TruSeq Nano DNA Libary Prep Reference Guide (15041110 D)

TruSeq Nano DNA Libary Prep Reference Guide (15041110 D)
TruSeq® Nano DNA 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
Enrich DNA Fragments
Validate Libraries
Normalize and Pool Libraries
Supporting Information
Technical Assistance
ILLUMINA PROPRIETARY
Part # 15041110 Rev. D
June 2015
Catalog # FC-121-9010DOC
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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,
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Part #
Revision
Date
15041110
D
June 2015
15041110
C
December
2014
15041110
B
November
2013
15041110
A
May 2013
TruSeq Nano DNA Library Prep Reference Guide
Description of Change
Updated Kit Contents:
• The kit contains either ERP2 or ERP3 and either ATL
or ATL2
• Removed box and tube part numbers
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
Corrected volume to 100 µl when running ERP thermal
cycling program to Convert Overhangs
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
Corrected Make CFP procedure in HS protocol to clarify
that the DNA plate is a midi plate
Modified Clean Up PCR HS protocol to omit sample
transfer to a midi plate
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
Added Microseal 'A' film to Consumables and Equipment
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
Added recommended thermal cycler settings to
Consumables and Equipment
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® Nano DNA 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 Nano DNA 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
} Index adapter tags for all samples
• Additional adapters and primers are not necessary
• Each TruSeq Nano DNA 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 Nano DNA 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 single sample sequencing or lower indexing pooling
levels.
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Part # 15041110 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 100 ng input gDNA.
} For a 550 bp insert size, use 200 ng 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.
Positive Control
Illumina recommends using Coriell Human-1 DNA (NA 18507) or Promega Human
Genomic DNA (G3041) as a positive control sample for this protocol.
TruSeq Nano DNA Library Prep Reference Guide
5
DNA Input Recommendations
DNA Input Recommendations
Additional Resources
The following documentation is available for download from the Illumina website.
Resource
Description
TruSeq Nano DNA Library Prep
Protocol Guide (part # 15075697)
Provides only protocol instructions. The protocol guide is
intended for experienced users.
TruSeq Nano DNA Library Prep
Checklist (part # 15075698)
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 Nano DNA 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 Nano DNA LT Library Prep Kit support page or TruSeq Nano DNA 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.
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Part # 15041110 Rev. D
This section describes the TruSeq Nano DNA 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 1 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 Nano DNA LT Library Prep Kit contains enough reagents to prepare up to
24 samples. When used together, TruSeq Nano DNA 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 6 for
information on how to access TruSeq Nano DNA 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 27.
TruSeq Nano DNA Library Prep Reference Guide
7
Protocol Introduction
Protocol Introduction
Tips and Techniques
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.
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Part # 15041110 Rev. D
Library Prep Workflow
Library Prep Workflow
Figure 1 TruSeq Nano DNA Library Prep Workflow
TruSeq Nano DNA Library Prep Reference Guide
9
Prepare for Pooling
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.
10
Part # 15041110 Rev. D
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
• 100 ng per sample for a 350 bp insert size
• 200 ng 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
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]
TruSeq Nano DNA Library Prep Reference Guide
11
Fragment DNA
Fragment DNA
Procedure
Normalize gDNA
1
Quantify gDNA using a fluorometric-based method.
2
Normalize gDNA samples with RSB to a final volume of 52.5 µl in the DNA plate.
• 100 ng for a 350 bp insert size
• 200 ng 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 2 350 bp Insert Settings
Covaris Setting
Duty Factor (%)
Intensity
Peak/Displayed Power (W)
Cycles/Burst
Duration (seconds)
Mode
Temperature (°C)
Table 3 550 bp Insert Settings
Covaris Setting
Duty Factor (%)
Intensity
Peak/Displayed Power (W)
Cycles/Burst
Duration (seconds)
Mode
Temperature (°C)
12
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.
Part # 15041110 Rev. D
Fragment DNA
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 62.5 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 60 µl supernatant to the corresponding well of the IMP plate.
TruSeq Nano DNA Library Prep Reference Guide
13
Repair Ends and Select Library Size
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)
Barcode labels
• ALP (Adapter Ligation Plate)
• CEP (Clean Up End Repair Plate)
Freshly prepared 80% ethanol (EtOH)
PCR grade water
Tube
• For ≤ 6 samples—1.7 ml microcentrifuge tube
• For > 6 samples—15 ml conical tube
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.
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
RSB
SPB
14
Storage
Instructions
-25°C to -15°C Thaw at room temperature, and then place on ice.
Return to storage after use.
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
Part # 15041110 Rev. D
Apply barcodes to label plates as follows.
• ALP [midi or PCR plate]
• CEP [midi or PCR plate]
Procedure
Convert Overhangs
1
Centrifuge ERP2 or ERP3 at 600 × g for 5 seconds.
2
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.
3
[HS] Centrifuge at 280 × g for 1 minute.
4
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 4 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 5 Diluted SPB for a 550 bp Insert Size
Formula
SPB
PCR grade water
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.
7
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.
TruSeq Nano DNA Library Prep Reference Guide
15
Repair Ends and Select Library Size
4
9
Discard remaining diluted SPB.
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 20 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 17.5 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.
16
Part # 15041110 Rev. D
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)
} [HS] Microseal 'B' adhesive seal
Preparation
1
Prepare the following consumables.
Item
ATL or ATL2
RSB
Storage
Instructions
-25°C to -15°C Thaw at room temperature.
Return to storage after use.
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
1
Centrifuge ATL or ATL2 at 600 × g for 5 seconds.
2
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.
3
Centrifuge at 280 × g for 1 minute.
4
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.
b Centrifuge at 280 × g for 1 minute.
TruSeq Nano DNA Library Prep Reference Guide
17
Adenylate 3ʹ Ends
Adenylate 3ʹ Ends
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)
Barcode labels
• CAP (Clean Up ALP Plate)
• DAP (DNA Adapter Plate)
• PCR (Polymerase Chain Reaction 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
}
}
}
}
}
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
Storage
Instructions
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.
18
2
[HS] Preheat a microheating system to 30°C.
3
[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
Part # 15041110 Rev. D
Ligate Adapters
4
Apply barcodes to label plates as follows.
• CAP [midi or PCR]
• PCR [Hard-Shell PCR or PCR]
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
Remove LIG2 from -25°C to -15°C storage.
5
Add the following reagents in the order listed to each well, and then mix thoroughly
as follows.
Reagent
RSB
LIG2
DNA adapters
Volume (µl)
2.5
2.5
2.5
• [HS] Shake at 1800 rpm for 2 minutes.
• [LS] Pipette up and down.
6
Centrifuge at 280 × g for 1 minute.
7
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.
8
Centrifuge STL at 600 × g for 5 seconds.
9
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.
10 [HS] Centrifuge at 280 × g for 1 minute.
Clean Up Ligated Fragments
1
Vortex SPB until well-dispersed.
TruSeq Nano DNA Library Prep Reference Guide
19
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
27.5 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 25 µl supernatant to the corresponding well of the PCR plate.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at -25°C to -15°C for up to 7 days.
20
Part # 15041110 Rev. D
This process uses PCR to selectively enrich those DNA fragments that have adapter
molecules on both ends and to amplify the amount of DNA in the library. PCR is
performed with a PCR Primer Cocktail that anneals to the ends of the adapters.
Minimize the number of PCR cycles to avoid skewing the representation of the library.
NOTE
PCR enriches for fragments that have adapters ligated on both ends. Fragments with only
1 or no adapters on their ends are by-products of inefficiencies in the ligation reaction.
Neither can be used to make clusters. Fragments with no adapters cannot hybridize to
surface-bound primers in the flow cell. Fragments with an adapter on 1 end can hybridize
to surface bound primers, but cannot form clusters.
Consumables
}
}
}
}
}
}
}
EPM (Enhanced PCR Mix)
PPC (PCR Primer Cocktail)
RSB (Resuspension Buffer)
SPB (Sample Purification Beads)
TSP1 (Target Sample Plate) barcode label
Freshly prepared 80% ethanol (EtOH)
Choose from the following containers:
• [HS] 96-well Hard-Shell 0.3 ml PCR plate (1)
• [LS] 96-well 0.3 ml PCR plate, semiskirted or skirtless (1)
} [HS] Microseal 'A' film
} Microseal 'B' adhesive seals
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.
Reagent Storage
Instructions
PPC
-25°C to -15°C Thaw at room temperature.
Invert to mix, then centrifuge at 600 × g for 1 minute. Do not
vortex.
Return to storage after use.
EPM
-25°C to -15°C Thaw on ice.
Invert to mix, then centrifuge at 600 × g for 1 minute. Do not
vortex.
Return to storage after use.
SPB
2°C to 8°C
Let stand for 30 minutes to bring to room temperature.
RSB
2°C to 8°C
Let stand for 30 minutes to bring to room temperature.
TruSeq Nano DNA Library Prep Reference Guide
21
Enrich DNA Fragments
Enrich DNA Fragments
2
Save the following PCRNano program on the thermal cycler:
• Choose the preheat lid option and set to 100°C
— 95°C for 3 minutes
• 8 cycles of:
— 98°C or 20 seconds
— 60°C for 15 seconds
— 72°C for 30 seconds
• 72°C for 5 minutes
• Hold at 4°C
3
Apply the TSP1 barcode to label a Hard-Shell PCR or PCR plate.
Procedure
Amplify DNA Fragments
1
Place on ice and add 5 µl PPC to each well.
2
Add 20 µl EPM to each well, and then mix thoroughly as follows.
• [HS] Shake at 1600 rpm for 20 seconds.
• [LS] Pipette up and down.
3
Centrifuge at 280 × g for 1 minute.
4
Place on the thermal cycler and run the PCRNano program. Each well contains
50 µl.
Clean Up Amplified DNA
1
Centrifuge at 280 × g for 1 minute.
2
Vortex SPB until well-dispersed.
3
Add SPB to each well. The volume depends on the type of adapter used.
Adapter Type
Adapter tubes
DAP
Volume SPB
50 µl
47.5 µl
4
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.
7
Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
8
Remove and discard all supernatant from each well.
9
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.
10 Use a 20 µl pipette to remove residual EtOH from each well.
22
Part # 15041110 Rev. D
Enrich DNA Fragments
11 Air-dry on the magnetic stand for 5 minutes.
12 Add 32.5 µl RSB to each well.
13 Remove from the magnetic stand, and then mix thoroughly as follows.
• [HS] Shake at 1800 rpm for 2 minutes.
• [LS] Pipette up and down.
14 Incubate at room temperature for 2 minutes.
15 [HS] Centrifuge at 280 × g for 1 minute.
16 Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
17 Transfer 30 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.
TruSeq Nano DNA Library Prep Reference Guide
23
Validate Libraries
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 a
fluorometric quantification method that uses dsDNA binding dyes or qPCR.
TruSeq Nano DNA Library Prep library quantification has been validated using the
KAPA Library Quantification Kit specified in the Consumables and Equipment on page 30.
Follow the KAPA instructions with the KAPA standard. To calculate the library
concentration in nM, perform the following insert size adjustment:
• For 350 bp libraries, use 470 bp for the average fragment length
• For 550 bp libraries, use 670 bp for the average fragment length
You can download the KAPA Library Quantification Kits for Illumina sequencing platforms
Technical Data Sheet from the Kapa Biosystems website (www.kapabiosystems.com).
Check Library Quality
Verify fragment size by checking the library size distribution. Run samples on an Agilent
Technologies 2100 Bioanalyzer for qualitative purposes only.
1
Do the following:
• If using a High Sensitivity DNA chip:
— Dilute the DNA library 1:100 with water.
— Run 1 µl diluted DNA library.
• If using a DNA 7500 chip, run 1 µl undiluted DNA library.
Figure 2 Example 350 bp Insert Library Distribution
Figure 3 Example 550 bp Insert Library Distribution
24
Part # 15041110 Rev. D
This process describes how to prepare DNA templates for cluster generation. Indexed
DNA libraries are normalized to 10 nM in the DCT plate and then pooled in equal
volumes in the PDP plate. Non-indexed DNA libraries are normalized to 10 nM in the
DCT plate.
Consumables
} 1.7 ml microcentrifuge tube (1) (when processing > 48 samples)
} Choose from the following containers:
• [HS]
— 96-well midi plates (2) (second plate for pooling)
• [LS]
— 96-well midi plates (2) (second plate for pooling > 40 samples)
— 96-well 0.3 ml PCR plate, semiskirted or skirtless (1) (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 [midi plate]
• [For pooling only] PDP [midi (> 40 samples) or PCR (≤ 40 samples) plate]
Procedure
Make DCT
1
Transfer 10 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 10 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 10–400 µl.
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.
TruSeq Nano DNA Library Prep Reference Guide
25
Normalize and Pool Libraries
Normalize and Pool Libraries
Make PDP
1
If pooling 2–24 samples, transfer 10 µl of each normalized library to a single well of
the PDP plate.
2
If pooling 25–48 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
If pooling 49–96 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 a 1.7 ml microcentrifuge tube.
4
Mix thoroughly as follows.
• [HS] Shake plate at 1800 rpm for 2 minutes or vortex the tube.
• [LS] Pipette up and down.
5
[HS] Centrifuge at 280 × g for 1 minute.
6
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 or cap the tube and store at -25°C to -15°C for up to
7 days.
26
Part # 15041110 Rev. D
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
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
DAP
DNA Adapter Plate
DCT
Diluted Cluster Template Plate
DNA
Customer Sample DNA Plate
EPM
Enhanced PCR Mix
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
PPC
PCR Primer Cocktail
RSB
Resuspension Buffer
SPB
Sample Purification Beads
STL
Stop Ligation Buffer
TSP1
Target Sample Plate 1
TruSeq Nano DNA Library Prep Reference Guide
27
Supporting Information
Supporting Information
Kit Contents
Make sure that you have all the reagents identified in this section before starting the
protocol.
The TruSeq Nano DNA LT Library Prep Kit is available in a Set A and a Set B. Each
TruSeq Nano DNA 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 6 TruSeq Nano DNA Library Prep Kits
Kit Name
TruSeq Nano DNA LT Library
Prep Kit - Set A
TruSeq Nano DNA LT Library
Prep Kit - Set B
TruSeq Nano DNA HT Library
Prep Kit
Catalog #
Number of
Indexes
FC-121-4001
Number of
Samples
Supported
24
FC-121-4002
24
12
FC-121-4003
96
96
12
TruSeq Nano DNA LT Library Prep Kit
The TruSeq Nano DNA 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.
28
Part # 15041110 Rev. D
Supporting Information
Set A
Quantity
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
STL
PPC
EPM
AD002
AD004
AD005
AD006
AD007
AD012
AD013
AD014
AD015
AD016
AD018
AD019
Description
Resuspension Buffer
End Repair Mix
A-Tailing Mix
Ligation Mix 2
Stop Ligation Buffer
PCR Primer Cocktail
Enhanced PCR Mix
DNA Adapter Index 2
DNA Adapter Index 4
DNA Adapter Index 5
DNA Adapter Index 6
DNA Adapter Index 7
DNA Adapter Index 12
DNA Adapter Index 13
DNA Adapter Index 14
DNA Adapter Index 15
DNA Adapter Index 16
DNA Adapter Index 18
DNA Adapter Index 19
Set B
Quantity
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
STL
PPC
EPM
AD001
AD003
AD008
AD009
AD010
AD011
AD020
AD021
AD022
AD023
AD025
AD027
Description
Resuspension Buffer
End Repair Mix
A-Tailing Mix
Ligation Mix 2
Stop Ligation Buffer
PCR Primer Cocktail
Enhanced PCR Mix
DNA Adapter Index 1
DNA Adapter Index 3
DNA Adapter Index 8
DNA Adapter Index 9
DNA Adapter Index 10
DNA Adapter Index 11
DNA Adapter Index 20
DNA Adapter Index 21
DNA Adapter Index 22
DNA Adapter Index 23
DNA Adapter Index 25
DNA Adapter Index 27
24 Samples - SP Beads Box, Store at 2°C to 8°C
Quantity
1
Reagent
SPB
TruSeq Nano DNA Library Prep Reference Guide
Description
Sample Purification Beads
29
TruSeq Nano DNA HT Library Prep Kit
The TruSeq Nano DNA 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 7 TruSeq Nano DNA HT Library Prep Kit, 96 Samples (Box 1 of 2), part # 15041877
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
STL
Stop Ligation Buffer
2
PPC
PCR Primer Cocktail
2
EPM
Enhanced PCR Mix
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
6
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 8 User-Supplied Consumables
30
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
Part # 15041110 Rev. D
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
One of the following:
• DNA 7500 Kit
• High Sensitivity DNA Kit
Agilent Technologies, part #: • 5067-1506
• 5067-4626
Ethanol 200 proof (absolute) for molecular
biology (500 ml)
Sigma-Aldrich, part # E7023
Ice bucket
General lab supplier
KAPA Library Quantification Kit Illumina/Universal
KAPA Biosystems, part # KK4824
Microseal 'A' film
Bio-Rad, part # MSA-5001
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 dsDNA HS Assay Kit
Life Technologies, catalog # Q32851
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
[Optional] Fluorometric quantification with
dsDNA binding dye reagents
General lab supplier
TruSeq Nano DNA Library Prep Reference Guide
Supporting Information
Consumable
31
Table 9 User-Supplied Consumables - Additional Items for HS Workflow
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 10 User-Supplied Equipment
Equipment
Supplier
2100 Bioanalyzer Desktop System
Agilent Technologies, part # G2940CA
96-well thermal cycler (with heated lid)
See Thermal Cyclers on page 33.
General lab 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
Vortexer
General lab supplier
qPCR system
See qPCR Systems on page 33.
General lab supplier
[Optional] Fluorometer for quantification
with dsDNA binding dyes
General lab supplier
Table 11 User-Supplied Equipment - Additional Items for HS Workflow
32
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
Part # 15041110 Rev. D
The following table lists the recommended settings for the Illumina recommended
thermal cycler, and other comparable models. If your lab has a thermal cycler that is not
listed, validate the thermal cycler before performing the TruSeq Nano DNA Library Prep
protocol.
Thermal Cycler
Temp
Mode
Bio-Rad DNA Engine Tetrad 2
Calculated
Heated, constant at
100°C
Plate
MJ Research PTC-225 DNA Engine
Tetrad
Calculated
Heated, constant at
100°C
Plate
Bio-Rad S1000
N/A
Heated, constant at
100°C
Plate
Lid Temp
Vessel
Type
qPCR Systems
The following table lists the validated qPCR systems for the TruSeq Nano DNA 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 Nano DNA 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.
TruSeq Nano DNA Library Prep Reference Guide
33
Supporting Information
Thermal Cyclers
Table 12 TruSeq Nano DNA 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 13 TruSeq Nano DNA LT Library Prep Kit Set B Indexed Adapter Sequences
Adapter
AD001
AD003
AD008
AD009
AD010
AD011
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)
Indexed Adapter Plate Sequences
The DAP in the TruSeq Nano DNA 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 14 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 15 Indexed Adapter 2 Sequences
Adapter
D501
D502
D503
D504
34
Sequence
TATAGCCT
ATAGAGGC
CCTATCCT
GGCTCTGA
Part # 15041110 Rev. D
Supporting Information
Figure 4
DAP Dual-Indexed Layout
TruSeq Nano DNA Library Prep Reference Guide
35
Notes
Notes
Notes
For technical assistance, contact Illumina Technical Support.
Table 16 Illumina General Contact Information
Website
Email
www.illumina.com
[email protected]
Table 17 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 Nano DNA Library Prep Reference Guide
Technical Assistance
Technical Assistance
*15041110*
Part # 15041110 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
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