TruSeq DNA Sample Prep Guide (15005180 C)

TruSeq DNA Sample Prep Guide (15005180 C)
TruSeq™ DNA
Sample Preparation Guide
FOR RESEARCH USE ONLY
ILLUMINA PROPRIETARY
Catalog # PE-940-2001
Part # 15005180 Rev. C
June 2011
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Part # 15005180 Rev. C
Revision History
Part #
Revision
15005180
C
Date
Description of Change
June • Corrected Ligation Mix (LIG) and Stop Ligation Buffer (STL) reagent
definitions:
2011
• Consumables:
• Replaced 4X gel loading dye with 6X gel loading dye for use during
Purify Ligation Products (BioLabs, catalog # B7021S).
• Changed Covaris tubes to MicroTube (6x16mm), AFA Fiber with
Crimp-Cap (Covaris, part # 520052).
• Added Microseal ‘A’ film for Enrich DNA Fragments procedure
(BioRad, part # MSA-5001).
• Changed TCY type plate name to Hardshell plate (HSP) and corrected
part number to Bio-Rad, part # HSP-9601.
• Added Master Mix Reagent Handling section and removed notes at the
beginning of procedures with aliquoting instructions.
• Qubit reagent system:
• Recommended for Input DNA Library Quantitation
• Parts added Consumables and Equipment lists
• Make CFP - Added step to quantify gDNA samples using a
fluorometric-based method such as Qubit or Picogreen
• Renamed "Pool Libraries" procedure to "Normalize and Pool Libraries" to
clarify that, even when not pooling, the normalization steps in this
procedure must be completed.
• Changed references to TruSeq Exome Enrichment to TruSeq Enrichment
to incorporate both exome and custom enrichment.
• Enrichment gel-free method insert size changed to 100-900 bp.
• AMPure XP Handling best practices and protocol clean up procedures
modified. Following the EtOH washes, the plate remains on the magnetic
stand while the samples air dry.
• HT clean up AMPure XP mixing procedures removed centrifugation step.
• Size Separate SSP and Clean Up PCR - final steps specify options for
whole-genome resequencing or enrichment.
• Validate Library - clarified that the procedure in this guide is for wholegenome resequencing and for enrichment, proceed to the TruSeq
Enrichment Guide for instructions.
TruSeq DNA Sample Preparation Guide
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Part #
Revision
15005180
B
15005180
A
Date
Description of Change
• Updated PCR Primer Cocktail (PPC) part number
• In-Line Control DNA- changed reference to RTA software version
1.9
• Updated sample sheet multiplexing information and field
definitions
• Changed High Speed Micro Plate Shaker part number
• Changed Illumina Equipment part numbers to corresponding
catalog numbers
• Added "using a single channel or multichannel pipette" consistently
throughout procedures
• Added step to centrifuge plate after resuspending AMPure XP
Beads in Resuspension Buffer
• All plates dry on magnetic stand
• Changed reference to qPCR Quantification Guide to its new name
Sequencing Library qPCR Quantification Guide
• Added DNA plate to Fragment Genomic DNA step in LT workflow
diagram and Fragment DNA procedure
• Added Appendix A - Alternate Fragmentation Protocols, containing
nebulizer protocol
• Changed CFP plate from TCY to MIDI
• Added pre-heat thermal cycler in LT protocol
• Incorporated gel-free method as primary procedure for
enrichment, with gel method as an option
• Changed time on magnetic stand from 2 minutes to 5 minutes
• Add ATL - removed "to the bottom of" when adding reagents to
each well
• Resuspension - HT protocol - made incubation step consistently a
separate step in the protocol and not part of the mixing procedure
• Added Index
November Initial Release
2010
January
2011
Part # 15005180 Rev. C
Table of Contents
Revision History
Table of Contents
List of Tables
Chapter 1 Overview
Introduction
Audience and Purpose
iii
v
vii
1
2
3
Chapter 2 Standard Operating Procedures
5
Introduction
Acronyms
Best Practices
DNA Input Recommendations
In-Line Control DNA
Tracking Tools
Kit Contents
Consumables and Equipment
6
7
9
13
15
17
20
23
Chapter 3 Low-Throughput (LT) Protocol
29
Introduction
Sample Prep Workflow
Fragment DNA
Perform End Repair
Adenylate 3' Ends
Ligate Adapters
Purify Ligation Products (gel method only)
Enrich DNA Fragments
Validate Library
Normalize and Pool Libraries
30
31
32
35
39
41
46
50
54
56
TruSeq DNA Sample Preparation Guide
v
Chapter 4 High-Throughput (HT) Protocol
Introduction
Sample Prep Workflow
Fragment DNA
Perform End Repair
Adenylate 3' Ends
Ligate Adapters
Purify Ligation Products (gel method only)
Enrich DNA Fragments
Validate Library
Normalize and Pool Libraries
Appendix A Alternate Fragmentation Protocols
Introduction
Index
Technical Assistance
vi
59
60
61
62
65
69
71
77
81
86
88
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92
97
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Part # 15005180 Rev. C
List of Tables
Table 1 Protocol Features
Table 2 Fragmentation Method Options
Table 3 TruSeq DNA Sample Preparation Acronyms
Table 4 In-Line Control Functions
Table 5 Sample Sheet Fields
Table 6 User-Supplied Consumables
Table 7 User-Supplied Consumables - Additional Items for LT Processing
Table 8 User-Supplied Consumables - Additional Items for HT Processing
Table 9 User-Supplied Equipment
Table 10 User-Supplied Equipment - Additional Items for HT Processing
Table 11 Fragmentation Method Options
Table 12 Size Selection Options
Table 13 Pooled Sample Volumes
Table 14 Fragmentation Method Options
Table 15 Size Selection Options
Table 16 Pooled Sample Volumes
Table 17 Illumina General Contact Information
Table 18 Illumina Customer Support Telephone Numbers
TruSeq DNA Sample Preparation Guide
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3
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26
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46
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62
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101
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Part # 15005180 Rev. C
Chapter 1 Overview
Introduction
Audience and Purpose
TruSeq DNA Sample Preparation Guide
2
3
1
Chapter 1
Overview
Overview
Introduction
This protocol explains how to prepare 12 pooled indexed paired-end libraries of
genomic DNA (gDNA) for subsequent cluster generation and DNA sequencing using
the reagents provided in the Illumina ® TruSeq™ DNA Sample Preparation Kit. The
goal of this protocol is to add adapter sequences onto the ends of DNA fragments to
generate multiplexed single read or paired-end sequencing libraries.
The sample preparation protocol offers:
Streamlined Workflow
} Master-mixed reagents to reduce reagent containers and pipetting
} Universal adapter for preparation of single read, paired-end, and multiplexing
Gel-free option for enrichment with optimized SPRI conditions
Optimized shearing for whole-genome resequencing
Higher Throughput
} Simultaneous preparation of 96 multiplexed DNA samples
} Volumes optimized for standard 96-well plate
Improved Troubleshooting
} Process control checks built-in for quality control
Universal index adapter tags all samples
} Additional adapters and primers not necessary
} Enables multiplexing earlier in the process
The protocol is compatible with no indexing or a lower indexing pooling level. The
libraries generated do not require PCR amplification to enable cluster generation,
although PCR is recommended in the standard protocol to robustly meet the yield
requirements of most standard applications.
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Part # 15005180 Rev. C
This guide documents the sample preparation protocol using the Illumina TruSeq DNA
Sample Prep Kit.
} Chapter 3, explains how to perform the TruSeq DNA Sample Preparation using the
Low Throughput (LT) Protocol
} Chapter 4, explains how to perform the TruSeq DNA Sample Preparation using the
High Throughput (HT) Protocol
Equivalent results can be expected from either protocol and their distinguishing
elements are as follows:
Table 1 Protocol Features
Number of Samples
Processed
Plate Type
Low Throughput
48 or fewer
with indexed adapters
96-well 0.3 ml PCR
96-well MIDI
Incubation Equipment
96-well thermal cycler
Mixing Method
Pipetting
High Throughput
More than 48
with indexed adapters
96-well HSP
96-well MIDI
96-well thermal cycler
Microheating system
Micro plate shaker
The TruSeq DNA Sample Prep fragmentation process is optimized to obtain final
libraries, with the following differences
Table 2 Fragmentation Method Options
Whole-genome
Resequencing
Gel Method
Covaris
Shearing
Duration
Insert Size
TruSeq DNA Sample Preparation Guide
40 seconds
300–400 bp
TruSeq Exome Enrichment
Gel-free Method
Gel Method
120 seconds
100–900 bp
200–300 bp
3
Audience and Purpose
Audience and Purpose
4
Part # 15005180 Rev. C
Chapter 2 Standard Operating Procedures
Chapter 2
Standard Operating
Procedures
Introduction
Acronyms
Best Practices
DNA Input Recommendations
In-Line Control DNA
Tracking Tools
Kit Contents
Consumables and Equipment
TruSeq DNA Sample Preparation Guide
6
7
9
13
15
17
20
23
5
Standard Operating Procedures
Introduction
This chapter explains standard operating procedures and precautions for performing
the TruSeq DNA Sample Preparation. You will also find lists of standard equipment
and consumables.
The sample preparation protocols described in the rest of this guide assume that you
are familiar with the contents of this chapter, have implemented all the
recommendations, and have obtained all of the requisite equipment and consumables.
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Part # 15005180 Rev. C
Acronyms
Acronyms
Table 3 TruSeq DNA Sample Preparation Acronyms
Acronym
Definition
ALP
Adapter Ligation Plate
ATL
A-Tailing Mix
CAP
Clean Up ALP Plate
CFP
Covaris Fragmentation Plate
CPP
Clean Up PCR Plate
CTA
A-Tailing Control
CTE
End Repair Control
CTL
Ligase Control
DCT
Diluted Cluster Template
dsDNA
double-stranded DNA
ERP
End Repair Mix
EUC
Experienced User Card
gDNA
genomic DNA
HSP
Hardshell Plate
HT
High Throughput
IMP
Insert Modification Plate
ISP
Intermediate Source Plate
LIG
Ligation Mix
LT
Low Throughput
TruSeq DNA Sample Preparation Guide
7
Standard Operating Procedures
Acronym
8
Definition
LTF
Lab Tracking Form
PCR
Polymerase Chain Reaction
PDP
Pooled Dilution Plate
PMM
PCR Master Mix
PPC
PCR Primer Cocktail
RSB
Resuspension Buffer
SSM
Second Strand Master Mix
SSP
Size Separate Plate
STL
Stop Ligation Buffer
TSP
Target Sample Plate
Part # 15005180 Rev. C
When preparing gDNA libraries for sequencing, you should always adhere to good
molecular biology practices.
Liquid Handling
Good liquid handling measures are essential, particularly when quantifying libraries or
diluting concentrated libraries for making clusters.
} Small differences in volumes (±0.5 µl) can sometimes give rise to very large
differences in cluster numbers (~100,000).
} Small volume pipetting can be a source of potential error in protocols that require
generation of standard curves, such as PicoGreen assays or qPCR, or those that
require small but precise volumes, such as the Agilent Bioanalyzer.
} If small volumes are unavoidable, then due diligence should be taken to ensure that
pipettes are correctly calibrated.
} Ensure that pipettes are not used at the volume extremes of their performance
specifications.
} Care should be taken with solutions of high molecular weight double-stranded
DNA (dsDNA). These can be viscous and not evenly dispersed, resulting in aliquot
measurements that are not representative of the true concentration of the solution.
} To minimize pipetting errors, especially with small volume enzyme additions,
prepare the reagents for multiple samples simultaneously. As a result, pipette once
from the reagent tubes with a larger volume, rather than many times with 1 µl
volumes. This will allow you to aliquot in a single pipetting movement to
individual samples and standardize across multiple samples.
Master Mix Reagent Handling
When handling the master mix reagents:
} Minimize freeze-thaw cycles. If you do not intend to consume the reagents in one
use, dispense the reagent into aliquots after the initial thaw and refreeze the aliquots
in order to avoid excessive freeze-thaw cycles. However, if you aliquot, you may not
have enough reagents for 48 reactions over multiple uses.
TruSeq DNA Sample Preparation Guide
9
Best Practices
Best Practices
Standard Operating Procedures
} Add reagents in the order indicated and avoid making master-mixes containing the
in-line controls.
} Take care while adding the A-Tailing Mix (ATL) and Ligation Mix (LIG) due to the
viscosity of the reagents.
AMPure XP Handling
Following appropriate handling methods when working with Agencourt AMPure XP
Beads:
NOTE
Cleanup procedures have only been verified using a 300 µl 96-well PCR or
MIDI plate. Comparable performance is not guaranteed when using a
microcentrifuge tube or other formats..
NOTE
Cleanup procedures have only been tested and validated using the magnetic
stand specified in Consumables and Equipment on page 23 . Comparable
performance is not guaranteed when using other magnets.
} Prior to use, allow the beads to come to room temperature.
} Immediately prior to use, vortex the beads until they are well dispersed. The color of
the liquid should appear homogeneous.
} When performing the LT protocol, after adding the beads to the reaction, mix the
solution gently and thoroughly by pipetting up and down 10 times, making sure
the liquid comes in contact with the beads and that the beads are resuspended
homogeneously..
} When performing the HT protocol, after adding the beads to the reaction, seal the
plate and shake the plate on a microplate shaker at 1,800 rpm for 2 minutes.
Repeat, if necessary, until the color of the mixture appears homogeneous after
mixing.
} Take care to minimize bead loss which can impact final yields.
} Change the tips for each sample.
} Let the mixed samples incubate for 15 minutes at room temperature for maximum
recovery.
} When aspirating the cleared solution from the reaction plate and wash step, it is
important to keep the plate on the magnetic stand and to not disturb the separated
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Part # 15005180 Rev. C
}
}
}
}
}
}
}
Avoid Cross-Contamination
Practice the following to avoid cross-contaimination:
} Open only one adapter at the time.
} Pipette carefully to avoid spillage.
} Clean pipettes and change gloves between handling different adapter stocks.
} Clean work surfaces thoroughly before and after the procedure.
Potential DNA Contaminants
Avoid potential DNA contaminants:
} Incorrect DNA quantitation may result from DNA contamination, for example, by
interference from superfluous nucleic acids in a sample (e.g., RNA, small nucleic
acid fragments, nucleotides, single-stranded DNA), excess proteins, or other
contaminating materials.
TruSeq DNA Sample Preparation Guide
11
Best Practices
}
magnetic beads. Aspirate slowly to prevent the beads from sliding down the sides
of the wells and into the pipette tips.
To prevent the carryover of beads after elution, approximately 2.5 µl of supernatant
are left when the eluates are removed from the bead pellet.
For the wash steps, prepare fresh 80% ethanol. Ethanol tends to absorb water from
the air, therefore, fresh 80% ethanol should be prepared for optimal results.
Be sure to remove all of the ethanol from the bottom of the wells, as it may contain
residual contaminants.
Keep the reaction plate on the magnetic stand and let it air-dry at room temperature
to prevent potential bead loss due to electrostatic forces. Allow for the complete
evaporation of residual ethanol, as the presence of ethanol will impact the
performance of the subsequent reactions. Illumina recommends at least 15 minutes
drying time, but a longer drying time may be required.
Use the Resuspension Buffer (RSB) for DNA elution.
Resuspend the dried pellets using a single channel or multichannel pipette.
When removing and discarding supernatant from the wells, use a single channel or
multichannel pipette and take care not to disturb the beads.
To maximize DNA recovery during elution, incubate the DNA/bead mix for 2
minutes at room temperature before placing the samples onto the magnet.
Standard Operating Procedures
} DNA quality may also affect the quantity of usable DNA in a sample. For example,
if the DNA is damaged (e.g., heavily nicked or containing extensive
apurinic/apyrimidinic sites), then many of these fragments may fail during library
preparation.
} High molecular weight dsDNA derived from host genomes can also interfere with
accurate quantitation. For example, bacterial artificial chromosomes (BACs) and
other bacterially-derived plasmids usually contain a small percentage of the
chromosomal DNA from the host cells, despite the best purification efforts. These
sequences may ultimately give rise to unwanted clusters on a flow cell lane.
However, this contamination can be accurately quantified by analyzing aligned
reads generated during sequencing against known bacterial sequences and
subtracting these out. High molecular weight contamination may also be estimated
prior to library preparation using qPCR assays designed to target unique
chromosomal markers.
Temperature Considerations
Temperature is an important consideration for making gDNA libraries:
} Keep libraries at temperatures<37°C.
} Avoid elevated temperatures, particularly in the steps preceding the adapter
ligation.
} DNA fragments that have a high AT content are more likely to denature into single
strands than GC-rich fragments, which can result in an increased probability of
creating a bias in the sequencing coverage.
} Take care not to denature the library prior to the agarose gel electrophoresis process,
because single-stranded DNA has a different migration rate.
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Part # 15005180 Rev. C
It is important to quantitate the input DNA and assess the DNA quality prior to
performingTruSeq DNA Sample Preparation.
Input DNA Quantitation
Follow these gDNA input recommendations:
} Correct quantification of genomic DNA is essential.
} Illumina recommends 1 µg input DNA.
} The ultimate success or failure of a library preparation strongly depends on using
an accurately quantified amount of input DNA.
} Illumina recommends using fluorometric based methods for quantification
including Qubit or PicoGreen to provide accurate quantification for dsDNA. UVspec based methods, such as the Nanodrop, will measure any nucleotides present
in the sample including RNA, dsDNA, ssDNA, and free nucleotides which can give
an inaccurate measurement of gDNA.
} DNA quantification methods that rely on intercalating fluorescent dyes measure
only double-stranded DNA and are less subject to excess nucleic acids.
• These methods require the preparation of calibration curves and are highly
sensitive to pipetting error.
• Ensure that pipettes are correctly calibrated and are not used at the volume
extremes of their performance specifications.
Assessing DNA Quality
} Absorbance measurements at 260 nm are commonly used to assess DNA quality:
• The ratio of absorbance at 260 nm to absorbance at 280 nm is used as an
indication of sample purity, and values of 1.8–2.0 are considered indicative of
relatively pure DNA.
• Both absorbance measurements can be compromised by the presence of RNA or
small nucleic acid fragments such as nucleotides.
• Genomic DNA samples should be carefully collected to ensure that they are free
of contaminants.
TruSeq DNA Sample Preparation Guide
13
DNA Input Recommendations
DNA Input Recommendations
Standard Operating Procedures
} Gel electrophoresis is a powerful means for revealing the condition (including the
presence or absence) of DNA in a sample.
• Impurities, such as detergents or proteins, can be revealed by smearing of DNA
bands.
• RNA, which interferes with 260 nm readings, is often visible at the bottom of a
gel.
• A ladder or smear below a band of interest may indicate nicking or other
damage to DNA.
• Where possible, or necessary, a gel should be run to assess the condition of the
DNA sample.
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Part # 15005180 Rev. C
The End Repair Control, A-Tailing Control, and Ligase Control reagents contain DNA
fragments used as controls for the enzymatic activities of the End Repair Mix, A-Tailing
Mix, and Ligation Mix, respectively. Each reagent contains dsDNA fragments designed
to report the success or failure of a specific enzymatic activity used in the library
preparation process. Readout is determined by sequencing. If a control's sequence
appears in the final sequencing data, it indicates that its corresponding step was
successful. If it does not, or if it appears in substantially diminished numbers, it
indicates the step failed. The controls are intended for troubleshooting and are useful for
identifying the specific mode of failure, but are uninformative in cases where
sequencing data is not generated from a library.
NOTE
The use of these controls is optional and they can be replaced with the same
volume of Resuspension Buffer.
The control molecules work through the design of their ends (Table 4). Controls are
added to the reactions just prior to their corresponding step in the protocol. Their end
structures match those of a DNA molecule that has not gone through the step. If the
step is successful, the control molecule will be modified to participate in downstream
reactions of library generation and resulting in sequencing data. If the step fails, the
control molecule will not go forward in the process and no sequencing data will be
generated. Using 1 µg of starting material, the controls yield approximately 0.2% of
clusters, although this can vary based on library yield.
Table 4 In-Line Control Functions
Reagent
Function
End Repair Mix
End Repair Mix
A-Tailing Mix
TruSeq DNA Sample Preparation Guide
End repair: Generate blunt ended
fragments by 3'–>5' exonuclease
and polymerase activities
End repair: Add 5'-phosphate
groups needed for downstream
ligation
A-tailing: Make fragments
compatible with adapters and
prevent self-ligation by adding a
3'-A overhang
Control
End Repair
Control 1*
End Repair
Control 2*
A-Tailing
Control
Structure of Control
DNA Ends
5' overhang at one end,
3' overhang at other
end
Blunt with 5'-OH group
Blunt with 5'-phosphate
group
15
In-Line Control DNA
In-Line Control DNA
Standard Operating Procedures
Reagent
Ligation Mix
Function
Ligation: Join adapters to inserts
Control
Ligase
Control
Structure of Control
DNA Ends
Single-base 3' 'A' base
overhang
*End Repair Control 1 and End Repair Control 2 are separate controls included in the End Repair
Control reagent
The control reagents can be used for a variety of library insert sizes. Each is provided in
ladders ranging from approximately 150–850 bp in 100 bp increments. Each control
molecule has a unique DNA sequence, indicating both its function and size. The RTA
software (version 1.9 and higher) recognizes these sequences and isolates the control
sequences from the main body of sequencing reads and reports their counts per lane in
the controls tab of the RTA status.html page.
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Part # 15005180 Rev. C
Illumina provides the following tools for sample tracking and guidance in the lab:
} Experienced User Cards to guide you through the protocol, but with less detail
than provided in this user guide.
} Lab Tracking Forms to record lab equipment start and stop times and record the
barcode of each reagent and plate used in the protocol.
} Sample Sheet Template to record information about your samples for later use in
data analysis.
NOTE
All of these documents can be downloaded via
http://www.illumina.com/support/documentation.ilmn.
Lab Tracking Form
Create a copy of the lab tracking form for each run. Use it to track information about
your sample preparation such as operator information, start and stop times, reagent lot
numbers, and barcodes. This form can be filled out and saved online or printed and
filled in by hand.
Sample Sheet
The sample sheet is a file that describes the samples in each lane, including the indexes
used, and is required for demultiplexing following sequencing. For instructions on
using the sample sheet to direct demultiplexing, see the analysis pipeline
documentation.
The sample sheet is a comma-separated values (*.csv) file that contains the sample
name and related information, as shown below. Create the sample sheet using Excel or
another text editing tool that supports .csv files. Fill in your sample sheet according to
the guidelines provided in this section.
Include lanes with multiplexed samples in the sample sheet, listing the information
below. Lanes with a single sample can be left out of the sample sheet. These singlesample lanes can then be aligned to a reference genome, as specified in the CASAVA
config.template.txt file.
TruSeq DNA Sample Preparation Guide
17
Tracking Tools
Tracking Tools
Standard Operating Procedures
Figure 1 Example: Sample Sheet
The sample sheet has the following fields:
Table 5 Sample Sheet Fields
18
Column
Header
Description
FCID
The flow cell ID
Lane
A positive integer indicating the lane number (1–8)
Sample ID
The sample ID. This can be used to specify samples in the CASAVA
config.template.txt file.
Sample Ref
The reference sequence for the sample. This can be used to specify a
reference genome in the CASAVA config.template.txt.
Index
The index sequence
Description
The sample description
Control
Y indicates the lane is a control lane
N indicates a sample
Recipe
The recipe used during sequencing
Operator
The name or ID of the operator
Part # 15005180 Rev. C
Tracking Tools
NOTE
To avoid misidentifying samples, ensure that the sample IDs entered in the
sample sheet correctly correspond to the DNA samples used.
TruSeq DNA Sample Preparation Guide
19
Standard Operating Procedures
Kit Contents
Check to ensure that you have all of the reagents identified in this section before
proceeding. Each TruSeq DNA Sample Prep Kit can be used to process up to 48
samples. The kit cartons also contains plate barcode labels.
Kit Contents, Boxes A and B
You will choose to receive either box A or B with the kit depending on the index
pooling level you require.
Store at -15° to -25°C
These boxes are shipped on dry ice. As soon as you receive them, store the following
components at -15° to -25°C.
Figure 2 TruSeq DNA Sample Prep Kit, Box A, part # 15012999
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1
Resuspension Buffer (RSB), part # 15012547
2
End Repair Mix (ERP), part # 15012494
3
A-Tailing Mix (ATL), part # 15012495
4
Ligation Mix (LIG), part # 15012496
5
End Repair Control (CTE), part # 15012360
6
A-Tailing Control (CTA), part # 15012361
7
Ligase Control (CTL), part # 15012362
8
Stop Ligation Buffer (STL), part # 15012546
9
DNA Adapter Index 2 (AD002), part # 15012348
Part # 15005180 Rev. C
Kit Contents
10 DNA Adapter Index 4 (AD004), part # 15012350
11 DNA Adapter Index 5 (AD005), part # 15012351
12 DNA Adapter Index 6 (AD006), part # 15012352
13 DNA Adapter Index 7 (AD007), part # 15012353
14 DNA Adapter Index 12 (AD012), part # 15012358
Figure 3 TruSeq DNA Sample Prep Kit, Box B, part # 15013001
1
Resuspension Buffer (RSB), part # 15012547
2
End Repair Mix (ERP), part # 15012494
3
A-Tailing Mix (ATL), part # 15012495
4
Ligation Mix (LIG), part # 15012496
5
End Repair Control (CTE), part # 15012360
6
A-Tailing Control (CTA), part # 15012361
7
Ligase Control (CTL), part # 15012362
8
Stop Ligation Buffer (STL), part # 15012546
9
DNA Adapter Index 1 (AD001), part # 15012347
10 DNA Adapter Index 3 (AD003), part # 15012349
11 DNA Adapter Index 8 (AD008), part # 15012354
12 DNA Adapter Index 9 (AD009), part # 15012355
13 DNA Adapter Index 10 (AD010), part # 15012356
14 DNA Adapter Index 11 (AD011), part # 15012357
TruSeq DNA Sample Preparation Guide
21
Standard Operating Procedures
Kit Contents, PCR Prep Box
Store at -15° to -25°C
This box is shipped on dry ice. As soon as you receive it, store the following
components at -15° to -25°C.
Figure 4 TruSeq DNA Sample Prep Kit, PCR Prep Box, part # 15012995
22
1
PCR Master Mix (PMM), part # 15013681
2
PCR Primer Cocktail (PPC), part # 15021792
Part # 15005180 Rev. C
Check to ensure that you have all of the necessary user-supplied consumables and
equipment before proceeding to sample preparation. These consumables and equipment
are Illumina recommended for the TruSeq DNA Sample Preparation protocols. The
requirement of some supplies are dependent upon the protocol performed (LT or HT)
and these items are specified in separate tables below.
Table 6 User-Supplied Consumables
Consumable
Supplier
10 µl barrier pipette tips
General lab supplier
10 µl multichannel pipettes
General lab supplier
10 µl single channel pipettes
General lab supplier
1000 µl barrier pipette tips
General lab supplier
1000 µl multichannel pipettes
General lab supplier
1000 µl single channel pipettes
General lab supplier
2 µl barrier pipette tips
General lab supplier
2 µl multichannel pipettes
General lab supplier
2 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
6X gel loading dye
BioLabs, catalog # B7021S
50 X TAE buffer
Bio-Rad, part # 161-0743
96-well storage plates, round well, 0.8
ml (“MIDI” plate)
Fisher Scientific, part # AB-0859
TruSeq DNA Sample Preparation Guide
23
Consumables and Equipment
Consumables and Equipment
Standard Operating Procedures
24
Consumable
Supplier
Agencourt AMPure XP 60 ml kit
Beckman Coulter Genomics, part #
A63881
BenchTop 100 bp DNA ladder
Promega, part # G829B
Certified low-range ultra agarose
Bio-Rad, part # 161-3107
Clean scalpels
General lab supplier
MicroTube (6x16mm), AFA fiber with
crimp-cap
Covaris, part # 520052
Distilled water
General lab supplier
DNase/RNase zapper (to decontaminate
surfaces)
General lab supplier
Ethanol 200 proof (absolute) for
molecular biology (500 ml)
Sigma Aldrich, part # E7023
Freshly prepared 80% ethanol
General lab supplier
Microseal ‘A’ film
BioRad, part # MSA-5001
Microseal ‘B’ adhesive seals
BioRad, part # MSB-1001
MinElute Gel Extraction Kit
QIAGEN, part# 28604
Paired-End Sample Prep Kit
(for alternative fragmentation by
nebulization only)
Illumina, catalog # PE-102-1001
(10 samples), or
Illumina, catalog # PE-102-1001
(40 samples)
PCR grade water (for gel-free method)
General lab supplier
QIAquick PCR Purification Kit
(for alternative fragmentation by
nebulization only)
QIAGEN, part # 28104
Qubit dsDNA BR Assay Kit
Life Technologies
100 assays, catalog # Q32850
500 assays, catalog # Q32853
Part # 15005180 Rev. C
Supplier
Qubit assay tubes or
Axygen PCR-05-C tubes
Life Technologies, catalog # Q32856 or
VWR, part # 10011-830
RNase/DNase-free multichannel
reagent reservoirs, disposable
VWR, part # 89094-658
RNase/DNase-free 8-well PCR strip
tubes and caps
General lab supplier
RNase/DNase zapper (to decontaminate
surfaces)
General lab supplier
SyBr Gold Nucleic acid gel stain
Invitrogen, part # S11494
Tris-Cl 10 mM, pH8.5 with 0.1% Tween
20, or QIAGEN EB
General lab supplier, or
QIAGEN, part # 19086
Tris-Cl 10 mM, pH8.5 with 0.1% Tween
20
General lab supplier
Tween 20
Sigma, part # P7949
Ultra pure water
General lab supplier
Consumables and Equipment
Consumable
Table 7 User-Supplied Consumables - Additional Items for LT Processing
Consumable
Supplier
96-well 0.3 ml skirtless PCR plates
E&K Scientific, part # 480096
Table 8 User-Supplied Consumables - Additional Items for HT Processing
Consumable
Supplier
Microseal 96-well PCR plates
(“HSP” plate)
Bio-Rad, part # HSP-9601
TruSeq DNA Sample Preparation Guide
25
Standard Operating Procedures
Table 9 User-Supplied Equipment
Equipment
Supplier
96-well thermal cycler (with
heated lid)
General lab supplier
Covaris S2 System, or
Covaris E210 System
Covaris, part # S2, or
Covaris, part # E210
Dark reader
transilluminator
Clare Chemical Research, part # D195M
Electrophoresis power
supply
General lab supplier
Magnetic stand-96
Ambion, part # AM10027
Microplate centrifuge
General lab supplier
Qubit 2.0 Fluorometer
Life Technologies, catalog # Q32866
http://products.invitrogen.com/ivgn/product/Q32866
Thermo Scientific Owl B2
EasyCast Mini Gel System
(US) Thermo Scientific, part # B2, or
Fisher Scientific, part # 09-528-110B
(Other Regions) Fisher Scientific,
part # OWL-130-101J B
Vortexer
General lab supplier
Table 10 User-Supplied Equipment - Additional Items for HT Processing
26
Consumable
Supplier
High Speed Micro Plate Shaker
VWR, catalog # 13500-890 (110V/120V)
VWR, catalog # 14216-214 (230V)
Part # 15005180 Rev. C
Supplier
MIDI plate insert for heating system
Illumina, catalog # BD-60-601
Stroboscope
(to calibrate the micro plate shaker)
General lab supplier
Tru Temp Microheating System
Illumina, catalog # SC-60-503 (115V)
Illumina, catalog # SC-60-504 (220V)
TruSeq DNA Sample Preparation Guide
Consumables and Equipment
Consumable
27
28
Part # 15005180 Rev. C
Chapter 3 Low-Throughput (LT) Protocol
Chapter 3
Low-Throughput (LT)
Protocol
Introduction
Sample Prep Workflow
Fragment DNA
Perform End Repair
Adenylate 3' Ends
Ligate Adapters
Purify Ligation Products (gel method only)
Enrich DNA Fragments
Validate Library
Normalize and Pool Libraries
TruSeq DNA Sample Preparation Guide
30
31
32
35
39
41
46
50
54
56
29
Low-Throughput (LT) Protocol
Introduction
This chapter describes the TruSeq DNA Sample Preparation low-throughput (LT)
protocol. Illumina recommends this protocol for processing 48 or fewer samples. Follow
the protocol in the order shown. For optimal sample tracking and quality control, fill
out the Lab Tracking Form as you perform the sample preparation.
When processing more than 48 samples, Illumina recommends following the protocol
described in Chapter 4 High-Throughput (HT) Protocol.
30
Part # 15005180 Rev. C
The following figure illustrates the processes of the LT TruSeq DNA Sample Preparation
protocol to prepare a template composed of 12 pooled indexed libraries.
Figure 5 TruSeq DNA Sample Preparation LT Workflow
TruSeq DNA Sample Preparation Guide
31
Sample Prep Workflow
Sample Prep Workflow
Low-Throughput (LT) Protocol
Fragment DNA
This process describes how to optimally fragment the gDNA depending on the
downstream application. Covaris shearing generates dsDNA fragments with 3' or 5'
overhangs. The fragmentation process described below was optimized to obtain final
libraries with the following differences:
Table 11 Fragmentation Method Options
Whole-genome
Resequencing
Gel Method
Covaris Shearing
Duration
Insert Size
40 seconds
300–400 bp
TruSeq Enrichment
Gel-free
Gel Method
Method
120 seconds
100–900 bp
200–300 bp
NOTE
If fragmenting using a nebulization technique, skip this procedure and
perform the Appendix A Alternate Fragmentation Protocols. The
nebulization procedures have only been validated for whole-genome
resequencing or enrichment with the gel-method.
Calculate the amount of DNA to be fragmented based on 1 µg input DNA for each
sample.
Illumina-Supplied Consumables
}
}
}
}
Resuspension Buffer (RSB) (1 tube)
CFP (Covaris Fragmentation Plate) barcode label
DNA (DNA Plate) barcode label
IMP (Insert Modification Plate) barcode label
User-Supplied Consumables
} 96-well 0.3 ml PCR plates
} Covaris Tubes
} DNA
32
Part # 15005180 Rev. C
} Review DNA Input Recommendations on page 13.
} Remove one tube of Resuspension Buffer from -15° to -25°C storage and thaw it at
room temperature.
} Turn on the Covaris instrument at least 30 minutes before starting.
} Following the manufacturer’s instructions, de-gas and pre-chill the water to a
temperature of 3° to 6°C. You may start the fragmentation procedure at 6°C.
} Apply a CFP barcode label to the Covaris tube plate.
} Apply a DNA barcode label to a new 96-well 0.3 ml PCR plate.
} Apply a IMP barcode label to a new 96-well 0.3 ml PCR plate.
Make CFP
1
Illumina recommends to quantify gDNA samples using a fluorometric-based
method such as Qubit or PicoGreen.
2
Illumina recommends to normalize the gDNA samples to 55 µl at 20 ng/µl into
each well of the new 0.3 ml PCR plate labeled with the DNA barcode.
Fragment DNA
1
Shear 1 µg of gDNA sample by transferring 52.5 µl of each DNA from the DNA
plate to each Covaris tube in the new 0.3 ml PCR plate labeled with CFP barcode.
NOTE
Load the DNA into the Covaris tube very slowly to avoid creating air
bubbles. However, they may not be preventable during the process run.
2
Fragment the DNA using the following settings:
Duty cycle
Intensity
Bursts per second
Duration
Mode
TruSeq DNA Sample Preparation Guide
Whole-genome Resequencing
TruSeq Enrichment
10%
5.0
200
40 seconds
10%
5.0
200
120 seconds
Frequency sweeping
Frequency sweeping
33
Fragment DNA
Preparation
Low-Throughput (LT) Protocol
Power
Temperature
Whole-genome Resequencing
TruSeq Enrichment
23W
5.5° to 6°C
23W
5.5° to 6°C
3
Seal the Covaris tubes and centrifuge to 600 xg for 1 minute.
4
Transfer 50 µl of fragmented DNA from each Covaris tube in the CFP plate to the
corresponding well of the new 0.3 ml PCR plate labeled with the IMP barcode using
a single channel pipette.
NOTE
When indexing libraries, Illumina recommends arranging samples that will
be combined into a common pool in the same row. Each column should
contain a common index. This will facilitate pipetting operations when
dispensing indexed adapters and pooling indexed libraries later in the
protocol.
34
Part # 15005180 Rev. C
This process converts the overhangs resulting from fragmentation into blunt ends using
an End Repair Mix. The 3' to 5' exonuclease activity of this mix removes the 3'
overhangs and the polymerase activity fills in the 5' overhangs.
Illumina-Supplied Consumables
}
}
}
}
(Optional) End Repair Control (CTE) (1 tube per 48 reactions)
End Repair Mix (ERP) (1 tube per 48 reactions)
Resuspension Buffer (RSB) (1 tube)
ALP (Adapter Ligation Plate) barcode label
User-Supplied Consumables
}
}
}
}
}
}
}
96-well 0.3 ml PCR plate
AMPure XP Beads
Freshly Prepared 80% Ethanol (EtOH)
Microseal ‘B’ Adhesive Seal
PCR Grade Water (for gel-free method for enrichment only)
RNase/DNase-free Reagent Reservoirs (if using multichannel pipettes)
RNase/DNase-free Strip Tubes and Caps (if using multichannel pipettes)
Preparation
} Remove the following from -15° to -25°C storage and thaw them at room
temperature:
• End Repair Control or Resuspension Buffer
• End Repair Mix
NOTE
The use of the End Repair Control is optional and it can be replaced with the
same volume of Resuspension Buffer.
} Review AMPure XP Handling on page 10.
} Remove the AMPure XP beads from storage and let stand for at least 30 minutes to
bring them to room temperature.
} Pre-heat the thermal cycler to 30°C.
TruSeq DNA Sample Preparation Guide
35
Perform End Repair
Perform End Repair
Low-Throughput (LT) Protocol
} Apply a ALP barcode label to a new 96-well 0.3 ml PCR plate.
Make IMP
1
Do one of the following:
• If using the in-line control reagent:
— Briefly centrifuge the thawed End Repair Control tube to 600 xg for 5
seconds.
— Add 10 µl of thawed End Repair Control to each well of the IMP plate that
contains 50 µl of fragmented DNA. Change the tip after each sample.
• If not using the in-line control reagent, add 10 µl of Resuspension Buffer to each
well of the IMP plate that contains 50 µl of fragmented DNA. Change the tip
after each sample.
2
Add 40 µl of End Repair Mix to each well of the IMP plate containing the
fragmented DNA. Change the tip after each sample.
3
Adjust the pipette to 100 µl, then gently pipette the entire volume of each pooled
library up and down 10 times to mix thoroughly. Change the tip after each sample.
4
Seal the IMP plate with a Microseal ‘B’ adhesive seal.
Incubate 1 IMP
1
Incubate the IMP plate on the pre-heated thermal cycler, with the lid closed, for 30
minutes at 30°C.
2
Remove the IMP plate from the thermal cycler.
Clean Up IMP
NOTE
Before performing clean up, review AMPure XP Handling on page 10 when
working with AMPure XP Beads.
36
1
Remove the adhesive seal from the IMP plate.
2
Vortex the AMPure XP Beads until they are well dispersed.
Part # 15005180 Rev. C
Do one of the following:
• If using the gel-free method:
— Prepare a diluted bead mixture by combining 125 µl of well-mixed AMPure
XP Beads with 35 µl of PCR grade water.
— Add 160 µl of the diluted bead mixture to each well of the IMP plate
containing 100 µl of End Repair Mix.
• If using the gel method, add 160 µl well-mixed AMPure XP Beads to each well
of the IMP plate containing 100 µl of End Repair Mix.
4
Adjust the pipette to 200 µl, then gently pipette the entire volume up and down 10
times to mix thoroughly. Change the tip after each sample.
5
Incubate the IMP plate at room temperature for 15 minutes.
6
Place the IMP plate on the magnetic stand at room temperature for 15 minutes or
until the liquid appears clear.
7
Using a 200 µl single channel or multichannel pipette set to 127.5 µl, remove and
discard 127.5 µl of the supernatant from each well of the IMP plate. Change the tip
after each sample.
8
Repeat step 7 once.
NOTE
Leave the IMP plate on the magnetic stand while performing the following
80% EtOH wash steps (9–11).
9
With the IMP plate on the magnetic stand, add 200 µl of freshly prepared 80%
EtOH to each well with a sample without disturbing the beads.
10 Incubate the IMP plate at room temperature for 30 seconds, then remove and
discard all of the supernatant from each well. Take care not to disturb the beads.
Change the tip after each sample.
11 Repeat steps 9 and 10 once for a total of two 80% EtOH washes.
12 Let the IMP plate stand at room temperature for 15 minutes to dry, then remove the
plate from the magnetic stand.
13 Resuspend the dried pellet in each well with 17.5 µl Resuspension Buffer. Gently
pipette the entire volume up and down 10 times to mix thoroughly.
14 Incubate the IMP plate at room temperature for 2 minutes.
TruSeq DNA Sample Preparation Guide
37
Perform End Repair
3
Low-Throughput (LT) Protocol
15 Place the IMP plate on the magnetic stand at room temperature for 5 minutes or
until the liquid appears clear.
16 Transfer 15 µl of the clear supernatant from each well of the IMP plate to the
corresponding well of the new 0.3 ml PCR plate labeled with the ALP barcode.
Change the tip after each sample.
SAFE STOPPING POINT
If you do not plan to proceed to Adenylate 3' Ends on page 39
immediately, the protocol can be safely stopped here. If you are
stopping, seal the ALP plate with a Microseal ‘B’ adhesive seal and store
at -15° to -25°C for up to seven days.
38
Part # 15005180 Rev. C
A single ‘A’ nucleotide is added to the 3’ ends of the blunt fragments to prevent them
from ligating to one another during the adapter ligation reaction. A corresponding
single ‘T’ nucleotide on the 3’ end of the adapter provides a complementary overhang
for ligating the adapter to the fragment. This strategy ensures a low rate of chimera
(concatenated template) formation.
Illumina-Supplied Consumables
} (Optional) A-Tailing Control (CTA) (1 tube per 48 reactions)
} A-Tailing Mix (ATL) (1 tube per 48 reactions)
} Resuspension Buffer (RSB)
User-Supplied Consumables
} Microseal ‘B’ Adhesive Seal
} RNase/DNase-free Reagent Reservoirs (if using multichannel pipettes)
} RNase/DNase-free Strip Tubes and Caps (if using multichannel pipettes)
Preparation
} Remove the following from -15° to -25°C storage and thaw them at room
temperature:
• A-Tailing Control or Resuspension Buffer
• A-Tailing Mix
NOTE
The use of the A-Tailing Control is optional and it can be replaced with the
same volume of Resuspension Buffer.
} Remove the ALP plate from -15° to -25°C storage, if it was stored at the conclusion
of Perform End Repair on page 35 and let stand to thaw at room temperature.
• Briefly centrifuge the thawed ALP plate to 280 xg for 1 minute
• Remove the adhesive seal from the ALP plate.
} Pre-heat the thermal cycler to 37°C.
TruSeq DNA Sample Preparation Guide
39
Adenylate 3' Ends
Adenylate 3' Ends
Low-Throughput (LT) Protocol
Add ATL
1
Do one of the following:
• If using the in-line control reagent, add 2.5 µl of thawed A-Tailing Control to
each well of the ALP plate.
• If not using the in-line control reagent, add 2.5 µl of Resuspension Buffer to
each well of the ALP plate.
2
Add 12.5 µl of thawed A-Tailing Mix to each well of the ALP plate.
3
Adjust the pipette to 30 µl, then gently pipette the entire volume up and down 10
times to mix thoroughly. Change the tip after each sample.
Incubate 1 ALP
40
1
Incubate the ALP plate on the pre-heated thermal cycler, with the lid closed, for 30
minutes at 37°C.
2
Immediately remove the ALP plate from the thermal cycler, then proceed
immediately to Ligate Adapters on page 41.
Part # 15005180 Rev. C
This process ligates multiple indexing adapters to the ends of the DNA fragments,
preparing them for hybridization onto a flow cell.
Illumina-Supplied Consumables
} Ligation Mix (LIG) (1 tube per 48 reactions)
} DNA Adapter Indexes 1–12 (AD001–AD012)
(1 tube per column of 8 reactions, depending on the DNA Adapter Indexes being
used)
} (Optional) Ligase Control (CTL) (1 tube per 48 reactions)
} Resuspension Buffer (RSB)
} Stop Ligation Buffer (STL) (1 tube per 48 reactions)
} CAP (Clean Up ALP Plate) barcode label
} PCR (Polymerase Chain Reaction) barcode label (for gel-free method only)
} SSP (Size Separate Plate) barcode label (for gel method only)
User-Supplied Consumables
}
}
}
}
}
}
96-well 0.3 ml PCR plates (2)
AMPure XP Beads
Freshly Prepared 80% Ethanol (EtOH)
Microseal ‘B’ Adhesive Seals
RNase/DNase-free Reagent Reservoirs (if using multichannel pipettes)
RNase/DNase-free Strip Tubes and Caps (if using multichannel pipettes)
Preparation
} Remove the following from -15° to -25°C storage and thaw them at room
temperature:
• Appropriate DNA Adapter Index tubes (AD001–AD012, depending on the DNA
Adapter Indexes being used)
• Stop Ligation Buffer
• Ligase Control or Resuspension Buffer
TruSeq DNA Sample Preparation Guide
41
Ligate Adapters
Ligate Adapters
Low-Throughput (LT) Protocol
NOTE
The use of the Ligase Control is optional and it can be replaced with the
same volume of Resuspension Buffer.
} Review AMPure XP Handling on page 10.
} Remove the AMPure XP beads from storage and let stand for at least 30 minutes to
bring them to room temperature.
} Pre-heat the thermal cycler to 30°C.
} Apply a CAP barcode label to a new 96-well 0.3 ml PCR plate.
} Do one of the following:
• If using the gel-free method, apply a PCR barcode label to a new 96-well 0.3 ml
PCR plate.
• If using the gel method, apply a SSP barcode label to a new 96-well 0.3 ml PCR
plate.
NOTE
When indexing libraries, Illumina recommends arranging samples that will
be combined into a common pool in the same row. Each column should
contain a common index. This will facilitate pipetting operations when
dispensing indexed adapters and pooling indexed libraries later in the
protocol.
Add LIG
42
1
Briefly centrifuge the thawed DNA Adapter Index tubes (AD001–AD012 depending
on the DNA Adapter Indexes being used), Ligase Control (if using Ligase Control),
and Stop Ligation Buffer tubes to 600 xg for 5 seconds.
2
Immediately before use, remove the Ligation Mix tube from -15° to -25°C storage.
3
Remove the adhesive seal from the ALP plate.
4
Do one of the following:
• If using the in-line control reagent, add 2.5 µl of thawed Ligase Control to each
well of the ALP plate.
• If not using the in-line control reagent, add 2.5 µl of Resuspension Buffer to
each well of the ALP plate.
5
Add 2.5 µl of Ligation Mix to each well of the ALP plate.
6
Return the Ligation Mix tube back to -15° to -25°C storage immediately after use.
Part # 15005180 Rev. C
Add 2.5 µl of the appropriate/desired thawed DNA Adapter Index (AD001–AD012
depending on the DNA Adapter Indexes being used) to each well of the ALP plate.
8
Adjust the pipette to 37.5 µl, then gently pipette the entire volume up and down 10
times to mix thoroughly. Change the tip after each sample.
9
Seal the ALP plate with a Microseal ‘B’ adhesive seal.
Incubate 2 ALP
1
Incubate the ALP plate on the pre-heated thermal cycler, with the lid closed, at 30°C
for 10 minutes.
2
Remove the ALP plate from the thermal cycler.
1
Remove the adhesive seal from the ALP plate.
2
Add 5 µl of Stop Ligation Buffer to each well of the ALP plate to inactivate the
ligation.
3
Adjust the pipette to 42.5 µl, then gently pipette the entire volume up and down 10
times to mix thoroughly. Change the tip after each sample.
Add STL
Clean Up ALP
NOTE
Before performing clean up, review AMPure XP Handling on page 10 when
working with AMPure XP Beads.
1
Vortex the AMPure XP Beads until they are well dispersed, then add 42.5 µl of
mixed AMPure XP Beads to each well of the ALP plate.
2
Adjust the pipette to 85 µl, then gently pipette the entire volume up and down 10
times to mix thoroughly. Change the tip after each sample.
3
Incubate the ALP plate at room temperature for 15 minutes.
4
Place the ALP plate on the magnetic stand at room temperature for 5 minutes or
until the liquid appears clear.
TruSeq DNA Sample Preparation Guide
43
Ligate Adapters
7
Low-Throughput (LT) Protocol
5
Remove and discard 80 µl of the supernatant from each well of the ALP plate.
Change the tip after each sample.
NOTE
Leave the ALP plate on the magnetic stand while performing the following
80% EtOH wash steps (6–8).
6
With the ALP plate remaining on the magnetic stand, add 200 µl of freshly
prepared 80% EtOH to each well without disturbing the beads.
7
Incubate the ALP plate at room temperature for 30 seconds, then remove and
discard all of the supernatant from each well. Change the tip after each sample.
8
Repeat steps 6 and 7 once for a total of two 80% EtOH washes.
9
While keeping the ALP plate on the magnetic stand, let the samples air dry at room
temperature for 15 minutes and then remove the plate from the magnetic stand.
10 Resuspend the dried pellet in each well with 52.5 µl Resuspension Buffer. Gently
pipette the entire volume up and down 10 times to mix thoroughly. Change the tip
after each sample.
11 Incubate the ALP plate at room temperature for 2 minutes.
12 Place the ALP plate on the magnetic stand at room temperature for 5 minutes or
until the liquid appears clear.
13 Transfer 50 µl of the clear supernatant from each well of the ALP plate to the
corresponding well of the new 0.3 ml PCR plate labeled with the CAP barcode.
Change the tip after each sample.
14 Vortex the AMPure XP Beads until they are well dispersed, then add 50 µl of mixed
AMPure XP Beads to each well of the CAP plate for a second clean up.
15 Adjust the pipette to 100 µl, then gently pipette the entire volume up and down 10
times to mix thoroughly. Change the tip after each sample.
16 Incubate the CAP plate at room temperature for 15 minutes.
17 Place the CAP plate on the magnetic stand at room temperature for 5 minutes or
until the liquid appears clear.
18 Remove and discard 95 µl of the supernatant from each well of the CAP plate.
Change the tip after each sample.
44
Part # 15005180 Rev. C
19 With the CAP plate remaining on the magnetic stand, add 200 µl of freshly
prepared 80% EtOH to each well without disturbing the beads.
20 Incubate the CAP plate at room temperature for 30 seconds, then remove and
discard all of the supernatant from each well. Change the tip after each sample.
21 Repeat steps 19 and 20 once for a total of two 80% EtOH washes.
22 While keeping the CAP plate on the magnetic stand, let the samples air dry at room
temperature for 15 minutes and then remove the plate from the magnetic stand.
23 Resuspend the dried pellet in each well with 22.5 µl Resuspension Buffer. Gently
pipette the entire volume up and down 10 times to mix thoroughly. Change the tip
after each sample.
24 Incubate the CAP plate at room temperature for 2 minutes.
25 Place the CAP plate on the magnetic stand at room temperature for 5 minutes or
until the liquid appears clear.
26 Do one of the following:
• If using the gel-free method:
— Transfer 20 µl of the clear supernatant from each well of the CAP plate to
the corresponding well of the new 0.3 ml PCR plate labeled with the PCR
barcode. Change the tip after each sample.
— Proceed to Enrich DNA Fragments on page 50.
• If using the gel method:
— Transfer 20 µl of the clear supernatant from each well of the CAP plate to
the corresponding well of the new 0.3 ml PCR plate labeled with the SSP
barcode. Change the tip after each sample.
— Proceed to Purify Ligation Products (gel method only) on page 46.
SAFE STOPPING POINT
If you do not plan to proceed to Enrich DNA Fragments on page 50 or Purify
Ligation Products (gel method only) on page 46 immediately, the protocol can be
safely stopped here. If you are stopping, seal the PCR or SSP plate with a
Microseal ‘B’ adhesive seal and store at -15° to -25°C for up to seven days.
TruSeq DNA Sample Preparation Guide
45
Ligate Adapters
NOTE
Leave the CAP plate on the magnetic stand while performing the following
80% EtOH wash steps (19–21)
Low-Throughput (LT) Protocol
Purify Ligation Products (gel method only)
This process is only performed when using the gel method. If you are running the gelfree method for enrichment, proceed to Enrich DNA Fragments on page 50.
This process purifies the products of the ligation reaction on a gel and removes
unligated adapters, as well as any adapters that may have ligated to one another, and
selects a size-range of sequencing library appropriate for cluster generation.
Illumina suggests the following gel insert size targets and slice locations. The gel slice
locations account for the length of the adapter sequences flanking the inserts. For other
applications, other size ranges may be desired and the cut size adjusted accordingly.
Table 12 Size Selection Options
Insert Size Target
3 mm Slice Location
Whole-genome
Resequencing
300–400 bpa
400–500 bp
TruSeq Enrichment
200–300 bp
300–400 bp
a. +/- 1 standard deviation of 20 bp, i.e, a < 20% variance for read lengths of 2 × 75 bp or shorter
Illumina-Supplied Consumables
} PCR (Polymerase Chain Reaction Plate) barcode label
} Resuspension Buffer (RSB) (1 tube)
User-Supplied Consumables
}
}
}
}
}
}
}
}
}
46
50 X TAE Buffer
96-well 0.3 ml PCR plate
BenchTop 100 bp DNA Ladder
Clean Scalpels
Certified Low-range Ultra Agarose
Distilled Water
6X Gel Loading Dye
MinElute Gel Extraction Kit
SyBr Gold Nucleic Acid Gel Stain
Part # 15005180 Rev. C
Preparation
} Prepare 1X TAE buffer (> 1 L)
} Apply a PCR barcode label to a new 96-well 0.3 ml PCR plate.
} Remove the SSP plate from -15° to -25°C storage, if it was stored at the conclusion of
Ligate Adapters on page 41 and let stand to thaw at room temperature.
• Briefly centrifuge the thawed SSP plate to 280 xg for 1 minute.
• Remove the adhesive seal from the thawed SSP plate.
} Clean the tray, the comb, and the gel tank with ethanol and rinse them thoroughly
with deionized water to avoid cross contamination.
Size Separate SSP
1
Prepare a 150 ml, 2% agarose with SyBr Gold gel using 1 X TAE Buffer as follows:
a Add 3 g of agarose powder in 150 ml of 1X TAE buffer.
b Microwave the gel buffer until the agarose powder is completely dissolved.
c Cool the gel buffer on the bench for 5 minutes, and then add 15 µl of SyBr Gold.
Swirl to mix.
d Pour the entire gel buffer to the gel tray.
NOTE
The final concentration of SyBr Gold should be 1X in the agarose gel buffer.
WARNING
It is very important to pre-stain your gel with SyBr Gold. When using other
staining dyes or staining the gel after running, the DNA will migrate more
slowly than the ladder. This will result in cutting out the wrong size
fragments.
2
Remove the adhesive seal from the thawed SSP plate.
3
Add 4 µl of 6X Gel Loading Dye to each well of the SSP plate.
TruSeq DNA Sample Preparation Guide
47
Purify Ligation Products (gel method only)
NOTE
Illumina strongly recommends using the user-supplied consumables
specified. Any deviation from these materials may result in incorrect sizeexcision or require additional user optimization
Low-Throughput (LT) Protocol
4
Add 17 µl Resuspension Buffer and 4 µl of 6X Gel Loading Dye to 3 µl of DNA
ladder.
WARNING
Do not to overload the DNA ladder. Without clear and distinct bands, it is
difficult to excise the correct fragment size. Also, an overloaded ladder may
run faster than the DNA sample library.
5
When the agarose gel is set, put it in the gel electrophoresis unit and fill the tank
with 1X TAE Buffer to the maximum fill mark.
Dimensions recommended for the electrophoresis unit;
12 cm x 14 cm (W x L), 800 ml buffer volume
6
Load all of the ladder solution onto one lane of the gel.
7
Load the samples from each well of the SSP plate onto the other lanes of the gel,
leaving a gap of at least one empty lane between samples and ladders.
NOTE
Flanking the library on both sides with ladders may make the library
excision easier.
NOTE
When handling multiple samples, to avoid the risk of cross-contamination
between libraries, leave a gap of at least one empty lane between samples
and use ladders on the first and last well of the gel to help locate the gel area
to be excised.
8
Run the gel at 120 V constant voltage for 120 minutes.
9
View the gel on a Dark Reader transilluminator.
10 Do one of the following:
• For whole-genome resequencing, excise a band from the gel spanning the width
of the lane and ranging in size from 400-500 bp using a clean scalpel. Use the
DNA ladder as a guide.
• For enrichment, excise a band from the gel spanning the width of the lane and
ranging in size from 300-400 bp using a clean scalpel. Use the DNA ladder as a
guide. For more information, see the TruSeq Exome Enrichment Guide.
48
Part # 15005180 Rev. C
NOTE
Use a clean scalpel per sample to avoid sample cross-contamination.
Size Separate Gel
1
Follow the instructions in the MinElute Gel Extraction Kit to purify each sample.
Incubate the gel slices in the QG solution at room temperature (not at 50°C as
instructed) until the gel slices have completely dissolved, while vortexing every 2
minutes.
2
Follow the instructions in the MinElute Gel Extraction Kit to purify on one
MinElute spin column, eluting in 25 µl of QIAGEN EB.
3
Transfer 20 µl of each sample from the MinElute collection tube to the new 0.3 ml
PCR plate labeled with the PCR barcode using a single channel pipette.
SAFE STOPPING POINT
If you do not plan to proceed to Enrich DNA Fragments on page 50
immediately, the protocol can be safely stopped here. If you are
stopping, seal the PCR plate with a Microseal ‘B’ adhesive seal and store
at -15° to -25°C for up to seven days.
TruSeq DNA Sample Preparation Guide
49
Purify Ligation Products (gel method only)
NOTE
Cutting a band between 400–500 bp will result in an insert size of
approximately 300–400 bp, accounting for the size of the adapters. Adapters
add approximately 120 bp to each fragment. The sequencing read length
should be considered when cutting fragment sizes. Sequencing reads that
over-reach into the adapter will cause chimeric reads, unalignable to the
reference sequence.
Low-Throughput (LT) Protocol
Enrich DNA Fragments
This process uses PCR to selectively enrich those DNA fragments that have adapter
molecules on both ends and to amplify the amount of DNA in the library. The PCR is
performed with a PCR primer cocktail that anneals to the ends of the adapters. The
number of PCR cycles should be minimized to avoid skewing the representation of the
library.
NOTE
PCR enriches for fragments that have adapters ligated on both ends.
Fragments with only one or no adapters on their ends are by-products of
inefficiencies in the ligation reaction. Neither species can be used to make
clusters, as fragments without any adapters cannot hybridize to surfacebound primers in the flow cell, and fragments with an adapter on only one
end can hybridize to surface bound primers but cannot form clusters.
Illumina-Supplied Consumables
}
}
}
}
PCR Master Mix (PMM) (1 tube per 48 reactions)
PCR Primer Cocktail (PPC) (1 tube per 48 reactions)
Resuspension Buffer (RSB)
TSP1 (Target Sample Plate) barcode label
User-Supplied Consumables
}
}
}
}
}
}
}
96-well 0.3 ml PCR plate
AMPure XP Beads
Freshly Prepared 80% Ethanol (EtOH)
Microseal ‘A’ Film
Microseal ‘B’ Adhesive Seals
RNase/DNase-free Reagent Reservoirs (if using multichannel pipettes)
RNase/DNase-free Strip Tubes and Caps (if using multichannel pipettes)
Preparation
} Remove one tube each of PCR Master Mix and PCR Primer Cocktail from -15° to
-25°C storage and thaw them at room temperature. Once thawed, keep the tubes on
ice.
50
Part # 15005180 Rev. C
NOTE
Illumina recommends 10 cycles of PCR for robust protocol performance.
However, to optimize yield versus cycle number, a titration of PCR cycles
may also be performed.
} Pre-heat the thermal cycler lid to 100°C.
} Apply a TSP1 barcode label to a new 96-well 0.3 ml PCR plate.
Make PCR
The following procedure assumes 1 µg of input DNA to library preparation and is
designed to result in high library yields.
1
Add 5 µl of thawed PCR Primer Cocktail to each well of the PCR plate. Change the
tip after each sample.
2
Add 25 µl of thawed PCR Master Mix to each well of the PCR plate. Change the tip
after each sample.
TruSeq DNA Sample Preparation Guide
51
Enrich DNA Fragments
} Briefly centrifuge the thawed PCR Master Mix and PCR Primer Cocktail tubes to
600 xg for 5 seconds.
} Review AMPure XP Handling on page 10.
} Remove the AMPure XP beads from storage and let stand for at least 30 minutes to
bring them to room temperature.
} Remove the PCR plate from -15° to -25°C storage, if it was stored at the conclusion
of Ligate Adapters on page 41 or Purify Ligation Products (gel method only) on page 46
and let stand to thaw at room temperature.
• Briefly centrifuge the thawed PCR plate to 280 xg for 1 minute.
• Remove the adhesive seal from the thawed PCR plate.
} Pre-program the thermal cycler as follows:
• 98°C for 30 seconds
• 10 cycles of:
— 98°C for 10 seconds
— 60°C for 30 seconds
— 72°C for 30 seconds
• 72°C for 5 minutes
• Hold at 4°C
Low-Throughput (LT) Protocol
3
Adjust the pipette to 40 µl, then gently pipette the entire volume up and down 10
times to mix thoroughly.
4
Seal the PCR plate with a Microseal ‘A’ film.
WARNING
Using a Microseal "B" adhesive seal for PCR may cause droplets of the
sample to splash onto the film when peeling the film off the plate.
Amp PCR
1
Amplify the PCR plate in the pre-programmed thermal cycler, with the lid closed,
as follows:
a 98°C for 30 seconds
b 10 cycles of:
98°C for 10 seconds
60°C for 30 seconds
72°C for 30 seconds
c 72°C for 5 minutes
d Hold at 4°C
Clean Up PCR
NOTE
Before performing clean up, review AMPure XP Handling on page 10 when
working with AMPure XP Beads.
52
1
Remove the adhesive seal from the PCR plate.
2
Vortex the AMPure XP Beads until they are well dispersed, then add 50 µl of the
mixed AMPure XP Beads to each well of the PCR plate containing 50 µl of the PCR
amplified library. Gently pipette the entire volume up and down 10 times to mix
thoroughly. Change the tip after each sample.
3
Incubate the PCR plate at room temperature for 15 minutes.
4
Place the PCR plate on the magnetic stand at room temperature for 5 minutes or
until the liquid appears clear.
Part # 15005180 Rev. C
Remove and discard 95 µl of the supernatant from each well of the PCR plate.
Change the tip after each sample.
NOTE
Leave the PCR plate on the magnetic stand while performing the following
80% EtOH wash steps (6–8).
6
With the PCR plate remaining on the magnetic stand, add 200 µl of freshly
prepared 80% EtOH to each well without disturbing the beads.
7
Incubate the PCR plate at room temperature for 30 seconds, then remove and
discard all of the supernatant from each well. Change the tip after each sample.
8
Repeat steps 6 and 7 once for a total of two 80% EtOH washes.
9
While keeping the PCR plate on the magnetic stand, let the samples air dry at room
temperature for 15 minutes and then remove the plate from the magnetic stand.
10 Resuspend the dried pellet in each well with 32.5 µl Resuspension Buffer. Gently
pipette the entire volume up and down 10 times to mix thoroughly. Change the tip
after each sample.
11 Incubate the PCR plate at room temperature for 2 minutes.
12 Place the PCR plate on the magnetic stand at room temperature for 5 minutes or
until the liquid appears clear.
13 Transfer 30 µl of the clear supernatant from each well of the PCR plate to the
corresponding well of the new 0.3 ml PCR plate labeled with the TSP1 barcode.
Change the tip after each sample.
14 Do one of the following:
• If performing whole-genome resequencing, proceed to Validate Library on page
54.
• If performing enrichment, proceed to the TruSeq Enrichment Guide for
instructions on how to quantify and qualify your library.
SAFE STOPPING POINT
If you do not plan to proceed to Validate Library on page 54 or TruSeq
Enrichment immediately, the protocol can be safely stopped here.If you
are stopping, seal the TSP1 plate with a Microseal ‘B’ adhesive seal and
store at -15° to -25°C for up to seven days.
TruSeq DNA Sample Preparation Guide
53
Enrich DNA Fragments
5
Low-Throughput (LT) Protocol
Validate Library
Illumina recommends performing the following procedures for quality control analysis
on your whole-genome resequencing sample library and quantification of the DNA
library templates. If performing enrichment, proceed directly to the TruSeq Enrichment
Guide for instructions on how to quantify and qualify your library.
Quantify Libraries
In order to achieve the highest quality of data on Illumina sequencing platforms, it is
important to create optimum cluster densities across every lane of every flow cell. This
requires accurate quantitation of DNA library templates. Quantify your libraries using
qPCR according to the Illumina Sequencing Library qPCR Quantification Guide.
Quality Control (Optional)
To verify the size of your PCR enriched fragments, check the template size distribution
by running an aliquot of the DNA library on a gel or on a Agilent Technologies 2100
Bioanalyzer using a High Sensitivity DNA chip.
} If validating by gel, load 10% of the volume of the library on a gel and check that
the size range is as expected: a narrow smear similar in size to the DNA excised
from the gel after the ligation.
} If using the Agilent Bioanalyzer with a high sensitivity DNA chip, make a 1:50
dilution of the library using water and load 1 µl of the diluted library on the Agilent
High Sensitivity DNA chip. Running samples on a Bioanalyzer should be used for
qualitative purposes only.
Figure 6 Example of DNA Library Size Distribution for Whole-Genome Resequencing
54
Part # 15005180 Rev. C
Validate Library
Figure 7 DNA PCR Product
NOTE
If the DNA is not a narrow smear, but is comprised of a long smear of
several hundred base pairs, or contains an intense 126 bp fragment (adapterdimer), then another purification step is recommended. Repeat Purify
Ligation Products (gel method only) on page 46.
TruSeq DNA Sample Preparation Guide
55
Low-Throughput (LT) Protocol
Normalize and Pool Libraries
This process describes how to prepare DNA templates that will be applied to cluster
generation. Multiplexed DNA libraries are normalized to 10 nM in the DCT plate and
then pooled in equal volumes in the PDP plate. Non-multiplexed DNA libraries are
normalized to 10 nM in the DCT plate without pooling.
Illumina-Supplied Consumables
} DCT (Diluted Cluster Template) barcode label
} PDP (Pooled DCT Plate) barcode label (for multiplexing only)
User-Supplied Consumables
}
}
}
}
96-well 0.3 ml PCR plate (for multiplexing only)
96-well MIDI plate
Microseal ‘B’ Adhesive seals
Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20
Preparation
} Apply a DCT barcode label to a new 96-well MIDI plate.
} Apply a PDP barcode label to a new 96-well 0.3 ml PCR plate (for multiplexing
only).
} Remove the TSP1 plate from -15° to -25°C storage, if it was stored at the conclusion
of Enrich DNA Fragments on page 50, and let stand to thaw at room temperature.
• Briefly centrifuge the thawed TSP1 plate to 280 xg for 1 minute.
• Remove the adhesive seal from the thawed TSP1 plate.
Make DCT
56
1
Transfer 10 µl of sample library from each well of the TSP1 plate to the
corresponding well of the new MIDI plate labeled with the DCT barcode. Change
the tip after each sample.
2
Normalize the concentration of sample library in each well of DCT plate to 10 nM
using Tris-Cl 10 mM, pH 8.5 with 0.1% Tween 20.
Part # 15005180 Rev. C
3
Gently pipette the entire normalized sample library volume up and down 10 times
to mix thoroughly.
4
Depending on the type of library you want to generate, do one of the following:
• For non-multiplexed paired-end libraries, the protocol stops here. Do one of the
following:
— Proceed to cluster generation. See the Illumina Cluster Generation User Guide.
— Seal the DCT plate with a Microseal ‘B’ adhesive seal and store at -15° to 25°C.
• For multiplexed paired-end libraries, proceed to Make PDP (for multiplexing only)
on page 57.
Make PDP (for multiplexing only)
NOTE
Do not make a PDP plate if there is no pooling.
1
Determine the number of samples to be combined together for each pool.
2
Transfer 10 µl of each normalized sample library to be pooled from the DCT plate
to one well of the new 0.3 ml PCR plate labeled with the PDP barcode.
The total volume in each well of the PDP plate should be 10X the number of
combined sample libraries and will be 10–120 µl (1–12 libraries).
Table 13 Pooled Sample Volumes
Number of pooled
samples
Volume
(µl)
1
10
2
20
3
30
TruSeq DNA Sample Preparation Guide
57
Normalize and Pool Libraries
NOTE
Depending on the yield quantification data of each sample library, the final
volume in the DCT plate may vary from 10-400 µl.
Low-Throughput (LT) Protocol
Number of pooled
samples
Volume
(µl)
4
40
5
50
6
60
7
70
8
80
9
90
10
100
11
110
12
120
NOTE
Keep track of which sample goes into which well, to avoid pooling two
samples with the same index.
58
3
Gently pipette the entire volume up and down 10 times to mix thoroughly.
4
Do one of the following:
• Proceed to cluster generation. See the Illumina Cluster Generation User Guide.
• Seal the PDP plate with a Microseal ‘B’ adhesive seal and store at -15° to -25°C.
Part # 15005180 Rev. C
Chapter 4 High-Throughput (HT) Protocol
Chapter 4
High-Throughput (HT)
Protocol
Introduction
Sample Prep Workflow
Fragment DNA
Perform End Repair
Adenylate 3' Ends
Ligate Adapters
Purify Ligation Products (gel method only)
Enrich DNA Fragments
Validate Library
Normalize and Pool Libraries
TruSeq DNA Sample Preparation Guide
60
61
62
65
69
71
77
81
86
88
59
High-Throughput (HT) Protocol
Introduction
This chapter describes the TruSeq DNA Sample Preparation high-throughput (HT)
protocol. Illumina recommends this protocol when processing more than 48 samples.
Follow the protocols in the order shown. For optimal sample tracking and quality
control, fill out the Lab Tracking Form as you perform the sample preparation.
When processing 48 or fewer samples, Illumina recommends following the Chapter 3
Low-Throughput (LT) Protocol.
The HT protocol requires shaking and heating equipment to mix reagents and for
incubation (see Consumables and Equipment on page 23).
60
Part # 15005180 Rev. C
The following figure illustrates the processes of the HT TruSeq DNA Sample
Preparation protocol to prepare a template composed of 12 pooled indexed libraries.
Figure 8 TruSeq DNA Sample Preparation HT Workflow
TruSeq DNA Sample Preparation Guide
61
Sample Prep Workflow
Sample Prep Workflow
High-Throughput (HT) Protocol
Fragment DNA
This process describes how to optimally fragment the gDNA depending on the
downstream application. Covaris shearing generates dsDNA fragments with 3' or 5'
overhangs. The fragmentation process described below was optimized to obtain final
libraries with the following differences:
Table 14 Fragmentation Method Options
Whole-genome
Resequencing
Gel Method
Covaris Shearing
Duration
Insert Size
40 seconds
300–400 bp
TruSeq Enrichment
Gel-free
Gel Method
Method
120 seconds
100–900 bp
200–300 bp
NOTE
If fragmenting using a nebulization technique, skip this procedure and
perform the Appendix A Alternate Fragmentation Protocols. The
nebulization procedures have only been validated for whole-genome
resequencing or enrichment with the gel-method.
Calculate the amount of DNA to be fragmented based on 1 µg input DNA for each
sample.
Illumina-Supplied Consumables
}
}
}
}
Resuspension Buffer (RSB) (1 tube)
CFP (Covaris Fragmentation Plate) barcode label
DNA (DNA Plate) barcode label
IMP (Insert Modification Plate) barcode label
User-Supplied Consumables
} 96-well MIDI plates
} Covaris Tubes
} DNA
62
Part # 15005180 Rev. C
} Review DNA Input Recommendations on page 13.
} Remove one tube of Resuspension Buffer from -15° to -25°C storage and thaw it at
room temperature.
} Turn on the Covaris instrument at least 30 minutes before starting.
} Following the manufacturer’s instructions, de-gas and pre-chill the water to a
temperature of 3° to 6°C. You may start the fragmentation procedure at 6°C.
} Apply a CFP barcode label to the Covaris tube plate.
} Apply a DNA barcode label to a new 96-well MIDI plate.
} Apply a IMP barcode label to a new 96-well MIDI plate.
Make CFP
1
Illumina recommends to quantify gDNA samples using a fluorometric-based
method such as Qubit or PicoGreen.
2
Illumina recommends to normalize the gDNA samples to 55 µl at 20 ng/µl into
each well of the new MIDI plate labeled with the DNA barcode.
Fragment DNA
1
Shear 1 µg of gDNA sample by transferring 52.5 µl of each DNA from the DNA
plate to each Covaris tube in the new HSP plate labeled with CFP barcode.
NOTE
Load the DNA into the Covaris tube very slowly to avoid creating air
bubbles. However, they may not be preventable during the process run.
2
Fragment the DNA using the following settings:
Duty cycle
Intensity
Bursts per second
Duration
Mode
TruSeq DNA Sample Preparation Guide
Whole-genome Resequencing
TruSeq Enrichment
10%
5.0
200
40 seconds
10%
5.0
200
120 seconds
Frequency sweeping
Frequency sweeping
63
Fragment DNA
Preparation
High-Throughput (HT) Protocol
Power
Temperature
Whole-genome Resequencing
TruSeq Enrichment
23W
5.5° to 6°C
23W
5.5° to 6°C
3
Seal the Covaris tubes and centrifuge to 600 xg for 1 minute.
4
Transfer 50 µl of fragmented DNA from each Covaris tube in the CFP plate to the
corresponding well of the new MIDI plate labeled with the IMP barcode using a
single channel pipette.
NOTE
When indexing libraries, Illumina recommends arranging samples that will
be combined into a common pool in the same row. Each column should
contain a common index. This will facilitate pipetting operations when
dispensing indexed adapters and pooling indexed libraries later in the
protocol.
64
Part # 15005180 Rev. C
This process converts the overhangs resulting from fragmentation into blunt ends using
an End Repair Mix. The 3' to 5' exonuclease activity of this mix removes the 3'
overhangs and the polymerase activity fills in the 5' overhangs.
Illumina-Supplied Consumables
}
}
}
}
(Optional) End Repair Control (CTE) (1 tube per 48 reactions)
End Repair Mix (ERP) (1 tube per 48 reactions)
Resuspension Buffer (RSB) (1 tube)
ALP (Adapter Ligation Plate) barcode label
User-Supplied Consumables
}
}
}
}
}
}
}
96-well MIDI plate
AMPure XP Beads
Freshly Prepared 80% Ethanol (EtOH)
Microseal ‘B’ Adhesive Seal
PCR Grade Water (for gel-free method only)
RNase/DNase-free Reagent Reservoirs
RNase/DNase-free Strip Tubes and Caps
Preparation
} Remove the following from -15° to -25°C storage and thaw them at room
temperature:
• End Repair Control or Resuspension Buffer
• End Repair Mix
NOTE
The use of the End Repair Control is optional and it can be replaced with the
same volume of Resuspension Buffer.
} Review AMPure XP Handling on page 10.
} Remove the AMPure XP beads from storage and let stand for at least 30 minutes to
bring them to room temperature.
} Pre-heat the microheating system to 30°C.
TruSeq DNA Sample Preparation Guide
65
Perform End Repair
Perform End Repair
High-Throughput (HT) Protocol
} Calibrate the microplate shaker with a stroboscope and set it to 1,800 rpm.
} Apply a ALP barcode label to a new 96-well MIDI plate.
Make IMP
1
Do one of the following:
• If using the in-line control reagent:
— Briefly centrifuge the thawed End Repair Control tube to 600 xg for 5
seconds.
— Add 10 µl of thawed End Repair Control to each well of the IMP plate that
contains 50 µl of fragmented DNA. Change the tip after each sample.
• If not using the in-line control reagent, add 10 µl of Resuspension Buffer to each
well of the IMP plate that contains 50 µl of fragmented DNA. Change the tip
after each sample.
2
Add 40 µl of End Repair Mix to each well of the IMP plate containing the
fragmented DNA. Mix thoroughly as follows:
a Seal the IMP plate with a Microseal ‘B’ adhesive seal.
b Shake the IMP plate on a microplate shaker at 1,800 rpm for 2 minutes.
c Centrifuge the IMP plate to 280 xg for 1 minute.
Incubate 1 IMP
1
Incubate the IMP plate on the pre-heated microheating system, with the lid closed,
for 30 minutes at 30°C.
2
Remove the IMP plate from the microheating system.
Clean Up IMP
NOTE
Before performing clean up, review AMPure XP Handling on page 10 when
working with AMPure XP Beads.
66
1
Remove the adhesive seal from the IMP plate.
2
Vortex the AMPure XP Beads until they are well dispersed.
Part # 15005180 Rev. C
Do one of the following:
• If using the gel-free method for enrichment:
— Prepare a diluted bead mixture by combining 125 µl of well-mixed AMPure
XP Beads with 35 µl of PCR grade water.
— Add 160 µl of the diluted bead mixture to each well of the IMP plate
containing 100 µl of End Repair Mix.
• If using the gel method, add 160 µl well-mixed AMPure XP Beads to each well
of the IMP plate containing 100 µl of End Repair Mix.
4
Mix thoroughly as follows:
a Seal the IMP plate with a Microseal ‘B’ adhesive seal.
b Shake the IMP plate on a microplate shaker at 1,800 rpm for 2 minutes.
5
Incubate the IMP plate at room temperature for 15 minutes.
6
Place the IMP plate on the magnetic stand at room temperature for 15 minutes or
until the liquid appears clear.
7
Remove the adhesive seal from the IMP plate.
8
Using a 200 µl single channel or multichannel pipette set to 127.5 µl, remove and
discard 127.5 µl of the supernatant from each well of the IMP plate. Change the tip
after each sample.
9
Repeat step 8 once.
NOTE
Leave the IMP plate on the magnetic stand while performing the following
80% EtOH wash steps (10–12).
10 With the IMP plate on the magnetic stand, add 200 µl of freshly prepared 80%
EtOH to each well with a sample without disturbing the beads.
11 Incubate the IMP plate at room temperature for 30 seconds, then remove and
discard all of the supernatant from each well. Change the tip after each sample.
12 Repeat steps 10 and 11 once for a total of two 80% EtOH washes.
13 Let the IMP plate stand at room temperature for 15 minutes to dry, then remove the
plate from the magnetic stand.
TruSeq DNA Sample Preparation Guide
67
Perform End Repair
3
High-Throughput (HT) Protocol
14 Resuspend the dried pellet in each well with 17.5 µl Resuspension Buffer. Mix
thoroughly as follows:
a Seal the IMP plate with a Microseal ‘B’ adhesive seal.
b Shake the IMP plate on a microplate shaker at 1,800 rpm for 2 minutes.
c Centrifuge the IMP plate to 280 xg for 1 minute.
15 Incubate the IMP plate at room temperature for 2 minutes.
16 Place the IMP plate on the magnetic stand at room temperature for 5 minutes or
until the liquid appears clear.
17 Remove the adhesive seal from the IMP plate.
18 Transfer 15 µl of the clear supernatant from each well of the IMP plate to the
corresponding well of the new MIDI plate labeled with the ALP barcode. Change
the tip after each sample.
SAFE STOPPING POINT
If you do not plan to proceed to Adenylate 3' Ends on page 69
immediately, the protocol can be safely stopped here. If you are
stopping, seal the ALP plate with a Microseal ‘B’ adhesive seal and store
at -15° to -25°C for up to seven days.
68
Part # 15005180 Rev. C
A single ‘A’ nucleotide is added to the 3’ ends of the blunt fragments to prevent them
from ligating to one another during the adapter ligation reaction. A corresponding
single ‘T’ nucleotide on the 3’ end of the adapter provides a complementary overhang
for ligating the adapter to the fragment. This strategy ensures a low rate of chimera
(concatenated template) formation.
Illumina-Supplied Consumables
} (Optional) A-Tailing Control (CTA) (1 tube per 48 reactions)
} A-Tailing Mix (ATL) (1 tube per 48 reactions)
} Resuspension Buffer (RSB)
User-Supplied Consumables
} Microseal ‘B’ Adhesive Seal
} RNase/DNase-free Reagent Reservoirs
} RNase/DNase-free Strip Tubes and Caps
Preparation
} Remove the following from -15° to -25°C storage and thaw them at room
temperature:
• A-Tailing Control or Resuspension Buffer
• A-Tailing Mix
NOTE
The use of the A-Tailing Control is optional and it can be replaced with the
same volume of Resuspension Buffer.
} Remove the ALP plate from -15° to -25°C storage, if it was stored at the conclusion
of Perform End Repair on page 65 and let stand to thaw at room temperature.
• Briefly centrifuge the thawed ALP plate to 280 xg for 1 minute
• Remove the adhesive seal from the ALP plate.
} Pre-heat the microheating system to 37°C.
TruSeq DNA Sample Preparation Guide
69
Adenylate 3' Ends
Adenylate 3' Ends
High-Throughput (HT) Protocol
Add ATL
1
Do one of the following:
• If using the in-line control reagent, add 2.5 µl of thawed A-Tailing Control to
each well of the ALP plate.
• If not using the in-line control reagent, add 2.5 µl of Resuspension Buffer to
each well of the ALP plate.
2
Add 12.5 µl of thawed A-Tailing Mix to each well of the ALP plate. Mix thoroughly
as follows:
a Seal the ALP plate with a Microseal ‘B’ adhesive seal.
b Shake the ALP plate on a microplate shaker at 1,800 rpm for 2 minutes.
c Centrifuge the ALP plate to 280 xg for 1 minute.
Incubate 1 ALP
70
1
Incubate the ALP plate on the pre-heated microheating system, with the lid closed,
for 30 minutes at 37°C.
2
Immediately remove the ALP plate from the microheating system, then proceed
immediately to Ligate Adapters on page 71.
Part # 15005180 Rev. C
This process ligates multiple indexing adapters to the ends of the DNA fragments,
preparing them for hybridization onto a flow cell.
Illumina-Supplied Consumables
} Ligation Mix (LIG) (1 tube per 48 reactions)
} DNA Adapter Indexes 1–12 (AD001–AD012)
(1 tube per column of 8 reactions, depending on the DNA Adapter Indexes being
used)
} (Optional) Ligase Control (CTL) (1 tube per 48 reactions)
} Resuspension Buffer (RSB)
} Stop Ligation Buffer (STL) (1 tube per 48 reactions)
} CAP (Clean Up ALP Plate) barcode label
} PCR (Polymerase Chain Reaction) barcode label (for gel-free method only)
} SSP (Size Separate Plate) barcode label (for gel method only)
User-Supplied Consumables
}
}
}
}
}
}
}
96-well MIDI plate
96-well HSP plate
AMPure XP Beads
Freshly Prepared 80% Ethanol (EtOH)
Microseal ‘B’ Adhesive Seals
RNase/DNase-free Reagent Reservoirs
RNase/DNase-free Strip Tubes and Caps
Preparation
} Remove the following from -15° to -25°C storage and thaw them at room
temperature:
• Appropriate DNA Adapter Index tubes (AD001–AD012, depending on the DNA
Adapter Indexes being used)
• Stop Ligation Buffer
• Ligase Control or Resuspension Buffer
TruSeq DNA Sample Preparation Guide
71
Ligate Adapters
Ligate Adapters
High-Throughput (HT) Protocol
NOTE
The use of the Ligase Control is optional and it can be replaced with the
same volume of Resuspension Buffer.
} Review AMPure XP Handling on page 10.
} Remove the AMPure XP beads from storage and let stand for at least 30 minutes to
bring them to room temperature.
} Pre-heat the microheating system to 30°C.
} Apply a CAP barcode label to a new 96-well MIDI plate.
} Do one of the following:
• If using the gel-free method for enrichment, apply a PCR barcode label to a new
96-well HSP plate.
• If using the gel method, apply a SSP barcode label to a new 96-well HSP plate.
NOTE
When indexing libraries, Illumina recommends arranging samples that will
be combined into a common pool in the same row. Each column should
contain a common index. This will facilitate pipetting operations when
dispensing indexed adapters and pooling indexed libraries later in the
protocol.
Add LIG
72
1
Briefly centrifuge the thawed DNA Adapter Index tubes (AD001–AD012 depending
on the DNA Adapter Indexes being used), Ligase Control (if using Ligase Control),
and Stop Ligation Buffer tubes to 600 xg for 5 seconds.
2
Immediately before use, remove the Ligation Mix tube from -15° to -25°C storage.
3
Remove the adhesive seal from the ALP plate.
4
Do one of the following:
• If using the in-line control reagent, add 2.5 µl of thawed Ligase Control to each
well of the ALP plate.
• If not using the in-line control reagent, add 2.5 µl of Resuspension Buffer to
each well of the ALP plate.
5
Add 2.5 µl of Ligation Mix to each well of the ALP plate.
6
Return the Ligation Mix tube back to -15° to -25°C storage immediately after use.
Part # 15005180 Rev. C
Add 2.5 µl of the appropriate/desired thawed DNA Adapter Index (AD001–AD012
depending on the DNA Adapter Indexes being used) to each well of the ALP plate.
Mix thoroughly as follows:
a Seal the ALP plate with a Microseal ‘B’ adhesive seal.
b Shake the ALP plate on a microplate shaker at 1,800 rpm for 2 minutes.
c Centrifuge the ALP plate to 280 xg for 1 minute.
Incubate 2 ALP
1
Incubate the ALP plate on the pre-heated microheating system, with the lid closed,
at 30°C for 10 minutes.
2
Remove the ALP plate from the microheating system.
1
Remove the adhesive seal from the ALP plate.
2
Add 5 µl of Stop Ligation Buffer to each well of the ALP plate to inactivate the
ligation mix. Mix thoroughly as follows:
a Seal the ALP plate with a Microseal ‘B’ adhesive seal.
b Shake the ALP plate on a microplate shaker at 1,800 rpm for 2 minutes.
c Centrifuge the ALP plate to 280 xg for 1 minute.
Add STL
Clean Up ALP
NOTE
Before performing clean up, review AMPure XP Handling on page 10 when
working with AMPure XP Beads.
1
Remove the adhesive seal from the ALP plate.
2
Vortex the AMPure XP Beads until they are well dispersed, then add 42.5 µl of
mixed AMPure XP Beads to each well of the ALP plate. Mix thoroughly as follows:
a Seal the ALP plate with a Microseal ‘B’ adhesive seal.
b Shake the ALP plate on a microplate shaker at 1,800 rpm for 2 minutes.
3
Incubate the ALP plate at room temperature for 15 minutes.
TruSeq DNA Sample Preparation Guide
73
Ligate Adapters
7
High-Throughput (HT) Protocol
4
Place the ALP plate on the magnetic stand at room temperature for 5 minutes or
until the liquid appears clear.
5
Remove the adhesive seal from the ALP plate.
6
Remove and discard 80 µl of the supernatant from each well of the ALP plate.
Change the tip after each sample.
NOTE
Leave the ALP plate on the magnetic stand while performing the following
80% EtOH wash steps (7–9).
7
With the ALP plate remaining on the magnetic stand, add 200 µl of freshly
prepared 80% EtOH to each well without disturbing the beads.
8
Incubate the ALP plate at room temperature for 30 seconds, then remove and
discard all of the supernatant from each well. Change the tip after each sample.
9
Repeat steps 7 and 8 once for a total of two 80% EtOH washes.
10 While keeping the ALP plate on the magnetic stand, let the samples air dry at room
temperature for 15 minutes.
11 Resuspend the dried pellet in each well with 52.5 µl Resuspension Buffer. Mix
thoroughly as follows:
a Seal the ALP plate with a Microseal ‘B’ adhesive seal.
b Shake the ALP plate on a microplate shaker at 1,800 rpm for 2 minutes.
12 Incubate the ALP plate at room temperature for 2 minutes.
13 Place the ALP plate on the magnetic stand at room temperature for 5 minutes or
until the liquid appears clear.
14 Remove the adhesive seal from the ALP plate.
15 Transfer 50 µl of the clear supernatant from each well of the ALP plate to the
corresponding well of the new MIDI plate labeled with the CAP barcode. Change
the tip after each sample.
16 Vortex the AMPure XP Beads until they are well dispersed, then add 50 µl of mixed
AMPure XP Beads to each well of the CAP plate. Mix thoroughly as follows:
a Seal the CAP plate with a Microseal ‘B’ adhesive seal.
b Shake the CAP plate on a microplate shaker at 1,800 rpm for 2 minutes.
74
Part # 15005180 Rev. C
18 Place the CAP plate on the magnetic stand at room temperature for 5 minutes or
until the liquid appears clear.
19 Remove the adhesive seal from the CAP plate.
20 Remove and discard 95 µl of the supernatant from each well of the CAP plate. Take
care not to disturb the beads. Change the tip after each sample.
NOTE
Leave the CAP plate on the magnetic stand while performing the following
80% EtOH wash steps (21–23)
21 With the CAP plate remaining on the magnetic stand, add 200 µl of freshly
prepared 80% EtOH to each well without disturbing the beads.
22 Incubate the CAP plate at room temperature for 30 seconds, then remove and
discard all of the supernatant from each well. Take care not to disturb the beads.
Change the tip after each sample.
23 Repeat steps 21 and 22 once for a total of two 80% EtOH washes.
24 While keeping the CAP plate on the magnetic stand, let the samples air dry at room
temperature for 15 minutes.
25 Resuspend the dried pellet in each well with 22.5 µl Resuspension Buffer. Mix
thoroughly as follows:
a Seal the CAP plate with a Microseal ‘B’ adhesive seal.
b Shake the CAP plate on a microplate shaker at 1,800 rpm for 2 minutes.
26 Incubate the CAP plate at room temperature for 2 minutes.
27 Place the CAP plate on the magnetic stand at room temperature for 5 minutes or
until the liquid appears clear.
28 Remove the adhesive seal from the CAP plate.
29 Do one of the following:
• If using the gel-free method for enrichment:
— Transfer 20 µl of the clear supernatant from each well of the CAP plate to
the corresponding well of the new HSP plate labeled with the PCR barcode.
Change the tip after each sample.
TruSeq DNA Sample Preparation Guide
75
Ligate Adapters
17 Incubate the CAP plate at room temperature for 15 minutes.
High-Throughput (HT) Protocol
— Proceed to Enrich DNA Fragments on page 81.
• If using the gel method:
— Transfer 20 µl of the clear supernatant from each well of the CAP plate to
the corresponding well of the new HSP plate labeled with the SSP barcode.
Change the tip after each sample.
— Proceed to Purify Ligation Products (gel method only) on page 77.
SAFE STOPPING POINT
If you do not plan to proceed to Enrich DNA Fragments on page 81 or
Purify Ligation Products (gel method only) on page 77 immediately, the
protocol can be safely stopped here. If you are stopping, seal the PCR or
SSP plate with a Microseal ‘B’ adhesive seal and store at -15° to -25°C for
up to seven days.
76
Part # 15005180 Rev. C
This process is only performed when using the gel method. If you are running the gelfree method for enrichment, proceed to Enrich DNA Fragments on page 81.
This process purifies the products of the ligation reaction on a gel and removes
unligated adapters, as well as any adapters that may have ligated to one another, and
selects a size-range of sequencing library appropriate for cluster generation.
Illumina suggests the following gel insert size targets and slice locations. The gel slice
locations account for the length of the adapter sequences flanking the inserts. For other
applications, other size ranges may be desired and the cut size adjusted accordingly.
Table 15 Size Selection Options
Insert Size Target
3 mm Slice Location
Whole-genome
Resequencing
300–400 bpa
400–500 bp
TruSeq Enrichment
200–300 bp
300–400 bp
a. +/- 1 standard deviation of 20 bp, i.e, a < 20% variance for read lengths of 2 × 75 bp or shorter
Illumina-Supplied Consumables
} PCR (Polymerase Chain Reaction Plate) barcode label
} Resuspension Buffer (RSB) (1 tube)
User-Supplied Consumables
}
}
}
}
}
}
}
}
}
50 X TAE Buffer
96-well HSP plate
BenchTop 100 bp DNA Ladder
Clean Scalpels
Certified Low-range Ultra Agarose
Distilled Water
6X Gel Loading Dye
MinElute Gel Extraction Kit
SyBr Gold Nucleic Acid Gel Stain
TruSeq DNA Sample Preparation Guide
77
Purify Ligation Products (gel method only)
Purify Ligation Products (gel method only)
High-Throughput (HT) Protocol
NOTE
Illumina strongly recommends using the user-supplied consumables
specified. Any deviation from these materials may result in incorrect sizeexcision or require additional user optimization
Preparation
} Prepare 1X TAE buffer (> 1 L)
} Apply a PCR barcode label to a new 96-well HSP plate.
} Remove the SSP plate from -15° to -25°C storage, if it was stored at the conclusion of
Ligate Adapters on page 71 and let stand to thaw at room temperature.
• Briefly centrifuge the thawed SSP plate to 280 xg for 1 minute.
• Remove the adhesive seal from the thawed SSP plate.
} Clean the tray, the comb, and the gel tank with ethanol and rinse them thoroughly
with deionized water to avoid cross contamination.
Size Separate SSP
1
Prepare a 150 ml, 2% agarose with SyBr Gold gel using 1 X TAE Buffer as follows:
a Add 3 g of agarose powder in 150 ml of 1X TAE buffer.
b Microwave the gel buffer until the agarose powder is completely dissolved.
c Cool the gel buffer on the bench for 5 minutes, and then add 15 µl of SyBr Gold.
Swirl to mix.
d Pour the entire gel buffer to the gel tray.
NOTE
The final concentration of SyBr Gold should be 1X in the agarose gel buffer.
WARNING
It is very important to pre-stain your gel with SyBr Gold. When using other
staining dyes or staining the gel after running, the DNA will migrate more
slowly than the ladder. This will result in cutting out the wrong size
fragments.
78
2
Remove the adhesive seal from the thawed SSP plate.
3
Add 4 µl of 6X Gel Loading Dye to each well of the SSP plate.
Part # 15005180 Rev. C
Add 17 µl Resuspension Buffer and 4 µl of 6X Gel Loading Dye to 3 µl of DNA
ladder.
WARNING
Do not to overload the DNA ladder. Without clear and distinct bands, it is
difficult to excise the correct fragment size. Also, an overloaded ladder may
run faster than the DNA sample library.
5
When the agarose gel is set, put it in the gel electrophoresis unit and fill the tank
with 1X TAE Buffer to the maximum fill mark.
Dimensions recommended for the electrophoresis unit;
12 cm x 14 cm (W x L), 800 ml buffer volume
6
Load all of the ladder solution onto one lane of the gel.
7
Load the samples from each well of the SSP plate onto the other lanes of the gel,
leaving a gap of at least one empty lane between samples and ladders.
NOTE
Flanking the library on both sides with ladders may make the library
excision easier.
NOTE
When handling multiple samples, to avoid the risk of cross-contamination
between libraries, leave a gap of at least one empty lane between samples
and use ladders on the first and last well of the gel to help locate the gel area
to be excised.
8
Run the gel at 120 V constant voltage for 120 minutes.
9
View the gel on a Dark Reader transilluminator.
10 Do one of the following:
• For whole-genome resequencing, excise a band from the gel spanning the width
of the lane and ranging in size from 400-500 bp using a clean scalpel. Use the
DNA ladder as a guide.
• For enrichment, excise a band from the gel spanning the width of the lane and
ranging in size from 300-400 bp using a clean scalpel. Use the DNA ladder as a
guide. For more information, see the TruSeq Exome Enrichment Guide.
TruSeq DNA Sample Preparation Guide
79
Purify Ligation Products (gel method only)
4
High-Throughput (HT) Protocol
NOTE
Cutting a band between 400–500 bp will result in an insert size of
approximately 300–400 bp, accounting for the size of the adapters. Adapters
add approximately 120 bp to each fragment. The sequencing read length
should be considered when cutting fragment sizes. Sequencing reads that
over-reach into the adapter will cause chimeric reads, unalignable to the
reference sequence.
NOTE
Use a clean scalpel per sample to avoid sample cross-contamination.
Size Separate Gel
1
Follow the instructions in the MinElute Gel Extraction Kit to purify each sample.
Incubate the gel slices in the QG solution at room temperature (not at 50°C as
instructed) until the gel slices have completely dissolved, while vortexing every 2
minutes.
2
Follow the instructions in the MinElute Gel Extraction Kit to purify on one
MinElute spin column, eluting in 25 µl of QIAGEN EB.
3
Transfer 20 µl of each sample from the MinElute collection tube to the new HSP
plate labeled with the PCR barcode using a single channel pipette.
SAFE STOPPING POINT
If you do not plan to proceed to Enrich DNA Fragments on page 81
immediately, the protocol can be safely stopped here. If you are
stopping, seal the PCR plate with a Microseal ‘B’ adhesive seal and store
at -15° to -25°C for up to seven days.
80
Part # 15005180 Rev. C
This process uses PCR to selectively enrich those DNA fragments that have adapter
molecules on both ends and to amplify the amount of DNA in the library. The PCR is
performed with a PCR primer cocktail that anneals to the ends of the adapters. The
number of PCR cycles should be minimized to avoid skewing the representation of the
library.
NOTE
PCR enriches for fragments that have adapters ligated on both ends.
Fragments with only one or no adapters on their ends are by-products of
inefficiencies in the ligation reaction. Neither species can be used to make
clusters, as fragments without any adapters cannot hybridize to surfacebound primers in the flow cell, and fragments with an adapter on only one
end can hybridize to surface bound primers but cannot form clusters.
Illumina-Supplied Consumables
}
}
}
}
}
PCR Master Mix (PMM) (1 tube per 48 reactions)
PCR Primer Cocktail (PPC) (1 tube per 48 reactions)
Resuspension Buffer (RSB)
CPP (Clean Up PCR Plate) barcode label
TSP1 (Target Sample Plate) barcode label
User-Supplied Consumables
}
}
}
}
}
}
}
}
96-well MIDI plate
96-well HSP plate
AMPure XP Beads
Freshly Prepared 80% Ethanol (EtOH)
Microseal ‘A’ Film
Microseal ‘B’ Adhesive Seals
RNase/DNase-free Reagent Reservoirs
RNase/DNase-free Strip Tubes and Caps
Preparation
} Remove one tube each of PCR Master Mix and PCR Primer Cocktail from -15° to
-25°C storage and thaw them at room temperature. Once thawed, keep the tubes on
TruSeq DNA Sample Preparation Guide
81
Enrich DNA Fragments
Enrich DNA Fragments
High-Throughput (HT) Protocol
ice.
} Briefly centrifuge the thawed PCR Master Mix and PCR Primer Cocktail tubes to
600 xg for 5 seconds.
} Review AMPure XP Handling on page 10.
} Remove the AMPure XP beads from storage and let stand for at least 30 minutes to
bring them to room temperature.
} Remove the PCR plate from -15° to -25°C storage, if it was stored at the conclusion
of Ligate Adapters on page 71 or Purify Ligation Products (gel method only) on page 77
and let stand to thaw at room temperature.
• Briefly centrifuge the thawed PCR plate to 280 xg for 1 minute.
• Remove the adhesive seal from the thawed PCR plate.
} Pre-program the thermal cycler as follows:
• 98°C for 30 seconds
• 10 cycles of:
— 98°C for 10 seconds
— 60°C for 30 seconds
— 72°C for 30 seconds
• 72°C for 5 minutes
• Hold at 4°C
NOTE
Illumina recommends 10 cycles of PCR for robust protocol performance.
However, to optimize yield versus cycle number, a titration of PCR cycles
may also be performed.
} Pre-heat the thermal cycler lid to 100°C.
} Apply a CPP barcode label to a new 96-well MIDI plate.
} Apply a TSP1 barcode label to a new 96-well HSP plate.
Make PCR
The following procedure assumes 1 µg of input DNA to library preparation and is
designed to result in high library yields.
1
82
Add 5 µl of thawed PCR Primer Cocktail to each well of the PCR plate. Change the
tip after each sample.
Part # 15005180 Rev. C
Add 25 µl of thawed PCR Master Mix to each well of the PCR plate. Change the tip
after each sample. Mix thoroughly as follows:
a Seal the PCR plate with a Microseal ‘A’ film.
b Shake the PCR plate on a microplate shaker at 1,600 rpm for 20 seconds.
c Centrifuge the PCR plate to 280 xg for 1 minute.
WARNING
Using a Microseal "B" adhesive seal for PCR may cause droplets of the
sample to splash onto the film when peeling the film off the plate.
Amp PCR
1
Amplify the PCR plate in the pre-programmed thermal cycler, with the lid closed,
as follows:
a 98°C for 30 seconds
b 10 cycles of:
98°C for 10 seconds
60°C for 30 seconds
72°C for 30 seconds
c 72°C for 5 minutes
d Hold at 4°C
Clean Up PCR
NOTE
Before performing clean up, review AMPure XP Handling on page 10 when
working with AMPure XP Beads.
1
Remove the adhesive seal from the PCR plate.
2
Vortex the AMPure XP Beads until they are well dispersed, then add 50 µl of the
mixed AMPure XP Beads to each well of the new MIDI plate labeled with the CPP
barcode. Change the tip after each sample.
3
Transfer the entire contents from each well of the PCR plate to the corresponding
well of the CPP plate containing 50 µl of mixed AMPure XP Beads. Mix thoroughly
as follows:
a Seal the CPP plate with a Microseal ‘B’ adhesive seal.
TruSeq DNA Sample Preparation Guide
83
Enrich DNA Fragments
2
High-Throughput (HT) Protocol
b
Shake the CPP plate on a microplate shaker at 1,800 rpm for 2 minutes.
4
Incubate the CPP plate at room temperature for 15 minutes.
5
Place the CPP plate on the magnetic stand at room temperature for 5 minutes or
until the liquid appears clear.
6
Remove the adhesive seal from the CPP plate.
7
Remove and discard 95 µl of the supernatant from each well of the CPP plate. Take
care not to disturb the beads. Change the tip after each sample.
NOTE
Leave the CPP plate on the magnetic stand while performing the following
80% EtOH wash steps (8–10).
8
With the CPP plate remaining on the magnetic stand, add 200 µl of freshly
prepared 80% EtOH to each well without disturbing the beads.
9
Incubate the CPP plate at room temperature for 30 seconds, then remove and
discard all of the supernatant from each well. Take care not to disturb the beads.
Change the tip after each sample.
10 Repeat steps 8 and 9 once for a total of two 80% EtOH washes.
11 While keeping the CPP plate on the magnetic stand, let the samples air dry at room
temperature for 15 minutes.
12 Resuspend the dried pellet in each well with 32.5 µl Resuspension Buffer. Mix
thoroughly as follows:
a Seal the CPP plate with a Microseal ‘B’ adhesive seal.
b Shake the CPP plate on a microplate shaker at 1,800 rpm for 2 minutes.
13 Incubate the CPP plate at room temperature for 2 minutes.
14 Place the CPP plate on the magnetic stand at room temperature for 5 minutes or
until the liquid appears clear.
15 Remove the adhesive seal from the CPP plate.
16 Transfer 30 µl of the clear supernatant from each well of the CPP plate to the
corresponding well of the new HSP plate labeled with the TSP1 barcode. Change the
tip after each sample.
84
Part # 15005180 Rev. C
SAFE STOPPING POINT
If you do not plan to proceed to Validate Library on page 86 or TruSeq
Enrichment immediately, the protocol can be safely stopped here.If you
are stopping, seal the TSP1 plate with a Microseal ‘B’ adhesive seal and
store at -15° to -25°C for up to seven days.
TruSeq DNA Sample Preparation Guide
85
Enrich DNA Fragments
17 Do one of the following:
• If performing whole-genome resequencing, proceed to Validate Library on page
86.
• If performing enrichment, proceed to the TruSeq Enrichment Guide for
instructions on how to quantify and qualify your library.
High-Throughput (HT) Protocol
Validate Library
Illumina recommends performing the following procedures for quality control analysis
on your whole-genome resequencing sample library and quantification of the DNA
library templates. If performing enrichment, proceed directly to the TruSeq Enrichment
Guide for instructions on how to quantify and qualify your library.
Quantify Libraries
In order to achieve the highest quality of data on Illumina sequencing platforms, it is
important to create optimum cluster densities across every lane of every flow cell. This
requires accurate quantitation of DNA library templates. Quantify your libraries using
qPCR according to the Illumina Sequencing Library qPCR Quantification Guide.
Quality Control (Optional)
To verify the size of your PCR enriched fragments, check the template size distribution
by running an aliquot of the DNA library on a gel or on a Agilent Technologies 2100
Bioanalyzer using a High Sensitivity DNA chip.
} If validating by gel, load 10% of the volume of the library on a gel and check that
the size range is as expected: a narrow smear similar in size to the DNA excised
from the gel after the ligation.
} If using the Agilent Bioanalyzer with a high sensitivity DNA chip, make a 1:50
dilution of the library using water and load 1 µl of the diluted library on the Agilent
High Sensitivity DNA chip. Running samples on a Bioanalyzer should be used for
qualitative purposes only.
Figure 9 Example of DNA Library Size Distribution for Whole-Genome Resequencing
86
Part # 15005180 Rev. C
Validate Library
Figure 10 DNA PCR Product
NOTE
If the DNA is not a narrow smear, but is comprised of a long smear of
several hundred base pairs, or contains an intense 126 bp fragment (adapterdimer), then another purification step is recommended. Repeat Purify
Ligation Products (gel method only) on page 77.
TruSeq DNA Sample Preparation Guide
87
High-Throughput (HT) Protocol
Normalize and Pool Libraries
This process describes how to prepare DNA templates that will be applied to cluster
generation. Multiplexed DNA libraries are normalized to 10 nM in the DCT plate and
then pooled in equal volumes in the PDP plate. Non-multiplexed DNA libraries are
normalized to 10 nM in the DCT plate without pooling.
Illumina-Supplied Consumables
} DCT (Diluted Cluster Template) barcode label
} PDP (Pooled DCT Plate) barcode label (for multiplexing only)
User-Supplied Consumables
}
}
}
}
96-well HSP plate (for multiplexing only)
96-well MIDI plate
Microseal ‘B’ Adhesive seals
Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20
Preparation
} Apply a DCT barcode label to a new 96-well MIDI plate.
} Apply a PDP barcode label to a new 96-well HSP plate (for multiplexing only).
} Remove the TSP1 plate from -15° to -25°C storage, if it was stored at the conclusion
of Enrich DNA Fragments on page 81, and let stand to thaw at room temperature.
• Briefly centrifuge the thawed TSP1 plate to 280 xg for 1 minute.
• Remove the adhesive seal from the thawed TSP1 plate.
Make DCT
88
1
Transfer 10 µl of sample library from each well of the TSP1 plate to the
corresponding well of the new MIDI plate labeled with the DCT barcode. Change
the tip after each sample.
2
Normalize the concentration of sample library in each well of DCT plate to 10 nM
using Tris-Cl 10 mM, pH 8.5 with 0.1% Tween 20.
Part # 15005180 Rev. C
3
Mix the DCT plate as follows:
a Seal the DCT plate with a Microseal ‘B’ adhesive seal.
b Shake the DCT plate on a microplate shaker at 1,000 rpm for 2 minutes.
c Centrifuge the DCT plate to 280 xg for 1 minute.
d Remove the adhesive seal from the DCT plate.
4
Depending on the type of library you want to generate, do one of the following:
• For non-multiplexed paired-end libraries, the protocol stops here. Do one of the
following:
— Proceed to cluster generation. See the Illumina Cluster Generation User Guide.
— Seal the DCT plate with a Microseal ‘B’ adhesive seal and store at -15° to 25°C.
• For multiplexed paired-end libraries, proceed to Make PDP (for multiplexing only)
on page 89.
Make PDP (for multiplexing only)
NOTE
Do not make a PDP plate if there is no pooling.
1
Determine the number of samples to be combined together for each pool.
2
Transfer 10 µl of each normalized sample library to be pooled from the DCT plate
to one well of the new HSP plate labeled with the PDP barcode.
The total volume in each well of the PDP plate should be 10X the number of
combined sample libraries and will be 10–120 µl (1–12 libraries).
Table 16 Pooled Sample Volumes
Number of pooled
samples
Volume
(µl)
1
10
TruSeq DNA Sample Preparation Guide
89
Normalize and Pool Libraries
NOTE
Depending on the yield quantification data of each sample library, the final
volume in the DCT plate may vary from 10-400 µl.
High-Throughput (HT) Protocol
Number of pooled
samples
Volume
(µl)
2
20
3
30
4
40
5
50
6
60
7
70
8
80
9
90
10
100
11
110
12
120
NOTE
Keep track of which sample goes into which well, to avoid pooling two
samples with the same index.
90
3
Mix the PDP plate as follows:
a Seal the PDP plate with a Microseal ‘B’ adhesive seal.
b Shake the PDP plate on a microplate shaker at 1,800 rpm for 2 minutes.
4
Do one of the following:
• Proceed to cluster generation. See the Illumina Cluster Generation User Guide.
• Seal the PDP plate with a Microseal ‘B’ adhesive seal and store at -15° to -25°C.
Part # 15005180 Rev. C
Appendix A Alternate Fragmentation Protocols
Introduction
TruSeq DNA Sample Preparation Guide
92
91
Appendix A
Alternate Fragmentation
Protocols
Alternate Fragmentation Protocols
Introduction
An alternative fragmentation method for TruSeq DNA Sample Preparation to the
procedures described in Fragment DNA on page 32 for the LT protocol or Fragment DNA
on page 62 for the HT protocol is using a nebulization technique, which breaks up DNA
into pieces less than 800 bp in minutes using a disposable device. This process
generates double-stranded DNA fragments containing 3' or 5' overhangs.
NOTE
These nebulization procedures have only been validated for whole-genome
resequencing or enrichment with the gel-method.
Illumina-Supplied Consumables
} IMP (Insert Modification Plate) barcode label
User-Supplied Consumables
} 96-well 0.3 ml PCR plate (for LT protocol), or
} 96-well MIDI plate (for HT protocol)
} The following consumables are provided in the Paired-End Sample Preparation Kit:
• Nebulizers (box of 10 nebulizers and vinyl accessory tubes)
• Nebulization Buffer (7 ml)
• TE Buffer
} QIAquick PCR Purification Kit
} Purified DNA (0.1–2 µg, 2 µg recommended)
DNA should be as intact as possible, with an OD260/280 ratio of 1.8–2.0
} Compressed Air of at least 32 psi
Do not use CO which could alter the pH of the nebulizer buffer
2
} PVC tubing
Dimensions: 1/4 inch ID, 3/8 inch OD, 1/16 inch wall, 1 meter length
Procedure
The DNA sample to be processed should be highly pure, having an
OD260/280 ratio of between 1.8 and 2.0, and should be as intact as possible.
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Part # 15005180 Rev. C
1
Remove a nebulizer from the plastic packaging and unscrew the blue lid.
Figure 11 Remove the Nebulizer Lid
2
Using gloves, remove a piece of vinyl tubing from the packaging and slip it over
the central atomizer tube. Push it all the way to the inner surface of the blue lid.
Figure 12 Assemble the Nebulizer
A
B
C
Blue Lid
Atomizer
Vinyl Tubing
3
Add 0.1–2 µg of Purified DNA in a total volume of 50 µl of TE Buffer to the
nebulizer.
4
Add 700 µl Nebulization Buffer to the DNA and mix well.
5
Screw the lid back on (finger-tight).
TruSeq DNA Sample Preparation Guide
93
Introduction
NOTE
If you are not familiar with this shearing method, Illumina recommends that
you test this procedure on test samples and practice assembling the
nebulizer before proceeding with your sample DNA.
Alternate Fragmentation Protocols
Figure 13 Replace the Nebulizer Lid
6
Chill the nebulizer containing the DNA solution on ice while performing the next
step.
7
Connect the compressed air source to the inlet port on the top of the nebulizer with
the PVC tubing, ensuring a tight fit.
Figure 14 Connect Compressed Air
8
Bury the nebulizer in an ice bucket and place it in a fume hood.
9
Use the regulator on the compressed air source to ensure the air is delivered at
32-35 psi.
10 Nebulize for 6 minutes. You may notice vapor rising from the nebulizer; this is
normal. Also, the Nebulization Buffer may turn white or appear frozen.
11 Centrifuge the nebulizer at 450 xg for 2 minutes to collect the droplets from the side
of the nebulizer. If necessary, use an old nebulizer as a counter-balance.
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Part # 15005180 Rev. C
13 Measure the recovered volume. Typically, you should recover 400–600 µl.
14 Follow the instructions in the QIAquick PCR Purification Kit to purify the sample
solution and concentrate it on one QIAquick column, eluting in 50 µl of QIAGEN
EB.
15 Transfer all of the 50 µl of fragmented DNA to each well of the new plate labeled
with the IMP barcode using a single channel pipette.
16 Do one of the following:
• For LT processing, proceed to Perform End Repair on page 35.
• For HT processing, proceed to Perform End Repair on page 65.
SAFE STOPPING POINT
If you do not plan to proceed to Perform End Repair immediately, the
protocol can be safely stopped here. If you are stopping, store the
samples at -15° to -25°C overnight or longer. When proceeding, thaw
the samples on ice.
TruSeq DNA Sample Preparation Guide
95
Introduction
12 If a centrifuge is not available, then use 2 ml of the binding buffer (PB or PBI buffer)
from the QIAquick PCR Purification Kit to rinse the sides of the nebulizer and
collect the DNA solution at the base of the nebulizer.
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Part # 15005180 Rev. C
Index
Index
5
50 X TAE Buffer
46, 77
6
6X gel loading dye
46, 77
A
Covaris instrument
Covaris shearing
Covaris tubes
cross-contamination
CTA
CTE
CTL
customer support
Acronyms
7
Add ATL
40, 70
Add LIG
42, 72
Add STL
43, 73
Agilent Bioanalyzer
9
ALP
35, 65
Amp PCR
52, 83
AMPure XP beads 35, 41, 50, 65, 71,
81
AMPure XP handling
10
ATL
39, 69
D
B
ERP
EUC
BenchTop 100 bp DNA ladder
46,
77
C
CAP
41, 71
certified low-range ultra agarose 46,
77
CFP
32, 62
Clean Up ALP
73
Clean Up IMP
36, 66
Clean Up PCR
52, 83
cluster generation
2, 58, 90
contamination
11
TruSeq DNA Sample Preparation Guide
DCT
DNA Adapter Indexes
DNA PCR product
DNA Plate (DNA)
DNA sequencing
documentation
dsDNA
33, 63
3, 32, 62
32, 62
11
39, 69
35, 65
41, 71
99
56, 88
41, 71
54, 86
32, 62
2
99
9, 92
E
35, 65
17
F
Fragment DNA
fragmentation
33, 63
92
G
gDNA
gel-free method
gel insert size
gel method
2
2, 37, 67
3, 32, 62
3, 37, 67
97
Index
H
help, technical
High Throughput (HT)
HSP
99
3
3
9
50, 81
57, 89
50, 81
92
I
Q
IMP
32, 36, 62, 66, 92
in-line control DNA
15
Incubate 1 ALP
40, 70
Incubate 1 IMP
36, 66
QIAquick PCR Purification Kit
92
qPCR
9
quality control
54, 86
quantify libraries
54, 86
quantitation
13
quantity and quality
13
L
Lab Tracking Form (LTF)
LIG
liquid handling
Low Throughput (LT)
17
41, 71
9
3
R
33, 63
56, 88
36, 66
51, 82
57, 89
2
3
3
3
46, 77
S
M
Make CFP
Make DCT
Make IMP
Make PCR
Make PDP
master-mixed reagents
micro plate shaker
microheating system
MIDI
MinElute Gel Extraction Kit
N
nebulization
nebulizer
normalize gDNA
92
92
33, 63
P
paired-end
PCR
PCR grade water
PDP
98
PicoGreen
PMM
pooled sample volumes
PPC
purified DNA
2
2, 41, 46, 71, 77
35, 65
56, 88
Reagent reservoirs 35, 39, 41, 50, 65,
69, 71, 81
RSB
32, 35, 39, 41, 46, 62, 65, 69,
71, 77, 81
sample sheet
shear gDNA
shearing
single read
Size Separate Gel
Size Separate SSP
SPRI
SSP
STL
Strip tubes and caps
SyBr Gold gel stain
17
33, 63
2
2
49, 80
47, 78
2
41, 71
41, 71
35, 39, 41, 50,
65, 69, 71, 81
46, 77
T
TE buffer
92
technical assistance
99
temperature
12
thermal cycler
3
Tris-Cl
56, 88
TruSeq Enrichment 3, 32, 46, 62, 77
Part # 15005180 Rev. C
Index
TSP1
50, 56, 81, 88
W
whole-genome resequencing 3, 32, 46,
62, 77
workflow diagram
31, 61
TruSeq DNA Sample Preparation Guide
99
Index
100
Part # 15005180 Rev. C
For technical assistance, contact Illumina Customer Support.
Table 17 Illumina General Contact Information
Illumina Website
Email
http://www.illumina.com
[email protected]
Table 18 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
Contact Number
North America
1.800.809.4566
Italy
800.874909
Austria
0800.296575
Netherlands
0800.0223859
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
MSDSs
Material safety data sheets (MSDSs) are available on the Illumina website at
http://www.illumina.com/msds.
Product Documentation
If you require additional product documentation, you can obtain PDFs from the
Illumina website. Go to http://www.illumina.com/support/documentation.ilmn. When
you click on a link, you will be asked to log in to iCom. After you log in, you can
view or save the PDF. To register for an iCom account, please visit
https://icom.illumina.com/Account/Register.
TruSeq DNA Sample Preparation Guide
101
Technical Assistance
Technical Assistance
Illumina, Inc.
9885 Towne Centre Drive
San Diego, CA 92121-1975
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com
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