Truseq Nano Dna Library Prep Guide 15041110 D PDF

TruSeq® Nano DNA Library Prep
Reference Guide
For Research Use Only. Not for use in diagnostic procedures.
Revision History 3
Introduction 4
DNA Input Recommendations 5
Additional Resources 6
Protocol Introduction 7
Tips and Techniques 8
Library Prep Workflow 9
Prepare for Pooling 10
Fragment DNA 11
Repair Ends and Select Library Size 14
Adenylate 3ʹ Ends 17
Ligate Adapters 18
Enrich DNA Fragments 21
Validate Libraries 24
Normalize and Pool Libraries 25
Supporting Information 27
Technical Assistance 37
ILLUMINA PROPRIETARY
Part # 15041110 Rev. D
June 2015
Catalog # FC-121-9010DOC
This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for the
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Revision History
Revision History
Part # Revision Date Description of Change
15041110 D June 2015 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
15041110 C December Kit names changed from 'sample' prep to 'library' prep
2014 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
15041110 B November Renamed Incubate 1 IMP to Incubate IMP
2013 Added recommended thermal cycler settings to
Consumables and Equipment
15041110 A May 2013 Initial Release
TruSeq Nano DNA Library Prep Reference Guide

3
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.
4 Part # 15041110 Rev. D

DNA Input Recommendations
DNA Input Recommendations
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.

5
Additional Resources
The following documentation is available for download from the Illumina website.
Resource Description
TruSeq Nano DNA Library Prep Provides only protocol instructions. The protocol guide is
Protocol Guide (part # 15075697) intended for experienced users.
TruSeq Nano DNA Library Prep Provides a checklist of the protocol steps. The checklist is
Checklist (part # 15075698) intended for experienced users.
Dual Index Sequencing with Provides guidelines for preparing for dual-indexing
TruSeq HT Library Prep sequencing when using a TruSeq Nano DNA HT Library
(part # 15059916) Prep Kit.
TruSeq Library Prep Pooling Provides TruSeq pooling guidelines for preparing libraries
Guide (part # 15042173) for Illumina sequencing systems that require balanced index
combinations. Review this guide before beginning library
preparation.
Illumina Experiment Manager Provide information about creating and editing appropriate
Guide (part # 15031335) and IEM sample sheets for Illumina sequencing systems and analysis
TruSeq DNA, RNA, or ChIP software and record parameters for your sample plate.
Quick Reference Card
(part # 15037152)
BaseSpace help Provides information about the BaseSpace® sequencing data

(help.basespace.illumina.com) 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.
6 Part # 15041110 Rev. D

Protocol Introduction
Protocol Introduction
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 HS LS
LT Kit - Number of samples > 24 with index ≤ 24 with index
processed at the same time adapter tubes* adapter tubes*
HT Kit - Number of samples > 24 with index ≤ 24 with index
processed at the same time adapter plate adapter plate
Plate Type 96-well Hard-Shell 96-well 0.3 ml PCR
PCR 96-well midi
96-well midi
Incubation Equipment Microheating systems 96-well
thermal cycler
Mixing Method 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.

7
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.
8 Part # 15041110 Rev. D

Library Prep Workflow
Library Prep Workflow
Figure 1 TruSeq Nano DNA Library Prep Workflow

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

Fragment DNA
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
• 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 Storage Instructions

RSB -25°C to -15°C Thaw at room temperature.
Store at 2°C to 8°C after the initial thaw.
SPB 2°C to 8°C 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]

11
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 M220 S220 S2 E210
Duty Factor (%) 20 5 10
Intensity — — 5.0
Peak/Displayed Power (W) 50 175 23 14
Cycles/Burst 200
Duration (seconds) 65 50 45
Mode — Frequency sweeping
Temperature (°C) 20 5.5–6
Table 3 550 bp Insert Settings

Covaris Setting M220 S220 S2 E210
Duty Factor (%) 20 5 10
Intensity — — 2.0
Peak/Displayed Power (W) 50 175 9 7
Cycles/Burst 200
Duration (seconds) 45 25 45
Mode — Frequency sweeping
Temperature (°C) 20 5.5–6
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.
12 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.
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.
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.

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)
} Barcode labels
• ALP (Adapter Ligation Plate)
• CEP (Clean Up End Repair Plate)
} PCR grade water
} Tube
• For ≤ 6 samples—1.7 ml microcentrifuge tube
• For > 6 samples—15 ml conical tube
• [HS] 96-well midi plates (2)
} Microseal 'B' adhesive seals
About Reagents
} The kit contains either ERP2 or ERP3.
Preparation

ERP2 or ERP3 -25°C to -15°C Thaw at room temperature, and then place on ice.
Return to storage after use.
RSB 2°C to 8°C Let stand for 30 minutes to bring to room
temperature.
SPB 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
14 Part # 15041110 Rev. D

Repair Ends and Select Library Size
• 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.
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

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 Example Amount
Your Calculation
per 12 samples
SPB # of samples X 109.25 µl 1311 µl
PCR grade water # of samples X 74.75 µl 897 µl
Table 5 Diluted SPB for a 550 bp Insert Size

Formula Example Amount
Your Calculation
per 12 samples
SPB # of samples X 92 µl 1104 µl
PCR grade water # of samples X 92 µl 1104 µl
3 Vortex diluted SPB until well-dispersed.

4 Add 160 µl diluted SPB to each well, and then mix thoroughly as follows.
8 Transfer 250 µl supernatant to the corresponding well of the CEP plate.

15
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.
10 Add 20 µl RSB to each well.
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

Adenylate 3ʹ Ends
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)
} [HS] Microseal 'B' adhesive seal
Preparation

ATL or ATL2 -25°C to -15°C Thaw at room temperature.
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:
• 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.
3 Centrifuge at 280 × g for 1 minute.
[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.

17
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)
} STL (Stop Ligation Buffer)
} Barcode labels
• CAP (Clean Up ALP Plate)
• DAP (DNA Adapter Plate)
• PCR (Polymerase Chain Reaction Plate)
• [HS] 96-well midi plate (1) and 96-well Hard-Shell 0.3 ml PCR plate (1)
} [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.
Preparation

DNA Adapters -25°C to -15°C Thaw at room temperature for 10 minutes.
The DAP can undergo up to 4 freeze-thaw cycles.
temperature.
STL -25°C to -15°C Thaw at room temperature.
SPB 2°C to 8°C Let stand for 30 minutes to bring to room
temperature.
2 [HS] Preheat a microheating system to 30°C.

3 [LS] Save the following LIG program on the thermal cycler:
• Hold at 4°C
18 Part # 15041110 Rev. D

Ligate Adapters
• 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 Speed Duration

Adapter tubes 600 × g 5 seconds
DAP 280 × g 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 Volume (µl)
RSB 2.5
LIG2 2.5
DNA adapters 2.5
• [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.
Clean Up Ligated Fragments

19
2 Perform steps 2a through 2m using the Round 1 volumes.
a Add SPB to each well, and then mix thoroughly as follows.
Round 1 Round 2
SPB 42.5 µl 50 µl
— [HS] Shake at 1800 rpm for 2 minutes.

— [LS] Pipette up and down.
b Incubate at room temperature for 5 minutes.
c [HS] Centrifuge at 280 × g for 1 minute.
d Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
e Remove and discard all supernatant from each well.
f 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.
g Use a 20 µl pipette to remove residual EtOH from each well.
h Air-dry on the magnetic stand for 5 minutes.
i Add RSB to each well.
Round 1 Round 2
RSB 52.5 µl 27.5 µl
j Remove from the magnetic stand, and then mix thoroughly as follows.
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

20 Part # 15041110 Rev. D

Enrich DNA Fragments
This process uses PCR to selectively enrich those DNA fragments that have adapter
molecules on both ends and to amplify the amount of DNA in the library. 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)
} TSP1 (Target Sample Plate) barcode label
• [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
About Reagents
Preparation
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.
EPM -25°C to -15°C Thaw on ice.
Invert to mix, then centrifuge at 600 × g for 1 minute. Do not
vortex.
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.

21
2 Save the following PCRNano program on the thermal cycler:
— 95°C for 3 minutes
• 8 cycles of:
— 98°C or 20 seconds
— 60°C for 15 seconds
— 72°C for 30 seconds
• 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.
4 Place on the thermal cycler and run the PCRNano program. Each well contains
50 µl.
Clean Up Amplified DNA

3 Add SPB to each well. The volume depends on the type of adapter used.
Adapter Type Volume SPB

Adapter tubes 50 µl
DAP 47.5 µl
4 Mix thoroughly, as follows.

22 Part # 15041110 Rev. D

12 Add 32.5 µl RSB to each well.
17 Transfer 30 µl supernatant to the corresponding well of the TSP1 plate.
SAFE STOPPING POINT


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

Normalize and Pool Libraries
Normalize and Pool Libraries
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)
• [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)
} 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
• 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.
NOTE
Depending on the yield quantification data of each library, the final volume of each
well can vary from 10–400 µl.
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.

25
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.
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.
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.
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

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 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

27
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 Catalog # Number of Number of
Samples Indexes
Supported
TruSeq Nano DNA LT Library FC-121-4001 24 12
Prep Kit - Set A
TruSeq Nano DNA LT Library FC-121-4002 24 12
Prep Kit - Set B
TruSeq Nano DNA HT Library FC-121-4003 96 96
Prep Kit
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

Set A
Quantity Reagent Description
1 RSB Resuspension Buffer
1 ERP2 or ERP3 End Repair Mix
1 ATL or ATL2 A-Tailing Mix
1 LIG2 Ligation Mix 2
1 STL Stop Ligation Buffer
1 PPC PCR Primer Cocktail
1 EPM Enhanced PCR Mix
1 AD002 DNA Adapter Index 2
Set B
24 Samples - SP Beads Box, Store at 2°C to 8°C

1 SPB 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
96 Samples - Adapter Plate Box, Store at -25°C to -15°C

1 DAP DNA Adapter Plate, 96plex
96 Samples - SP Beads Box, Store at 2°C to 8°C

6 SPB 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
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
30 Part # 15041110 Rev. D

Consumable Supplier
96-well storage plates, round well, Fisher Scientific,

0.8 ml (midi plate) part # AB-0859
One of the following: Agilent Technologies, part #:

• DNA 7500 Kit • 5067-1506
• High Sensitivity DNA Kit • 5067-4626
Ethanol 200 proof (absolute) for molecular Sigma-Aldrich, part # E7023

biology (500 ml)
Ice bucket General lab supplier
KAPA Library Quantification Kit - KAPA Biosystems, part # KK4824

Illumina/Universal
Microseal 'A' film Bio-Rad, part # MSA-5001
Microseal 'B' adhesive seals Bio-Rad, part # MSB-1001
microTUBE AFA Fiber 6x16mm with Covaris, part #

• Crimp-Cap or • 520052 or
• Pre-Slit Snap-Cap (for use with Covaris • 520045
M220)
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 VWR, part # 89094-658

reservoirs, disposable
Tris-HCl 10 mM, pH 8.5 General lab supplier
Tween 20 Sigma-Aldrich, part # P7949
[Optional] Fluorometric quantification with General lab supplier

dsDNA binding dye reagents

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 E&K Scientific, part # 480096 or

Twin.tec 96-well PCR plates 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) General lab supplier

See Thermal Cyclers on page 33.
One of the following Covaris systems: Covaris M220, part # 500295

• S2 For all other models, contact Covaris
• S220
• E210
• M220
Magnetic stand-96 Life Technologies, catalog # AM10027
Microplate centrifuge General lab supplier
Vortexer General lab supplier
qPCR system General lab supplier
See qPCR Systems on page 33.
[Optional] Fluorometer for quantification General lab supplier

with dsDNA binding dyes
Table 11 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 Illumina, catalog #

Note: Two systems are recommended • SC-60-503 (110 V) or
to support successive heating procedures. • SC-60-504 (220 V)
Midi plate insert for heating system Illumina, catalog # BD-60-601

Note: Two inserts are recommended
to support successive heating procedures.
Stroboscope General lab supplier
32 Part # 15041110 Rev. D

Thermal Cyclers
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 Vessel

Lid Temp
Mode Type
Bio-Rad DNA Engine Tetrad 2 Calculated Heated, constant at Plate

100°C
MJ Research PTC-225 DNA Engine Calculated Heated, constant at Plate

Tetrad 100°C
Bio-Rad S1000 N/A Heated, constant at Plate

100°C
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 Bio-Rad, part # 185-5195
System *
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.

33
Table 12 TruSeq Nano DNA LT Library Prep Kit Set A Indexed Adapter Sequences
Adapter Sequence Adapter Sequence
AD002 CGATGT(A) AD013 AGTCAA(C)
AD004 TGACCA(A) AD014 AGTTCC(G)
AD005 ACAGTG(A) AD015 ATGTCA(G)
AD006 GCCAAT(A) AD016 CCGTCC(C)
AD007 CAGATC(A) AD018 GTCCGC(A)
AD012 CTTGTA(A) AD019 GTGAAA(C)
Table 13 TruSeq Nano DNA LT Library Prep Kit Set B Indexed Adapter Sequences
AD001 ATCACG(A) AD020 GTGGCC(T)
AD003 TTAGGC(A) AD021 GTTTCG(G)
AD008 ACTTGA(A) AD022 CGTACG(T)
AD009 GATCAG(A) AD023 GAGTGG(A)
AD010 TAGCTT(A) AD025 ACTGAT(A)
AD011 GGCTAC(A) AD027 ATTCCT(T)
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

D701 ATTACTCG D707 CTGAAGCT
D702 TCCGGAGA D708 TAATGCGC
D703 CGCTCATT D709 CGGCTATG
D704 GAGATTCC D710 TCCGCGAA
D705 ATTCAGAA D711 TCTCGCGC
D706 GAATTCGT D712 AGCGATAG
Table 15 Indexed Adapter 2 Sequences

D501 TATAGCCT D505 AGGCGAAG
D502 ATAGAGGC D506 TAATCTTA
D503 CCTATCCT D507 CAGGACGT
D504 GGCTCTGA D508 GTACTGAC
34 Part # 15041110 Rev. D

Figure 4 DAP Dual-Indexed Layout

35
Notes
Notes
Notes
Technical Assistance
Technical Assistance
For technical assistance, contact Illumina Technical Support.
Table 16 Illumina General Contact Information

Website www.illumina.com
Email techsupport@illumina.com
Table 17 Illumina Customer Support Telephone Numbers

Region Contact Number Region Contact Number
North America 1.800.809.4566 Italy 800.874909
Australia 1.800.775.688 Netherlands 0800.0223859
Austria 0800.296575 New Zealand 0800.451.650
Belgium 0800.81102 Norway 800.16836
Denmark 80882346 Spain 900.812168
Finland 0800.918363 Sweden 020790181
France 0800.911850 Switzerland 0800.563118
Germany 0800.180.8994 United Kingdom 0800.917.0041
Ireland 1.800.812949 Other countries +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.

*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)
techsupport@illumina.com
www.illumina.com

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TruSeq® Nano DNA Library Prep

For Research Use Only. Not for use in diagnostic procedures.

TruSeq Nano DNA Library Prep Reference Guide

4 Part # 15041110 Rev. D

Quantify Input DNA

Assess DNA Quality

TruSeq Nano DNA Library Prep Reference Guide

BaseSpace help Provides information about the BaseSpace® sequencing data

6 Part # 15041110 Rev. D

Table 1 Workflow Options

TruSeq Nano DNA Library Prep Reference Guide

Sealing the Plate

8 Part # 15041110 Rev. D

TruSeq Nano DNA Library Prep Reference Guide

10 Part # 15041110 Rev. D

Item Storage Instructions

2 Turn on and set up the Covaris instrument according to manufacturer guidelines.

TruSeq Nano DNA Library Prep Reference Guide

Table 2 350 bp Insert Settings

Table 3 550 bp Insert Settings

4 Centrifuge at 280 × g for 5 seconds.

12 Part # 15041110 Rev. D

TruSeq Nano DNA Library Prep Reference Guide

Item Storage Instructions

2 [HS] Preheat the microheating system to 30°C.

14 Part # 15041110 Rev. D

Remove Large DNA Fragments

Table 5 Diluted SPB for a 550 bp Insert Size

3 Vortex diluted SPB until well-dispersed.

TruSeq Nano DNA Library Prep Reference Guide

Remove Small DNA Fragments

SAFE STOPPING POINT

16 Part # 15041110 Rev. D

Item Storage Instructions

TruSeq Nano DNA Library Prep Reference Guide

Item Storage Instructions

2 [HS] Preheat a microheating system to 30°C.

18 Part # 15041110 Rev. D

Add Index Adapters

Reagent Speed Duration

3 [HT kit] Prepare the DAP as follows.

TruSeq Nano DNA Library Prep Reference Guide

— [HS] Shake at 1800 rpm for 2 minutes.

SAFE STOPPING POINT

20 Part # 15041110 Rev. D

Reagent Storage Instructions

TruSeq Nano DNA Library Prep Reference Guide

Clean Up Amplified DNA

Adapter Type Volume SPB

4 Mix thoroughly, as follows.

22 Part # 15041110 Rev. D

SAFE STOPPING POINT

TruSeq Nano DNA Library Prep Reference Guide

Check Library Quality

Figure 2 Example 350 bp Insert Library Distribution

Figure 3 Example 550 bp Insert Library Distribution

24 Part # 15041110 Rev. D