TruSeq DNA PCR-Free Library Prep Reference Guide (15036187 D)

TruSeq DNA PCR-Free Library Prep Reference Guide (15036187 D)
TruSeq® DNA PCR-Free Library Prep
Reference Guide
For Research Use Only. Not for use in diagnostic procedures.
Revision History
Introduction
DNA Input Recommendations
Additional Resources
Protocol Introduction
Tips and Techniques
Library Prep Workflow
Prepare for Pooling
Fragment DNA
Repair Ends and Select Library Size
Adenylate 3ʹ Ends
Ligate Adapters
Validate Libraries
Normalize and Pool Libraries
Supporting Information
Technical Assistance
ILLUMINA PROPRIETARY
Part # 15036187 Rev. D
June 2015
Catalog # FC-121-9006DOC
3
4
5
7
8
9
10
11
12
15
18
20
23
26
28
37
This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for the
contractual use of its customer in connection with the use of the product(s) described herein and for no other purpose. This
document and its contents shall not be used or distributed for any other purpose and/or otherwise communicated, disclosed,
or reproduced in any way whatsoever without the prior written consent of Illumina. Illumina does not convey any license
under its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this document.
The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order
to ensure the proper and safe use of the product(s) described herein. All of the contents of this document must be fully read
and understood prior to using such product(s).
FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN
MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND
DAMAGE TO OTHER PROPERTY.
ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S)
DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE).
© 2015 Illumina, Inc. All rights reserved.
Illumina, 24sure, BaseSpace, BeadArray, BlueFish, BlueFuse, BlueGnome, cBot, CSPro, CytoChip, DesignStudio,
Epicentre, GAIIx, Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, HiSeq X, Infinium,
iScan, iSelect, MiSeq, NeoPrep, Nextera, NextBio, NextSeq, Powered by Illumina, SeqMonitor, SureMDA, TruGenome,
TruSeq, TruSight, Understand Your Genome, UYG, VeraCode, verifi, VeriSeq, the pumpkin orange color, and the
streaming bases design are trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/or other countries. All other
names, logos, and other trademarks are the property of their respective owners.
Part #
Revision
Date
15036187
D
June 2015
15036187
C
December
2014
15036187
B
November
2013
15036187
A
January
2013
TruSeq DNA PCR-Free Library Prep Reference Guide
Description of Change
Updated Kit Contents with reagent changes and
removed box and tube part numbers
Corrected CTA and CTL volumes to 2.5 µl
Removed SAV for viewing in In-Line Control DNA
Changed title of this document to Reference Guide
Updated design of workflow diagram
Renamed and combined some procedures as needed to
improve continuity
Combined HS and LS protocol options into a single
workflow
Simplified consumables information at the beginning of
each section
Revised step-by-step instructions to be more succinct
Removed reference to obsolete Experienced User Cards
and added reference to new protocol guide and
checklist
Changed BaseSpace resource reference to helpcenter
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 (eg, IMP plate), except for
first instance and last instance in each procedure
Bead cleanup procedures modified to remove EtOH
before air-drying samples
Added centrifuge step before each thermal cycler
incubation in the LS protocol
Added well volume to heat incubation procedures
Updated Prepare for Pooling 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 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
3
Revision History
Revision History
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 libraries for single-read or paired-end 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
} Single workflow with options 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 lower indexing pooling levels. The
libraries generated do not require PCR amplification to enable cluster generation.
4
Part # 15036187 Rev. D
For best results, follow the input recommendations. Quantify the input gDNA and assess
the gDNA quality before beginning library preparation.
} For a 350 bp insert size, use 1 µg input gDNA.
} For a 550 bp insert size, use 2 µg input gDNA.
} Input amounts lower than those specified results in low yield and increased
duplicates.
Quantify Input DNA
Use the following recommendations to quantify input DNA:
} Successful library preparation depends on accurate quantification of input DNA. To
verify results, use multiple methods.
} Use fluorometric-based methods for quantification, such as Qubit or PicoGreen.
} 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.
} Do not use spectrophotometric-based methods, such as NanoDrop, which measure
the presence of nucleotides and can result in an inaccurate measurement of gDNA.
} Quantification methods depend on accurate pipetting methods. Do not use pipettes
at the extremes of volume specifications. Make sure that pipettes are calibrated.
Assess 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. Values of 1.8–2.0 indicate relatively pure DNA.
} The presence of RNA or small nucleic acid fragments, such as nucleotides, can
compromise both absorbance measurements.
} Make sure that samples are free of contaminants.
In-Line Control DNA
The CTE (End Repair Control), CTA (A-Tailing Control), and CTL (Ligation Control)
contain DNA fragments used as controls for the enzymatic activities of the ERP 2 (End
Repair Mix 2), ATL (A-Tailing Mix), and LIG 2 (Ligation Mix 2). Each inline control
contains dsDNA fragments designed to report the success or failure of a specific
enzymatic activity.
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.
TruSeq DNA PCR-Free Library Prep Reference Guide
5
DNA Input Recommendations
DNA Input Recommendations
Table 1 In-Line Control Functions
Reagent
Function
ERP 2
End repair: Generate blunt ended fragments
by 3'–> 5' exonuclease and 5'–> 3' polymerase
activities
ERP 2
End repair: Add 5'-phosphate groups needed
for downstream ligation
ATL
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
LIG 2
Control
End
Repair
Control
1*
End
Repair
Control
2*
ATailing
Control
Ligation
Control
Structure of
Control DNA
Ends
5' overhang at
1 end, 3' overhang
at other end
Blunt with 5'-OH
group
Blunt with 5'phosphate group
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
Inline controls 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 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.
Inline controls are used for troubleshooting and used to identify the specific mode of
failure, but are uninformative in cases where sequencing data are not generated from a
library. Using the controls is optional and they can be replaced with the same volume of
RSB.
6
Part # 15036187 Rev. D
Additional Resources
Additional Resources
The following documentation is available for download from the Illumina website.
Resource
Description
TruSeq DNA PCR-Free Library
Prep Protocol Guide (part #
15075699)
Provides only protocol instructions. The protocol guide is
intended for experienced users.
TruSeq DNA PCR-Free Library
Prep Checklist (part # 15075700)
Provides a checklist of the protocol steps. The checklist is
intended for experienced users.
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 help
(help.basespace.illumina.com)
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 PCRFree 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.
TruSeq DNA PCR-Free Library Prep Reference Guide
7
Protocol Introduction
This section describes the TruSeq DNA PCR-Free Library Prep protocol.
} Follow the protocol in the order described, using the specified volumes and
incubation parameters.
} The protocol provides a single workflow with options for using different plate types
as containers.
• Differences for each option are designated with [HS] or [LS].
• Follow the instructions for the container that you are using.
• You can expect equivalent results from either option. However, the [HS] option
can yield more consistent results between samples.
• The distinguishing elements of the protocol options are as follows.
Table 2 Workflow Options
Workflow Designator
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
HS
> 24 with index
adapter tubes*
LS
≤ 24 with index
adapter tubes*
> 24 with index
adapter plate
≤ 24 with index
adapter plate
96-well Hard-Shell
96-well 0.3 ml PCR
PCR
96-well midi
96-well midi
Microheating systems 96-well
thermal cycler
Microplate shaker
Pipetting
* 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.
} Review Best Practices before proceeding. See Additional Resources on page 7 for
information on how to access TruSeq DNA PCR-Free Library Prep Best Practices on
the Illumina website.
} Before proceeding, confirm kit contents and make sure that you have the required
equipment and consumables. For more information, see Supporting Information on
page 28.
8
Part # 15036187 Rev. D
Unless a safe stopping point is specified in the protocol, proceed immediately to the next
step.
Avoiding Cross-Contamination
} When adding or transferring samples, change tips between each sample.
} When adding adapters or primers, change tips between each row and each column.
} Remove unused index adapter tubes from the working area.
Sealing the Plate
} Always seal the 96-well plate before the following steps in the protocol:
• Shaking steps
• Centrifuge steps
• Thermal cycling steps
} Apply the adhesive seal to cover the plate and seal with a rubber roller.
} Microseal 'B' adhesive seals are effective at -40°C to 110°C, and suitable for skirted or
semiskirted PCR plates.
} Microseal 'A' adhesive film is effective for thermal cycling and easy to cut when
using fewer than 96 wells.
Plate Transfers
} When transferring volumes between plates, transfer the specified volume from each
well of a plate to the corresponding well of the other plate.
TruSeq DNA PCR-Free Library Prep Reference Guide
9
Tips and Techniques
Tips and Techniques
Library Prep Workflow
Figure 1 TruSeq DNA PCR-Free Library Prep Workflow
10
Part # 15036187 Rev. D
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 the BaseSpace Prep tab to organize samples, libraries, pools, and a run for
Illumina sequencing systems and analysis software.
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.
TruSeq DNA PCR-Free Library Prep Reference Guide
11
Prepare for Pooling
Prepare for Pooling
Fragment DNA
This process describes how to optimally fragment gDNA to a 350 bp, or 550 bp insert
size. Covaris shearing generates dsDNA fragments with 3' or 5' overhangs.
Consumables
} gDNA samples
• 1 µg per sample for a 350 bp insert size
• 2 µg per sample for a 550 bp insert size
} RSB (Resuspension Buffer)
} SPB (Sample Purification Beads)
} Barcode labels
• CFP (Covaris Fragmentation Plate)
• CSP (Clean Up Sheared DNA Plate)
• DNA (DNA Plate)
• IMP (Insert Modification Plate)
} Freshly prepared 80% ethanol (EtOH)
} Choose from the following containers:
• [HS] 96-well midi plates (3) and 96-well Hard-Shell 0.3 ml PCR plate (1)
• [LS] 96-well 0.3 ml PCR plates, semiskirted or skirtless (4)
} Covaris tubes (1 per sample)
} Microseal 'B' adhesive seal
About Reagents
} Vortex SPB before each use.
} Vortex SPB frequently to make sure that beads are evenly distributed.
} Aspirate and dispense SPB slowly due to the viscosity of the solution.
Preparation
1
Prepare the following consumables.
Item
RSB
SPB
12
Storage
-25°C to -15°C
2°C to 8°C
Instructions
Thaw at room temperature.
Store at 2°C to 8°C after the initial thaw.
Let stand for 30 minutes to bring to room temperature.
Keep at room temperature for later use in the protocol.
2
Turn on and set up the Covaris instrument according to manufacturer guidelines.
3
[HS] Calibrate the microplate shaker with a stroboscope and set it to 1800 rpm.
4
Apply barcodes to label plates as follows.
• DNA [midi or PCR plate]
• CFP [Hard-Shell PCR or PCR plate]
• CSP [midi or PCR plate]
• IMP [midi or PCR plate]
Part # 15036187 Rev. D
Normalize gDNA
1
Quantify gDNA using a fluorometric-based method.
2
Normalize gDNA samples with RSB to a final volume of 55 µl in the DNA plate.
• 20 ng/µl for a 350 bp insert size
• 40 ng/µl for a 550 bp insert size
3
Mix thoroughly as follows.
• [HS] Shake at 1800 rpm for 2 minutes.
• [LS] Pipette up and down.
4
Centrifuge as follows.
• [HS] Centrifuge at 280 × g for 1 minute.
• [LS] Centrifuge briefly.
Fragment DNA
1
Transfer 52.5 µl DNA samples to separate Covaris tubes.
Use the wells of the CFP plate to hold Covaris tubes upright.
2
Centrifuge at 280 × g for 5 seconds.
3
Fragment the DNA using the following Covaris settings.
Table 3 350 bp Insert Settings
Covaris Setting
Duty Factor (%)
Intensity
Peak/Displayed Power (W)
Cycles/Burst
Duration (seconds)
Mode
Temperature (°C)
Table 4 550 bp Insert Settings
Covaris Setting
Duty Factor (%)
Intensity
Peak/Displayed Power (W)
Cycles/Burst
Duration (seconds)
Mode
Temperature (°C)
M220
S220
20
—
50
5
—
175
S2
E210
10
5.0
23
14
200
65
—
20
50
45
Frequency sweeping
5.5–6
M220
S220
S2
E210
20
—
50
5
—
175
45
—
20
200
25
45
Frequency sweeping
5.5–6
10
2.0
9
7
4
Centrifuge at 280 × g for 5 seconds.
5
Transfer 50 µl supernatant from each Covaris tube to the corresponding well of the
CSP plate.
TruSeq DNA PCR-Free Library Prep Reference Guide
13
Fragment DNA
Procedure
Clean Up Fragmented DNA
1
Vortex SPB until well-dispersed.
2
Add 80 µl SPB to each well, and then mix thoroughly as follows.
• [HS] Shake at 1800 rpm for 2 minutes.
• [LS] Pipette up and down.
3
Incubate at room temperature for 5 minutes.
4
[HS] Centrifuge at 280 × g for 1 minute.
5
Place on a magnetic stand and wait until the liquid is clear (~8 minutes).
6
Remove and discard all supernatant from each well.
7
Wash 2 times as follows.
a Add 200 µl freshly prepared 80% EtOH to each well.
b Incubate on the magnetic stand for 30 seconds.
c Remove and discard all supernatant from each well.
8
Use a 20 µl pipette to remove residual EtOH from each well.
9
Air-dry on the magnetic stand for 5 minutes.
10 Add 52.5 µl RSB to each well.
11 Remove from the magnetic stand, and then mix thoroughly as follows.
• [HS] Shake at 1800 rpm for 2 minutes.
• [LS] Pipette up and down.
12 Incubate at room temperature for 2 minutes.
13 [HS] Centrifuge at 280 × g for 1 minute.
14 Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
15 Transfer 50 µl supernatant to the corresponding well of the IMP plate.
14
Part # 15036187 Rev. D
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 SPB (Sample Purification
Beads).
Consumables
}
}
}
}
}
}
}
}
}
}
ERP2 or ERP3 (End Repair Mix)
RSB (Resuspension Buffer)
SPB (Sample Purification Beads)
[Optional] CTE (End Repair Control)
Barcode labels
• ALP (Adapter Ligation Plate)
• CEP (Clean Up End Repair Plate)
15 ml conical tube
Freshly prepared 80% ethanol (EtOH)
PCR grade water
Choose from the following containers:
• [HS] 96-well midi plates (2)
• [LS] 96-well 0.3 ml PCR plates, semiskirted or skirtless (2)
Microseal 'B' adhesive seals
About Reagents
}
}
}
}
}
The kit contains either ERP2 or ERP3.
Using CTE is optional. Use RSB as a substitute.
Vortex SPB before each use.
Vortex SPB frequently to make sure that beads are evenly distributed.
Aspirate and dispense SPB slowly due to the viscosity of the solution.
Preparation
1
Prepare the following consumables.
Item
ERP2 or ERP3
CTE
RSB
SPB
Storage
Instructions
-25°C to -15°C Thaw at room temperature, and then place on ice.
Return to storage after use.
-25°C to -15°C Thaw at room temperature, and then place on ice.
2°C to 8°C
Let stand for 30 minutes to bring to room
temperature.
2°C to 8°C
Let stand for 30 minutes to bring to room
temperature.
2
[HS] Preheat the microheating system to 30°C.
3
[LS] Save the following ERP program on the thermal cycler:
• Choose the preheat lid option and set to 100°C
• 30°C for 30 minutes
• Hold at 4°C
TruSeq DNA PCR-Free Library Prep Reference Guide
15
Repair Ends and Select Library Size
Repair Ends and Select Library Size
4
Apply barcodes to label plates as follows.
• ALP [midi or PCR plate]
• CEP [midi or PCR plate]
Procedure
Convert Overhangs
1
Centrifuge CTE at 600 × g for 5 seconds.
2
Add 10 µl CTE to each well.
3
Centrifuge ERP2 or ERP3 at 600 × g for 5 seconds.
4
Add 40 µl ERP2 or ERP3 to each well, and then mix thoroughly as follows.
• [HS] Shake at 1800 rpm for 2 minutes.
• [LS] Pipette up and down.
5
[HS] Centrifuge at 280 × g for 1 minute.
6
Incubate as follows.
• [HS] Place on the 30°C microheating system with the heated lid closed for
30 minutes, and then place on ice.
• [LS] Place on the thermal cycler and run the ERP program. Each well contains
100 µl.
Remove Large DNA Fragments
1
Vortex SPB until well-dispersed.
2
Dilute SPB with PCR grade water to 160 µl per 100 µl of end-repaired sample.
• When processing ≤ 6 samples, use a new 1.7 ml microcentrifuge tube.
• When processing > 6 samples, use a new 15 ml conical tube.
Determine the volumes using the following formulas, which include 15% excess for
multiple samples.
Table 5 Diluted SPB for a 350 bp Insert Size
Formula
SPB
PCR grade water
# of samples X 109.25 µl
# of samples X 74.75 µl
Table 6 Diluted SPB for a 550 bp Insert Size
Formula
SPB
PCR grade water
16
Example Amount
per 12 samples
1311 µl
897 µl
# of samples X 92 µl
# of samples X 92 µl
Example Amount
per 12 samples
1104 µl
1104 µl
Your Calculation
Your Calculation
3
Vortex diluted SPB until well-dispersed.
4
Add 160 µl diluted SPB to each well, and then mix thoroughly as follows.
• [HS] Shake at 1800 rpm for 2 minutes.
• [LS] Pipette up and down.
5
Incubate at room temperature for 5 minutes.
6
[HS] Centrifuge at 280 × g for 1 minute.
Part # 15036187 Rev. D
Place on a magnetic stand and wait until the liquid is clear (~5 minutes).
8
Transfer 250 µl supernatant to the corresponding well of the CEP plate.
9
Discard remaining diluted SPB.
Repair Ends and Select Library Size
7
Remove Small DNA Fragments
1
Vortex undiluted SPB until well-dispersed.
2
Add 30 µl undiluted SPB to each well, and then mix thoroughly as follows.
• [HS] Shake at 1800 rpm for 2 minutes.
• [LS] Pipette up and down.
3
Incubate at room temperature for 5 minutes.
4
[HS] Centrifuge at 280 × g for 1 minute.
5
Place on a magnetic stand and wait until the liquid is clear (~5 minutes).
6
Remove and discard all supernatant from each well.
7
Wash 2 times as follows.
a Add 200 µl freshly prepared 80% EtOH to each well.
b Incubate on the magnetic stand for 30 seconds.
c Remove and discard all supernatant from each well.
8
Use a 20 µl pipette to remove residual EtOH from each well.
9
Air-dry on the magnetic stand for 5 minutes.
10 Add 17.5 µl RSB to each well.
11 Remove from the magnetic stand, and then mix thoroughly as follows.
• [HS] Shake at 1800 rpm for 2 minutes.
• [LS] Pipette up and down.
12 Incubate at room temperature for 2 minutes.
13 [HS] Centrifuge at 280 × g for 1 minute.
14 Place on a magnetic stand and wait until the liquid is clear (~5 minutes).
15 Transfer 15 µl supernatant to the corresponding well of the ALP plate.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at -25°C to -15°C for up to 7 days.
TruSeq DNA PCR-Free Library Prep Reference Guide
17
Adenylate 3ʹ Ends
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
}
}
}
}
ATL or ATL2 (A-Tailing Mix)
RSB (Resuspension Buffer)
[Optional] CTA (A-Tailing Control)
[HS] Microseal 'B' adhesive seal
About Reagents
} The kit contains either ATL or ATL2.
} Using CTA is optional. Use RSB as a substitute.
Preparation
1
Prepare the following consumables.
Item
ATL or ATL2
CTA
RSB
Storage
Instructions
-25°C to -15°C Thaw at room temperature.
Return to storage after use.
-25°C to -15°C Thaw at room temperature, and then place on ice.
2°C to 8°C
Let stand for 30 minutes to bring to room
temperature.
2
[HS] Preheat 2 microheating systems, the first to 37°C and the second to 70°C.
3
[LS] Save the following ATAIL70 program on the thermal cycler:
• 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
Procedure
18
1
Centrifuge CTA at 600 × g for 5 seconds.
2
Add 2.5 µl CTA to each well.
3
Centrifuge ATL or ATL2 at 600 × g for 5 seconds.
4
Add 12.5 µl ATL or ATL2 to each well, and then mix thoroughly as follows.
• [HS] Shake at 1800 rpm for 2 minutes.
• [LS] Pipette up and down.
Part # 15036187 Rev. D
Incubate as follows.
[HS]
a Place on the 37°C microheating system with the lid closed for 30 minutes.
b Move to the 70°C microheating system with the lid closed for 5 minutes.
c Place on ice for 5 minutes.
[LS]
a Place on the thermal cycler and run the ATAIL70 program. Each well contains
30 µl.
TruSeq DNA PCR-Free Library Prep Reference Guide
19
Adenylate 3ʹ Ends
5
Ligate Adapters
This process ligates multiple indexing adapters to the ends of the DNA fragments,
preparing them for hybridization onto a flow cell.
Consumables
}
}
}
}
}
}
}
DNA Adapters (tubes or DAP)
LIG2 (Ligation Mix 2)
RSB (Resuspension Buffer)
SPB (Sample Purification Beads)
STL (Stop Ligation Buffer)
[Optional] CTL (Ligation Control)
Barcode labels
• CAP (Clean Up ALP Plate)
• DAP (DNA Adapter Plate)
• TSP1 (Target Sample Plate)
} Freshly prepared 80% ethanol (EtOH)
} Choose from the following containers:
• [HS] 96-well midi plate (1) and 96-well Hard-Shell 0.3 ml PCR plate (1)
• [LS] 96-well 0.3 ml PCR plates, semiskirted or skirtless (2)
} [HS] Microseal 'B' adhesive seals
About Reagents
}
}
}
}
}
}
Using CTL is optional. Use RSB as a substitute.
Do not remove the LIG2 from storage until instructed to do so in the procedure.
Return LIG2 to storage immediately after use.
Vortex SPB before each use.
Vortex SPB frequently to make sure that beads are evenly distributed.
Aspirate and dispense SPB slowly due to the viscosity of the solution.
Preparation
1
Prepare the following consumables.
Item
CTL
Storage
Instructions
-25°C to -15°C Thaw at room temperature.
Return to storage after use.
DNA Adapters -25°C to -15°C Thaw at room temperature for 10 minutes.
Return to storage after use.
The DAP can undergo up to 4 freeze-thaw cycles.
RSB
2°C to 8°C
Let stand for 30 minutes to bring to room
temperature.
STL
-25°C to -15°C Thaw at room temperature.
Return to storage after use.
SPB
2°C to 8°C
Let stand for 30 minutes to bring to room
temperature.
2
20
[HS] Preheat a microheating system to 30°C.
Part # 15036187 Rev. D
[LS] Save the following LIG program on the thermal cycler:
• Choose the preheat lid option and set to 100°C
• 30°C for 10 minutes
• Hold at 4°C
4
Apply barcodes to label plates as follows.
• CAP [midi or PCR]
• TSP1 [Hard-Shell PCR or PCR]
Ligate Adapters
3
Procedure
Add Index Adapters
1
[HT kit] Remove the tape seal from the DAP.
2
Centrifuge the DNA adapters as follows.
Reagent
Adapter tubes
DAP
Speed
600 × g
280 × g
Duration
5 seconds
1 minute
3
[HT kit] Prepare the DAP as follows.
a Remove the plastic cover. Save the cover if you are not processing the entire
plate at the same time.
b Apply the DAP barcode.
4
Centrifuge CTL at 600 × g for 5 seconds.
5
Remove LIG2 from -25°C to -15°C storage.
6
Add the following reagents in the order listed to each well, and then mix thoroughly
as follows.
Reagent
CTL
LIG2
DNA adapters
Volume (µl)
2.5
2.5
2.5
• [HS] Shake at 1800 rpm for 2 minutes.
• [LS] Pipette up and down.
7
Centrifuge at 280 × g for 1 minute.
8
Incubate as follows.
• [HS] Place on the 30°C microheating system with the lid closed for 10 minutes,
and then place on ice.
• [LS] Place on the thermal cycler and run the LIG program. Each well contains
37.5 µl.
9
Centrifuge STL at 600 × g for 5 seconds.
10 Add 5 µl STL to each well, and then mix thoroughly as follows.
• [HS] Shake at 1800 rpm for 2 minutes.
• [LS] Pipette up and down.
11 [HS] Centrifuge at 280 × g for 1 minute.
TruSeq DNA PCR-Free Library Prep Reference Guide
21
Clean Up Ligated Fragments
1
Vortex SPB until well-dispersed.
2
Perform steps 2a through 2m using the Round 1 volumes.
a Add SPB to each well, and then mix thoroughly as follows.
SPB
b
c
d
e
f
g
h
i
Round 1
42.5 µl
Round 2
50 µl
— [HS] Shake at 1800 rpm for 2 minutes.
— [LS] Pipette up and down.
Incubate at room temperature for 5 minutes.
[HS] Centrifuge at 280 × g for 1 minute.
Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
Remove and discard all supernatant from each well.
Wash 2 times as follows.
— Add 200 µl freshly prepared 80% EtOH to each well.
— Incubate on the magnetic stand for 30 seconds.
— Remove and discard all supernatant from each well.
Use a 20 µl pipette to remove residual EtOH from each well.
Air-dry on the magnetic stand for 5 minutes.
Add RSB to each well.
RSB
Round 1
52.5 µl
Round 2
22.5 µl
j
Remove from the magnetic stand, and then mix thoroughly as follows.
— [HS] Shake at 1800 rpm for 2 minutes.
— [LS] Pipette up and down.
k Incubate at room temperature for 2 minutes.
l
[HS] Centrifuge at 280 × g for 1 minute.
m Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
3
Transfer 50 µl supernatant to the corresponding well of the CAP plate.
4
Repeat steps 2a through 2m with the new plate using the Round 2 volumes.
5
Transfer 20 µl supernatant to the corresponding well of the TSP1 plate.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at -25°C to -15°C for up to 7 days.
22
Part # 15036187 Rev. D
Perform the following procedures to quantify libraries and check library quality.
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 quantification of DNA libraries. Quantify the libraries using
qPCR.
} TruSeq DNA PCR-Free Library Prep library quantification has been validated using
the KAPA Library Quantification Kit specified in the Consumables and Equipment on
page 31.
} 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.
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:
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 quantification.
} Perform 2 additional independent (not serial) 1:10,000 and 1:20,000 dilutions using
at least 2 µl of the initial diluted libraries to evaluate quantification 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 7 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 7–Table 8.
Table 7 350 bp Library Concentration Calculation
Calculated
Average
Size
Dilution
by qPCR
diluted
adjusted
Factor
instrument (pM)*
library
diluted
(pM)
library (pM)
1:10,000
A1
A2
A=
W1 =
(A1 + A2)/2
A x (452/470)
1:20,000
B1
B2
B=
W2 =
(B1 + B2)/2
B x (452/470)
Table 8 550 bp Library Concentration Calculation
Calculated
Average
Size
Dilution
by qPCR
diluted
adjusted
Factor
instrument (pM)*
library
diluted
(pM)
library (pM)
1:10,000
C1
C2
C=
W3 =
(C1 + C2)/2
C x (452/670)
1:20,000
D1
D2
D=
W4 =
(D1 + D2)/2
D x (452/670)
Undiluted
library (pM)
*
C1 =
W1 x 10,000
C2 =
W2 x 20,000
Undiluted
library (pM)
*
C3 =
W3 x 10,000
C4 =
W4 x 20,000
Undiluted
library
(pM)
(C1 + C2)/2
Undiluted
library
(pM)
(C3 + C4)/2
*Duplicate data points
TruSeq DNA PCR-Free Library Prep Reference Guide
23
Validate Libraries
Validate Libraries
} Obtain the calculated concentration of the 1:10,000 and 1:20,000 library dilutions, 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).
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 (eg, 1:10,000 and 1:20,000). Use the average of the replicate data points
corresponding to each library DNA dilution to calculate the concentration of the
undiluted library.
} If a replicate is an outlier, it can be omitted from the calculation. If multiple replicates
are outliers, repeat the assay.
Quality Control
Verify fragment size by checking the library size distribution. Run samples on an Agilent
Technologies 2100 Bioanalyzer for qualitative purposes only.
1
Dilute the DNA library 1:5 with water.
2
Run 1 µl diluted DNA library on a High Sensitivity DNA chip.
Library fragment sizes measured on the Bioanalyzer are substantially larger than would
be predicted or derived from sequencing data. The larger size is because of the
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 2–Figure 3 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.
24
Part # 15036187 Rev. D
Validate Libraries
Figure 2 Example 350 bp Insert Library Distribution
A
B
Bioanalyzer
Paired-End Alignment
Figure 3 Example 550 bp Insert Library Distribution
A
B
Bioanalyzer
Paired-End Alignment
TruSeq DNA PCR-Free Library Prep Reference Guide
25
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
} Choose from the following containers:
• [HS]
— 96-well midi plate (1) (for pooling > 40 samples)
— 96-well Hard-Shell 0.3 ml PCR plates (2) (second plate for pooling ≤ 40
samples)
• [LS]
— 96-well midi plate (1) (for pooling > 40 samples)
— 96-well 0.3 ml PCR plates, semiskirted or skirtless (2) (second plate for
pooling ≤ 40 samples)
} Microseal 'B' adhesive seals
} Tris-HCl 10 mM, pH8.5 with 0.1% Tween 20
} Barcode labels
• DCT (Diluted Cluster Template)
• PDP (Pooled DCT Plate) (for pooling only)
Preparation
1
Apply barcodes to label plates as follows.
• DCT [PCR or Hard-Shell PCR plate]
• [For pooling only] PDP [midi (> 40 samples) or PCR (≤ 40 samples) or Hard-Shell
PCR plate]
Procedure
Make DCT
1
Transfer 5 µl library to the corresponding well of the DCT plate.
2
Normalize the library concentration with Tris-HCl 10 mM, pH 8.5 with 0.1%
Tween 20 to 2 nM, and then mix thoroughly as follows.
• [HS] Shake at 1000 rpm for 2 minutes.
• [LS] Pipette up and down.
NOTE
Depending on the yield quantification data of each library, the final volume of each
well can vary from 5–100 µl.
26
3
[HS] Centrifuge at 280 × g for 1 minute.
4
Do the following,
• To pool libraries, proceed to the next step in the workflow.
• For libraries that are not pooled, proceed to cluster generation. For more
information, see the system guide for your Illumina platform.
Part # 15036187 Rev. D
1
If pooling 2–24 samples, transfer 5 µl of each normalized library to a single well of
the PDP plate.
2
If pooling 25–96 samples, do the following.
a Transfer 5 µl of each column of normalized library to column 1 of the PDP plate,
and then mix thoroughly as follows.
— [HS] Shake at 1800 rpm for 2 minutes.
— [LS] Pipette up and down.
b [HS] Centrifuge at 280 × g for 1 minute.
c Transfer the contents of each well of column 1 to well A2.
3
Mix thoroughly as follows.
• [HS] Shake plate at 1800 rpm for 2 minutes.
• [LS] Pipette up and down.
4
[HS] Centrifuge at 280 × g for 1 minute.
5
Proceed to cluster generation. For more information, see the system guide for your
Illumina sequencing platform.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at -25°C to -15°C.
TruSeq DNA PCR-Free Library Prep Reference Guide
27
Normalize and Pool Libraries
Make PDP
Supporting Information
The protocols provided in this guide assume that you are familiar with the contents of
this section and that you have the required equipment and consumables.
Acronyms
Acronym
28
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
ERP
End Repair Mix
HS
High Sample
HT
High Throughput
IEM
Illumina Experiment Manager
IMP
Insert Modification Plate
LIG
Ligation Mix
LS
Low Sample
LT
Low Throughput
PDP
Pooled Dilution Plate
RSB
Resuspension Buffer
SPB
Sample Purification Beads
STL
Stop Ligation Buffer
TSP1
Target Sample Plate 1
Part # 15036187 Rev. D
Make sure that you have all the reagents identified in this section before starting the
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 9 TruSeq DNA PCR-Free Library Prep Kits
Kit Name
Catalog #
Number of
Indexes
FC-121-3001
Number of
Samples
Supported
24
TruSeq DNA PCR-Free LT Library
Prep Kit - Set A
TruSeq DNA PCR-Free LT Library
Prep Kit - Set B
TruSeq DNA PCR-Free HT Library
Prep Kit
FC-121-3002
24
12
FC-121-3003
96
96
12
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, Store at -25°C to -15°C
You receive either box A or B with the kit depending on the set you ordered. These boxes
also contain plate barcode labels.
NOTE
The kit contains either ERP2 or ERP3 and either ATL or ATL2.
TruSeq DNA PCR-Free Library Prep Reference Guide
29
Supporting Information
Kit Contents
Set A
Quantity
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Reagent
RSB
ERP2 or ERP3
ATL or ATL2
LIG2
CTE
CTA
CTL
STL
AD002
AD004
AD005
AD006
AD007
AD012
AD013
AD014
AD015
AD016
AD018
AD019
Description
Resuspension Buffer
End Repair Mix
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
Set B
Quantity
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Reagent
RSB
ERP2 or ERP3
ATL or ATL2
LIG2
CTE
CTA
CTL
STL
AD001
AD003
AD008
AD009
AD010
AD011
AD020
AD021
AD022
AD023
AD025
AD027
Description
Resuspension Buffer
End Repair Mix
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
Quantity
1
30
Reagent
SPB
Description
Sample Purification Beads
Part # 15036187 Rev. D
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 - (Box 1 of 2), Store at -25°C to -15°C
This box also contains plate barcode labels.
NOTE
The kit contains either ERP2 or ERP3 and either ATL or ATL2.
Table 10 TruSeq DNA PCR-Free HT Library Prep Kit, 96 Samples (Box 1 of 2), part #
15037059
Quantity
Reagent
Description
1
RSB
Resuspension Buffer
2
ERP2 or ERP3
End Repair Mix
2
ATL or ATL2
A-Tailing Mix
2
LIG2
Ligation Mix 2
2
CTE
End Repair Control
2
CTA
A-Tailing Control
2
CTL
Ligation Control
2
STL
Stop Ligation Buffer
96 Samples - Adapter Plate Box, Store at -25°C to -15°C
Quantity
1
Reagent
DAP
Description
DNA Adapter Plate, 96plex
96 Samples - SP Beads Box, Store at 2°C to 8°C
Quantity
5
Reagent
SPB
Description
Sample Purification Beads
Consumables and Equipment
Make sure that you have the required user-supplied consumables and equipment before
starting the protocol. Some items required depend on the workflow performed (HS or LS)
and these items are specified in separate tables.
The protocol has been optimized and validated using the items listed. Comparable
performance is not guaranteed when using alternate consumables and equipment.
Table 11 User-Supplied Consumables
Consumable
Supplier
1.7 ml microcentrifuge tubes
General lab supplier
15 ml conical tubes
General lab supplier
10 µl barrier pipette tips
General lab supplier
10 µl multichannel pipettes
General lab supplier
TruSeq DNA PCR-Free Library Prep Reference Guide
31
Supporting Information
TruSeq DNA PCR-Free HT Library Prep Kit
32
Consumable
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
96-well storage plates, round well,
0.8 ml (midi plate)
Fisher Scientific,
part # AB-0859
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
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-tube 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
Part # 15036187 Rev. D
Consumable
Supplier
96-well Hard-Shell 0.3 ml PCR plate
Bio-Rad, part # HSP-9601
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 13 User-Supplied Equipment
Equipment
Supplier
2100 Bioanalyzer Desktop System
Agilent Technologies, part # G2940CA
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 34.
General lab supplier
Table 14 User-Supplied Equipment - Additional Items for HS Workflow
Equipment
Supplier
High-Speed Microplate Shaker
VWR, catalog # • 13500-890 (110 V/120 V) or
• 14216-214 (230 V)
SciGene TruTemp Heating System
Note: Two systems are recommended
to support successive heating procedures.
Illumina, catalog # • SC-60-503 (110 V) or
• SC-60-504 (220 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
Table 15 User-Supplied Equipment - Additional Items for LS Workflow
Equipment
Supplier
96-well thermal cycler
(with heated lid)
General lab supplier
TruSeq DNA PCR-Free Library Prep Reference Guide
33
Supporting Information
Table 12 User-Supplied Consumables - Additional Items for HS Workflow
qPCR Systems
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 *
Bio-Rad, part # 185-5195
Mx3000P qPCR System
Agilent, part # 401511
* Use 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.
Indexed Adapter Sequences
This section describes the indexed adapter sequences.
Indexed Adapter Tube Sequences
The TruSeq DNA PCR-Free LT Library Prep Kit contains the following indexed adapter
sequences.
} The index numbering is not contiguous. There is no Index 17, 24, or 26.
} The sequence contains 7 bases. The seventh base, shown in parenthesis (), is not
included in the Index Read. Record only the first 6 bases in a sample sheet. For
indexes 13 and above, the seventh base (in parentheses) might not be A, which is
seen in the cycle 7 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 16 TruSeq DNA PCR-Free LT Library Prep Kit Set A Indexed Adapter Sequences
Adapter
AD002
AD004
AD005
AD006
AD007
AD012
Sequence
CGATGT(A)
TGACCA(A)
ACAGTG(A)
GCCAAT(A)
CAGATC(A)
CTTGTA(A)
Adapter
AD013
AD014
AD015
AD016
AD018
AD019
Sequence
AGTCAA(C)
AGTTCC(G)
ATGTCA(G)
CCGTCC(C)
GTCCGC(A)
GTGAAA(C)
Table 17 TruSeq DNA PCR-Free LT Library Prep Kit Set B Indexed Adapter Sequences
Adapter
AD001
AD003
AD008
AD009
AD010
AD011
34
Sequence
ATCACG(A)
TTAGGC(A)
ACTTGA(A)
GATCAG(A)
TAGCTT(A)
GGCTAC(A)
Adapter
AD020
AD021
AD022
AD023
AD025
AD027
Sequence
GTGGCC(T)
GTTTCG(G)
CGTACG(T)
GAGTGG(A)
ACTGAT(A)
ATTCCT(T)
Part # 15036187 Rev. D
The DAP in the TruSeq DNA PCR-Free HT Library Prep Kit contains the following
indexed adapter sequences.
The indexed adapter sequence recorded in the sample sheet contains 8 bases, and all 8
bases are sequenced during the Index Read.
Table 18 Indexed Adapter 1 Sequences
Adapter
D701
D702
D703
D704
D705
D706
Sequence
ATTACTCG
TCCGGAGA
CGCTCATT
GAGATTCC
ATTCAGAA
GAATTCGT
Adapter
D707
D708
D709
D710
D711
D712
Sequence
CTGAAGCT
TAATGCGC
CGGCTATG
TCCGCGAA
TCTCGCGC
AGCGATAG
Adapter
D505
D506
D507
D508
Sequence
AGGCGAAG
TAATCTTA
CAGGACGT
GTACTGAC
Table 19 Indexed Adapter 2 Sequences
Adapter
D501
D502
D503
D504
Figure 4
Sequence
TATAGCCT
ATAGAGGC
CCTATCCT
GGCTCTGA
DAP Dual-Indexed Layout
TruSeq DNA PCR-Free Library Prep Reference Guide
35
Supporting Information
Indexed Adapter Plate Sequences
Notes
Notes
Notes
For technical assistance, contact Illumina Technical Support.
Table 20 Illumina General Contact Information
Website
Email
www.illumina.com
[email protected]
Table 21 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
North America
1.800.809.4566
Italy
Australia
1.800.775.688
Netherlands
Austria
0800.296575
New Zealand
Belgium
0800.81102
Norway
Denmark
80882346
Spain
Finland
0800.918363
Sweden
France
0800.911850
Switzerland
Germany
0800.180.8994
United Kingdom
Ireland
1.800.812949
Other countries
Contact Number
800.874909
0800.0223859
0800.451.650
800.16836
900.812168
020790181
0800.563118
0800.917.0041
+44.1799.534000
Safety Data Sheets
Safety data sheets (SDSs) are available on the Illumina website at
support.illumina.com/sds.html.
Product Documentation
Product documentation in PDF is available for download from the Illumina website. Go
to support.illumina.com, select a product, then select Documentation & Literature.
TruSeq DNA PCR-Free Library Prep Reference Guide
Technical Assistance
Technical Assistance
*15036187*
Part # 15036187 Rev. D
Illumina
San Diego, California 92122 U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Download PDF

advertisement