TruSeq Nano DNA Sample Preparation Guide 15041110 B

TruSeq Nano DNA Sample Preparation Guide 15041110 B
TruSeq® Nano DNA
Sample Preparation Guide
FOR RESEARCH USE ONLY
ILLUMINA PROPRIETARY
Catalog # FC-121-9010DOC
Part # 15041110 Rev. B
November 2013
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Part # 15041110 Rev. B
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TruSeq Nano DNA Sample Preparation Guide
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Part # 15041110 Rev. B
Revision History
Part #
Revision
Date
15041110
B
November
2013
15041110
A
May 2013
TruSeq Nano DNA Sample Preparation Guide
Description of Change
• Renamed Incubate 1 IMP to Incubate IMP
• Added recommended thermal cycler settings to Consumables and
Equipment
Initial Release
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Part # 15041110 Rev. B
Table of Contents
Revision History
Table of Contents
List of Tables
Chapter 1 Overview
Introduction
Protocol Features
DNA Input Recommendations
Positive Control
Additional Resources
Chapter 2 Low Sample (LS) Protocol
Introduction
Sample Prep Workflow
Prepare Adapter Setup
Fragment DNA
Perform End Repair and Size Selection
Adenylate 3' Ends
Ligate Adapters
Enrich DNA Fragments
Validate Library
Normalize and Pool Libraries
Chapter 3 High Sample (HS) Protocol
Introduction
Sample Prep Workflow
Prepare Adapter Setup
Fragment DNA
Perform End Repair and Size Selection
Adenylate 3' Ends
Ligate Adapters
Enrich DNA Fragments
Validate Library
Normalize and Pool Libraries
Appendix A Supporting Information
TruSeq Nano DNA Sample Preparation Guide
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Introduction
Acronyms
Kit Contents
Consumables and Equipment
Indexed Adapter Sequences
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87
89
95
100
Index
103
Technical Assistance
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Part # 15041110 Rev. B
List of Tables
Table 1 Protocol Features
Table 2 Insert Size Options
Table 3 Insert Size Options
Table 4 Covaris S220 Settings
Table 5 Covaris M220 Settings
Table 6 Covaris S2 and E210 Settings
Table 7 Diluted Bead Mixture for a 350 bp Insert Size
Table 8 Diluted Bead Mixture for a 550 bp Insert Size
Table 9 Insert Size Options
Table 10 Covaris S220 Settings
Table 11 Covaris M220 Settings
Table 12 Covaris S2 and E210 Settings
Table 13 Diluted Bead Mixture for a 350 bp Insert Size
Table 14 Diluted Bead Mixture for a 550 bp Insert Size
Table 15 TruSeq Nano DNA Sample Preparation Acronyms
Table 16 TruSeq Nano DNA Sample Prep Kits
Table 17 User-Supplied Consumables
Table 18 User-Supplied Consumables - Additional Items for LS Processing
Table 19 User-Supplied Consumables - Additional Items for HS Processing
Table 20 User-Supplied Equipment
Table 21 User-Supplied Equipment - Additional Items for LS Processing
Table 22 User-Supplied Equipment - Additional Items for HS Processing
Table 23 TruSeq Nano DNA LT Sample Prep Kit Set A Indexed Adapter Sequences
Table 24 TruSeq Nano DNA LT Sample Prep Kit Set B Indexed Adapter Sequences
Table 25 TruSeq Nano DNA HT Sample Prep Kit Indexed Adapter 1 Sequences
Table 26 TruSeq Nano DNA HT Sample Prep Kit Indexed Adapter 2 Sequences
Table 27 Illumina General Contact Information
Table 28 Illumina Customer Support Telephone Numbers
TruSeq Nano DNA Sample Preparation Guide
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Part # 15041110 Rev. B
Chapter 1 Overview
Introduction
Protocol Features
DNA Input Recommendations
Positive Control
Additional Resources
TruSeq Nano DNA Sample Preparation Guide
2
3
4
5
6
1
Chapter 1
Overview
Overview
Introduction
This protocol explains how to prepare up to 96 pooled, indexed paired-end libraries of
genomic DNA (gDNA) for subsequent cluster generation and DNA sequencing using the
reagents provided in the Illumina® TruSeq® Nano DNA Sample Prep Kits (low-throughput
(LT) and high-throughput (HT)). The goal of this protocol is to add adapter sequences onto
the ends of DNA fragments to generate indexed single read or paired-end sequencing
libraries.
The sample preparation protocol offers:
Streamlined Workflow
} Master-mixed reagents to reduce reagent containers and pipetting
} Universal adapter for preparation of single read, paired-end, and indexing
Optimized shearing for whole-genome resequencing with 350 bp and 550 bp insert size
workflows
Bead-based size selection reagents included in each kit
Optimized workflows for processing low sample (LS) and high sample (HS) numbers in
parallel
Compatibility with LT and HT kit configurations
High Throughput
} Adapter plate allows for simultaneous preparation of 96 dual-indexed DNA samples
} Volumes optimized for standard 96-well plate
Index Adapter Tags All Samples
} Additional adapters and primers not necessary
} Each TruSeq Nano DNA LT Sample Prep Kit contains adapter index tubes
recommended for preparing up to 24 samples for sequencing. Together kits A and B
allow for pooling up to 24 samples
} The TruSeq Nano DNA HT Sample Prep Kit contains a 96-well plate with 96 uniquely
indexed adapter combinations designed for manual or automated preparation of 96
uniquely indexed samples
The protocol is compatible with single sample sequencing or lower indexing pooling levels.
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Part # 15041110 Rev. B
This guide documents the TruSeq Nano DNA Sample Preparation protocol using a TruSeq
Nano DNA LT Sample Prep Kit or TruSeq Nano DNA HT Sample Prep Kit.
} Chapter 2 Low Sample (LS) Protocol explains how to perform the TruSeq Nano DNA
Sample Preparation using the Low Sample Protocol
} Chapter 3 High Sample (HS) Protocol explains how to perform the TruSeq Nano DNA
Sample Preparation using the High Sample Protocol
Equivalent results can be expected from either protocol, however the HS protocol can yield
more consistent results between samples. Their distinguishing elements are as follows:
Table 1 Protocol Features
LT Kit - Number of samples
processed at one time
HT Kit - Number of samples
processed at one time
Plate Type
Incubation Equipment
Mixing Method
Low Sample
≤ 24 with indexed
adapter tubes*
High Sample
> 24 with indexed
adapter tubes*
≤ 24 with indexed
adapter plate
> 24 with indexed
adapter plate
96-well 0.3 ml PCR
96-well HSP
96-well MIDI
Microheating systems
Microplate shaker
96-well thermal cycler
Pipetting
* Each TruSeq Nano DNA LT Sample Prep Kit contains enough reagents to prepare up to 24 samples.
When used together, TruSeq Nano DNA LT Sample Prep Kits A and B allow for pooling up to 24
samples using the 12 different indices in each kit. Illumina does not recommend preparing more than
24 samples at a time using the LS protocol. An alternative to using the HS protocol for more than 24
samples is to perform separate library preparations to ensure robust performance.
The TruSeq Nano DNA Sample Preparation fragmentation process is optimized to obtain
final libraries, with the following average insert size.
Table 2 Insert Size Options
Insert Size
Input DNA Per Sample
Recommended Read Length
350 bp
550 bp
100 ng
≤ 2 x 101 bp
200 ng
≤ 2 x 151 bp*
* Read lengths greater than 2 x 151 bp produce a significantly higher percentage of overlapping readpairs.
TruSeq Nano DNA Sample Preparation Guide
3
Protocol Features
Protocol Features
Overview
DNA Input Recommendations
It is important to quantitate the input DNA and assess the DNA quality before performing
TruSeq Nano DNA Sample Preparation.
Input DNA Quantitation
Follow these DNA input recommendations:
} Correct quantification of gDNA is essential.
} 100 ng input DNA is recommended for the 350 bp insert size workflow and 200 ng for
the 550 bp insert size workflow.
} The ultimate success or failure of library preparation strongly depends on using an
accurately quantified amount of input DNA.
} Illumina recommends using fluorometric based methods for quantification including
Qubit or PicoGreen to provide accurate quantification of dsDNA. UV
spectrophotometric-based methods, such as the Nanodrop, measure any nucleotides
present in the sample including RNA, dsDNA, ssDNA, and free nucleotides, which can
give an inaccurate measurement of gDNA.
} Use multiple methods of quantification to verify results.
} DNA quantification methods that rely on intercalating fluorescent dyes measure only
double-stranded DNA and are less subject to the presence of excess nucleic acids.
• These methods require the preparation of calibration curves and are highly
sensitive to pipetting error.
• Make sure that pipettes are correctly calibrated and are not used at the volume
extremes of their performance specifications.
Assessing DNA Quality
Absorbance measurements at 260 nm are commonly used to assess DNA quality:
} The ratio of absorbance at 260 nm to absorbance at 280 nm is used as an indication of
sample purity, and values of 1.8–2.0 are considered indicative of relatively pure DNA.
} The presence of RNA or small nucleic acid fragments, such as nucleotides, can
compromise both absorbance measurements.
} Carefully collect gDNA samples to make sure that they are free of contaminants.
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Part # 15041110 Rev. B
In-line controls are not provided in TruSeq Nano DNA Sample Preparation kits. Therefore,
Illumina recommends using Coriell Human-1 DNA (NA 18507) or Promega Human
Genomic DNA (G3041) as a positive control sample for this protocol.
TruSeq Nano DNA Sample Preparation Guide
5
Positive Control
Positive Control
Overview
Additional Resources
The following resources are available for TruSeq Nano DNA Sample Preparation protocol
guidance and sample tracking. Access these and other resources on the Illumina website at
support.illumina.com/sequencing/kits.ilmn. Then, select TruSeq Nano DNA LT Sample
Prep Kit Support or TruSeq Nano DNA HT Sample Prep Kit Support.
Resource
Description
Training
Illustrates elements of the TruSeq Nano DNA Sample
Preparation process. Viewing these videos is recommended
for new and less experienced users before starting sample
preparation.
• Click Training on TruSeq Nano DNA LT Sample Prep
Kit Support or
• Click Training on TruSeq Nano DNA HT Sample Prep
Kit Support
Best Practices
Provides best practices specific to this protocol. Review these
best practices before starting sample preparation. Topics
include:
• Handling Liquids
• Handling Master Mix Reagents
• Handling Magnetic Beads
• Avoiding Cross-Contamination
• Potential DNA Contaminants
• Temperature Considerations
• Equipment
• Click Best Practices on TruSeq Nano DNA LT Sample
Prep Kit Support or
• Click Best Practices on TruSeq Nano DNA HT Sample
Prep Kit Support
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Part # 15041110 Rev. B
Description
TruSeq Nano DNA Sample
Preparation LS Protocol
Experienced User Card and Lab
Tracking Form
(part # 15041111)
Provides LS 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.
• Click Documentation & Literature on TruSeq Nano DNA
LT Sample Prep Kit Support or
• Click Documentation & Literature on TruSeq Nano DNA
HT Sample Prep Kit Support
TruSeq Nano DNA Sample
Preparation HS Protocol
Experienced User Card and Lab
Tracking Form
(part # 15041112)
Provides HS 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.
• Click Documentation & Literature on TruSeq Nano DNA
LT Sample Prep Kit Support or
• Click Documentation & Literature on TruSeq Nano DNA
HT Sample Prep Kit Support
TruSeq Sample Preparation
Pooling Guide (part # 15042173)
Provides TruSeq pooling guidelines for sample preparation.
Review this guide before beginning library preparation.
• Click Documentation & Literature on TruSeq Nano DNA
LT Sample Prep Kit Support or
• Click Documentation & Literature on TruSeq Nano DNA
HT Sample Prep Kit Support
Illumina Experiment Manager
(IEM)
Enables you to create and edit appropriate sample sheets for
Illumina sequencers and analysis software and record
parameters for your sample plate.
To download the software:
• Click Downloads on TruSeq Nano DNA LT Sample Prep
Kit Support or
• Click Downloads on TruSeq Nano DNA HT Sample Prep
Kit Support
To download the documentation:
• Click Documentation & Literature on TruSeq Nano DNA
LT Sample Prep Kit Support or
• Click Documentation & Literature on TruSeq Nano DNA
HT Sample Prep Kit Support
TruSeq Nano DNA Sample Preparation Guide
7
Additional Resources
Resource
8
Part # 15041110 Rev. B
Chapter 2 Low Sample (LS) Protocol
Introduction
Sample Prep Workflow
Prepare Adapter Setup
Fragment DNA
Perform End Repair and Size Selection
Adenylate 3' Ends
Ligate Adapters
Enrich DNA Fragments
Validate Library
Normalize and Pool Libraries
TruSeq Nano DNA Sample Preparation Guide
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11
12
13
19
26
28
36
41
43
9
Chapter 2
Low Sample (LS) Protocol
Low Sample (LS) Protocol
Introduction
This chapter describes the TruSeq Nano DNA Sample Preparation LS protocol. This
protocol is intended for preparing up to 24 samples at one time using either the LT or HT
kit.
} Follow the protocol in the order described, using the specified volumes and incubation
parameters.
} Review Best Practices before proceeding. See Additional Resources on page 6 for
information on how to access TruSeq Nano DNA Sample Preparation Best Practices on
the Illumina website.
} Review the TruSeq Sample Preparation Pooling Guide (part # 15042173) before proceeding.
See Additional Resources on page 6 for information on how to download the guide from
the Illumina website.
} Review Appendix A Supporting Information before proceeding, to confirm your kit
contents and make sure that you have obtained all of the requisite equipment and
consumables for the LS protocol.
10
Part # 15041110 Rev. B
The following figure illustrates the processes of the TruSeq Nano DNA Sample Preparation
LS protocol to prepare templates using indexed adapter tubes or a DAP.
Figure 1 TruSeq Nano DNA Sample Preparation LS Workflow
TruSeq Nano DNA Sample Preparation Guide
11
Sample Prep Workflow
Sample Prep Workflow
Low Sample (LS) Protocol
Prepare Adapter Setup
If you are pooling, record information about your samples before beginning library
preparation for later use in data analysis.
} Use IEM to create and edit a sample sheet for Illumina sequencers and analysis
software. See Additional Resources on page 6 for information on how to download IEM
software and documentation from the Illumina website.
} Review planning steps in the TruSeq Sample Preparation Pooling Guide (part # 15042173).
See Additional Resources on page 6 for information on how to download the guide from
the Illumina website.
If you are pooling using adapter index tubes, Illumina recommends arranging samples
that will be combined into a common pool in the same row. Include a common index in
each column. This arrangement facilitates pipetting operations when dispensing indexed
adapters and pooling indexed libraries later in the protocol.
If you are pooling with the DAP, arrange samples that will be pooled together in the same
orientation as the indices in the DAP.
12
Part # 15041110 Rev. B
Fragment DNA
Fragment DNA
This process describes how to optimally fragment the gDNA depending on the
downstream application. Covaris shearing generates dsDNA fragments with 3' or 5'
overhangs. The fragmentation process is optimized to obtain final libraries with the
following average insert sizes:
Table 3 Insert Size Options
Insert Size
Input DNA Per Sample
Recommended Read Length
350 bp
550 bp
100 ng
≤ 2 x 101 bp
200 ng
≤ 2 x 151 bp*
* Read lengths greater than 2 x 151 bp produce a significantly higher percentage of overlapping readpairs.
Consumables
Item
Quantity
Storage
Supplied By
Resuspension Buffer (RSB)
1 tube
-15°C to -25°C
(2°C to 8°C
after initial
thaw)
Illumina
Sample Purification Beads (SPB)
1 tube per 24
reactions
2°C to 8°C
Illumina
Barcode labels for:
• CFP (Covaris Fragmentation
Plate)
• CSP (Clean Up Sheared DNA
Plate)
• DNA (DNA Plate)
• IMP (Insert Modification
Plate)
1 label per plate
15°C to 30°C
Illumina
96-well 0.3 ml PCR plates
4
15°C to 30°C
User
TruSeq Nano DNA Sample Preparation Guide
13
Low Sample (LS) Protocol
Item
Quantity
Storage
Supplied By
Covaris tubes
1 per sample
15°C to 30°C
User
DNA samples
100 ng per sample
for a 350 bp insert
size
or
200 ng per sample
for a 550 bp insert
size
-15°C to -25°C
User
Freshly prepared 80% ethanol
(EtOH)
400 µl per sample
15°C to 30°C
User
Microseal ‘B’ adhesive seal
1
15°C to 30°C
User
Preparation
} Review DNA Input Recommendations on page 4.
} Review Best Practices for Handling Magnetic Beads. See Additional Resources on page 6
for information on how to access TruSeq Nano DNA Sample Preparation Best Practices
on the Illumina website.
} Remove the Sample Purification Beads from 2°C to 8°C storage and let stand for at
least 30 minutes to bring them to room temperature.
} Remove one tube of Resuspension Buffer from -15°C to -25°C storage and thaw it at
room temperature.
NOTE
The Resuspension Buffer can be stored at 2°C to 8°C after the initial thaw.
} Turn on the Covaris instrument and follow the manufacturer's guidelines to set up
your instrument.
} Apply a CFP barcode label to a new 96-well 0.3 ml PCR plate.
} Apply a CSP barcode label to a new 96-well 0.3 ml PCR plate.
} Apply a DNA barcode label to a new 96-well 0.3 ml PCR plate.
} Apply an IMP barcode label to a new 96-well 0.3 ml PCR plate.
14
Part # 15041110 Rev. B
1
Illumina recommends quantifying gDNA samples using a fluorometric quantification
method that uses dsDNA binding dyes.
2
Normalize the gDNA samples with Resuspension Buffer to one of the following in each
well of the new 0.3 ml PCR plate labeled with the DNA barcode:
• 100 ng in a final volume of 52.5 µl for a 350 bp insert size
• 200 ng in a final volume of 52.5 µl for a 550 bp insert size
Fragment DNA
1
Shear one of the following amounts of gDNA sample by transferring 52.5 µl of each
DNA sample from the DNA plate to a separate, new Covaris tube:
• 100 ng for a 350 bp insert size
• 200 ng for a 550 bp insert size
Use the wells of the new 0.3 ml PCR plate labeled with CFP barcode or another device
to hold the Covaris tubes upright.
NOTE
Load the DNA sample into the Covaris tube slowly to avoid creating air
bubbles. However, air bubbles might not be preventable.
2
Centrifuge the CFP plate at 600 × g for 5 seconds.
3
Fragment the DNA using the following settings:
Table 4 Covaris S220 Settings
Setting
350 bp Insert
Duty factor
5%
Peak Incident Power
175 W
Cycles per burst
Duration
Mode
Temperature
TruSeq Nano DNA Sample Preparation Guide
550 bp Insert
200
50 seconds
25 seconds
Frequency sweeping
5.5° to 6°C
15
Fragment DNA
Make CFP
Low Sample (LS) Protocol
Table 5 Covaris M220 Settings
Setting
350 bp Insert
550 bp Insert
Duty factor
20%
Peak Incident Power
50 W
Cycles per burst
Duration
200
65 seconds
45 seconds
Temperature
20°C
NOTE
The Covaris M220 settings are optimized for use with the Covaris microTUBE
AFA Fiber Pre-Slit Snap-Cap 6x16mm.
Table 6 Covaris S2 and E210 Settings
Setting
350 bp Insert
Duty cycle
Intensity
10%
5.0
Cycles per burst
Displayed Power
Temperature
16
2.0
200
Duration
Mode
550 bp Insert
45 seconds
Frequency sweeping
S2—23 W
S2—9 W
E210—14 W
E210—7 W
5.5° to 6°C
4
Centrifuge the CFP plate at 600 × g for 5 seconds.
5
Transfer 50 µl of fragmented DNA from each Covaris tube in the CFP plate to the
corresponding well of the new 0.3 ml PCR plate labeled with the CSP barcode, using a
single channel pipette.
Part # 15041110 Rev. B
Proceed immediately to Clean Up Fragmented DNA.
Clean Up Fragmented DNA
1
Vortex the room temperature Sample Purification Beads for at least 1 minute or until
they are well dispersed.
2
Add 80 µl well-mixed Sample Purification Beads to each well of the CSP plate
containing 50 µl of fragmented gDNA. Set a 200 µl pipette to 125 µl, and then gently
pipette the entire volume up and down 10 times to mix thoroughly.
NOTE
Vortex the Sample Purification Beads frequently to make sure that they are evenly
distributed. Illumina recommends the following:
• If using a single channel pipette, vortex the beads after processing four samples
• If using a multichannel pipette, vortex the beads after processing four columns
• If the beads are in a reagent reservoir, mix with a 1000 µl pipette.
NOTE
Keep the Sample Purification Beads tube at room temperature for later use in the
protocol.
3
Incubate the CSP plate at room temperature for 5 minutes.
4
Place the CSP plate on the magnetic stand at room temperature for 8 minutes or until
the liquid is clear.
5
Using a 200 µl single channel or multichannel pipette set to 125 µl, remove and
discard 125 µl of the supernatant from each well of the CSP plate.
NOTE
Leave the CSP plate on the magnetic stand while performing the following steps 6–10.
6
With the CSP plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to
each well without disturbing the beads.
7
Incubate the CSP plate at room temperature for 30 seconds, and then remove and
discard all of the supernatant from each well. Take care not to disturb the beads.
8
Repeat steps 6 and 7 one time for a total of two 80% EtOH washes.
9
With the CSP plate on the magnetic stand, let the samples air-dry at room temperature
for 5 minutes. Remove and discard any remaining EtOH with a 10 µl pipette.
TruSeq Nano DNA Sample Preparation Guide
17
Fragment DNA
6
Low Sample (LS) Protocol
10 With the CSP plate on the magnetic stand, add 62.5 µl Resuspension Buffer to each
well of the plate.
11 Remove the CSP plate from the magnetic stand.
12 Resuspend the beads in each well of the CSP plate by repeatedly dispensing the
Resuspension Buffer over the bead pellet until it is immersed in the solution. Gently
pipette the entire volume up and down 10 times to mix thoroughly.
13 Incubate the CSP plate at room temperature for 2 minutes.
14 Place the CSP plate on the magnetic stand at room temperature for 5 minutes or until
the liquid is clear.
15 Transfer 60 µl of the clear supernatant from each well of the CSP plate to the
corresponding well of the new 0.3 ml PCR plate labeled with the IMP barcode. Take
care not to disturb the beads.
NOTE
Make sure that you use a 0.3 ml PCR plate, because IMP plate volumes are greater than
a 0.2 ml PCR plate. Final volumes during size selection are up to 260 µl per well.
16 Proceed immediately to Perform End Repair and Size Selection on page 19.
18
Part # 15041110 Rev. B
This process converts the overhangs resulting from fragmentation into blunt ends using
End Repair Mix 2. The 3' to 5' exonuclease activity of this mix removes the 3' overhangs
and the 5' to 3' polymerase activity fills in the 5' overhangs. Following end repair, the
appropriate library size is selected using different ratios of the Sample Purification Beads.
Consumables
Item
Quantity
Storage
Supplied By
End Repair Mix 2 (ERP2)
LT kit - 1 tube per 24
reactions
or
HT kit - 1 tube per 48
reactions
-15°C to -25°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
Sample Purification Beads (SPB)
1 tube per 24
reactions
2°C to 8°C
Illumina
Barcode labels for:
• ALP (Adapter Ligation Plate)
• CEP (Clean Up End Repair
Plate)
1 label per plate
15°C to 30°C
Illumina
15 ml conical tube
(when processing > 6 samples
at a time)
or
1.7 ml microcentrifuge tube
(when processing ≤ 6 samples
at a time)
1
15°C to 30°C
User
96-well 0.3 ml PCR plates
2
15°C to 30°C
User
Freshly prepared 80% ethanol
(EtOH)
400 µl per sample
15°C to 30°C
User
TruSeq Nano DNA Sample Preparation Guide
19
Perform End Repair and Size Selection
Perform End Repair and Size Selection
Low Sample (LS) Protocol
Item
Quantity
Storage
Supplied By
Ice bucket
As needed
-15°C to -25°C
User
Microseal ‘B’ adhesive seals
2
15°C to 30°C
User
PCR grade water
1 bottle
15°C to 30°C
User
RNase/DNase-free eight-tube
strips and caps (if using
multichannel pipettes)
6
15°C to 30°C
User
RNase/DNase-free reagent
reservoirs (if using
multichannel pipettes)
6
15°C to 30°C
User
Preparation
} Prepare an ice bucket.
} Remove the End Repair Mix 2 from -15°C to -25°C storage and thaw it at room
temperature. Place the tube on ice.
} Remove the Sample Purification Beads and Resuspension Buffer from 2°C to 8°C
storage and bring them to room temperature.
} Review Best Practices for Handling Magnetic Beads. See Additional Resources on page 6
for information on how to access TruSeq Nano DNA Sample Preparation Best Practices
on the Illumina website.
} Pre-program the thermal cycler with the following program and save as ERP:
• Choose the thermal cycler pre-heat lid option and set to 100°C
• 30°C for 30 minutes
• Hold at 4°C
} Apply an ALP barcode label to a new 96-well 0.3 ml PCR plate.
} Apply a CEP barcode label to a new 96-well 0.3 ml PCR plate.
Make IMP
1
20
Centrifuge the thawed End Repair Mix 2 tube at 600 × g for 5 seconds.
Part # 15041110 Rev. B
Add 40 µl End Repair Mix 2 to each well of the IMP plate containing the fragmented
DNA. Set a 200 µl pipette to 95 µl, and then gently pipette the entire volume up and
down 10 times to mix thoroughly.
3
Seal the IMP plate with a Microseal ‘B’ adhesive seal.
4
Return the End Repair Mix 2 tube to -15°C to -25°C storage.
Incubate IMP
1
Place the sealed IMP plate on the pre-programmed thermal cycler. Close the lid then
select and run the ERP program.
a Choose the thermal cycler pre-heat lid option and set to 100°C
b 30°C for 30 minutes
c Hold at 4°C
2
Remove the IMP plate from the thermal cycler when the program reaches 4°C.
Clean Up IMP and Size Selection
1
Remove the adhesive seal from the IMP plate.
Remove Large DNA Fragments
1
Vortex the Sample Purification Beads for at least 1 minute or until they are well
dispersed.
TruSeq Nano DNA Sample Preparation Guide
21
Perform End Repair and Size Selection
2
Low Sample (LS) Protocol
2
Add the Sample Purification Beads and PCR grade water to one of the following tubes,
to create a diluted bead mixture of 160 µl per 100 µl of end-repaired sample:
• New 15 ml conical tube, when processing > 6 samples at a time
• New 1.7 ml microcentrifuge tube, when processing ≤ 6 samples at a time
Determine the volumes using the following formulas, which include 15% excess for
multiple samples:
Table 7 Diluted Bead Mixture for a 350 bp Insert Size
Formula
Sample Purification Beads
PCR grade water
# of samples X
109.25 µl
# of samples X
74.75 µl
Table 8 Diluted Bead Mixture for a 550 bp Insert Size
Formula
Sample Purification Beads
PCR grade water
# of samples X
92 µl
# of samples X
92 µl
Example
Amount
per 12
samples
Your
Calculation
1311 µl
897 µl
Example
Amount
per 12
samples
1104 µl
Your
Calculation
1104 µl
3
Vortex the diluted bead mixture for 5 seconds to make sure that the beads are evenly
dispersed.
4
Add 160 µl of the diluted bead mixture to each well of the IMP plate containing 100 µl
of the end repaired sample. Set a 200 µl pipette to 200 µl, and then gently pipette the
entire volume up and down 10 times to mix thoroughly.
NOTE
Aspirate the diluted bead mixture slowly and dispense it slowly due to the viscosity of
the solution. Changes in the volume of the diluted bead mixture affect the insert size of
your library.
22
Part # 15041110 Rev. B
5
Incubate the IMP plate at room temperature for 5 minutes.
6
Place the IMP plate on the magnetic stand at room temperature for 5 minutes or until
the liquid is clear.
7
Use a 200 µl single channel or multichannel pipette set to 125 µl to transfer 125 µl of
the supernatant, containing the DNA of interest, from each well of the IMP plate to
the corresponding well of the new 0.3 ml PCR plate labeled with the CEP barcode.
Take care not to disturb the beads.
NOTE
Transfer, do not discard, the supernatant. It contains the DNA of interest.
8
Repeat step 7 one time. Each CEP plate well now contains a total of 250 µl of DNA of
interest.
9
Discard the IMP plate containing the beads.
10 Discard any remaining diluted bead mixture.
Remove Small DNA Fragments
NOTE
In the following steps, use undiluted Sample Purification Beads.
1
Vortex the Sample Purification Beads for at least 1 minute or until they are well
dispersed.
2
Add 30 µl undiluted Sample Purification Beads to each well of the CEP plate
containing 250 µl of the supernatant with the DNA of interest. Set a 200 µl pipette to
200 µl, and then gently pipette the entire volume up and down 10 times to mix
thoroughly.
TruSeq Nano DNA Sample Preparation Guide
23
Perform End Repair and Size Selection
NOTE
Vortex the diluted bead mixture frequently. Illumina recommends the following:
• If using a single channel pipette, vortex the mixture after processing four samples
• If using a multichannel pipette, vortex the mixture after processing four columns
• If the mixture is in a reagent reservoir, mix with a 1000 µl pipette.
Low Sample (LS) Protocol
NOTE
Aspirate the Sample Purification Beads slowly and dispense them slowly due to the
viscosity of the solution. Changes in the volume of the bead mixture affect the insert size
of your library.
NOTE
Vortex the Sample Purification Beads frequently to make sure that they are evenly
distributed. Illumina recommends the following:
• If using a single channel pipette, vortex the beads after processing four samples
• If using a multichannel pipette, vortex the beads after processing four columns
• If the beads are in a reagent reservoir, mix with a 1000 µl pipette.
3
Incubate the CEP plate at room temperature for 5 minutes.
4
Place the CEP plate on the magnetic stand at room temperature for 5 minutes or until
the liquid is clear.
5
Using a 200 µl single channel or multichannel pipette set to 138 µl, remove and
discard 138 µl of the supernatant from each well of the CEP plate. Take care not to
disturb the beads.
6
Repeat step 5 one time, removing and discarding a total of 276 µl of the supernatant
from each well.
NOTE
Leave the CEP plate on the magnetic stand while performing the following steps 7–11.
7
With the CEP plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to
each well without disturbing the beads.
8
Incubate the CEP plate at room temperature for 30 seconds, and then remove and
discard all of the supernatant from each well. Take care not to disturb the beads.
9
Repeat steps 7 and 8 one time for a total of two 80% EtOH washes.
10 With the CEP plate on the magnetic stand, let the samples air-dry at room temperature
for 5 minutes. Remove and discard any remaining EtOH with a 10 µl pipette.
11 With the CEP plate on the magnetic stand, add 20 µl Resuspension Buffer to each well
of the plate.
12 Remove the CEP plate from the magnetic stand.
13 Resuspend the beads in each well of the CEP plate by repeatedly dispensing the
Resuspension Buffer over the bead pellet until it is immersed in the solution. Gently
pipette the entire volume up and down 10 times to mix thoroughly.
24
Part # 15041110 Rev. B
15 Place the CEP plate on the magnetic stand at room temperature for 5 minutes or until
the liquid is clear.
16 Transfer 17.5 µl of the clear supernatant from each well of the CEP plate to the
corresponding well of the new 0.3 ml PCR plate labeled with the ALP barcode.
SAFE STOPPING POINT
If you do not plan to proceed immediately to Adenylate 3' Ends on page 26, the protocol can
be safely stopped here. If you are stopping, seal the ALP plate with a Microseal ‘B’ adhesive
seal and store at -15°C to -25°C for up to 7 days.
TruSeq Nano DNA Sample Preparation Guide
25
Perform End Repair and Size Selection
14 Incubate the CEP plate at room temperature for 2 minutes.
Low Sample (LS) Protocol
Adenylate 3' Ends
A single ‘A’ nucleotide is added to the 3’ ends of the blunt fragments to prevent them from
ligating to one another during the adapter ligation reaction. A corresponding single
‘T’ nucleotide on the 3’ end of the adapter provides a complementary overhang for ligating
the adapter to the fragment. This strategy ensures a low rate of chimera (concatenated
template) formation.
Consumables
Item
Quantity
Storage
Supplied By
A-Tailing Mix (ATL)
LT kit - 1 tube per 24
reactions
or
HT kit - 1 tube per 48
reactions
-15°C to -25°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
Microseal ‘B’ adhesive seal
1
15°C to 30°C
User
RNase/DNase-free eight-tube
strips and caps
(if using multichannel pipettes)
2
15°C to 30°C
User
RNase/DNase-free reagent
reservoirs
(if using multichannel pipettes)
2
15°C to 30°C
User
Preparation
} Remove the A-Tailing Mix from -15°C to -25°C storage and thaw it at room
temperature:
} Remove the Resuspension Buffer from 2°C to 8°C storage and bring it to room
temperature.
} Remove the ALP plate from -15°C to -25°C storage, if it was stored at the conclusion of
Clean Up IMP and Size Selection on page 21.
• Let it thaw at room temperature.
26
Part # 15041110 Rev. B
Add ATL
1
Centrifuge the thawed A-Tailing Mix tube at 600 × g for 5 seconds.
2
Add 12.5 µl thawed A-Tailing Mix to each well of the ALP plate. Set a 20 µl pipette to
20 µl, then gently pipette the entire volume up and down 10 times to mix thoroughly.
3
Seal the ALP plate with a Microseal ‘B’ adhesive seal.
4
Return the A-Tailing Mix tube to -15° to -25°C storage.
Incubate 1 ALP
1
Place the sealed ALP plate, containing 30 µl of each sample, on the pre-programmed
thermal cycler. Close the lid, then select and run the ATAIL70 program.
a Choose the pre-heat lid option and set to 100°C
b 37°C for 30 minutes
c 70°C for 5 minutes
d 4°C for 5 minutes
e Hold at 4°C
2
When the thermal cycler temperature has been at 4°C for 5 minutes, remove the ALP
plate from the thermal cycler.
3
Centrifuge the ALP plate at 280 × g for 1 minute.
4
Proceed immediately to Ligate Adapters on page 28.
TruSeq Nano DNA Sample Preparation Guide
27
Adenylate 3' Ends
• Centrifuge the thawed ALP plate at 280 × g for 1 minute.
• Remove the adhesive seal from the ALP plate.
} Pre-program the thermal cycler with the following program and save as ATAIL70:
• Choose the pre-heat lid option and set to 100°C
• 37°C for 30 minutes
• 70°C for 5 minutes
• 4°C for 5 minutes
• Hold at 4°C
Low Sample (LS) Protocol
Ligate Adapters
This process ligates multiple indexing adapters to the ends of the DNA fragments,
preparing them for hybridization onto a flow cell.
Consumables
28
Item
Quantity
Storage
Supplied By
Choose from the following
depending on the kit you are
using:
• TruSeq Nano DNA LT
Sample Prep Kit contents:
• DNA Adapter Indices
(AD001–AD016, AD018–
AD023, AD025, AD027)
• TruSeq Nano DNA HT
Sample Prep Kit contents:
• DAP (DNA Adapter Plate)
1 tube of each index
being used, per
column of 8 reactions
or
1 DAP
-15°C to -25°C
Illumina
Ligation Mix 2 (LIG2)
LT kit - 1 tube per 24
reactions
or
HT kit - 1 tube per 48
reactions
-15°C to -25°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
Sample Purification Beads (SPB)
1 tube per 24
reactions
2°C to 8°C
Illumina
Stop Ligation Buffer (STL)
LT kit - 1 tube per 24
reactions
or
HT kit - 1 tube per 48
reactions
-15°C to -25°C
Illumina
Part # 15041110 Rev. B
Quantity
Storage
Supplied By
Barcode labels for:
• CAP (Clean Up ALP Plate)
• DAP (DNA Adapter Plate)
(if using the HT kit)
• PCR (Polymerase Chain
Reaction Plate)
1 label per plate
15°C to 30°C
Illumina
96-well 0.3 ml PCR plates
2
15°C to 30°C
User
Freshly prepared 80% ethanol
(EtOH)
800 µl per sample
15°C to 30°C
User
Microseal ‘B’ adhesive seals
2
15°C to 30°C
User
RNase/DNase-free eight-tube
strips and caps (if using
multichannel pipettes)
3–27
15°C to 30°C
User
RNase/DNase-free reagent
reservoirs (if using
multichannel pipettes)
3–27
15°C to 30°C
User
Preparation
} Remove the following from -15°C to -25°C storage and thaw them at room temperature:
• Appropriate DNA Adapter tubes (depending on the DNA Adapter Indices being
used) or the DAP.
NOTE
• Review the TruSeq Sample Preparation Pooling Guide (part # 15042173). See
Additional Resources on page 6 for information on how to download the guide
from the Illumina website.
• When indexing libraries using adapter index tubes, Illumina recommends
arranging samples that are going to be combined into a common pool in the
same row. Also, include a common index in each column. This arrangement
facilitates pipetting operations when dispensing indexed adapters and pooling
indexed libraries later in the protocol.
• When indexing libraries with the DAP, arrange samples that will be pooled
together in the same orientation as the indices in the DAP.
TruSeq Nano DNA Sample Preparation Guide
29
Ligate Adapters
Item
Low Sample (LS) Protocol
NOTE
When indexing libraries with the DAP:
• Review Handling Adapter Plate in the TruSeq Sample Preparation Pooling Guide (part
# 15042173). See Additional Resources on page 6 for information on how to
download the guide from the Illumina website.
• Illumina recommends that the DAP does not undergo more than four freezethaw cycles. To maximize the use of the DAP, process more than 24 samples at a
time. These samples can then be pooled in any supported configuration.
• Stop Ligation Buffer
NOTE
Do not remove the Ligation Mix 2 tube from -15°C to -25°C storage until instructed to
do so in the procedures.
} Remove the Resuspension Buffer from 2°C to 8°C storage and bring it to room
temperature.
} Review Best Practices for Handling Magnetic Beads. See Additional Resources on page 6
for information on how to access TruSeq Nano DNA Sample Preparation Best Practices
on the Illumina website.
} Remove the Sample Purification Beads from 2°C to 8°C storage and let stand for at
least 30 minutes to bring them to room temperature.
} Pre-program the thermal cycler with the following program and save as LIG:
• Choose the thermal cycler pre-heat lid option and set to 100°C
• 30°C for 10 minutes
• Hold at 4°C
} Apply a CAP barcode label to a new 96-well 0.3 ml PCR plate.
} Apply a PCR barcode label to a new 96-well 0.3 ml PCR plate.
Add LIG
1
30
Do one of the following:
• If using DNA Adapter tubes, centrifuge the thawed tubes at 600 × g for 5 seconds.
Part # 15041110 Rev. B
2
Centrifuge the Stop Ligation Buffer tube at 600 × g for 5 seconds.
3
Immediately before use, remove the Ligation Mix 2 tube from -15°C to -25°C storage.
4
Remove the adhesive seal from the ALP plate.
5
Add 2.5 µl Resuspension Buffer to each well of the ALP plate.
6
Add 2.5 µl Ligation Mix 2 to each well of the ALP plate.
7
Return the Ligation Mix 2 tube to -15°C to -25°C storage immediately after use.
8
Do one of the following:
• If using DNA Adapter tubes, add 2.5 µl thawed DNA Adapter Index to each well
of the ALP plate. Set a 200 µl pipette to 35 µl, then gently pipette the entire volume
up and down 10 times to mix thoroughly.
• If using a DAP:
— Place the DAP on the benchtop so that the part number barcode, on the long
side of the plate, is facing you and the clipped corner is on the lower left.
Figure 2 Correct DAP Orientation
TruSeq Nano DNA Sample Preparation Guide
31
Ligate Adapters
• If using a DAP:
— Thaw the plate for 10 minutes at room temperature on the benchtop. Visually
inspect the wells to make sure that they all are thawed.
— Remove the adapter plate tape seal.
— Centrifuge the plate at 280 × g for 1 minute to collect all of the adapter to the
bottom of the well.
— Remove the plastic cover. Save the cover if you are not processing the entire
plate at one time.
— If it is the first time using this DAP, apply the DAP barcode label to the plate.
Low Sample (LS) Protocol
— Do one of the following to pierce the foil seal:
— If using the entire plate at one time, use the bottom of a clean 96-well semiskirted PCR plate to pierce a hole in all of the well seals simultaneously.
Gently, but firmly, press the clean plate over the foil seal.
— If using only part of the plate, use the bottom of a clean eight-tube strip,
with caps attached, to pierce holes in the seals of the wells that will be
used for ligation. Repeat with a new, clean eight-tube strip, with caps
attached, for each row or column of adapters that will be used for ligation.
— Using an eight-tip multichannel pipette, transfer 2.5 µl thawed DNA Adapter
from the DAP well to each well of the ALP plate. Set a 200 µl pipette to 35 µl,
then gently pipette the entire volume up and down 10 times to mix thoroughly.
9
Seal the ALP plate with a Microseal ‘B’ adhesive seal.
10 Centrifuge the ALP plate at 280 × g for 1 minute.
Incubate 2 ALP
1
Place the sealed ALP plate, containing 37.5 µl of each sample, on the pre-programmed
thermal cycler. Close the lid then select and run the LIG program.
a Choose the thermal cycler pre-heat lid option and set to 100°C
b 30°C for 10 minutes
c Hold at 4°C
2
Remove the ALP plate from the thermal cycler when the program reaches 4°C.
1
Remove the adhesive seal from the ALP plate.
2
Add 5 µl Stop Ligation Buffer to each well of the ALP plate to inactivate the ligation.
Set a 200 µl pipette to 40 µl, then gently pipette the entire volume up and down
10 times to mix thoroughly.
Add STL
Clean Up ALP
1
32
Vortex the Sample Purification Beads for at least 1 minute or until they are well
dispersed.
Part # 15041110 Rev. B
Add 42.5 µl well-mixed Sample Purification Beads to each well of the ALP plate. Set a
200 µl pipette to 75 µl, and then gently pipette the entire volume up and down
10 times to mix thoroughly.
NOTE
Vortex the Sample Purification Beads frequently to make sure that they are evenly
distributed. Illumina recommends the following:
• If using a single channel pipette, vortex the beads after processing four samples
• If using a multichannel pipette, vortex the beads after processing four columns
• If the beads are in a reagent reservoir, mix with a 1000 µl pipette.
3
Incubate the ALP plate at room temperature for 5 minutes.
4
Place the ALP plate on the magnetic stand at room temperature for 5 minutes or until
the liquid is clear.
5
Remove and discard 80 µl of the supernatant from each well of the ALP plate. Take
care not to disturb the beads.
NOTE
Leave the ALP plate on the magnetic stand while performing the following steps 6–10.
6
With the ALP plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to
each well without disturbing the beads.
7
Incubate the ALP plate at room temperature for 30 seconds, and then remove and
discard all of the supernatant from each well. Take care not to disturb the beads.
8
Repeat steps 6 and 7 one time for a total of two 80% EtOH washes.
9
With the ALP plate on the magnetic stand, let the samples air-dry at room temperature
for 5 minutes. Remove and discard any remaining EtOH with a 10 µl pipette.
10 With the ALP plate on the magnetic stand, add 52.5 µl Resuspension Buffer to each
well of the plate.
11 Remove the ALP plate from the magnetic stand.
12 Resuspend the beads in each well of the ALP plate by repeatedly dispensing the
Resuspension Buffer over the bead pellet until it is immersed in the solution. Gently
pipette the entire volume up and down 10 times to mix thoroughly.
13 Incubate the ALP plate at room temperature for 2 minutes.
14 Place the ALP plate on the magnetic stand at room temperature for 5 minutes or until
the liquid is clear.
TruSeq Nano DNA Sample Preparation Guide
33
Ligate Adapters
2
Low Sample (LS) Protocol
15 Transfer 50 µl of the clear supernatant from each well of the ALP plate to the
corresponding well of the new 0.3 ml PCR plate labeled with the CAP barcode. Take
care not to disturb the beads.
16 Vortex the Sample Purification Beads until they are well dispersed.
17 Add 50 µl mixed Sample Purification Beads to each well of the CAP plate for a second
cleanup. Set a 200 µl pipette to 90 µl, and then gently pipette the entire volume up and
down 10 times to mix thoroughly.
18 Incubate the CAP plate at room temperature for 5 minutes.
19 Place the CAP plate on the magnetic stand at room temperature for 5 minutes or until
the liquid is clear.
20 Remove and discard 95 µl of the supernatant from each well of the CAP plate. Take
care not to disturb the beads.
NOTE
Leave the CAP plate on the magnetic stand while performing the following steps 21–25.
21 With the CAP plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to
each well. Take care not to disturb the beads.
22 Incubate the CAP plate at room temperature for 30 seconds, and then remove and
discard all of the supernatant from each well. Take care not to disturb the beads.
23 Repeat steps 21 and 22 one time for a total of two 80% EtOH washes.
24 With the CAP plate on the magnetic stand, let the samples air-dry at room temperature
for 5 minutes. Remove and discard any remaining EtOH with a 10 µl pipette.
25 With the CAP plate on the magnetic stand, add 27.5 µl Resuspension Buffer to each
well of the plate.
26 Remove the CAP plate from the magnetic stand.
27 Resuspend the beads in each well of the CAP plate by repeatedly dispensing the
Resuspension Buffer over the bead pellet until it is immersed in the solution. Gently
pipette the entire volume up and down 10 times to mix thoroughly.
28 Incubate the CAP plate at room temperature for 2 minutes.
29 Place the CAP plate on the magnetic stand at room temperature for 5 minutes or until
the liquid is clear.
34
Part # 15041110 Rev. B
SAFE STOPPING POINT
If you do not plan to proceed immediately to Enrich DNA Fragments on page 36, you can
safely stop the protocol here. If you are stopping, seal the PCR plate with a Microseal ‘B’
adhesive seal and store at -15°C to -25°C for up to 7 days.
TruSeq Nano DNA Sample Preparation Guide
35
Ligate Adapters
30 Transfer 25 µl of the clear supernatant from each well of the CAP plate to the
corresponding well of the new 0.3 ml PCR plate labeled with the PCR barcode. Take
care not to disturb the beads.
Low Sample (LS) Protocol
Enrich DNA Fragments
This process uses PCR to selectively enrich those DNA fragments that have adapter
molecules on both ends and to amplify the amount of DNA in the library. The PCR is
performed with a PCR Primer Cocktail that anneals to the ends of the adapters. Minimize
the number of PCR cycles to avoid skewing the representation of the library.
NOTE
PCR enriches for fragments that have adapters ligated on both ends. Fragments with only
one or no adapters on their ends are by-products of inefficiencies in the ligation reaction.
Neither species can be used to make clusters. Fragments without any adapters cannot
hybridize to surface-bound primers in the flow cell. Fragments with an adapter on only one
end can hybridize to surface bound primers, but cannot form clusters.
Consumables
36
Item
Quantity
Storage
Supplied By
Enhanced PCR Mix (EPM)
LT kit - 1 tube per 24
reactions
or
HT kit - 1 tube per 48
reactions
-15°C to -25°C
Illumina
PCR Primer Cocktail (PPC)
LT kit - 1 tube per 24
reactions
or
HT kit - 1 tube per 48
reactions
-15°C to -25°C
Illumina
Sample Purification Beads
(SPB)
1 tube per 24 reactions
2°C to 8°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
TSP1 (Target Sample Plate)
barcode label
1 label per plate
15°C to 30°C
Illumina
96-well 0.3 ml PCR plate
1
15°C to 30°C
User
Part # 15041110 Rev. B
Quantity
Storage
Supplied By
Freshly prepared 80%
ethanol (EtOH)
400 µl per sample
15°C to 30°C
User
Microseal ‘B’ adhesive seals
2
15°C to 30°C
User
RNase/DNase-free eighttube strips and caps
(if using multichannel
pipettes)
5
15°C to 30°C
User
RNase/DNase-free reagent
reservoirs
(if using multichannel
pipettes)
5
15°C to 30°C
User
Preparation
} Remove the Enhanced PCR Mix and PCR Primer Cocktail from -15°C to -25°C storage
and thaw them at room temperature.
} Centrifuge the thawed Enhanced PCR Mix and PCR Primer Cocktail tubes to 600 × g
for 5 seconds.
} Remove the Resuspension Buffer from 2°C to 8°C storage and bring it to room
temperature.
} Remove the Sample Purification Beads from 2°C to 8°C storage and let stand for at
least 30 minutes to bring them to room temperature.
} Review Best Practices for Handling Magnetic Beads. See Additional Resources on page 6
for information on how to access TruSeq Nano DNA Sample Preparation Best Practices
on the Illumina website.
} Remove the PCR plate from -15°C to -25°C storage, if it was stored at the conclusion of
Clean Up ALP on page 32.
• Let it thaw at room temperature.
• Centrifuge the thawed PCR plate at 280 × g for 1 minute.
• Remove the adhesive seal from the thawed PCR plate.
TruSeq Nano DNA Sample Preparation Guide
37
Enrich DNA Fragments
Item
Low Sample (LS) Protocol
} Pre-program the thermal cycler with the following program and save as PCRNano:
• Choose the pre-heat lid option and set to 100°C
• 95°C for 3 minutes
• 8 cycles of:
— 98°C for 20 seconds
— 60°C for 15 seconds
— 72°C for 30 seconds
• 72°C for 5 minutes
• Hold at 4°C
NOTE
Illumina recommends 8 cycles of PCR for robust protocol performance.
} Apply a TSP1 barcode label to a new 96-well 0.3 ml PCR plate.
Make PCR
The following procedure assumes the following amount of input DNA sample for library
preparation and is designed to result in high library yields:
} 100 ng for a 350 bp insert size
} 200 ng for a 550 bp insert size
38
1
Add 5 µl thawed PCR Primer Cocktail to each well of the PCR plate.
2
Add 20 µl thawed Enhanced PCR Mix to each well of the PCR plate. Set a 200 µl
pipette to 40 µl, then gently pipette the entire volume up and down 10 times to mix
thoroughly.
3
Seal the PCR plate with a Microseal ‘B’ adhesive seal.
Part # 15041110 Rev. B
1
Place the sealed PCR plate, containing 50 µl of each sample, on the pre-programmed
thermal cycler. Close the lid, then select and run PCRNano to amplify the plate.
a Choose the pre-heat lid option and set to 100°C
b 95°C for 3 minutes
c 8 cycles of:
— 98°C for 20 seconds
— 60°C for 15 seconds
— 72°C for 30 seconds
d 72°C for 5 minutes
e Hold at 4°C
Clean Up PCR
1
Centrifuge the PCR plate at 280 × g for 1 minute.
2
Remove the adhesive seal from the PCR plate.
3
Vortex the Sample Purification Beads until they are well dispersed.
4
Do one of the following, depending on the adapter type used:
• If using the DNA Adapter tubes, add 50 µl mixed Sample Purification Beads to
each well of the PCR plate containing 50 µl of the PCR amplified library. Set a 200
µl pipette to 90 µl, then gently pipette the entire volume up and down 10 times to
mix thoroughly.
• If using the DAP, add 47.5 µl mixed Sample Purification Beads to each well of the
PCR plate containing 50 µl of the PCR amplified library. Set a 200 µl pipette to 90
µl, then gently pipette the entire volume up and down 10 times to mix thoroughly.
5
Incubate the PCR plate at room temperature for 5 minutes.
6
Place the PCR plate on the magnetic stand at room temperature for 5 minutes or until
the liquid is clear.
7
Remove and discard 95 µl of the supernatant from each well of the PCR plate.
NOTE
Leave the PCR plate on the magnetic stand while performing the following 80% EtOH
wash steps (8–10).
TruSeq Nano DNA Sample Preparation Guide
39
Enrich DNA Fragments
Amp PCR
Low Sample (LS) Protocol
8
With the PCR plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to
each well without disturbing the beads.
9
Incubate the PCR plate at room temperature for 30 seconds, and then remove and
discard all of the supernatant from each well.
10 Repeat steps 8 and 9 one time for a total of two 80% EtOH washes.
11 With the PCR plate on the magnetic stand, let the samples air-dry at room temperature
for 5 minutes. Remove and discard any remaining EtOH from each well of the PCR
plate with a 10 µl pipette.
12 With the PCR plate on the magnetic stand, add 32.5 µl Resuspension Buffer to each
well of the PCR plate.
13 Remove the PCR plate from the magnetic stand.
14 Resuspend the beads in each well of the PCR plate by repeatedly dispensing the
Resuspension Buffer over the bead pellet until it is immersed in the solution. Gently
pipette the entire volume up and down 10 times to mix thoroughly.
15 Incubate the PCR plate at room temperature for 2 minutes.
16 Place the PCR plate on the magnetic stand at room temperature for 5 minutes or until
the liquid is clear.
17 Transfer 30 µl of the clear supernatant from each well of the PCR plate to the
corresponding well of the new 0.3 ml PCR plate labeled with the TSP1 barcode.
SAFE STOPPING POINT
If you do not plan to proceed immediately to Validate Library on page 41, you can safely stop
the protocol here. If you are stopping, seal the TSP1 plate with a Microseal ‘B’ adhesive seal
and store at -15°C to -25°C for up to 7 days.
40
Part # 15041110 Rev. B
Illumina recommends performing the following procedures for quality control analysis on
your sample library and quantification of the DNA library templates.
Quantify Libraries
To achieve the highest data quality on Illumina sequencing platforms, it is important to
create optimum cluster densities across every lane of a flow cell. Optimizing cluster
densities requires accurate quantitation of DNA library templates. Quantify your libraries
using a fluorometric quantification method that uses dsDNA binding dyes or qPCR.
NOTE
TruSeq Nano DNA Sample Prep library quantitation has been validated using the Eco RealTime PCR System and KAPA Library Quantification Kit specified in the Consumables and
Equipment on page 95. Follow the KAPA instructions with the KAPA standard. To calculate
the library concentration in nM, perform the following insert size adjustment:
• For 350 bp libraries, use 470 bp for the average fragment length
• For 550 bp libraries, use 670 bp for the average fragment length
NOTE
You can download the KAPA Library Quantification Kits for Illumina sequencing platforms
Technical Data Sheet from the Kapa Biosystems website (www.kapabiosystems.com).
[Optional] Quality Control
To verify the size of your fragments, check the template size distribution. Run samples on a
Bioanalyzer for qualitative purposes only.
Do one of the following:
• If using a High Sensitivity DNA chip:
— Prepare a 1:100 dilution of the DNA library with water.
— Run 1 µl of the diluted DNA library on an Agilent Technologies 2100
Bioanalyzer.
• If using a DNA 7500 chip, run 1 µl of undiluted DNA library on an Agilent
Technologies 2100 Bioanalyzer.
TruSeq Nano DNA Sample Preparation Guide
41
Validate Library
Validate Library
Low Sample (LS) Protocol
Figure 3 Example TruSeq Nano DNA Sample Preparation 350 bp Insert Library Distribution
Figure 4 Example TruSeq Nano DNA Sample Preparation 550 bp Insert Library Distribution
42
Part # 15041110 Rev. B
This process describes how to prepare DNA templates for cluster generation. Indexed DNA
libraries are normalized to 10 nM in the DCT plate and then pooled in equal volumes in
the PDP plate. DNA libraries not intended for pooling are normalized to 10 nM in the DCT
plate.
Consumables
Item
Quantity
Storage
Supplied By
Barcode labels for:
• DCT (Diluted Cluster
Template)
• PDP (Pooled DCT Plate)
(for pooling only)
1 label per plate
15°C to 30°C
Illumina
1.7 ml microcentrifuge tube
(when processing > 48 samples
at a time)
1
15°C to 30°C
User
96-well MIDI plates
2
(second plate for
pooling only, if
pooling > 40 samples)
15°C to 30°C
User
96-well 0.3 ml PCR plate
(for pooling only, if pooling
≤ 40 samples)
1
15°C to 30°C
User
Microseal ‘B’ adhesive seals
2
15°C to 30°C
User
Tris-HCl 10 mM, pH8.5
with 0.1% Tween 20
Enough to normalize
the concentration of
each sample library
to 10 nM
15°C to 30°C
User
TruSeq Nano DNA Sample Preparation Guide
43
Normalize and Pool Libraries
Normalize and Pool Libraries
Low Sample (LS) Protocol
Preparation
} Remove the TSP1 plate from -15°C to -25°C storage, if it was stored at the conclusion of
Clean Up PCR on page 39.
• Let it thaw at room temperature.
• Centrifuge the thawed TSP1 plate at 280 × g for 1 minute.
• Remove the adhesive seal from the thawed TSP1 plate.
} Apply a DCT barcode label to a new 96-well MIDI plate.
} [For pooling only] Apply a PDP barcode label to a new 96-well 0.3 ml PCR plate if
pooling ≤ 40 samples or a 96-well MIDI plate if pooling > 40 samples.
Make DCT
1
Transfer 10 µl of sample library from each well of the TSP1 plate to the corresponding
well of the new MIDI plate labeled with the DCT barcode.
2
Normalize the concentration of sample library in each well of the DCT plate to 10 nM
using Tris-HCl 10 mM, pH 8.5 with 0.1% Tween 20.
NOTE
Depending on the yield quantification data of each sample library, the final volume in
the DCT plate can vary from 10–400 µl.
3
Gently pipette the entire normalized sample library volume up and down 10 times to
mix thoroughly.
4
Depending on the type of library you want to generate, do one of the following:
• For non-pooled libraries, the protocol stops here. Do one of the following:
— Proceed to cluster generation. For more information, see the cluster generation
section of the user guide for your Illumina platform.
— Seal the DCT plate with a Microseal ‘B’ adhesive seal and store at
-15°C to -25°C.
• For pooled libraries, proceed to Make PDP (for pooling only).
Make PDP (for pooling only)
NOTE
Do not make a PDP plate if you are not pooling samples.
44
Part # 15041110 Rev. B
Determine the number of samples to be combined together for each pool.
NOTE
Note the sample that is in each well, to avoid pooling two samples with the same index.
2
Do one of the following:
• If pooling 2–24 samples:
— Transfer 10 µl of each normalized sample library to be pooled from the DCT
plate to one well of the new 0.3 ml PCR plate labeled with the PDP barcode.
The total volume in each well of the PDP plate is 10 X the number of combined
sample libraries and 20–240 µl (2–24 libraries). For example, the volume for 2
samples is 20 µl, the volume for 12 samples is 120 µl, or the volume for 24
samples is 240 µl.
• If pooling 25–48 samples:
— Using a multichannel pipette, transfer 5 µl of each normalized sample library
in column 1 of the DCT plate to column 1 of the new 0.3 ml PCR or MIDI plate
labeled with the PDP barcode.
— Transfer 5 µl of each normalized sample library in column 2 of the DCT plate
to column 1 of the PDP plate.
— Repeat the transfer for as many times as there are remaining columns in the
DCT plate. The result is a PDP plate with pooled samples in column 1. Gently
pipette the entire volume of each well of column 1 up and down 10 times to
mix thoroughly.
— Combine the contents of each well of column 1 into well A2 of the PDP plate
for the final pool.
• If pooling 49–96 samples:
— Using a multichannel pipette, transfer 5 µl of each normalized sample library
in column 1 of the DCT plate to column 1 of the new MIDI plate labeled with
the PDP barcode.
— Transfer 5 µl of each normalized sample library in column 2 of the DCT plate
to column 1 of the PDP plate.
— Repeat the transfer for as many times as there are remaining columns in the
DCT plate. The result is a PDP plate with pooled samples in column 1. Gently
pipette the entire volume of each well of column 1 up and down 10 times to
mix thoroughly.
— Combine the contents of each well of column 1 into a 1.7 ml microcentrifuge
tube for the final pool.
TruSeq Nano DNA Sample Preparation Guide
45
Normalize and Pool Libraries
1
Low Sample (LS) Protocol
46
3
Gently pipette the entire volume up and down 10 times to mix thoroughly.
4
Do one of the following:
• Proceed to cluster generation. For more information, see the user guide for your
Illumina sequencer.
• Do one of the following, depending on the item that contains the final pool:
— Seal the PDP plate with a Microseal ‘B’ adhesive seal and store at -15°C
to -25°C.
— Cap the 1.7 ml microcentrifuge tube and store at -15°C to -25°C.
Part # 15041110 Rev. B
Chapter 3 High Sample (HS) Protocol
Introduction
Sample Prep Workflow
Prepare Adapter Setup
Fragment DNA
Perform End Repair and Size Selection
Adenylate 3' Ends
Ligate Adapters
Enrich DNA Fragments
Validate Library
Normalize and Pool Libraries
TruSeq Nano DNA Sample Preparation Guide
48
49
50
51
57
64
66
74
79
81
47
Chapter 3
High Sample (HS) Protocol
High Sample (HS) Protocol
Introduction
This chapter describes the TruSeq Nano DNA Sample Preparation HS protocol. This
protocol is intended for preparing more than 24 samples at one time using either the LT or
HT kit.
} Follow the protocols in the order described, using the specified volumes and incubation
parameters.
} Review Best Practices before proceeding. See Additional Resources on page 6 for
information on how to access TruSeq Nano DNA Sample Preparation Best Practices on
the Illumina website.
} Review the TruSeq Sample Preparation Pooling Guide (part # 15042173) before proceeding.
See Additional Resources on page 6 for information on how to download the guide from
the Illumina website.
} Review Appendix A Supporting Information before proceeding, to confirm your kit
contents and make sure that you have obtained all of the requisite equipment and
consumables for the HS protocol.
} This HS protocol requires shaking and heating equipment to mix reagents and for
incubation (see User-Supplied Equipment - Additional Items for HS Processing on page 98).
48
Part # 15041110 Rev. B
The following figure illustrates the processes of the TruSeq Nano DNA Sample Preparation
HS protocol to prepare templates using indexed adapter tubes or a DAP.
Figure 5 TruSeq Nano DNA Sample Preparation HS Workflow
TruSeq Nano DNA Sample Preparation Guide
49
Sample Prep Workflow
Sample Prep Workflow
High Sample (HS) Protocol
Prepare Adapter Setup
If you are pooling, record information about your samples before beginning library
preparation for later use in data analysis.
} Use IEM to create and edit a sample sheet for Illumina sequencers and analysis
software. See Additional Resources on page 6 for information on how to download IEM
software and documentation from the Illumina website.
} Review planning steps in the TruSeq Sample Preparation Pooling Guide (part # 15042173).
See Additional Resources on page 6 for information on how to download the guide from
the Illumina website.
If you are pooling using adapter index tubes, Illumina recommends arranging samples
that will be combined into a common pool in the same row. Include a common index in
each column. This arrangement facilitates pipetting operations when dispensing indexed
adapters and pooling indexed libraries later in the protocol.
If you are pooling with the DAP, arrange samples that will be pooled together in the same
orientation as the indices in the DAP.
50
Part # 15041110 Rev. B
Fragment DNA
Fragment DNA
This process describes how to optimally fragment the gDNA depending on the
downstream application. Covaris shearing generates dsDNA fragments with 3' or 5'
overhangs. The fragmentation process is optimized to obtain final libraries with the
following average insert sizes:
Table 9 Insert Size Options
Insert Size
Input DNA Per Sample
Recommended Read Length
350 bp
550 bp
100 ng
≤ 2 x 101 bp
200 ng
≤ 2 x 151 bp*
* Read lengths greater than 2 x 151 bp produce a significantly higher percentage of overlapping readpairs.
Consumables
Item
Quantity
Storage
Supplied By
Resuspension Buffer (RSB)
1 tube
-15°C to -25°C
(2°C to 8°C
after initial
thaw)
Illumina
Sample Purification Beads (SPB)
1 tube per 24
reactions
2°C to 8°C
Illumina
Barcode labels for:
• CFP (Covaris Fragmentation
Plate)
• CSP (Clean Up Sheared DNA
Plate)
• DNA (DNA Plate)
• IMP (Insert Modification
Plate)
1 label per plate
15°C to 30°C
Illumina
96-well HSP plate
1
15°C to 30°C
User
TruSeq Nano DNA Sample Preparation Guide
51
High Sample (HS) Protocol
Item
Quantity
Storage
Supplied By
96-well MIDI plates
3
15°C to 30°C
User
Covaris tubes
1 per sample
15°C to 30°C
User
DNA samples
100 ng per sample
for a 350 bp insert
size
or
200 ng per sample
for a 550 bp insert
size
-15°C to -25°C
User
Freshly prepared 80% ethanol
(EtOH)
400 µl per sample
15°C to 30°C
User
Microseal ‘B’ adhesive seal
1
15°C to 30°C
User
Preparation
} Review DNA Input Recommendations on page 4.
} Review Best Practices for Handling Magnetic Beads. See Additional Resources on page 6
for information on how to access TruSeq Nano DNA Sample Preparation Best Practices
on the Illumina website.
} Remove the Sample Purification Beads from 2°C to 8°C storage and let stand for at
least 30 minutes to bring them to room temperature.
} Remove one tube of Resuspension Buffer from -15°C to -25°C storage and thaw it at
room temperature.
NOTE
The Resuspension Buffer can be stored at 2°C to 8°C after the initial thaw.
} Turn on the Covaris instrument and follow the manufacturer's guidelines to set up
your instrument.
} Calibrate the microplate shaker with a stroboscope and set it to 1800 rpm.
} Apply a CFP barcode label to a new 96-well HSP plate
} Apply a CSP barcode label to a new 96-well MIDI plate.
} Apply a DNA barcode label to a new 96-well MIDI plate.
} Apply an IMP barcode label to a new 96-well MIDI plate.
52
Part # 15041110 Rev. B
1
Illumina recommends quantifying gDNA samples using a fluorometric quantification
method that uses dsDNA binding dyes.
2
Normalize the gDNA samples with Resuspension Buffer to one of the following in each
well of the new 0.3 ml PCR plate labeled with the DNA barcode:
• 100 ng in a final volume of 52.5 µl for a 350 bp insert size
• 200 ng in a final volume of 52.5 µl for a 550 bp insert size
Fragment DNA
1
Shear one of the following amounts of gDNA sample by transferring 52.5 µl of each
DNA sample from the DNA plate to a separate, new Covaris tube:
• 100 ng for a 350 bp insert size
• 200 ng for a 550 bp insert size
Use the wells of the new HSP plate labeled with CFP barcode or another device to hold
the Covaris tubes upright.
NOTE
Load the DNA sample into the Covaris tube slowly to avoid creating air bubbles.
However, air bubbles might not be preventable.
2
Centrifuge the CFP plate at 600 × g for 5 seconds.
3
Fragment the DNA using the following settings:
Table 10 Covaris S220 Settings
Setting
350 bp Insert
Duty factor
5%
Peak Incident Power
175 W
Cycles per burst
Duration
Mode
Temperature
TruSeq Nano DNA Sample Preparation Guide
550 bp Insert
200
50 seconds
25 seconds
Frequency sweeping
5.5° to 6°C
53
Fragment DNA
Make CFP
High Sample (HS) Protocol
Table 11 Covaris M220 Settings
Setting
350 bp Insert
550 bp Insert
Duty factor
20%
Peak Incident Power
50 W
Cycles per burst
Duration
200
65 seconds
45 seconds
Temperature
20°C
NOTE
The Covaris M220 settings are optimized for use with the Covaris microTUBE AFA
Fiber Pre-Slit Snap-Cap 6x16mm.
Table 12 Covaris S2 and E210 Settings
Setting
350 bp Insert
Duty cycle
Intensity
10%
5.0
Cycles per burst
Displayed Power
Temperature
54
2.0
200
Duration
Mode
550 bp Insert
45 seconds
Frequency sweeping
S2—23 W
S2—9 W
E210—14 W
E210—7 W
5.5° to 6°C
4
Centrifuge the CFP plate at 600 × g for 5 seconds.
5
Transfer 50 µl of fragmented DNA from each Covaris tube in the CFP plate to the
corresponding well of the new MIDI plate labeled with the CSP barcode, using a single
channel pipette.
Part # 15041110 Rev. B
Proceed immediately to Clean Up Fragmented DNA.
Clean Up Fragmented DNA
1
Vortex the room temperature Sample Purification Beads for at least 1 minute or until
they are well dispersed.
2
Add 80 µl well-mixed Sample Purification Beads to each well of the CSP plate
containing 50 µl of fragmented gDNA. Mix thoroughly as follows:
a Seal the CSP plate with a Microseal ‘B’ adhesive seal.
b Shake the CSP plate on a microplate shaker at 1800 rpm for 2 minutes.
NOTE
Vortex the Sample Purification Beads frequently to make sure that they are evenly
distributed. Illumina recommends the following:
• If using a single channel pipette, vortex the beads after processing four samples
• If using a multichannel pipette, vortex the beads after processing four columns
• If the beads are in a reagent reservoir, mix with a 1000 µl pipette.
NOTE
Keep the Sample Purification Beads tube at room temperature for later use in the
protocol.
3
Incubate the CSP plate at room temperature for 5 minutes.
4
Centrifuge the CSP plate at 280 × g for 1 minute.
5
Remove the adhesive seal from the CSP plate.
6
Place the CSP plate on the magnetic stand at room temperature for 8 minutes or until
the liquid is clear.
7
Using a 200 µl single channel or multichannel pipette set to 125 µl, remove and
discard 125 µl of the supernatant from each well of the CSP plate. Take care not to
disturb the beads.
NOTE
Leave the CSP plate on the magnetic stand while performing the following steps 8–12.
8
With the CSP plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to
each well without disturbing the beads.
9
Incubate the CSP plate at room temperature for 30 seconds, and then remove and
discard all of the supernatant from each well. Take care not to disturb the beads.
TruSeq Nano DNA Sample Preparation Guide
55
Fragment DNA
6
High Sample (HS) Protocol
10 Repeat steps 8 and 9 one time for a total of two 80% EtOH washes.
11 With the CSP plate on the magnetic stand, let the samples air-dry at room temperature
for 5 minutes. Remove and discard any remaining EtOH with a 10 µl pipette.
12 With the CSP plate on the magnetic stand, add 62.5 µl Resuspension Buffer to each
well of the plate.
13 Remove the CSP plate from the magnetic stand.
14 Mix thoroughly as follows:
a Seal the CSP plate with a Microseal ‘B’ adhesive seal.
b Shake the CSP plate on a microplate shaker at 1800 rpm for 2 minutes.
15 Incubate the CSP plate at room temperature for 2 minutes.
16 Centrifuge the CSP plate at 280 × g for 1 minute.
17 Remove the adhesive seal from the CSP plate.
18 Place the CSP plate on the magnetic stand at room temperature for 5 minutes or until
the liquid is clear.
19 Transfer 60 µl of the clear supernatant from each well of the CSP plate to the
corresponding well of the new MIDI plate labeled with the IMP barcode. Take care not
to disturb the beads.
20 Proceed immediately to Perform End Repair and Size Selection on page 57.
56
Part # 15041110 Rev. B
This process converts the overhangs resulting from fragmentation into blunt ends using
End Repair Mix 2. The 3' to 5' exonuclease activity of this mix removes the 3' overhangs
and the 5' to 3' polymerase activity fills in the 5' overhangs. Following end repair, the
appropriate library size is selected using different ratios of the Sample Purification Beads.
Consumables
Item
Quantity
Storage
Supplied By
End Repair Mix 2 (ERP2)
LT kit - 1 tube per 24
reactions
or
HT kit - 1 tube per 48
reactions
-15°C to -25°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
Sample Purification Beads (SPB)
1 tube per 24
reactions
2°C to 8°C
Illumina
Barcode labels for:
• ALP (Adapter Ligation Plate)
• CEP (Clean Up End Repair
Plate)
1 label per plate
15°C to 30°C
Illumina
15 ml conical tube
(when processing > 6 samples
at a time)
or
1.7 ml microcentrifuge tube
(when processing ≤ 6 samples
at a time)
1
15°C to 30°C
User
96-well MIDI plates
2
15°C to 30°C
User
Freshly prepared 80% ethanol
(EtOH)
400 µl per sample
15°C to 30°C
User
TruSeq Nano DNA Sample Preparation Guide
57
Perform End Repair and Size Selection
Perform End Repair and Size Selection
High Sample (HS) Protocol
Item
Quantity
Storage
Supplied By
Ice bucket
As needed
-15°C to -25°C
User
Microseal ‘B’ adhesive seals
5
15°C to 30°C
User
PCR grade water
1 bottle
15°C to 30°C
User
RNase/DNase-free eight-tube
strips and caps (if using
multichannel pipettes)
5
15°C to 30°C
User
RNase/DNase-free reagent
reservoirs (if using
multichannel pipettes)
5
15°C to 30°C
User
Preparation
} Prepare an ice bucket.
} Remove the End Repair Mix 2 from -15°C to -25°C storage and thaw it at room
temperature. Place the tube on ice.
} Review Best Practices for Handling Magnetic Beads. See Additional Resources on page 6
for information on how to access TruSeq Nano DNA Sample Preparation Best Practices
on the Illumina website.
} Make sure that the Sample Purification Beads and Resuspension Buffer are at room
temperature.
} Pre-heat the microheating system to 30°C.
} Apply an ALP barcode label to a new 96-well MIDI plate.
} Apply a CEP barcode label to a new 96-well MIDI plate.
Make IMP
58
1
Centrifuge the thawed End Repair Mix 2 tube at 600 × g for 5 seconds.
2
Add 40 µl End Repair Mix 2 to each well of the IMP plate containing the fragmented
DNA. Mix thoroughly as follows:
a Seal the IMP plate with a Microseal ‘B’ adhesive seal.
b Shake the IMP plate on a microplate shaker at 1800 rpm for 2 minutes.
3
Centrifuge the IMP plate at 280 × g for 1 minute.
Part # 15041110 Rev. B
Return the End Repair Mix 2 tube to -15°C to -25°C storage.
Incubate IMP
1
Place the sealed IMP plate on the pre-heated microheating system. Close the lid and
incubate at 30°C for 30 minutes.
2
Remove the IMP plate from the microheating system and place the plate on ice until
you are ready for the next step.
Clean Up IMP and Size Selection
1
Remove the adhesive seal from the IMP plate.
Remove Large DNA Fragments
1
Vortex the Sample Purification Beads for at least 1 minute or until they are well
dispersed.
TruSeq Nano DNA Sample Preparation Guide
59
Perform End Repair and Size Selection
4
High Sample (HS) Protocol
2
Add the Sample Purification Beads and PCR grade water to one of the following tubes,
to create a diluted bead mixture of 160 µl per 100 µl of end-repaired sample:
• New 15 ml conical tube, when processing > 6 samples at a time
• New 1.7 ml microcentrifuge tube, when processing ≤ 6 samples at a time
Determine the volumes using the following formulas, which include 15% excess for
multiple samples:
Table 13 Diluted Bead Mixture for a 350 bp Insert Size
Formula
Sample Purification Beads
PCR grade water
# of samples X
109.25 µl
# of samples X
74.75 µl
Table 14 Diluted Bead Mixture for a 550 bp Insert Size
Formula
Sample Purification Beads
PCR grade water
# of samples X
92 µl
# of samples X
92 µl
Example
Amount
per 12
samples
Your
Calculation
1311 µl
897 µl
Example
Amount
per 12
samples
1104 µl
Your
Calculation
1104 µl
3
Vortex the diluted bead mixture for 5 seconds to make sure that the beads are evenly
dispersed.
4
Add 160 µl of the diluted bead mixture to each well of the IMP plate containing 100 µl
of the end repaired sample. Mix thoroughly as follows:
a Seal the IMP plate with a Microseal ‘B’ adhesive seal.
b Shake the IMP plate on a microplate shaker at 1800 rpm for 2 minutes.
NOTE
Aspirate the diluted bead mixture slowly and dispense it slowly due to the viscosity of
the solution. Changes in the volume of the diluted bead mixture affect the insert size of
your library.
60
Part # 15041110 Rev. B
5
Incubate the IMP plate at room temperature for 5 minutes.
6
Centrifuge the IMP plate at 280 × g for 1 minute.
7
Remove the adhesive seal from the IMP plate.
8
Place the IMP plate on the magnetic stand at room temperature for 5 minutes or until
the liquid is clear.
9
Use a 200 µl single channel or multichannel pipette set to 125 µl to transfer 125 µl of
the supernatant, containing the DNA of interest, from each well of the IMP plate to
the corresponding well of the new MIDI plate labeled with the CEP barcode. Take
care not to disturb the beads.
NOTE
Transfer, do not discard, the supernatant. It contains the DNA of interest.
10 Repeat step 9 one time. Each CEP plate well now contains a total of 250 µl of DNA of
interest.
11 Discard the IMP plate containing the beads.
12 Discard any remaining diluted bead mixture.
Remove Small DNA Fragments
NOTE
In the following steps, use undiluted Sample Purification Beads.
1
Vortex the Sample Purification Beads for at least 1 minute or until they are well
dispersed.
2
Add 30 µl undiluted Sample Purification Beads to each well of the CEP plate
containing 250 µl of supernatant with the DNA of interest. Mix thoroughly as follows:
a Seal the CEP plate with a Microseal ‘B’ adhesive seal.
b Shake the CEP plate on a microplate shaker at 1800 rpm for 2 minutes.
TruSeq Nano DNA Sample Preparation Guide
61
Perform End Repair and Size Selection
NOTE
Vortex the diluted bead mixture frequently. Illumina recommends the following:
• If using a single channel pipette, vortex the mixture after processing four samples
• If using a multichannel pipette, vortex the mixture after processing four columns
• If the mixture is in a reagent reservoir, mix with a 1000 µl pipette.
High Sample (HS) Protocol
NOTE
Aspirate the Sample Purification Beads slowly and dispense them slowly due to the
viscosity of the solution. Changes in the volume of the bead mixture affect the insert size
of your library.
NOTE
Vortex the Sample Purification Beads frequently to make sure that they are evenly
distributed. Illumina recommends the following:
• If using a single channel pipette, vortex the beads after processing four samples
• If using a multichannel pipette, vortex the beads after processing four columns
• If the beads are in a reagent reservoir, mix with a 1000 µl pipette.
3
Incubate the CEP plate at room temperature for 5 minutes.
4
Centrifuge the CEP plate at 280 × g for 1 minute.
5
Remove the adhesive seal from the CEP plate.
6
Place the CEP plate on the magnetic stand at room temperature for 5 minutes or until
the liquid is clear.
7
Using a 200 µl single channel or multichannel pipette set to 138 µl, remove and
discard 138 µl of the supernatant from each well of the CEP plate. Take care not to
disturb the beads.
8
Repeat step 7 one time, removing and discarding a total of 276 µl of supernatant from
each well.
NOTE
Leave the CEP plate on the magnetic stand while performing the following steps 9–13.
9
With the CEP plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to
each well without disturbing the beads.
10 Incubate the CEP plate at room temperature for 30 seconds, and then remove and
discard all of the supernatant from each well. Take care not to disturb the beads.
11 Repeat steps 9 and 10 one time for a total of two 80% EtOH washes.
12 With the CEP plate on the magnetic stand, let the samples air-dry at room temperature
for 5 minutes. Remove and discard any remaining EtOH with a 10 µl pipette.
13 With the CEP plate on the magnetic stand, add 20 µl Resuspension Buffer to each well
of the plate.
14 Remove the CEP plate from the magnetic stand.
62
Part # 15041110 Rev. B
16 Incubate the CEP plate at room temperature for 2 minutes.
17 Centrifuge the CEP plate at 280 × g for 1 minute.
18 Remove the adhesive seal from the CEP plate.
19 Place the CEP plate on the magnetic stand at room temperature for 5 minutes or until
the liquid is clear.
20 Transfer 17.5 µl of the clear supernatant from each well of the CEP plate to the
corresponding well of the new MIDI plate labeled with the ALP barcode.
SAFE STOPPING POINT
If you do not plan to proceed to Adenylate 3' Ends on page 64 immediately, the protocol can
be safely stopped here. If you are stopping, seal the ALP plate with a Microseal ‘B’ adhesive
seal and store at -15°C to -25°C for up to 7 days.
TruSeq Nano DNA Sample Preparation Guide
63
Perform End Repair and Size Selection
15 Mix thoroughly as follows:
a Seal the CEP plate with a Microseal ‘B’ adhesive seal.
b Shake the CEP plate on a microplate shaker at 1800 rpm for 2 minutes.
High Sample (HS) Protocol
Adenylate 3' Ends
A single ‘A’ nucleotide is added to the 3’ ends of the blunt fragments to prevent them from
ligating to one another during the adapter ligation reaction. A corresponding single
‘T’ nucleotide on the 3’ end of the adapter provides a complementary overhang for ligating
the adapter to the fragment. This strategy ensures a low rate of chimera (concatenated
template) formation.
Consumables
Item
Quantity
Storage
Supplied By
A-Tailing Mix (ATL)
LT kit - 1 tube per 24
reactions
or
HT kit - 1 tube per 48
reactions
-15°C to -25°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
Ice bucket
As needed
-15°C to -25°C
User
Microseal ‘B’ adhesive seal
1
15°C to 30°C
User
RNase/DNase-free eight-tube
strips and caps
(if using multichannel pipettes)
2
15°C to 30°C
User
RNase/DNase-free reagent
reservoirs
(if using multichannel pipettes)
2
15°C to 30°C
User
Preparation
} Prepare an ice bucket.
} Remove the A-Tailing Mix from -15°C to -25°C storage and thaw it at room
temperature:
} Remove the Resuspension Buffer from 2°C to 8°C storage and bring it to room
temperature.
64
Part # 15041110 Rev. B
Add ATL
1
Centrifuge the thawed A-Tailing Mix tube at 600 × g for 5 seconds.
2
Add 12.5 µl thawed A-Tailing Mix to each well of the ALP plate. Mix thoroughly as
follows:
a Seal the ALP plate with a Microseal ‘B’ adhesive seal.
b Shake the ALP plate on a microplate shaker at 1800 rpm for 2 minutes.
3
Centrifuge the ALP plate at 280 × g for 1 minute.
4
Return the A-Tailing Mix tube to -15° to -25°C storage.
Incubate 1 ALP
1
Place the sealed ALP plate on the pre-heated microheating system 1. Close the lid and
incubate at 37°C for 30 minutes.
2
Immediately after the 37°C incubation, remove the ALP plate from system 1 and place
the plate on the pre-heated microheating system 2. Close the lid and incubate at 70°C
for 5 minutes.
3
Set the microheating system 1 to 30°C in preparation for Ligate Adapters.
4
Immediately remove the ALP plate from the microheating system 2 and place the plate
on ice for 5 minutes.
5
Proceed immediately to Ligate Adapters on page 66.
TruSeq Nano DNA Sample Preparation Guide
65
Adenylate 3' Ends
} Remove the ALP plate from -15°C to -25°C storage, if it was stored at the conclusion of
Clean Up IMP and Size Selection on page 59.
• Let it thaw at room temperature.
• Centrifuge the thawed ALP plate at 280 × g for 1 minute.
• Remove the adhesive seal from the ALP plate.
} Pre-heat two microheating systems: system 1 to 37°C and system 2 to 70°C.
High Sample (HS) Protocol
Ligate Adapters
This process ligates indexing adapters to the ends of the DNA fragments, preparing them
for hybridization onto a flow cell.
Consumables
66
Item
Quantity
Storage
Supplied By
Choose from the following
depending on the kit you
are using:
• TruSeq Nano DNA LT
Sample Prep Kit contents:
• DNA Adapter Indices
(AD001–AD016,
AD018–AD023, AD025,
AD027)
• TruSeq Nano DNA HT
Sample Prep Kit contents:
• DAP (DNA Adapter
Plate)
1 tube of each index being
used, per column of 8
reactions
or
1 DAP
-15°C to -25°C
Illumina
Ligation Mix 2 (LIG2)
LT kit - 1 tube per 24
reactions
or
HT kit - 1 tube per 48
reactions
-15°C to -25°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
Sample Purification Beads
(SPB)
1 tube per 24 reactions
2°C to 8°C
Illumina
Stop Ligation Buffer (STL)
LT kit - 1 tube per 24
reactions
or
HT kit - 1 tube per 48
reactions
-15°C to -25°C
Illumina
Part # 15041110 Rev. B
Quantity
Storage
Supplied By
Barcode labels for:
• CAP (Clean Up ALP
Plate)
• DAP (DNA Adapter
Plate)
(if using the HT kit)
• PCR (Polymerase Chain
Reaction Plate)
1 label per plate
15°C to 30°C
Illumina
96-well HSP plate
1
15°C to 30°C
User
96-well MIDI plate
1
15°C to 30°C
User
Freshly prepared 80%
ethanol (EtOH)
800 µl per sample
15°C to 30°C
User
Ice bucket
As needed
-15°C to -25°C
User
Microseal ‘B’ adhesive seals
7
15°C to 30°C
User
RNase/DNase-free eighttube strips and caps (if
using multichannel
pipettes)
3–27
15°C to 30°C
User
RNase/DNase-free reagent
reservoirs (if using
multichannel pipettes)
3–27
15°C to 30°C
User
Preparation
} Prepare an ice bucket.
} Remove the following from -15°C to -25°C storage and thaw them at room temperature:
• Appropriate DNA Adapter tubes (depending on the DNA Adapter Indices being
used) or the DAP.
TruSeq Nano DNA Sample Preparation Guide
67
Ligate Adapters
Item
High Sample (HS) Protocol
NOTE
• Review the TruSeq Sample Preparation Pooling Guide (part # 15042173). See
Additional Resources on page 6 for information on how to download the guide
from the Illumina website.
• When indexing libraries using adapter index tubes, Illumina recommends
arranging samples that are going to be combined into a common pool in the
same row. Also, include a common index in each column. This arrangement
facilitates pipetting operations when dispensing indexed adapters and pooling
indexed libraries later in the protocol.
• When indexing libraries with the DAP, arrange samples that will be pooled
together in the same orientation as the indices in the DAP.
NOTE
When indexing libraries with the DAP:
• Review Handling Adapter Plate in the TruSeq Sample Preparation Pooling Guide (part
# 15042173). See Additional Resources on page 6 for information on how to
download the guide from the Illumina website.
• Illumina recommends that the DAP does not undergo more than four freezethaw cycles. To maximize the use of the DAP, process more than 24 samples at a
time. These samples can then be pooled in any supported configuration.
• Stop Ligation Buffer
NOTE
Do not remove the Ligation Mix 2 tube from -15°C to -25°C storage until instructed to
do so in the procedures.
} Remove the Resuspension Buffer from 2°C to 8°C storage and bring it to room
temperature.
} Review Best Practices for Handling Magnetic Beads. See Additional Resources on page 6
for information on how to access TruSeq Nano DNA Sample Preparation Best Practices
on the Illumina website.
} Remove the Sample Purification Beads from 2°C to 8°C storage and let stand for at
least 30 minutes to bring them to room temperature.
} Pre-heat the microheating system 1 to 30°C.
} Apply a CAP barcode label to a new 96-well MIDI plate.
} Apply a PCR barcode label to a new 96-well HSP plate.
68
Part # 15041110 Rev. B
1
Do one of the following:
• If using DNA Adapter tubes, centrifuge the thawed tubes at 600 × g for 5 seconds.
• If using a DAP:
— Thaw the plate for 10 minutes at room temperature on the benchtop. Visually
inspect the wells to make sure that they all are thawed.
— Remove the adapter plate tape seal.
— Centrifuge the plate at 280 × g for 1 minute to collect all of the adapter to the
bottom of the well.
— Remove the plastic cover. Save the cover if you are not processing the entire
plate at one time.
— If it is the first time using this DAP, apply the DAP barcode label to the plate.
2
Centrifuge the Stop Ligation Buffer tube at 600 × g for 5 seconds.
3
Immediately before use, remove the Ligation Mix 2 tube from -15°C to -25°C storage.
4
Remove the adhesive seal from the ALP plate.
5
Add 2.5 µl Resuspension Buffer to each well of the ALP plate.
6
Add 2.5 µl Ligation Mix 2 to each well of the ALP plate.
7
Return the Ligation Mix 2 tube to -15°C to -25°C storage immediately after use.
8
Do one of the following:
• If using DNA Adapter tubes, add 2.5 µl thawed DNA Adapter Index to each well
of the ALP plate.
• If using a DAP:
— Place the DAP on the benchtop so that the part number barcode, on the long
side of the plate, is facing you and the clipped corner is on the lower left.
Figure 6 Correct DAP Orientation
TruSeq Nano DNA Sample Preparation Guide
69
Ligate Adapters
Add LIG
High Sample (HS) Protocol
— Do one of the following to pierce the foil seal:
— If using the entire plate at one time, use the bottom of a clean 96-well semiskirted PCR plate to pierce a hole in all of the well seals simultaneously.
Gently, but firmly, press the clean plate over the foil seal.
— If using only part of the plate, use the bottom of a clean eight-tube strip,
with caps attached, to pierce holes in the seals of the wells that will be
used for ligation. Repeat with a new, clean eight-tube strip, with caps
attached, for each row or column of adapters that will be used for ligation.
— Using an eight-tip multichannel pipette, transfer 2.5 µl thawed DNA Adapter
from the DAP well to each well of the ALP plate.
9
Mix thoroughly as follows:
a Seal the ALP plate with a Microseal ‘B’ adhesive seal.
b Shake the ALP plate on a microplate shaker at 1800 rpm for 2 minutes.
10 Centrifuge the ALP plate at 280 × g for 1 minute.
Incubate 2 ALP
1
Incubate the ALP plate on the pre-heated microheating system, with the lid closed, at
30°C for 10 minutes.
2
Remove the ALP plate from the microheating system and place the plate on ice until
you are ready for the next step.
1
Remove the adhesive seal from the ALP plate.
2
Add 5 µl Stop Ligation Buffer to each well of the ALP plate to inactivate the ligation
mix. Mix thoroughly as follows:
a Seal the ALP plate with a Microseal ‘B’ adhesive seal.
b Shake the ALP plate on a microplate shaker at 1800 rpm for 2 minutes.
3
Centrifuge the ALP plate at 280 × g for 1 minute.
Add STL
Clean Up ALP
1
70
Remove the adhesive seal from the ALP plate.
Part # 15041110 Rev. B
Vortex the Sample Purification Beads for at least 1 minute or until they are well
dispersed.
3
Add 42.5 µl well-mixed Sample Purification Beads to each well of the ALP plate. Mix
thoroughly as follows:
a Seal the ALP plate with a Microseal ‘B’ adhesive seal.
b Shake the ALP plate on a microplate shaker at 1800 rpm for 2 minutes.
NOTE
Vortex the Sample Purification Beads frequently to make sure that they are evenly
distributed. Illumina recommends the following:
• If using a single channel pipette, vortex the beads after processing four samples
• If using a multichannel pipette, vortex the beads after processing four columns
• If the beads are in a reagent reservoir, mix with a 1000 µl pipette.
4
Incubate the ALP plate at room temperature for 5 minutes.
5
Centrifuge the ALP plate at 280 × g for 1 minute.
6
Remove the adhesive seal from the ALP plate.
7
Place the ALP plate on the magnetic stand at room temperature for 5 minutes or until
the liquid is clear.
8
Remove and discard 80 µl of the supernatant from each well of the ALP plate. Take
care not to disturb the beads.
NOTE
Leave the ALP plate on the magnetic stand while performing the following steps 9–13.
9
With the ALP plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to
each well without disturbing the beads.
10 Incubate the ALP plate at room temperature for 30 seconds, and then remove and
discard all of the supernatant from each well. Take care not to disturb the beads.
11 Repeat steps 9 and 10 one time for a total of two 80% EtOH washes.
12 With the ALP plate on the magnetic stand, let the samples air-dry at room temperature
for 5 minutes. Remove and discard any remaining EtOH with a 10 µl pipette.
13 With the ALP plate on the magnetic stand, add 52.5 µl Resuspension Buffer to each
well of the plate.
14 Remove the ALP plate from the magnetic stand.
TruSeq Nano DNA Sample Preparation Guide
71
Ligate Adapters
2
High Sample (HS) Protocol
15 Mix thoroughly as follows:
a Seal the ALP plate with a Microseal ‘B’ adhesive seal.
b Shake the ALP plate on a microplate shaker at 1800 rpm for 2 minutes.
16 Incubate the ALP plate at room temperature for 2 minutes.
17 Centrifuge the ALP plate at 280 × g for 1 minute.
18 Remove the adhesive seal from the ALP plate.
19 Place the ALP plate on the magnetic stand at room temperature for 5 minutes or until
the liquid is clear.
20 Transfer 50 µl of the clear supernatant from each well of the ALP plate to the
corresponding well of the new MIDI plate labeled with the CAP barcode. Take care not
to disturb the beads.
21 Vortex the Sample Purification Beads until they are well dispersed.
22 Add 50 µl mixed Sample Purification Beads to each well of the CAP plate. Mix
thoroughly as follows:
a Seal the CAP plate with a Microseal ‘B’ adhesive seal.
b Shake the CAP plate on a microplate shaker at 1800 rpm for 2 minutes.
23 Incubate the CAP plate at room temperature for 5 minutes.
24 Centrifuge the CAP plate at 280 × g for 1 minute.
25 Remove the adhesive seal from the CAP plate.
26 Place the CAP plate on the magnetic stand at room temperature for 5 minutes or until
the liquid is clear.
27 Remove and discard 95 µl of the supernatant from each well of the CAP plate. Take
care not to disturb the beads.
NOTE
Leave the CAP plate on the magnetic stand while performing the following steps 28–32.
28 With the CAP plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to
each well. Take care not to disturb the beads.
29 Incubate the CAP plate at room temperature for 30 seconds, and then remove and
discard all of the supernatant from each well. Take care not to disturb the beads.
30 Repeat steps 28 and 29 one time for a total of two 80% EtOH washes.
72
Part # 15041110 Rev. B
32 With the CAP plate on the magnetic stand, add 27.5 µl Resuspension Buffer to each
well of the plate.
33 Remove the CAP plate from the magnetic stand.
34 Mix thoroughly as follows:
a Seal the CAP plate with a Microseal ‘B’ adhesive seal.
b Shake the CAP plate on a microplate shaker at 1800 rpm for 2 minutes.
35 Incubate the CAP plate at room temperature for 2 minutes.
36 Centrifuge the CAP plate at 280 × g for 1 minute.
37 Remove the adhesive seal from the CAP plate.
38 Place the CAP plate on the magnetic stand at room temperature for 5 minutes or until
the liquid is clear.
39 Transfer 25 µl of the clear supernatant from each well of the CAP plate to the
corresponding well of the new HSP plate labeled with the PCR barcode. Take care not
to disturb the beads.
SAFE STOPPING POINT
If you do not plan to proceed immediately to Enrich DNA Fragments on page 74, the protocol
can be safely stopped here. If you are stopping, seal the PCR plate with a Microseal ‘B’
adhesive seal and store at -15°C to -25°C for up to 7 days.
TruSeq Nano DNA Sample Preparation Guide
73
Ligate Adapters
31 With the CAP plate on the magnetic stand, let the samples air-dry at room temperature
for 5 minutes. Remove and discard any remaining EtOH with a 10 µl pipette.
High Sample (HS) Protocol
Enrich DNA Fragments
This process uses PCR to selectively enrich those DNA fragments that have adapter
molecules on both ends and to amplify the amount of DNA in the library. The PCR is
performed with a PCR Primer Cocktail that anneals to the ends of the adapters. Minimize
the number of PCR cycles to avoid skewing the representation of the library.
NOTE
PCR enriches for fragments that have adapters ligated on both ends. Fragments with only
one or no adapters on their ends are by-products of inefficiencies in the ligation reaction.
Neither species can be used to make clusters. Fragments without any adapters cannot
hybridize to surface-bound primers in the flow cell. Fragments with an adapter on only one
end can hybridize to surface bound primers, but cannot form clusters.
Consumables
74
Item
Quantity
Storage
Supplied By
Enhanced PCR Mix (EPM)
LT kit - 1 tube per 24
reactions
or
HT kit - 1 tube per 48
reactions
-15°C to -25°C
Illumina
PCR Primer Cocktail (PPC)
LT kit - 1 tube per 24
reactions
or
HT kit - 1 tube per 48
reactions
-15°C to -25°C
Illumina
Sample Purification Beads (SPB)
1 tube per 24
reactions
2°C to 8°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
Barcode labels for:
• CPP (Clean Up PCR Plate)
• TSP1 (Target Sample Plate)
1 label per plate
15°C to 30°C
Illumina
96-well HSP plate
1
15°C to 30°C
User
Part # 15041110 Rev. B
Quantity
Storage
Supplied By
96-well MIDI plate
1
15°C to 30°C
User
Freshly prepared 80% ethanol
(EtOH)
400 µl per sample
15°C to 30°C
User
Microseal ‘A’ film
1
15°C to 30°C
User
Microseal ‘B’ adhesive seals
3
15°C to 30°C
User
RNase/DNase-free eight-tube
strips and caps
(if using multichannel pipettes)
5
15°C to 30°C
User
RNase/DNase-free reagent
reservoirs
(if using multichannel pipettes)
5
15°C to 30°C
User
Preparation
} Remove the Enhanced PCR Mix and PCR Primer Cocktail from -15°C to -25°C storage
and thaw them at room temperature.
} Centrifuge the thawed Enhanced PCR Mix and PCR Primer Cocktail tubes to 600 × g
for 5 seconds.
} Remove the Resuspension Buffer from 2°C to 8°C storage and bring it to room
temperature.
} Remove the Sample Purification Beads from 2°C to 8°C storage and let stand for at
least 30 minutes to bring them to room temperature.
} Review Best Practices for Handling Magnetic Beads. See Additional Resources on page 6
for information on how to access TruSeq Nano DNA Sample Preparation Best Practices
on the Illumina website.
} Remove the PCR plate from -15°C to -25°C storage, if it was stored at the conclusion of
Clean Up ALP on page 70.
• Let it thaw at room temperature.
• Centrifuge the thawed PCR plate at 280 × g for 1 minute.
• Remove the adhesive seal from the thawed PCR plate.
TruSeq Nano DNA Sample Preparation Guide
75
Enrich DNA Fragments
Item
High Sample (HS) Protocol
} Pre-program the thermal cycler with the following program and save as PCRNano:
• Choose the pre-heat lid option and set to 100°C
• 95°C for 3 minutes
• 8 cycles of:
— 98°C for 20 seconds
— 60°C for 15 seconds
— 72°C for 30 seconds
• 72°C for 5 minutes
• Hold at 4°C
NOTE
Illumina recommends 8 cycles of PCR for robust protocol performance.
} Apply a CPP barcode label to a new 96-well MIDI plate.
} Apply a TSP1 barcode label to a new 96-well HSP plate.
Make PCR
The following procedure assumes the following amount of input DNA sample for library
preparation and is designed to result in high library yields:
} 100 ng for a 350 bp insert size
} 200 ng for a 550 bp insert size
1
Add 5 µl thawed PCR Primer Cocktail to each well of the PCR plate.
2
Add 20 µl thawed Enhanced PCR Mix to each well of the PCR plate.
a Seal the PCR plate with a Microseal ‘A’ film.
WARNING
Follow vendor instructions for applying Microseal "A" sealing films. Improper use could
lead to inefficient sealing (evaporation of sample or cross-contamination) or too efficient
sealing (parts of the seal remain in the well after removing the whole seal).
b
3
76
Shake the PCR plate on a microplate shaker at 1600 rpm for 20 seconds.
Centrifuge the PCR plate at 280 × g for 1 minute.
Part # 15041110 Rev. B
1
Place the sealed PCR plate, containing 50 µl of each sample, on the pre-programmed
thermal cycler. Close the lid, then select and run PCRNano to amplify the plate.
a Choose the pre-heat lid option and set to 100°C
b 95°C for 3 minutes
c 8 cycles of:
— 98°C for 20 seconds
— 60°C for 15 seconds
— 72°C for 30 seconds
d 72°C for 5 minutes
e Hold at 4°C
Clean Up PCR
1
Centrifuge the PCR plate at 280 × g for 1 minute.
2
Remove the adhesive seal from the PCR plate.
3
Vortex the Sample Purification Beads until they are well dispersed.
4
Do one of the following, depending on the adapter type used:
• If using the DNA Adapter tubes, add 50 µl mixed Sample Purification Beads to
each well of the new MIDI plate labeled with the CPP barcode.
• If using the DAP, add 47.5 µl mixed Sample Purification Beads to each well of the
new MIDI plate labeled with the CPP barcode.
5
Transfer the entire contents from each well of the PCR plate to the corresponding well
of the CPP plate containing 50 µl mixed Sample Purification Beads. Mix thoroughly as
follows:
a Seal the CPP plate with a Microseal ‘B’ adhesive seal.
b Shake the CPP plate on a microplate shaker at 1800 rpm for 2 minutes.
6
Incubate the CPP plate at room temperature for 5 minutes.
7
Centrifuge the CPP plate at 280 × g for 1 minute.
8
Remove the adhesive seal from the CPP plate.
TruSeq Nano DNA Sample Preparation Guide
77
Enrich DNA Fragments
Amp PCR
High Sample (HS) Protocol
9
Place the CPP plate on the magnetic stand at room temperature for 5 minutes or until
the liquid is clear.
10 Remove and discard 95 µl of the supernatant from each well of the CPP plate.
NOTE
Leave the CPP plate on the magnetic stand while performing the following 80% EtOH
wash steps (11–13).
11 With the CPP plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to
each well without disturbing the beads.
12 Incubate the CPP plate at room temperature for 30 seconds, and then remove and
discard all of the supernatant from each well.
13 Repeat steps 11 and 12 one time for a total of two 80% EtOH washes.
14 With the CPP plate on the magnetic stand, let the samples air-dry at room temperature
for 5 minutes. Remove and discard any remaining EtOH from each well of the CPP
plate with a 10 µl pipette.
15 With the CPP plate on the magnetic stand, add 32.5 µl Resuspension Buffer to each
well of the CPP plate.
16 Remove the CPP plate from the magnetic stand.
17 Mix thoroughly as follows:
a Seal the CPP plate with a Microseal ‘B’ adhesive seal.
b Shake the CPP plate on a microplate shaker at 1800 rpm for 2 minutes.
18 Incubate the CPP plate at room temperature for 2 minutes.
19 Centrifuge the CPP plate at 280 × g for 1 minute.
20 Remove the adhesive seal from the CPP plate.
21 Place the CPP plate on the magnetic stand at room temperature for 5 minutes or until
the liquid is clear.
22 Transfer 30 µl of the clear supernatant from each well of the CPP plate to the
corresponding well of the new HSP plate labeled with the TSP1 barcode.
SAFE STOPPING POINT
If you do not plan to proceed immediately to Validate Library on page 79, you can safely stop
the protocol here. If you are stopping, seal the TSP1 plate with a Microseal ‘B’ adhesive seal
and store at -15°C to -25°C for up to 7 days.
78
Part # 15041110 Rev. B
Illumina recommends performing the following procedures for quality control analysis on
your sample library and quantification of the DNA library templates.
Quantify Libraries
To achieve the highest data quality on Illumina sequencing platforms, it is important to
create optimum cluster densities across every lane of a flow cell. Optimizing cluster
densities requires accurate quantitation of DNA library templates. Quantify your libraries
using a fluorometric quantification method that uses dsDNA binding dyes or qPCR.
NOTE
TruSeq Nano DNA Sample Prep library quantitation has been validated using the Eco RealTime PCR System and KAPA Library Quantification Kit specified in the Consumables and
Equipment on page 95. Follow the KAPA instructions with the KAPA standard. To calculate
the library concentration in nM, perform the following insert size adjustment:
• For 350 bp libraries, use 470 bp for the average fragment length
• For 550 bp libraries, use 670 bp for the average fragment length
NOTE
You can download the KAPA Library Quantification Kits for Illumina sequencing platforms
Technical Data Sheet from the Kapa Biosystems website (www.kapabiosystems.com).
[Optional] Quality Control
To verify the size of your fragments, check the template size distribution. Run samples on a
Bioanalyzer for qualitative purposes only.
Do one of the following:
• If using a High Sensitivity DNA chip:
— Prepare a 1:100 dilution of the DNA library with water.
— Run 1 µl of the diluted DNA library on an Agilent Technologies 2100
Bioanalyzer.
• If using a DNA 7500 chip, run 1 µl of undiluted DNA library on an Agilent
Technologies 2100 Bioanalyzer.
TruSeq Nano DNA Sample Preparation Guide
79
Validate Library
Validate Library
High Sample (HS) Protocol
Figure 7 Example TruSeq Nano DNA Sample Preparation 350 bp Insert Library Distribution
Figure 8 Example TruSeq Nano DNA Sample Preparation 550 bp Insert Library Distribution
80
Part # 15041110 Rev. B
This process describes how to prepare DNA templates for cluster generation. Indexed DNA
libraries are normalized to 10 nM in the DCT plate and then pooled in equal volumes in
the PDP plate. DNA libraries not intended for pooling are normalized to 10 nM in the DCT
plate.
Consumables
Item
Quantity
Storage
Supplied By
Barcode labels for:
• DCT (Diluted Cluster
Template)
• PDP (Pooled DCT Plate)
(for pooling only)
1 label per plate
15°C to 30°C
Illumina
1.7 ml microcentrifuge tube
(when processing > 48 samples
at a time)
1
15°C to 30°C
User
96-well MIDI plates
2
(second plate for
pooling only, if
pooling > 40 samples)
15°C to 30°C
User
96-well 0.3 ml PCR plate
(for pooling only, if pooling
≤ 40 samples)
1
15°C to 30°C
User
Microseal ‘B’ adhesive seals
2
15°C to 30°C
User
Tris-HCl 10 mM, pH8.5
with 0.1% Tween 20
Enough to normalize
the concentration of
each sample library
to 10 nM
15°C to 30°C
User
TruSeq Nano DNA Sample Preparation Guide
81
Normalize and Pool Libraries
Normalize and Pool Libraries
High Sample (HS) Protocol
Preparation
} Remove the TSP1 plate from -15°C to -25°C storage, if it was stored at the conclusion of
Clean Up PCR on page 39.
• Let it thaw at room temperature.
• Centrifuge the thawed TSP1 plate at 280 × g for 1 minute.
• Remove the adhesive seal from the thawed TSP1 plate.
} Apply a DCT barcode label to a new 96-well MIDI plate.
} [For pooling only] Apply a PDP barcode label to a new 96-well 0.3 ml PCR plate if
pooling ≤ 40 samples or a 96-well MIDI plate if pooling > 40 samples.
Make DCT
1
Transfer 10 µl of sample library from each well of the TSP1 plate to the corresponding
well of the new MIDI plate labeled with the DCT barcode.
2
Normalize the concentration of sample library in each well of the DCT plate to 10 nM
using Tris-HCl 10 mM, pH 8.5 with 0.1% Tween 20.
NOTE
Depending on the yield quantification data of each sample library, the final volume in
the DCT plate can vary from 10–400 µl.
82
3
Mix the DCT plate as follows:
a Seal the DCT plate with a Microseal ‘B’ adhesive seal.
b Shake the DCT plate on a microplate shaker at 1000 rpm for 2 minutes.
4
Centrifuge the DCT plate at 280 × g for 1 minute.
5
Remove the adhesive seal from the DCT plate.
6
Depending on the type of library you want to generate, do one of the following:
• For non-pooled libraries, the protocol stops here. Do one of the following:
— Proceed to cluster generation. For more information, see the cluster generation
section of the user guide for your Illumina platform.
— Seal the DCT plate with a Microseal ‘B’ adhesive seal and store at
-15°C to -25°C.
• For pooled libraries, proceed to Make PDP (for pooling only).
Part # 15041110 Rev. B
NOTE
Do not make a PDP plate if you are not pooling samples.
1
Determine the number of samples to be combined together for each pool.
NOTE
Make a note of which sample goes into which well, to avoid pooling two samples with
the same index.
2
Do one of the following:
• If pooling 2–24 samples:
— Transfer 10 µl of each normalized sample library to be pooled from the DCT
plate to one well of the new MIDI plate labeled with the PDP barcode.
— The total volume in each well of the PDP plate is 10X the number of combined
sample libraries and 20–240 µl (2–24 libraries). For example, the volume for
2 samples is 20 µl, the volume for 12 samples is 120 µl, or the volume for
24 samples is 240 µl.
• If pooling 25–48 samples:
— Using a multichannel pipette, transfer 5 µl of each normalized sample library
in column 1 from the DCT plate to column 1 of the new MIDI plate labeled
with the PDP barcode.
— Transfer 5 µl of each normalized sample library in column 2 of the DCT plate
to column 1 of the PDP plate.
— Repeat the transfer for as many times as there are remaining columns in the
DCT plate. The result is a PDP plate with pooled samples in column 1. Mix the
PDP plate as follows:
— Seal the PDP plate with a Microseal ‘B’ adhesive seal.
— Shake the PDP plate on a microplate shaker at 1800 rpm for 2 minutes.
— Centrifuge the PDP plate at 280 × g for 1 minute.
— Remove the adhesive seal from the PDP plate.
— Combine the contents of each well of column 1 into well A2 of the PDP plate
for the final pool.
TruSeq Nano DNA Sample Preparation Guide
83
Normalize and Pool Libraries
Make PDP (for pooling only)
High Sample (HS) Protocol
• If pooling 49–96 samples:
— Using a multichannel pipette, transfer 5 µl of each normalized sample library
in column 1 of the DCT plate to column 1 of the new MIDI plate labeled with
the PDP barcode.
— Transfer 5 µl of each normalized sample library in column 2 of the DCT plate
to column 1 of the PDP plate.
— Repeat the transfer for as many times as there are remaining columns in the
DCT plate. The result is a PDP plate with pooled samples in column 1. Mix the
PDP plate as follows:
— Seal the PDP plate with a Microseal ‘B’ adhesive seal.
— Shake the PDP plate on a microplate shaker at 1800 rpm for 2 minutes.
— Centrifuge the PDP plate at 280 × g for 1 minute.
— Remove the adhesive seal from the PDP plate.
— Combine the contents of each well of column 1 into a 1.7 ml microcentrifuge
tube for the final pool.
84
3
Mix as follows, depending on the item that contains the final pool:
• For the PDP plate:
— Seal the PDP plate with a Microseal ‘B’ adhesive seal.
— Shake the PDP plate on a microplate shaker at 1800 rpm for 2 minutes.
— Centrifuge the PDP plate at 280 × g for 1 minute.
• For the 1.7 ml microcentrifuge tube:
— Cap the tube.
— Vortex the tube to mix thoroughly.
4
Do one of the following:
• Proceed to cluster generation. For more information, see the cluster generation
section of the user guide for your Illumina platform.
• Store the sealed PDP plate or capped 1.7 ml microcentrifuge tube at -15°C to -25°C.
Part # 15041110 Rev. B
Appendix A Supporting Information
Introduction
Acronyms
Kit Contents
Consumables and Equipment
Indexed Adapter Sequences
TruSeq Nano DNA Sample Preparation Guide
86
87
89
95
100
85
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.
86
Part # 15041110 Rev. B
Acronyms
Acronyms
Table 15 TruSeq Nano DNA Sample Preparation Acronyms
Acronym
Definition
ALP
Adapter Ligation Plate
ATL
A-Tailing Mix
CAP
Clean Up ALP Plate
CEP
Clean Up End Repair Plate
CFP
Covaris Fragmentation Plate
CPP
Clean Up PCR Plate
CSP
Clean Up Sheared DNA Plate
DAP
DNA Adapter Plate
DCT
Diluted Cluster Template
DNA
Customer Sample DNA Plate
dsDNA
double-stranded DNA
EPM
Enhanced PCR Mix
ERP2
End Repair Mix 2
EUC
Experienced User Card
gDNA
genomic DNA
HSP
Hard-Shell Plate
HS
High Sample
HT
High Throughput
TruSeq Nano DNA Sample Preparation Guide
87
Supporting Information
Acronym
88
Definition
IEM
Illumina Experiment Manager
IMP
Insert Modification Plate
LIG2
Ligation Mix 2
LS
Low Sample
LT
Low Throughput
LTF
Lab Tracking Form
PCR
Polymerase Chain Reaction
PDP
Pooled Dilution Plate
PPC
PCR Primer Cocktail
RSB
Resuspension Buffer
SPB
Sample Purification Beads
STL
Stop Ligation Buffer
TSP
Target Sample Plate
Part # 15041110 Rev. B
Check to make sure that you have all of the reagents identified in this section before
starting the TruSeq Nano DNA Sample Preparation protocol. The TruSeq Nano DNA LT
Sample Prep Kits are available as Set A and B. Each TruSeq Nano DNA LT Sample Prep
Kit contains enough reagents to prepare up to 24 samples. When used together, TruSeq
Nano DNA LT Sample Prep Kits A and B allow for pooling up to 24 samples using the 12
different indices in each kit.
Table 16 TruSeq Nano DNA Sample Prep Kits
Kit Name
Catalog #
Number
of
Samples
Supported
Number
of
Indices
TruSeq Nano DNA LT Sample
Prep Kit - Set A
FC-121-4001
24
12
TruSeq Nano DNA LT Sample
Prep Kit - Set B
FC-121-4002
24
12
TruSeq Nano DNA HT Sample
Prep Kit
FC-121-4003
96
96
TruSeq Nano DNA LT Sample Prep Kit
The TruSeq Nano DNA LT Sample Prep Kit contains two boxes: a Set A or Set B box and
an SP Beads box.
24 Samples - Set A or Set B Box
You receive either box A or B with the kit depending on the set you ordered. These boxes
also contain plate barcode labels.
Store at -15°C to -25°C
These boxes are shipped on dry ice. As soon as you receive them, store the following
components at -15°C to -25°C.
TruSeq Nano DNA Sample Preparation Guide
89
Kit Contents
Kit Contents
Supporting Information
Set A
Figure 9 TruSeq Nano DNA LT Sample Prep Kit, 24 Samples-Set A (Box 1 of 2), part # 15041757
Slot
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
90
Reagent
RSB
ERP2
ATL
LIG2
STL
PPC
EPM
AD002
AD004
AD005
AD006
AD007
AD012
AD013
AD014
AD015
AD016
AD018
AD019
Part #
15026770
15036418
15012495
15036183
15012546
15031748
15041700
15026621
15026623
15026624
15026625
15026627
15026632
15024641
15024642
15024643
15024644
15024646
15024647
Description
Resuspension Buffer
End Repair Mix 2
A-Tailing Mix
Ligation Mix 2
Stop Ligation Buffer
PCR Primer Cocktail
Enhanced PCR Mix
DNA Adapter Index 2
DNA Adapter Index 4
DNA Adapter Index 5
DNA Adapter Index 6
DNA Adapter Index 7
DNA Adapter Index 12
DNA Adapter Index 13
DNA Adapter Index 14
DNA Adapter Index 15
DNA Adapter Index 16
DNA Adapter Index 18
DNA Adapter Index 19
Part # 15041110 Rev. B
Figure 10 TruSeq Nano DNA LT Sample Prep Kit, 24 Samples-Set B (Box 1 of 2), part # 15041759
Slot
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Reagent
RSB
ERP2
ATL
LIG2
STL
PPC
EPM
AD001
AD003
AD008
AD009
AD010
AD011
AD020
AD021
AD022
AD023
AD025
AD027
Part #
15026770
15036418
15012495
15036183
15012546
15031748
15041700
15026620
15026622
15026628
15026629
15026630
15026631
15024648
15024649
15024650
15024651
15024653
15024654
Description
Resuspension Buffer
End Repair Mix 2
A-Tailing Mix
Ligation Mix 2
Stop Ligation Buffer
PCR Primer Cocktail
Enhanced PCR Mix
DNA Adapter Index 1
DNA Adapter Index 3
DNA Adapter Index 8
DNA Adapter Index 9
DNA Adapter Index 10
DNA Adapter Index 11
DNA Adapter Index 20
DNA Adapter Index 21
DNA Adapter Index 22
DNA Adapter Index 23
DNA Adapter Index 25
DNA Adapter Index 27
24 Samples - SP Beads Box Store at 2°C to 8°C
This box is shipped at 2°C to 8°C. As soon as you receive it, store the components at 2°C to
8°C.
TruSeq Nano DNA Sample Preparation Guide
91
Kit Contents
Set B
Supporting Information
Figure 11 TruSeq Nano DNA LT Sample Prep Kit, 24 Samples SP Beads (Box 2 of 2),
part # 15041758
Slot
1
Reagent
SPB
Part #
15041032
Description
Sample Purification Beads
TruSeq Nano DNA HT Sample Prep Kit
The TruSeq Nano DNA HT Sample Prep Kit contains three boxes: a core reagent box, an
Adapter Plate box, and an SP Beads box.
96 Samples - Core Reagents Box
Store at -15°C to -25°C
This box is shipped on dry ice. As soon as you receive it, store the following components at
-15°C to -25°C. This box also contains plate barcode labels.
Figure 12 TruSeq Nano DNA HT Sample Prep Kit, 96 Samples (Box 1 of 2), part # 15041877
92
Part # 15041110 Rev. B
Reagent
RSB
ERP2
ATL
LIG2
STL
PPC
EPM
Part #
15026770
15036182
15012495
15036184
15012546
15031748
15041027
Kit Contents
Slot
1–2
3–4
5–6
7–8
9–10
11–12
13–14
Description
Resuspension Buffer
End Repair Mix 2
A-Tailing Mix
Ligation Mix 2
Stop Ligation Buffer
PCR Primer Cocktail
Enhanced PCR Mix
96 Samples - Adapter Plate Box
Store at -15°C to -25°C
This box is shipped on dry ice. As soon as you receive it, store the contents at -15°C
to -25°C.
Figure 13 TruSeq Nano DNA HT Sample Prep Kit, 96, Adapter Plate Box, part # 15032317
Slot
1
Reagent
DAP
Part #
Description
15016426 DNA Adapter Plate, 96plex
96 Samples - SP Beads Box Store at 2°C to 8°C
This box is shipped at 2°C to 8°C. As soon as you receive it, store the components at 2°C to
8°C.
TruSeq Nano DNA Sample Preparation Guide
93
Supporting Information
Figure 14 TruSeq Nano DNA HT Sample Prep Kit, 96 Samples SP Beads (Box 2 of 2),
part # 15041878
Slot
1–4
94
Reagent
SPB
Part #
15041032
Description
Sample Purification Beads
Part # 15041110 Rev. B
Check to make sure that you have all of the necessary user-supplied consumables and
equipment before starting the TruSeq Nano DNA Sample Preparation protocol. The
requirement for some supplies is dependent upon the protocol performed (LS or HS) and
these items are specified in separate tables.
NOTE
The TruSeq Nano DNA Sample Preparation protocol has been optimized and validated using
the items listed. Comparable performance is not guaranteed when using alternate
consumables and equipment.
Table 17 User-Supplied Consumables
Consumable
Supplier
1.7 ml microcentrifuge tubes
General lab supplier
15 ml conical tubes
General lab supplier
10 µl barrier pipette tips
General lab supplier
10 µl multichannel pipettes
General lab supplier
10 µl single channel pipettes
General lab supplier
1000 µl barrier pipette tips
General lab supplier
1000 µl multichannel pipettes
General lab supplier
1000 µl single channel pipettes
General lab supplier
20 µl barrier pipette tips
General lab supplier
20 µl multichannel pipettes
General lab supplier
20 µl single channel pipettes
General lab supplier
200 µl barrier pipette tips
General lab supplier
200 µl multichannel pipettes
General lab supplier
200 µl single channel pipettes
General lab supplier
TruSeq Nano DNA Sample Preparation Guide
95
Consumables and Equipment
Consumables and Equipment
Supporting Information
96
Consumable
Supplier
96-well storage plates, round well,
0.8 ml (“MIDI” plate)
Fisher Scientific,
part # AB-0859
Distilled water
General lab supplier
Ethanol 200 proof (absolute)
for molecular biology (500 ml)
Sigma-Aldrich,
part # E7023
[Optional] Fluorometric quantitation with dsDNA binding
dye reagents
General lab supplier
Ice bucket
General lab supplier
[Optional] KAPA Library Quantification Kit Illumina/Universal
KAPA Biosystems, part #
KK4824
Microseal ‘B’ adhesive seals
Bio-Rad,
part # MSB-1001
microTUBE AFA Fiber 6x16mm with
• Crimp-Cap, or
• Pre-Slit Snap-Cap (for use with Covaris M220)
Covaris,
• part # 520052, or
• part # 520045
PCR grade water
General lab supplier
RNaseZap
(to decontaminate surfaces)
General lab supplier
RNase/DNase-free eight-tubes strips and caps
General lab supplier
RNase/DNase-free multichannel reagent reservoirs,
disposable
VWR, part # 89094-658
Tris-HCl 10 mM, pH 8.5
General lab supplier
Tween 20
Sigma-Aldrich,
part # P7949
Part # 15041110 Rev. B
Consumables and Equipment
Table 18 User-Supplied Consumables - Additional Items for LS Processing
Consumable
Supplier
96-well 0.3 ml skirtless PCR plates, or
Twin.tec 96-well PCR plates
E&K Scientific, part # 480096
Eppendorf, part # 951020303
Table 19 User-Supplied Consumables - Additional Items for HS Processing
Consumable
Supplier
Hard-Shell 96-well PCR Plates (“HSP” plate)
Bio-Rad, part # HSP-9601
Table 20 User-Supplied Equipment
Equipment
Supplier
[Optional] 2100 Bioanalyzer Desktop System
Agilent, part # G2940CA
[Optional] Agilent DNA 7500 Kit
Agilent, part # 5067-1506
[Optional] Agilent High Sensitivity DNA Kit
Agilent, part # 5067-4626
One of the following Covaris systems:
• S2
• S220
• E210
• M220
Covaris M220, part # 500295
For all other models, contact
Covaris
[Optional] Eco ™ Real-Time PCR System
Illumina, catalog #:
EC-100-1000 (110 V)
EC-100-1001 (220 V)
[Optional] Fluorometer for quantitation with
dsDNA binding dyes
General lab supplier
Magnetic stand-96
Life Technologies,
catalog # AM10027
Microplate centrifuge
General lab supplier
Vortexer
General lab supplier
TruSeq Nano DNA Sample Preparation Guide
97
Supporting Information
Table 21 User-Supplied Equipment - Additional Items for LS Processing
Equipment
Supplier
96-well thermal cycler
(with heated lid)
See Thermal Cyclers on page 99.
General lab supplier
Table 22 User-Supplied Equipment - Additional Items for HS Processing
98
Equipment
Supplier
High-Speed Microplate Shaker
VWR, catalog # 13500-890 (110 V/120 V)
VWR, catalog # 14216-214 (230 V)
MIDI plate insert for heating system
Note: Two inserts are recommended
to support successive heating procedures.
Illumina, catalog # BD-60-601
Stroboscope
General lab supplier
SciGene TruTemp Heating System
Note: Two systems are recommended
to support successive heating procedures.
Illumina, catalog # SC-60-503 (115 V)
Illumina, catalog # SC-60-504 (220 V)
Part # 15041110 Rev. B
The following table lists the recommended settings for the Illumina recommended thermal
cycler, as well as other comparable models. If your lab has a thermal cycler that is not
listed, validate the thermal cycler before performing the TruSeq Nano DNA Sample
Preparation protocol.
Thermal Cycler
Temp
Mode
Bio-Rad DNA Engine Tetrad 2
Calculated
Heated, constant at
100°C
Plate
MJ Research PTC-225 DNA Engine
Tetrad
Calculated
Heated, constant at
100°C
Plate
Bio-Rad S1000
N/A
Heated, constant at
100°C
Plate
TruSeq Nano DNA Sample Preparation Guide
Lid Temp
Vessel
Type
99
Consumables and Equipment
Thermal Cyclers
Supporting Information
Indexed Adapter Sequences
This section details the indexed adapter sequences.
TruSeq Nano DNA LT Sample Prep Kit Indexed Adapter Sequences
The TruSeq Nano DNA LT Sample Prep Kit contains the following indexed adapter
sequences.
NOTE
• The index numbering is not contiguous. There is no Index 17, 24, or 26.
• The base in parentheses () indicates the base for the seventh cycle and is not considered as
part of the index sequence. Record the index in the sample sheet as only six bases. For
indices 13 and above, the seventh base (in parentheses) might not be A, which is seen in
the seventh cycle of the index read.
• For more information on the number of cycles used to sequence the index read,
reference your instrument user guide.
Table 23 TruSeq Nano DNA LT Sample Prep Kit Set A Indexed Adapter Sequences
Adapter
100
Sequence
Adapter
Sequence
AD002
CGATGT(A)
AD013
AGTCAA(C)
AD004
TGACCA(A)
AD014
AGTTCC(G)
AD005
ACAGTG(A)
AD015
ATGTCA(G)
AD006
GCCAAT(A)
AD016
CCGTCC(C)
AD007
CAGATC(A)
AD018
GTCCGC(A)
AD012
CTTGTA(A)
AD019
GTGAAA(C)
Part # 15041110 Rev. B
Adapter
Sequence
Adapter
Sequence
AD001
ATCACG(A)
AD020
GTGGCC(T)
AD003
TTAGGC(A)
AD021
GTTTCG(G)
AD008
ACTTGA(A)
AD022
CGTACG(T)
AD009
GATCAG(A)
AD023
GAGTGG(A)
AD010
TAGCTT(A)
AD025
ACTGAT(A)
AD011
GGCTAC(A)
AD027
ATTCCT(T)
TruSeq Nano DNA HT Sample Prep Kit Indexed Adapter Sequences
The DAP in the TruSeq Nano DNA HT Sample Prep Kit contains the following indexed
adapter sequences:
NOTE
The Index recorded in the sample sheet is the full 8 bases and 8 bases are sequenced per
indexed read.
Table 25 TruSeq Nano DNA HT Sample Prep Kit Indexed Adapter 1 Sequences
Adapter
Sequence
Adapter
Sequence
D701
ATTACTCG
D707
CTGAAGCT
D702
TCCGGAGA
D708
TAATGCGC
D703
CGCTCATT
D709
CGGCTATG
D704
GAGATTCC
D710
TCCGCGAA
D705
ATTCAGAA
D711
TCTCGCGC
D706
GAATTCGT
D712
AGCGATAG
TruSeq Nano DNA Sample Preparation Guide
101
Indexed Adapter Sequences
Table 24 TruSeq Nano DNA LT Sample Prep Kit Set B Indexed Adapter Sequences
Supporting Information
Table 26 TruSeq Nano DNA HT Sample Prep Kit Indexed Adapter 2 Sequences
102
Adapter
Sequence
Adapter
Sequence
D501
TATAGCCT
D505
AGGCGAAG
D502
ATAGAGGC
D506
TAATCTTA
D503
CCTATCCT
D507
CAGGACGT
D504
GGCTCTGA
D508
GTACTGAC
Part # 15041110 Rev. B
A
Acronyms 87
Add ATL 27, 65
Add LIG 30, 69
Add STL 32, 70
ALP 19, 57
Amp PCR 39, 77
ATL 26, 64
Index
Index
EtOH 14, 19, 29, 52, 57, 67
experienced user card (EUC) 7
F
Fragment DNA 15, 53
G
gDNA 2
B
H
Best Practices 6
help, technical 105
High Sample (HS) 3
HSP 3
C
CAP 29, 67
CEP 19, 57
CFP 13, 51
Clean Up ALP 32, 70
Clean Up IMP 21, 59
Clean Up PCR 39, 77
cluster generation 2, 46, 84
Covaris instrument 14, 52
Covaris shearing 13, 51
Covaris tubes 14, 52
CPP 74
CSP 13, 51
customer support 105
D
DAP 28, 66
DCT 43, 81
DNA Adapter Indices 28, 66
DNA Plate (DNA) 13, 51
DNA sequencing 2
documentation 105
E
EPM 36, 74
ERP2 19, 57
TruSeq Nano DNA Sample Preparation Guide
I
IEM 7
IMP 13, 51
Incubate 1 ALP 27, 65
Incubate 1 IMP 21, 59
Incubate 2 ALP 32, 70
indexed adapter 100-101
insert size 13, 51
L
lab tracking form (LTF) 7
LIG2 28, 66
Low Sample (LS) 3
M
Make CFP 15, 53
Make DCT 44, 82
Make IMP 20, 58
Make PCR 38, 76
Make PDP 45, 83
master-mixed reagents 2
micro plate shaker 3
microheating system 3
103
Index
MIDI 3
N
normalize gDNA 15, 53
W
workflow diagram 11, 49
P
paired-end 2
PCR 29, 67
PCR grade water 20, 58
PDP 43, 81
pooled sample volumes 45, 83
pooling guidelines 7
positive control 5
PPC 36, 74
Q
qPCR 41, 79
quality control 41, 79
quantify libraries 41, 79
quantitation 4
quantity and quality 4
R
Reagent Reservoirs 20, 26, 29, 37, 58,
64, 67, 75
RSB 13, 19, 26, 28, 36, 51, 57, 64, 66, 74
S
shear gDNA 15, 53
shearing 2
single read 2
Size Selection 21, 59
SPB 13, 19, 28, 36, 51, 57, 66, 74
STL 28, 66
strip tubes and caps 20, 26, 29, 37, 58,
64, 67, 75
T
technical assistance 105
thermal cycler 3
Training 6
Tris-HCl 43, 81
TSP1 36, 44, 74, 82
104
Part # 15041110 Rev. B
For technical assistance, contact Illumina Technical Support.
Table 27 Illumina General Contact Information
Illumina Website
Email
www.illumina.com
[email protected]
Table 28 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
North America
1.800.809.4566
Italy
Austria
0800.296575
Netherlands
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
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
www.illumina.com/msds.
Product Documentation
Product documentation in PDF is available for download from the Illumina website. Go
to www.illumina.com/support, select a product, then click Documentation & Literature.
TruSeq Nano DNA Sample Preparation Guide
105
Technical Assistance
Technical Assistance
Illumina
San Diego, California, U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com
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