Nextera XT DNA Library Preparation Guide Part - Support

Nextera XT DNA
Library Preparation Guide
FOR RESEARCH USE ONLY
ILLUMINA PROPRIETARY
Part # 15031942 Rev. E
January 2015
Catalog # FC-131-9002DOC
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Nextera XT DNA Library Preparation Guide
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Revision History
Part #
Revision
Date
15031942
E
15031942
D
January
2015
September
2014
15031942
C
October
2012
15031942
B
July 2012
15031942
A
May 2012
Nextera XT DNA Library Preparation Guide
Description of Change
• Corrected info for Nextera XT DNA Library Preparation Index Kit
v2 Set A (FC-131-2001) to include index N715.
• Added info for new index kits that enable preparation of up to 384
indexed paired-end libraries.
• Updated DNA Input Recommendations for diluting starting material
and the potential results of incomplete tagmentation.
• Added new Nextera XT Quality Metrics on page 30 with new
information on how to troubleshoot fluctuations in cluster density.
• Removed Dual Indexing Principle and Low Plexity Pooling
Guidelines sections. This information can be found in the Nextera
Low-Plex Pooling Guidelines Tech Note on the Nextera XT DNA
Library Preparation support page.
• References to read lengths on the MiSeq were updated for v3
chemistry.
• Added instructions for alternate tip if processing fewer than 24
samples while transferring LNB1 beads in Library Normalization.
• Added NaOH 1N pH > 12.5 to the Consumables and Equipment list
as a user-supplied consumable.
• Removed Tween 20 from Consumables and Equipment list.
Consumable not used in protocol.
• Modifications were added in PCR Clean-Up for 2x300 runs on the
MiSeq.
• New section for clustering samples on the HiSeq, HiScanSQ, and
GAIIx. See Clustering Nextera XT Samples for HiSeq, HiScanSQ, and
GAIIx on page 24.
• The Dual Indexing Principle section listed incorrect catalog numbers
for the Nextera XT Index kits. The correct catalog numbers are
now listed.
• Added emphasis on making sure the NT (Neutralize Tagment
Buffer) and LNS1 (Library Normalization Storage Buffer 1)
reagents are at room temperature before use in the protocol.
• Removed reference to Tris-Cl 10mM, pH8.5 with 0.1% Tween 20
from the User-Supplied Consumables table because it is not used
in this library preparation.
• Added emphasis on making sure the NT (Neutralize Tagment
Buffer) and LNS1 (Library Normalization Storage Buffer 1)
reagents are at room temperature before use in the protocol.
• Removed reference to Tris-Cl 10mM, pH8.5 with 0.1% Tween 20
from the User-Supplied Consumables table because it is not used
in this library preparation.
• Initial Release
v
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Part # 15031942 Rev. E
Table of Contents
Revision History
Table of Contents
Chapter 1 Overview
Introduction
DNA Input Recommendations
Additional Resources
Chapter 2 Protocol
Introduction
Nextera XT DNA Library Preparation Workflow
Tagmentation of Input DNA
PCR Amplification
PCR Clean-Up
Validate Library
Library Normalization
Library Pooling and MiSeq Sample Loading
Clustering Nextera XT Samples for HiSeq, HiScanSQ, and GAIIx
Appendix A Supporting Information
Introduction
How does the Nextera XT Assay Work?
Nextera XT Quality Metrics
Acronyms
Nextera XT DNA Library Preparation Kit
Consumables and Equipment
Technical Assistance
Nextera XT DNA Library Preparation Guide
v
vii
1
2
3
4
5
6
7
8
10
14
17
18
22
24
27
28
29
30
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Chapter 1 Overview
Introduction
DNA Input Recommendations
Additional Resources
Nextera XT DNA Library Preparation Guide
2
3
4
1
Chapter 1
Overview
Overview
Introduction
This protocol explains how to prepare up to 384 indexed paired-end libraries from various
DNA for subsequent cluster generation and DNA sequencing. This protocol uses the
reagents provided in the Illumina Nextera®XT DNA Library Preparation Kit. The goal of
this protocol is to fragment and add adapter sequences onto template DNA with a single
tube Nextera XT tagmentation reaction to generate multiplexed sequencing libraries.
The Nextera XT DNA Library Preparation protocol offers:
Sequencing's fastest and easiest preparation
} Single well enzymatic reaction both fragments and adds adapter in only 15 minutes,
no mechanical fragmentation/shearing required
} Master mixed reagents to reduce reagent containers, pipetting and hands-on time
} Innovative sample normalization that eliminates the need for library quantification
before sample pooling and sequencing
Lowest DNA input
} Only 1 ng input DNA needed
Single kit for many applications
} Easily prepare amplicons, small genomes, and plasmids
} Fastest method to prepare libraries for any Illumina sequencing system
Flexible throughput
} 384 indexes available and supported on all Illumina sequencing systems
} Master mixed reagents and automation-friendly configurations
Table 1 Example of Applications for Different Nextera Kits
Nextera (FC-131-1031)
Nextera XT (FC-131-1096)
Large / complex genomes
Small genomes, amplicons, plasmids
Human genomes
PCR Amplicons (> 300 bp)*
non-human mammalian genomes (e.g. mouse,
rat, bovine)
Plasmids
Plant genomes (e.g. Arabidopsis, maize, rice)
Microbial Genomes (e.g. Prokaryotes, archaea)
Invertebrates genomes (e.g. Drosophila)
Concatenated Amplicons
double-stranded cDNA
* Illumina recommends > 300 bp to ensure even coverage across the length of the DNA fragment. An
expected drop off in sequencing coverage about 50 bp from each distal end of a fragment can be
seen. The drop off is because the tagmentation reaction cannot add an adapter right at the distal end
of a fragment. This enzymatic clipping of PCR primers avoids wasted sequencing output on noninformative bases that do not contain genomic inserts. If you wish to sequence the genomic loci
contained within a PCR primer, simply design your amplicons to be ~100 bases larger than the
desired insert to be sequenced.
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Part # 15031942 Rev. E
The Nextera XT DNA Library Preparation Kit protocol is optimized for 1 ng of input DNA
total. Illumina strongly recommends quantifying the starting genomic material.
NOTE
Illumina recommends diluting starting material in molecular grade water or 10 mM Tris-HCl,
pH 7.5–8.5.
Input DNA Quantitation
Nextera XT DNA Library Preparation library preps use an enzymatic DNA fragmentation
step and thus can be more sensitive to DNA input compared to mechanical fragmentation
methods. The ultimate success of the assay strongly depends on using an accurately
quantified amount of input DNA library. Therefore, the correct quantitation of the DNA
library is essential.
To obtain an accurate quantification of the DNA library, quantify the starting DNA library
using a fluorometric based method. The method chosen must be for duplex DNA such as
the Qubit dsDNA BR Assay system. Illumina recommends using 2 μl of each DNA sample
with 198 μl of the Qubit working solution for sample quantification. Avoid methods that
measure total nucleic acid content (e.g. nanodrop or other UV absorbance methods).
Common contaminants such as ssDNA, RNA, and oligos are not substrates for the Nextera
XT assay.
Assessing DNA Quality
UV absorbance is a commonly used method to assess the quality of a DNA sample. The
ratio of absorbance at 260 nm to absorbance at 280 nm is used as an indication of sample
purity. This protocol is optimized for DNA with absorbance ratio values of 1.8–2.0, which
eliminates the presence of EDTA in your samples.
Place your starting DNA in either elution buffer or water. Incomplete tagmentation
potentially leads to library preparation failure, poor clustering, or a higher than expected
scaffold number.
Nextera XT DNA Library Preparation Guide
3
DNA Input Recommendations
DNA Input Recommendations
Overview
Additional Resources
The following documentation is available for download from the Illumina website.
Resource
Description
Nextera XT DNA Library
Preparation Experienced User
Card and Lab Tracking Form (part
# 15031943)
Provides protocol instructions, but with less detail than what is
provided in this user guide. New or less experienced users
are advised to follow this user guide and not the EUC and
LTF.
Sample Sheet Guide and IEM
Nextera Quick Reference Card
(part # 15037155)
Provide information about creating and editing appropriate
sample sheets for Illumina sequencing systems and analysis
software and record parameters for your sample plate.
Analysis Software Guide
Provides information about the BaseSpace® sequencing data
analysis tool that also enables you to organize samples,
libraries, pools, and sequencing runs in a single environment.
Nextera XT DNA Library
Preparation Low-Plex Pooling
Guidelines Tech Note
Provides pooling guidelines and dual indexing strategies for
Nextera XT DNA Library Preparation library preparation.
Visit the Nextera XT DNA Library Preparation support page on the Illumina website for
access to additional documentation, software downloads, online training, frequently asked
questions, and best practices.
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Chapter 2 Protocol
Introduction
Nextera XT DNA Library Preparation Workflow
Tagmentation of Input DNA
PCR Amplification
PCR Clean-Up
Validate Library
Library Normalization
Library Pooling and MiSeq Sample Loading
Clustering Nextera XT Samples for HiSeq, HiScanSQ, and GAIIx
Nextera XT DNA Library Preparation Guide
6
7
8
10
14
17
18
22
24
5
Chapter 2
Protocol
Protocol
Introduction
This chapter describes the Nextera XT DNA Library Preparation protocol.
} Review Best Practices before proceeding. See Additional Resources on page 4 for
information on how to access Nextera XT DNA Library Preparation Best Practices on
the Illumina website.
} Review Appendix A Supporting Information to confirm your kit contents and make
sure that you have obtained all of the requisite equipment and consumables.
} Follow the protocols in the order shown, using the specified volumes and incubation
parameters.
} If you are pooling, record information about your samples before beginning library
preparation for later use in data analysis.
• Do one of the following:
— Use IEM to create and edit sample sheets for Illumina sequencing systems and
analysis software. See Additional Resources on page 4 for information on how to
download IEM software and documentation from the Illumina website.
— Use BaseSpace to organize samples, libraries, pools, and a run for Illumina
sequencing systems and analysis software. See Additional Resources on page 4
for information on how to access BaseSpace or download BaseSpace
documentation from the Illumina website.
• Include a common index in each column to facilitate pipetting operations when
dispensing indexed adapters and pooling indexed libraries. See Additional Resources
on page 4 for information on how to download the Nextera Low-Plex Pooling
Guidelines Tech Note.
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Nextera XT DNA Library Preparation Workflow
Nextera XT DNA Library Preparation Workflow
The following diagram illustrates the workflow using the Nextera XT DNA Library
Preparation Kit. Safe stopping points are marked between steps.
Figure 1 Nextera XT DNA Library Preparation Workflow (For 8 samples)
Nextera XT DNA Library Preparation Guide
7
Protocol
Tagmentation of Input DNA
During this step, input DNA is tagmented (tagged and fragmented) by the Nextera XT
transposome. The Nextera XT transposome simultaneously fragments the input DNA and
adds adapter sequences to the ends, allowing amplification by PCR in subsequent steps.
Estimated Time (8 reactions)
} Hands-on: 7 minutes
} Total duration: 17 minutes
Consumables
Item
Quantity
Storage
Supplied By
ATM (Amplicon Tagment Mix)
1 tube
-25°C to -15°C
Illumina
TD (Tagment DNA Buffer)
1 tube
-25°C to -15°C
Illumina
NT (Neutralize Tagment
Buffer)
1 tube
Room
temperature
Illumina
-25°C to -15°C
User
Input DNA (0.2 ng/μl)
96-well hard shell TCY plate
1 plate
Microseal 'B' adhesive film
User
User
Preparation
1
Remove the ATM, TD, and input DNA from -25°C to -15°C storage and thaw on ice.
2
Visually inspect NT to make sure that there is no precipitate. If there is precipitate,
vortex until all particulates are resuspended.
3
After thawing, mix reagents by gently inverting the tubes 3–5 times, followed by a brief
spin in a microcentrifuge.
Make NTA
NOTE
Make sure that the reaction is assembled in the order described for optimal kit
performance. You do not need to assemble the reaction on ice.
1
Label a new 96-well TCY plate NTA (Nextera XT Tagment Amplicon Plate).
2
Add 10 μl TD Buffer to each well to be used in this assay. Change tips between
samples.
NOTE
Calculate the total volume of TD for all reactions, and divide the volume equally among
the wells of a PCR eight-tube strip. Use a multichannel pipette to dispense into the NTA
plate.
3
Add 5 μl input DNA at 0.2 ng/μl (1 ng total) to each sample well of the NTA plate.
NOTE
It is critical to use the full amount of input DNA.
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Part # 15031942 Rev. E
Using a multichannel pipette, gently pipette up and down 5 times to mix. Change tips
between samples.
5
Add 5 μl ATM to the wells containing input DNA and TD Buffer. Change tips between
samples.
NOTE
Calculate the total volume of ATM for all reactions, and divide the volume equally
among the wells of a PCR eight-tube strip. Use a multichannel pipette to dispense into
the NTA plate.
6
Using a multichannel pipette, gently pipette up and down 5 times to mix. Change tips
between samples.
7
Seal the NTA plate with a Microseal 'B' adhesive seal.
NOTE
For instructions on viewing a video demonstration of this process, see Additional
Resources on page 4.
8
Centrifuge at 280 × g at 20°C for 1 minute.
9
Place the NTA plate in a thermal cycler and run the following program:
NOTE
Make sure that the thermal cycler lid is heated during the incubation.
• 55°C for 5 minutes
• Hold at 10°C
10 When the sample reaches 10°C, proceed immediately to Neutralize NTA as the
transposome is still active.
Neutralize NTA
NOTE
Calculate the total volume of NT for all reactions, and divide the volume equally among
the wells of a PCR eight-tube strip. Use a multichannel pipette to dispense into the NTA
plate.
1
Carefully remove the Microseal 'B' seal and add 5 μl NT Buffer to each well of the
NTA plate. Change tips between samples.
NOTE
For instructions on viewing a video demonstration of this process, see Additional
Resources on page 4.
2
Using a multichannel pipette, gently pipette up and down 5 times to mix. Change tips
between samples.
3
Seal the NTA plate with a Microseal 'B' adhesive seal.
4
Centrifuge at 280 × g at 20°C for 1 minute.
5
Place the NTA plate at room temperature for 5 minutes.
6
[Optional] Assess tagmentation by running 1 μl of sample on an HS bioanalyzer chip.
Nextera XT DNA Library Preparation Guide
9
Tagmentation of Input DNA
4
Protocol
PCR Amplification
In this step, the tagmented DNA is amplified via a limited-cycle PCR program. The PCR
step also adds index 1 (i7) and index 2 (i5) and sequences required for cluster formation. It
is critical to use the full amount of recommended input DNA and not add extra cycles of
PCR cycles to ensure libraries that produce high-quality sequencing results.
Estimated Time (8 reactions)
} Hands-on: 7 minutes
} Cycle time: 38 minutes
} Total duration: 45 minutes
Consumables
Item
Quantity
Storage
Supplied By
NPM (Nextera
PCR Master Mix)
1 tube
-25°C to -15°C
Illumina
Index 1 primers (N7XX)
1 tube each index
-25°C to -15°C
Illumina
Index 2 primers (S5XX)
1 tube each index
-25°C to -15°C
Illumina
TruSeq Index Plate Fixture
Illumina
Microseal 'A' film
User
Preparation
1
If preparing the full set of 24/96 libraries for pooling and sequencing, proceed to step 2.
If less than a full set of libraries is pooled for sequencing, make sure that the correct
index 1 (i7) and index 2 (i5) primers have been selected. Use the Illumina Experiment
Manager and the Nextera XT DNA Library Preparation Low-Plex Pooling Guidelines Tech
Note to confirm the selected index primers.
NOTE
For instructions on viewing a video demonstration of this process, see Additional
Resources on page 4.
10
2
Remove NPM and the index primers (i5 and i7) from -25°C to -15°C storage and thaw
on a bench at room temperature.
Allow approximately 20 minutes to thaw NPM and index primers.
3
After all reagents are thawed, gently invert each tube 3–5 times to mix and briefly
centrifuge the tubes in a microcentrifuge. Use 1.7 ml Eppendorf tubes as adapters for
the microcentrifuge.
4
For 24 libraries arrange the index primers in the TruSeq Index Plate Fixture using the
following arrangement:
a Arrange index 1 (i7) primers (orange caps) in order horizontally.
b Arrange index 2 (i5) primers (white caps) in order vertically.
c To avoid index cross-contamination, discard the original caps and apply new caps
provided in the kit.
d Record their positions on the Experienced User Card.
Part # 15031942 Rev. E
PCR Amplification
Figure 2 TruSeq Index Plate Fixture Arrangement (24 libraries)
A
B
C
5
Index primer 1 (i7) (orange caps)
Index primer 2 (i5) (white caps)
NTA plate
For 96 libraries arrange the index primers in the TruSeq Index Plate Fixture using the
following arrangement:
a Arrange index 1 (i7) primer tubes (orange caps) in order horizontally.
b Arrange index 2 (i5) primers (white caps) in order vertically.
c To avoid index cross-contamination, discard the original caps and apply new caps
provided in the kit.
d Record their positions on the Experienced User Card.
Nextera XT DNA Library Preparation Guide
11
Protocol
Figure 3 TruSeq Index Plate Fixture (96 libraries)
A
B
C
Index primer 1 (i7) (orange caps)
Index primer 2 (i5) (white caps)
NTA plate
Amplify NTA
1
Place the NTA plate in the TruSeq Index Plate Fixture.
2
Add 15 μl NPM to each well of the NTA plate containing index primers. Change tips
between samples.
NOTE
Calculate the total volume of NPM for all reactions, and divide the volume equally
among the wells of a PCR eight-tube strip. Use a multichannel pipette to dispense into
the NTA plate.
12
3
Using a multichannel pipette, add 5 μl index 2 primers (white caps) to each column of
the NTA plate. Changing tips between columns is required to avoid cross-contamination.
4
Using a multichannel pipette, add 5 μl index 1 primers (orange caps) to each row of
the NTA plate. Tips must be changed after each row to avoid index cross-contamination.
5
To avoid index cross-contamination, discard the original white caps and apply new
white caps provided in the kit.
6
To avoid index cross-contamination, discard the original orange caps and apply new
orange caps provided in the kit. Remove all the index primer tubes from the working
area.
7
Using a multichannel pipette, gently pipette up and down 5 times to mix. Change tips
between samples to avoid index and sample cross-contamination.
8
Cover the NTA plate with Microseal 'A' film and seal with a rubber roller.
Part # 15031942 Rev. E
PCR Amplification
9
Centrifuge the NTA plate at 280 × g at 20°C for 1 minute.
10 Perform PCR using the following program on a thermal cycler:
NOTE
Make sure that the thermal cycler lid is heated during the incubation.
• 72°C for 3 minutes
• 95°C for 30 seconds
• 12 cycles of:
— 95°C for 10 seconds
— 55°C for 30 seconds
— 72°C for 30 seconds
• 72°C for 5 minutes
• Hold at 10°C
SAFE STOPPING POINT
If you do not plan to proceed immediately to PCR Clean-Up, there are two options for
storage. The NTA plate can remain on the thermal cycler overnight or you can store it at
2°C to 8°C for up to two days.
Nextera XT DNA Library Preparation Guide
13
Protocol
PCR Clean-Up
This step uses AMPure XP beads to purify the library DNA, and provides a size selection
step that removes short library fragments from the population.
NOTE
For instructions on viewing a video demonstration of this process, see Additional Resources
on page 4.
Estimated Time (8 reactions)
} Hands-on: 15 minutes
} Total duration: 30 minutes
Consumables
Item
Quantity
Storage
Supplied By
RSB (Resuspension Buffer)
1 tube
-25°C to -15°C
Illumina
2°C to 8°C
User
AMPure XP beads
Freshly prepared 80% ethanol
(EtOH)
User
96-well MIDI plates
1 plate
User
96-well TCY plates
1 plate
User
Preparation
NOTE
Review the Best Practices section at the beginning of this protocol regarding the
handling of magnetic beads and washing with 80% ethanol during the PCR clean-up.
1
Bring the AMPure XP beads to room temperature.
2
Prepare fresh 80% ethanol from absolute ethanol.
NOTE
Always prepare fresh 80% ethanol for wash steps. Ethanol can absorb water from the air
impacting your results.
Make CAN
1
Centrifuge the NTA plate at 280 × g for 1 min (20˚C) to collect condensation.
2
Label a new MIDI plate CAA (Clean Amplified Plate).
3
Using a multichannel pipette set to 50 μl, transfer the PCR product from the NTA plate
to the CAA plate. Change tips between samples.
NOTE
The ratio of PCR product to volume of beads is set at 3:2. (For example, 50 μl PCR
product to 30 μl AMPure.) If you pull less than 50 μl of PCR product, adjust your ratio of
AMPure beads accordingly.
4
14
Vortex the AMPure XP beads for 30 seconds to make sure that the beads are evenly
dispersed. Add an appropriate volume of beads to a trough.
Part # 15031942 Rev. E
Using a multichannel pipette, add 30 μl AMPure XP beads to each well of the CAA
plate.
For 2x250 or 2x300 runs on the MiSeq, add 25 μl of AMPure XP beads to each well of
the CAA plate.
Smaller amplicon inputs into Nextera XT preps typically yield smaller insert size
ranges. To maximize recovery of smaller fragments out of the SPRI cleanup, Illumina
recommends the following conditions:
Size of Largest Amplicon in
Pool
AMPure XP
Recommendation
AMPure XP Volume
< 300 bp
1.8x AMPure XP*
90 μl
300–500 bp
1.8x AMPure XP
90 μl
> 500 bp
0.6x AMPure XP
(0.5x AMPure XP for 2x250
runs on the MiSeq)
30 μl
(25 μl for 2x250 or 2x300 runs
on the MiSeq)
* Illumina recommends > 300 bp to ensure even coverage across the length of the DNA fragment. An
expected drop off in sequencing coverage about 50 bp from each distal end of a fragment can be
seen. The drop off is because the tagmentation reaction cannot add an adapter right at the distal end
of a fragment. This enzymatic clipping of PCR primers avoids wasted sequencing output on noninformative bases that do not contain genomic inserts. If you wish to sequence the genomic loci
contained within a PCR primer, simply design your amplicons to be ~100 bases larger than the
desired insert to be sequenced.
6
Gently pipette mix up and down 10 times.
NOTE
Alternatively the solution can be mixed by shaking the CAA plate on a microplate
shaker at 1800 rpm for 2 minutes.
7
Incubate at room temperature without shaking for 5 minutes.
8
Place the plate on a magnetic stand for 2 minutes or until the supernatant has cleared.
9
With the CAA plate on the magnetic stand, use a multichannel pipette to remove and
discard the supernatant carefully. Change tips between samples.
NOTE
If any beads are inadvertently aspirated into the tips, dispense the beads back to the
plate. Then let the plate rest on the magnet for 2 minutes and confirm that the
supernatant has cleared.
10 With the CAA plate on the magnetic stand, wash the beads with freshly prepared 80%
ethanol as follows:
a Using a multichannel pipette, add 200 μl freshly prepared 80% ethanol to each
sample well. Do not resuspend the beads yet.
b Incubate the plate on the magnetic stand for 30 seconds.
c Carefully remove and discard the supernatant.
11 With the CAA plate on the magnetic stand, perform a second ethanol wash as follows:
a Using a multichannel pipette, add 200 μl freshly prepared 80% ethanol to each
sample well.
b Incubate the plate on the magnetic stand for 30 seconds.
c Carefully remove and discard the supernatant.
d Use a P20 multichannel pipette with fine pipette tips to remove excess ethanol.
Nextera XT DNA Library Preparation Guide
15
PCR Clean-Up
5
Protocol
12 With the CAA plate still on the magnetic stand, allow the beads to air-dry for
15 minutes.
13 Remove the CAA plate from the magnetic stand. Using a multichannel pipette, add
52.5 μl RSB to each well of the CAA plate.
14 Gently pipette mix up and down 10 times, changing tips after each column.
NOTE
Alternatively the solution can be mixed by shaking the CAA plate on a microplate
shaker at 1800 rpm for 2 minutes.
15 Incubate at room temperature for 2 minutes.
16 Place the plate on the magnetic stand for 2 minutes or until the supernatant has
cleared.
17 Label a new TCY plate CAN (Clean Amplified NTA Plate).
18 Using a multichannel pipette, carefully transfer 50 μl of the supernatant from the CAA
plate to the CAN plate. Change tips between samples to avoid cross-contamination.
19 [Optional] See Nextera XT Quality Metrics on page 30 for information on how to
troubleshoot fluctuations in cluster density.
SAFE STOPPING POINT
If you do not plan to proceed to Library Normalization immediately, seal the CAN plate
with Microseal “B” adhesive seal and store it at -25°C to -15°C for up to a week.
16
Part # 15031942 Rev. E
It is optional to check the size distribution for some/all libraries by running 1 μl of
undiluted library on an Agilent Technology 2100 Bioanalyzer using a High Sensitivity
DNA chip. Figure 4 shows example traces of successfully sequenced libraries. Typical
libraries show a broad size distribution from ~250 bp to 1000 bp, as in the top panel. A
wide variety of libraries can be sequenced, with average fragment sizes as small as 250 bp
to as large as 1000–1500 bp.
Figure 4 Successful Human Genomic DNA Library Size Distributions Sequenced on HiSeq
Nextera XT DNA Library Preparation Guide
17
Validate Library
Validate Library
Protocol
Library Normalization
This process normalizes the quantity of each library to ensure more equal library
representation in your pooled sample.
NOTE
For instructions on viewing a video demonstration of this process, see Additional Resources
on page 4.
Estimated Time (96 reactions)
} Total duration: 1 hour 20 minutes
} Hands-on: 30 minutes
Consumables
Item
Quantity
Storage
Supplied By
LNA1 (Library Normalization
Additives 1)
1 tube
-25°C to -15°C
Illumina
LNB1 (Library Normalization
Beads 1)
1 tube
2°C to 8°C
Illumina
LNW1 (Library Normalization
Wash 1)
2 tubes
2°C to 8°C
Illumina
LNS1 (Library Normalization
Storage Buffer 1)
1 tube
Room
temperature
Illumina
0.1 N NaOH (less than one
week old)
3 ml per 96 samples
User
96-well MIDI plate
1 plate
User
96-well TCY plate
1 plate
User
15 ml conical tube
1 tube
User
WARNING
This set of reagents contains formamide, an aliphatic amide that is a probable
reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact,
and eye contact. Dispose of containers and any unused contents in accordance with the
governmental safety standards for your region.
For more information, see the SDS for this kit, at support.illumina.com/sds.html.
Preparation
NOTE
Illumina recommends performing the LNA1 preparation step under a fume hood.
1
Remove LNA1 from -25°C to -15°C storage and bring to room temperature. Use a 20°C
to 25°C water bath as needed.
NOTE
LNA1 might form visible precipitates or crystals. Before use, vortex vigorously, and
then hold the tube in front of a light and visually inspect to make sure that all precipitate
has dissolved.
18
Part # 15031942 Rev. E
Remove LNB1 and LNW1 from 2°C to 8°C storage and bring to room temperature.
Use a 20°C to 25°C water bath as needed.
3
Vigorously vortex LNB1 for at least 1 minute with intermittent inversion. Repeat this
step until the beads are well-resuspended and no pellet is found at the bottom of the
tube when the tube is inverted.
4
Make sure that LNS1 is at room temperature before use.
Elute LNP
1
Label a new MIDI plate LNP (Library Normalization Plate).
2
Using a P20 multichannel pipette and fine tips, carefully transfer 20 μl of the
supernatant from the CAN plate to the LNP plate. Change tips between samples to
avoid cross-contamination.
3
For 96 samples, add 4.4 ml LNA1 to a fresh 15 ml conical tube.
4
Use a P1000 pipette set to 1000 μl to resuspend LNB1 thoroughly. Pipette up and down
15–20 times, until the bead pellet at the bottom is resuspended.
WARNING
It is critical to resuspend the LNB1 bead pellet at the bottom of the tube completely. The
use of a P1000 ensures that the beads are homogeneously resuspended and that there is
no bead mass at the bottom of the tube. This resuspension is essential for achieving
consistent cluster density on the flow cell.
5
Immediately after LNB1 is thoroughly resuspended, use a P1000 pipette to transfer
800 μl LNB1 to the 15 ml conical tube containing LNA1. Mix well by inverting the tube
15–20 times. The resulting LNA1/LNB1 bead mix is enough for 96 samples. Pour the
bead mix into a trough and use it immediately in the next step.
NOTE
If you do not plan to use full tubes for 96 samples, a P1000 set to 1000 μl is still required
to resuspend the beads completely in step 4. Mix only the required amounts of LNA1
and LNB1 for the current experiment. Never use a P200 pipette to handle LNB1. Store
the remaining LNA1 and LNB1 separately at their respective recommended
temperatures. If not used immediately, never freeze or mix the LNB1 beads with LNA1
to preserve stability.
NOTE
If you are processing fewer than 24 samples, use a wide bore P200 tip or a P200 tip with
the end cut off.
6
Using a multichannel pipette, add 45 μl combined LNA1/LNB1 to each well of the
LNP plate containing libraries. If you use care to avoid cross-contamination, changing
tips between columns is not required.
7
Seal the LNP plate with a Microseal 'B' adhesive seal.
8
Shake the LNP plate on a microplate shaker at 1800 rpm for 30 minutes.
NOTE
The 30 minute incubation is critical for proper library normalization. Incubations of
greater or less than 30 minutes can affect library representation and cluster density.
9
Place the plate on a magnetic stand for 2 minutes and confirm that the supernatant has
cleared.
Nextera XT DNA Library Preparation Guide
19
Library Normalization
2
Protocol
10 With the LNP plate on the magnetic stand, use a multichannel pipette set to 80 μl to
remove the supernatant and then discard in an appropriate hazardous waste
container.
NOTE
If any beads are aspirated into the tips, dispense the beads back to the plate and let the
plate rest on the magnet for 2 minutes, and then make sure that the supernatant is clear.
11 Remove the LNP plate from the magnetic stand and wash the beads with LNW1, as
follows:
a Using a multichannel pipette, add 45 μl LNW1 to each sample well.
Changing tips is not required if you use care to avoid cross-contamination.
b Seal the LNP plate with a Microseal 'B' adhesive seal.
c Shake the LNP plate on a microplate shaker at 1800 rpm for 5 minutes.
d Place the plate on the magnetic stand for 2 minutes or until the supernatant has
cleared.
e Carefully remove and discard the supernatant in an appropriate hazardous waste
container.
12 Remove the LNP plate from the magnetic stand and repeat the wash with LNW1, as
follows:
a Using a multichannel pipette, add 45 μl LNW1 to each well.
Changing tips between columns is not required if you use care to avoid crosscontamination.
b Seal the LNP plate with a Microseal 'B' adhesive seal.
c Shake the LNP plate on a microplate shaker at 1800 rpm for 5 minutes.
d Place the plate on the magnetic stand for 2 minutes or until the supernatant has
cleared.
e Carefully remove and discard the supernatant in an appropriate hazardous waste
container.
13 Remove the LNP plate from the magnetic stand and add 30 μl 0.1 N NaOH (fresh
stock solution) to each well to elute the sample.
14 Seal the LNP plate with a Microseal 'B' adhesive seal.
15 Shake the LNP plate on a microplate shaker at 1800 rpm for 5 minutes.
16 During the 5 minute elution, apply the SGP (Storage Plate) barcode plate sticker to a
new 96-well PCR plate.
17 Add 30 μl LNS1 to each well to be used in the SGP plate.
18 After the 5 minute elution, make sure that all samples in the LNP plate are
resuspended completely. If the samples are not resuspended, gently pipette those
samples up and down or lightly tap the plate on the bench to resuspend the beads.
Then shake for another 5 minutes.
19 Place the LNP plate on the magnetic stand for 2 minutes or until the liquid is clear.
20 Using a multichannel pipette set to 30 μl, transfer the supernatant from the LNP plate
to the SGP plate. Change tips between samples to avoid cross-contamination.
NOTE
If any beads are aspirated into the tips, dispense the beads back to the plate and let the
plate rest on the magnet for 2 minutes, and then make sure that the supernatant is clear.
21 Seal the SGP plate with Microseal 'B' and then centrifuge to 1000 × g for 1 minute.
20
Part # 15031942 Rev. E
SAFE STOPPING POINT
If you do not plan to proceed to Library Pooling and MiSeq Sample Loading following the
completion of Library Normalization, store the sealed SGP plate at -25°C to -15°C for up
to one week.
Nextera XT DNA Library Preparation Guide
21
Library Normalization
NOTE
Because the final library pool consists of single-stranded DNA, it does not resolve well
on an agarose gel or Bioanalyzer chip. qPCR can be used for quality control if desired.
For more information, please see the Sequencing Library qPCR Quantification Guide (part #
11322363).
Protocol
Library Pooling and MiSeq Sample Loading
In preparation for cluster generation and sequencing, equal volumes of normalized library
are combined, diluted in Hybridization Buffer, and heat denatured before sequencing.
Estimated Time (8 reactions)
} Total duration: 5 minutes
} Hands-on: 5 minutes
Estimated Time (24 reactions)
} Total duration: 10 minutes
} Hands-on: 10 minutes
Consumables
Item
Quantity
Storage
Supplied By
HT1 (Hybridization Buffer)
1 tube
-25°C to -15°C
Illumina
MiSeq reagent cartridge
1 cartridge
-25°C to -15°C
Illumina
Eppendorf LoBind
Microcentrifuge Tube
1 tube
User
PCR 8-tube strip
1
User
2.5 L Ice bucket
1
User
Preparation
1
Set a heat block suitable for 1.5 ml centrifuge tubes to 96°C.
2
Remove a MiSeq reagent cartridge from -25°C to -15°C storage and thaw at room
temperature.
3
In an ice bucket, prepare an ice-water bath by combining 3 parts ice and 1 part water.
Make DAL
NOTE
Prepare fresh DAL for each use.
22
1
If the SGP plate was stored frozen, thaw the plate at room temperature.
2
Centrifuge the SGP plate at 1000 × g for 1 minute at 20°C to collect condensation.
3
If the SGP plate was stored frozen, mix each library to be sequenced by pipetting up
and down 3–5 times using a P200 multichannel pipette. Change tips between samples.
4
Using a P20 multichannel pipette, transfer 5 μl of each library to be sequenced from the
SGP plate, column by column, to a PCR 8-tube strip. Change tips after each column to
avoid cross-contamination.
5
Label a fresh Eppendorf tube PAL (Pooled Amplicon Library).
Part # 15031942 Rev. E
Combine and transfer the contents of the PCR 8-tube strip into the PAL tube. Mix PAL
well.
7
Label a fresh Eppendorf tube DAL (Diluted Amplicon Library).
8
Add 576 μl HT1 to the DAL tube.
9
Transfer 24 μl PAL to the DAL tube containing HT1. Using the same tip, pipette up
and down 3–5 times to rinse the tip and ensure complete transfer.
NOTE
This recommendation is for both MiSeq v2 and v3 reagents. The recommended volumes
for diluting PAL with HT1 represent a 25-fold dilution. This dilution ratio was established
by using the recommended equipment and following the normalization procedure
strictly under typical laboratory conditions (e.g. 20°C to 25°C). An example of
recommended equipment is a plate shaker that is calibrated for shaking speed. If cluster
density is found to be too high or too low, change this dilution ratio to better suit the
equipment, temperature, and user handling in your laboratory after validation.
10 Mix DAL by vortexing the tube at top speed.
11 Using a heat block, incubate the DAL tube at 96°C for 2 minutes.
12 After the incubation, invert DAL 1–2 times to mix and immediately place in the icewater bath.
13 Keep the DAL tube in the ice-water bath for 5 minutes.
14 Load DAL into a thawed MiSeq reagent cartridge into the Load Samples reservoir.
NOTE
It is required to perform this heat denaturation step immediately before loading DAL
into the MiSeq reagent cartridge to ensure efficient template loading on the MiSeq flow
cell.
15 Cap the PAL tube and seal the SGP plate with a Microseal ‘B’ adhesive seal.
16 Store the PAL tube and the SGP plate at -25°C to -15°C for up to one week.
17 Sequence your library as indicated in the MiSeq System User Guide (part # 15027617).
Nextera XT DNA Library Preparation Guide
23
Library Pooling and MiSeq Sample Loading
6
Protocol
Clustering Nextera XT Samples for HiSeq, HiScanSQ,
and GAIIx
In preparation for cluster generation and sequencing, equal volumes of normalized library
are combined, diluted in Hybridization Buffer, and heat denatured prior to MiSeq
sequencing. Libraries can be quantified after PCR Clean-Up. See Nextera XT Quality Metrics
on page 30 for additional information. The steps in this section describe BBN for HiSeq,
HiScanSQ, and GAIIx.
Estimated Time (8 reactions)
} Total duration: 5 minutes
} Hands-on: 5 minutes
Consumables
Item
Quantity
Storage
Supplied By
HT1 (Hybridization Buffer)
1 tube
-25°C to -15°C
Illumina
Eppendorf tubes (screw cap
recommended)
2 tubes
User
PCR 8-tube strip
2
User
Make DAL
NOTE
Prepare fresh DAL for each use.
1
If the SGP plate was stored frozen, thaw the SGP plate at room temperature.
2
Centrifuge the SGP plate at 1,000 x g for 1 minute at 20°C to collect condensation.
3
If the SGP plate was stored frozen, mix each library to be sequenced by pipetting up
and down 3–5 times using a P200 multichannel pipette. Change tips between samples.
4
Using a P20 multichannel pipette, transfer 5 μl of each library to be sequenced from the
SGP plate, column by column, to a PCR 8-tube strip. Change tips after each column to
avoid cross-contamination.
5
Label a fresh Eppendorf tube PAL (Pooled Amplicon Library).
6
Combine and transfer the contents of the PCR 8-tube strip into the PAL tube. Mix PAL
well.
7
Label a fresh Eppendorf tube DAL (Diluted Amplicon Library).
8
Add 585 μl of HT1 to the DAL tube.
9
Transfer 15 μl of PAL to the DAL tube containing HT1. Using the same tip, pipette up
and down 3–5 times to rinse the tip and ensure complete transfer.
NOTE
24
Part # 15031942 Rev. E
10 Mix DAL by vortexing the tube at top speed.
11 Transfer 120 μl of the DAL tube into each well of the PCR 8-tube strip that is loaded
onto the cBot for clustering.
NOTE
It is not required to perform heat denaturation before cBot loading because the
clustering process includes a heat denaturation step.
12 Store the PAL tube and sealed SGP plate at -25°C to -15°C for up to a week.
13 Proceed to clustering and sequencing your library as indicated in the following guides:
} For all high output runs see the cBot User Guide.
} For HiSeq rapid runs, see the associated instrument guide.
Nextera XT DNA Library Preparation Guide
25
Clustering Nextera XT Samples for HiSeq,
For both MiSeq v2 and v3 reagents, the recommended volumes for diluting PAL with
HT1 represent a 40-fold dilution. This dilution ratio was established by using the
recommended equipment (e.g. plate shaker calibrated for shaking speed) and following
the normalization procedure strictly under typical laboratory conditions (e.g. 20°C–
25°C). If cluster density is found to be too high or too low, change this dilution ratio.
Change the dilution ratio to better suit the equipment, temperature, and user handling
in your laboratory after validation.
26
Part # 15031942 Rev. E
Appendix A Supporting Information
Introduction
How does the Nextera XT Assay Work?
Nextera XT Quality Metrics
Acronyms
Nextera XT DNA Library Preparation Kit
Consumables and Equipment
Nextera XT DNA Library Preparation Guide
28
29
30
32
33
36
27
Appendix A
Supporting Information
Supporting Information
Introduction
The protocols described in this guide assume that you have reviewed the contents of this
appendix, confirmed your kit contents, and obtained all of the requisite consumables and
equipment.
28
Part # 15031942 Rev. E
The Nextera XT DNA Library Preparation Kit uses an engineered transposome to
simultaneously fragment and tag ("tagment") input DNA, adding unique adapter sequences
in the process. A limited-cycle PCR reaction uses the adapter sequences to amplify the
insert DNA. The PCR reaction also adds index sequences on both ends of the DNA, thus
enabling dual-indexed sequencing of pooled libraries on any Illumina Sequencing System.
A
B
C
Nextera XT transposome with adapters is combined with template DNA
Tagmentation to fragment and add adapters
Limited cycle PCR to add sequencing primer sequences and indexes
Nextera XT DNA Library Preparation Guide
29
How does the Nextera XT Assay Work?
How does the Nextera XT Assay Work?
Supporting Information
Nextera XT Quality Metrics
Two factors can cause cluster density fluctuations in libraries prepared with the Nextera XT
DNA Library Preparation kit:
} The average sample size is too large or too small after tagmentation.
} The final sample concentration is too low due to a low yield when starting the beadbased normalization step.
To troubleshoot fluctuations in cluster density, consider checking library size and library
concentration.
Check Library Size
Larger molecules cluster less efficiently than smaller molecules. If the fragment size after
tagmentation is larger than expected, low cluster numbers are possible. The inverse is also
true. The average expected library size after tagmentation is between 400 bp and 1.2 kb.
Illumina recommends that you check the library size with a high sensitivity bioanalyzer
trace after the PCR cleanup step. Look for a long low plateau. Alternatively, PCR amplify
the library with the KAPA QPCR primers and run the product on an agarose gel.
} Short libraries indicate too little input DNA—Illumina recommends requantifying the
input DNA with a fluorometric method. If necessary, start with 10%–25% more input
DNA. If the library peak is below 400 bp and you want to continue with this library,
dilute the library further. Rather than 1:25, increase the final PAL dilution to 1:26 or
1:27 before sequencing.
} Long libraries indicate too much input DNA or the presence of inhibitors—Make sure
that the input DNA is free from inhibitors, repeat quantification, and start with less
input DNA. If you want to sequence a longer library, lower the final PAL dilution to
1:24 before sequencing.
Check Library Concentration
Bead-based normalization is most efficient when the library yield after PCR is 10–15 nM, or
higher. Measure library concentration using HS dsDNA Qubit after PCR clean up, and
measure library size with a Bioanalyzer to calculate molarity.
Typically, low library concentration after LNB normalization causes low input starting the
normalization step. If you are starting with high-quality DNA and see low yield after PCR
clean up, possible causes are issues with AMPure clean up or issues during PCR.
If your results show either condition, confirm proper storage of the PCR master mix at
-25°C to -15°C in a no-frost freezer. Make sure that there were minimal freeze-thaw cycles.
For more information, Illumina recommends the following:
} Best practices for bead handling—From the Nextera XT DNA Library Preparation kit
support page, click Best Practices tab and review Handling Magnetic Beads.
} Online training module—Review section 2.4 of the TruSeq: Sample Purification Bead Size
Selection and Best Practices, which is a short training with guidance on bead handling.
To access this training, click the Training tab on the Nextera XT DNA Library
Preparation kit support page.
After normalization, you can expect constant final library concentration. However, certain
issues can affect cluster density. To ensure quality results, check the library concentration
after library normalization using high sensitivity ssDNA Qubit. Keep in mind that the
library is single stranded for this calculation. Understanding library concentration after
30
Part # 15031942 Rev. E
Nextera XT Quality Metrics
library normalization helps in determining the appropriate final PAL dilution before
sequencing.
Nextera XT DNA Library Preparation Guide
31
Supporting Information
Acronyms
Table 2 Nextera XT DNA Library Preparation Acronyms
Acronym
32
Definition
ATM
Amplicon Tagment Mix
CAA
Clean Amplified Plate
CAN
Clean Amplified NTA Plate
DAL
Diluted Amplicon Libraries
HT1
Hybridization Buffer
LNA1
Library Normalization Additives 1
LNB1
Library Normalization Beads 1
LNS1
Library Normalization Storage Buffer 1
LNW1
Library Normalization Wash 1
LNP
Library Normalization Plate
NT
Neutralize Tagment Buffer
NPM
Nextera PCR Master Mix
NTA
Nextera XT Tagment Amplicon Plate
PAL
Pooled Amplicon Library
RSB
Resuspension Buffer
SGP
Storage Plate
TD
Tagment DNA Buffer
Part # 15031942 Rev. E
The Nextera XT DNA Library Preparation Kit is packaged in 96 or 24 sample boxes and
shipped on dry ice unless specified otherwise. Each kit has a corresponding Index Kit that
contains 96 or 24 indexes. Illumina offers multiple 96 index kits that allow up to 384 index
kit combinations.
NOTE
Certain components of the kit are stored at a different temperature than the
temperature at which they are shipped. As soon as you receive your kit, store the kit
components at the specified temperature.
CAUTION
If sequencing Nextera XT libraries with HiSeq 2500/2000/1500/1000, HiScanSQ,
or GAIIx, you must be sure to use the TruSeq Dual Index Sequencing Primer
Boxes (Single Read or Paired End, as appropriate) for all sequencing run types:
non-indexed, single-indexed, and dual-indexed. These sequencing primers are
included for the MiSeq, NextSeq, and HiSeq rapid run mode. If sequencing a
Nextera XT library with the MiSeq System these add-on kits are not
required.
96 Samples
Consumable
Nextera XT DNA Library Preparation Kit
Nextera XT DNA Library Preparation Index Kit (96 Indexes, 384
Samples)
Nextera XT DNA Library Preparation Index Kit v2 Set A (96 Indexes,
384 samples)
Nextera XT DNA Library Preparation Index Kit v2 Set B (96 Indexes,
384 samples)
Nextera XT DNA Library Preparation Index Kit v2 Set C (96 Indexes,
384 samples)
Nextera XT DNA Library Preparation Index Kit v2 Set D (96 Indexes,
384 samples)
Catalog #
FC-131-1096
FC-131-1002
FC-131-2001
FC-131-2002
FC-131-2003
FC-131-2004
24 Samples
Consumable
Nextera XT DNA Library Preparation Kit
Nextera XT DNA Library Preparation Index Kit (24 Indexes, 96
Samples)
Catalog #
FC-131-1024
FC-131-1001
TruSeq Index Plate Fixture Kit
Illumina recommends using the index plate fixture to help with correctly arranging the
index primers during the PCR Amplification steps. Each kit contains two fixtures and can
be used for both the 24-sample kit and 96-sample kit.
Consumable
TruSeq Index Plate Fixture Kit
Nextera XT DNA Library Preparation Guide
Catalog #
FC-130-1005
33
Nextera XT DNA Library Preparation Kit
Nextera XT DNA Library Preparation Kit
Supporting Information
96 Sample Kit Contents (FC-131-1096)
Nextera XT DNA Library Preparation Kit
} Box 1
Quantity
1
2
1
4
1
2
1
Acronym
ATM
TD
NPM
RSB
LNA1
LNW1
HT1
Reagent Name
Amplicon Tagment Mix, 96 RXN
Tagment DNA Buffer
Nextera PCR Master Mix
Resuspension Buffer
Library Normalization Additives 1
Library Normalization Wash 1
Hybridization Buffer
Storage Temperature
-25°C to -15°C
-25°C to -15°C
-25°C to -15°C
-25°C to -15°C
-25°C to -15°C
2°C to 8°C
-25°C to -15°C
Acronym
NT
LNB1
LNS1
Reagent Name
Neutralize Tagment Buffer
Library Normalization Beads 1
Library Normalization Storage Buffer 1
Storage Temperature
Room temperature
2°C to 8°C
Room temperature
} Box 2
Quantity
1
1
1
Nextera XT DNA Library Preparation Index Kit (96 Indexes, 384 Samples)
(FC-131-1002)
Quantity
8 tubes
12 tubes
Reagent Name
Index Primers, S502 to S508, and S517
Index Primers, N701 to N712
Storage Temperature
-25°C to -15°C
-25°C to -15°C
24 Sample Kit Contents (FC-131-1024)
Nextera XT DNA Library Preparation Kit
} Box 1
Quantity
1
1
1
1
1
1
1
Acronym
ATM
TD
NPM
RSB
LNA1
LNW1
HT1
Reagent Name
Amplicon Tagment Mix, 24 RXN
Tagment DNA Buffer
Nextera PCR Master Mix
Resuspension Buffer
Library Normalization Additives 1
Library Normalization Wash 1
Hybridization Buffer
Storage Temperature
-25°C to -15°C
-25°C to -15°C
-25°C to -15°C
-25°C to -15°C
-25°C to -15°C
2°C to 8°C
-25°C to -15°C
Acronym
NT
LNB1
LNS1
Reagent Name
Neutralize Tagment Buffer
Library Normalization Beads 1
Library Normalization Storage Buffer 1
Storage Temperature
Room temperature
2°C to 8°C
Room temperature
} Box 2
Quantity
1
1
1
34
Part # 15031942 Rev. E
Quantity
4 tubes
6 tubes
Reagent Name
Index Primers, S502 to S504, and S517
Index Primers, N701 to N706
Storage Temperature
-25°C to -15°C
-25°C to -15°C
Nextera XT DNA Library Preparation Index Kit v2 Set A (FC-131-2001)
Quantity
8 tubes
12 tubes
Reagent Name
Index Primers, S502, S503, S505 to S508, S510, and S511
Index Primers, N701 to N707, N710 to N712, N714, and
N715
Storage Temperature
-25°C to -15°C
-25°C to -15°C
Nextera XT DNA Library Preparation Index Kit v2 Set B (FC-131-2002)
Quantity
8 tubes
12 tubes
Reagent Name
Index Primers, S502, S503, S505 to S508, S510, and S511
Index Primers, N716, N718 to N724, and N726 to N729
Storage Temperature
-25°C to -15°C
-25°C to -15°C
Nextera XT DNA Library Preparation Index Kit v2 Set C (FC-131-2003)
Quantity
8 tubes
12 tubes
Reagent Name
Index Primers, S513, S515 to S518, and S520 to S522
Index Primers, N701 to N707, N710 to N712, N714, and
N715
Storage Temperature
-25°C to -15°C
-25°C to -15°C
Nextera XT DNA Library Preparation Index Kit v2 Set D (FC-131-2004)
Quantity
8 tubes
12 tubes
Reagent Name
Index Primers, S513, S515 to S518, and S520 to S522
Index Primers, N716, N718 to N724, and N726 to N729
Nextera XT DNA Library Preparation Guide
Storage Temperature
-25°C to -15°C
-25°C to -15°C
35
Nextera XT DNA Library Preparation Kit
Nextera XT DNA Library Preparation Index Kit (24 Indexes, 96 Samples)(FC131-1001)
Supporting Information
Consumables and Equipment
Check to make sure that you have all of the necessary user-supplied consumables and
equipment before proceeding to library preparation. These consumables and equipment are
Illumina recommended for the Nextera XT DNA Library Preparation protocols.
Table 3 User-Supplied Consumables
36
Consumable
Supplier
10 μl pipette tips
General lab supplier
10 μl multichannel pipettes
General lab supplier
10 μl single channel pipettes
General lab supplier
1000 μl pipette tips
General lab supplier
1000 μl multichannel pipettes
General lab supplier
1000 μl single channel pipettes
General lab supplier
200 μl pipette tips
General lab supplier
200 μl multichannel pipettes
General lab supplier
200 μl single channel pipettes
General lab supplier
96-well storage plates, round well,
0.8 ml (“MIDI” plate)
Fisher Scientific,
part # AB-0859
Agencourt AMPure XP 60 ml kit
Beckman Coulter
Genomics,
part # A63881
Distilled water
General lab supplier
Ethanol 200 proof (absolute)
for molecular biology (500 ml)
Sigma-Aldrich,
part # E7023
Microseal ‘A’ film
Bio-Rad,
part # MSA-5001
Microseal ‘B’ adhesive seals
Bio-Rad,
part # MSB-1001
NaOH 1N pH > 12.5
General lab supplier
PCR grade water (for gel-free method)
General lab supplier
RNase/DNase-free multichannel reagent reservoirs,
disposable
VWR, part # 89094-658
Ultra pure water
General lab supplier
Microseal 96-well PCR plates
(“TCY” plate)
Bio-Rad, part # HSP-9601
Part # 15031942 Rev. E
Equipment
Supplier
96-well thermal cycler
(with heated lid)
See table in Thermal Cycler section.
Heat Block for 1.5 ml centrifuge tubes
General lab supplier
High-Speed microplate shaker
VWR, catalog # 13500-890 (110 V/120 V)
VWR, catalog # 14216-214 (230 V)
Magnetic stand-96
Ambion, part # AM10027
Microplate centrifuge
General lab supplier
Vortexer
General lab supplier
Thermal Cycler
The following table lists the recommended settings for selected thermal cycler models. If
your lab has not yet performed the Nextera XT DNA Library Preparation protocol, Illumina
recommends that you validate any thermal cyclers not listed.
Thermal Cycler
Temp Mode
Lid Temp
Vessel Type
Bio-Rad DNA Engine
Tetrad 2
Calculated
Heated, Constant
at 100°C
Polypropylene plates
and tubes
MJ Research DNA
Engine Tetrad
Calculated
Heated
Plate
Eppendorf Mastercycler
Pro S
Gradient S,
Simulated Tube
Heated
Plate
Nextera XT DNA Library Preparation Guide
37
Consumables and Equipment
Table 4 User-Supplied Equipment
38
Part # 15031942 Rev. E
For technical assistance, contact Illumina Technical Support.
Table 5 Illumina General Contact Information
Website
Email
www.illumina.com
[email protected]
Table 6 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
North America
1.800.809.4566
Italy
Australia
1.800.775.688
Netherlands
Austria
0800.296575
New Zealand
Belgium
0800.81102
Norway
Denmark
80882346
Spain
Finland
0800.918363
Sweden
France
0800.911850
Switzerland
Germany
0800.180.8994
United Kingdom
Ireland
1.800.812949
Other countries
Contact Number
800.874909
0800.0223859
0800.451.650
800.16836
900.812168
020790181
0800.563118
0800.917.0041
+44.1799.534000
Safety Data Sheets
Safety data sheets (SDSs) are available on the Illumina website at
support.illumina.com/sds.html.
Product Documentation
Product documentation in PDF is available for download from the Illumina website. Go
to support.illumina.com, select a product, then click Documentation & Literature.
Nextera XT DNA Library Preparation Guide
39
Technical Assistance
Technical Assistance
Illumina
San Diego, California 92122 U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com

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