fragmentation and tagging, the Agilent NGS Workstation purifies the prepared DNA using AMPure XP beads.

This step uses the SureSelectQXT Reagent Kit components listed in Table 11 in addition to reagents prepared for use on page 34 to page 34.

Prepare the workstation

1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes. 2 Gently wipe down the Labware MiniHub, Bravo decks, and BenchCel with a DNA Away

decontamination wipe. 3 Pre-set the temperature of Bravo deck position 4 to 52C and position 6 to 4C using the

Inheco Multi TEC control touchscreen, as described in Setting the Temperature of Bravo Deck Heat Blocks. On the control touchscreen, Bravo deck position 4 corresponds to CPAC 2, position 1 and Bravo deck position 6 corresponds to CPAC 2, position 2.

4 Turn on the ThermoCube, set to 0C, at position 9 of the Bravo deck. Be sure that the chiller reservoir contains at least 300 mL of 25% ethanol.

5 Place red PCR plate inserts at Bravo deck positions 4 and 9. 6 Load tip boxes for the run in the BenchCel Microplate Handling Workstation according to

Table 12.

7 Load the workstation MiniHub with the empty plates and other labware components for the run, using the positions shown in the Workstation Setup region of the VWorks Form. Use the plate orientations shown in Figure 2 on page 21.

Table 11 Reagents for DNA fragmentation and adaptor-tagging

Kit Component Storage Location Where Used

SureSelect QXT Buffer SureSelect QXT Library Prep Kit Box 2, 20C page 35

SureSelect QXT Enzyme Mix ILM SureSelect QXT Library Prep Kit Box 2, 20C page 35

Table 12 Initial BenchCel configuration for LibraryPrep_QXT_ILM_v1.0.rst

No. of Columns Processed Rack 1 Rack 2 Rack 3 Rack 4

1 1 Tip box Empty Empty Empty

2 2 Tip boxes Empty Empty Empty

3 2 Tip boxes Empty Empty Empty

4 3 Tip boxes Empty Empty Empty

6 4 Tip boxes Empty Empty Empty

12 8 Tip boxes Empty Empty Empty

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 35

Prepare the purification reagents

8 Verify that the AMPure XP bead suspension is at room temperature. Do not freeze the beads at any time.

9 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color.

10 Prepare a Nunc DeepWell source plate for the beads by adding 55 L of homogeneous AMPure XP beads per well, for each well to be processed. Place the bead source plate on shelf 2 of cassette 3 of the workstation MiniHub.

11 Prepare a Thermo Scientific reservoir containing 15 mL of nuclease-free water. Place the water reservoir on shelf 2 of cassette 2 of the workstation MiniHub.

12 Prepare a separate Thermo Scientific reservoir containing 45 mL of freshly-prepared 70% ethanol. Place the ethanol reservoir on shelf 1 of cassette 2 of the workstation MiniHub.

Prepare the Library Prep Master Mix and Stop Solution source plates

13 Prepare the Stop Solution source plate using an Eppendorf twin.tec full-skirted PCR plate. Add 35 L of 1X SureSelect QXT Stop Solution per well, for each well to be processed. Place the source plate on shelf 4 of cassette 4 of the workstation MiniHub.

14 Before use, vortex the SureSelect QXT Buffer and SureSelect QXT Enzyme Mix ILM tubes vigorously at high speed.

These components are in liquid form when removed from 20C storage and should be returned to 20C storage promptly after use.

15 Prepare the appropriate volume of Library Prep Master Mix, according to Table 13. Mix well by vortexing for 20 seconds and then keep on ice.

CAUTION Minor variations in volumes of the solutions combined in step 15 below may result in DNA fragment size variation.

The SureSelect QXT Buffer and Enzyme Mix solutions are highly viscous. Thorough mixing of the reagents is critical for optimal performance.

Table 13 Preparation of Library Prep Master Mix

SureSelectQXT Reagent Volume for 1 Library

Volume for 1 Column

Volume for 2 Columns

Volume for 3 Columns

Volume for 4 Columns

Volume for 6 Columns

Volume for 12 Columns

SureSelect QXT Buffer 17.0 L 216.8 L 361.3 L 505.8 L 650.3 L 939.3 L 1878.5 L

SureSelect QXT Enzyme Mix ILM

2.0 L 25.5 L 42.5 L 59.5 L 76.5 L 110.5 L 221.0 L

Total Volume 19 L 242.3 L 403.8 L 565.3 L 726.8 L 1049.8 L 2099.5 L

36 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

16 Prepare the Library Prep master mix source plate using a Nunc DeepWell plate, containing the mixture from step 15. Add the volume indicated in Table 14 to all wells of column 1 of the Nunc DeepWell plate. Keep the master mix on ice during the aliquoting steps. The final configuration of the master mix source plate is shown in Figure 4.

Figure 4 Configuration of the master mix source plate for LibraryPrep_QXT_ILM_v1.0.rst

17 Seal the master mix source plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165C and 1.0 sec.

18 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate any bubbles. Keep the master mix source plate on ice.

Table 14 Preparation of the Master Mix Source Plate for LibraryPrep_QXT_ILM_v1.0.rst

Master Mix Solution

Position on Source Plate

Volume of Master Mix added per Well of Nunc Deep Well Source Plate

1-Column Runs

2-Column Runs

3-Column Runs

4-Column Runs

6-Column Runs

12-Column Runs

Library Prep Master Mix

Column 1 (A1-H1)

27.9 L 48.1 L 68.3 L 88.5 L 128.8 L 260.1 L

NOTE The presence of bubbles in source plate solutions may cause inaccurate volume transfer by the Bravo liquid handling platform. Ensure that the source plate is sealed and centrifuged prior to use in a run.

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 37

Load the Agilent NGS Workstation

19 Verify that the Labware MiniHub has been loaded as shown in Table 15.

20 Load the Bravo deck according to Table 16.

Run VWorks runset LibraryPrep_QXT_ILM_v1.0.rst

21 On the SureSelect setup form, under Select Protocol to Run, select LibraryPrep_QXT_ILM_v1.0.rst.

22 Select the number of columns of samples to be processed. Runs must include 1, 2, 3, 4, 6, or 12 columns.

23 Click Display Initial Workstation Setup.

Table 15 Initial MiniHub configuration for LibraryPrep_QXT_ILM_v1.0.rst

Vertical Shelf Position Cassette 1 Cassette 2 Cassette 3 Cassette 4

Shelf 5 (Top) Empty Empty Nunc DeepWell plate

Empty Empty

Shelf 4 Empty Empty Empty Stop Solution source plate from step 13

Shelf 3 Empty Empty Eppendorf plate

Empty Empty

Shelf 2 New tip box Nuclease-free water reservoir from step 11

AMPure XP beads in Nunc DeepWell plate from step 10

Empty

Shelf 1 (Bottom) Empty tip box 70% ethanol reservoir from step 12

Empty Empty tip box

Table 16 Initial Bravo deck configuration for LibraryPrep_QXT_ILM_v1.0.rst

Location Content

1 Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)

4 Empty red insert

6 Library Prep Master Mix source plate (unsealed)

7 gDNA samples (5 L of 10 ng/L DNA per well) in Eppendorf plate (unsealed)

9 Empty Eppendorf plate on red insert

38 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

24 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form.

25 When verification is complete, click Run Selected Protocol.

26 When ready to begin the run, click OK in the following window.

Running the LibraryPrep_QXT_ILM_v1.0.rst runset takes approximately 1 hour. Once complete, the purified, adaptor-ligated DNA samples are located in the Eppendorf plate at position 7 of the Bravo deck.

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 39

Step 3. Amplify adaptor-ligated libraries In this step, the Agilent NGS Workstation completes the liquid handling steps for amplification of the adaptor-ligated DNA samples using automation protocol Pre-CapturePCR_QXT_ILM_v1.0.pro. Afterward, you transfer the PCR plate to a thermal cycler for amplification.

This step uses the SureSelectQXT Reagent Kit components listed in Table 17.

Prepare the workstation

1 Turn on the ThermoCube, set to 0C, at position 9 of the Bravo deck. Be sure that the chiller reservoir contains at least 300 mL of 25% ethanol.

2 Leave tip boxes on shelves 1 and 2 in cassette 1 of the Labware MiniHub from the previous LibraryPrep_QXT_ILM_v1.0.rst run. Otherwise, clear the remaining positions of the MiniHub and BenchCel of plates and tip boxes.

3 Pre-set the temperature of Bravo deck position 6 to 4C using the Inheco Multi TEC control touchscreen, as described in Setting the Temperature of Bravo Deck Heat Blocks. Bravo deck position 6 corresponds to CPAC 2, position 2 on the Multi TEC control touchscreen.

4 Load tip boxes for the run in the BenchCel Microplate Handling Workstation according to Table 18.

Table 17 Reagents for precapture amplification

Kit Component Storage Location Where Used

Herculase II Fusion DNA Polymerase SureSelect QXT Library Prep Kit Box 2, 20C page 41

Herculase II 5 Reaction Buffer SureSelect QXT Library Prep Kit Box 2, 20C page 41

100 mM dNTP Mix (25 mM each dNTP) SureSelect QXT Library Prep Kit Box 2, 20C page 41

SureSelect QXT Primer Mix SureSelect QXT Hyb Module Box 2, 20C page 41

DMSO Transferred to Room Temperature storage on page 34

page 41

CAUTION To avoid cross-contaminating libraries, set up PCR master mixes in a dedicated clean area or PCR hood with UV sterilization and positive air flow.

Table 18 Initial BenchCel configuration for Pre-CapturePCR_QXT_ILM_v1.0.pro

No. of Columns Processed

Rack 1 Rack 2 Rack 3 Rack 4

1 1 Tip box Empty Empty Empty

2 1 Tip box Empty Empty Empty

3 1 Tip box Empty Empty Empty

4 1 Tip box Empty Empty Empty

6 1 Tip box Empty Empty Empty

12 1 Tip box Empty Empty Empty

40 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Prepare the pre-capture PCR master mix and master mix source plate

5 Prepare the appropriate volume of pre-capture PCR Master Mix, according to Table 19 Mix well using a vortex mixer and keep on ice.

6 Using the same Nunc DeepWell master mix source plate that was used for the LibraryPrep_QXT_ILM_v1.0.rst run, add the volume of PCR Master Mix indicated in Table 20 to all wells of column 2 of the master mix source plate. The final configuration of the master mix source plate is shown in Figure 5.

Table 19 Preparation of Pre-Capture PCR Master Mix

SureSelectQXT Reagent Volume for 1 Library

Volume for 1 Column

Volume for 2 Columns

Volume for 3 Columns

Volume for 4 Columns

Volume for 6 Columns

Volume for 12 Columns

Nuclease-free water 13.5 L 172.1 L 286.9 L 401.6 L 516.4 L 745.9 L 1491.8 L

Herculase II 5X Reaction Buffer

10.0 L 127.5 L 212.5 L 297.5 L 382.5 L 552.5 L 1105 L

DMSO 2.5 L 31.9 L 53.1 L 74.4 L 95.6 L 138.1 L 276.3 L

dNTP mix 0.5 L 6.4 L 10.6 L 14.9 L 19.1 L 27.6 L 55.3 L

SureSelect QXT Primer Mix

1.0 L 12.8 L 21.3 L 29.8 L 38.3 L 55.3 L 110.5 L

Herculase II Fusion DNA Polymerase

1.0 L 12.8 L 21.3 L 29.8 L 38.3 L 55.3 L 110.5 L

Total Volume 28.5 L 363.4 L 605.6 L 847.9 L 1090.1 L 1574.6 L 3149.3 L

Table 20 Preparation of the Master Mix Source Plate for Pre-CapturePCR_QXT_ILM_v1.0.pro

Master Mix Solution

Position on Source Plate

Volume of Master Mix added per Well of Nunc Deep Well Source Plate

1-Column Runs

2-Column Runs

3-Column Runs

4-Column Runs

6-Column Runs

12-Column Runs

Pre-Capture PCR Master Mix

Column 2 (A2-H2)

41.9 L 72.1 L 102.4 L 132.7 L 193.3 L 390.1 L

NOTE If you are using a new DeepWell plate for the pre-capture PCR source plate, leave column 1 empty and add the PCR Master Mix to column 2 of the new plate.

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 41

Figure 5 Configuration of the master mix source plate for Pre-CapturePCR_QXT_ILM_v1.0.pro. Column 1 was used to dispense master mix during the previous protocol.

7 Seal the master mix source plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165C and 1.0 sec.

8 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate any bubbles.

NOTE The presence of bubbles in source plate solutions may cause inaccurate volume transfer by the Bravo liquid handling platform. Ensure that the source plate is sealed and centrifuged prior to use in a run.

42 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Load the Agilent NGS Workstation

9 Load the Labware MiniHub according to Table 21.

10 Load the Bravo deck according to Table 22.

Run VWorks protocol Pre-CapturePCR_QXT_ILM_v1.0.pro

11 On the SureSelect setup form, under Select Protocol to Run, select Pre-CapturePCR_QXT_ILM_v1.0.pro.

12 Under Select PCR plate labware for Thermal Cycling, select the specific type of PCR plate used at position 6 of the Bravo deck.

13 Select the number of columns of samples to be processed. Runs must include 1, 2, 3, 4, 6, or 12 columns.

14 Click Display Initial Workstation Setup.

Table 21 Initial MiniHub configuration for Pre-CapturePCR_QXT_ILM_v1.0.pro

Vertical Shelf Position

Cassette 1 Cassette 2 Cassette 3 Cassette 4

Shelf 5 (Top) Empty Empty Empty Empty

Shelf 4 Empty Empty Empty Empty

Shelf 3 Empty Empty Empty Empty

Shelf 2 Clean tip box*

* The clean tip box (Cassette 1, Shelf 2) and waste tip box (Cassette 1, Shelf 1) are retained from the LibraryPrep_QXT_ILM_v1.0.rst run and reused here.

Empty Empty Empty

Shelf 1 (Bottom) Waste tip box* Empty Empty Empty tip box

NOTE If you are using a new box of tips on shelf 2 of cassette 1, first remove the tips from column 1 of the tip box. Any tips present in column 1 of the tip box may be inappropriately loaded onto the Bravo platform pipette head and may interfere with automated processing steps.

Table 22 Initial Bravo deck configuration for Pre-CapturePCR_QXT_ILM_v1.0.pro

Location Content

6 Empty PCR plate seated in red insert (PCR plate type must be specified on setup form under step 2)

7 Adaptor-ligated DNA samples in Eppendorf plate

9 Master mix plate containing PCR Master Mix in Column 2 (unsealed)

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 43

15 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form.

16 When verification is complete, click Run Selected Protocol.

Running the Pre-CapturePCR_QXT_ILM_v1.0.pro protocol takes approximately 15 minutes. Once complete, the PCR-ready samples, containing prepped DNA and PCR master mix, are located in the PCR plate at position 6 of the Bravo deck. 17 When you see the following prompt, remove the PCR plate from position 6 of the Bravo deck

and seal the plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165C and 3.0 seconds.

18 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate air bubbles.

19 Transfer the PCR plate to a thermal cycler and run the PCR amplification program shown in Table 23.

44 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Step 4. Purify amplified DNA using AMPure XP beads In this step, the Agilent NGS Workstation transfers AMPure XP beads and amplified adaptor-ligated DNA to a Nunc DeepWell plate and then collects and washes the bead-bound DNA.

Prepare the workstation and reagents

1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes. 2 Verify that the AMPure XP bead suspension is at room temperature. (If necessary, allow the

bead solution to come to room temperature for at least 30 minutes.) Do not freeze the beads at any time.

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

4 Prepare a Nunc DeepWell source plate for the beads by adding 55 l of homogeneous AMPure XP beads per well, for each well to be processed.

5 Prepare a Thermo Scientific reservoir containing 20 mL of nuclease-free water. 6 Prepare a separate Thermo Scientific reservoir containing 45 mL of freshly-prepared 70%

ethanol. 7 Centrifuge the amplified DNA sample plate for 30 seconds to drive the well contents off the

walls and plate seal.

Table 23 Pre-Capture PCR cycling program

Segment Number Number of Cycles Temperature Time

1 1 68C 2 minutes

2 1 98C 2 minutes

3 8 98C 30 seconds

57C 30 seconds

72C 1 minute

4 1 72C 5 minutes

5 1 4C Hold

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 45

8 Load the Labware MiniHub according to Table 24, using the plate orientations shown in Figure 2 on page 21.

9 Load the Bravo deck according to Table 25.

10 Load the BenchCel Microplate Handling Workstation according to Table 26.

Table 24 Initial MiniHub configuration for AMPureXP_QXT_ILM_v1.0.pro:Pre-Capture PCR

Vertical Shelf Position Cassette 1 Cassette 2 Cassette 3 Cassette 4

Shelf 5 (Top) Empty Nunc DeepWell plate

Empty Empty Empty

Shelf 4 Empty Empty Empty Empty

Shelf 3 Empty Empty Eppendorf Plate

Empty Empty

Shelf 2 Empty Nuclease-free water reservoir from step 5

AMPure XP beads in Nunc DeepWell plate from step 4

Empty

Shelf 1 (Bottom) Empty 70% ethanol reservoir from step 6

Empty Empty tip box

Table 25 Initial Bravo deck configuration for AMPureXP_QXT_ILM_v1.0.pro:Pre-Capture PCR

Location Content

1 Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)

9 Amplified DNA libraries in unsealed PCR plate seated in red insert (PCR plate type must be specified on setup form under step 2)

Table 26 Initial BenchCel configuration for AMPureXP_QXT_ILM_v1.0.pro:Pre-Capture PCR

No. of Columns Processed

Rack 1 Rack 2 Rack 3 Rack 4

1 1 Tip box Empty Empty Empty

2 1 Tip box Empty Empty Empty

3 2 Tip boxes Empty Empty Empty

4 2 Tip boxes Empty Empty Empty

6 3 Tip boxes Empty Empty Empty

12 6 Tip boxes Empty Empty Empty

46 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Run VWorks protocol AMPureXP_QXT_ILM_v1.0.pro:Pre-Capture PCR

11 On the SureSelect setup form, under Select Protocol to Run, select AMPureXP_QXT_ILM_v1.0.pro:Pre-Capture PCR.

12 Under Select PCR plate labware for Thermal Cycling, select the specific type of PCR plate containing the amplified libraries at position 9.

13 Select the number of columns of samples to be processed. Runs must include 1, 2, 3, 4, 6, or 12 columns.

14 Click Display Initial Workstation Setup.

15 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form.

16 When verification is complete, click Run Selected Protocol.

The purification protocol takes approximately 45 minutes. When complete, the purified DNA samples are in the Eppendorf plate located on Bravo deck position 7.

NOTE AMPureXP purification protocols are used during multiple steps of the SureSelect automation workflow. Be sure to select the correct workflow step when initiating the automation protocol.

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 47

Step 5. Assess Library DNA quantity and quality Measure the concentration of each library using one of the methods detailed below.

Option 1: Analysis using the Agilent 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 245 to 325 bp. Sample electropherograms are shown in Figure 6. Variability of fragmentation profiles may be observed.

4 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.

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.

NOTE The presence of magnetic beads in the samples may adversely impact the Bioanalyzer results. If you suspect bead contamination in the samples, place the plate or strip tube on the magnetic rack before withdrawing samples for analysis.

NOTE A peak DNA fragment size significantly less than 245 bp may indicate too little gDNA in the fragmentation reaction and may be associated with increased duplicates in the sequencing data. In contrast, a peak DNA fragment size significantly greater than 325 bp may indicate too much gDNA in the fragmentation reaction and may be associated with decreased percent-on-target performance in sequencing results.

48 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Figure 6 Representative sample electropherograms showing pre-capture analysis of amplified library DNA using the Agilent 2100 Bioanalyzer and a DNA 1000 Assay.

Option 2: Analysis using an Agilent TapeStation and D1000 ScreenTape

Use a D1000 ScreenTape and associated reagent kit. Perform the assay according to 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.

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.

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.

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 49

3 Verify that the electropherogram shows the peak of DNA fragment size positioned between 245 to 325 bp. Sample electropherograms are shown in Figure 7. Variability of fragmentation profiles may be observed.

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

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

Figure 7 Representative sample electropherograms showing pre-capture analysis of amplified library DNA using a D1000 ScreenTape.

NOTE A peak DNA fragment size significantly less than 245 bp may indicate too little gDNA in the fragmentation reaction and may be associated with increased duplicates in the sequencing data. In contrast, a peak DNA fragment size significantly greater than 325 bp may indicate too much gDNA in the fragmentation reaction and may be associated with decreased percent-on-target performance in sequencing results.

50 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

SureSelectQXT Automated Target Enrichment for the Illumina Platform Protocol

4 Hybridization Step 1. Aliquot prepped DNA samples for hybridization 52 Step 2. Hybridize the gDNA library and probe 55 Step 3. Capture the hybridized DNA 64

This chapter describes the steps to combine the prepped library with the blocking agents and the Probe Capture Library. Each DNA library sample must be hybridized and captured individually prior to addition of the indexing tag by PCR.

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

51

Step 1. Aliquot prepped DNA samples for hybridization For each sample library prepared, do one hybridization and capture. Do not pool samples at this stage.

The amount of prepared gDNA library used in the hybridization reaction varies according to the design size of the probe used for hybridization as outlined in Table 27 below. Use the maximum possible amount of each prepped DNA, within the range listed in Table 27.

Using the DNA concentration for each sample determined on page 48 to page 50, calculate the volume of each sample to be used for hybridization using the appropriate formula below:

Volume (l) = 750 ng/concentration (ng/L)

OR

Volume (l) = 1500 ng/concentration (ng/L)

If the concentration of any sample is not sufficient to allow use of the recommended amount of DNA (750 ng for probes 3.0 Mb or 1500 ng for probes >3.0 Mb), then use the full remaining volume of DNA sample (approximately 12 L) for the hybridization step.

The automation protocol Aliquot_Libraries_v1.0.pro is used to prepare a new sample plate containing the appropriate amount of each DNA sample for hybridization. Before running the automation protocol, you must create a table containing instructions for the Agilent NGS Workstation indicating the volume of each sample to aliquot, as described in the steps below. 1 Create a .csv (comma separated value) file with the headers shown in Figure 8. The header

text must not contain spaces. The table may be created using a spreadsheet application, such as Microsoft Excel software, and then saved in .csv format. The file must include rows for all 96 wells of the plate.

2 Enter the information requested in the header for each DNA sample. In the SourceBC field, enter the sample plate description or barcode. The SourceBC field

contents must be identical for all rows. In the SourceWell and DestinationWell fields, enter each well position for the plate.

SourceWell and DestinationWell field contents must be identical for a given sample. In the Volume field, enter the volume (in L) of each DNA sample to be used in the

hybridization step (see page 52 for guidelines). For all empty wells on the plate, enter the value 0, as shown in Figure 8; do not delete rows for empty wells.

Table 27 Amount of adaptor-tagged DNA libraries used for hybridization

Probe Capture Library Size Amount of prepared gDNA library used in hybridization

Probes 3.0 Mb 750 to 1500 ng DNA

Probes 3.0 Mb 500 to 750 ng DNA

52 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Figure 8 Sample spreadsheet for 1-column run.

3 Load the .csv file onto the PC containing the VWorks software into a suitable folder, such as C: > VWorks Workspace > NGS Option B > QXT_ILM_v1.0 > Aliquot Library Input Files.

4 Turn on the chiller, set to 0C, at position 9 of the Bravo deck. Be sure that the chiller reservoir contains at least 300 mL of 25% ethanol.

5 Load the Bravo deck according to Table 28.

6 On the SureSelect setup form, under Select Protocol to Run, select Aliquot_Libraries_v1.0.pro.

7 Click Display Initial Workstation Setup.

NOTE You can find a sample spreadsheet in the directory C: > VWorks Workspace > NGS Option B > QXT_ILM_v1.0 > Aliquot Library Input Files > Aliquot_Libraries_full_plate_template.csv.

The Aliquot_Libraries_full_plate_template.csv file may be copied and used as a template for creating the .csv files for each Aliquot_Libraries_v1.0.pro run. If you are using the sample file as a template for runs with fewer than 12 columns, be sure to retain rows for all 96 wells, and populate the Volume column with 0 for unused wells.

Table 28 Initial Bravo deck configuration for Aliquot_Libraries_v1.0.pro

Location Content

5 Empty Eppendorf plate

6 Empty tip box

8 New tip box

9 Prepped library DNA in Eppendorf plate

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 53

8 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form.

9 When verification is complete, click Run Selected Protocol.

10 When prompted by the dialog below, browse to the .csv file created for the source plate of the current run, and then click OK to start the run.

The library aliquoting protocol takes approximately 1 hour for 96 samples. When complete, the DNA sample plate is on Bravo deck position 5.

11 Remove the sample plate from the Bravo deck and use a vacuum concentrator to dry the samples at 45C.

12 Reconstitute each dried sample with 12 L of nuclease-free water. Pipette up and down along the sides of each well for optimal recovery.

13 Seal the plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165C and 1.0 sec.

14 Vortex the plate for 30 seconds to ensure complete reconstitution, then centrifuge the plate for 1 minute to drive the well contents off the walls and plate seal.

54 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Step 2. Hybridize the gDNA library and probe In this step, automation protocol Hybridization_QXT_v1.0.pro is used to complete the liquid handling steps to set up the hybridization reactions. Afterward, you transfer the sample plate to a thermal cycler, held at 65C, to allow hybridization of the DNA samples to the Probe Capture Library.

This step uses the SureSelectQXT Reagent Kit components listed in Table 29. Thaw each component under the conditions indicated in the table. Vortex each reagent to mix, then spin briefly to collect the liquid.

Program the thermal cycler

1 Pre-program the thermal cycler for the Hybridization workflow by entering the thermal cycling program shown in Table 30 below.

It is critical to pre-program the thermal cycler before starting the automation protocol for Hybridization, in order to maintain the required sample and reagent temperatures during the workflow.

Table 29 Reagents for Hybridization and Capture

Kit Component Storage Location Thawing Conditions Where Used

SureSelect Fast Hybridization Buffer

SureSelect QXT Hyb Module Box 2, 20C

Warm to Room Temperature (RT), then keep at RT

page 58

SureSelect QXT Fast Blocker Mix

SureSelect QXT Hyb Module Box 2, 20C

Thaw on ice page 56

SureSelect RNase Block

SureSelect QXT Hyb Module Box 2, 20C

Thaw on ice page 57 or page 58

Probe Capture Library 80C Thaw on ice page 57 or page 58

Table 30 Thermal cycler program for Hybridization*

* When setting up the thermal cycling program, use a reaction volume setting of 35 l (final volume of hybridization reactions during cycling in Segment 4).

Segment Number Purpose Number of Cycles Temperature Time

1 Denaturation 1 95C 5 minutes

2 Blocking 1 65C 10 minutes

3 Hold for NGS workstation steps

Samples are transferred to the NGS Workstation during this Hold step when prompted by the VWorks soft- ware.

1 65C Hold

4 Hybridization 60 65C 1 minute

37C 3 seconds

5 Hold until start of Capture

Samples are held at 65C until they are processed in the Capture & Wash automation protocol that begins on page 64.

1 65C Hold

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 55

Prepare the workstation

2 Clear the Labware MiniHub and BenchCel of all plates and tip boxes. 3 Gently wipe down the Labware MiniHub, Bravo decks, and BenchCel with a DNA Away

decontamination wipe. 4 Turn on the chiller, set to 0C, at position 9 of the Bravo deck. Be sure that the chiller reservoir

contains at least 300 mL of 25% ethanol. 5 Place a red PCR plate insert at Bravo deck position 4. 6 Place the silver Nunc DeepWell plate insert on position 6 of the Bravo deck. This insert is

required to facilitate heat transfer to DeepWell source plate wells during the Hybridization protocol.

7 Place an empty tip box on shelf 2 of cassette 4 of the workstation MiniHub. 8 Load tip boxes for the run in the BenchCel Microplate Handling Workstation according to

Table 31.

Prepare the Block Master Mix

9 Prepare the appropriate volume of Block Master Mix, on ice, as indicated in Table 32.

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

Table 31 Initial BenchCel configuration for Hybridization_QXT_v1.0.pro

No. of Columns Processed Rack 1 Rack 2 Rack 3 Rack 4

1 2 Tip boxes Empty Empty Empty

2 2 Tip boxes Empty Empty Empty

3 2 Tip boxes Empty Empty Empty

4 3 Tip boxes Empty Empty Empty

6 3 Tip boxes Empty Empty Empty

12 5 Tip boxes Empty Empty Empty

Table 32 Preparation of Block Master Mix

SureSelectQXT Reagent Volume for 1 Library

Volume for 1 Column

Volume for 2 Columns

Volume for 3 Columns

Volume for 4 Columns

Volume for 6 Columns

Volume for 12 Columns

Nuclease-free water 2.5 L 31.9 L 53.1 L 74.4 L 95.6 L 138.1 L 276.3 L

SureSelect QXT Fast Blocker Mix (blue cap)

5.0 L 63.8 L 106.3 L 148.8 L 191.3 L 276.3 L 552.5 L

Total Volume 7.5 L 95.6 L 159.4 L 223.1 L 286.9 L 414.4 L 828.8 L

56 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Prepare one or more Capture Library Master Mixes

10 Prepare the appropriate volume of Capture Library Master Mix for each of the Probes that will be used for hybridization as indicated in Table 33 to Table 36. Mix thoroughly by vortexing at high speed then spin down briefly. Keep the Capture Library Master Mix(es) on ice.

a For runs that use a single Probe for all rows, prepare a Master Mix as described in Table 33 or Table 34, based on the Mb target size of your design.

NOTE Each row of the prepped gDNA sample plate may be hybridized to a different Probe. However, Probes of different sizes require different post-capture amplification cycles. Plan experiments such that similar-sized Probes are hybridized on the same plate.

For runs that use a single Probe for all rows of the plate, prepare the master mix as described in Step a (Table 33 or Table 34) on page 57.

For runs that use different Probes for individual rows, prepare each master mix as described in Step b (Table 35 or Table 36) on page 58.

Table 33 Preparation of Capture Library Master Mix for Probes<3 Mb, 8 rows of wells

Target size <3.0 Mb

Reagent Volume for 1 Library

Volume for 1 Column

Volume for 2 Columns

Volume for 3 Columns

Volume for 4 Columns

Volume for 6 Columns

Volume for 12 Columns

Nuclease-free water 4.5 L 76.5 L 114.8 L 153.0 L 191.3 L 306.0 L 592.9 L

RNase Block (purple cap)

0.5 L 8.5 L 12.8 L 17.0 L 21.3 L 34.0 L 65.9 L

Probe (with design <3 Mb

2.0 L 34.0 L 51.0 L 68.0 L 85.0 L 136.0 L 263.5 L

Total Volume 7.0 L 119.0 L 178.6 L 238.0 L 297.6 L 476.0 L 922.3 L

Table 34 Preparation of Capture Library Master Mix for Probes3 Mb, 8 rows of wells

Target size 3.0 Mb

Reagent Volume for 1 Library

Volume for 1 Column

Volume for 2 Columns

Volume for 3 Columns

Volume for 4 Columns

Volume for 6 Columns

Volume for 12 Columns

Nuclease-free water 1.5 L 25.5 L 38.3 L 51.0 L 63.8 L 102.0 L 197.6 L

RNase Block (purple cap)

0.5 L 8.5 L 12.8 L 17.0 L 21.3 L 34.0 L 65.9 L

Probe (with design 3 Mb)

5.0 L 85.0 L 127.5 L 170.0 L 212.5 L 340.0 L 658.8 L

Total Volume 7.0 L 119.0 L 178.6 L 238.0 L 297.6 L 476.0 L 922.3 L

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 57

b For runs that use different Probes in individual rows, prepare a Master Mix for each Probe as listed in Table 35 or Table 36, based on the Mb target size of each design. The volumes listed in Table 35 and Table 36 are for a single row of sample wells. If a given Probe will be hybridized in multiple rows, multiply each of the values below by the number of rows assigned to that Probe.

Prepare the Hybridization Buffer master mix

11 Prepare the appropriate volume of Hybridization Buffer Master Mix, at room temperature, as indicated in Table 37.

Table 35 Preparation of Capture Library Master Mix for Probes <3 Mb, single row of wells

Target size <3.0 Mb

Reagent Volume for 1 Library

Volume for 1 Column

Volume for 2 Columns

Volume for 3 Columns

Volume for 4 Columns

Volume for 6 Columns

Volume for 12 Columns

Nuclease-free water 4.5 L 9.0 L 13.8 L 18.6 L 23.3 L 37.7 L 73.5 L

RNase Block (purple cap)

0.5 L 1.0 L 1.5 L 2.1 L 2.6 L 4.2 L 8.2 L

Probe (with design <3 Mb

2.0 L 4.0 L 6.1 L 8.3 L 10.4 L 16.8 L 32.7 L

Total Volume 7.0 L 14.0 L 21.4 L 28.9 L 36.3 L 58.6 L 114.4 L

Table 36 Preparation of Capture Library Master Mix for Probes3 Mb, single row of wells

Target size >3.0 Mb

Reagent Volume for 1 Library

Volume for 1 Column

Volume for 2 Columns

Volume for 3 Columns

Volume for 4 Columns

Volume for 6 Columns

Volume for 12 Columns

Nuclease-free water 1.5 L 3.0 L 4.6 L 6.2 L 7.8 L 12.6 L 24.5 L

RNase Block (purple cap)

0.5 L 1.0 L 1.5 L 2.1 L 2.6 L 4.2 L 8.2 L

Probe (with design 3 Mb)

5.0 L 10.0 L 15.3 L 20.6 L 25.9 L 41.9 L 81.7 L

Total Volume 7.0 L 14.0 L 21.4 L 28.9 L 36.3 L 58.6 L 114.4 L

Table 37 Preparation of Hybridization Buffer Master Mix

SureSelectQXT Reagent Volume for 1 Library

Volume for 1 Column

Volume for 2 Columns

Volume for 3 Columns

Volume for 4 Columns

Volume for 6 Columns

Volume for 12 Columns

Nuclease-free water 2.5 L 53.1 L 74.4 L 95.6 L 116.9 L 159.4 L 297.5 L

SureSelect Fast Hybridization Buffer (bottle)

6.0 L 127.5 L 178.5 L 229.5 L 280.5 L 382.5 L 714.0 L

Total Volume 8.5 L 180.6 L 252.9 L 325.1 L 397.4 L 541.9 L 1011.5 L

58 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Prepare the master mix source plate

12 In a Nunc DeepWell plate, prepare the master mix source plate containing the master mixes prepared in step 9 to step 11 at room temperature. Add the volumes indicated in Table 38 of each master mix to each well of the indicated column of the Nunc DeepWell plate. When using multiple Probes in a run, add each Capture Library Master Mix to the appropriate row(s) of the Nunc DeepWell plate. The final configuration of the master mix source plate is shown in Figure 9.

Figure 9 Configuration of the master mix source plate for Hybridization_QXT_v1.0.pro.

13 Seal the master mix source plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165C and 1.0 sec.

Table 38 Preparation of the Master Mix Source Plate for Hybridization_QXT_v1.0.pro

Master Mix Solution

Position on Source Plate

Volume of Master Mix added per Well of Nunc Deep Well Source Plate

1-Column Runs

2-Column Runs

3-Column Runs

4-Column Runs

6-Column Runs

12-Column Runs

Block Master Mix Column 1 (A1-H1)

11.0 L 19.0 L 27.0 L 34.9 L 50.9 L 102.7 L

Capture Library Master Mix

Column 2 (A2-H2)

14.0 L 21.4 L 28.9 L 36.3 L 58.6 L 114.4 L

Hybridization Buffer Master Mix

Column 3 (A3-H3)

19.9 L 29.0 L 38.0 L 47.0 L 65.1 L 123.8 L

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 59

14 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate any bubbles. Keep the master mix plate at room temperature.

Load the Bravo deck

15 Load the Bravo deck according to Table 39.

Run VWorks protocol Hybridization_QXT_v1.0.pro

16 On the SureSelect setup form, under Select Protocol to Run, select Hybridization_QXT_v1.0.pro.

17 Under Select PCR plate labware for Thermal Cycling, select the specific type of PCR plate used at position 4 of the Bravo deck.

18 Select the number of columns of samples to be processed. Runs must include 1, 2, 3, 4, 6, or 12 columns.

19 Click Display Initial Workstation Setup.

20 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form.

21 When verification is complete, click Run Selected Protocol.

Table 39 Initial Bravo deck configuration for Hybridization_QXT_v1.0.pro

Location Content

4 Empty PCR plate seated in red insert (PCR plate type must be specified on setup form under step 2)

5 Empty Eppendorf plate

6 Master Mix source plate (unsealed) seated on silver Nunc DeepWell insert

8 Empty tip box

9 Prepared gDNA aliquots in Eppendorf plate (unsealed)

60 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

The Agilent NGS Workstation combines the prepped gDNA in the wells of the sample plate with the aliquotted SureSelect Block Master Mix. When this process is complete, you will be prompted to transfer the plate to the thermal cycler for sample denaturation and blocking prior to hybridization.

22 When prompted by VWorks as shown below, remove the PCR plate from position 4 of the Bravo deck, leaving the red insert in place. After removing the sample plate, click Continue.

23 Seal the sample plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165C and 3.0 sec.

24 Transfer the sealed plate to a thermal cycler and initiate the preprogrammed thermal cycling program described in Table 30 on page 55. The denaturation and blocking segments of the preprogrammed thermal cycler program are shown in Figure 10 below for reference.

Figure 10 Preprogrammed thermal cycler segments used for sample denaturation and blocking prior to hybridization.

While the sample plate incubates on the thermal cycler, the Agilent NGS Workstation combines aliquots of the Capture Library master mix and Hybridization Buffer master mix.

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 61

25 When the workstation has finished aliquoting the Capture Library and Hybridization Buffer master mixes, you will be prompted by VWorks as shown below. When the thermal cycler reaches the 65C hold step, click Continue. Leave the sample plate in the thermal cycler until you are notified to move it.

26 When prompted by VWorks as shown below, quickly remove the sample plate from the thermal cycler, unseal the plate carefully to avoid splashing, and transfer the plate to position 4 of the Bravo deck, seated in the red insert. Click Continue.

CAUTION You must complete step 25 to step 29 quickly, and immediately after being prompted by the VWorks software. It is important that sample temperature remains approximately 65C during transfers between the Agilent NGS Workstation and thermal cycler.

62 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

The Agilent NGS Workstation transfers the Capture Library-Hybridization Buffer mixture to the wells of the PCR plate that contain the mixture of prepped gDNA samples and blocking agents. 27 When prompted by VWorks as shown below, quickly remove the PCR sample plate from Bravo

deck position 4, leaving the red insert in place.

28 Seal the sample plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165C and 3.0 sec.

29 Quickly transfer the plate back to the thermal cycler, held at 65C. Press the Play button to initiate the hybridization segment of the pre-programmed thermal cycling program (segment 4 from Table 30 on page 55). During this step, the prepared DNA samples are hybridized to the Probe.

30 After initiating hybridization on the thermal cycler, click Continue on the VWorks screen. 31 To finish the VWorks protocol, click Continue in the Unused Tips and Empty Tip box dialogs,

and click Yes in the Protocol Complete dialog.

WARNING Warning

Bravo deck position 4 will be hot.

Use caution when handling components that contact heated deck positions.

CAUTION The thermal cycler is held at 65C using a heated lid at 105C. The lid of the thermal cycler is hot and can cause burns. Use caution when working near the lid.

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 63

Step 3. Capture the hybridized DNA This step uses runset SureSelectQXT_Capture&Wash_v1.0.rst to automate capture of the gDNA-probe hybrids using streptavidin-coated magnetic beads. Setup tasks for the Capture & Wash protocol (step 1, below, through step 16 on page 66) should be completed during the thermal cycler incubation for hybridization (approximately 1.5-hour duration) started on page 63.

The Capture & Wash runset uses the SureSelectQXT Reagent Kit components in Table 40 in addition to streptavidin-coated magnetic beads obtained from another supplier (see Table 1 on page 12).

Prepare the workstation

1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes. 2 Gently wipe down the Labware MiniHub, Bravo decks, and BenchCel with a DNA Away

decontamination wipe. 3 Pre-set the temperature of Bravo deck position 4 to 66C using the Inheco Multi TEC control

touchscreen, as described in Setting the Temperature of Bravo Deck Heat Blocks. Bravo deck position 4 corresponds to CPAC 2, position 1 on the Multi TEC control touchscreen.

4 Place a red PCR plate insert at Bravo deck position 4. 5 Place the silver Nunc DeepWell plate insert on position 6 of the Bravo deck. This insert is

required to facilitate heat transfer to DeepWell source plate wells during the Capture & Wash runset. When loading a source plate on the silver insert, make sure the plate is seated properly to ensure proper heat transfer.

Table 40 Reagents for hybrid capture

Kit Component Storage Location Where Used

SureSelect Binding Buffer SureSelect QXT Hyb Module Box 1, RT page 65

SureSelect Wash Buffer 1 SureSelect QXT Hyb Module Box 1, RT page 65

SureSelect Wash Buffer 2 SureSelect QXT Hyb Module Box 1, RT page 65

64 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Prepare the Dynabeads streptavidin beads

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

7 Wash the magnetic beads. a In a conical vial, combine the components listed in Table 41. The volumes below include

the required overage.

b Mix the beads on a vortex mixer for 5 seconds. c Put the vial into a magnetic separator device. d Remove and discard the supernatant. e Repeat step a through step d for a total of 3 washes. (Retain the beads after each wash and

combine with a fresh aliquot of the indicated volume of SureSelect Binding Buffer.) 8 Resuspend the beads in SureSelect Binding buffer, according to Table 42 below.

9 Prepare a Nunc DeepWell source plate for the washed streptavidin bead suspension. For each well to be processed, add 200 L of the homogeneous bead suspension to the Nunc DeepWell plate.

10 Place the streptavidin bead source plate at position 5 of the Bravo deck.

Prepare capture and wash solution source plates

11 Prepare a Thermo Scientific reservoir containing 15 mL of nuclease-free water. 12 Prepare an Eppendorf source plate labeled Wash #1. For each well to be processed, add

160 L of SureSelect Wash Buffer 1. 13 Prepare a Nunc DeepWell source plate labeled Wash #2. For each well to be processed, add

1150 L of SureSelect Wash Buffer 2.

Table 41 Magnetic bead washing mixture

Reagent Volume for 1 Library

Volume for 1 Column

Volume for 2 Columns

Volume for 3 Columns

Volume for 4 Columns

Volume for 6 Columns

Volume for 12 Columns

Dynabeads MyOne Streptavidin T1 bead suspension

50 L 425 L 825 L 1225 L 1.65 mL 2.5 mL 5.0 mL

SureSelect Binding Buffer

0.2 mL 1.7 mL 3.3 mL 4.9 mL 6.6 mL 10 mL 20 mL

Total Volume 0.25 mL 2.125 mL 4.125 mL 6.125 mL 8.25 mL 12.5 mL 25 mL

Table 42 Preparation of magnetic beads for SureSelectQXT_Capture&Wash_v1.0.rst

Reagent Volume for 1 Library

Volume for 1 Column

Volume for 2 Columns

Volume for 3 Columns

Volume for 4 Columns

Volume for 6 Columns

Volume for 12 Columns

SureSelect Binding Buffer

0.2 mL 1.7 mL 3.3 mL 4.9 mL 6.6 mL 10 mL 20 mL

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 65

Load the Agilent NGS Workstation

14 Load the Labware MiniHub according to Table 43, using the plate orientations shown in Figure 2.

15 Load the Bravo deck according to Table 44 (position 5 should already be loaded).

16 Load the BenchCel Microplate Handling Workstation according to Table 45.

Table 43 Initial MiniHub configuration for SureSelectQXT_Capture&Wash_v1.0.rst

Vertical Shelf Position Cassette 1 Cassette 2 Cassette 3 Cassette 4

Shelf 5 (Top) Empty Empty Empty Empty

Shelf 4 Empty Empty Empty Empty

Shelf 3 Empty Eppendorf plate

Empty Wash #1 Eppendorf source plate

Empty

Shelf 2 Empty Nuclease-free water reservoir

Empty Empty

Shelf 1 (Bottom) Empty Empty Empty Empty tip box

Table 44 Initial Bravo deck configuration for SureSelectQXT_Capture&Wash_v1.0.rst

Location Content

1 Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)

4 Empty red insert

5 Dynabeads streptavidin bead DeepWell source plate

6 Wash #2 DeepWell source plate seated on silver Nunc DeepWell insert

Table 45 Initial BenchCel configuration for SureSelectQXT_Capture&Wash_v1.0.rst

No. of Columns Processed Rack 1 Rack 2 Rack 3 Rack 4

1 1 Tip boxes Empty Empty Empty

2 2 Tip boxes Empty Empty Empty

3 3 Tip boxes Empty Empty Empty

4 4 Tip boxes Empty Empty Empty

6 6 Tip boxes Empty Empty Empty

12 10 Tip boxes 2 Tip boxes Empty Empty

66 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Run VWorks runset SureSelectQXT_Capture&Wash_v1.0.rst

Start the SureSelectQXT_Capture&Wash_v1.0.rst runset upon completion of the hybridization incubation (approximately 1 hour) that was started on page 63, when the thermal cycler program reaches the 65C Hold step in Segment 5.

The total duration of the SureSelectQXT_Capture&Wash_v1.0.rst runset is approximately 1.5 hours. A workstation operator must be present to complete two actions during the runset at the time points in the table below. The times provided are approximate; each action is completed in response to a VWorks prompt at the appropriate time in the runset.

17 On the SureSelect setup form, under Select Protocol to Run, select SureSelectQXT_Capture&Wash_v1.0.rst.

18 Under Select PCR plate labware for Thermal Cycling, select the specific type of PCR plate used for hybridization. This plate will be transferred from the thermal cycler to Bravo deck position 4 when prompted by VWorks.

19 Select the number of columns of samples to be processed. Runs must include 1, 2, 3, 4, 6, or 12 columns.

20 Click Display Initial Workstation Setup.

21 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form.

22 When verification is complete, click Run Selected Protocol.

Table 46

Operator action Approximate time after run start

Transfer hybridization reactions from thermal cycler to NGS workstation

<5 minutes

Remove PCR plate from red aluminum insert 510 minutes

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 67

23 When ready to begin the run, click OK in the following window. If the temperature of Bravo deck position 4 was not pre-set to 66C, the runset will pause while position 4 reaches temperature.

24 When prompted by VWorks as shown below, quickly remove the PCR plate, containing the hybridization reactions held at 65C, from the thermal cycler. Unseal the plate carefully to avoid splashing, and quickly transfer the plate to position 4 of the Bravo deck, seated in the red insert. Click Continue to resume the runset.

CAUTION It is important to complete step 24 quickly and carefully. Transfer the sample plate to the Bravo platform quickly to retain the 65C sample temperature. Unseal the plate without tilting or jerking the plate to avoid sample splashing. Make sure that the Agilent NGS Workstation is completely prepared, with deck platforms at temperature and all components in place, before you transfer the sample plate to the Bravo deck.

WARNING Warning

Bravo deck position 4 will be hot.

Use caution when handling components that contact heated deck positions.

68 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

25 When prompted by VWorks as shown below, remove the PCR plate from position 4 of the Bravo deck, leaving the red aluminum insert in place. When finished, click Continue to resume the runset.

The remainder of the SureSelectQXT_Capture&Wash_v1.0.rst runset takes approximately 1.5 hours. Once the runset is complete, the captured, bead-bound DNA samples are located in the Eppendorf plate at position 9 of the Bravo deck

When the runset is complete, seal the plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165C and 1.0 sec and store the plate on ice while setting up the next automation protocol.

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

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 69

70 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

SureSelectQXT Automated Target Enrichment for the Illumina Platform Protocol

5 Indexing and Sample Processing for Multiplexed Sequencing Step 1. Amplify the captured libraries to add index tags 72 Step 2. Purify the amplified indexed libraries using AMPure XP beads 78 Step 3. Assess indexed DNA quality 80 Step 4. Quantify each index-tagged library by QPCR (optional) 82 Step 5. Pool samples for Multiplexed Sequencing 82 Step 6. Prepare sequencing samples 83 Step 7. Set up the sequencing run and trim adaptors from the reads 88

This chapter describes the steps to add index tags by amplification, and to purify and assess quality and quantity of the indexed libraries. Sample pooling instructions are provided to prepare the indexed samples for multiplexed sequencing, and guidelines are provided for downstream sequencing steps.

71

Step 1. Amplify the captured libraries to add index tags In this step, the Agilent NGS Workstation completes the liquid handling steps for PCR-based addition of dual indexing tags to the SureSelect-enriched DNA samples using automation protocol Post-CapturePCR_QXT_ILM_v1.0.pro. After the PCR plate is prepared by the Agilent NGS Workstation, you transfer the plate to a thermal cycler for amplification.

This step uses the components listed in Table 47. Thaw then vortex to mix the reagents listed below and keep on ice.

Prepare the workstation

1 Turn on the ThermoCube, set to 0C, at position 9 of the Bravo deck. Be sure that the chiller reservoir contains at least 300 mL of 25% ethanol.

2 Clear the Labware MiniHub and BenchCel of plates and tip boxes. 3 Pre-set the temperature of Bravo deck positions 4 and 6 to 4C using the Inheco Multi TEC

control touchscreen, as described in Setting the Temperature of Bravo Deck Heat Blocks. Bravo deck position 4 corresponds to CPAC 2, position 1 and deck position 6 corresponds to CPAC 2, position 2 on the Multi TEC control touchscreen.

4 Place a red PCR plate insert at Bravo deck position 6.

Table 47 Reagents for post-capture indexing by PCR amplification

Kit Component Storage Location Where Used

Herculase II Fusion DNA Polymerase SureSelect QXT Library Prep Kit Box 2, 20C page 73

Herculase II 5 Reaction Buffer SureSelect QXT Library Prep Kit Box 2, 20C page 73

100 mM dNTP Mix (25 mM each dNTP) SureSelect QXT Library Prep Kit Box 2, 20C page 73

SureSelect QXT P7 and P5 dual indexing primers

SureSelect QXT Library Prep Kit Box 2, 20C page 74

72 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Prepare the PCR master mix

5 Prepare the appropriate volume of PCR master mix, according to Table 48. Mix well using a vortex mixer and keep on ice.

6 Using the same Nunc DeepWell master mix source plate that was used for the Hybridization_QXT_v1.0.pro protocol, add the volume of PCR master mix indicated in Table 49 to all wells of column 4 of the plate. Keep the source plate on ice until it is used on page 74.

CAUTION To avoid cross-contaminating libraries, set up PCR master mixes in a dedicated clean area or PCR hood with UV sterilization and positive air flow.

Table 48 Preparation of PCR Master Mix for Post-CapturePCR_QXT_ILM_v1.0.pro

SureSelectQXT Reagent Volume for 1 Library

Volume for 1 Column

Volume for 2 Columns

Volume for 3 Columns

Volume for 4 Columns

Volume for 6 Columns

Volume for 12 Columns

Nuclease-free water 9.5 L 121.1 L 201.9 L 282.6 L 363.4 L 524.9 L 1049.8 L

Herculase II 5 Reaction Buffer

10.0 L 127.5 L 212.5 L 297.5 L 382.5 L 552.5 L 1105.0 L

100 mM dNTP Mix 0.5 L 6.4 L 10.6 L 14.9 L 19.1 L 27.6 L 55.3 L

Herculase II Fusion DNA Polymerase

1.0 L 12.8 L 21.3 L 29.8 L 38.3 L 55.3 L 110.5 L

Total Volume 21.0 L 267.8 L 446.3 L 624.8 L 803.3 L 1160.3 L 2320.6 L

Table 49 Preparation of the Master Mix Source Plate for Post-CapturePCR_QXT_ILM_v1.0.pro

Master Mix Solution

Position on Source Plate

Volume of Master Mix added per Well of Nunc Deep Well Source Plate

1-Column Runs

2-Column Runs

3-Column Runs

4-Column Runs

6-Column Runs

12-Column Runs

PCR Master Mix Column 4 (A4-H4)

30.8 L 53.2 L 75.5 L 97.8 L 142.4 L 287.4 L

NOTE If you are using a new DeepWell plate for the post-capture PCR source plate, leave columns 1 to 3 empty and add the PCR Master Mix to column 4 of the new plate.

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 73

Assign and aliquot indexing primers

7 Determine the appropriate index assignments for each sample. See the Reference section for sequences of the index portion of the P7 (page 94) and P5 (page 94) indexing primers used to amplify the DNA libraries in this step.

Use the following guidelines for dual index assignments: Use a different indexing primer combination for each sample to be sequenced in the same

lane. All samples on the same row of the target-enriched DNA library plate must be assigned to

the same P5 indexing primer (P5 i13 through P5 i20). This design results from the automation protocol configuration in which the P5 indexing primer is dispensed from a single source plate column to all columns of the indexing PCR plate. Each row of samples may be assigned to the same or different P5 primers, depending on run size and multiplexing requirements. (See step 10, below, for details of P5 primer addition to the master mix source plate.)

The automation protocol configuration allows for any of the provided P7 indexing primers (P7 i1 through P7 i12) to be assigned to any sample position of the target-enriched DNA library plate. (See step 8 and step 9 below, for P7 primer source plate setup details.)

For sample multiplexing, Agilent recommends maximizing index diversity on both P7 and P5 primers as required for color balance. For example, when 8-plexing, use eight different P7 index primers with two P5 index primers. See Table 79 on page 96 for additional details.

8 Dilute each P7 indexing primer (P7 i1 through P7 i12) to be used in the run according to Table 50. The volumes below include the required excess.

9 In a fresh PCR plate, aliquot 5 l of the appropriate P7 indexing primer dilution from Table 50 to the intended sample indexing well position(s).

Keep the plate on ice. 10 Obtain the Nunc DeepWell master mix source plate containing the PCR Master Mix in column 4

(prepared in step 6, above). Add each P5 indexing primer (P5 i13 through P5 i20) to be used in the run to the master mix in the appropriate well of column 4. Add the volume listed in Table 51 to each well of column 4, according to the number of sample columns in the run. Each well of column 4 can contain the same or different P5 indexing primers. The final configuration of the master mix source plate is shown in Figure 11 on page 75.

Keep the source plate on ice.

Table 50 Preparation of P7 indexing primer dilutions

Reagent Volume to Index 1 Sample Volume to Index 8 Samples

Nuclease-free water 4.0 L 34 L

SureSelect QXT P7 dual indexing primer (P7 i1 to P7 i12)

1.0 L 8.5 L

Total Volume 5.0 L 42.5 L

74 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Figure 11 Configuration of the master mix source plate for Post-CapturePCR_QXT_ILM_v1.0.pro.

11 Seal the master mix source plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165C and 1.0 sec.

12 Vortex the plate to ensure complete mixing, then centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate any bubbles.

Table 51 Addition of P5 indexing primers to the post-capture PCR master mix source plate

Solution added to Source Plate

Position on Source Plate

Volume of Primer added per Well of Nunc Deep Well Source Plate

1-Column Runs

2-Column Runs

3-Column Runs

4-Column Runs

6-Column Runs

12-Column Runs

SureSelect QXT P5 dual indexing primer(s)*

Column 4 (A4-H4)

1.5 L 2.5 L 3.6 L 4.7 L 6.8 L 13.7 L

* Each well of column 4 may contain the same or different P5 indexing primer. Typical 12-column runs include all eight of the pro- vided SureSelect QXT P5 dual indexing primers (P5 i13 through P5 i20), resulting in a different P5 primer assignment to each row of the PCR indexing plate.

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 75

Load the Agilent NGS Workstation

13 Load the Labware MiniHub according to Table 52, using the plate orientations shown in Figure 2.

14 Load the Bravo deck according to Table 53.

15 Load the BenchCel Microplate Handling Workstation according to Table 54.

Table 52 Initial MiniHub configuration for Post-CapturePCR_QXT_ILM_v1.0.pro

Vertical Shelf Position Cassette 1 Cassette 2 Cassette 3 Cassette 4

Shelf 5 (Top) Empty Empty Empty Empty

Shelf 4 Empty Empty Empty Empty

Shelf 3 Empty Empty Empty Empty

Shelf 2 New tip box Empty Empty Empty

Shelf 1 (Bottom) Empty tip box Empty Empty Empty tip box

Table 53 Initial Bravo deck configuration for Post-CapturePCR_QXT_ILM_v1.0.pro

Location Content

4 Captured DNA bead suspensions in Eppendorf twin.tec plate

6 Diluted P7 indexing primers in PCR plate seated in red insert (PCR plate type must be specified on setup form under step 2)

9 Master mix plate containing P5 indexing primers and PCR Master Mix in Column 4 (unsealed)

Table 54 Initial BenchCel configuration for Post-CapturePCR_QXT_ILM_v1.0.pro

No. of Columns Processed

Rack 1 Rack 2 Rack 3 Rack 4

1 1 Tip box Empty Empty Empty

2 1 Tip box Empty Empty Empty

3 1 Tip box Empty Empty Empty

4 1 Tip box Empty Empty Empty

6 1 Tip box Empty Empty Empty

12 1 Tip box Empty Empty Empty

76 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Run VWorks protocol Post-CapturePCR_QXT_ILM_v1.0.pro

16 On the SureSelect setup form, under Select Protocol to Run, select Post-CapturePCR_QXT_ILM_v1.0.pro.

17 Under Select PCR plate labware for Thermal Cycling, select the specific type of PCR plate used at position 6 of the Bravo deck.

18 Select the number of columns of samples to be processed. Runs must include 1, 2, 3, 4, 6, or 12 columns.

19 Click Display Initial Workstation Setup.

20 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form.

21 When verification is complete, click Run Selected Protocol.

Running the Post-CapturePCR_QXT_ILM_v1.0.pro protocol takes approximately 15 minutes. Once complete, the PCR-ready samples, containing captured DNA and PCR master mix, are located in the PCR plate at position 6 of the Bravo deck.

When you see the following prompt, remove the PCR plate from position 6 of the Bravo deck and seal the plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165C and 3.0 seconds.

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 77

22 Transfer the PCR plate to a thermal cycler and run the PCR amplification program shown in Table 55.

Step 2. Purify the amplified indexed libraries using AMPure XP beads In this step, the Agilent NGS Workstation transfers AMPure XP beads to the indexed DNA sample plate and then collects and washes the bead-bound DNA.

Prepare the workstation and reagents

1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes. 2 Gently wipe down the Labware MiniHub, Bravo decks, and BenchCel with a DNA Away

decontamination wipe. 3 Verify that the AMPure XP bead suspension is at room temperature. (If necessary, allow the

bead solution to come to room temperature for at least 30 minutes.) Do not freeze the beads at any time.

4 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color.

5 Turn on the ThermoCube, set to 4C, at position 9 of the Bravo deck. Be sure that the chiller reservoir contains at least 300 mL of 25% ethanol.

6 Pre-set the temperature of Bravo deck position 4 to 45C using the Inheco Multi TEC control touchscreen, as described in Setting the Temperature of Bravo Deck Heat Blocks. Bravo deck position 4 corresponds to CPAC 2, position 1 on the Multi TEC control touchscreen.

7 Prepare a Nunc DeepWell source plate containing AMPure XP beads. For each well to be processed, add 65 l of homogeneous AMPure XP beads per well to the Nunc DeepWell plate.

8 Prepare a Thermo Scientific reservoir containing 15 mL of nuclease-free water. 9 Prepare a separate Thermo Scientific reservoir containing 45 mL of freshly-prepared 70%

ethanol. 10 Centrifuge the indexed DNA sample plate for 30 seconds to drive the well contents off the

walls and plate seal.

Table 55 Post-Capture PCR cycling program

Segment Number of Cycles Temperature Time

1 1 98C 2 minutes

2 Probes3 Mb: 10 Cycles

Probes 1 to 3 Mb: 12 Cycles Probes1 Mb: 14 Cycles

98C 30 seconds

58C 30 seconds

72C 1 minute

3 1 72C 10 minutes

4 1 4C Hold

78 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

11 Load the Labware MiniHub according to Table 56, using the plate orientations shown in Figure 2.

12 Load the Bravo deck according to Table 57.

13 Load the BenchCel Microplate Handling Workstation according to Table 58.

Run VWorks protocol AMPureXP_QXT_ILM_v1.0.pro:Post-Capture PCR

14 On the SureSelect setup form, under Select Protocol to Run, select AMPureXP_QXT_ILM_v1.0.pro:Post-Capture PCR.

Table 56 Initial MiniHub configuration for AMPureXP_QXT_ILM_v1.0.pro:Post-Capture PCR

Vertical Shelf Position Cassette 1 Cassette 2 Cassette 3 Cassette 4

Shelf 5 (Top) Empty Nunc DeepWell plate

Empty Empty Empty

Shelf 4 Empty Empty Empty Empty

Shelf 3 Empty Empty Eppendorf Plate

Empty Empty

Shelf 2 Empty Nuclease-free water reservoir from step 8

AMPure XP beads in Nunc DeepWell plate from step 7

Empty

Shelf 1 (Bottom) Empty 70% ethanol reservoir from step 9

Empty Empty tip box

Table 57 Initial Bravo deck configuration for AMPureXP_QXT_ILM_v1.0.pro:Post-Capture PCR

Location Content

1 Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)

9 Indexed library samples in unsealed PCR plate seated in red insert (PCR plate type must be specified on setup form under step 2)

Table 58 Initial BenchCel configuration for AMPureXP_QXT_ILM_v1.0.pro:Post-Capture PCR

No. of Columns Processed Rack 1 Rack 2 Rack 3 Rack 4

1 1 Tip box Empty Empty Empty

2 1 Tip box Empty Empty Empty

3 2 Tip boxes Empty Empty Empty

4 2 Tip boxes Empty Empty Empty

6 3 Tip boxes Empty Empty Empty

12 6 Tip boxes Empty Empty Empty

NOTE AMPureXP purification protocols are used during multiple steps of the SureSelect automation workflow. Be sure to select the correct workflow step when initiating the automation protocol.

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 79

15 Under Select PCR plate labware for Thermal Cycling, select the specific type of PCR plate containing the indexed libraries at position 9.

16 Select the number of columns of samples to be processed. Runs must include 1, 2, 3, 4, 6, or 12 columns.

17 Click Display Initial Workstation Setup.

18 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form.

19 When verification is complete, click Run Selected Protocol.

The purification protocol takes approximately 45 minutes. When complete, the amplified DNA samples are in the Eppendorf plate located on Bravo deck position 7.

Step 3. Assess indexed DNA quality

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

Use the Bioanalyzer High Sensitivity DNA Assay to analyze the amplified indexed 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 Prepare the chip, samples and ladder as instructed in the reagent kit guide, using 1 L of each

sample for the analysis.

4 Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation.

5 Verify that the electropherogram shows the peak of DNA fragment size positioned between 325 and 450 bp. A sample electropherogram is shown in Figure 12.

NOTE For some samples, Bioanalyzer results are improved by diluting 1 l of the sample in 9 L of 10 mM Tris, 1 mM EDTA prior to analysis. Be sure to mix well by vortexing at 2000 rpm on the IKA vortex supplied with the Bioanalyzer before analyzing the diluted samples.

80 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

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.

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

Use a High Sensitivity D1000 ScreenTape and associated reagent kit. Perform the assay according to 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.

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 325 and 450 bp. A sample electropherogram is shown in Figure 13.

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

Figure 13 Post-capture library DNA analyzed using a High Sensitivity D1000 ScreenTape.

Figure 12 Post-capture analysis of library DNA using the High Sensitivity DNA Assay.

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.

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 81

Step 4. Quantify each index-tagged library by QPCR (optional) Refer to the protocol that is included with the Agilent QPCR NGS Library Quantification Kit (p/n G4880A) for more details to do this step. 1 Use the Agilent QPCR NGS Library Quantification Kit (for Illumina) to determine the

concentration of each index-tagged captured library. 2 Prepare a standard curve using the quantification standard included in the kit, according to the

instructions provided in the user guide. 3 Dilute each index-tagged captured library such that it falls within the range of the standard

curve.

Typically this corresponds to approximately a 1:1000 to 1:10,000 dilution of the captured DNA. 4 Prepare the QPCR master mix with Illumina adaptor-specific PCR primers according to

instructions provided in the kit. 5 Add an aliquot of the master mix to PCR tubes and add template. 6 On a QPCR system, such as the Mx3005p, run the thermal profile outlined in the QPCR NGS

Library Quantification kit user guide. Use the SYBR Green instrument setting. 7 Use the standard curve to determine the concentration of each unknown index-tagged library,

in nM.

The concentration will be used to accurately pool samples for multiplexed sequencing.

Step 5. Pool samples for Multiplexed Sequencing 1 Combine the libraries such that each index-tagged sample is present in equimolar amounts in

the pool. For each library, use the formula below to determine the amount of indexed sample to use.

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

# is the number of indexes, and

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

Table 59 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.

Volume of Index V f C f # C i

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

82 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

If the final volume of the combined index-tagged samples is less than the desired final volume, V(f), add Low TE to bring the volume to the desired level.

If the final volume of the combined index-tagged samples is greater than the final desired volume, V(f), lyophilize and reconstitute to the desired volume.

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

Step 6. Prepare sequencing samples Proceed to cluster amplification using the appropriate Illumina Paired-End Cluster Generation Kit. See Table 60 for kit configurations compatible with the recommended read length plus reads for the SureSelectQXT 8-bp dual indexes.

The optimal seeding concentration for SureSelectQXT target-enriched libraries varies according to sequencing platform, run type and Illumina kit version. See Table 60 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.

To do this step, refer to the manufacturers instructions, using the modifications described on page 84 for use of the SureSelectQXT Read Primers with the Illumina Paired-End Cluster Generation Kits. Follow Illuminas recommendation for a PhiX control in a low-concentration spike-in for improved sequencing quality control.

Table 59 Example of indexed sample volume calculation for total volume of 20 L

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

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 83

Using the SureSelectQXT Read Primers with Illuminas Paired-End Cluster Generation Kits

To sequence the SureSelectQXT libraries on Illuminas sequencing platforms, you need to use the following custom sequencing primers, provided in SureSelect QXT Library Prep Kit Box 2: SureSelect QXT Read Primer 1 SureSelect QXT Read Primer 2 SureSelect QXT Index 1 Read Primer SureSelect QXT Index 2 Read Primer (this primer is used only for HiSeq 3000, HiSeq 4000, and

NextSeq platforms and for NovaSeq platform runs using v1.5 chemistry)

These SureSelectQXT custom sequencing primers are provided at 100 M and must be diluted in the corresponding Illumina primer solution, using the platform-specific instructions below:

For the HiSeq 2500 platform, combine the primers as shown in Table 61 or Table 62 on page 85.

For the HiSeq 3000 or HiSeq 4000 platform, combine the primers as shown in Table 63 on page 85.

For the MiSeq platform, combine the primers as shown in Table 64 on page 86.

For the NextSeq platform, combine the primers as shown in Table 65 or Table 66 on page 86.

For the NovaSeq platform, combine the primers as shown in Table 67 through Table 70 on page 86.

Table 60 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 1013 pM

HiSeq 2500 High Output 2 100 bp 250 Cycle Kit v4 1316 pM

MiSeq All Runs 2 100 bp or 2 150 bp

300 Cycle Kit v2 1013 pM

MiSeq All Runs 2 75 bp 150 Cycle Kit v3 1419 pM

NextSeq 500/550 All Runs 2 100 bp or 2 150 bp

300 Cycle Kit v2.5 1.31.8 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

* If your application requires a different read length, verify that you have sufficient sequencing reagents to complete Reads 1 and 2 in addition to the dual 8-bp index reads.

84 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

NOTE It is important to combine the primers precisely in the indicated ratios. Carefully follow the instructions indicated in Table 61 to Table 70. Where specified, add the custom primer volume directly to the solution already in cBot reagent plate wells. Otherwise, combine measured volumes of each solution; do not rely on volumes reported on vial labels or in Illumina literature. Vortex each mixture vigorously to ensure homogeneity for proper detection of the indexes using the custom read primers.

Table 61 HiSeq 2500 High Output custom sequencing primer preparation

Sequencing Read Volume of SureSelectQXT Primer Volume of Illumina TruSeq Primer

Total Volume

Read 1 6 L SureSelect QXT Read Primer 1 (brown cap) 1194 L HP10 1.2 mL*

Index 15 L SureSelect QXT Index 1 Read Primer (clear cap) 2985 L HP12 3 mL

Read 2 15 L SureSelect QXT Read Primer 2 (black cap) 2985 L HP11 3 mL

* Aliquot the mixture as directed for HP6 or HP10 in Illuminas cluster generation protocol.

Table 62 HiSeq 2500 Rapid Mode custom sequencing primer preparation

Sequencing Read Volume of SureSelectQXT Primer Volume of Illumina TruSeq Primer

Total Volume

Read 1 8.8 L SureSelect QXT Read Primer 1 (brown cap) 1741.2 L HP10 1.75 mL

Index 8.8 L SureSelect QXT Index 1 Read Primer (clear cap) 1741.2 L HP12 1.75 mL

Read 2 8.8 L SureSelect QXT Read Primer 2 (black cap) 1741.2 L HP11 1.75 mL

Table 63 HiSeq 3000 and HiSeq 4000 custom sequencing primer preparation

Sequencing Read Volume of SureSelectQXT Primer Volume of Illumina TruSeq Primer

Total Volume

Reagent Rack Position

Read 1 1.5 L SureSelect QXT Read Primer 1 (brown cap) 298.5 L HP10* 0.3 mL per well

cBot Column 11

Read 2 15 L SureSelect QXT Read Primer 2 (black cap) 2985 L HP11 3 mL 16

Index 1+ Index 2 22.5 L SureSelect QXT Index 1 Read Primer (clear cap) + 22.5 L SureSelect QXT Index 2 Read Primer (purple cap)

4455 L HP14 4.5 mL 17

* Use cBot recipe HiSeq_3000_4000_HD_Exclusion_Amp_v1.0. Add 1.5 L SureSelect QXT Read Primer 1 to the 298.5 L of HP10 in each well of column 11 in the cBot reagent plate.

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 85

Table 64 MiSeq platform custom sequencing primer preparation

Sequencing Read Volume of SureSelectQXT Primer Volume of Illumina TruSeq Primer

Total Volume

Final Cartridge Position

Read 1 3 L SureSelect QXT Read Primer 1 (brown cap) 597 L HP10 (well 12)

0.6 mL well 18

Index 3 L SureSelect QXT Index 1 Read Primer (clear cap) 597 L HP12 (well 13)

0.6 mL well 19

Read 2 3 L SureSelect QXT Read Primer 2 (black cap) 597 L HP11 (well 14)

0.6 mL well 20

Table 65 NextSeq 500/550 High-Output v2 Kit custom sequencing primer preparation

Sequencing Read Volume of SureSelectQXT Primer Volume of Illumina Primer

Total Volume

Final Cartridge Position

Read 1 3.9 L SureSelect QXT Read Primer 1 (brown cap) 1296.1 L BP10 (from well 20)

1.3 mL well 7

Read 2 4.2 L SureSelect QXT Read Primer 2 (black cap) 1395.8 L BP11 (from well 21)

1.4 mL well 8

Index 1+ Index 2 6 L SureSelect QXT Index 1 Read Primer (clear cap) + 6 L SureSelect QXT Index 2 Read Primer (purple cap)

1988 L BP14 (from well 22)

2 mL well 9

Table 66 NextSeq 500/550 Mid-Output v2 Kit custom sequencing primer preparation

Sequencing Read Volume of SureSelectQXT Primer Volume of Illumina Primer

Total Volume

Final Cartridge Position

Read 1 2.7 L SureSelect QXT Read Primer 1 (brown cap) 897.3 L BP10 (from well 20)

0.9 mL well 7

Read 2 3.3 L SureSelect QXT Read Primer 2 (black cap) 1096.7 L BP11 (from well 21)

1.1 mL well 8

Index 1+ Index 2 4.8 L SureSelect QXT Index 1 Read Primer (clear cap) + 4.8 L SureSelect QXT Index 2 Read Primer (purple cap)

1590.4 L BP14 (from well 22)

1.6 mL well 9

Table 67 NovaSeq 6000 using SP/S1/S2 flowcell with v1.0 chemistry custom sequencing primer preparation

Sequencing Read Volume of SureSelectQXT Primer Volume of Illumina Primer

Total Volume

Final Cartridge Position

Read 1 12 L SureSelect QXT Read Primer 1 (brown cap) 3988 L BP10 (well 24)

4 mL 5

Index 15 L SureSelect QXT Index 1 Read Primer (clear cap)

4985 L BP14 (well 23)

5 mL 7

Read 2 6 L SureSelect QXT Read Primer 2 (black cap) 1994 L BP11 (well 13)

2 mL 6

86 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Table 68 NovaSeq 6000 using S4 flowcell with v1.0 chemistry custom sequencing primer preparation

Sequencing Read Volume of SureSelectQXT Primer Volume of Illumina Primer

Total Volume

Final Cartridge Position

Read 1 21.9 L SureSelect QXT Read Primer 1 (brown cap) 7278.1 L BP10 (well 24)

7.3 mL 5

Index 15 L SureSelect QXT Index 1 Read Primer (clear cap)

4985 L BP14 (well 23)

5 mL 7

Read 2 10.5 L SureSelect QXT Read Primer 2 (black cap) 3489.5 L BP11 (well 13)

3.5 mL 6

Table 69 NovaSeq 6000 using SP/S1/S2 flowcell with v1.5 chemistry custom sequencing primer preparation

Sequencing Read Volume of SureSelectQXT Primer Volume of Illumina Primer

Total Volume

Final Cartridge Position

Read 1 12 L SureSelect QXT Read Primer 1 (brown cap) 3988 L BP10 (well 24)

4 mL 5

Index 1+ Index 2 15 L SureSelect QXT Index 1 Read Primer (clear cap) + 15 L SureSelect QXT Index 2 Read Primer (purple cap)

4970 L VP14 (well 23)

5 mL 7

Read 2 6 L SureSelect QXT Read Primer 2 (black cap) 1994 L BP11 (well 13)

2 mL 6

Table 70 NovaSeq 6000 using S4 flowcell with v1.5 chemistry custom sequencing primer preparation

Sequencing Read Volume of SureSelectQXT Primer Volume of Illumina Primer

Total Volume

Final Cartridge Position

Read 1 21.9 L SureSelect QXT Read Primer 1 (brown cap) 7278.1 L BP10 (well 24)

7.3 mL 5

Index 1+ Index 2 15 L SureSelect QXT Index 1 Read Primer (clear cap) + 15 L SureSelect QXT Index 2 Read Primer (purple cap)

4970 L VP14 (well 23)

5 mL 7

Read 2 10.5 L SureSelect QXT Read Primer 2 (black cap) 3489.5 L BP11 (well 13)

3.5 mL 6

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 87

Step 7. Set up the sequencing run and trim adaptors from the reads Refer to Illumina protocols to set up custom sequencing primer runs, using the additional guidelines outlined below.

For SureSelectQXT dual index sequence information, see page 94.

Before aligning reads to the reference genome, SureSelectQXT adaptor sequences must be trimmed from the reads. You can use SureCall, Agilent's NGS data analysis software, to perform adaptor trimming, alignment of reads and variant calling of FASTQ sequencing data files generated by Illumina sequencers. To download SureCall free-of-charge and for additional information, including tutorials on this software, visit the SureCall page at www.agilent.com.

To use SureCall to analyze SureSelectQXT library data, you first need to define an analysis workflow. This analysis workflow identifies the libraries as SureSelectQXT libraries and enables automated adaptor trimming. The trimmed FASTQ files can then be used for alignment to generate BAMs for downstream analysis.

To create the analysis workflow, refer to Figure 14 on page 88. Upon starting SureCall, click the Analysis Workflow tab. Choose the appropriate analysis type (single sample, paired, or trio analysis), and then click the Import Unaligned Files button. Using the menus near the top of the screen, choose the appropriate design description from the Design menu, select Default SureSelect Method from the Analysis Method menu, and select QXT from the Library Prep menu. Within the Select Unaligned Sample Files window, specify your read 1 and read 2 files using the Add buttons. Once done, refer to the SureCall guide for next steps on alignment and variant calling.

If using another pipeline for alignment and downstream analysis, refer to the platform-specific guidelines starting on page 89.

Figure 14 Analysis workflow setup in SureCall

88 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

MiSeq platform sequencing run setup and adaptor trimming guidelines

Use the Illumina Experiment Manager (IEM) software to generate a custom primer Sample Sheet.

Set up the run to include adapter trimming using the IEM Sample Sheet Wizard. When prompted by the wizard, select the Use Adapter Trimming option, and specify CTGTCTCTTGATCACA as the adapter sequence. This enables the MiSeq Reporter software to identify the adaptor sequence and trim the adaptor from reads.

HiSeq/NextSeq/NovaSeq platform sequencing run setup and adaptor trimming guidelines

Set up sequencing runs using the settings shown in Table 71. For HiSeq runs, select Dual Index on the Run Configuration screen of the instrument control software interface. Since custom primers are spiked into the standard sequencing primer tubes, no additional specialized settings are required to accommodate the use of custom primers in the run.

For the NextSeq or NovaSeq platform, Cycle Number and custom sequencing primer settings can be specified on the Run Configuration screen of the instrument control software interface.

After the sequencing run is complete, generate demultiplexed FASTQ data following Illuminas instructions and then trim adaptor sequences from the reads using the Trimmer utility of the Agilent Genomics NextGen Toolkit (AGeNT). For additional information and to download this toolkit free-of-charge, visit the AGeNT page at www.agilent.com.

Table 71 Run Configuration screen Cycle Number settings

Run Segment Cycle Number

Read 1 100

Index 1 (i7) 8

Index 2 (i5) 8

Read 2 100

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 89

90 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

SureSelectQXT Automated Target Enrichment for the Illumina Platform Protocol

6 Reference Kit Contents 92 Nucleotide Sequences of SureSelectQXT Dual Indexes 94 Guidelines for Multiplexing with Dual-Indexed Samples 96

This chapter contains reference information, including component kit contents and reference information for use during the downstream sample sequencing steps.

91

Kit Contents

SureSelectQXT Reagent Kits contain the following component kits:

The contents of each of the component kits listed in Table 72 are described in Table 73 to Table 75 below.

Table 72 SureSelectQXT Reagent Kit Contents

Component Kits Storage Condition Part Number

SureSelect QXT Library Prep Kit, ILM, Box 2*

* SureSelect QXT Library Prep Kit, ILM, Box 1 is not required for the workflow described in this manual.

20C 5500-0127

SureSelect QXT Target Enrichment Kit, ILM Hyb Module, Box 1 Room Temperature 5190-7335

SureSelect QXT Target Enrichment Kit, ILM Hyb Module, Box 2 20C 5190-7334

Table 73 SureSelect QXT Library Prep Kit Box 2 Content

Kit Component Format

SureSelect QXT Buffer bottle

SureSelect QXT Enzyme Mix ILM tube with orange cap

Herculase II Fusion DNA Polymerase tube with red cap

Herculase II 5 Reaction Buffer tube with clear cap

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

DMSO tube with green cap

SureSelect QXT Read Primer 1 tube with amber cap

SureSelect QXT Read Primer 2 tube with black cap

SureSelect QXT Index 1 Read Primer tube with clear cap

SureSelect QXT Index 2 Read Primer tube with purple cap

SureSelect QXT P7 dual indexing primers P7 i1 through P7 i12 provided in 12 tubes with yellow caps (one tube per primer)

SureSelect QXT P5 dual indexing primers P5 i13 through P5 i20 provided in 8 tubes with blue caps (one tube per primer)

Table 74 SureSelect QXT Hyb Module Box 1 Content

Kit Component Format

SureSelect QXT Stop Solution bottle

SureSelect Binding Buffer bottle

SureSelect Wash Buffer 1 bottle

SureSelect Wash Buffer 2 bottle

92 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Table 75 SureSelect QXT Hyb Module Box 2 Content

Kit Component Format

SureSelect Fast Hybridization Buffer bottle

SureSelect QXT Fast Blocker Mix tube with blue cap

SureSelect QXT Primer Mix tube with clear cap

SureSelect RNase Block tube with purple cap

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 93

Nucleotide Sequences of SureSelectQXT Dual Indexes

The nucleotide sequence of each SureSelectQXT index is provided in the tables below.

Note that some index number assignments of the SureSelectQXT P5 and P7 indexes differ from the index number assignments used by Illumina for indexes of similar or identical sequence.

Each index is 8 bases in length. Refer to Illuminas sequencing run setup instructions for sequencing libraries using 8-base indexes.

Table 76 SureSelectQXT P7 Indexes 1 to 12

Index Number Sequence

P7 Index 1 (P7 i1) TAAGGCGA

P7 Index 2 (P7 i2) CGTACTAG

P7 Index 3 (P7 i3) AGGCAGAA

P7 Index 4 (P7 i4) TCCTGAGC

P7 Index 5 (P7 i5) GTAGAGGA

P7 Index 6 (P7 i6) TAGGCATG

P7 Index 7 (P7 i7) CTCTCTAC

P7 Index 8 (P7 i8) CAGAGAGG

P7 Index 9 (P7 i9) GCTACGCT

P7 Index 10 (P7 i10) CGAGGCTG

P7 Index 11 (P7 i11) AAGAGGCA

P7 Index 12 (P7 i12) GGACTCCT

Table 77 SureSelectQXT P5 Indexes 13 to 20 for HiSeq 2500, MiSeq, or NovaSeq (v1.0 chemistry) platform

Index Number Sequence

P5 Index 13 (P5 i13) TAGATCGC

P5 Index 14 (P5 i14) CTCTCTAT

P5 Index 15 (P5 i15) TATCCTCT

P5 Index 16 (P5 i16) AGAGTAGA

P5 Index 17 (P5 i17) GTAAGGAG

P5 Index 18 (P5 i18) ACTGCATA

P5 Index 19 (P5 i19) AAGGAGTA

P5 Index 20 (P5 i20) CTAAGCCT

94 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Table 78 SureSelectQXT P5 Indexes 13 to 20 for HiSeq 3000/4000, NextSeq, or NovaSeq (v1.5 chemistry) platform*

Index Number Sequence

P5 Index 13 (P5 i13) GCGATCTA

P5 Index 14 (P5 i14) ATAGAGAG

P5 Index 15 (P5 i15) AGAGGATA

P5 Index 16 (P5 i16) TCTACTCT

P5 Index 17 (P5 i17) CTCCTTAC

P5 Index 18 (P5 i18) TATGCAGT

P5 Index 19 (P5 i19) TACTCCTT

P5 Index 20 (P5 i20) AGGCTTAG

* When doing runs on these platforms through BaseSpace, use the reverse complement sequences provided in Table 77.

SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing 95

Guidelines for Multiplexing with Dual-Indexed Samples

Agilent recommends following the dual index sample pooling guidelines and shown in Table 79. These are designed to maintain color balance at each cycle of the index reads on both ends. They also provide flexibility of demultiplexing as single or dual indexed samples in low-plexity experiments. One-base mismatches should be allowed during demultiplexing.

Table 79 Dual index sample pooling guidelines for 96 Reaction Kits

Plexity of Sample Pool Recommended SureSelectQXT P7 Indexes Recommended SureSelectQXT P5 Indexes

1-plex Any P7 index i1 to i12 Any P5 index (i13 to i20)

2-plex P7 i1 and P7 i2 OR P7 i2 and P7 i4

P5 i13 and P5 i14 OR P5 i15 and P5 i16 OR P5 i17 and P5 i18

3-plex P7 i1, P7 i2 and P7 i4 OR P7 i3, P7 i4 and P7 i6 OR P7 i5, P7 i7 and P7 i8

P5 i13 and P5 i14 OR P5 i15 and P5 i16 OR P5 i17 and P5 i18 (as needed)

4-plex P7 i1, P7 i2, P7 i3* and P7 i4 OR P7 i3, P7 i4, P7 i5* and P7 i6 OR P7 i5, P7 i6*, P7 i7 and P7 i8

P5 i13 and P5 i14 OR P5 i15 and P5 i16 OR P5 i17 and P5 i18 (as needed)

5-plex P7 i1, P7 i2, P7 i3*, P7 i4 and P7 i5* OR P7 i3, P7 i4, P7 i5*, P7 i6 and p7 i7* OR P7 i5, P7 i6*, P7 i7, P7 i8 and p7 i9*

P5 i13 and P5 i14 OR P5 i15 and P5 i16 OR P5 i17 and P5 i18 (as needed)

6- to 12-plex Any combination of P7 indexes i1 to i12 using each index only once

P5 i13 and P5 i14 OR P5 i15 and P5 i16 OR P5 i17 and P5 i18 (as needed)

13- to 96-plex All twelve P7 indexes (i1 to i12) P5 i13 and P5 i14 and any other P5 index OR P5 i15 and P5 i16 and any other P5 index OR P5 i17 and P5 i18 and any other P5 index (as needed)

* The indicated indexes may be substituted with another index, as long as the substitute index differs from all others used in the sample pool.

96 SureSelectQXT AutomatedTarget Enrichment for Illumina Multiplexed Sequencing

Manualsnet FAQs

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