Nextera™ DNA Flex Library Prep Reference Guide (1000000025416) Illumina Nextera 1000000025416 01 20180523

User Manual:

Open the PDF directly: View PDF .
Page Count: 30

Nextera™ DNA Flex Library Prep

Reference Guide

Document # 1000000025416 v01

April 2018

ILLUMINA PROPRIETARY

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

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, AND WILL VOID ANY WARRANTY APPLICABLE TO THE PRODUCT(S).

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

DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE).

© 2018 Illumina, Inc. All rights reserved.

All trademarks are the property of Illumina, Inc. or their respective owners. For specific trademark information, see

www.illumina.com/company/legal.html.

Document # 1000000025416 v01

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

ii

Nextera™ DNA Flex Library Prep Reference Guide

Revision History

Document Date Description of Change

Document #

1000000025416 v01

April

2018

Replaced references to the

Nextera DNA Flex Pooling Guide (document

# 1000000031471)

with the

Index Adapters Pooling Guide (document #

1000000041074)

. Pooling information is consolidated into the

Index

Adapters Pooling Guide.

Document #

1000000025416 v00

October

2017

Initial release.

Document # 1000000025416 v01

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

iii

Nextera™ DNA Flex Library Prep Reference Guide

Table of Contents

Revision History iii

Chapter 1 Overview 1

Introduction 1

Genomic DNA Input Recommendations 1

Additional Resources 2

Chapter 2 Protocol 3

Introduction 3

Nextera DNA Flex Library Prep Workflow 4

Tips and Techniques 4

Tagment Genomic DNA 5

Post Tagmentation Cleanup 6

Amplify Tagmented DNA 7

Clean up Libraries 9

Pooling Libraries 11

Chapter 3 Sequencing 14

Appendix A Supporting Information 15

Introduction 15

How the Nextera DNA Flex Assay Works 16

Acronyms 17

Product Contents 17

Consumables and Equipment 19

Blood Lysis (Optional) 21

Saliva Lysis (Optional) 23

Technical Assistance 25

Document # 1000000025416 v01

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

iv

Chapter 1 Overview

Introduction 1

Genomic DNA Input Recommendations 1

Additional Resources 2

Introduction

This protocol explains how to prepare up to 96 indexed paired-end libraries from genomic DNA for

subsequent sequencing on an Illumina sequencing system.

The Nextera™ DNAFlex Library Prep protocol:

uUses an enzymatic reaction, called tagmentation, to fragment DNA and add adapter sequences in only

15 minutes

uInnovative sample normalization at inputs > 100 ng

uStreamlines sample pooling and sequencing

uReduces excessive pipetting and overall hands-on time, while optimizing use of consumables by using

master mix reagents

uGenerates libraries from as little as 1 ng input

uPrepares libraries directly from blood or saliva samples

Genomic DNA Input Recommendations

The Nextera DNA Flex Library Prep protocol is compatible with DNA inputs ranging from 1–500 ng, or higher.

For human DNA samples and other large complex genomes, the recommended DNA input is between 100–

500 ng. For small genomes, the DNA input amount can be reduced to as low as 1ng (modifying the PCR

cycling conditions accordingly).

For DNA inputs between 100–500 ng, accurate quantification of the initial DNA sample is not required, and

normalization of the final yield is expected.

If you are using less than 100 ng DNA input, we recommend quantification of the initial DNA sample to

determine the number of PCR cycles required. In this case, because final libraries yields from low inputs are

not normalized by this library prep method, quantification and normalization of libraries before sequencing is

recommended.

Total DNA Input

(ng)

Quantification of Input DNA

Recommended

Recommended # of PCR

Cycles

Normalized Library

Yield

1–9

Yes

12

No

10–24 8

25–49 6

50–99 5

100–500 No 5 Yes

Blood/Saliva No 5 Yes

Table 1 DNA Input Recommendations

Document # 1000000025416 v01

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

1

NOTE

Protocols specific to blood and saliva are included in the

Supporting Information

section of this guide. The

blood protocol requires the Flex Lysis Reagent Kit, which is not provided with the Nextera DNA Flex Library

Prep Kit. This kit is sold separately—refer to Illumina catalog #20015884.

Input DNA Quantification (less than 100 ng)

When input is less than 100 ng, use a fluorometric-based method to quantify input DNA. Avoid methods that

measure total nucleic acid, such as NanoDrop or other UV absorbance methods.

If you use the Qubit dsDNA BR Assay Kit and/or HSKit, use 2 µl of each DNA sample with 198 µl of the Qubit

Working Solution.

Assess DNA Quality

UV absorbance is a common method used for assessing the quality of a DNA sample. The ratio of

absorbance at 260 nm to absorbance at 280 nm provides an indication of sample purity. This protocol is

optimized for DNA with absorbance ratio values of 1.8–2.0, which indicates a pure DNA sample. Target a

260/280 ratio of 2.0–2.2. Values outside this range indicate the presence of contaminants that may cause

incomplete tagmentation and adversely impact the final library yield. For a complete list of contaminants,

including sources, avoidance, and effects on the library, see the

Nextera XT Troubleshooting Technical Note

.

NOTE

Incomplete tagmentation caused by contaminants may result in library preparation failure, poor clustering, or

an unexpectedly high scaffold number.

Additional Resources

The Nextera DNA Flex Library Prep support pages on the Illumina website provide additional resources.

These resources include software, training, compatible products, best practices, and the following

documentation. Always check the support pages for the latest versions.

Resource Description

Custom Protocol Selector

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.

Index Adapter Pooling Guide (document #

1000000041074)

Provides pooling guidelines and dual indexing strategies for using the Nextera

DNA Flex Library Prep kit.

Nextera™ DNA Flex Library Prep Checklist

(document # 1000000033561)

Provides a checklist of the protocol steps. The checklist is intended for

experienced users.

Nextera™ DNA Flex Library Prep

Consumables and Equipment List

(document # 1000000033564)

Provides an interactive checklist of user-provided consumables and equipment.

Document # 1000000025416 v01

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

2

Nextera™ DNA Flex Library Prep Reference Guide

Chapter 2 Protocol

Introduction 3

Nextera DNA Flex Library Prep Workflow 4

Tips and Techniques 4

Tagment Genomic DNA 5

Post Tagmentation Cleanup 6

Amplify Tagmented DNA 7

Clean up Libraries 9

Pooling Libraries 11

Introduction

This chapter describes the Nextera DNA Flex Library Prep protocol.

uReview Best Practices before proceeding. See

Additional Resources

on page 2 for information on how to

access Nextera DNA Flex Library Prep Best Practices on the Illumina website.

uBefore proceeding, confirm kit contents and make sure that you have the required equipment and

consumables. See

Supporting Information

on page 15.

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

Prepare for Pooling

If you plan to pool libraries, record information about your samples before beginning library prep. For more

information, see the Nextera DNA Flex Library Prep support page.

Document # 1000000025416 v01

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

3

Nextera DNA Flex Library Prep Workflow

Figure 1 Nextera DNA Flex Library Prep Workflow

Tips and Techniques

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

Avoiding Cross-Contamination

uWhen adding or transferring samples or reagent master mixes, change tips between

each sample

.

uWhen adding index adapters, change tips between

each row

and

each column

.

uRemove unused index adapter tubes from the working area.

uOpen only one index adapter tube at a time, to prevent misplacing caps.

Sealing the Plate

uAlways seal the 96-well plate before any thermal cycling steps in the protocol.

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

Document # 1000000025416 v01

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

4

Nextera™ DNA Flex Library Prep Reference Guide

uMicroseal ‘F’ adhesive foils are effective at temperatures down to -70°C and are recommended for long-

term storage of the 96-well plates containing the final libraries.

uMicroseal 'B' adhesive seals are effective at -40°C to 110°C, and suitable for skirted or semiskirted PCR

plates. Microseal 'B' seals can be for thermal cycling or short-term storage.

uMicroseal 'A' adhesive film is effective for thermal cycling.

Handling Bead-Linked Transposomes (BLT)

uStore the BLT stock tube upright in the refrigerator to make sure that the beads are always submerged in

the buffer.

uVortex the BLT stock tube thoroughly to make sure that the beads remain in suspension while in aliquot.

Centrifugation before pipetting is not recommended.

uIf beads are adhered to the side or top of a 96-well plate, centrifugation is acceptable. Fully resuspend

the bead pellet by pipetting until thoroughly mixed.

uWhen performing wash steps:

uUse an appropriate magnet for the plate. See

Consumables and Equipment

on page 19.

uKeep the plate on the magnet, unless instructed otherwise.

uAvoid agitating the plate, or disturbing the bead pellet, while the plate is on the magnet.

uIf beads are accidentally aspirated into the pipette tip, dispense all liquid back into the well and leave

until clear to allow the beads to settle.

uDispense tagment wash buffer directly onto the beads.

uIf liquid becomes adhered to the side or top of the tube or well, pulse centrifuge briefly to pull volume

into solution.

Handling Tagment Wash Buffer (TWB)

uMinimize the potential of TWB foaming during tagmentation with a deliberately gentle pipetting technique.

Tagment Genomic DNA

This step uses the Bead-Linked Transposomes BLT to tagment DNA. This process fragments and tags the

DNA with adapter sequences.

Consumables

uBLT(Bead-Linked Transposomes)

uTB1 (Tagmentation Buffer)

uNuclease-free water

u96-well PCR plate

uMicroseal 'B' adhesive seal

u1.7 ml microcentrifuge tubes

Preparation

1 Prepare the following consumables:

Document # 1000000025416 v01

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

5

Nextera™ DNA Flex Library Prep Reference Guide

Item Storage Instructions

BLT 2°C to 8°C

NOTE:Storing at temperatures below 2°C renders the BLT unusable.

Bring to room temperature. Vortex to

mix.

TB1 -25°C to -15°C Bring to room temperature. Vortex to

mix.

2 Save the TAG program on the thermal cycler:

uChoose the preheat lid option and set to 100°C

u55°C for 15 minutes

u10°C hold

uEach well or tube contains 50 µl

See

Genomic DNA Input Recommendations

on page 1 for recommended DNA input amounts and sample

types.

Procedure

1 Transfer between 2 µl and 30 µl of DNA into the wells of a 96-well PCR plate, so that the total input

amount (ng) is within the desired range.

2 Add nuclease-free water to the DNA samples to bring the total volume to 30 µl.

3 Vortex the BLT vigorously for 10 seconds, then visually check the beads for complete resuspension.

Repeat as necessary.

4 Prepare tagmentation master mix. For each reaction use:

Reagent Volume per reaction (µl)

BLT 11µl

TB1 11 µl

5 Vortex the tagmentation master mix thoroughly to make sure the BLT beads are evenly resuspended BLT

in the buffer.

NOTE

Make sure that the tagmentation master mix is vortexed well and the beads remain evenly resuspended

in the mix while it is being aliquoted into the 96-well plate.

6 Using fresh tips, transfer 20 µl of tagmentation master mix to each well containing a sample.

7 Pipette mix the 50 µl reaction mix to resuspend.

8 Seal the plate with Microseal 'B', place on the preprogrammed thermal cycler and run the TAG program.

Post Tagmentation Cleanup

This step washes the adapter-tagged DNA on the BLT before PCR amplification.

Consumables

uTSB (Tagment Stop Buffer)

uTWB (Tagment Wash Buffer)

u96-well plate magnet

uMicroseal 'B' adhesive seal

Document # 1000000025416 v01

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

6

Nextera™ DNA Flex Library Prep Reference Guide

Preparation

Prepare the following consumables:

Item Storage Instructions

ST2 15°C to 30°C Check for any precipitates. If present, heat the buffer at 37°C for 10 minutes, and

vortex until they dissolve. Use at room temperature.

TWB 15°C to 30°C Use at room temperature.

Procedure

1 Add 10 µl of TSBto the tagmentation reaction.

2 Gently pipette mix the entire volume to resuspend the beads.

3 Seal the plate and incubate at 37°C for 15 minutes on a thermal cycler with heated lid set at 100° C and

volume reaction of 60 µl. Then hold at 10° C.

4 Place the plate on the magnet for 3 minutes or until solution is clear.

5 Using a multichannel pipette, remove supernatant and discard.

NOTE

If beads became disturbed during aspiration, redisperse solution into the wells. Keep plate on the

magnet to let beads settle.

6 Remove the plate from the magnet and add 100 µl TWB. Gently pipette mix until beads are fully

resuspended.

7 Place the plate on the magnet for 3 minutes or until solution is clear.

8 Remove supernatant with a multichannel pipette and discard.

9 Repeat steps 6through 8for a total of 2 washes.

10 Remove the plate from the magnet and add 100 μl TWB. Gently pipette mix until beads are fully

resuspended.

11 Seal the plate and place the plate with TWBon the magnet and allow it to incubate until step 3in the

Amplify Tagmented DNA

. The plate should incubate for at least 3 minutes or until clear.

NOTE

Keep the pellet in TWB to help prevent any chance of over drying the beads.

12 While the samples are incubating, continue with the protocol.

Amplify Tagmented DNA

This step amplifies the tagmented DNA using a limited-cycle PCR program. The PCR step adds Index 1 (i7)

adapters, Index 2 (i5) adapters, and sequences required for sequencing cluster formation.

Consumables

uEPM (Enhanced PCR Mix)

uNextera DNA Flex index adapters

uNuclease-free water

Document # 1000000025416 v01

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

7

Nextera™ DNA Flex Library Prep Reference Guide

uMicroseal 'A' adhesive seal

uMicroseal 'B' adhesive seal

u1.7 ml microcentrifuge tubes

uP20 multichannel pipette

uP200 multichannel pipette

Preparation

Prepare the following consumables:

Item Storage Instructions

EPM -25°C to

-15°C

Thaw on ice. Invert to mix, then briefly centrifuge.

Nextera DNA Flex

Indexes

-25°C to

-15°C

Thaw at room temperature.For index tubes: Vortex to mix, then centrifuge

briefly.

For plates: Spin briefly before use.

To yield robust libraries, use this number of PCR cycles, depending on expected initial DNA input amount:

DNAInput (ng) Number of PCRCycles

1–9 12

10–24 8

25–49 6

50–100 5

>100 5

Blood/Saliva 5

Procedure

1 Prepare the PCR master mix.

Reagent Volume per reaction

EPM 22 µl

Nuclease-free water 22 µl

2 Vortex and spin down the PCR master mix.

3 Remove the third TWB wash from thesamples while on the magnet. Use a P20 multichannel pipette to

remove any excess liquid from the plate.

Any remaining foam on the well walls does not adversely affect the library.

4 Remove the plate from the magnet. Proceed

immediately

to the next step to prevent excessive drying of

the beads.

5 Add 40 µl of the PCR master mix to each sample well. Pipette mix to make sure that the beads are

thoroughly resuspended.

Document # 1000000025416 v01

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

8

Nextera™ DNA Flex Library Prep Reference Guide

6 Add index adapters to each sample. Check volumes in the following table. For low-plexity conditions,

refer to the

Index Adapters Pooling Guide (document # 1000000041074)

.

Index Kit Type Kit Configuration Volume of Index Adapter

per Sample

24 plex (dual index) Individual tubes 5 µl i5 adapter

5 µl i7 adapter

96 plex (dual index) 96-well plate 10 µl of primer mix

NOTE

For tubes,open only one index adapter tube at a time to prevent misplacing caps; alternatively, use

fresh caps after opening each tube.

For plates, each well of the index plate is for a single use only.

7 Using a pipette set to 40 µl, pipette mix a minimum of 10 times to mix the entire reaction volume.

8 Seal the plate, place in the thermal cycler and run the program.

u68°C, 3 min

u98°C, 3 min

uRepeat cycling conditions below for the total number of cycles listed in

DNA Volume-Based

Recommendations

:

u98°C, 45 sec

u62°C, 30 sec

u68°C, 2 min

u68°C, 1 min

u10°C hold

9 Remove the plate from the thermal cycler when the PCR program completes.

10 Centrifuge for 1 minute at 280 × g to make sure all the liquid is at the bottom of the well.

SAFE STOPPING POINT

If you are stopping, seal the plate with a Microseal 'B' adhesive seal, and store at 2°Cto8°C for up to 3 days.

Clean up Libraries

This step purifies the amplified libraries through a double-sided bead purification procedure.

Consumables

uPurification Beads

uFreshly prepared 80% ethanol (EtOH)

uRSB (Resuspension Buffer)

uMidi plates (2)

u96-well PCR plate

uMicroseal 'B' adhesive seal

uMicroseal 'F' foil seals

u1.7 ml microcentrifuge tubes

u96-well plate magnet

Document # 1000000025416 v01

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

9

Nextera™ DNA Flex Library Prep Reference Guide

uNuclease-free water

Preparation

1 Prepare the following consumables:

Item Storage Instructions

PB 2°C to 8°C Let stand at room temperature for 30 minutes. Vortex and invert to mix.

RSB -25°C to -15°C Thaw and bring to room temperature. Vortex to mix.

Procedure

1 Place the plate on the magnet for 5 minutes or until the supernatant is clear.

2 Transfer 45 µl of the PCR supernatant into a fresh midi plate.

3 Vortex and invert PB multiple times to ensure full resuspension.

4 Prepare a master mix of diluted SPB:

Reagent Volume per reaction (µl)

PB 45 µl

Nuclease-free water 40 µl

5 Vortex the diluted PB master mix thoroughly and add 85 µl mix to each PCR product.

6 Pipette mix a minimum of 10 times or until thoroughly mixed.

WARNING

Complete mixing is critical to proper size distribution of libraries.

7 Seal the plate and incubate at room temperature for 5 minutes.

8 Place the midi plate on a plate magnet for 5 minutes or until supernatant is clear.

9 During incubation, vortex the PB (

undiluted

stock tube) thoroughly, and then add 15 µl to each well in a

new

midi plate.

10 Transfer 125 µl of supernatant from the first midi plate into the second midi plate (containing the 15 µl of

PB).

11 Pipette mix 10 times.

NOTE

Mixing is critical for proper size distribution of libraries.

12 Seal the second midi plate and incubate at room temperature for 5 minutes.

13 Place midi plate on a magnet for 5 minutes or until clear.

14 Remove and discard supernatant without disrupting the beads.

15 With the plate on the magnet, add 200 µl of fresh 80% ethanol without mixing and incubate for 30

seconds.

16 Pipette to remove the ethanol.

17 Repeat steps 15 and 16 for a total of 2 washes.

18 Use a P20 pipette to remove any excess liquid from the midi plate.

Document # 1000000025416 v01

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

10

Nextera™ DNA Flex Library Prep Reference Guide

19 Air-dry on the magnetic stand until dry (~5 minutes).

20 Remove the midi plate from the magnet and add 32 µl of RSB to the beads.

21 Pipette mix until thoroughly resuspended.

22 Incubate at room temperature on the bench for 2 minutes.

23 Place the midi plate back on the magnet for 2 minutes or until clear.

24 Transfer 30 µl of the supernatant into a new 96-well PCR plate.

SAFE STOPPING POINT

If you are stopping, seal the plate with a Microseal 'F' foil seal and store at -25°Cto-15°C for up to 30 days.

Pooling Libraries

When using DNA inputs of 100–500 ng, the normalization features of this library protocol mean that

quantification and normalization of individual libraries, generated within a single experiment, is not necessary.

There may, however, be slight variations in final yields between different library preparation events, so to

achieve optimal cluster density we advise you to pool your libraries with equal volume and quantify the pool

prior to sequencing.

HiSeqX and HiSeq 3000/4000 users

The wider range in acceptable clustering concentrations on these instruments means that if the

Qubit/PicoGreen quantification for the library pool (as described in the section below) is between 9.5 ng/µl

and 12.5 ng/µl, a 1:11 dilution (10 µl pooled library + 100 µl RSB) can be made to take the pool to the

concentration required for sequencing (ie, 2–3 nM).

For DNA inputs of 100–500 ng

1 Pool 5 µl per sample of up to 96 libraries into a single 1.5 ml microcentrifuge tube. Mix by vortexing then

spin down in a microfuge.

2 Quantify the single pooled library using a dsDNA specific fluorescent dye method such as Qubit or

PicoGreen.

For DNA inputs of <100 ng

1 Quantify each library individually using Qubit or PicoGreen.

For Libraries generated from blood or saliva

The volumes of blood and saliva used in this protocol were developed to provide normalization for most

samples; however, it should be noted that blood and saliva are heterogeneous sample types. The ability of

Nextera DNA Flex to generate normalized libraries depends on the total amount of DNA obtained from the

lysed sample, and this can be adversely affected by numerous factors independent of kit performance.

These factors include, but are not limited to:

uviscosity of the saliva samples

ublood sample age

ustorage conditions

uunderlying medical conditions affecting white blood cell counts

Document # 1000000025416 v01

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

11

Nextera™ DNA Flex Library Prep Reference Guide

Assuming you start with adequate DNA input (>100 ng), expect normalization of the libraries equal to that

observed in 100–500 ng gDNA input. For improved confidence, you can quantify each library individually prior

to pooling using Qubit or PicoGreen.

Check Library Quality (Optional)

Run 1 µl of either the pooled library, or the individual libraries, on an Advanced Analytical Fragment Analyzer

with the HS-NGS High Sensitivity 474 kit or an Agilent Technology 2100 Bioanalyzer using a High Sensitivity

DNA kit. Typical library size profiles are shown below with the average fragment size expected to be around

600 bp (when analyzed using a size range of 150–1500 bp).

Figure 2 Final Library Run on Fragment Analyzer Using High Sensitivity NGS Kit

Figure 3 Final Library Run on Bioanalyzer Using High Sensitivity DNA Kit

Document # 1000000025416 v01

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

12

Nextera™ DNA Flex Library Prep Reference Guide

Calculate Molarity

1 Calculate the molarity of the individual, or pooled library, using the following formula:

NOTE

If the individual, or pooled libraries, were run on a Bioanalyzer or Fragment Analyzer, use the average

size obtained for the sample, otherwise use 600 bp as the average fragment length in the calculation.

2 After the molarity has been calculated:

aFor libraries that were pooled prior to quantification

uDilute the pool with RSB to .

bFor libraries that have been quantified individually

uDilute the individual samples with RSB to , then pool 10 µl of each diluted sample in a single tube.

Sequencer Dilute To Template Loading

Concentration

Denaturation

Instructions

Document Number

NovaSeq See document #

1000000019358

See document #

1000000019358

1000000019358

HiSeqX 2–3 nM 200–300 pM 15006165

HiSeq 3000/ 4000 2–3 nM 200–300 pM 15006165

HiSeq 2000/ 2500 High Output

Mode

2 nM 12 pM 15006165

HiSeq 2500 Rapid Run Mode 2 nM 8.5 pM 15050107

NextSeq 2 nM 1.2–1.3 pM 15048776

MiSeq 4 nM 12 pM 15039740

MiniSeq 2 nM 1.2–1.3 pM 1000000002697

Table 2 Recommended Loading Concentrations

NOTE

Use these recommended loading concentrations only as a general guideline.

Optimize the loading concentrations for your workflow and quantification method.

Document # 1000000025416 v01

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

13

Nextera™ DNA Flex Library Prep Reference Guide

Chapter 3 Sequencing

Nextera DNA Flex supports read lengths up to 2 x 151 cycles..

Sequencer Read Length

NovaSeq, HiSeq X, HiSeq 3000/4000, NextSeq, MiSeq, MiniSeq, iSeq 2 x 151

HiSeq 2000, HiSeq 2500 High Output 2 x 126

HiSeq Rapid Run 2 x 101

Table 3 Recommended Read Length on Illumina Sequencers

Document # 1000000025416 v01

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

14

Appendix A Supporting Information

Supporting Information

Introduction 15

How the Nextera DNA Flex Assay Works 16

Acronyms 17

Product Contents 17

Consumables and Equipment 19

Blood Lysis (Optional) 21

Saliva Lysis (Optional) 23

Introduction

The protocol described in this guide assumes that you have reviewed the contents of this section, confirmed

workflow contents, and obtained all required consumables and equipment.

Document # 1000000025416 v01

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

15

How the Nextera DNA Flex Assay Works

The Nextera DNA Flex library prep kit uses an innovative, bead-based transposome complex to tagment

genomic DNA by fragmenting and adding adapter tag sequences in a single reaction step. Once saturated

with input DNA, the bead-based transposome complex fragments a set number of DNA molecules, providing

flexibility to use a wide DNAinput range, consistent tight fragment size distribution, and normalized libraries

Following the tagmentation step, a limited-cycle PCR step adds Nextera DNA Flex-specific index adapter

sequences to the ends of a DNA fragment, enabling capability across all Illumina sequencing platforms. A

subsequent Sample Purification Bead (SPB) cleanup step then purifies libraries for use on an Illumina

sequencer.

Figure 4 Nextera DNA Flex Workflow

Document # 1000000025416 v01

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

16

Nextera™ DNA Flex Library Prep Reference Guide

Acronyms

Acronym Definition

BLB Blood Lysis Buffer

BLT Bead Linked Transposome

EPM Enhanced PCRMix

EtOH Ethanol

PK1 Proteinase K

RSB Resuspension Buffer

SPB Sample Purification Beads

TB1 Tagmentation Buffer 1

TSB Tagment Stop Buffer

TWB Tagment Wash Buffer

Product Contents

Nextera DNA Flex Library Prep Kit Configurations

NOTE

Certain components of the kit are stored at a temperature that differs from the shipping temperature. Store

kit components at the temperature specified.

Nextera DNA Flex Library Prep - 24 Samples

Box 1 of 3

Quantity Acronym Description Storage Temperature

1 SPB Sample Purification Beads 2°C to 8°C

1 TSB Tagment Stop Buffer Room temperature

1 TWB Tagment Wash Buffer Room temperature

Box 2 of 3

Quantity Acronym Description Storage Temperature

1 RSB Resuspension Buffer -25°C to -15°C

1 TB1 Tagmentation Buffer 1 -25°C to -15°C

1 EPM Enhanced PCR Mix -25°C to -15°C

Box 3 of 3

Quantity Acronym Description Storage Temperature

1 BLT Bead-Linked Transposomes 2°C to 8°C

Document # 1000000025416 v01

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

17

Nextera™ DNA Flex Library Prep Reference Guide

Nextera DNA Flex Library Prep - 96 Samples

Box 1 of 3

Quantity Acronym Description Storage Temperature

1 SPB Sample Purification Beads 2°C to 8°C

4 TSB Tagment Stop Buffer Room temperature

1 TWB Tagment Wash Buffer Room temperature

Box 2 of 3

Quantity Acronym Description Storage Temperature

1 RSB Resuspension Buffer -25°C to -15°C

4 TB1 Tagmentation Buffer 1 -25°C to -15°C

4 EPM Enhanced PCR Mix -25°C to -15°C

Box 3 of 3

Quantity Acronym Description Storage Temperature

4 BLT Bead-Linked Transposomes 2°C to 8°C

Index Kit Contents

Make sure that you have all the reagents identified in this section before proceeding to the library preparation

procedures. Kits are available in the following configurations.

Consumable Catalog #

Nextera™ DNACD Indexes (24Indexes, 24 Samples) 20018707

Nextera™ DNA CDIndexes (96Indexes, 96 Samples) 20018708

NOTE

Certain components of the kit are stored at a temperature that differs from the shipping temperature. Store

kit components at the temperature specified in this protocol.

Document # 1000000025416 v01

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

18

Nextera™ DNA Flex Library Prep Reference Guide

24 Dual Index (Tube Format) - 24 Samples

Quantity Index Name Description Storage Temperature

1 H503 DNAAdapter -25°C to -15°C

1 H505 DNAAdapter -25°C to -15°C

1 H506 DNAAdapter -25°C to -15°C

1 H517 DNAAdapter -25°C to -15°C

1 H710 DNAAdapter -25°C to -15°C

1 H705 DNAAdapter -25°C to -15°C

1 H706 DNAAdapter -25°C to -15°C

1 H707 DNAAdapter -25°C to -15°C

1 H711 DNAAdapter -25°C to -15°C

1 H714 DNAAdapter -25°C to -15°C

96Dual Index (Plate Format) - 96 Samples

Quantity Description Storage Temperature

1 96 Dual Adapter Index Plate -25°C to -15°C

Blood Lysis Kit Contents

Confirm that all reagents identified in this section are available before proceeding to the library preparation

procedures.

Consumable Catalog #

Nextera DNA Flex Library Prep—FlexLysis Reagent Kit 20018706

Flex Lysis Reagent Kit

Quantity Acronym Description Storage Temperature

4 BLB Blood Lysis Buffer Room temperature

4 PK1 Proteinase K -25°C to -15°C

NOTE

Purification Beads are not included in this kit, however sufficient SPB to run the blood lysis workflow are

included in the 24-plex and 96-plex library kits.

Consumables and Equipment

Confirm that all required user-supplied consumables and equipment are present and available 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.

Document # 1000000025416 v01

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

19

Nextera™ DNA Flex Library Prep Reference Guide

Consumables

Consumable Supplier

10 µl pipette tips General lab supplier

10 µl multichannel pipettes General lab supplier

10 µl single channel pipettes General lab supplier

20 µl pipette tips General lab supplier

20 µl multichannel pipettes General lab supplier

20 µl single channel pipettes General lab supplier

200 µl pipette tips General lab supplier

200 µl pipette tips General lab supplier

200 µl single channel pipettes General lab supplier

1000 µl pipette tips General lab supplier

1000 µl single channel pipettes General lab supplier

96-well storage plates, round well, 0.8 ml (midi plate) xs Fisher Scientific, catalog #AB-0859

Hard-Shell 96-well PCR plates Bio-Rad, catalog # HSP-9601

Microseal 'A' film Bio-Rad, catalog #MSA-5001

Microseal 'B' adhesive seals Bio-Rad, catalog #MSB-1001

Microseal 'F' foil seals Bio-Rad, catalog #MSF-1001

RNase/DNase-free multichannel reagent reservoirs, disposable VWR, catalog # 89094-658

Ethanol 200proof (absolute) for molecular biology (500ml) Sigma-Aldrich, product # E7023

Nuclease-free water General lab supplier

[Optional]Agilent High Sensitivity DNAKit Agilent, catalog # 5067-4626

Qubit dsDNA HSAssay Kit ThermoFisher Scientific, catalog # Q32851 or Q32854

Quant-iT™PicoGreen® dsDNAAssay Kit ThermoFisher Scientific, catalog # P11496

[Optional] High Sensitivity NGS Fragment Analysis Kit Advanced Analytical, catalog # DNF-474

FlexLysis Reagent Kit Illumina, catalog # 20015884

EDTA Blood Collection tubes Becton Dickinson

Oragene DNA Collection Kitfor Saliva DNAGenotek, catalog # OGR-500 or OGD-510

Equipment

Equipment Supplier

Magnetic Stand-96 Thermo Fisher Scientific, catalog #AM10027

Microplate centrifuge General lab supplier

Microcentrifuge General lab supplier

Vortexer General lab supplier

[Optional] 2100 Bioanalyzer System Agilent, catalog # G2940CA

Qubit® Fluorometer 3.0 ThermoFisher Scientific, catalog # Q33216, Q33217 or Q33218

[Optional]Fragment Analyzer™ Analytical Advanced Analytical

Document # 1000000025416 v01

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

20

Nextera™ DNA Flex Library Prep Reference Guide

Thermal Cyclers

Use the recommended settings for the selected thermal cycler models listed. Before performing library prep,

validate any thermal cyclers not listed.

Thermal Cycler Temp Mode Lid Temp Vessel Type

Bio-Rad C-1000 Touch thermal cycler Calculated Heated Plate

Bio-Rad DNA Engine Tetrad 2 Calculated Heated Polypropylene

plates and

tubes

MJ Research DNA Engine Tetrad Calculated Heated Plate

Eppendorf Mastercycler Pro S Gradient S, Simulated Tube Heated Plate

Blood Lysis (Optional)

This protocol has been validated using fresh whole blood collected in EDTA collection tubes. Following

collection, store the blood at 4°C and process it within 3 days.

NOTE

The use of frozen blood has not been validated, therefore cannot be recommended.

CAUTION

Blood is a potential source of infectious diseases. Follow site specific procedures to ensure the safe handling

of blood samples. During the lysis protocol, ensure that the entire blood sample is fully lysed (ie, brown in

color following the heat incubation step) before proceeding to subsequent steps.

Consumables

uBlood samples collected into EDTA collection tubes

uSPB(Sample Purification Beads)

uBLB(Blood Lysis Buffer)

uPK1 (Proteinase K)

uNuclease-free water

uFreshly prepared 80% ethanol (EtOH)

u96-well PCR plate

u96-well plate magnet

Preparation

1 Prepare the following consumables:

Item Storage Instructions

BLB 15°C to

30°C*

BLB must be at room temperature for optimal use. Check for any precipitates. If present, heat at

37°C for 10 minutes and vortex until resuspended.

SPB 2°C to

8°C**

Let stand for 30 minutes to bring to room temperature. Keep at room temperature for later use in the

protocol.

PK1 -25°C

to -

15°C

Place on ice until needed.

Document # 1000000025416 v01

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

21

Nextera™ DNA Flex Library Prep Reference Guide

*BLB is shipped frozen but should be stored at room temperature.

**SPB is included in the Nextera DNA Flex Library Prep Kit.

Procedure

1 Create a lysis master mix containing the following volumes for each prep:

Reagent Volume per reaction (µl)

BLB 7 µl

PK1 2 µl

Nuclease-free water 31 µl

2 Make sure that the EDTA blood tube is thoroughly mixed by inverting the tube.

3 Transfer 10 µl of blood into the well of a 96-well PCRplate.

4 Vortex and spin down the lysis master mix.

5 Add 40 µl of the master mix to each sample.

6 Vortex and invert SPB multiple times to ensure full resuspension.

7 Add 20 µl of SPB to the sample well.

8 Use a pipette set to 50 µl and gently mix 10 times to ensure that the beads are thoroughly mixed with the

sample.

9 Seal the plate and incubate at 56°C for 10 minutes at on a thermal cycler with heated lid to 100°C.

10 Place the plate on a magnet for 5 minutes.

NOTE

The beads are not visible at this point due to the darker brown color of the blood from the lysis reaction.

Allow the sample to sit on the magnet for 5 minutes to make sure that the beads fully migrate to the

magnet.

11 Carefully pipette off supernatant without disturbing the beads. Check for the presence of the bead pellet

within the sample well before discarding the supernatant.

NOTE

If the beads are accidentally aspirated, the sample can be dispensed back in the well and allowed to

settle again before the supernatant is removed.

12 Add 150 µl of 80% ethanol (EtOH)and incubate for 30 seconds on the magnet.

13 Remove and discard all of the EtOH.

14 Use a P20 pipette to remove any residual EtOH.

15 Remove the plate from magnet.

16 Resuspend the beads in 30 µl of water and pipette mix until resuspended.

17 Proceed directly to step 3to add the tagmentation master mix directly to the sample well containing 30 µl

of water and the beads.

SAFE STOPPING POINT

If you are stopping, seal the plate with a Microseal 'B' adhesive seal, and store at 2°Cto8°C for up to 3 days.

Document # 1000000025416 v01

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

22

Nextera™ DNA Flex Library Prep Reference Guide

Saliva Lysis (Optional)

This protocol is validated for saliva collected only in Oragene DNA Saliva collection tubes. Following collection,

the saliva is mixed with the Oragene DX Solution contained in the collection tube, making it stable at room

temperature.

NOTE

Prior to performing the saliva lysis, the Oragene tubes containing the samples need to be incubated for at

least 1 hour at 50°C in water or an air incubator (as recommended by DNA Genotek) to lyse the cells. This

incubation step can be performed at any time after the sample is collected. Once heat treated, the samples

can be stored at room temperature.

This protocol is expected to generate >100 ng of DNAoutput at the end of the saliva lysis step.

CAUTION

Saliva is a potential source of infectious diseases. Follow site specific procedures to ensure the safe handling

of saliva samples.

Consumables

uSaliva samples (collected in Oragene DNA collection tubes and heat treated)

uSPB (Sample Purification Beads)

uNuclease-free water

uFreshly prepared 80% ethanol (EtOH)

u96-well PCR plate

u96-well plate magnet

Preparation

1 Prepare the following consumable:

Item Storage Instructions

SPB 2°C to

8°C*

Let stand for 30 minutes to bring to room temperature. Keep at room temperature for later use in the

protocol.

*SPB is included in the Nextera DNA Flex Library Prep Kit.

Procedure

1 Transfer 20 µl of water into a 96-well PCR plate (1 well per sample).

2 Vortex the heat treatedOragene DNAcollection tube to make sure that sample is thoroughly mixed.

3 Transfer 30 µl of the sample into a 96-well plate containing water and slowly pipette mix.

NOTE

Some saliva samples can be viscous so using wide-bored pipette tips can allow more accurate

pipetting.

4 Vortex and invert SPB multiple times to ensure full resuspension.

5 Add 20 µl of SPB to the sample well.

Document # 1000000025416 v01

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

23

Nextera™ DNA Flex Library Prep Reference Guide

6 Use a pipette set to 50 µl and gently mix 10 times to ensure the beads are thoroughly mixed with the

sample.

7 Leave the plate at room temperature for 5 minutes.

8 Place the plate on the magnet for 5 minutes.

9 Carefully pipette off supernatant without disturbing the beads. Check for the presence of the bead pellet

within the sample well before discarding the supernatant.

NOTE

If the beads are accidentally aspirated, the sample can be dispensed back in the well and allowed to

settle again before the supernatant is removed.

10 Add 150 µl of 80% ethanol (EtOH)to the SPB pellet and leave for 30 seconds on the magnet.

11 Remove and discard all the EtOH.

12 Use a P20 pipette to remove any residual EtOH.

13 Remove the plate from the magnet.

14 Resuspend the beads in 30 µl water and pipette mix until fully resuspended.

15 Proceed directly to step 3of the

Tagment Genomic DNA Procedure

on page 6 to add the tagmentation

master mix directly to the sample well containing 30 µl of water and the beads.

SAFE STOPPING POINT

If you are stopping, seal the plate with a Microseal 'B' adhesive seal, and store at 2°Cto8°C for up to 3 days.

Document # 1000000025416 v01

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

24

Nextera™ DNA Flex Library Prep Reference Guide

Technical Assistance

For technical assistance, contact Illumina Technical Support.

Website: www.illumina.com

Email: techsupport@illumina.com

Illumina Customer Support Telephone Numbers

Region Toll Free Regional

North America +1.800.809.4566

Australia +1.800.775.688

Austria +43 800006249 +43 19286540

Belgium +32 80077160 +32 34002973

China 400.066.5835

Denmark +45 80820183 +45 89871156

Finland +358 800918363 +358 974790110

France +33 805102193 +33 170770446

Germany +49 8001014940 +49 8938035677

Hong Kong 800960230

Ireland +353 1800936608 +353016950506

Italy +39 800985513 +39 236003759

Japan 0800.111.5011

Netherlands +31 8000222493 +31 207132960

New Zealand 0800.451.650

Norway +47 800 16836 +47 21939693

Singapore +1.800.579.2745

Spain +34 911899417 +34 800300143

Sweden +46 850619671 +46 200883979

Switzerland +41 565800000 +41 800200442

Taiwan 00806651752

United Kingdom +44 8000126019 +44 2073057197

Other countries +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.

Document # 1000000025416 v01

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

25

Illumina

5200 Illumina Way

San Diego, California 92122 U.S.A.

+1.800.809.ILMN (4566)

+1.858.202.4566 (outside North America)

techsupport@illumina.com

www.illumina.com

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

© 2018 Illumina, Inc. All rights reserved.

Document # 1000000025416 v01