Nextera Rapid Capture Enrichment Reference Guide (15037436 v01)

Nextera Rapid Capture Enrichment Reference Guide (15037436 v01)

Nextera

®

Rapid Capture

Enrichment Reference Guide

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

ILLUMINA PROPRIETARY

Document # 15037436 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).

FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN

MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND

DAMAGE TO OTHER PROPERTY.

ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S)

DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE).

© 2016 Illumina, Inc. All rights reserved.

Illumina, 24sure, BaseSpace, BeadArray, BlueFish, BlueFuse, BlueGnome, cBot, CSPro, CytoChip, DesignStudio,

Epicentre, ForenSeq, Genetic Energy, GenomeStudio, GoldenGate, HiScan, HiSeq, HiSeq X, Infinium, iScan, iSelect,

MiSeq, MiSeqDx, MiSeq FGx, NeoPrep, NextBio, Nextera, NextSeq, Powered by Illumina, SureMDA, TruGenome,

TruSeq, TruSight, Understand Your Genome, UYG, VeraCode, verifi, VeriSeq, the pumpkin orange color, and the streaming bases design are trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/or other countries. All other names, logos, and other trademarks are the property of their respective owners.

ii

Document # 15037436 v01

Revision History

Document

Document # 15037436 v01

15037436 Rev. J

Date

January

2016

June 2015

Description of Change

• Renamed and combined some procedures as needed to improve continuity

• Added reference to the Custom Protocol Selector

• Corrected quantity of ET2 for 6 plex and 9 plex in Box 1 of the

Exome 8 Reaction kit

• Removed step to remove plate from magnetic stand before adding RSB in:

• Clean Up Amplified DNA

• Clean Up Amplified Enriched Library

• Changed SMB mixing to invert tube instead of vortex tube

• Updated Kit Contents:

• Removed individual kit configurations

• Removed box and tube part numbers

• Removed Nextera from NEM and NLM descriptions

• Corrected High Sensitivity DNA Kit part #

• Changed title of this document to Reference Guide

• 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

• Changed BaseSpace resource reference to helpcenter

Nextera Rapid Capture Enrichment Reference Guide iii

iv

Document

15037436 Rev. H

15037436 Rev. G

Date

September

2014

May 2014

Description of Change

• Removed List of Tables

• Updated Additional Resources to remove web navigation instructions and written urls

• Removed use of plate name (eg NLT plate), except for first instance and last instance in each procedure

• Change 'index primer' to 'index adapter' to correspond to reagent labeling

• Added instructions to use new caps on index adapter tubes after use

• Replaced Nextera Rapid Capture Custom Enrichment Kit (96

Samples) (catalog # FC-140-1008), Box 3 (part # 15055302) with part # 15055366

• Added a more Streptavidin Magnetic Beads tube to the following kits to make sure that there are sufficient reagents:

• Nextera Rapid Capture, 8 rxn × 3 plex (Box 1 of 3), part # 15050491

• Nextera Rapid Capture, 8 rxn × 6 plex (Box 1 of 3), part # 15050492

• Nextera Rapid Capture, 8 rxn × 9 plex (Box 1 of 3), part # 15050493

• Corrected slot number for Elute Target Buffer 2 and Stop

Tagment Buffer in Nextera Rapid Capture, 8 rxn × 1 plex (Box 1 of 3), part # 15050019

• Removed mention of Index Adapter Replacement Caps in the

Nextera Rapid Capture Custom Enrichment Kit (288 Samples), catalog # FC-140-1009

• Updated SDS link to support.illumina.com/sds.html

• Replaced the E501 and E502 Index Adapters with E505 and E506

Index Adapters in the exome and expanded exome 12 plex kits, custom 48 and 96 samples kits, and updated the kit Box 3 part numbers

• Added E502 tubes to the exome 8 rxn × 6 plex and 8 rxn × 9 plex kits

• Replaced the E501 Index Adapter with the E517 Index Adapter in the custom 288 samples kit, updated the kit Box 3 part number, and corrected the kit configuration

• Revised the following sections regarding the index adapter replacements:

• Amplify Tagmented DNA

• Kit Contents

• Index Sequences

• Corrected the hyperlink to the Nextera Rapid Capture

Enrichment Low-Plex Pooling Guidelines Tech Note in

Additional Resources

• Changed 'sample' prep to 'library' prep

Document # 15037436 v01

Document

15037436 Rev. F

15037436 Rev. E

15037436 Rev. D

15037436 Rev. C

Date

January

2014

November

2013

August

2013

June 2013

Description of Change

• Added Nextera ® Rapid Capture Exome Enrichment 8 rxn × 3 plex, 8 rxn × 6 plex, and 8 rxn × 9 plex kits. Revised the following sections regarding these kits:

• Protocol Introduction

• Amplify Tagmented DNA

• Kit Contents

• Index Sequences

• Make DNA Stock Plate

• Modified description of existing exome and expanded exome kits to denote the reactions and plexity of each kit

• Changed the following plate names to clarify library vs.

sample:

• NES (Nextera Enrichment Sample Plate) is now NEL

(Nextera Enrichment Library Plate)

• NLS (Nextera Library Sample Plate) is now NIL (Nextera

Index Library Plate)

• Added DNA Input Recommendations for an optional cleanup step followed by requantitation of DNA samples

• Added references to BaseSpace

® for organizing samples, libraries, pools, and runs

• Removed Low-Plex Pooling Guidelines, which are now available in a Nextera Rapid Capture Enrichment Low-Plex

Pooling Guidelines Tech Note

• Moved content from the Sequencing section to Quantify

Libraries and the Nextera Rapid Capture Enrichment support pages on the Illumina website

• Corrected the 8 Samples - Oligos box configuration

• Added guidance on EDTA to DNA Input Recommendations

• Added training and qPCR information to Additional Resources

• Renamed the EUC to EUC and LTF

• Added Nextera Rapid Capture Exome Enrichment (8 Samples)

Kit. Revised the following sections regarding this kit:

• Protocol Introduction

• Amplify Tagmented DNA

• Kit Contents

• Index Sequences

• Make DNA Stock Plate

• Corrected the 96 Samples - Box 2 configuration

• Providing 8 Index 2 (i5) adapters in Box 3 of the Nextera Rapid

Capture Custom Enrichment Kits (288 Samples). Revised the following sections regarding the additional indexes:

• 288 Samples Kit Contents

• Low-Plex Pooling Guidelines

• Index Sequences

• Appended 'Enrichment' to product name

Nextera Rapid Capture Enrichment Reference Guide v

Document

15037436 Rev. B

15037436 Rev. A

Date

May 2013

February

2013

Description of Change

• Added Nextera Rapid Capture Custom Kit information

• Changed protocol for small-size capture panels

• Reorganized Getting Started content and move some topics to the Appendix

• Replaced Best Practices with reference to content on the

Illumina website

• Removed recommendation for a paired-end 76 cycle sequencing run

Initial Release vi

Document # 15037436 v01

Table of Contents

Revision History

Table of Contents

Chapter 1 Overview

Introduction

DNA Input Recommendations

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

Kit Contents

Consumables and Equipment

Index Sequences

DNA Quantification

Technical Assistance

iii

vii

1

2

3

4

5

19

22

23

25

27

28

30

11

13

15

17

8

9

6

7

31

32

33

34

42

45

46

51

Nextera Rapid Capture Enrichment Reference Guide vii

viii

Document # 15037436 v01

  Chapter 1  Overview

Overview

Introduction

DNA Input Recommendations

Additional Resources

2

3

4

Nextera Rapid Capture Enrichment Reference Guide

1

Introduction

This protocol explains how to prepare up to 96 indexed, paired-end libraries, followed by enrichment using exome or custom probe panels and reagents provided in an Illumina

Nextera

®

®

Rapid Capture Exome, Expanded Exome, or Custom Enrichment kit. The libraries are prepared for subsequent cluster generation and DNA sequencing. The goal of this protocol is to fragment and add adapter sequences onto template DNA to generate indexed sequencing libraries that can be carried through enrichment for targeted resequencing applications.

The Nextera Rapid Capture Enrichment protocol offers:

} Excellent data quality with low input of 50 ng

}

Fast and easy preparation of up to 96 enriched libraries in ~1.5 days, including

~5 hours of hands-on time

}

High throughput, automation-friendly procedures

2

Document # 15037436 v01

DNA Input Recommendations

Nextera Rapid Capture Enrichment library preparation uses an enzymatic DNA fragmentation step and thus can be more sensitive to DNA input compared to mechanical fragmentation methods. The ultimate success of enrichment strongly depends on using an accurately quantified amount of input DNA. Therefore, accurate quantification of the gDNA is essential.

Use a fluorometric-based method to quantify input gDNA specific for double-stranded

DNA (dsDNA), such as QuantiFluor or Qubit, and run samples in triplicate for confident measurements.

} 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 assay.

}

Make sure that the starting DNA does not contain more than 1 mM EDTA and is free of organic contaminants, such as phenol and ethanol. For more information, see

DNA

Quantification on page 46

.

} DNA samples can contain substances that interfere with the Nextera tagmentation reaction and result in unexpected library insert sizes. To make sure that conditions are optimal before you begin library preparation, perform an optional sample cleanup, and then requantify the DNA samples.

The Nextera Rapid Capture Enrichment 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, which can affect enrichment performance. A lower mass input of gDNA or low quality gDNA in the tagmentation reaction can generate smaller than expected insert sizes.

Smaller inserts can be lost during subsequent cleanup steps and result in lower diversity.

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

Nextera Rapid Capture Enrichment Reference Guide

3

Additional Resources

Visit the Nextera Rapid Capture Enrichment kit 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

Nextera Rapid Capture

Enrichment Protocol Guide

(document # 15075701)

Nextera Rapid Capture

Enrichment Checklist (document #

15075702)

Nextera Rapid Capture

Enrichment Low-Plex Pooling

Guidelines Tech Note

Sequencing with Nextera Rapid

Capture Enrichment Kits

(document # 15037435)

Sequencing Library qPCR

Quantification Guide

(document # 11322363)

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.

Provides a checklist of the protocol steps.

The checklist is intended for experienced users.

Provides pooling guidelines and dual indexing strategies for

Nextera Rapid Capture Enrichment library preparation.

Provides guidelines for preparing for sequencing when using a Nextera Rapid Capture Enrichment kit.

Describes a qPCR method for quantifying sequencing by synthesis (SBS) libraries generated using the Illumina library prep protocols.

4

Document # 15037436 v01

  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

17

19

22

23

9

11

13

15

25

27

28

30

6

7

8

Nextera Rapid Capture Enrichment Reference Guide

5

Introduction

This section describes the Nextera Rapid Capture Enrichment protocol.

}

Follow the protocol in the order described using the specified parameters.

}

Review Best Practices before proceeding. See

Additional Resources on page 4

for information on how to access Nextera Rapid Capture Best Practices on the Illumina website.

}

Before proceeding, confirm kit contents and make sure that you have the required equipment and consumables. See

Consumables and Equipment on page 42

.

}

Include a common index in each column. A common index facilitates pipetting operations when dispensing index adapters and pooling indexed libraries. For more information, see the Nextera Rapid Capture Enrichment Low-Plex Pooling Guidelines Tech

Note.

}

Nextera Rapid Capture kits support the following reactions and plexity. For more information on the kit configurations, see

Kit Contents on page 34

.

Samples Plexity

8

24

48

72

24

48

96

288

Enrichment

Reactions

8

8

2

4

8

8

8

24

1

3

6

9

12

12

12

12

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 Nextera Rapid Capture Enrichment support page for more information.

6

Document # 15037436 v01

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.

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.

Nextera Rapid Capture Enrichment Reference Guide

7

Library Prep Workflow

The following diagram illustrates the workflow using a Nextera Rapid Capture Enrichment kit. Safe stopping points are marked between steps.

Figure 1 Nextera Rapid Capture Enrichment Workflow

8

Document # 15037436 v01

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)

} ST2 (Stop Tagment Buffer 2)

}

TD (Tagment DNA Buffer)

}

TDE1 (Tagment DNA Enzyme)

} gDNA (50 ng per sample)

} 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. Gently invert the thawed tubes 3–5 times, and then centrifuge briefly.

TD -25°C to -15°C Thaw on ice. Gently invert the thawed tubes 3–5 times, and then centrifuge briefly.

TDE1 -25°C to -15°C Thaw on ice. Gently invert the thawed tubes 3–5 times, and then centrifuge briefly. Set aside on ice.

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

ST

Keep at room temperature for later use in the protocol.

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.

3 Use IEM to determine what index adapters to use or BaseSpace Prep tab to organize your samples, libraries, pools, and run.

4 Label a new midi plate NLT with a marker.

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).

Nextera Rapid Capture Enrichment Reference Guide

9

Tagment DNA

1 Add the following items in the order listed to each well of the NLT plate.

Item

Normalized gDNA

TD

TDE1

Volume (µl)

10

25

15

2 Shake at 1800 rpm for 1 minute.

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.

10

Document # 15037436 v01

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 Storage

RSB -25°C to -15°C

SPB 2°C to 8°C

Instructions

Thaw at room temperature.

RSB can be stored at 2°C to 8°C after the initial thaw.

Let stand for 30 minutes to bring to room temperature.

2 Prepare fresh 80% ethanol from absolute ethanol.

3 Label a new Hard-Shell PCR plate NLA with a marker.

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 (2–5 minutes).

6 Remove and discard all supernatant from each well.

7 Wash 2 times as follows.

a Add 200 μl freshly prepared 80% EtOH to each well.

b Incubate on the magnetic stand for 30 seconds.

c Remove and discard all supernatant from each well.

8 Using a 20 μl pipette, remove residual 80% EtOH from each well.

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

10 Remove from the magnetic stand.

Nextera Rapid Capture Enrichment Reference Guide

11

11 Add 22.5 μl RSB to each well.

12 Shake at 1800 rpm for 1 minute.

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 (2–5 minutes).

16 Transfer 20 μl supernatant to the corresponding well of the NLA plate.

17 [Optional] Run 1 μl of the reaction on an Agilent Technologies 2100 Bioanalyzer using a High Sensitivity DNA chip. You can expect to see a broad distribution of DNA fragments with a size range from ~150 bp to ~1 kbp.

Figure 2 Example Post-Tagmentation Library Distribution

12

Document # 15037436 v01

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.

Nextera Rapid Capture Enrichment Reference Guide

13

Figure 3 TruSeq Index Plate Fixture (96 libraries)

14

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 # 15037436 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% ethanol from absolute ethanol.

3 Label plates with a marker as follows.

} NIL - Hard-Shell PCR

}

NLC - midi

Procedure

1 Centrifuge at 280 × g for 1 minute.

2 Transfer 50 μl supernatant to the corresponding well of the NLC 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 (2–5 minutes).

8 Remove and discard all supernatant from each well.

9 Wash 2 times as follows.

a Add 200 μl freshly prepared 80% EtOH to each well.

b Incubate on the magnetic stand for 30 seconds.

c Remove and discard all supernatant from each well.

Nextera Rapid Capture Enrichment Reference Guide

15

10 Using a 20 μl pipette, remove residual 80% EtOH from each well.

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

12 Add 27 μl RSB to each well.

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 the NIL plate.

18 Quantify the library using a fluorometric method, such as QuantiFluor or Qubit. For an example protocol using the Promega QuantiFluor method, see

DNA Quantification on page 46 .

NOTE

Inaccurate quantification and pooling can result in a higher representation of some samples compared to others in the same pool.

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

DNA 1000 chip. You can expect to see a distribution of DNA fragments with a size range from ~150 bp to ~1 kbp.

Figure 4 Example of Post-PCR, Pre-Enriched Library Distribution

16

NOTE

The sample peak must not be significantly shifted compared to the example shown in

Figure 4 , although traces can differ depending on sample quality. A larger peak

distribution (> 350 bp) can indicate > 50 ng gDNA input going into tagmentation, which can result in lower on-target reads. Conversely, a smaller sample peak distribution

(< 225 bp) can indicate < 50 ng gDNA or low quality gDNA, which can result in reduced library diversity or elevated duplicates.

SAFE STOPPING POINT

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

Document # 15037436 v01

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)

} One of the following, depending on the kit you are using:

}

CEX (Coding Exome Oligos)

}

EEX (Expanded Exome Oligos)

} RCO (Rapid Capture Oligos)

} RSB (Resuspension Buffer)

}

96-well Hard-Shell 0.3 ml PCR plate (1)

}

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

One of the following, depending on the kit you are using:

• CEX

• EEX

• RCO

EHB

RSB

Storage Instructions

-25°C to -15°C Thaw at room temperature.

-25°C to -15°C Thaw at room temperature.

2°C to 8°C 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

NOTE

Incubate the plate at the 58°C holding temperature for at least 90 minutes and up to a maximum of 24 hours.

3 Label a new Hard-Shell PCR plate NEH1 with a marker.

Nextera Rapid Capture Enrichment Reference Guide

17

Pool Libraries

NOTE

Pooling is not performed with the Nextera Rapid Capture Enrichment Kit (8 rxn × 1 plex).

For custom enrichment projects, avoid low levels of sample multiplexing (< 6-plex) for a small capture target size (< 2 Mb). The final enriched sample yield can be insufficient for clustering and subsequent sequencing. For more information, see the Nextera Rapid Capture

Low-Plex Pooling Guidelines Tech Note.

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.

}

If you are using an Amicon Ultra-0.5 centrifugal filter unit (0.5 ml, 30 kDa), you do not need to rinse the device before use. Most of the volume filters through in

5 minutes. Larger starting volumes can take up to 30 minutes to filter.

} 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 the NEH1 plate.

Item

DNA library sample or pool

EHB

CEX, EEX, or RCO

Volume (µl)

40

50

10

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.

18

Document # 15037436 v01

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.

3 Label plates with a marker as follows.

} NEW1 - midi

} NEH2 - Hard-Shell PCR

Nextera Rapid Capture Enrichment Reference Guide

19

Procedure

First Bind

1 Centrifuge at 280 × g for 1 minute.

2 Transfer all (~100 μl) to the corresponding well of the NEW1 plate.

NOTE

If you see a greater than 15% sample loss, do not proceed with the protocol. Poor sealing or insufficient heating of the lid can cause sample loss.

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 (2–5 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 in a 1.7 ml microcentrifuge tube, and then vortex to mix.

}

EE1 (28.5 μl)

} HP3 (1.5 μl)

2 Add 23 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 the NEH2 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.

20

Document # 15037436 v01

SAFE STOPPING POINT

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

Nextera Rapid Capture Enrichment Reference Guide

21

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)

}

One of the following, depending on the kit you are using:

}

CEX (Coding Exome Oligos)

} EEX (Expanded Exome Oligos)

} RCO (Rapid Capture 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

One of the following, depending on the kit you are using:

• CEX

• EEX

• RCO

EHB

RSB

Storage Instructions

-25°C to -15°C Thaw at room temperature.

-25°C to -15°C Thaw at room temperature.

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

Procedure

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

Reagent

RSB

EHB

CEX, EEX, or RCO

Volume (µl)

15

50

10

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.

22

Document # 15037436 v01

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.

3 Label plates with a marker as follows.

}

NEW2 - midi

}

NEC1 - midi

Procedure

Second Bind

1 Centrifuge at 280 × g for 1 minute.

Nextera Rapid Capture Enrichment Reference Guide

23

24

2 Transfer all (~100 μl) supernatant to the corresponding well of the NEW2 plate.

NOTE

If you see a greater than 15% sample loss, do not proceed with the protocol. Poor sealing or insufficient heating of the lid can cause sample loss.

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 (2–5 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 in a 1.7 ml microcentrifuge tube, and then vortex to mix.

}

EE1 (28.5 μl)

}

HP3 (1.5 μl)

2 Add 23 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 the NEC1 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 # 15037436 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% ethanol from absolute ethanol.

3 Label a new Hard-Shell PCR plate NEA with a marker.

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 Remove from the magnetic stand.

11 Add 27.5 μl RSB to each well.

12 Shake at 1800 rpm for 1 minute.

Nextera Rapid Capture Enrichment Reference Guide

25

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 25 μl supernatant to the corresponding well of the NEA plate.

SAFE STOPPING POINT

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

26

Document # 15037436 v01

Amplify Enriched Library

This step uses a 10-cycle or 12-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

EPM

PPC

Storage

-25°C to -15°C

-25°C to -15°C

Instructions

Thaw on ice.

Thaw at room temperature.

2 Determine the appropriate number of PCR cycles:

}

For capture target sizes > 2 Mb, perform 10 cycle

}

For capture target sizes < 2 Mb, perform 12 cycles

3 Save the following AMP10 or AMP12 program on the thermal cycler:

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

}

98°C for 30 seconds

}

10 or 12 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 AMP10 or AMP12 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.

Nextera Rapid Capture Enrichment Reference Guide

27

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% ethanol from absolute ethanol.

3 Label plates with a marker as follows.

}

NEC2 - midi

}

NEL - Hard-Shell PCR

Procedure

1 Centrifuge at 280 × g for 1 minute.

2 Transfer 50 μl to the corresponding well of the NEC2 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 (2–5 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.

28

Document # 15037436 v01

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

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

12 Remove from the magnetic stand.

13 Add 32 μl RSB to each well.

14 Shake at 1800 rpm for 1 minute.

15 Incubate at room temperature for 2 minutes.

16 Centrifuge at 280 × g for 1 minute.

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

18 Transfer 30 μl supernatant to the corresponding well of the NEL plate.

SAFE STOPPING POINT

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

Nextera Rapid Capture Enrichment Reference Guide

29

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.

1 Quantify the enriched library using a fluorometric method.

For an example protocol using the Promega QuantiFluor method, see

DNA

Quantification on page 46

.

2 Use the following formula to convert from ng/μl to nM. Assume a 400 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 * 400) x 10^6 = 57 nM

Alternatively, you can quantify libraries using qPCR according to the Sequencing Library

qPCR Quantification Guide (part # 11322363).

Assess Quality

[Optional]

1 If the library concentration is higher than the supported quantitative range for the High

Sensitivity DNA chip, dilute the library 1:10 with RSB.

2 Run 1 μl of 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.

Figure 5 Example Post Enrichment (12-plex Enrichment) Library Distribution

30

NOTE

A second minor peak at ~100 bp can be present and likely corresponds to residual singlestranded probes in the sample. The presence of these residual probes does not affect downstream clustering and sequencing of your enriched sample.

Document # 15037436 v01

Appendix A  Supporting Information

Supporting Information

Introduction

Acronyms

Kit Contents

Consumables and Equipment

Index Sequences

DNA Quantification

32

33

34

42

45

46

Nextera Rapid Capture Enrichment Reference Guide

31

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.

32

Document # 15037436 v01

Acronyms

RSB

SMB

SPB

ST

NLM

NLT

PPC

RCO

TD

TDE1

NEH2

NEL

NEM

NEW1

NEW2

NIL

NLA

NLC

Acronym

CEX

EE1

EEX

EHB

ET2

EWS

HP3

NEA

NEC1

NEC2

NEH1

Definition

Coding Exome Oligos

Enrichment Elution Buffer 1

Expanded Exome Oligos

Enrichment Hybridization Buffer

Elute Target Buffer 2

Enrichment Wash Solution

2N NaOH

Nextera Enrichment Amplification Plate

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

Rapid Capture Oligos

Resuspension Buffer

Streptavidin Magnetic Beads

Sample Purification Beads

Stop Tagment Buffer

Tagment DNA Buffer

Tagment DNA Enzyme TDE

Nextera Rapid Capture Enrichment Reference Guide

33

Kit Contents

Check to make sure that you have all the reagents identified in this section before proceeding to the library preparation and enrichment procedures. The following Nextera

Rapid Capture Enrichment kits are available.

Table 1 Nextera Rapid Capture Exome Enrichment Kits

Kit Name

Nextera Rapid Capture Exome Enrichment Kit (8 rxn × 1 plex)

Nextera Rapid Capture Exome Enrichment Kit (8 rxn × 3 plex)

Nextera Rapid Capture Exome Enrichment Kit (8 rxn × 6 plex)

Nextera Rapid Capture Exome Enrichment Kit (8 rxn × 9 plex)

Nextera Rapid Capture Exome Enrichment Kit (2 rxn × 12 plex)

Nextera Rapid Capture Exome Enrichment Kit (4 rxn × 12 plex)

Nextera Rapid Capture Exome Enrichment Kit (8 rxn × 12 plex)

Catalog #

FC-140-1000

FC-140-1083

FC-140-1086

FC-140-1089

FC-140-1001

FC-140-1002

FC-140-1003

Table 2 Nextera Rapid Capture Expanded Exome Enrichment Kits

Kit Name

Nextera Rapid Capture Expanded Exome Enrichment Kit (2 rxn × 12 plex)

Nextera Rapid Capture Expanded Exome Enrichment Kit (4 rxn × 12 plex)

Nextera Rapid Capture Expanded Exome Enrichment Kit (8 rxn × 12 plex)

Catalog #

FC-140-1004

FC-140-1005

FC-140-1006

Table 3 Nextera Rapid Capture Custom Enrichment Kits

Kit Name

Nextera Rapid Capture Custom Enrichment Kit (48 Samples)

Nextera Rapid Capture Custom Enrichment Kit (96 Samples)

Nextera Rapid Capture Custom Enrichment Kit (288 Samples)

Enrichment

Reactions

4

8

24

Catalog #

FC-140-1007

FC-140-1008

FC-140-1009

Box Configurations

This section details the contents of each Nextera Rapid Capture Enrichment box.

Nextera Rapid Capture Exome Enrichment Kits (8 rxn)

Each kit contains 3 boxes of reagents, 1 box of oligos, and 1 box of index replacement caps.

Box 1, Store as specified

Quantity per 8 rxn kit

1 plex 3 plex 6 plex

1

1

1

4

1

4

1

1

2

4

1

1

9 plex

1

1

2

4

Reagent

SPB

SMB

ET2

ST

Description

Sample Purification Beads

Streptavidin Magnetic Beads

Elute Target Buffer 2

Stop Tagment Buffer

34

Document # 15037436 v01

Box 2, Store at -25°C to -15°C

Quantity per 8 rxn kit

1

1

4

1

2

1

1

2

1

1 plex 3 plex 6 plex

1 1 1

1

1

1

2

1

1

1

2

1

1

2

2

1

1

3

1

1

3

1

1

3

3

2

1

2

1

3

9 plex

1

Reagent

RSB

EWS

TDE1

EE1

TD

NLM

EHB

HP3

PPC

NEM

Description

Resuspension Buffer

Enrichment Wash Solution

Tagment DNA Enzyme

Enrichment Elution Buffer 1

Tagment DNA Buffer

Library Amp Mix

Enrichment Hybridization Buffer

2N NaOH

PCR Primer Cocktail

Enrichment Amp Mix

Box 3, Store at -25°C to -15°C

Quantity per 8 rxn kit

1

1 plex

1

1

1

1

3 plex

1

1

1

1

1

1

1

6 plex 9 plex

2 3

1

1

1

1

1

1

1

1

1

Reagent

E502

N701

N702

N703

N704

N705

N707

N710

N711

N712

Description

E502 Index Adapter

N701 Index Adapter

N702 Index Adapter

N703 Index Adapter

N704 Index Adapter

N705 Index Adapter

N707 Index Adapter

N710 Index Adapter

N711 Index Adapter

N712 Index Adapter

8 Reaction - Oligos, Store at -25°C to -15°C

Quantity

4

Reagent

CEX

Description

Coding Exome Oligos

Index Adapter Replacement Caps, Store at 15°C to 30°C

Description

i7 Index Tube Caps, Orange i5 Index Tube Caps, White

Nextera Rapid Capture Exome and Expanded Exome Enrichment Kits

(2 rxn x 12 plex)

These kits contain enough reagents to support 24 samples in 2 x 12-plex enrichment reactions. Each kit contains 4 boxes of reagents and 1 box of index replacement caps. The

Exome and Expanded Exome Enrichment kits differ in the oligo contents of Box 4.

Nextera Rapid Capture Enrichment Reference Guide

35

36

Box 1, Store as specified

Reagent

SPB

SMB

ET2

ST

Description

Sample Purification Beads

Streptavidin Magnetic Beads

Elute Target Buffer 2

Stop Tagment Buffer

Box 2, Store at -25°C to -15°C

Reagent

RSB

TDE1

TD

EWS

NLM

EHB

HP3

PPC

NEM

EE1

Description

Resuspension Buffer

Tagment DNA Enzyme

Tagment DNA Buffer

Enrichment Wash Solution

Library Amp Mix

Enrichment Hybridization Buffer

2N NaOH

PCR Primer Cocktail

Enrichment Amp Mix

Enrichment Elution Buffer 1

Storage Temperature

2°C to 8°C

2°C to 8°C

2°C to 8°C

15°C to 30°C

Box 3, Store at -25°C to -15°C

Reagent

N707

N708

N709

N710

N711

N712

E505

E506

N701

N702

N703

N704

N705

N706

Description

E505 Index Adapter

E506 Index Adapter

N701 Index Adapter

N702 Index Adapter

N703 Index Adapter

N704 Index Adapter

N705 Index Adapter

N706 Index Adapter

N707 Index Adapter

N708 Index Adapter

N709 Index Adapter

N710 Index Adapter

N711 Index Adapter

N712 Index Adapter

Box 4, Store at -25°C to -15°C

You receive 1 of the following, depending on the kit: Exome or Expanded Exome.

Reagent

CEX

Description

Coding Exome Oligos

Reagent

EEX

Description

Expanded Exome Oligos

Document # 15037436 v01

Index Adapter Replacement Caps, Store at 15°C to 30°C

Description

i7 Index Tube Caps, Orange i5 Index Tube Caps, White

Nextera Rapid Capture - Exome, Expanded Exome, and Custom

Enrichment (4 rxn × 12 plex/48 Samples)

These kits contain enough reagents to support 48 samples in 4 x 12-plex enrichment reactions. Each kit contains 4 boxes of reagents and 1 box of index replacement caps. The kits differ in the oligo contents of Box 4.

Box 1, Store as specified

Quantity Reagent

1

1

2

2

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, Store at -25°C to -15°C

Reagent

RSB

TDE1

EWS

TD

NLM

EHB

HP3

PPC

NEM

EE1

Description

Resuspension Buffer

Tagment DNA Enzyme

Enrichment Wash Solution

Tagment DNA Buffer

Library Amp Mix

Enrichment Hybridization Buffer

2N NaOH

PCR Primer Cocktail

Enrichment Amp Mix

Enrichment Elution Buffer 1

Nextera Rapid Capture Enrichment Reference Guide

37

38

Box 3, Store at -25°C to -15°C

Reagent

N707

N708

N709

N710

N711

N712

E505

E506

N701

N702

N703

N704

N705

N706

Description

E505 Index Adapter

E506 Index Adapter

N701 Index Adapter

N702 Index Adapter

N703 Index Adapter

N704 Index Adapter

N705 Index Adapter

N706 Index Adapter

N707 Index Adapter

N708 Index Adapter

N709 Index Adapter

N710 Index Adapter

N711 Index Adapter

N712 Index Adapter

Box 4, Store at -25°C to -15°C

You receive 1 of the following, depending on the kit: Exome, Expanded Exome, or Custom.

Quantity

2

Reagent

CEX

Description

Coding Exome Oligos

Quantity

2

Reagent

EEX

Description

Expanded Exome Oligos

Quantity

1

Reagent

RCO

Description

Rapid Capture Oligos

The lot number for the custom kit box is specific to each customer order. The box contains

20, 120, or 240 slots, depending on the pulldowns that were ordered with your custom kit.

The number of occupied slots depends on the number of pulldowns. Each tube is labeled with the Project ID.

Index Adapter Replacement Caps, Store at 15°C to 30°C

Description

i7 Index Tube Caps, Orange i5 Index Tube Caps, White

Nextera Rapid Capture - Exome, Expanded Exome, and Custom

Enrichment (8 rxn x 12 plex/96 Samples)

These kits contain enough reagents to support 96 samples in 8 x 12-plex enrichment reactions. Each kit contains 4 boxes of reagents and 1 box of index replacement caps. The kits differ in the oligo contents of Box 4.

Document # 15037436 v01

Box 1, Store as specified

Quantity Reagent

1

1

4

4

SPB

SMB

ET2

ST

Description

Sample Purification Beads

Streptavidin Magnetic Beads

Elute Target Buffer 2

Stop Tagment Buffer

Box 2, Store at -25°C to -15°C

Quantity

1

1

4

2

4

2

1

1

2

1

2

1

Reagent

RSB

EWS

TDE1

TD

NLM

EHB

HP3

PPC

NEM

EWS

NEM

EE1

Description

Resuspension Buffer

Enrichment Wash Solution

Tagment DNA Enzyme

Tagment DNA Buffer

Library Amp Mix

Enrichment Hybridization Buffer

2N NaOH

PCR Primer Cocktail

Enrichment Amp Mix

Enrichment Wash Solution

Enrichment Amp Mix

Enrichment Elution Buffer 1

Box 3, Store at -25°C to -15°C

Quantity

1

1

1

1

1

1

1

1

1

1

1

1

2

2

Reagent

N707

N708

N709

N710

N711

N712

E505

E506

N701

N702

N703

N704

N705

N706

Description

E505 Index Adapter

E506 Index Adapter

N701 Index Adapter

N702 Index Adapter

N703 Index Adapter

N704 Index Adapter

N705 Index Adapter

N706 Index Adapter

N707 Index Adapter

N708 Index Adapter

N709 Index Adapter

N710 Index Adapter

N711 Index Adapter

N712 Index Adapter

Storage

Temperature

2°C to 8°C

2°C to 8°C

2°C to 8°C

15°C to 30°C

Nextera Rapid Capture Enrichment Reference Guide

39

40

Box 4, Store at -25°C to -15°C

You receive 1 of the following, depending on the kit: Exome, Expanded Exome, or Custom.

Quantity

4

Reagent

CEX

Description

Coding Exome Oligos

Quantity

4

Reagent

EEX

Description

Expanded Exome Oligos

Quantity

2

Reagent

RCO

Description

Rapid Capture Oligos

The lot number for the custom kit box is specific to each customer order. The box contains

20, 120, or 240 slots, depending on the pulldowns that were ordered with your custom kit.

The number of occupied slots depends on the number of pulldowns. Each tube is labeled with the Project ID.

Index Adapter Replacement Caps, Store at 15°C to 30°C

Description

i7 Index Tube Caps, Orange i5 Index Tube Caps, White

Nextera Rapid Capture Custom Enrichment Kits (288 Samples)

The Nextera Rapid Capture Custom Enrichment (288 Samples) Kit contains enough reagents to support 288 samples in 24 x 12-plex enrichment reactions. Each kit contains 9 boxes of reagents and an index replacement caps box.

Box 1, 2, and Index Adapter Replacement Caps

The Nextera Rapid Capture Custom Enrichment 288 Samples Kit contains 3 each of the

Nextera Rapid Capture Custom Enrichment 96 Samples Kit boxes 1 and 2 and an Index

Adapter Replacement Caps Box. For the contents of these boxes, see

Nextera Rapid Capture -

Exome, Expanded Exome, and Custom Enrichment (8 rxn x 12 plex/96 Samples) on page 38 .

Document # 15037436 v01

Box 3, Store at -25°C to -15°C

The Nextera Rapid Capture Custom Enrichment 288 Samples Kit contains 2 of these boxes.

E508

N701

N702

N703

N704

N705

N706

N707

Reagent

E517

E502

E503

E504

E505

E506

E507

N708

N709

N710

N711

N712

Description

E517 Index Adapter

E502 Index Adapter

E503 Index Adapter

E504 Index Adapter

E505 Index Adapter

E506 Index Adapter

E507 Index Adapter

E508 Index Adapter

N701 Index Adapter

N702 Index Adapter

N703 Index Adapter

N704 Index Adapter

N705 Index Adapter

N706 Index Adapter

N707 Index Adapter

N708 Index Adapter

N709 Index Adapter

N710 Index Adapter

N711 Index Adapter

N712 Index Adapter

Box 4, Store at -25°C to -15°C

Quantity

6

Reagent

RCO

Description

Rapid Capture Oligos

The lot number for the custom kit box is specific to each customer order. The box contains

20, 120, or 240 slots, depending on the pulldowns that were ordered with your custom kit.

The number of occupied slots depends on the number of pulldowns. Each tube is labeled with the Project ID.

Nextera Rapid Capture Enrichment 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 flat clear bottom black micro plates

Note: Used when quantifying samples with a SpectraMax M5 Micro Plate Reader.

96-well storage plates, round well, 0.8 ml

(midi plate)

Hard-Shell 96-well PCR Plates

Aluminum foil

Conical centrifuge tubes (15 ml or 50 ml)

Distilled water

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

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

Corning, catalog # 3904

Fisher Scientific, catalog # AB-0859

Bio-Rad, catalog # HSP-9601

General lab supplier

General lab supplier

General lab supplier

Sigma-Aldrich, product # E7023

Bio-Rad, catalog # MSA-5001

Bio-Rad, part # MSB-1001

General lab supplier

VWR, catalog # 89094-658

42

Document # 15037436 v01

Consumable

Tris-HCl 10 mM, pH 8.5

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

Note: Use to concentrate a pooled library.

Otherwise, use a vacuum concentrator.

[Optional] DNA 1000 Kit

[Optional] High Sensitivity DNA Kit

Supplier

General lab supplier

Millipore, catalog # UFC503008

Agilent Technologies, catalog # 5067-1504

Agilent Technologies, catalog # 5067-4626

Equipment

Equipment

DNA Engine Multi-Bay Thermal Cycler

See

Thermal Cyclers on page 44 .

High-Speed Microplate Shaker

Magnetic stand-96

Microcentrifuge

Microheating System-SciGene TruTemp

Heating System

Supplier

• Bio-Rad, catalog #  PTC-0240G or

• PTC-0220G, with Alpha Unit, catalog

# ALS-1296GC

VWR, catalog # 

• 13500-890 (110 V/120 V) or

• 14216-214 (230 V)

Life Technologies, catalog # 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

Promega, catalog # E2670

Microplate centrifuge

Midi plate insert for microheating system

QuantiFluor dsDNA System or similar fluorometric-based DNA quantification system

SpectraMax M5 Multi-Mode Micro Plate

Reader or similar fluorometric-based DNA quantification system

Vortexer

[Optional] 2100 Bioanalyzer Desktop System

[Optional] TruSeq Index Plate Fixture Kit

Note: Recommended for setting up indexed adapters. This part is reusable.

[Optional] Vacuum concentrator

Note: Use to concentrate a pooled library.

Otherwise, use Amicon Ultra-0.5 centrifugal filter units.

Molecular Devices, part # M5

General lab supplier

Agilent Technologies, catalog # G2940CA

Illumina, catalog # FC-130-1005

General lab supplier

Nextera Rapid Capture Enrichment Reference Guide

43

Thermal Cyclers

Use the following recommended settings for selected thermal cycler models. Before performing library prep, validate any thermal cyclers not listed.

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

44

Document # 15037436 v01

Index Sequences

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

There are 12 different Index 1 (i7) adapters (eg, N705) and up to 8 different Index 2 (i5) adapters (eg, E505), depending on the kit you are using. In the Index adapter name:

} 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

NOTE

See

Kit Contents on page 34

to determine which indexes are provided in your Nextera Rapid

Capture Enrichment kit.

Use the following bases for entry on your sample sheet.

Table 4 Index Adapter Sequences

Index 1 (i7) Sequence

N701

N702

N703

N704

N705

N706

N707

N708

N709

N710

N711

N712

TAAGGCGA

CGTACTAG

AGGCAGAA

TCCTGAGC

GGACTCCT

TAGGCATG

CTCTCTAC

CAGAGAGG

GCTACGCT

CGAGGCTG

AAGAGGCA

GTAGAGGA

Index 2 (i5)

E502

E503

E504

E505

E506

E507

E508

E517

Sequence

CTCTCTAT

TATCCTCT

AGAGTAGA

GTAAGGAG

ACTGCATA

AAGGAGTA

CTAAGCCT

GCGTAAGA

Nextera Rapid Capture Enrichment Reference Guide

45

DNA Quantification

Perform the QuantiFluor dsDNA assay to quantify dsDNA samples. The assay can quantify small DNA volumes and measure DNA directly. Other techniques can pick up contaminates, such as RNA and proteins. Use a spectrofluorometer for DNA-specific quantification. Spectrophotometry can also measure RNA and yield values that are too high.

Consumables

} 1X TE

}

96-well flat clear bottom black microplates (2)

}

96-well midi plates (2)

} Aluminum foil

} Conical centrifuge tube (15 ml or 50 ml)

}

Lambda DNA

}

Microseal 'B' adhesive seals

} QuantiFluor dsDNA dye

} RNase/DNase-free Reagent Reservoir

About Reagents

} QuantiFluor dsDNA dye often crystallizes at room temperature. Make sure that the dye is thawed and liquid.

Preparation

1 Remove the QuantiFluor dsDNA dye from to 2°C to 8°C and let stand at room temperature for 60 minutes in a light-impermeable container.

Procedure

Make Lambda DNA Stock Plate

1 Dilute lambda DNA in well A1 of a new midi plate to 1 ng/μl in a final volume of

300 μl. Pipette to mix.

}

Use the following formula to calculate the amount of lambda DNA to add to A1:

(300 μl) X (1 ng/μl)

(stock Lambda DNA concentration)

= μl of stock Lambda DNA to add to

A1

}

Dilute DNA in well A1 using the following formula:

(300 μl) - (μl of stock Lambda DNA in well A1) = μl of 1X TE to add to A1

2 Add 150 μl 1X TE to wells B, C, D, E, F, G, and H of column 1.

46

Document # 15037436 v01

Figure 6 Dilution of Stock Lambda DNA Standard

3 Transfer 150 μl lambda DNA from well A1 to well B1. Pipette to mix.

4 Transfer 150 μl from well B1 to well C1. Pipette to mix.

5 Repeat the transfer for wells D1, E1, F1, and G1, changing tips each time. Well H1 serves as the blank 0 ng/μl Lambda DNA.

Table 5 Concentrations of Lambda DNA

Row-Column

A1

B1

Concentration (ng/µl)

1

0.5

C1

D1

E1

F1

G1

H1

0.25

0.125

0.0625

0.03125

0.015625

0

Final Volume in Well (µl)

150

150

150

150

150

150

300

150

Figure 7 Serial Dilutions of Lambda DNA

Nextera Rapid Capture Enrichment Reference Guide

47

48

Make DNA Stock Plate

In a new midi plate, prepare the appropriate dilutions of your DNA samples using 1X TE.

Measure each sample in triplicate. Make sure that at least 50 μl of diluted sample is prepared for quantification with the QuantiFluor dsDNA dye. Scale for replicate measurements.

1 Dilute using 1 of the following options, depending on the sample quality or library type:

}

High-quality gDNA—Dilute 1:1000. For example: 2 μl of gDNA + 1998 μl of 1X TE.

}

Pre-enriched Nextera Rapid Capture libraries—Dilute 1:200. For example: 2 μl of library sample + 398 μl of 1X TE.

} Post-enriched Nextera Rapid Capture library dilution:

}

1-plex, 3-plex, 6-plex, and 9-plex (8 reaction kits)—Dilute 1:50. For example: 2 μl of postenriched library + 98 μl of 1X TE.

}

12-plex—Dilute 1:100. For example: 2 μl of postenriched library + 198 μl of 1X

TE.

2 Shake at 1200 rpm for 1 minute.

3 Centrifuge at 280 × g for 1 minute

Dilute QuantiFluor dsDNA Dye

1 Prepare a 1:200 dilution of QuantiFluor dsDNA dye in 1X TE in a conical centrifuge tube wrapped in aluminum foil.

Run each sample and standard in triplicate. For each measurement, 40 μl of diluted

QuantiFluor dye is required. Scale as appropriate.

2 Vortex to mix.

Make Lambda DNA Quant Plate

1 Pour the diluted QuantiFluor dsDNA dye/1X TE into a new reagent reservoir.

2 Transfer 40 μl diluted QuantiFluor dsDNA dye/1X TE into each well of columns 1–3 of a new microplate.

3 Transfer 40 μl from each well of the lambda DNA stock plate to columns 1–3.

Document # 15037436 v01

Figure 8 Lambda DNA Quant Plate with QuantiFluor dsDNA Dye/1X TE

4 Shake at 1200 rpm for 1 minute.

5 Centrifuge at 280 × g for 1 minute

6 Protect from light until read by the spectrofluorometer.

Make DNA Quant Plate

1 Transfer 40 μl QuantiFluor dsDNA reagent/1X TE dilution to each well of the microplate.

2 Transfer 40 μl DNA sample in the DNA stock plate to the microplate.

3 Shake at 1200 rpm for 1 minute.

4 Centrifuge at 280 × g for 1 minute

5 Protect from light until read by the spectrofluorometer.

Read Quant Plate

1 Measure fluorescence (485 nm Ex / 538 nm Em) of both the Lambda DNA quant and

DNA quant plates according to the spectrofluorometer/software recommendations.

2 Calculate the DNA concentration of your unknown samples using the fluorescence values determined from step

1

as follows: a Calculate the average relative fluorescence units (RFU) of the Lambda DNA standards run in triplicate on the lambda DNA quant plate.

b Calculate an Adjusted RFU by subtracting the RFU of the blank Lambda DNA standard (0 ng/μl) Row H from all unknown and standard samples.

c Create a scatter plot of the lambda DNA standard curve values with the Adjusted

RFU on the Y axis and DNA concentration (ng/μl) on the X axis.

d Determine the equation of the line for the lambda DNA standard curve values, which is in the format of y = mx + b is equivalent to RFU = (slope*concentration) + y_int.

e Calculate the concentration for each unknown sample by using the RFU for each sample for y in the equation and determining the value for x in ng/μl.

f Multiply the resulting concentration by the appropriate dilution factor.

Nextera Rapid Capture Enrichment Reference Guide

49

g Use the following formula to convert from ng/μl to nM.

(concentration in ng/μl)

(660 g/mol × average library size)

For example:

15 ng/μl

(660 g/mol × 400)

× 10 6

× 10 6

=

= concentration in nM

57 nM

50

Document # 15037436 v01

Technical Assistance

For technical assistance, contact Illumina Technical Support.

Table 6 Illumina General Contact Information

Website

www.illumina.com

Email

[email protected]

Table 7 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.

Nextera Rapid Capture Enrichment Reference Guide

51

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

Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project