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

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Nextera™ DNA Flex Library Prep
Reference Guide

Document # 1000000025416 v01
April 2018

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

ILLUMINA PROPRIETARY

Nextera™ DNA Flex Library Prep Reference Guide

This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for
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www.illumina.com/company/legal.html.

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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

Table of Contents
Revision History

iii

Chapter 1 Overview

Introduction
Genomic DNA Input Recommendations
Additional Resources

1
1
2

Chapter 2 Protocol

Introduction
Nextera DNA Flex Library Prep Workflow
Tips and Techniques
Tagment Genomic DNA
Post Tagmentation Cleanup
Amplify Tagmented DNA
Clean up Libraries
Pooling Libraries

3
4
4
5
6
7
9
11

Chapter 3 Sequencing

Appendix A Supporting Information

Introduction
How the Nextera DNA Flex Assay Works
Acronyms
Product Contents
Consumables and Equipment
Blood Lysis (Optional)
Saliva Lysis (Optional)

Technical Assistance

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16
17
17
19
21
23

Chapter 1 Overview
Introduction
Genomic DNA Input Recommendations
Additional Resources

1
1
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:
u

Uses an enzymatic reaction, called tagmentation, to fragment DNA and add adapter sequences in only
15 minutes

Innovative sample normalization at inputs > 100 ng

Streamlines sample pooling and sequencing

Reduces excessive pipetting and overall hands-on time, while optimizing use of consumables by using
master mix reagents

Generates libraries from as little as 1 ng input

Prepares 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.
Table 1 DNA Input Recommendations
Total DNA Input
(ng)

Quantification of Input DNA
Recommended

Recommended # of PCR
Cycles

1–9

10–24

25–49

Yes

50–99

Normalized Library
Yield

100–500

Yes

Blood/Saliva

Yes

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Nextera™ DNA Flex Library Prep Reference Guide

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.

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Chapter 2 Protocol
Introduction
Nextera DNA Flex Library Prep Workflow
Tips and Techniques
Tagment Genomic DNA
Post Tagmentation Cleanup
Amplify Tagmented DNA
Clean up Libraries
Pooling Libraries

3
4
4
5
6
7
9
11

Introduction
This chapter describes the Nextera DNA Flex Library Prep protocol.
u

Review 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.

Before proceeding, confirm kit contents and make sure that you have the required equipment and
consumables. See Supporting Information on page 15.

Follow 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.

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Nextera™ DNA Flex Library Prep Reference Guide

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
u

When adding or transferring samples or reagent master mixes, change tips between each sample .

When adding index adapters, change tips between each row and each column.

Remove unused index adapter tubes from the working area.

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

Sealing the Plate
u

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

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

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Nextera™ DNA Flex Library Prep Reference Guide

Microseal ‘F’ adhesive foils are effective at temperatures down to -70°C and are recommended for longterm storage of the 96-well plates containing the final libraries.

Microseal '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.

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

Handling Bead-Linked Transposomes (BLT)
u

Store the BLT stock tube upright in the refrigerator to make sure that the beads are always submerged in
the buffer.

Vortex the BLT stock tube thoroughly to make sure that the beads remain in suspension while in aliquot.
Centrifugation before pipetting is not recommended.

If 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.

When performing wash steps:
u Use an appropriate magnet for the plate. See Consumables and Equipment on page 19.
u Keep the plate on the magnet, unless instructed otherwise.
u Avoid agitating the plate, or disturbing the bead pellet, while the plate is on the magnet.
u If 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.
u Dispense tagment wash buffer directly onto the beads.
u If 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)
u

Minimize 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
u

BLT (Bead-Linked Transposomes)

TB1 (Tagmentation Buffer)

Nuclease-free water

96-well PCR plate

Microseal 'B' adhesive seal

1.7 ml microcentrifuge tubes

Preparation
1

Prepare the following consumables:

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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.

Save the TAG program on the thermal cycler:
u Choose the preheat lid option and set to 100°C
u 55°C for 15 minutes
u 10°C hold
u Each 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.

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

Vortex the BLT vigorously for 10 seconds, then visually check the beads for complete resuspension.
Repeat as necessary.

Prepare tagmentation master mix. For each reaction use:

Reagent

Volume per reaction (µl)

BLT

11 µl

TB1

11 µl

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.

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

Pipette mix the 50 µl reaction mix to resuspend.

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
u

TSB (Tagment Stop Buffer)

TWB (Tagment Wash Buffer)

96-well plate magnet

Microseal 'B' adhesive seal

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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.

Gently pipette mix the entire volume to resuspend the beads.

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.

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

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.

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

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

Remove supernatant with a multichannel pipette and discard.

Repeat steps 6 through 8 for 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 3 in 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
u

EPM (Enhanced PCR Mix)

Nextera DNA Flex index adapters

Nuclease-free water

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Nextera™ DNA Flex Library Prep Reference Guide

Microseal 'A' adhesive seal

Microseal 'B' adhesive seal

1.7 ml microcentrifuge tubes

P20 multichannel pipette

P200 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

10–24

25–49

50–100

>100

Blood/Saliva

Procedure
1

Prepare the PCR master mix.
Reagent

Volume per reaction

EPM

22 µl

Nuclease-free water

22 µl

Vortex and spin down the PCR master mix.

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.

Remove the plate from the magnet. Proceed immediately to the next step to prevent excessive drying of
the beads.

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

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Nextera™ DNA Flex Library Prep Reference Guide

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).
Volume of Index Adapter
per Sample

Index Kit Type

Kit Configuration

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.

Seal the plate, place in the thermal cycler and run the program.
u 68°C, 3 min
u 98°C, 3 min
u Repeat cycling conditions below for the total number of cycles listed in DNA Volume-Based
Recommendations :
u 98°C, 45 sec
u 62°C, 30 sec
u 68°C, 2 min
u 68°C, 1 min
u 10°C hold

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
u

Purification Beads

Freshly prepared 80% ethanol (EtOH)

RSB (Resuspension Buffer)

Midi plates (2)

96-well PCR plate

Microseal 'B' adhesive seal

Microseal 'F' foil seals

1.7 ml microcentrifuge tubes

96-well plate magnet

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Nextera™ DNA Flex Library Prep Reference Guide

Nuclease-free water

Preparation
1

Prepare the following consumables:
Item

Storage

Instructions

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.

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

Vortex and invert PB multiple times to ensure full resuspension.

Prepare a master mix of diluted SPB:
Reagent

Volume per reaction (µl)

45 µl

Nuclease-free water

40 µl

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

Pipette mix a minimum of 10 times or until thoroughly mixed.
WARNING
Complete mixing is critical to proper size distribution of libraries.

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

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

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.

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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.

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:
u

viscosity of the saliva samples

blood sample age

storage conditions

underlying medical conditions affecting white blood cell counts

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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

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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:
a
b

For libraries that were pooled prior to quantification
u Dilute the pool with RSB to .
For libraries that have been quantified individually
u Dilute the individual samples with RSB to , then pool 10 µl of each diluted sample in a single tube.

Table 2 Recommended Loading Concentrations
Denaturation
Instructions
Document Number

Sequencer

Dilute To

Template Loading
Concentration

NovaSeq

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

NOTE
Use these recommended loading concentrations only as a general guideline.
Optimize the loading concentrations for your workflow and quantification method.

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Chapter 3 Sequencing
Nextera DNA Flex supports read lengths up to 2 x 151 cycles..
Table 3 Recommended Read Length on Illumina Sequencers
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

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Appendix A Supporting Information

Supporting Information
Introduction
How the Nextera DNA Flex Assay Works
Acronyms
Product Contents
Consumables and Equipment
Blood Lysis (Optional)
Saliva Lysis (Optional)

15
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19
21
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.

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Nextera™ DNA Flex Library Prep Reference Guide

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

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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

SPB

Sample Purification Beads

2°C to 8°C

TSB

Tagment Stop Buffer

Room temperature

TWB

Tagment Wash Buffer

Room temperature

Box 2 of 3
Quantity

Acronym

Description

Storage Temperature

RSB

Resuspension Buffer

-25°C to -15°C

TB1

Tagmentation Buffer 1

-25°C to -15°C

EPM

Enhanced PCR Mix

-25°C to -15°C

Box 3 of 3
Quantity

Acronym

Description

Storage Temperature

BLT

Bead-Linked Transposomes

2°C to 8°C

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Nextera™ DNA Flex Library Prep Reference Guide

Nextera DNA Flex Library Prep - 96 Samples
Box 1 of 3
Quantity

Acronym

Description

Storage Temperature

SPB

Sample Purification Beads

2°C to 8°C

TSB

Tagment Stop Buffer

Room temperature

TWB

Tagment Wash Buffer

Room temperature

Box 2 of 3
Quantity

Acronym

Description

Storage Temperature

RSB

Resuspension Buffer

-25°C to -15°C

TB1

Tagmentation Buffer 1

-25°C to -15°C

EPM

Enhanced PCR Mix

-25°C to -15°C

Box 3 of 3
Quantity

Acronym

Description

Storage Temperature

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.

Nextera™ DNA Flex Library Prep Reference Guide

24 Dual Index (Tube Format) - 24 Samples
Quantity

Index Name

Description

Storage Temperature

H503

DNA Adapter

-25°C to -15°C

H505

DNA Adapter

-25°C to -15°C

H506

DNA Adapter

-25°C to -15°C

H517

DNA Adapter

-25°C to -15°C

H710

DNA Adapter

-25°C to -15°C

H705

DNA Adapter

-25°C to -15°C

H706

DNA Adapter

-25°C to -15°C

H707

DNA Adapter

-25°C to -15°C

H711

DNA Adapter

-25°C to -15°C

H714

DNA Adapter

-25°C to -15°C

96 Dual Index (Plate Format) - 96 Samples
Quantity

Description

Storage Temperature

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

BLB

Blood Lysis Buffer

Room temperature

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.

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.

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
u

Blood samples collected into EDTA collection tubes

SPB (Sample Purification Beads)

BLB (Blood Lysis Buffer)

PK1 (Proteinase K)

Nuclease-free water

Freshly prepared 80% ethanol (EtOH)

96-well PCR plate

96-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.

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

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

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

Vortex and spin down the lysis master mix.

Add 40 µl of the master mix to each sample.

Vortex and invert SPB multiple times to ensure full resuspension.

Add 20 µl of SPB to the sample well.

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

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.

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
u

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

SPB (Sample Purification Beads)

Nuclease-free water

Freshly prepared 80% ethanol (EtOH)

96-well PCR plate

96-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).

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

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.

Vortex and invert SPB multiple times to ensure full resuspension.

Add 20 µl of SPB to the sample well.

Document # 1000000025416 v01

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

Nextera™ DNA Flex Library Prep Reference Guide

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

Leave the plate at room temperature for 5 minutes.

Place the plate on the magnet for 5 minutes.

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 3 of 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.

Technical Assistance
For technical assistance, contact Illumina Technical Support.
Website:
Email:

www.illumina.com
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

New Zealand

0800.451.650

Norway

+47 800 16836

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

Other countries

+44.1799.534000

+31 207132960

+47 21939693

+44 2073057197

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.

Document # 1000000025416 v01

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

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Create Date                     : 2018:04:02 15:19:53-07:00
Modify Date                     : 2018:04:02 15:19:53-07:00
Title                           : Nextera™ DNA Flex Library Prep Reference Guide (1000000025416)
Author                          : Illumina
Subject                         : Instructions for preparing samples using the Nextera™ DNA Flex Library Prep Kit.

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

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