WGS protocol
TruSeq® DNA PCR-Free
Library Prep Guide
FOR RESEARCH USE ONLY
ILLUMINA PROPRIETARY
Part # 15036187 Rev. C
December 2014
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TruSeq DNA PCR-Free Library Prep Guide
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Part # 15036187 Rev. C
Revision History
Part #
Revision
Date
15036187
C
December
2014
15036187
B
November
2013
15036187
A
January
2013
Description of Change
• Kit names changed from 'sample' prep to 'library' prep
• Added references to BaseSpace® for organizing samples, libraries,
pools, and runs
• Removed use of plate name (e.g., IMP plate), except for first
instance and last instance in each procedure
• Bead cleanup procedures modified to remove EtOH before placing
plate on the magnetic stand to dry.
• Added centrifuge step before each thermal cycler incubation in the
LS protocol
• Added well volume to heat incubation procedures
• Updated Prepare Adapter Setup sections
• Updated Additional Resources
• Removed List of Tables
• Updated SDS link to support.illumina.com/sds.html
• Renamed Incubate 1 IMP to Incubate IMP
• HS protocol
• Corrected Make DCT procedure to clarify that the DCT plate is
an HSP plate
• Moved centrifuge steps after incubate
• EUC and LTF merged into a single document per protocol
• Created new appendix of Supporting Information containing
Acronyms, Kit Contents, Consumables and Equipment, and Indexed
Adapter Sequences
• Replaced Best Practices section with a reference to content on the
Illumina website
• Replaced Adapter Options and Pooling Guidelines sections with a
reference to the TruSeq Sample Preparation Pooling Guide (part #
15042173)
• Removed Usage Guidelines
Initial Release
vi
Part # 15036187 Rev. C
Table of Contents
Revision History
Table of Contents
Chapter 1 Overview
Introduction
Protocol Features
DNA Input Recommendations
Additional Resources
Chapter 2 Low Sample (LS) Protocol
Introduction
Library Prep Workflow
Prepare Adapter Setup
Fragment DNA
Perform End Repair and Size Selection
Adenylate 3' Ends
Ligate Adapters
Validate Libraries
Normalize and Pool Libraries
Chapter 3 High Sample (HS) Protocol
Introduction
Library Prep Workflow
Prepare Adapter Setup
Fragment DNA
Perform End Repair and Size Selection
Adenylate 3' Ends
Ligate Adapters
Validate Library
Normalize and Pool Libraries
Appendix A Supporting Information
Introduction
Acronyms
Kit Contents
Consumables and Equipment
Indexed Adapter Sequences
Technical Assistance
v
vii
1
2
3
4
6
7
8
9
10
11
16
21
23
29
32
35
36
37
38
39
44
49
51
57
60
63
64
65
67
72
76
79
viii
Part # 15036187 Rev. C
Chapter 1 Overview
Overview
Introduction
Protocol Features
DNA Input Recommendations
Additional Resources
TruSeq DNA PCR-Free Library Prep Guide
2
3
4
6
1
Overview
Introduction
This protocol explains how to prepare up to 96 uniquely indexed paired-end libraries of
genomic DNA (gDNA) using Illumina® TruSeq® DNA PCR-Free Library Prep kits. The
purpose of the protocol is to add adapter sequences onto the ends of DNA fragments to
generate indexed single read or paired-end sequencing libraries. The libraries are ready for
subsequent cluster generation and sequencing.
The TruSeq DNA PCR-Free Library Prep 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
} Low-throughput (LT) and high-throughput (HT) kit configurations
} High throughput
• Adapter plate allows for simultaneous preparation of 96 dual-indexed DNA
samples
• Volumes optimized for standard 96-well plate
} Advanced troubleshooting
• Process control checks built-in for quality control
} Index adapter tags for all samples
• Additional adapters and primers are not necessary
• Each TruSeq DNA PCR-Free LT Library 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 DNA PCR-Free HT Library 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 no indexing or a lower indexing pooling level. The
libraries generated do not require PCR amplification to enable cluster generation.
2
Part # 15036187 Rev. C
This guide documents the TruSeq DNA PCR-Free Library Prep protocol using a TruSeq
DNA PCR-Free LT Library Prep Kit or TruSeq DNA PCR-Free HT Library Prep Kit.
} Chapter 2 Low Sample (LS) Protocol—Explains how to perform TruSeq DNA PCR-Free
Library Prep using the Low Sample Protocol
} Chapter 3 High Sample (HS) Protocol—Explains how to perform TruSeq DNA PCRFree Library Prep 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 the same time
HT Kit - Number of samples processed
at the same 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 midi
96-well thermal cycler
Pipetting
96-well HSP
96-well midi
Microheating systems
Microplate shaker
* Each TruSeq DNA PCR-Free LT Library Prep Kit contains enough reagents to prepare up to 24 samples.
When used together, TruSeq DNA PCR-Free LT Library Prep Kits A and B allow for pooling up to 24
samples using the 12 different indexes 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 DNA PCR-Free Library Prep 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
1 µg
≤ 2 x 101 bp
2 µg
≤ 2 x 151 bp*
* Read lengths greater than 2 x 151 bp produce a significantly higher percentage of overlapping readpairs.
TruSeq DNA PCR-Free Library Prep Guide
3
Protocol Features
Protocol Features
Overview
DNA Input Recommendations
It is important to quantify the input DNA and assess the DNA quality before beginning
TruSeq DNA PCR-Free Library Prep.
Input DNA Quantification
Follow these DNA input recommendations:
} Correct quantification of gDNA is essential.
} 1 µg input DNA is recommended for the 350 bp insert size workflow.
} 2 µg input DNA is recommended 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.
} Use multiple methods of quantification to verify results.
} Illumina recommends using fluorometric based methods for quantification, such as
Qubit or PicoGreen, to provide accurate quantification of dsDNA. UV
spectrophotometric-based methods, such as Nanodrop, measure any nucleotides
present in the sample including RNA, dsDNA, ssDNA, and free nucleotides. These
methods can give an inaccurate measurement of gDNA.
} 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.
In-Line Control DNA
The End Repair Control, A-Tailing Control, and Ligation Control contain DNA fragments
used as controls for the enzymatic activities of the End Repair Mix 2, A-Tailing Mix, and
Ligation Mix 2, respectively. Each reagent contains dsDNA fragments designed to report
the success or failure of a specific enzymatic activity used in the library preparation
process. Sequencing determines the readout. If the sequence of an in-line control is in the
final sequencing data viewed in the Sequence Analysis Viewer (SAV), it indicates that its
corresponding step was successful. If it does not, or if it is in substantially diminished
numbers, it indicates the step failed. The controls are intended for troubleshooting and are
useful for identifying the specific mode of failure, but are uninformative in cases where
sequencing data are not generated from a library.
NOTE
The use of these controls is optional and they can be replaced with the same volume of
Resuspension Buffer.
4
Part # 15036187 Rev. C
Table 3 In-Line Control Functions
Reagent
Function
End Repair Mix 2
End Repair Mix 2
A-Tailing Mix
Ligation Mix 2
End repair: Generate blunt ended
fragments by 3'–> 5' exonuclease
and 5'–> 3' polymerase activities
End repair: Add 5'-phosphate
groups needed for downstream
ligation
A-tailing: Make fragments
compatible with adapters and
prevent self-ligation by adding a
3'-A overhang
Ligation: Join 3'-T overhang
adapters to 3'-A overhang inserts
Control
Structure of
Control DNA
Ends
End
5' overhang at 1
Repair
end, 3' overhang at
Control 1* other end
End
Blunt with 5'-OH
Repair
group
Control 2*
A-Tailing
Blunt with 5'Control
phosphate group
Ligation
Control
Single-base 3' 'A'
base overhang
*End Repair Control 1 and End Repair Control 2 are separate controls included in the End Repair Control
reagent
The control reagents can be used for various library insert sizes. Each is provided in
ladders ranging from approximately 150–850 bp in 100 bp increments. Each control
molecule has a unique DNA sequence, indicating both its function and size. The RTA
software (v1.9, and later) recognizes these sequences and isolates the control sequences
from the main body of sequencing reads. RTA reports the control sequences counts per lane
in the controls tab of the RTA status.html page. The CTE1 and CTE2 controls show a
narrow distribution of sizes while the CTA and CTL controls show a broad size
distribution, because the size selection step is before A-Tailing. For more information
regarding the control read-out in the SAV, see the Sequence Analysis Viewer User Guide (part #
15020619).
TruSeq DNA PCR-Free Library Prep Guide
5
DNA Input Recommendations
The control molecules work through the design of their ends. Controls are added to the
reactions before their corresponding step in the protocol. Their end structures match the
end structures of a DNA molecule that has not gone through the step. If the step is
successful, the control molecule is modified to participate in downstream reactions of
library generation and resulting in sequencing data. If the step fails, the control molecule
does not go forward in the process and no sequencing data are generated.
Overview
Additional Resources
The following documentation is available for download from the Illumina website.
Resource
Description
TruSeq DNA PCR-Free Library
Prep LS Protocol Experienced
User Card (part # 15036190)
Provides LS protocol instructions, but with less detail than
what is provided in this guide. New or less experienced users
are advised to follow this guide and not the Experienced
User Card (EUC).
TruSeq DNA PCR-Free Library
Prep HS Protocol Experienced
User Card (part # 15036191)
Provides HS protocol instructions, but with less detail than
what is provided in this guide. New or less experienced users
are advised to follow this guide and not the Experienced
User Card (EUC).
Dual Index Sequencing with
TruSeq HT Library Prep
(part # 15059916)
Provides guidelines for preparing for dual-indexing
sequencing when using a TruSeq DNA PCR-Free HT Library
Prep Kit.
TruSeq Library Prep Pooling
Guide (part # 15042173)
Provides TruSeq pooling guidelines for preparing libraries for
Illumina sequencing systems that require balanced index
combinations. Review this guide before beginning library
preparation.
Illumina Experiment Manager
Guide (part # 15031335) and IEM
TruSeq DNA, RNA, or ChIP
Quick Reference Card
(part # 15037152)
Provide information about creating and editing appropriate
sample sheets for Illumina sequencing systems and analysis
software and record parameters for your sample plate.
BaseSpace User Guide
(part # 15044182)
Provides information about the BaseSpace® sequencing data
analysis tool that also enables you to organize samples,
libraries, pools, and sequencing runs in a single environment.
Visit the TruSeq DNA PCR-Free LT Library Prep Kit support page or TruSeq DNA PCR-Free
HT Library Prep Kit support page on the Illumina website for access to requirements and
compatibility, additional documentation, software downloads, online training, frequently
asked questions, and best practices.
6
Part # 15036187 Rev. C
Chapter 2 Low Sample (LS) Protocol
Introduction
Library Prep Workflow
Prepare Adapter Setup
Fragment DNA
Perform End Repair and Size Selection
Adenylate 3' Ends
Ligate Adapters
Validate Libraries
Normalize and Pool Libraries
TruSeq DNA PCR-Free Library Prep Guide
8
9
10
11
16
21
23
29
32
7
Chapter 2
Low Sample (LS) Protocol
Low Sample (LS) Protocol
Introduction
This chapter describes the TruSeq DNA PCR-Free Library Prep LS protocol. This protocol is
intended for preparing up to 24 samples at the same 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 DNA PCR-Free Library Prep Best Practices on 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.
8
Part # 15036187 Rev. C
Library Prep Workflow
Library Prep Workflow
The following figure illustrates theTruSeq DNA PCR-Free Library Prep LS workflow.
Figure 1 TruSeq DNA PCR-Free Library Prep LS Workflow
TruSeq DNA PCR-Free Library Prep Guide
9
Low Sample (LS) Protocol
Prepare Adapter Setup
If you are pooling, use IEM or BaseSpace to record information about your samples before
beginning library prep.
} Use IEM to create and edit sample sheets for Illumina sequencing systems and analysis
software.
} Use BaseSpace to organize samples, libraries, pools, and a run for Illumina sequencing
systems and analysis software.
NOTE
For information about IEM and BaseSpace software documentation on the Illumina website,
see Additional Resources on page 6.
Review the planning steps in the TruSeq Library Prep Pooling Guide (part # 15042173) when
preparing libraries for Illumina sequencing systems that require balanced index
combinations.
10
Part # 15036187 Rev. C
This process describes how to optimally fragment the gDNA depending on the Covaris
platform used. 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 4 Insert Size Options
Insert Size
Input DNA Per Sample
Recommended Read Length
350 bp
550 bp
1 µg
≤ 2 x 101 bp
2 µg
≤ 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
-25°C to -15°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
Covaris tubes
1 per sample
15°C to 30°C
User
DNA samples
• 1 µg per sample
for a 350 bp insert
size
• 2 µg per sample
for a 550 bp insert
size
-25°C to -15°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.
TruSeq DNA PCR-Free Library Prep Guide
11
Fragment DNA
Fragment DNA
Low Sample (LS) Protocol
} Review best practices for handling magnetic beads. See Additional Resources on page 6
for information about TruSeq DNA PCR-Free Library Prep 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 the Resuspension Buffer from -25°C to -15°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 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.
Make CFP
1
Illumina recommends quantifying gDNA samples using a fluorometric-based method
such as Qubit or PicoGreen.
2
Illumina recommends normalizing gDNA samples with Resuspension Buffer, in a final
volume of 55 µl in each well of the new 0.3 ml PCR plate labeled with the DNA
barcode:
• 20 ng/µl for a 350 bp insert size
• 40 ng/µl for a 550 bp insert size
3
Gently pipette the entire volume up and down 10 times, then centrifuge briefly.
Fragment DNA
1
Shear the gDNA sample by transferring 52.5 µl of each DNA sample from the DNA
plate to a separate, new Covaris tube:
• 1 µg for a 350 bp insert size
• 2 µg 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
12
Centrifuge the plate at 280 × g for 5 seconds.
Part # 15036187 Rev. C
Fragment DNA
3
Fragment the DNA using the following settings:
Table 5 Covaris S220 Settings
Setting
350 bp Insert
550 bp Insert
Duty factor
5%
Peak Incident Power
175 W
Cycles per burst
Duration
200
50 seconds
Mode
25 seconds
Frequency sweeping
Temperature
5.5° to 6°C
NOTE
The Covaris M220 settings are optimized for use with the Covaris microTUBE AFA
Fiber Pre-Slit Snap-Cap 6x16mm.
Table 6 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
Table 7 Covaris S2 and E210 Settings
Setting
350 bp Insert
Duty cycle
Intensity
10%
5.0
2.0
Cycles per burst
200
Duration
Mode
Displayed Power
Temperature
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 plate at 280 × 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.
TruSeq DNA PCR-Free Library Prep Guide
13
Low Sample (LS) Protocol
6
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 mixed Sample Purification Beads to each well. 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 4 samples
• If using a multichannel pipette, vortex the beads after processing 4 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 plate at room temperature for 5 minutes.
4
Place the plate on the magnetic stand 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 clear supernatant from each well.
NOTE
Leave the plate on the magnetic stand while performing the following steps 6–11.
6
With the plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to each
well. Do not disturb the beads.
7
Incubate the 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 to perform an 80% EtOH wash 2 times.
9
Remove and discard any remaining EtOH from each well with a 10 µl pipette.
10 With the plate on the magnetic stand, let the samples air-dry at room temperature for
5 minutes. Do not allow the beads to crack.
11 With the plate on the magnetic stand, add 52.5 µl Resuspension Buffer to each well.
Make sure the Resuspension Buffer runs over the beads. Do not touch the beads with
the pipette tip.
12 Remove the plate from the magnetic stand.
13 Resuspend the beads in each well by repeatedly dispensing the Resuspension Buffer
over the beads until they are immersed in the solution. Do not touch the beads with the
pipette tip. Gently pipette the entire volume up and down 10 times to mix thoroughly.
14 Incubate the plate at room temperature for 2 minutes.
15 Place the plate on the magnetic stand for 5 minutes or until the liquid is clear.
14
Part # 15036187 Rev. C
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.
17 Proceed immediately to Perform End Repair and Size Selection.
TruSeq DNA PCR-Free Library Prep Guide
15
Fragment DNA
16 Transfer 50 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.
Low Sample (LS) Protocol
Perform End Repair and Size Selection
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
-25°C to -15°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
[Optional] End Repair Control
(CTE)
1 tube per 48
reactions
-25°C to -15°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
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
Ice bucket
As needed
-25°C to -15°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 8-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.
16
Part # 15036187 Rev. C
NOTE
The use of the End Repair Control is optional and it can be replaced with the same
volume of Resuspension Buffer.
} Review best practices for handling magnetic beads. See Additional Resources on page 6
for information about TruSeq DNA PCR-Free Library Prep best practices on the
Illumina website.
} Remove the Sample Purification Beads and Resuspension Buffer from 2°C to 8°C
storage and let stand for at least 30 minutes to bring them to room temperature.
} Preprogram the thermal cycler with the following program and save as ERP:
• Choose the thermal cycler preheat 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
Do the following:
• If using the in-line control reagent:
— Centrifuge the thawed End Repair Control tube at 600 × g for 5 seconds.
— Add 10 µl thawed End Repair Control to each well.
• If not using the in-line control reagent, add 10 µl Resuspension Buffer to each well.
2
Centrifuge the thawed End Repair Mix 2 tube at 600 × g for 5 seconds.
3
Add 40 µl End Repair Mix 2 to each well. Set a 200 µl pipette to 75 µl and then gently
pipette the entire volume up and down 10 times to mix thoroughly.
4
Seal the IMP plate with a Microseal ‘B’ adhesive seal.
5
Return the End Repair Mix 2 tube to -25°C to -15°C storage.
Incubate IMP
1
Centrifuge the IMP plate at 280 × g for 1 minute.
2
Place the plate on the preprogrammed thermal cycler. Close the lid then select and run
the ERP program. Each well contains 50 µl of sample.
a Choose the thermal cycler preheat lid option and set to 100°C
b 30°C for 30 minutes
c Hold at 4°C
3
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 DNA PCR-Free Library Prep Guide
17
Perform End Repair and Size Selection
} Remove the End Repair Control from -25°C to -15°C storage and thaw it at room
temperature.
Low Sample (LS) Protocol
2
Combine Sample Purification Beads and PCR grade water in a tube to create a diluted
bead mixture of 160 µl per 100 µl of end-repaired sample:
• When processing > 6 samples at a time, use a new 15 ml conical tube
• When processing ≤ 6 samples at a time, use a new 1.7 ml microcentrifuge tube
Determine the volumes using the following formulas, which include 15% excess for
multiple samples:
Table 8 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 9 Diluted Bead Mixture for a 550 bp Insert Size
Formula
Sample Purification Beads
PCR grade water
Example
Amount
per 12
samples
# of samples X
92 µl
# of samples X
92 µl
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. 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.
NOTE
Vortex the diluted bead mixture frequently. Illumina recommends the following:
• If using a single channel pipette, vortex the mixture after processing 4 samples
• If using a multichannel pipette, vortex the mixture after processing 4 columns
• If the mixture is in a reagent reservoir, mix with a 1000 µl pipette.
5
Incubate the plate at room temperature for 5 minutes.
6
Place the plate on the magnetic stand for 5 minutes or until the liquid is clear.
7
Set a 200 µl single channel or multichannel pipette to 125 µl. Transfer 125 µl of the
supernatant, which contains 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
18
Repeat step 7. Each CEP plate well now contains a total of 250 µl of DNA of interest.
Part # 15036187 Rev. C
Discard the IMP plate that contains 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. 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 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
and yield 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 4 samples
• If using a multichannel pipette, vortex the beads after processing 4 columns
• If the beads are in a reagent reservoir, mix with a 1000 µl pipette
3
Incubate the plate at room temperature for 5 minutes.
4
Place the plate on the magnetic stand 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. Take care not to disturb the beads.
6
Repeat step 5 to remove and discard a total of 276 µl of the supernatant from each
well.
NOTE
Leave the plate on the magnetic stand while performing the following steps 7–12.
7
With the plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to each
well. Do not disturb the beads.
8
Incubate the 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 to perform an 80% EtOH wash 2 times.
10 Remove and discard any remaining EtOH with a 10 µl pipette.
11 With the plate on the magnetic stand, let the samples air-dry at room temperature for
5 minutes. Do not allow the beads to crack.
12 With the plate on the magnetic stand, add 17.5 µl Resuspension Buffer to each well.
Make sure the Resuspension Buffer runs over the beads. Do not touch the beads with
the pipette tip.
13 Remove the plate from the magnetic stand.
14 Resuspend the beads in each well 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.
TruSeq DNA PCR-Free Library Prep Guide
19
Perform End Repair and Size Selection
9
Low Sample (LS) Protocol
15 Incubate the plate at room temperature for 2 minutes.
16 Place the plate on the magnetic stand for 5 minutes or until the liquid is clear.
17 Transfer 15 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.
NOTE
Illumina recommends performing 2 consecutive transfers. This technique reduces
sample loss by making sure that all of the liquid is transferred without disturbing the
beads.
1. Using a 10 µl single channel or multichannel pipette set to 10 µl, transfer 10 µl of the
clear supernatant from each well of the IMP plate to the corresponding well of the
ALP plate.
2. Set the pipette to 5 µl and use the same pipette tip to transfer 5 µl of the clear
supernatant from each well of the IMP plate to the corresponding well of the ALP
plate.
SAFE STOPPING POINT
If you do not plan to proceed immediately to Adenylate 3' Ends, you can safely stop the
protocol here. If you are stopping, seal the ALP plate with a Microseal ‘B’ adhesive seal and
store at -25°C to -15°C for up to 7 days.
20
Part # 15036187 Rev. C
A single ‘A’ nucleotide is added to the 3’ ends of the blunt fragments to prevent them from
ligating to each other 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
-25°C to -15°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
[Optional] A-Tailing Control
(CTA)
1 tube per 48
reactions
-25°C to -15°C
Illumina
Microseal ‘B’ adhesive seal
1
15°C to 30°C
User
RNase/DNase-free 8-tube strips
and caps
(if using multichannel pipettes)
3
15°C to 30°C
User
RNase/DNase-free reagent
reservoirs
(if using multichannel pipettes)
3
15°C to 30°C
User
Preparation
} Remove the following from -25°C to -15°C storage and thaw them at room temperature.
• A-Tailing Control
NOTE
The use of the A-Tailing Control is optional and it can be replaced with the same
volume of Resuspension Buffer.
• A-Tailing Mix
} Remove the Resuspension Buffer from 2°C to 8°C storage and bring it to room
temperature.
} Remove the ALP plate from -25°C to -15°C storage, if it was stored at the conclusion of
Clean Up IMP and Size Selection.
• Let it thaw at room temperature.
• Centrifuge the thawed plate at 280 × g for 1 minute.
• Remove the adhesive seal from the plate.
TruSeq DNA PCR-Free Library Prep Guide
21
Adenylate 3' Ends
Adenylate 3' Ends
Low Sample (LS) Protocol
} Preprogram the thermal cycler with the following program and save as ATAIL70:
• Choose the preheat 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
Add ATL
1
Centrifuge the thawed A-Tailing Control (if using A-Tailing Control) and A-Tailing Mix
tubes at 600 × g for 5 seconds.
2
Do the following:
• If using the in-line control reagent, add 2.5 µl thawed A-Tailing Control to each
well.
• If not using the in-line control reagent, add 2.5 µl Resuspension Buffer to each well.
3
Add 12.5 µl thawed A-Tailing Mix to each well. Set a 200 µl pipette to 20 µl, then
gently pipette the entire volume up and down 10 times to mix thoroughly.
4
Seal the ALP plate with a Microseal ‘B’ adhesive seal.
Incubate 1 ALP
22
1
Centrifuge the ALP plate at 280 × g for 1 minute.
2
Place the plate on the preprogrammed thermal cycler. Close the lid, then select and run
the ATAIL70 program. Each well contains 30 µl of sample.
a Choose the preheat 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
3
When the thermal cycler temperature has been at 4°C for 5 minutes, remove the plate
from the thermal cycler.
4
Centrifuge the ALP plate at 280 × g for 1 minute.
5
Proceed immediately to Ligate Adapters.
Part # 15036187 Rev. C
This process ligates multiple indexing adapters to the ends of the DNA fragments,
preparing them for hybridization onto a flow cell.
Consumables
Item
Quantity
Storage
Supplied By
Choose from the following
depending on the kit you are
using:
• TruSeq DNA PCR-Free LT
Library Prep Kit contents:
• DNA Adapter Indexes
(AD001–AD016, AD018–
AD023, AD025, AD027)
• TruSeq DNA PCR-Free HT
Library Prep Kit contents:
• DAP (DNA Adapter Plate)
1 tube of each index
being used, per
column of 8 reactions
or
1 DAP
-25°C to -15°C
Illumina
Ligation Mix 2 (LIG2)
LT kit - 1 tube per 24
reactions
or
HT kit - 1 tube per 48
reactions
-25°C to -15°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
-25°C to -15°C
Illumina
[Optional] Ligation Control
(CTL)
1 tube per 48
reactions
-25°C to -15°C
Illumina
Barcode labels for:
• CAP (Clean Up ALP Plate)
• DAP (DNA Adapter Plate)
(if using the HT kit)
• TSP1 (Target Sample 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
TruSeq DNA PCR-Free Library Prep Guide
23
Ligate Adapters
Ligate Adapters
Low Sample (LS) Protocol
Item
Quantity
Storage
Supplied By
RNase/DNase-free 8-tube strips
and caps (if using multichannel
pipettes)
5–28
15°C to 30°C
User
RNase/DNase-free reagent
reservoirs (if using
multichannel pipettes)
5–28
15°C to 30°C
User
Preparation
} Remove the following from -25°C to -15°C storage and thaw them at room temperature:
• Appropriate DNA Adapter tubes (depending on the DNA Adapter Indexes being
used) or the DAP.
NOTE
• Review the TruSeq Library Prep Pooling Guide (part # 15042173).
• 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:
• Review Handling Adapter Plate in the TruSeq Library Prep Pooling Guide
(part # 15042173).
• Arrange samples that will be pooled together in the same orientation as the
indexes in the DAP.
• Illumina recommends that the RAP does not undergo more than 4 freezethaw cycles. To maximize the use of the RAP, 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 -25°C to -15°C storage until instructed to
do so in the procedure.
• Ligation Control
NOTE
The use of the Ligation Control is optional and it can be replaced with the same
volume of Resuspension Buffer.
} 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 about TruSeq DNA PCR-Free Library Prep best practices on the
Illumina website.
} Preprogram the thermal cycler with the following program and save as LIG:
• Choose the thermal cycler preheat 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 TSP1 barcode label to a new 96-well 0.3 ml PCR plate.
24
Part # 15036187 Rev. C
1
Do 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 tape seal from the plate.
— 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 the same time.
— If it is the first time using this DAP, apply the DAP barcode label to the plate.
2
Centrifuge the Ligation Control (if using Ligation Control) and Stop Ligation Buffer
tubes at 600 × g for 5 seconds.
3
Immediately before use, remove the Ligation Mix 2 tube from -25°C to -15°C storage.
4
Remove the adhesive seal from the ALP plate.
5
Do the following:
• If using the in-line control reagent, add 2.5 µl thawed Ligation Control to each well.
• If not using the in-line control reagent, add 2.5 µl Resuspension Buffer to each well.
6
Add 2.5 µl Ligation Mix 2 to each well.
7
Return the Ligation Mix 2 tube to -25°C to -15°C storage immediately after use.
8
Do the following:
• If using DNA Adapter tubes, add 2.5 µl thawed DNA Adapter Index to each well.
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
— Do the following to pierce the foil seal:
— If using the entire plate at the same time, use the bottom of a clean 96-well
semi-skirted 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 8-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 8-tube strip, with caps attached, for each
row or column of adapters that will be used for ligation.
TruSeq DNA PCR-Free Library Prep Guide
25
Ligate Adapters
Add LIG
Low Sample (LS) Protocol
— Using an 8-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 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 on the preprogrammed thermal cycler. Close the lid then
select and run the LIG program. Each well contains 37.5 µl of sample.
a Choose the thermal cycler preheat 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 to inactivate the ligation. Set a 200 µl pipette
to 30 µl, then gently pipette the entire volume up and down 10 times to mix
thoroughly.
Add STL
Clean Up ALP
1
Vortex the Sample Purification Beads for at least 1 minute or until they are well
dispersed.
2
Add 42.5 µl mixed Sample Purification Beads to each well. Set a 200 µl pipette to
60 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 4 samples
• If using a multichannel pipette, vortex the beads after processing 4 columns
• If the beads are in a reagent reservoir, mix with a 1000 µl pipette
3
Incubate the plate at room temperature for 5 minutes.
4
Place the plate on the magnetic stand for 5 minutes or until the liquid is clear.
5
Remove and discard 80 µl of the supernatant from each well. Take care not to disturb
the beads.
NOTE
Leave the plate on the magnetic stand while performing the following steps 6–11.
26
6
With the plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to each
well. Do not disturb the beads.
7
Incubate the 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 to perform an 80% EtOH wash 2 times.
Part # 15036187 Rev. C
Remove and discard any remaining EtOH from each well with a 10 µl pipette.
10 With the plate on the magnetic stand, let the samples air-dry at room temperature for
5 minutes. Do not allow the beads to crack.
11 With the plate on the magnetic stand, add 52.5 µl Resuspension Buffer to each well.
Make sure the Resuspension Buffer runs over the beads. Do not touch the beads with
the pipette tip.
12 Remove the plate from the magnetic stand.
13 Resuspend the beads in each well 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.
14 Incubate the plate at room temperature for 2 minutes.
15 Place the plate on the magnetic stand for 5 minutes or until the liquid is clear.
16 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.
17 Vortex the Sample Purification Beads for at least 1 minute or until they are well
dispersed.
18 Add 50 µl mixed Sample Purification Beads to each well for a second cleanup. Set a
200 µl pipette to 75 µl, and then gently pipette the entire volume up and down
10 times to mix thoroughly.
19 Incubate the plate at room temperature for 5 minutes.
20 Place the plate on the magnetic stand for 5 minutes or until the liquid is clear.
21 Remove and discard 95 µl of the supernatant from each well. Take care not to disturb
the beads.
NOTE
Leave the plate on the magnetic stand while performing the following steps 22–27.
22 With the plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to each
well. Do not disturb the beads.
23 Incubate the 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.
24 Repeat steps 22 and 23 to perform an 80% EtOH wash 2 times.
25 Remove and discard any remaining EtOH from each well with a 10 µl pipette.
26 With the plate on the magnetic stand, let the samples air-dry at room temperature for
5 minutes. Do not allow the beads to crack.
27 With the plate on the magnetic stand, add 22.5 µl Resuspension Buffer to each well.
Make sure the Resuspension Buffer runs over the beads. Do not touch the beads with
the pipette tip.
28 Remove the plate from the magnetic stand.
29 Resuspend the beads in each well 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.
30 Incubate the plate at room temperature for 2 minutes.
TruSeq DNA PCR-Free Library Prep Guide
27
Ligate Adapters
9
Low Sample (LS) Protocol
31 Place the plate on the magnetic stand for 5 minutes or until the liquid is clear.
32 Transfer 20 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 TSP1 barcode. Take
care not to disturb the beads.
33 Return the following to -25°C to -15°C storage:
• Ligation Control
• DNA Adapter Indexes
• DAP
• Stop Ligation Buffer
SAFE STOPPING POINT
If you do not plan to proceed immediately to Validate Libraries, you can safely stop the
protocol here. If you are stopping, seal the TSP1 plate with a Microseal ‘B’ adhesive seal and
store at -25°C to -15°C for up to 7 days.
28
Part # 15036187 Rev. C
Illumina recommends performing the following procedures for quality control analysis on
the sample libraries and quantification of the DNA libraries.
Quantify Libraries
To achieve the highest quality data on Illumina sequencing platforms, it is important to
create optimum cluster densities across every lane of the flow cell. Optimizing cluster
densities requires accurate quantitation of DNA libraries. qPCR must be used to quantify
TruSeq DNA PCR-Free Library Prep libraries.
NOTE
Methods other than qPCR quantify molecules that do not have adapters on both ends and
do not form clusters. More of these nonclusterable molecules can be present due to the
absence of PCR enrichment and quantification by methods other than qPCR can be
inaccurate.
NOTE
TruSeq DNA PCR-Free Library Prep library quantitation has been validated using the KAPA
Library Quantification Kit specified in the Consumables and Equipment on page 72.
Follow qPCR instructions included in the KAPA Library Quantification Kits for Illumina
sequencing platforms Technical Data Sheet using the KAPA standard
(www.kapabiosystems.com), with the following modifications:
NOTE
You can download the KAPA Library Quantification Kits for Illumina sequencing platforms
Technical Data Sheet from the Kapa Biosystems website (www.kapabiosystems.com).
} Use at least 2 µl of the original library stock in the library dilution step to ensure
accurate and reproducible quantitation.
} Illumina recommends 2 further independent (not serial) 1:10,000 and 1:20,000 dilutions
using at least 2 µl of the initial diluted libraries to evaluate quantitation precision.
NOTE
For guidance on handling small liquid volumes, see Handling Liquids in the TruSeq DNA
PCR-Free Library Prep Best Practices. See Additional Resources on page 6 for information
TruSeq DNA PCR-Free Library Prep Best Practices on the Illumina website.
The concentration of each library is calculated as indicated in Table 10 and Table 11:
} Obtain the calculated concentration of the 1:10,000 and 1:20,000 dilutions of the library,
as determined by qPCR, in relation to the concentrations of the correctly annotated
KAPA DNA Standards 1–6. Use the average of the replicate data points to determine
the concentration of the diluted library.
} Perform a size adjustment calculation to account for the difference in size between the
average fragment length of the library and the KAPA DNA Standard (452 bp):
• 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
Do not use the average fragment length of the library insert size based on the
Bioanalyzer results. PCR-free library fragment sizes measured on the Bioanalyzer
are substantially larger than would be predicted or derived from sequencing data.
} Calculate the concentration of the undiluted library by taking account of the relevant
dilution factor (e.g. 1:10,000 and 1:20,000). Use the average of the replicate data points
TruSeq DNA PCR-Free Library Prep Guide
29
Validate Libraries
Validate Libraries
Low Sample (LS) Protocol
corresponding to each library DNA dilution to calculate the concentration of the
undiluted library.
} If 1 of the replicates appears to be an outlier, it can be omitted from the calculation. If
more than 1 of replicates appears to be outliers, repeat the assay.
Table 10 350 bp Library Concentration Calculation
Calculated
Average
Dilution
by qPCR
diluted
Factor
instrument (pM)*
library (pM)
1:10,000
A1
A2
A=
(A1 + A2)/2
1:20,000
B1
B2
B=
(B1 + B2)/2
Size adjusted
diluted
library (pM)
W1 =
A x (452/470)
W2 =
B x (452/470)
Table 11 550 bp Library Concentration Calculation
Calculated
Average
Dilution
by qPCR
diluted
Factor
instrument (pM)*
library (pM)
1:10,000
C1
C2
C=
(C1 + C2)/2
1:20,000
D1
D2
D=
(D1 + D2)/2
Size adjusted
diluted
library (pM)
W3 =
C x (452/670)
W4 =
D x (452/670)
Undiluted
library (pM)*
Undiluted
library (pM)
C1 =
W1 x 10,000
C2 =
W2 x 20,000
(C1 + C2)/2
Undiluted
library (pM)*
Undiluted
library (pM)
C3 =
W3 x 10,000
C4 =
W4 x 20,000
(C3 + C4)/2
*Duplicate data points
Quality Control
To verify the size of your fragments, check the template size distribution.
1
Prepare a 1:5 dilution of the DNA library with water.
2
Run 1 µl of the diluted DNA library on an Agilent Technologies 2100 Bioanalyzer
using a High Sensitivity DNA chip.
Run samples on a Bioanalyzer for qualitative purposes only.
When performing quality control on TruSeq DNA PCR-Free Library Prep libraries, PCR-Free
library fragment sizes measured on the Bioanalyzer are substantially larger than would be
predicted or derived from sequencing data. The larger size is due to anomalous migration
of fragments on the chip due to the presence of certain structural features, which would
normally be removed if a subsequent PCR-enrichment step were performed. Figure 3 and
Figure 4 show a comparison between library fragment sizes derived by a Bioanalyzer and
the corresponding insert sizes derived from the alignment of paired-end reads to a suitable
reference sequence.
30
Part # 15036187 Rev. C
Validate Libraries
Figure 3 Example TruSeq DNA PCR-Free Library Prep 350 bp Insert Library Distribution
A
B
Bioanalyzer
Paired-End Alignment
Figure 4 Example TruSeq DNA PCR-Free Library Prep 550 bp Insert Library Distribution
A
B
Bioanalyzer
Paired-End Alignment
TruSeq DNA PCR-Free Library Prep Guide
31
Low Sample (LS) Protocol
Normalize and Pool Libraries
This process describes how to prepare DNA templates for cluster generation. Indexed DNA
libraries are normalized to 2 nM in the DCT plate and then pooled in equal volumes in the
PDP plate. Non-indexed DNA libraries are normalized to 2 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
96-well midi plate
(for pooling only, if pooling
> 40 samples)
1
15°C to 30°C
User
96-well 0.3 ml PCR plates
2
(second plate for
pooling only, if
pooling ≤ 40 samples)
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 library to 2 nM
15°C to 30°C
User
Preparation
} Remove the TSP1 plate from -25°C to -15°C storage, if it was stored at the conclusion of
Clean Up ALP.
• Let it thaw at room temperature.
• Centrifuge the thawed plate at 280 × g for 1 minute.
• Remove the adhesive seal from the thawed plate.
} Apply a DCT barcode label to a new 96-well 0.3 ml PCR plate.
} [For pooling only] Review the TruSeq Library Prep Pooling Guide (part # 15042173).
} [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 5 µl of the library from each well of the TSP1 plate to the corresponding well
of the new 0.3 ml PCR plate labeled with the DCT barcode.
2
Normalize the library concentration in each well to 2 nM using Tris-HCl 10 mM,
pH 8.5 with 0.1% Tween 20.
NOTE
Depending on the yield quantification data of each library, the final volume in the plate
can vary from 5–100 µl.
3
32
Gently pipette the entire volume up and down 10 times to mix thoroughly.
Part # 15036187 Rev. C
Do the following, depending on the type of library you want to generate:
• For libraries that are not pooled, the protocol stops here. Do the following:
— To continue immediately to sequencing, proceed to cluster generation. For more
information, see the cluster generation section of the system guide for your
Illumina platform.
— To stop here before sequencing, seal the DCT plate with a Microseal ‘B’
adhesive seal and store at -25°C to -15°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.
1
Determine the number of samples to be combined together for each pool.
NOTE
Avoid pooling 2 samples with the same index.
2
Do the following:
• If pooling 2–24 samples:
— Transfer 5 µl of each normalized library to be pooled from the DCT plate to
1 well of the new 0.3 ml PCR plate labeled with the PDP barcode.
The total volume in each well is 5 X the number of combined sample libraries and
10–120 µl (2–24 libraries). For example, the volume for 2 samples is 10 µl, the
volume for 12 samples is 60 µl, or the volume for 24 samples is 120 µl.
• If pooling 25–96 samples:
— Using a multichannel pipette, transfer 5 µl of each normalized 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 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.
3
Gently pipette the entire volume up and down 10 times to mix thoroughly.
4
Do the following:
• To continue immediately to sequencing, proceed to cluster generation. For more
information, see the system guide for your Illumina sequencing platform.
• To stop here before sequencing, seal the PDP plate with a Microseal ‘B’ adhesive
seal and store at -25°C to -15°C.
TruSeq DNA PCR-Free Library Prep Guide
33
Normalize and Pool Libraries
4
34
Part # 15036187 Rev. C
Chapter 3 High Sample (HS) Protocol
Introduction
Library Prep Workflow
Prepare Adapter Setup
Fragment DNA
Perform End Repair and Size Selection
Adenylate 3' Ends
Ligate Adapters
Validate Library
Normalize and Pool Libraries
TruSeq DNA PCR-Free Library Prep Guide
36
37
38
39
44
49
51
57
60
35
Chapter 3
High Sample (HS) Protocol
High Sample (HS) Protocol
Introduction
This chapter describes the TruSeq DNA PCR-Free Library Prep HS protocol. This protocol is
intended for preparing more than 24 samples at the same 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 DNA PCR-Free Library Prep Best Practices on 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 74).
36
Part # 15036187 Rev. C
The following figure illustrates theTruSeq DNA PCR-Free Library Prep HS workflow.
Figure 5 TruSeq DNA PCR-Free Library Prep HS Workflow
TruSeq DNA PCR-Free Library Prep Guide
37
Library Prep Workflow
Library Prep Workflow
High Sample (HS) Protocol
Prepare Adapter Setup
If you are pooling, use IEM or BaseSpace to record information about your samples before
beginning library prep.
} Use IEM to create and edit sample sheets for Illumina sequencing systems and analysis
software.
} Use BaseSpace to organize samples, libraries, pools, and a run for Illumina sequencing
systems and analysis software.
NOTE
For information about IEM and BaseSpace software documentation on the Illumina website,
see Additional Resources on page 6.
Review the planning steps in the TruSeq Library Prep Pooling Guide (part # 15042173) when
preparing libraries for Illumina sequencing systems that require balanced index
combinations.
38
Part # 15036187 Rev. C
This process describes how to optimally fragment the gDNA depending on the Covaris
platform used. 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 12 Insert Size Options
Insert Size
Input DNA Per Sample
Recommended Read Length
350 bp
550 bp
1 µg
≤ 2 x 101 bp
2 µg
≤ 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
-25°C to -15°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
96-well midi plates
3
15°C to 30°C
User
Covaris tubes
1 per sample
15°C to 30°C
User
DNA samples
• 1 µg per sample
for a 350 bp insert
size
• 2 µg per sample
for a 550 bp insert
size
-25°C to -15°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
TruSeq DNA PCR-Free Library Prep Guide
39
Fragment DNA
Fragment DNA
High Sample (HS) Protocol
Preparation
} Review DNA Input Recommendations on page 4.
} Review best practices for handling magnetic beads. See Additional Resources on page 6
for information about TruSeq DNA PCR-Free Library Prep 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 the Resuspension Buffer from -25°C to -15°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 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.
Make CFP
1
Illumina recommends quantifying gDNA samples using a fluorometric-based method
such as Qubit or PicoGreen.
2
Illumina recommends normalizing gDNA samples with Resuspension Buffer, to a final
volume of 55 µl in each well of the new midi plate labeled with the DNA barcode:
• 20 ng/µl for a 350 bp insert size
• 40 ng/µl for a 550 bp insert size
3
Mix thoroughly as follows:
a Seal the plate with a Microseal ‘B’ adhesive seal.
b Shake the plate on a microplate shaker at 1800 rpm for 2 minutes.
4
Centrifuge the DNA plate at 280 × g for 1 minute.
Fragment DNA
1
Remove the adhesive seal from the DNA plate.
2
Shear the gDNA sample by transferring 52.5 µl of each DNA sample from the DNA
plate to a separate, new Covaris tube:
• 1 µg for a 350 bp insert size
• 2 µg 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.
3
40
Centrifuge the CFP plate at 280 × g for 5 seconds.
Part # 15036187 Rev. C
Fragment DNA
4
Fragment the DNA using the following settings:
Table 13 Covaris S220 Settings
Setting
350 bp Insert
550 bp Insert
Duty factor
5%
Peak Incident Power
175 W
Cycles per burst
Duration
200
50 seconds
Mode
25 seconds
Frequency sweeping
Temperature
5.5° to 6°C
NOTE
The Covaris M220 settings are optimized for use with the Covaris microTUBE AFA
Fiber Pre-Slit Snap-Cap 6x16mm.
Table 14 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
Table 15 Covaris S2 and E210 Settings
Setting
350 bp Insert
Duty cycle
Intensity
10%
5.0
2.0
Cycles per burst
200
Duration
Mode
Displayed Power
Temperature
550 bp Insert
45 seconds
Frequency sweeping
S2—23 W
S2—9 W
E210—14 W
E210—7 W
5.5° to 6°C
5
Centrifuge the plate at 280 × g for 5 seconds.
6
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.
TruSeq DNA PCR-Free Library Prep Guide
41
High Sample (HS) Protocol
7
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 mixed Sample Purification Beads to each well. Mix thoroughly as follows:
a Seal the plate with a Microseal ‘B’ adhesive seal.
b Shake the 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 4 samples
• If using a multichannel pipette, vortex the beads after processing 4 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 plate at room temperature for 5 minutes.
4
Centrifuge the plate at 280 × g for 1 minute.
5
Remove the adhesive seal from the plate.
6
Place the plate on the magnetic stand 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 clear supernatant from each well. Take care not to disturb the
beads.
NOTE
Leave the plate on the magnetic stand while performing the following steps 8–13.
8
With the plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to each
well. Do not disturb the beads.
9
Incubate the 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.
10 Repeat steps 8 and 9 to perform an 80% EtOH wash 2 times.
11 Remove and discard any remaining EtOH from each well with a 10 µl pipette.
12 With the plate on the magnetic stand, let the samples air-dry at room temperature for
5 minutes. Do not allow the beads to crack.
13 With the plate on the magnetic stand, add 52.5 µl Resuspension Buffer to each well.
Make sure the Resuspension Buffer runs over the beads. Do not touch the beads with
the pipette tip.
14 Remove the plate from the magnetic stand.
15 Mix thoroughly as follows:
a Seal the plate with a Microseal ‘B’ adhesive seal.
b Shake the plate on a microplate shaker at 1800 rpm for 2 minutes.
16 Incubate the plate at room temperature for 2 minutes.
17 Centrifuge the plate at 280 × g for 1 minute.
42
Part # 15036187 Rev. C
19 Place the plate on the magnetic stand for 5 minutes or until the liquid is clear.
20 Transfer 50 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.
21 Proceed immediately to Perform End Repair and Size Selection.
TruSeq DNA PCR-Free Library Prep Guide
43
Fragment DNA
18 Remove the adhesive seal from the plate.
High Sample (HS) Protocol
Perform End Repair and Size Selection
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
-25°C to -15°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
[Optional] End Repair Control
(CTE)
1 tube per 48
reactions
-25°C to -15°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
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
Ice bucket
As needed
-25°C to -15°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 8-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 -25°C to -15°C storage. Thaw it at room
temperature and then place it on ice.
} Remove the End Repair Control from -25°C to -15°C storage and thaw it at room
44
Part # 15036187 Rev. C
NOTE
The use of the End Repair Control is optional and it can be replaced with the same
volume of Resuspension Buffer.
} Remove the Sample Purification Beads and Resuspension Buffer 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 about TruSeq DNA PCR-Free Library Prep best practices on the
Illumina website.
} Preheat 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
1
Do the following:
• If using the in-line control reagent:
— Centrifuge the thawed End Repair Control tube at 600 × g for 5 seconds.
— Add 10 µl thawed End Repair Control to each well.
• If not using the in-line control reagent, add 10 µl Resuspension Buffer to each well.
2
Centrifuge the thawed End Repair Mix 2 tube at 600 × g for 5 seconds.
3
Add 40 µl End Repair Mix to each well. Mix thoroughly as follows:
a Seal the plate with a Microseal ‘B’ adhesive seal.
b Shake the plate on a microplate shaker at 1800 rpm for 2 minutes.
4
Centrifuge the IMP plate at 280 × g for 1 minute.
5
Return the End Repair Mix 2 tube to -25°C to -15°C storage.
Incubate IMP
1
Place the IMP plate on the preheated microheating system. Close the lid and incubate
at 30°C for 30 minutes. Each well contains 50 µl of sample.
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 DNA PCR-Free Library Prep Guide
45
Perform End Repair and Size Selection
temperature.
High Sample (HS) Protocol
2
Combine Sample Purification Beads and PCR grade water in a tube to create a diluted
bead mixture of 160 µl per 100 µl of end-repaired sample:
• When processing > 6 samples at a time, use a new 15 ml conical tube
• When processing ≤ 6 samples at a time, use a new 1.7 ml microcentrifuge tube
Determine the volumes using the following formulas, which include 15% excess for
multiple samples:
Table 16 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 17 Diluted Bead Mixture for a 550 bp Insert Size
Formula
Sample Purification Beads
PCR grade water
Example
Amount
per 12
samples
# of samples X
92 µl
# of samples X
92 µl
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. Mix thoroughly
as follows:
a Seal the plate with a Microseal ‘B’ adhesive seal.
b Shake the 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.
NOTE
Vortex the diluted bead mixture frequently. Illumina recommends the following:
• If using a single channel pipette, vortex the mixture after processing 4 samples
• If using a multichannel pipette, vortex the mixture after processing 4 columns
• If the mixture is in a reagent reservoir, mix with a 1000 µl pipette.
46
5
Incubate the plate at room temperature for 5 minutes.
6
Centrifuge the plate at 280 × g for 1 minute.
7
Remove the adhesive seal from the plate.
8
Place the plate on the magnetic stand for 5 minutes or until the liquid is clear.
Part # 15036187 Rev. C
Set a 200 µl single channel or multichannel pipette to 125 µl. Transfer 125 µl of the
supernatant, which contains 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. Each CEP plate well now contains a total of 250 µl of DNA of interest.
11 Discard the IMP plate that contains 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. Mix
thoroughly as follows:
a Seal the plate with a Microseal ‘B’ adhesive seal.
b Shake the plate on a microplate shaker at 1800 rpm for 2 minutes.
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. If the beads are in a reagent reservoir, Illumina recommends mixing with a
1000 µl pipette.
3
Incubate the plate at room temperature for 5 minutes.
4
Centrifuge the plate at 280 × g for 1 minute.
5
Remove the adhesive seal from the plate.
6
Place the plate on the magnetic stand 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. Take care not to disturb the beads.
8
Repeat step 7 to remove and discard a total of 276 µl of supernatant from each well.
NOTE
Leave the plate on the magnetic stand while performing the following steps 9–14.
9
With the plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to each
well. Do not disturb the beads.
10 Incubate the 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 to perform an 80% EtOH wash 2 times.
12 Remove and discard any remaining EtOH with a 10 µl pipette.
13 With the plate on the magnetic stand, let the samples air-dry at room temperature for
5 minutes. Do not allow the beads to crack.
TruSeq DNA PCR-Free Library Prep Guide
47
Perform End Repair and Size Selection
9
High Sample (HS) Protocol
14 With the plate on the magnetic stand, add 17.5 µl Resuspension Buffer to each well.
Make sure the Resuspension Buffer runs over the beads. Do not touch the beads with
the pipette tip.
15 Remove the plate from the magnetic stand.
16 Mix thoroughly as follows:
a Seal the plate with a Microseal ‘B’ adhesive seal.
b Shake the plate on a microplate shaker at 1800 rpm for 2 minutes.
17 Incubate the plate at room temperature for 2 minutes.
18 Centrifuge the plate at 280 × g for 1 minute.
19 Remove the adhesive seal from the plate.
20 Place the plate on the magnetic stand for 5 minutes or until the liquid is clear.
21 Transfer 15 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.
NOTE
Illumina recommends performing 2 consecutive transfers. This technique reduces
sample loss by making sure that all of the liquid is transferred without disturbing the
beads.
1. Using a 10 µl single channel or multichannel pipette set to 10 µl, transfer 10 µl of the
clear supernatant from each well of the IMP plate to the corresponding well of the
ALP plate.
2. Set the pipette to 5 µl and use the same pipette tip to transfer 5 µl of the clear
supernatant from each well of the IMP plate to the corresponding well of the ALP
plate.
SAFE STOPPING POINT
If you do not plan to proceed to Adenylate 3' Ends immediately, you can safely stop the
protocol here. If you are stopping, seal the ALP plate with a Microseal ‘B’ adhesive seal and
store at -25°C to -15°C for up to 7 days.
48
Part # 15036187 Rev. C
A single ‘A’ nucleotide is added to the 3’ ends of the blunt fragments to prevent them from
ligating to each other 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
-25°C to -15°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
[Optional] A-Tailing Control
(CTA)
1 tube per 48
reactions
-25°C to -15°C
Illumina
Microseal ‘B’ adhesive seal
1
15°C to 30°C
User
RNase/DNase-free 8-tube strips
and caps
(if using multichannel pipettes)
3
15°C to 30°C
User
RNase/DNase-free reagent
reservoirs
(if using multichannel pipettes)
3
15°C to 30°C
User
Preparation
} Remove the following from -25°C to -15°C storage and thaw them at room temperature.
• A-Tailing Control
NOTE
The use of the A-Tailing Control is optional and it can be replaced with the same
volume of Resuspension Buffer.
• A-Tailing Mix
} Remove the Resuspension Buffer from 2°C to 8°C storage and bring it to room
temperature.
} Remove the ALP plate from -25°C to -15°C storage, if it was stored at the conclusion of
Clean Up IMP and Size Selection.
• Let it thaw at room temperature.
• Centrifuge the thawed plate at 280 × g for 1 minute.
• Remove the adhesive seal from the plate.
} Preheat 2 microheating systems: system 1 to 37°C and system 2 to 70°C.
Add ATL
1
Centrifuge the thawed A-Tailing Control (if using A-Tailing Control) and A-Tailing Mix
tubes at 600 × g for 5 seconds.
TruSeq DNA PCR-Free Library Prep Guide
49
Adenylate 3' Ends
Adenylate 3' Ends
High Sample (HS) Protocol
2
Do the following:
• If using the in-line control reagent, add 2.5 µl thawed A-Tailing Control to each
well.
• If not using the in-line control reagent, add 2.5 µl Resuspension Buffer to each well.
3
Add 12.5 µl thawed A-Tailing Mix to each well. Mix thoroughly as follows:
a Seal the plate with a Microseal ‘B’ adhesive seal.
b Shake the plate on a microplate shaker at 1800 rpm for 2 minutes.
4
Centrifuge the ALP plate at 280 × g for 1 minute.
Incubate 1 ALP
50
1
Place the sealed ALP plate on the preheated microheating system 1. Close the lid and
incubate at 37°C for 30 minutes. Each well contains 30 µl of sample.
2
Immediately after the 37°C incubation, remove the plate from system 1 and place the
plate on the preheated 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.
Part # 15036187 Rev. C
This process ligates indexing adapters to the ends of the DNA fragments, preparing them
for hybridization onto a flow cell.
Consumables
Item
Quantity
Storage
Supplied By
Choose from the following
depending on the kit you are
using:
• TruSeq DNA PCR-Free LT
Library Prep Kit contents:
• DNA Adapter Indexes
(AD001–AD016, AD018–
AD023, AD025, AD027)
• TruSeq DNA PCR-Free HT
Library Prep Kit contents:
• DAP (DNA Adapter Plate)
1 tube of each index
being used, per
column of 8 reactions
or
1 DAP
-25°C to -15°C
Illumina
Ligation Mix 2 (LIG2)
LT kit - 1 tube per 24
reactions
or
HT kit - 1 tube per 48
reactions
-25°C to -15°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
-25°C to -15°C
Illumina
[Optional] Ligation Control
(CTL)
1 tube per 48
reactions
-25°C to -15°C
Illumina
Barcode labels for:
• CAP (Clean Up ALP Plate)
• DAP (DNA Adapter Plate)
(if using the HT kit)
• 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
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
-25°C to -15°C
User
Microseal ‘B’ adhesive seals
7
15°C to 30°C
User
TruSeq DNA PCR-Free Library Prep Guide
51
Ligate Adapters
Ligate Adapters
High Sample (HS) Protocol
Item
Quantity
Storage
Supplied By
RNase/DNase-free 8-tube strips
and caps (if using multichannel
pipettes)
5–28
15°C to 30°C
User
RNase/DNase-free reagent
reservoirs (if using
multichannel pipettes)
5–28
15°C to 30°C
User
Preparation
} Prepare an ice bucket.
} Remove the following from -25°C to -15°C storage and thaw them at room temperature:
• Appropriate DNA Adapter tubes (depending on the DNA Adapter Indexes being
used) or the DAP.
NOTE
• Review the TruSeq Library Prep Pooling Guide (part # 15042173).
• 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:
• Review Handling Adapter Plate in the TruSeq Library Prep Pooling Guide
(part # 15042173).
• Arrange samples that will be pooled together in the same orientation as the
indexes in the DAP.
• Illumina recommends that the RAP does not undergo more than 4 freezethaw cycles. To maximize the use of the RAP, 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 -25°C to -15°C storage until instructed to
do so in the procedure.
• Ligation Control
NOTE
The use of the Ligation Control is optional and it can be replaced with the same
volume of Resuspension Buffer.
} 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 about TruSeq DNA PCR-Free Library Prep best practices on the
Illumina website.
} Preheat the microheating system 1 to 30°C.
} Apply a CAP barcode label to a new 96-well midi plate.
} Apply a TSP1 barcode label to a new 96-well HSP plate.
52
Part # 15036187 Rev. C
1
Do 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 tape seal from the plate.
— 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 the same time.
— If it is the first time using this DAP, apply the DAP barcode label to the plate.
2
Centrifuge the Ligation Control (if using Ligation Control) and Stop Ligation Buffer
tubes at 600 × g for 5 seconds.
3
Immediately before use, remove the Ligation Mix 2 tube from -25°C to -15°C storage.
4
Remove the adhesive seal from the ALP plate.
5
Do the following:
• If using the in-line control reagent, add 2.5 µl thawed Ligation Control to each well.
• If not using the in-line control reagent, add 2.5 µl Resuspension Buffer to each well.
6
Add 2.5 µl Ligation Mix 2 to each well.
7
Return the Ligation Mix 2 tube to -25°C to -15°C storage immediately after use.
8
Do the following:
• If using DNA Adapter tubes, add 2.5 µl thawed DNA Adapter Index to each well.
• 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
— Do the following to pierce the foil seal:
— If using the entire plate at the same time, use the bottom of a clean 96-well
semi-skirted 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 8-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 8-tube strip, with caps attached, for each
row or column of adapters that will be used for ligation.
— Using an 8-tip multichannel pipette, transfer 2.5 µl thawed DNA Adapter from
the DAP well to each well of the ALP plate.
TruSeq DNA PCR-Free Library Prep Guide
53
Ligate Adapters
Add LIG
High Sample (HS) Protocol
9
Mix thoroughly as follows:
a Seal the plate with a Microseal ‘B’ adhesive seal.
b Shake the 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
Place the sealed ALP plate on the preheated microheating system. Close the lid and
incubate at 30°C for 10 minutes. Each well contains 37.5 µl of sample.
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 to inactivate the ligation mix. Mix
thoroughly as follows:
a Seal the plate with a Microseal ‘B’ adhesive seal.
b Shake the 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
Remove the adhesive seal from the ALP plate.
2
Vortex the Sample Purification Beads for at least 1 minute or until they are well
dispersed.
3
Add 42.5 µl mixed Sample Purification Beads to each well. Mix thoroughly as follows:
a Seal the plate with a Microseal ‘B’ adhesive seal.
b Shake the 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 4 samples
• If using a multichannel pipette, vortex the beads after processing 4 columns
• If the beads are in a reagent reservoir, mix with a 1000 µl pipette
4
Incubate the plate at room temperature for 5 minutes.
5
Centrifuge the plate at 280 × g for 1 minute.
6
Remove the adhesive seal from the plate.
7
Place the plate on the magnetic stand for 5 minutes or until the liquid is clear.
8
Remove and discard 80 µl of the supernatant from each well. Take care not to disturb
the beads.
NOTE
Leave the plate on the magnetic stand while performing the following steps 9–14.
9
54
With the plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to each
well. Do not disturb the beads.
Part # 15036187 Rev. C
11 Repeat steps 9 and 10 to perform an 80% EtOH wash 2 times.
12 Remove and discard any remaining EtOH from each well with a 10 µl pipette.
13 With the plate on the magnetic stand, let the samples air-dry at room temperature for
5 minutes. Do not allow the beads to crack.
14 With the plate on the magnetic stand, add 52.5 µl Resuspension Buffer to each well.
Make sure the Resuspension Buffer runs over the beads. Do not touch the beads with
the pipette tip.
15 Remove the plate from the magnetic stand.
16 Mix thoroughly as follows:
a Seal the plate with a Microseal ‘B’ adhesive seal.
b Shake the plate on a microplate shaker at 1800 rpm for 2 minutes.
17 Incubate the plate at room temperature for 2 minutes.
18 Centrifuge the plate at 280 × g for 1 minute.
19 Remove the adhesive seal from the plate.
20 Place the plate on the magnetic stand for 5 minutes or until the liquid is clear.
21 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.
22 Vortex the Sample Purification Beads for at least 1 minute or until they are well
dispersed.
23 Add 50 µl mixed Sample Purification Beads to each well. Mix thoroughly as follows:
a Seal the plate with a Microseal ‘B’ adhesive seal.
b Shake the plate on a microplate shaker at 1800 rpm for 2 minutes.
24 Incubate the plate at room temperature for 5 minutes.
25 Centrifuge the plate at 280 × g for 1 minute.
26 Remove the adhesive seal from the plate.
27 Place the plate on the magnetic stand for 5 minutes or until the liquid is clear.
28 Remove and discard 95 µl of the supernatant from each well. Take care not to disturb
the beads.
NOTE
Leave the plate on the magnetic stand while performing the following steps 29–34.
29 With the plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to each
well. Take care not to disturb the beads.
30 Incubate the 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.
31 Repeat steps 29 and 30 to perform an 80% EtOH wash 2 times.
32 Remove and discard any remaining EtOH from each well with a 10 µl pipette.
33 With the plate on the magnetic stand, let the samples air-dry at room temperature for
5 minutes. Do not allow the beads to crack.
TruSeq DNA PCR-Free Library Prep Guide
55
Ligate Adapters
10 Incubate the 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.
High Sample (HS) Protocol
34 With the plate on the magnetic stand, add 22.5 µl Resuspension Buffer to each well.
Make sure the Resuspension Buffer runs over the beads. Do not touch the beads with
the pipette tip.
35 Remove the plate from the magnetic stand.
36 Mix thoroughly as follows:
a Seal the plate with a Microseal ‘B’ adhesive seal.
b Shake the plate on a microplate shaker at 1800 rpm for 2 minutes.
37 Incubate the plate at room temperature for 2 minutes.
38 Centrifuge the plate at 280 × g for 1 minute.
39 Remove the adhesive seal from the plate.
40 Place the plate on the magnetic stand for 5 minutes or until the liquid is clear.
41 Transfer 20 µl of the clear supernatant from each well of the CAP plate to the
corresponding well of the new HSP plate labeled with the TSP1 barcode. Take care not
to disturb the beads.
42 Return the following to -25°C to -15°C storage:
• Ligation Control
• DNA Adapter Indexes
• DAP
• Stop Ligation Buffer
SAFE STOPPING POINT
If you do not plan to proceed immediately to Validate Library, you can safely stop the
protocol here. If you are stopping, seal the TSP1 plate with a Microseal ‘B’ adhesive seal and
store at -25°C to -15°C for up to 7 days.
56
Part # 15036187 Rev. C
Illumina recommends performing the following procedures for quality control analysis on
the sample libraries and quantification of the DNA library templates.
Quantify Libraries
To achieve the highest quality data on Illumina sequencing platforms, it is important to
create optimum cluster densities across every lane of the flow cell. Optimizing cluster
densities requires accurate quantitation of DNA libraries. qPCR must be used to quantify
TruSeq DNA PCR-Free Library Prep libraries.
NOTE
Methods other than qPCR quantify molecules that do not have adapters on both ends and
do not form clusters. More of these nonclusterable molecules can be present due to the
absence of PCR enrichment and quantification by methods other than qPCR can be
inaccurate.
NOTE
TruSeq DNA PCR-Free Library Prep library quantitation has been validated using the KAPA
Library Quantification Kit specified in the Consumables and Equipment on page 72.
Follow qPCR instructions included in the KAPA Library Quantification Kits for Illumina
sequencing platforms Technical Data Sheet using the KAPA standard
(www.kapabiosystems.com), with the following modifications:
NOTE
You can download the KAPA Library Quantification Kits for Illumina sequencing platforms
Technical Data Sheet from the Kapa Biosystems website (www.kapabiosystems.com).
} Use at least 2 µl of the original library stock in the library dilution step to ensure
accurate and reproducible quantitation.
} Illumina recommends 2 further independent (not serial) 1:10,000 and 1:20,000 dilutions
using at least 2 µl of the initial diluted libraries to evaluate quantitation precision.
NOTE
For guidance on handling small liquid volumes, see Handling Liquids in the TruSeq DNA
PCR-Free Library Prep Best Practices. See Additional Resources on page 6 for information
TruSeq DNA PCR-Free Library Prep Best Practices on the Illumina website.
The concentration of each library is calculated as indicated in Table 18 and Table 19:
} Obtain the calculated concentration of the 1:10,000 and 1:20,000 dilutions of the library,
as determined by qPCR, in relation to the concentrations of the correctly annotated
KAPA DNA Standards 1–6. Use the average of the replicate data points to determine
the concentration of the diluted library.
} Perform a size adjustment calculation to account for the difference in size between the
average fragment length of the library and the KAPA DNA Standard (452 bp):
• 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
Do not use the average fragment length of the library insert size based on the
Bioanalyzer results. PCR-free library fragment sizes measured on the Bioanalyzer
are substantially larger than would be predicted or derived from sequencing data.
} Calculate the concentration of the undiluted library by taking account of the relevant
dilution factor (e.g. 1:10,000 and 1:20,000). Use the average of the replicate data points
TruSeq DNA PCR-Free Library Prep Guide
57
Validate Library
Validate Library
High Sample (HS) Protocol
corresponding to each library DNA dilution to calculate the concentration of the
undiluted library.
} If 1 of the replicates appears to be an outlier, it can be omitted from the calculation. If
more than 1 of replicates appears to be outliers, repeat the assay.
Table 18 350 bp Library Concentration Calculation
Calculated
Average
Dilution
by qPCR
diluted
Factor
instrument (pM)*
library (pM)
1:10,000
A1
A2
A=
(A1 + A2)/2
1:20,000
B1
B2
B=
(B1 + B2)/2
Size adjusted
diluted
library (pM)
W1 =
A x (452/470)
W2 =
B x (452/470)
Table 19 550 bp Library Concentration Calculation
Calculated
Average
Dilution
by qPCR
diluted
Factor
instrument (pM)*
library (pM)
1:10,000
C1
C2
C=
(C1 + C2)/2
1:20,000
D1
D2
D=
(D1 + D2)/2
Size adjusted
diluted
library (pM)
W3 =
C x (452/670)
W4 =
D x (452/670)
Undiluted
library (pM)*
Undiluted
library (pM)
C1 =
W1 x 10,000
C2 =
W2 x 20,000
(C1 + C2)/2
Undiluted
library (pM)*
Undiluted
library (pM)
C3 =
W3 x 10,000
C4 =
W4 x 20,000
(C3 + C4)/2
*Duplicate data points
Quality Control
To verify the size of your fragments, check the template size distribution.
1
Prepare a 1:5 dilution of the DNA library with water.
2
Run 1 µl of the diluted DNA library on an Agilent Technologies 2100 Bioanalyzer
using a High Sensitivity DNA chip.
Run samples on a Bioanalyzer for qualitative purposes only.
When performing quality control on TruSeq DNA PCR-Free Library Prep libraries, PCR-Free
library fragment sizes measured on the Bioanalyzer are substantially larger than would be
predicted or derived from sequencing data. The larger size is due to anomalous migration
of fragments on the chip due to the presence of certain structural features, which would
normally be removed if a subsequent PCR-enrichment step were performed. Figure 7 and
Figure 8 show a comparison between library fragment sizes derived by a Bioanalyzer and
the corresponding insert sizes derived from the alignment of paired-end reads to a suitable
reference sequence.
58
Part # 15036187 Rev. C
A
B
Bioanalyzer
Paired-End Alignment
Figure 8 Example TruSeq DNA PCR-Free Library Prep 550 bp Insert Library Distribution
A
B
Bioanalyzer
Paired-End Alignment
TruSeq DNA PCR-Free Library Prep Guide
59
Validate Library
Figure 7 Example TruSeq DNA PCR-Free Library Prep 350 bp Insert Library Distribution
High Sample (HS) Protocol
Normalize and Pool Libraries
This process describes how to prepare DNA templates for cluster generation. Indexed DNA
libraries are normalized to 2 nM in the DCT plate and then pooled in equal volumes in the
PDP plate. Non-indexed DNA libraries are normalized to 2 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
96-well HSP plate
2
(second plate for
pooling only, if
pooling ≤ 40 samples)
15°C to 30°C
User
96-well midi plate
(for pooling only, if pooling
> 40 samples)
1
15°C to 30°C
User
Microseal ‘B’ adhesive seals
5
15°C to 30°C
User
Tris-HCl 10 mM, pH8.5 with
0.1% Tween 20
Enough to normalize
the concentration of
each library to 2 nM
15°C to 30°C
User
Preparation
} Remove the TSP1 plate from -25°C to -15°C storage, if it was stored at the conclusion of
Clean Up ALP.
• Let it thaw at room temperature.
• Centrifuge the thawed plate at 280 × g for 1 minute.
• Remove the adhesive seal from the thawed plate.
} [For pooling only] Review the TruSeq Library Prep Pooling Guide (part # 15042173).
} Apply a DCT barcode label to a new 96-well HSP plate.
} [For pooling only] Apply a PDP barcode label to a new 96-well HSP plate if pooling
≤ 40 samples or a 96-well midi plate if pooling > 40 samples.
Make DCT
1
Transfer 5 µl of the library from each well of the TSP1 plate to the corresponding well
of the new HSP plate labeled with the DCT barcode.
2
Normalize the library concentration in each well to 2 nM using Tris-HCl 10 mM,
pH 8.5 with 0.1% Tween 20.
NOTE
Depending on the yield quantification data of each library, the final volume in the plate
can vary from 5–100 µl.
60
Part # 15036187 Rev. C
Mix thoroughly as follows:
a Seal the plate with a Microseal ‘B’ adhesive seal.
b Shake the plate on a microplate shaker at 1000 rpm for 2 minutes.
4
Centrifuge the plate at 280 × g for 1 minute.
5
Remove the adhesive seal from the plate.
6
Do the following, depending on the type of library you want to generate:
• For libraries that are not pooled, the protocol stops here. Do the following:
— To continue immediately to sequencing, proceed to cluster generation. For more
information, see the cluster generation section of the system guide for your
Illumina platform.
— To stop here before sequencing, seal the DCT plate with a Microseal ‘B’
adhesive seal and store at -25°C to -15°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.
1
Determine the number of samples to be combined together for each pool.
NOTE
Avoid pooling 2 samples with the same index.
2
Do the following:
• If pooling 2–24 samples:
— Transfer 5 µl of each normalized library to be pooled from the DCT plate to
1 well of the new HSP plate labeled with the PDP barcode.
— The total volume in each well is 5X the number of combined sample libraries
and 10–120 µl (2–24 libraries). For example, the volume for 2 samples is 10 µl,
the volume for 12 samples is 60 µl, or the volume for 24 samples is 120 µl.
• If pooling 25–96 samples:
— Using a multichannel pipette, transfer 5 µl of each normalized library in
column 1 of the DCT plate to column 1 of the new HSP or midi plate labeled
with the PDP barcode.
— Transfer 5 µl of each normalized 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 plate with a Microseal ‘B’ adhesive seal.
— Shake the plate on a microplate shaker at 1800 rpm for 2 minutes.
— Centrifuge the plate at 280 × g for 1 minute.
— Remove the adhesive seal from the plate.
— Combine the contents of each well of column 1 into well A2 of the plate for the
final pool.
3
Mix thoroughly as follows:
a Seal the plate with a Microseal ‘B’ adhesive seal.
b Shake the plate on a microplate shaker at 1800 rpm for 2 minutes.
4
Centrifuge the plate at 280 × g for 1 minute.
TruSeq DNA PCR-Free Library Prep Guide
61
Normalize and Pool Libraries
3
High Sample (HS) Protocol
5
62
Do the following:
• To continue immediately to sequencing, proceed to cluster generation. For more
information, see the system guide for your Illumina sequencing platform.
• To stop here before sequencing, store the sealed PDP plate at -25°C to -15°C.
Part # 15036187 Rev. C
Appendix A Supporting Information
Introduction
Acronyms
Kit Contents
Consumables and Equipment
Indexed Adapter Sequences
TruSeq DNA PCR-Free Library Prep Guide
64
65
67
72
76
63
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.
64
Part # 15036187 Rev. C
Acronyms
Acronyms
Table 20 TruSeq DNA PCR-Free Library Prep 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
CSP
Clean Up Sheared DNA Plate
CTA
A-Tailing Control
CTE
End Repair Control
CTL
Ligation Control
DAP
DNA Adapter Plate
DCT
Diluted Cluster Template Plate
DNA
Customer Sample DNA Plate
dsDNA
double-stranded DNA
ERP2
End Repair Mix 2
EUC
Experienced User Card
gDNA
genomic DNA
HS
High Sample
HSP
Hard-Shell Plate
HT
High Throughput
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
TruSeq DNA PCR-Free Library Prep Guide
65
Supporting Information
Acronym
66
Definition
RSB
Resuspension Buffer
SPB
Sample Purification Beads
STL
Stop Ligation Buffer
TSP1
Target Sample Plate 1
Part # 15036187 Rev. C
Check to make sure that you have all of the reagents identified in this section before
starting the TruSeq DNA PCR-Free Library Prep protocol.
The TruSeq DNA PCR-Free LT Library Prep Kit is available in a Set A and a Set B. Each
TruSeq DNA PCR-Free LT Library Prep Kit contains enough reagents to prepare up to
24 samples. When used together, sets A and B allow for pooling up to 24 samples using
the 12 different indexes in each kit.
Table 21 TruSeq DNA PCR-Free Library Prep Kits
Kit Name
Catalog #
Number
of
Samples
Supported
Number
of
Indexes
TruSeq DNA PCR-Free LT
Library Prep Kit - Set A
FC-121-3001
24
12
TruSeq DNA PCR-Free LT
Library Prep Kit - Set B
FC-121-3002
24
12
TruSeq DNA PCR-Free HT
Library Prep Kit
FC-121-3003
96
96
TruSeq DNA PCR-Free LT Library Prep Kit
The TruSeq DNA PCR-Free LT Library Prep Kit contains 2 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 -25°C to -15°C
These boxes are shipped on dry ice. As soon as you receive them, store the following
components at -25°C to -15°C.
TruSeq DNA PCR-Free Library Prep Guide
67
Kit Contents
Kit Contents
Supporting Information
Set A
Figure 9 TruSeq DNA PCR-Free LT Library Prep Kit, 24 Samples-Set A (Box 1 of 2),
part # 15037063
Slot
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
68
Reagent
RSB
ERP2
ATL
LIG2
CTE
CTA
CTL
STL
AD002
AD004
AD005
AD006
AD007
AD012
AD013
AD014
AD015
AD016
AD018
AD019
Part #
15026770
15036418
15012495
15036183
15026774
15026775
15026776
15012546
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
End Repair Control
A-Tailing Control
Ligation Control
Stop Ligation Buffer
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 # 15036187 Rev. C
Kit Contents
Set B
Figure 10 TruSeq DNA PCR-Free LT Library Prep Kit, 24 Samples-Set B (Box 1 of 2),
part # 15037061
Slot
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Reagent
RSB
ERP2
ATL
LIG2
CTE
CTA
CTL
STL
AD001
AD003
AD008
AD009
AD010
AD011
AD020
AD021
AD022
AD023
AD025
AD027
Part #
15026770
15036418
15012495
15036183
15026774
15026775
15026776
15012546
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
End Repair Control
A-Tailing Control
Ligation Control
Stop Ligation Buffer
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.
Figure 11 TruSeq DNA PCR-Free LT Library Prep Kit, 24 Samples SP Beads (Box 2 of 2),
part # 15037158
Slot
1
Reagent
SPB
TruSeq DNA PCR-Free Library Prep Guide
Part #
15037172
Description
Sample Purification Beads
69
Supporting Information
TruSeq DNA PCR-Free HT Library Prep Kit
The TruSeq DNA PCR-Free HT Library Prep Kit contains 3 boxes: a core reagent box, an
Adapter Plate box, and an SP Beads box.
96 Samples - Core Reagents Box
Store at -25°C to -15°C
This box is shipped on dry ice. As soon as you receive it, store the following components at
-25°C to -15°C. This box also contains plate barcode labels.
Figure 12 TruSeq DNA PCR-Free HT Library Prep Kit, 96 Samples (Box 1 of 2), part # 15037059
Slot
1–2
3–4
5–6
7–8
9–10
11–12
13–14
15–16
Reagent
RSB
ERP2
ATL
LIG2
CTE
CTA
CTL
STL
Part #
15026770
15036182
15012495
15036184
15026774
15026775
15026776
15012546
Description
Resuspension Buffer
End Repair Mix 2
A-Tailing Mix
Ligation Mix 2
End Repair Control
A-Tailing Control
Ligation Control
Stop Ligation Buffer
96 Samples - Adapter Plate Box
Store at -25°C to -15°C
This box is shipped on dry ice. As soon as you receive it, store the contents at
-25°C to -15°C.
Figure 13 TruSeq DNA PCR-Free HT Library Prep Kit, 96, Adapter Plate Box, part # 15032317
Slot
1
70
Reagent
DAP
Part #
Description
15016426 DNA Adapter Plate, 96plex
Part # 15036187 Rev. C
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.
Figure 14 TruSeq DNA PCR-Free HT Library Prep Kit, 96 Samples SP Beads (Box 2 of 2),
part # 15037163
Slot
1–4
Reagent
SPB
TruSeq DNA PCR-Free Library Prep Guide
Part #
15037172
Description
Sample Purification Beads
71
Kit Contents
96 Samples - SP Beads Box Supporting Information
Consumables and Equipment
Check to make sure that you have all of the necessary user-supplied consumables and
equipment before starting the TruSeq DNA PCR-Free Library Prep 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 DNA PCR-Free Library Prep protocol has been optimized and validated using
the items listed. Comparable performance is not guaranteed when using alternate
consumables and equipment.
Table 22 User-Supplied Consumables
72
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
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
1000 µl barrier pipette tips
General lab supplier
1000 µl multichannel pipettes
General lab supplier
1000 µl single channel pipettes
General lab supplier
Ethanol 200 proof (absolute)
for molecular biology (500 ml)
Sigma-Aldrich,
part # E7023
High Sensitivity DNA Kit
Agilent Technologies, part #
5067-4626
Ice bucket
General lab supplier
KAPA Library Quantification Kit Illumina/Universal
KAPA Biosystems, part #
KK4824
Microseal ‘B’ adhesive seals
Bio-Rad,
part # MSB-1001
Part # 15036187 Rev. C
Supplier
microTUBE AFA Fiber 6x16mm with
• Crimp-Cap or
• Pre-Slit Snap-Cap (for use with Covaris M220)
Covaris, part #
• 520052 or
• 520045
PCR grade water
General lab supplier
Qubit assay tubes or
Axygen PCR-05-C tubes
Life Technologies,
catalog # Q32856 or
VWR, part # 10011-830
Qubit dsDNA BR Assay Kit
Life Technologies, catalog # • 100 assays, Q32850
• 500 assays, Q32853
RNaseZap
(to decontaminate surfaces)
General lab supplier
RNase/DNase-free 8-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
Ultra pure water
General lab supplier
Consumables and Equipment
Consumable
Table 23 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 or
Eppendorf, part # 951020303
Table 24 User-Supplied Consumables - Additional Items for HS Processing
Consumable
Supplier
96-well storage plates, round well,
0.8 ml (“midi” plate)
Fisher Scientific,
part # AB-0859
Hard-Shell 96-well PCR Plates (“HSP” plate)
Bio-Rad, part # HSP-9601
Table 25 User-Supplied Equipment
Equipment
Supplier
2100 Bioanalyzer Desktop System
Agilent Technologies,
part # G2940CA
TruSeq DNA PCR-Free Library Prep Guide
73
Supporting Information
Equipment
Supplier
One of the following Covaris systems:
• S2
• S220
• E210
• M220
Covaris M220, part # 500295
For all other models, contact
Covaris
Magnetic stand-96
Life Technologies,
catalog # AM10027
Microplate centrifuge
General lab supplier
Qubit 2.0 Fluorometer
Life Technologies, catalog # Q32866
Vortexer
General lab supplier
qPCR system
See qPCR Systems on page 75.
General lab supplier
Table 26 User-Supplied Equipment - Additional Items for LS Processing
Equipment
Supplier
96-well thermal cycler
(with heated lid)
General lab supplier
Table 27 User-Supplied Equipment - Additional Items for HS Processing
74
Equipment
Supplier
High-Speed Microplate Shaker
VWR, catalog # • 13500-890 (110 V/120 V) or
• 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) or
• SC-60-504 (220 V)
Part # 15036187 Rev. C
The following table lists the validated qPCR systems for the TruSeq DNA PCR-Free Library
Prep protocol.
Equipment
Supplier
CFX96 Touch Real-Time PCR Detection System 1
Bio-Rad, part # 185-5195
Mx3000P qPCR System
Agilent, part # 401511
1. Illumina recommends using CFX Manager software version 3.0 with Cq Determination mode: Single
Threshold; Baseline Setting:Baseline Subtracted Curve Fit and Apply Fluorescent Drift Correction for
data analysis. This setting can correct for abnormalities in fluorescence intensity of the standard curve
caused by the instrument. For software installation, contact Bio-Rad.
TruSeq DNA PCR-Free Library Prep Guide
75
Consumables and Equipment
qPCR Systems
Supporting Information
Indexed Adapter Sequences
This section details the indexed adapter sequences.
TruSeq DNA PCR-Free LT Library Prep Kit Indexed Adapter Sequences
The TruSeq DNA PCR-Free LT Library 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 6 bases. For
indexes 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, see the
system guide for your Illumina sequencing platform.
Table 28 TruSeq DNA PCR-Free LT Library Prep Kit Set A Indexed Adapter Sequences
Adapter
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)
Table 29 TruSeq DNA PCR-Free LT Library Prep Kit Set B Indexed Adapter Sequences
Adapter
76
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)
Part # 15036187 Rev. C
The DAP in the TruSeq DNA PCR-Free HT Library 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 30 TruSeq DNA PCR-Free HT Library 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
Table 31 TruSeq DNA PCR-Free HT Library Prep Kit Indexed Adapter 2 Sequences
Adapter
Sequence
Adapter
Sequence
D501
TATAGCCT
D505
AGGCGAAG
D502
ATAGAGGC
D506
TAATCTTA
D503
CCTATCCT
D507
CAGGACGT
D504
GGCTCTGA
D508
GTACTGAC
The DAP dual-index layout:
Figure 15
DAP Dual-Indexed Layout
TruSeq DNA PCR-Free Library Prep Guide
77
Indexed Adapter Sequences
TruSeq DNA PCR-Free HT Library Prep Kit Indexed Adapter Sequences
78
Part # 15036187 Rev. C
For technical assistance, contact Illumina Technical Support.
Table 32 Illumina General Contact Information
Address
5200 Illumina Way
San Diego, CA 92122 USA
Website
www.illumina.com
Email
[email protected]
Table 33 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
support.illumina.com/sds.html.
Product Documentation
Product documentation in PDF is available for download from the Illumina website. Go
to support.illumina.com, select a product, then click Documentation & Literature.
TruSeq DNA PCR-Free Library Prep Guide
79
Technical Assistance
Technical Assistance
Illumina
5200 Illumina Way
San Diego, California 92122 U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com
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