TruSight One Sequencing Panel Reference Guide (15046431 01)

TruSight One Sequencing Panel Reference Guide (15046431 01)

TruSight

®

One Sequencing

Panel Reference Guide

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

ILLUMINA PROPRIETARY

Document # 15046431 v01

January 2016

Customize a short end-to-end workflow guide with the Custom Protocol Selector support.illumina.com/custom-protocol-selector.html

This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for the contractual use of its customer in connection with the use of the product(s) described herein and for no other purpose. This document and its contents shall not be used or distributed for any other purpose and/or otherwise communicated, disclosed, or reproduced in any way whatsoever without the prior written consent of Illumina. Illumina does not convey any license under its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this document.

The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product(s) described herein. All of the contents of this document must be fully read and understood prior to using such product(s).

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© 2016 Illumina, Inc. All rights reserved.

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ii

Document # 15046431 v01

Revision History

Document

Document # 15046433 v01

Part # 15046433 Rev. A

Date

January

2

016

October

2013

Description of Change

• Corrected storage of MiSeq Reagent Kit v3 Box 2 to 2°C to 8°C.

• Updated design of workflow diagram.

• Renamed and combined some procedures as needed to improve continuity.

• Simplified consumables information at the beginning of each section.

• Revised step-by-step instructions to be more succinct.

• Removed reference to obsolete Experienced User Cards and added reference to new protocol guide and checklist.

• Removed Preparing Your Libraries for Sequencing on a MiSeq section.

Initial release.

TruSight One Sequencing Panel Reference Guide iii

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Document # 15046431 v01

Table of Contents

Revision History

Table of Contents

Chapter 1 Overview

Introduction

DNA Input Recommendations

Critical Steps for Successful Enrichment and Coverage

Additional Resources

Chapter 2 Protocol

Introduction

Tips and Techniques

Library Prep Workflow

Tagment Genomic DNA

Clean Up Tagmented DNA

Amplify Tagmented DNA

Clean Up Amplified DNA

Hybridize Probes

Capture Hybridized Probes

Perform Second Hybridization

Perform Second Capture

Clean Up Captured Library

Amplify Enriched Library

Clean Up Amplified Enriched Library

Check Enriched Libraries

Appendix A Supporting Information

Introduction

Acronyms

Alternative Thermal Cycler Steps for Successful Enrichment

Kit Contents

Consumables and Equipment

Index Sequences

Technical Assistance

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TruSight One Sequencing Panel Reference Guide v

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Document # 15046431 v01

 Chapter 1  Overview

Overview

Introduction

DNA Input Recommendations

Critical Steps for Successful Enrichment and Coverage

Additional Resources

2

3

4

5

TruSight One Sequencing Panel Reference Guide

1

Introduction

This protocol explains how to prepare up to 36 indexed, paired-end libraries, followed by enrichment using the TruSight ® One Sequencing Panel and reagents provided in an

Illumina TruSight One Sequencing Panel kit. This protocol fragments and adds adapter sequences onto template DNA to generate indexed libraries that can be carried through enrichment for targeted resequencing applications.

The TruSight One Sequencing Panel protocol offers:

} Fast and easy sample preparation

}

Prepare up to 36 enriched libraries in approximately 1.5 days with approximately 5 hours of hands-on time

}

High throughput, automation-friendly procedures with no fragmentation bottlenecks

} Low DNA input and excellent data quality

}

Low input of 50 ng

}

Access precious samples with no affect on performance

}

Ability to archive samples for subsequent analysis

} High enrichment rates, low duplicates, and exceptional coverage uniformity

}

Efficient use of sequencing

}

Reliable variant calling

}

Reduced hands-on time with the most cost-effective, high-throughput workflow

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DNA Input Recommendations

Using an enzymatic DNA fragmentation step allows TruSight One library preparation to be more sensitive to DNA input than mechanical fragmentation methods. Accurate quantification of the starting gDNA is essential to enrichment success.

Quantify the starting gDNA using a fluorometric-based method specific for double-stranded

DNA (dsDNA) and run samples in triplicate. Avoid methods that measure total nucleic acid content, such as NanoDrop or other UV absorbance methods. Common contaminants such as ssDNA, RNA, and oligos are not substrates for the TruSight One Sequencing Panel.

The TruSight One protocol has been optimized for 50 ng of total gDNA. A higher mass input of gDNA can result in incomplete tagmentation and larger insert sizes, and can affect enrichment performance. Conversely, a lower mass input of gDNA or low quality gDNA in the tagmentation reaction can generate smaller than expected insert sizes, which can be lost during subsequent cleanup steps and result in lower diversity.

To minimize gDNA sample input variability into the tagmentation step, perform a 2-step method of gDNA normalization. After the initial quantification, gDNA samples are normalized to 10 ng/µl. Samples are then quantified using a similar fluorometric-based method and normalized to a final 5 ng/µl.

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Critical Steps for Successful Enrichment and Coverage

To ensure robust performance from the TruSight One Sequencing Panel, use a microheating system with a midi plate insert for the enrichment wash steps. The enrichment wash steps reduce nonspecific DNA binding and require that samples are maintained at the indicated temperature. Too low or too high temperatures can result in lower percent enrichments and decreased yields. If a microheating system is not available, a thermal cycler can be used with some modifications. See

Alternative Thermal Cycler Steps for Successful Enrichment

on page 38

for instructions using a thermal cycler.

Obtaining Desired Reads Per Sample

The number of resulting reads for each sample of a pool depend on the following factors:

}

Accurate quantification of tagmented samples before pooling for enrichment. Inaccurate quantification can lead to uneven pooling between samples in the enrichment and can result in less than expected reads for a given sample.

}

Accurate quantification of final enriched library pools. Use the same dilution of final library for both quantification and clustering. Inaccurate quantification can result in lower than targeted cluster densities, less reads passing filter and/or inefficient demultiplexing if overclustered.

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Additional Resources

Visit the TruSight One Sequencing Panel support page on the Illumina website for documentation, software downloads, training resources, and information about compatible

Illumina products.

The following documentation is available for download from the Illumina website.

Resource

Custom Protocol Selector

TruSight One Sequencing Panel

Protocol Guide (document #

1000000006694)

TruSight One Sequencing Panel

Checklist (document #

1000000006696)

Description

http://support.illumina.com/custom-protocol-selector.html

A wizard for generating customized end-to-end documentation that is tailored to the library prep method, run parameters, and analysis method used for the sequencing run.

Provides only protocol instructions.

The protocol guide is intended for experienced users. For new or less experienced users, see the TruSight One Sequencing

Panel Reference Guide.

Provides a checklist of the protocol steps.

The checklist is intended for experienced users. For new or less experienced users, see the TruSight One Sequencing Panel

Reference Guide.

TruSight One Sequencing Panel Reference Guide

5

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  Chapter 2  Protocol

Protocol

Introduction

Tips and Techniques

Library Prep Workflow

Tagment Genomic DNA

Clean Up Tagmented DNA

Amplify Tagmented DNA

Clean Up Amplified DNA

Hybridize Probes

Capture Hybridized Probes

Perform Second Hybridization

Perform Second Capture

Clean Up Captured Library

Amplify Enriched Library

Clean Up Amplified Enriched Library

Check Enriched Libraries

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Introduction

This chapter describes the TruSight One protocol.

}

Follow the protocols in the order shown, using the specified volumes and incubation parameters.

} Review Best Practices from the TruSight One support page on the Illumina website.

} Include a common index in each column. A common index facilitates pipetting operations when dispensing index adapters and pooling indexed libraries later in the protocol.

Prepare for Pooling

If you plan to pool libraries, record information about your samples before beginning library prep. Different methods are available depending on the sequencing instrument you are using. See the TruSight One Sequencing Panel support page for more information.

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Tips and Techniques

Unless a safe stopping point is specified in the protocol, proceed immediately to the next step.

Avoiding Cross-Contamination

} When adding or transferring samples, change tips between

each sample

.

} When adding adapters or primers, change tips between

each row

and

each column

.

}

Remove unused index adapter tubes from the working area.

Sealing the Plate

}

Always seal the 96-well plate before the following steps in the protocol:

}

Shaking steps

}

Vortexing steps

} Centrifuge steps

}

Thermal cycling steps

}

Apply the adhesive seal to cover the plate and seal with a rubber roller.

}

Microseal 'B' adhesive seals are effective at -40°C to 110°C, and suitable for skirted or semiskirted PCR plates. Use Microseal 'B' for shaking, centrifuging, and long-term storage.

}

Microseal 'A' adhesive film is effective for thermal cycling and easy to cut when using fewer than 96 wells.

Plate Transfers

}

When transferring volumes between plates, transfer the specified volume from each well of a plate to the corresponding well of the other plate.

Centrifugation

}

Centrifuge at any step in the procedure to consolidate liquid or beads in the bottom of the well, and to prevent sample loss.

}

To pellet beads, centrifuge at 280 × g for 1 minute.

Handling Beads

}

Pipette bead suspension slowly.

}

When mixing, mix thoroughly.

} If beads are aspirated into the pipette tips, dispense back to the plate on the magnetic stand and wait until the liquid is clear (~2 minutes).

}

When washing beads:

}

Use the appropriate magnet for the plate.

} Dispense liquid so that beads on the side of the wells are wetted.

} Keep the plate on the magnet until the instructions specify to remove it.

}

Do not agitate the plate while on the magnetic stand. Do not disturb the bead pellet.

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Library Prep Workflow

The following diagram illustrates the workflow using a TruSight One Sequencing Panel kit.

Safe stopping points are marked between steps.

Figure 1 TruSight One Sequencing Panel Workflow

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Tagment Genomic DNA

This step uses the Nextera transposome to tagment gDNA, which is a process that fragments DNA and then tags the DNA with adapter sequences in a single step.

Consumables

} SPB (Sample Purification Beads)

} ST (Stop Tagment Buffer)

}

TD (Tagment DNA Buffer)

}

TDE1 (Tagment DNA Enzyme)

} gDNA (50 ng per sample)

} PCR-grade water

}

Tris-HCl 10 mM, pH 8.5

}

96-well midi plate (1)

} Microseal 'B' adhesive seals

Preparation

1 Prepare the following consumables.

Item Storage Instructions

gDNA -25°C to -15°C Thaw on ice. Invert to mix, and then centrifuge briefly.

TD

SPB

-25°C to -15°C Thaw on ice. Invert to mix, and then centrifuge briefly.

TDE1 -25°C to -15°C Thaw on ice. Invert to mix, and then centrifuge briefly. Set aside on ice.

2°C to 8°C Let stand for 30 minutes to bring to room temperature. Set aside at room temperature.

ST 15°C to 30°C Check for precipitates. If present, vortex until all particulates are resuspended.

2 Preheat a microheating system with midi plate insert to 58°C.

Procedure

Quantify and Normalize gDNA

1 Quantify gDNA using a fluorometric method, such as QuantiFluor or Qubit.

2 Normalize gDNA in Tris-HCl 10 mM, pH 8.5 to 10 ng/µl.

3 Requantify the normalized gDNA using the same fluorometric quantification method.

4 Dilute the normalized gDNA in Tris-HCl 10 mM, pH 8.5 to a final volume of 10 µl at

5 ng/µl (50 ng total).

Tagment DNA

1 Add the following items in the order listed to each well of a new midi plate.

}

Normalized gDNA (10 µl)

} TD (25 µl)

} TDE1 (5 µl)

}

PCR-grade water (10 µl)

2 Shake at 1800 rpm for 1 minute.

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11

3 Centrifuge at 280 × g for 1 minute.

4 Place on the 58°C microheating system with the lid closed for 10 minutes.

5 Add 15 µl ST to each well.

6 Shake at 1800 rpm for 1 minute.

7 Centrifuge at 280 × g for 1 minute.

8 Incubate at room temperature for 4 minutes.

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Clean Up Tagmented DNA

This step uses SPB (Sample Purification Beads) to purify the tagmented DNA from the

Nextera transposome. The cleanup step removes the Nextera transposome that can otherwise bind to DNA ends and interfere with downstream processes.

Consumables

} RSB (Resuspension Buffer)

} SPB (Sample Purification Beads)

}

Freshly prepared 80% ethanol (EtOH)

}

96-well Hard-Shell 0.3 ml PCR plate

} Microseal 'B' adhesive seals

About Reagents

}

Vortex SPB before each use.

} Vortex SPB frequently to make sure that beads are evenly distributed.

}

Aspirate and dispense SPB slowly due to the viscosity of the solution.

Preparation

1 Prepare the following consumables:

Item

RSB

SPB

Storage

2°C to 8°C

2°C to 8°C

Instructions

Let stand for 30 minutes to bring to room temperature.

Let stand for 30 minutes to bring to room temperature.

2 Prepare fresh 80% EtOH.

Procedure

1 Add 65 µl SPB to each well.

2 Shake at 1800 rpm for 1 minute.

3 Incubate at room temperature for 8 minutes.

4 Centrifuge at 280 × g for 1 minute.

5 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

6 Remove and discard all supernatant from each well.

7 Wash 2 times as follows.

a Add 200 µl fresh 80% EtOH to each well.

b Incubate on the magnetic stand for 30 seconds.

c Remove and discard all supernatant from each well.

8 Use a 20 µl pipette to remove residual EtOH from each well.

9 Air-dry on the magnetic stand for 10 minutes.

10 Remove from the magnetic stand.

11 Add 22.5 µl RSB to each well.

12 Shake at 1800 rpm for 1 minute.

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13 Incubate at room temperature for 2 minutes.

14 Centrifuge at 280 × g for 1 minute.

15 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

16 Transfer 20 µl supernatant to the corresponding well of a new Hard-Shell PCR plate.

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Amplify Tagmented DNA

This step amplifies purified tagmented DNA and adds index adapters using a 10-cycle

PCR program. This PCR step adds Index 1 (i7) adapters, Index 2 (i5) adapters, and sequencing adapters required for cluster amplification.

Consumables

} Index 1 (i7) adapters and orange tube caps

} Index 2 (i5) adapters and white tube caps

}

NLM (Library Amp Mix)

}

1.7 ml microcentrifuge tubes (1 per index adapter tube)

} Microseal 'A' film

} Microseal 'B' adhesive seal

}

[Optional] TruSeq Index Plate Fixture Kit

NOTE

Use Microseal 'A' when sealing the plate before placing on the thermal cycler. Use Microseal

'B' for other steps that require a sealed plate.

Preparation

1 Prepare the following consumables.

Item

Index adapters

(i5 and i7)

NLM

Storage Instructions

-25°C to -15°C Only remove adapters being used. Thaw at room temperature for 20 minutes.

Vortex each tube to mix. Centrifuge briefly using a 1.7 ml

Eppendorf tube.

-25°C to -15°C Thaw on ice.

2 Save the following NLM AMP program on the thermal cycler:

} Choose the preheat lid option and set to 100°C

} 72°C for 3 minutes

}

98°C for 30 seconds

}

10 cycles of:

} 98°C for 10 seconds

} 60°C for 30 seconds

}

72°C for 30 seconds

}

72°C for 5 minutes

} Hold at 10°C

Procedure

1 Arrange Index 1 (i7) adapters in columns 1–12 of the TruSeq Index Plate Fixture.

2 Arrange Index 2 (i5) adapters in rows A–H of the TruSeq Index Plate Fixture.

3 Place the plate on the TruSeq Index Plate Fixture.

TruSight One Sequencing Panel Reference Guide

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Figure 2 TruSeq Index Plate Fixture (96 libraries)

16

A Columns 1–12: Index 1 (i7) adapters (orange caps)

B Rows A–H: Index 2 (i5) adapters (white caps)

C 96-well plate

4 Using a multichannel pipette, add 5 µl of each Index 1 (i7) adapter down each column.

Replace the cap on each i7 adapter tube with a new orange cap.

5 Using a multichannel pipette, add 5 µl of each Index 2 (i5) adapter across each row.

Replace the cap on each i5 adapter tube with a new white cap.

6 Add 20 µl NLM to each well.

7 Shake at 1200 rpm for 1 minute.

8 Centrifuge at 280 × g for 1 minute.

9 Place on the preprogrammed thermal cycler and run the NLM AMP program.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at 2°C to 8°C for up to 2 days. Alternatively, leave on the thermal cycler overnight.

Document # 15046431 v01

Clean Up Amplified DNA

This step uses SPB (Sample Purification Beads) to purify the DNA library and remove unwanted products.

Consumables

} RSB (Resuspension Buffer)

} SPB (Sample Purification Beads)

}

Freshly prepared 80% ethanol (EtOH)

}

96-well Hard-Shell 0.3 ml PCR plate

} 96-well midi plate

} Microseal 'B' adhesive seals

About Reagents

} Vortex SPB before each use.

}

Vortex SPB frequently to make sure that beads are evenly distributed.

}

Aspirate and dispense SPB slowly due to the viscosity of the solution.

Preparation

1 Prepare the following consumables.

Item

RSB

SPB

Storage Instructions

2°C to 8°C Let stand for 30 minutes to bring to room temperature.

2°C to 8°C Let stand for 30 minutes to bring to room temperature.

2 Prepare fresh 80% EtOH.

Procedure

1 Centrifuge at 280 × g for 1 minute.

2 Transfer 50 µl supernatant to the corresponding well of a new midi plate.

3 Add 90 µl SPB to each well.

4 Shake at 1800 rpm for 1 minute.

5 Incubate at room temperature for 10 minutes.

6 Centrifuge at 280 × g for 1 minute.

7 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

8 Remove and discard all supernatant from each well.

9 Wash 2 times as follows.

a Add 200 µl fresh 80% EtOH to each well.

b Incubate on the magnetic stand for 30 seconds.

c Remove and discard all supernatant from each well.

10 Use a 20 µl pipette to remove residual EtOH from each well.

11 Air-dry on the magnetic stand for 10 minutes.

12 Add 27.5 µl RSB to each well.

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13 Shake at 1800 rpm for 1 minute.

14 Incubate at room temperature for 2 minutes.

15 Centrifuge at 280 × g for 1 minute.

16 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

17 Transfer 25 µl supernatant to the corresponding well of a new Hard-Shell PCR plate.

18 Quantify the library using a fluorometric method, such as QuantiFluor or Qubit.

19 [Optional] Run 1 µl of the library on an Agilent Technologies 2100 Bioanalyzer using a

DNA 1000 chip.

Expect a distribution of DNA fragments with a size range from ~300 bp to ~1 kbp.

A sharp peak is not necessary, but most of the fragments must fall within the desired range. Traces can vary from library to library. The following traces show examples of possible distributions, but are not inclusive of successful libraries.

Figure 3 Examples of Post-PCR, Pre-Enriched Library Distribution

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SAFE STOPPING POINT

If you are stopping, seal the plate and store at -25°C to -15°C for up to 14 days.

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Hybridize Probes

This step combines DNA libraries containing unique indexes into a single pool, and then binds targeted regions of the DNA with capture probes.

Consumables

} EHB (Enrichment Hybridization Buffer)

}

TOO (TruSight One Oligos)

}

RSB (Resuspension Buffer)

} 96-well Hard-Shell 0.3 ml PCR plate

} Microseal 'B' adhesive seal

}

[Optional] Amicon Ultra-0.5 centrifugal filter unit (0.5 ml, 30 kDa) (1 per pooled sample)

About Reagents

}

Before using EHB, vortex to resuspend the solution. Make sure that no crystal structures are present. If crystals and cloudiness are observed, vortex until the solution is clear.

Preparation

1 Prepare the following consumables.

Item Storage

TOO -25°C to -15°C

EHB -25°C to -15°C

RSB 2°C to 8°C

Instructions

Thaw at room temperature.

Thaw at room temperature.

Let stand for 30 minutes to bring to room temperature.

2 Save the NRC HYB program on the thermal cycler:

} Choose the preheat lid option and set to 100°C

}

95°C for 10 minutes

}

18 cycles of 1 minute each, starting at 94°C, then decreasing 2°C per cycle

} Hold at 58°C

Pool Libraries

1 Combine 500 ng of each DNA library. Make sure that each library has a unique index.

Library Pool

Complexity

Total DNA Library

Mass (ng)

Library Pool

Complexity

Total DNA Library

Mass (ng)

1-plex

2-plex

3-plex

4-plex

5-plex

6-plex

500

1000

1500

2000

2500

3000

7-plex

8-plex

9-plex

10-plex

11-plex

12-plex

3500

4000

4500

5000

5500

6000

}

If the total volume is > 40 µl, use a vacuum concentrator or Amicon Ultra-0.5

centrifugal filter unit (0.5 ml, 30 kDa) to concentrate the pooled sample to 40 µl.

} If you are using a vacuum concentrator, use a no heat setting and a medium drying rate.

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}

If you are using an Amicon Ultra-0.5 centrifugal filter unit (0.5 ml, 30 kDa), rinsing the device before use is not required. Most volume filters through in

5 minutes. Up to 30 minutes might be needed, depending on the starting volume.

}

If the total volume is < 40 µl, increase the volume to 40 µl with RSB.

Procedure

1 Add the following items in the order listed to each well of a new Hard-Shell PCR plate.

}

DNA library sample or pool (40 µl)

}

EHB (50 µl)

}

TOO (10 µl)

2 Shake at 1200 rpm for 1 minute.

3 Centrifuge at 280 × g for 1 minute.

4 Place on the preprogrammed thermal cycler and run the NRC HYB program. Each well contains 100 µl.

5 Keep at the 58°C holding temperature for at least 90 minutes and up to 24 hours.

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Capture Hybridized Probes

This step uses SMB (Streptavidin Magnetic Beads) to capture probes hybridized to the targeted regions of interest. Two heated washes remove nonspecific binding from the beads.

The enriched library is then eluted from the beads and prepared for a second round of hybridization.

Consumables

} EE1 (Enrichment Elution Buffer 1)

}

ET2 (Elute Target Buffer 2)

}

EWS (Enrichment Wash Solution)

} HP3 (2 N NaOH)

} SMB (Streptavidin Magnetic Beads)

}

96-well Hard-Shell 0.3 ml PCR plate

}

96-well midi plate

} 1.7 ml microcentrifuge tube

} Microseal 'B' adhesive seals

About Reagents

} EWS can be cloudy after reaching room temperature.

}

Vortex EWS before use.

}

Make sure that you use SMB (2 ml tube) and not SPB (15 ml tube) for this procedure.

}

Invert and vortex SMB to mix before use.

} Discard elution premix after use.

Preparation

1 Prepare the following consumables.

Item Storage

EE1 -25°C to -15°C

EWS

HP3

ET2

SMB

-25°C to -15°C

-25°C to -15°C

2°C to 8°C

2°C to 8°C

Instructions

Thaw at room temperature.

Return to storage after use.

Thaw at room temperature.

Return to storage after use.

Thaw at room temperature.

Return to storage after use.

Let stand at room temperature.

Return to storage after use.

Let stand for 30 minutes to bring to room temperature.

Return to storage after use.

2 Preheat a microheating system with midi plate insert to 50°C.

Procedure

First Bind

1 Centrifuge at 280 × g for 1 minute.

2 Transfer all volumes to the corresponding well of a new midi plate.

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3 Add 250 µl SMB to each well.

4 Shake at 1200 rpm for 5 minutes.

5 Incubate at room temperature for 25 minutes.

6 Centrifuge at 280 × g for 1 minute.

7 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

8 Remove and discard all supernatant from each well.

9 Remove from the magnetic stand.

First Wash

1 Wash 2 times as follows.

a Add 200 µl EWS to each well.

b Shake at 1800 rpm for 4 minutes.

c Pipette to resuspend the bead pellet further.

d Place on the 50°C microheating system with the lid closed for 30 minutes.

e Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

f Remove and discard all supernatant from each well.

g Remove from the magnetic stand.

First Elution

1 Create elution premix by combining the following volumes per sample in a 1.7 ml microcentrifuge tube, and then vortex.

} EE1 (28.5 µl)

}

HP3 (1.5 µl)

2 Add 23.5 µl elution premix to each well.

3 Shake at 1800 rpm for 2 minutes.

4 Incubate at room temperature for 2 minutes.

5 Centrifuge at 280 × g for 1 minute.

6 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

7 Transfer 21 µl supernatant to the corresponding well of a new Hard-Shell PCR plate.

8 Add 4 µl ET2 to each well.

9 Shake at 1200 rpm for 1 minute.

10 Centrifuge at 280 × g for 1 minute.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at -25°C to -15°C for up to 7 days.

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Perform Second Hybridization

This step binds targeted regions of the enriched DNA with capture probes a second time.

This second hybridization ensures high specificity of the captured regions.

Consumables

} EHB (Enrichment Hybridization Buffer)

}

TOO (TruSight One Oligos)

}

RSB (Resuspension Buffer)

} Microseal 'B' adhesive seals

About Reagents

}

Before using EHB, vortex to resuspend the solution. Make sure that no crystal structures are present. If crystals and cloudiness are observed, vortex until the solution is clear.

Preparation

1 Prepare the following consumables.

Item Storage

TOO -25°C to -15°C

EHB -25°C to -15°C

RSB 2°C to 8°C

Instructions

Thaw at room temperature.

Thaw at room temperature.

Let stand for 30 minutes to bring to room temperature.

Procedure

1 Add the following reagents in the order listed to each sample well.

} RSB (15 µl)

}

EHB (50 µl)

}

TOO (10 µl)

2 Shake at 1200 rpm for 1 minute.

3 Centrifuge at 280 × g for 1 minute.

4 Place on the preprogrammed thermal cycler and run the NRC HYB program. Each well contains 100 µl.

5 Keep at the 58°C holding temperature for at least 14.5 hours and up to 24 hours.

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Perform Second Capture

This step uses SMB (Streptavidin Magnetic Beads) to capture probes hybridized to the targeted regions of interest. Two heated washes remove nonspecific binding from the beads.

The enriched library is then eluted from the beads and prepared for sequencing.

Consumables

} EE1 (Enrichment Elution Buffer 1)

} ET2 (Elute Target Buffer 2)

}

EWS (Enrichment Wash Solution)

}

HP3 (2 N NaOH)

} SMB (Streptavidin Magnetic Beads)

} 96-well midi plates (2)

}

1.7 ml microcentrifuge tube

}

Microseal 'B' adhesive seals

About Reagents

}

EWS can be cloudy after reaching room temperature.

}

Vortex EWS before use.

} Invert SMB to mix before use.

}

Discard elution premix after use.

Preparation

1 Prepare the following consumables.

Item Storage

EE1 -25°C to -15°C

EWS

HP3

ET2

SMB

-25°C to -15°C

-25°C to -15°C

2°C to 8°C

2°C to 8°C

Instructions

Thaw at room temperature.

Return to storage after use.

Thaw at room temperature.

Return to storage after use.

Thaw at room temperature.

Return to storage after use.

Let stand at room temperature.

Return to storage after use.

Let stand for 30 minutes to bring to room temperature.

Return to storage after use.

2 Preheat a microheating system with midi plate insert to 50°C.

Procedure

Second Bind

1 Centrifuge at 280 × g for 1 minute.

2 Transfer supernatant to the corresponding well of a new midi plate.

3 Add 250 µl SMB to each well.

4 Shake at 1200 rpm for 5 minutes.

5 Incubate at room temperature for 25 minutes.

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25

26

6 Centrifuge at 280 × g for 1 minute.

7 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

8 Remove and discard all supernatant from each well.

9 Remove from the magnetic stand.

Second Wash

1 Wash 2 times as follows.

a Add 200 µl EWS to each well.

b Shake at 1800 rpm for 4 minutes.

c Pipette to resuspend the bead pellet further.

d Place on the 50°C microheating system with the lid closed for 30 minutes.

e Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

f Remove and discard all supernatant from each well.

g Remove from the magnetic stand.

Second Elution

1 Create elution premix by combining the following volumes per sample in a 1.7 ml microcentrifuge tube, and then vortex:

}

EE1 (28.5 µl)

}

HP3 (1.5 µl)

2 Add 23.5 µl elution premix to each well.

3 Shake at 1800 rpm for 2 minutes.

4 Incubate at room temperature for 2 minutes.

5 Centrifuge at 280 × g for 1 minute.

6 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

7 Transfer 21 µl supernatant to the corresponding well of a new midi plate.

8 Add 4 µl ET2 to each well.

9 Shake at 1800 rpm for 1 minute.

10 Centrifuge at 280 × g for 1 minute.

Document # 15046431 v01

Clean Up Captured Library

This step uses SPB (Sample Purification Beads) to purify the captured library before PCR amplification.

Consumables

} RSB (Resuspension Buffer)

} SPB (Sample Purification Beads)

}

Freshly prepared 80% ethanol (EtOH)

}

96-well Hard-Shell 0.3 ml PCR plate

} Microseal 'B' adhesive seals

About Reagents

}

Vortex SPB before each use.

} Vortex SPB frequently to make sure that beads are evenly distributed.

}

Aspirate and dispense SPB slowly due to the viscosity of the solution.

Preparation

1 Prepare the following consumables.

Item

RSB

SPB

Storage Instructions

2°C to 8°C Let stand for 30 minutes to bring to room temperature.

2°C to 8°C Let stand for 30 minutes to bring to room temperature.

2 Prepare fresh 80% EtOH.

Procedure

1 Add 45 µl SPB to each well.

2 Shake at 1800 rpm for 1 minute.

3 Incubate at room temperature for 10 minutes.

4 Centrifuge at 280 × g for 1 minute.

5 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

6 Remove and discard all supernatant from each well.

7 Wash 2 times as follows.

a Add 200 µl fresh 80% EtOH to each well.

b Incubate on the magnetic stand for 30 seconds.

c Remove and discard all supernatant from each well.

8 Use a 20 µl pipette to remove residual EtOH from each well.

9 Air-dry on the magnetic stand for 10 minutes.

10 Add 27.5 µl RSB to each well.

11 Shake at 1800 rpm for 1 minute.

12 Incubate at room temperature for 2 minutes.

13 Centrifuge at 280 × g for 1 minute.

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27

14 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

15 Transfer 25 µl supernatant to the corresponding well of a new Hard-Shell PCR plate.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at -25°C to -15°C for up to 7 days.

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Amplify Enriched Library

This step uses a 10-cycle PCR program to amplify the enriched library.

Consumables

} NEM (Enrichment Amp Mix)

} PPC (PCR Primer Cocktail)

}

Microseal 'A' film

}

Microseal 'B' adhesive seal

NOTE

Use Microseal 'A' when sealing the plate before placing on the thermal cycler. Use Microseal

'B' for other steps that require a sealed plate.

Preparation

1 Prepare the following consumables.

Item

NEM

PPC

Storage

-25°C to -15°C

-25°C to -15°C

Instructions

Thaw on ice.

Thaw on ice.

2 Save the following NEM AMP10 program on the thermal cycler:

}

Choose the preheat lid option and set to 100°C

}

98°C for 30 seconds

} 10 cycles of:

}

98°C for 10 seconds

}

60°C for 30 seconds

}

72°C for 30 seconds

} 72°C for 5 minutes

}

Hold at 10°C

Procedure

1 Add 5 µl PPC to each well.

2 Add 20 µl NEM to each well.

3 Shake at 1200 rpm for 1 minute.

4 Centrifuge at 280 × g for 1 minute.

5 Place on the preprogrammed thermal cycler and run the NEM AMP10 program. Each well contains 50 µl.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at 2°C to 8°C for up to 2 days.

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29

Clean Up Amplified Enriched Library

This step uses SPB (Sample Purification Beads) to purify the enriched library and remove unwanted products.

Consumables

} RSB (Resuspension Buffer)

}

SPB (Sample Purification Beads)

}

Freshly prepared 80% ethanol (EtOH)

} 96-well Hard-Shell 0.3 ml PCR plate

} 96-well midi plate

}

Microseal 'B' adhesive seals

About Reagents

}

Vortex SPB before each use.

}

Vortex SPB frequently to make sure that beads are evenly distributed.

}

Aspirate and dispense SPB slowly due to the viscosity of the solution.

Preparation

1 Prepare the following consumables.

Item

RSB

SPB

Storage Instructions

2°C to 8°C Let stand for 30 minutes to bring to room temperature.

2°C to 8°C Let stand for 30 minutes to bring to room temperature.

2 Prepare fresh 80% EtOH.

Procedure

1 Centrifuge at 280 × g for 1 minute.

2 Transfer 50 µl to the corresponding well of a new midi plate.

3 Add 90 µl SPB to each well.

4 Shake at 1800 rpm for 1 minute.

5 Incubate at room temperature for 10 minutes.

6 Centrifuge at 280 × g for 1 minute.

7 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

8 Remove and discard all supernatant from each well.

9 Wash 2 times as follows.

a Add 200 µl fresh 80% EtOH to each well.

b Incubate on the magnetic stand for 30 seconds.

c Remove and discard all supernatant from each well.

10 Use a 20 µl pipette to remove residual EtOH from each well.

11 Air-dry on the magnetic stand for 10 minutes.

12 Add 32.5 µl RSB to each well.

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Document # 15046431 v01

13 Shake at 1800 rpm for 1 minute.

14 Incubate at room temperature for 2 minutes.

15 Centrifuge at 280 × g for 1 minute.

16 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

17 Transfer 30 µl supernatant to the corresponding well of a new Hard-Shell PCR plate.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at -25°C to -15°C for up to 7 days.

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31

Check Enriched Libraries

Perform the following procedures to check enriched library quality.

Quantify Libraries

Accurately quantify DNA libraries to ensure optimum cluster densities on the flow cell.

Use a fluorometric dsDNA assay to quantify dsDNA libraries. Other techniques can introduce contamination such as RNA and proteins. Use a spectrofluorometer for DNAspecific quantification. Spectrophotometry can also measure RNA and yield values that are too high.

1 Dilute the postenriched library before quantification as follows:

}

For 3-plex to 12-plex enrichments, dilute by adding 2 µl library to 28 µl RSB in a new tube or well. Use this dilution for quantification and quality assessment, as well as sequencing.

} For 1-plex or 2-plex enrichments, a dilution is not needed.

2 Quantify using a fluorometric method.

Use the following formula to convert from ng/µl to nM. Assume a 650 bp library size or calculate based on the average size of the enriched library.

(concentration in ng/µl)

(660 g/mol * average library size) x 10^6 = concentration in nM

For example:

(15 ng/µl)

(660 g/mol * 650) x 10^6 = 34.9 nM

3 Do the following:

}

To assess library quality, proceed to the next step in the workflow.

}

To skip library quality assessment, proceed to cluster generation. For more information, see the system guide for your Illumina sequencing instrument.

Alternatively, you can quantify libraries using qPCR according to the

Sequencing Library qPCR Quantification Guide (part # 11322363)

.

Assess Quality

[Optional]

1 Run 1 µl post enriched library on an Agilent Technologies 2100 Bioanalyzer using a

High Sensitivity DNA chip.

Expect a distribution of DNA fragments with a size range from ~200 bp to ~1 kbp.

Depending on the level of indexing, insert size distribution can vary slightly. However, the sample peak must not be significantly shifted compared to the following example.

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Figure 4 Example Post Enrichment Library Distributions

NOTE

The blue lines indicate the boundaries that were manually created to determine average library size. In the first example, a second minor peak at ~2000 bp is visible. Do not include minor peaks in the determination of average library size. The presence of these larger fragments does not affect downstream clustering and sequencing of your enriched library.

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Appendix A  Supporting Information

Supporting Information

Introduction

Acronyms

Alternative Thermal Cycler Steps for Successful Enrichment

Kit Contents

Consumables and Equipment

Index Sequences

36

37

38

39

42

44

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35

Introduction

The protocols described in this guide assume that you have reviewed the contents of this appendix, confirmed your kit contents, and obtained all the required consumables and equipment.

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Document # 15046431 v01

Acronyms

RSB

SMB

SPB

ST

TD

TDE1

TOO

NEW1

NEW2

NIL

NLA

NLC

NLM

NLT

PPC

Acronym

EE1

EHB

ET2

EWS

HP3

NEC1

NEC2

NEH1

NEH2

NEL

NEM

Definition

Enrichment Elution Buffer 1

Enrichment Hybridization Buffer

Elute Target Buffer 2

Enrichment Wash Solution

2N NaOH

Nextera Enriched Clean Up Plate 1

Nextera Enriched Clean Up Plate 2

Nextera Enrichment Hyb Plate 1

Nextera Enrichment Hyb Plate 2

Nextera Enrichment Library Plate

Enrichment Amp Mix

Nextera Enrichment Wash Plate 1

Nextera Enrichment Wash Plate 2

Nextera Index Library Plate

Nextera Library Amplification Plate

Nextera Library Clean Up Plate

Library Amp Mix

Nextera Library Tagment Plate

PCR Primer Cocktail

Resuspension Buffer

Streptavidin Magnetic Beads

Sample Purification Beads

Stop Tagment Buffer

Tagment DNA Buffer

Tagment DNA Enzyme TDE

TruSight One Oligos

TruSight One Sequencing Panel Reference Guide

37

Alternative Thermal Cycler Steps for

Successful Enrichment

If using a thermal cycler instead of a microheating system during enrichment wash steps, perform the following steps.

1 Save the following WASH program on the thermal cycler:

} Choose the preheat lid option and set to 100°C

}

42°C for 30 minutes

2 Transfer samples and beads resuspended in EWS to a PCR plate (~200 µl).

3 Place on a thermal cycler and run the WASH program.

4 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

5 Remove and discard all supernatant from each well.

6 Remove from the magnetic stand.

7 Add 200 µl EWS to each well.

8 Shake at 1800 rpm for 4 minutes.

9 Pipette to resuspend the bead pellet further.

10 Repeat steps

3 – 6

for a total of 2 washes.

11 Continue to the elution step.

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Kit Contents

Make sure that you have all reagents identified in this section before proceeding to the library preparation procedures. TruSight One kits are available in the following configurations.

Kit Name

TruSight One Sequencing Panel (9

Samples)

TruSight One Sequencing Panel (36

Samples)

Catalog

#

FC-141-

1006

FC-141-

1007

*TG

Catalog #

TG-141-

1006

TG-141-

1007

Number of

Samples

9

36

* Consumables labeled TG include features to help reduce the frequency of revalidation. These consumables are available with a supply agreement and require providing a binding forecast.

For more information, contact your Illumina representative.

Note regarding biomarker patents and other patents unique to specific uses of products.

Some genomic variants, including some nucleic acid sequences, and their use in specific applications might be protected by patents. Customers are advised to determine whether they are required to obtain licenses from the party that owns or controls such patents to use the product for their specific application.

TruSight One Sequencing Panel Contents

(9 Samples) (FC-141-1006, TG-141-1006)

Box 1 - Rapid Capture Reagents

Quantity

1

1

1

2

Reagent

SPB

SMB

ET2

ST

Description

Sample Purification Beads

Streptavidin Magnetic Beads

Elute Target Buffer 2

Stop Tagment Buffer

Storage Temperature

2°C to 8°C

2°C to 8°C

2°C to 8°C

15°C to 30°C

Box 2 - Rapid Capture Reagents, Store at -25°C to -15°C

Quantity

1

1

1

1

1

2

1

1

1

1

Reagent

TDE1

EE1

TD

RSB

NLM

EHB

EWS

HP3

PPC

NEM

Description

Tagment DNA Enzyme

Enrichment Elution Buffer 1

Tagment DNA Buffer

Resuspension Buffer

Nextera Library Amplification Mix

Enrichment Hybridization Buffer

Enrichment Wash Solution

2N NaOH

PCR Primer Cocktail

Nextera Enrichment Amplification Mix

TruSight One Sequencing Panel Reference Guide

39

Box 3 - Indices, Store at -25°C to -15°C

Quantity

2

3

Reagent

i5 Index Primers, E503, E504 i7 Index Primers, N701, N705, N709

Box 4 - Oligos, Store at -25°C to -15°C

Quantity

1

Reagent

TruSight One Content Set

MiSeq Reagent Kit v3, Box 1,

Store at -25°C to -15°C

Quantity

3

3

Component

HT1 (Hybridization Buffer)

Reagent Cartridge

MiSeq Reagent Kit v3, Box 2, Store at 2°C to 8°C

Quantity

3

3

Component

MiSeq Flow Cell

Reagent Cartridge

TruSight One Sequencing Panel Contents

(36 Samples) (FC-141-1007, TG-141-1007)

Box 1 - Rapid Capture Reagents

Quantity

1

1

2

2

Reagent

SPB

SMB

ET2

ST

Description

Sample Purification Beads

Streptavidin Magnetic Beads

Elute Target Buffer 2

Stop Tagment Buffer

Storage Temperature

2°C to 8°C

2°C to 8°C

2°C to 8°C

15°C to 30°C

Box 2 - Rapid Capture Reagents, Store at -25°C to -15°C

Quantity

1

1

2

1

1

2

1

1

2

1

Reagent

TDE1

EE1

TD

RSB

NLM

EHB

EWS

HP3

PPC

NEM

Description

Tagment DNA Enzyme

Enrichment Elution Buffer 1

Tagment DNA Buffer

Resuspension Buffer

Nextera Library Amplification Mix

Enrichment Hybridization Buffer

Enrichment Wash Solution

2N NaOH

PCR Primer Cocktail

Nextera Enrichment Amplification Mix

40

Document # 15046431 v01

Box 3 - Indices, Store at -25°C to -15°C

Quantity

4

12

Reagent

i5 Index Primers, E502 to E505 i7 Index Primers, N701 to N712

Box 4 - Oligos, Store at -25°C to -15°C

Quantity

1

Reagent

TruSight One Content Set

TruSight One Sequencing Panel Reference Guide

41

Consumables and Equipment

Make sure that you have the required user-supplied consumables and equipment before starting the protocol.

The protocol has been optimized and validated using the items listed. Comparable performance is not guaranteed when using alternate consumables and equipment.

Consumables

Consumable

1.7 ml microcentrifuge tubes

20 µl barrier pipette tips

20 µl multichannel pipettes

20 µl single channel pipettes

200 µl barrier pipette tips

200 µl multichannel pipettes

200 µl single channel pipettes

1000 µl barrier pipette tips

1000 µl multichannel pipettes

1000 µl single channel pipettes

Adhesive seal roller

96-well storage plates, round well, 0.8 ml

(midi plate)

Hard-Shell 96-well PCR Plates

Ethanol 200 proof (absolute) for molecular biology (500 ml)

Microseal 'A' film

Microseal 'B' adhesive seals

RNase/DNase-free 8-tube strips and caps

RNase/DNase-free multichannel reagent reservoirs, disposable

Tris-HCl 10 mM, pH 8.5

PCR-grade water

[Optional] Amicon Ultra-0.5 centrifugal filter unit (0.5 ml, 30 kDa)*

[Optional] DNA 1000 Kit

[Optional] High Sensitivity DNA Kit

Supplier

General lab supplier

General lab supplier

General lab supplier

General lab supplier

General lab supplier

General lab supplier

General lab supplier

General lab supplier

General lab supplier

General lab supplier

General lab supplier

Fisher Scientific, part # AB-0859

Bio-Rad, part # HSP-9601

Sigma-Aldrich, part # E7023

Bio-Rad, part # MSA-5001

Bio-Rad, part # MSB-1001

General lab supplier

VWR, part # 89094-658

General lab supplier

General lab supplier

Millipore, part # UFC503008

Agilent Technologies, part # 5067-1504

Agilent Technologies, part # 5067-4626

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Document # 15046431 v01

* Use to concentrate a pooled library. Otherwise, use a vacuum concentrator.

Equipment

Equipment

DNA Engine Multi-Bay Thermal Cycler

See

Thermal Cyclers

on page 43 .

High-Speed Microplate Shaker

Magnetic stand-96

Microcentrifuge

Microheating System-SciGene TruTemp

Heating System

Microplate centrifuge

Midi plate insert for microheating system

Fluorometric quantification with dsDNA binding dye reagents

Vortexer

[Optional] 2100 Bioanalyzer Desktop System

[Optional] TruSeq Index Plate Fixture Kit¹

[Optional] Vacuum concentrator²

Supplier

Bio-Rad, part #  PTC-0240G or

PTC-0220G, with Alpha Unit, part # ALS-1296GC

VWR, catalog # 

• 13500-890 (110 V/120 V) or

• 14216-214 (230 V)

Life Technologies, part # AM10027

General lab supplier

Illumina, catalog #

• SC-60-503 (115 V) or

• SC-60-504 (220 V)

General lab supplier

Illumina, catalog # BD-60-601

General lab supplier

General lab supplier

Agilent Technologies, part # G2940CA

Illumina, catalog # FC-130-1005

General lab supplier

¹ Reusable and recommended for setting up indexed adapters.

² Use to concentrate a pooled library. Alternatively, use Amicon Ultra-0.5 centrifugal filter units.

Thermal Cyclers

The following table lists the recommended settings for the recommended thermal cycler, and other comparable models. If your lab has a thermal cycler that is not listed, validate the thermal cycler before performing the protocol.

Thermal Cycler

Bio-Rad DNA Engine

Tetrad 2

MJ Research DNA

Engine Tetrad

Eppendorf

Mastercycler Pro S

Temp Mode

Calculated

Calculated

Gradient S,

Simulated Tube

Lid Temp

Heated, Constant at 100°C

Heated

Heated

Vessel Type

Polypropylene plates and tubes

Plate

Plate

TruSight One Sequencing Panel Reference Guide

43

Index Sequences

The Illumina dual-index strategy adds 2 8-base indexes, Index 1 (i7) and Index 2 (i5), to each sample.

}

N refers to Nextera

}

E refers to Enrichment

} 7 refers to Index 1 (i7)

}

5 refers to Index 2 (i5)

}

01–12 refers to the Index number

Use the following bases for entry on your sample sheet.

Index 1 (i7)

N701

N702*

N703*

N704*

N705

N706*

N707*

N708*

N709

N710*

N711*

N712*

Sequence

TAAGGCGA

CGTACTAG

AGGCAGAA

TCCTGAGC

GGACTCCT

TAGGCATG

CTCTCTAC

CAGAGAGG

GCTACGCT

CGAGGCTG

AAGAGGCA

GTAGAGGA

Index 2 (i5)

E502*

E503

E504

E505*

Sequence

CTCTCTAT

TATCCTCT

AGAGTAGA

GTAAGGAG

* Only available in the TruSight One Sequencing Panel Kit (36 Samples).

NOTE

The E500 series Index 2 (i5) sequences in the TruSight One kits are identical to S500 series

Index 2 (i5) sequences in other kits. However, the Index 2 (i5) adapters are not interchangeable across kits.

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Document # 15046431 v01

Technical Assistance

For technical assistance, contact Illumina Technical Support.

Table 1 Illumina General Contact Information

Website

www.illumina.com

Email

[email protected]

Table 2 Illumina Customer Support Telephone Numbers

Region

North America

Australia

Austria

Belgium

China

Denmark

Finland

France

Germany

Hong Kong

Ireland

Italy

Contact Number

1.800.809.4566

1.800.775.688

0800.296575

0800.81102

400.635.9898

80882346

0800.918363

0800.911850

0800.180.8994

800960230

1.800.812949

800.874909

Region

Japan

Netherlands

New Zealand

Norway

Singapore

Spain

Sweden

Switzerland

Taiwan

United Kingdom

Other countries

Contact Number

0800.111.5011

0800.0223859

0800.451.650

800.16836

1.800.579.2745

900.812168

020790181

0800.563118

00806651752

0800.917.0041

+44.1799.534000

Safety data sheets (SDSs)

—Available on the Illumina website at support.illumina.com/sds.html

.

Product documentation

—Available for download in PDF from the Illumina website. Go to support.illumina.com, select a product, then select

Documentation & Literature

.

TruSight One Sequencing Panel Reference Guide

45

Illumina

5200 Illumina Way

San Diego, California 92122 U.S.A.

+1.800.809.ILMN (4566)

+1.858.202.4566 (outside North America) [email protected]

www.illumina.com

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