TruSeq Library Prep Pooling Guide (15042173 v01)

TruSeq Library Prep Pooling Guide (15042173 v01)
TruSeq Library Prep Pooling
Guide
For Research Use Only. Not for use in diagnostic procedures.
Revision History
Introduction
Index Adapter Sequences
Adapter Options
Pooling Guidelines
Technical Assistance
ILLUMINA PROPRIETARY
Document # 15042173 v01
November 2015
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Date
Document
Description of Change
Document # 15042173
v01
November
2015
Part # 15042173 Rev. D
March
2015
Part # 15042173 Rev. C
December
2014
• TruSeq product names changed from 'sample' prep to 'library'
prep
• Specify Illumina sequencing systems that require balanced index
combinations
• Added reference to BaseSpace in Pooling Preparation with Adapter
Plate
• Updated SDS link to support.illumina.com/sds.html
Part # 15042173 Rev. B
February
2014
• Added TruSeq Small RNA Library Prep Kit adapter sequences
and pooling guidelines
Part # 15042173 Rev. A
May 2013
Initial release.
TruSeq Library Prep Pooling Guide
• Added TruSeq Exome Library Prep Kit adapter sequences and
pooling guidelines
• Added NeoPrep adapter sequences and pooling guidelines
3
Revision History
Revision History
Introduction
Introduction
This document provides Illumina® pooling guidelines for performing TruSeq® library
prep for sequencing on the following systems:
} MiSeq® when running MiSeq Control Software 2.1 or earlier
} HiSeq® when running HiSeq Control Software 2.0.12 or earlier
} HiScanSQ™
} Genome Analyzer™
The document is a supplement to TruSeq library prep guides and assumes that you have
reviewed your kit guide for details on performing the protocol.
NOTE
You can download TruSeq library prep guides from the Illumina website at
www.illumina.com. Go to the support page for the TruSeq kit you are using, then click
Documentation & Literature.
TruSeq Library Prep Pooling Guide
4
Index Adapter Sequences
Index Adapter Sequences
TruSeq Small RNA Library Prep Kit Index Sequences
The TruSeq Small RNA Library Prep Kit contains the following index sequences:
Table 1 Indices A Box - Index Sequences 1–12
Index
RPI1
RPI2
RPI3
RPI4
RPI5
RPI6
Sequence
ATCACG
CGATGT
TTAGGC
TGACCA
ACAGTG
GCCAAT
Index
RPI7
RPI8
RPI9
RPI10
RPI11
RPI12
Sequence
CAGATC
ACTTGA
GATCAG
TAGCTT
GGCTAC
CTTGTA
Index
RPI19
RPI20
RPI21
RPI22
RPI23
RPI24
Sequence
GTGAAA
GTGGCC
GTTTCG
CGTACG
GAGTGG
GGTAGC
Index
RPI31
RPI32
RPI33
RPI34
RPI35
RPI36
Sequence
CACGAT
CACTCA
CAGGCG
CATGGC
CATTTT
CCAACA
Index
RPI43
RPI44
RPI45
RPI46
RPI47
RPI48
Sequence
TACAGC
TATAAT
TCATTC
TCCCGA
TCGAAG
TCGGCA
Table 2 Indices B Box - Index Sequences 13–24
Index
RPI13
RPI14
RPI15
RPI16
RPI17
RPI18
Sequence
AGTCAA
AGTTCC
ATGTCA
CCGTCC
GTAGAG
GTCCGC
Table 3 Indices C Box - Index Sequences 25–36
Index
RPI25
RPI26
RPI27
RPI28
RPI29
RPI30
Sequence
ACTGAT
ATGAGC
ATTCCT
CAAAAG
CAACTA
CACCGG
Table 4 Indices D Box - Index Sequences 37–48
Index
RPI37
RPI38
RPI39
RPI40
RPI41
RPI42
TruSeq Library Prep Pooling Guide
Sequence
CGGAAT
CTAGCT
CTATAC
CTCAGA
GACGAC
TAATCG
5
TruSeq LT or ChIP Kit Index Adapter Tube Sequences
A TruSeq LT or ChIP kit contains the following index adapter sequences.
} The index numbering is not contiguous. There is no Index 17, 24, or 26.
} The sequence contains 7 bases. The seventh base, shown in parenthesis (), is not
included in the Index Read. Record only the first 6 bases in a sample sheet. For
indexes 13 and above, the seventh base (in parentheses) might not be A, which is
seen in cycle 7 of the Index Read.
} For more information on the number of cycles used to sequence the Index Read, see
the system guide for your Illumina sequencing platform.
Table 5 Set A Index Adapter Sequences for DNA LT Kits
Adapter
AD002
AD004
AD005
AD006
AD007
AD012
Sequence
CGATGT(A)
TGACCA(A)
ACAGTG(A)
GCCAAT(A)
CAGATC(A)
CTTGTA(A)
Adapter
AD013
AD014
AD015
AD016
AD018
AD019
Sequence
AGTCAA(C)
AGTTCC(G)
ATGTCA(G)
CCGTCC(C)
GTCCGC(A)
GTGAAA(C)
Table 6 Set B Index Adapter Sequences for DNA LT Kits
Adapter
AD001
AD003
AD008
AD009
AD010
AD011
Sequence
ATCACG(A)
TTAGGC(A)
ACTTGA(A)
GATCAG(A)
TAGCTT(A)
GGCTAC(A)
Adapter
AD020
AD021
AD022
AD023
AD025
AD027
Sequence
GTGGCC(T)
GTTTCG(G)
CGTACG(T)
GAGTGG(A)
ACTGAT(A)
ATTCCT(T)
Table 7 Set A Index Adapter Sequences for RNA LT and ChIP Kits
Adapter
AR002
AR004
AR005
AR006
AR007
AR012
Sequence
CGATGT(A)
TGACCA(A)
ACAGTG(A)
GCCAAT(A)
CAGATC(A)
CTTGTA(A)
Adapter
AR013
AR014
AR015
AR016
AR018
AR019
Sequence
AGTCAA(C)
AGTTCC(G)
ATGTCA(G)
CCGTCC(C)
GTCCGC(A)
GTGAAA(C)
Table 8 Set B Index Adapter Sequences for RNA LT and ChIP Kits
Adapter
AR001
AR003
AR008
AR009
AR010
AR011
6
Sequence
ATCACG(A)
TTAGGC(A)
ACTTGA(A)
GATCAG(A)
TAGCTT(A)
GGCTAC(A)
Adapter
AR020
AR021
AR022
AR023
AR025
AR027
Sequence
GTGGCC(T)
GTTTCG(G)
CGTACG(T)
GAGTGG(A)
ACTGAT(A)
ATTCCT(T)
Document # 15042173 v01
TruSeq Exome Library Prep 8 rxn × 3 plex, 8 rxn × 6 plex, and 8 rxn × 9 plex kits contain
adapters in tubes with the following indexed adapter sequences.
} The sequence contains 7 bases. The seventh base, shown in parenthesis (), is not
included in the Index Read. Record only the first 6 bases in a sample sheet. For
indexes 13 and above, the seventh base (in parentheses) might not be A, which is
seen in the cycle 7 of the Index Read.
} For more information on the number of cycles used to sequence the Index Read, see
the system guide for your Illumina sequencing platform.
Table 9 TruSeq Exome Library Prep Index Adapter Sequences
Adapter
Sequence
AD002
AD004
AD005
AD006
AD007
AD012
AD013
AD014
AD015
AD019
CGATGT(A)
TGACCA(A)
ACAGTG(A)
GCCAAT(A)
CAGATC(A)
CTTGTA(A)
AGTCAA(C)
AGTTCC(G)
ATGTCA(G)
GTGAAA(C)
8×3
Reaction Kit
•
8×6
Reaction Kits
•
•
•
•
•
•
•
8×9
Reaction Kits
•
•
•
•
•
•
•
•
•
•
Index Adapter Plate Sequences
The adapter plate contains the following index adapter sequences. The index adapter
sequence recorded in the sample sheet contains 8 bases, and all 8 bases are sequenced
during the Index Read.
Table 10 Index Adapter 1 Sequences
Adapter
D701
D702
D703
D704
D705
D706
Sequence
ATTACTCG
TCCGGAGA
CGCTCATT
GAGATTCC
ATTCAGAA
GAATTCGT
Adapter
D707
D708
D709
D710
D711
D712
Sequence
CTGAAGCT
TAATGCGC
CGGCTATG
TCCGCGAA
TCTCGCGC
AGCGATAG
Adapter
D505
D506
D507
D508
Sequence
AGGCGAAG
TAATCTTA
CAGGACGT
GTACTGAC
Table 11 Index Adapter 2 Sequences
Adapter
D501
D502
D503
D504
TruSeq Library Prep Pooling Guide
Sequence
TATAGCCT
ATAGAGGC
CCTATCCT
GGCTCTGA
7
Index Adapter Sequences
TruSeq Exome Library Prep Kit Index Adapter Tube Sequences
Figure 1 Adapter Plate Dual-Index Layout
TruSeq Library Prep for NeoPrep Indexed Adapter Sequences
TruSeq library prep kits for NeoPrep contain the following indexed adapter sequences.
} The indexes are TruSeq LT single-index adapters.
} The index sequences have not changed, however the adapters are not the same as
manual TruSeq library prep adapters. Do not use them for manual TruSeq library
prep and do not use the adapters in the manual TruSeq library prep kits for NeoPrep
library prep.
} The index numbering is not contiguous. There is no Index 17, 24, or 26.
} The base in parentheses () indicates the base for the seventh cycle and is not
considered as part of the index sequence. Record the index in the sample sheet as
only 6 bases. For indexes 13 and above, the seventh base (in parentheses) might not
be A, which is seen in the seventh cycle of the Index Read.
} For more information on the number of cycles used to sequence the Index Read,
reference your Illumina sequencing system guide.
Table 12 TruSeq Library Prep for NeoPrep DNA Indexed Adapter Sequences
Adapter
ND001
ND002
ND003
ND004
ND005
ND006
ND007
ND008
ND009
ND010
ND011
ND012
8
Sequence
ATCACG(A)
CGATGT(A)
TTAGGC(A)
TGACCA(A)
ACAGTG(A)
GCCAAT(A)
CAGATC(A)
ACTTGA(A)
GATCAG(A)
TAGCTT(A)
GGCTAC(A)
CTTGTA(A)
Adapter
ND013
ND014
ND015
ND016
ND018
ND019
ND020
ND021
ND022
ND023
ND025
ND027
Sequence
AGTCAA(C)
AGTTCC(G)
ATGTCA(G)
CCGTCC(C)
GTCCGC(A)
GTGAAA(C)
GTGGCC(T)
GTTTCG(G)
CGTACG(T)
GAGTGG(A)
ACTGAT(A)
ATTCCT(T)
Document # 15042173 v01
Index Adapter Sequences
Table 13 TruSeq Library Prep for NeoPrep RNA Indexed Adapter Sequences
Adapter
NR001
NR002
NR003
NR004
NR005
NR006
NR007
NR008
NR009
NR010
NR011
NR012
TruSeq Library Prep Pooling Guide
Sequence
ATCACG(A)
CGATGT(A)
TTAGGC(A)
TGACCA(A)
ACAGTG(A)
GCCAAT(A)
CAGATC(A)
ACTTGA(A)
GATCAG(A)
TAGCTT(A)
GGCTAC(A)
CTTGTA(A)
Adapter
NR013
NR014
NR015
NR016
NR018
NR019
NR020
NR021
NR022
NR023
NR025
NR027
Sequence
AGTCAA(C)
AGTTCC(G)
ATGTCA(G)
CCGTCC(C)
GTCCGC(A)
GTGAAA(C)
GTGGCC(T)
GTTTCG(G)
CGTACG(T)
GAGTGG(A)
ACTGAT(A)
ATTCCT(T)
9
Illumina provides various methods for indexing samples to perform pooled sequencing
depending on the kit that you are using; adapter index tubes, an adapter plate, or a
reagent plate.
Adapter Tubes
Adapter index tubes can be used to perform pooled sequencing.
} Each tube contains a unique single 6 base index adapter on the P7 strand and
contains enough reagent for 18 reactions.
} Libraries prepared with these adapters can be sequenced on the MiSeq® using a
6 cycle Single Index Read and any other Illumina sequencing platform using a
7 cycle Single Index Read.
} Adapter index tubes are provided in the following kits:
} TruSeq Small RNA Library Prep Kit—Contains RNA PCR Primer Index tubes
} TruSeq LT library prep kits—Contain DNA or RNA Adapter Index tubes,
depending on the kit that you are using
} TruSeq Exome Library Prep kits (8x3, 8x6, and 8x9)—Contain DNA Adapter
Index tubes
} TruSeq ChIP Library Prep Kit—Contains RNA Adapter Index tubes
For more information on pooling guidelines when using adapter index tubes, see:
} TruSeq Small RNA Library Prep Kit Pooling Guidelines on page 15
} TruSeq LT Kit or ChIP Kit Adapter Tube Pooling Guidelines on page 16
} TruSeq Exome Library Prep Kit Adapter Tube Pooling Guidelines on page 17
For more information on sequencing libraries prepared using a TruSeq LT kit, see the
system guide for your Illumina sequencing platform.
Adapter Plate
A TruSeq HT or TruSeq Exome Library Prep Kit (8 rxn × 12 plex) contains a DAP or
RAP, depending on the kit that you are using. An adapter plate is a 96-well plate
containing 96 uniquely indexed adapter combinations designed for manual or
automated preparation of 96 uniquely indexed samples.
} Each well is single-use and the plate can undergo up to 4 freeze-thaw cycles.
} The DAP or RAP adapters provided in this plate are dual-indexed, meaning that
each adapter contains 2 indexes. The adapters are referred to as Index 1(i7), an 8
base Index on the P7 strand, and Index 2(i5), an 8 base Index on the P5 strand.
} There are 12 Index 1 sequences (D701–D712) arrayed across the columns and 8
Index 2 sequences (D501–D508) arrayed down the rows, to generate 96 uniquely
dual-indexed adapter combinations in the plate.
} If compatible, libraries prepared with these adapters can be sequenced on an
Illumina sequencing platform using the dual-indexed recipes for dual indexing or
the 8 cycle single-indexed recipe for single indexing.
For more information on pooling guidelines when using the DAP or RAP, see Adapter
Plate Pooling Guidelines on page 17
For more information on sequencing libraries prepared using a TruSeq HT or TruSeq
Exome Library Prep Kit (8 rxn × 12 plex), see the system guide for your Illumina
sequencing platform.
TruSeq Library Prep Pooling Guide
10
Adapter Options
Adapter Options
Table 14 Dual-Indexed Sequencing Platform Compatibility
Platform
Genome Analyzer
HiSeq
MiSeq
Compatibility
Requires TruSeq Dual Index Sequencing
Primer Box, Single Read for dual-indexed
sequencing on a single-read flow cell.a
Requires SCS 2.10/RTA 1.13 or later
Process with OLB 1.9.4 or later if offline base
call is needed
Process with CASAVA 1.8.2 or later
Requires TruSeq Dual Index Sequencing
Primer Box, Single Read for dual-indexed
sequencing on a v3 single-read flow cell.a
Requires HCS 1.5/RTA 1.13 or later
Process with OLB 1.9.3 or later if offline base
call is needed
Process with CASAVA 1.8.2 or later or HAS
(any version)
Full compatibility
a. Not required for sequencing on paired-end flow cells or Rapid 2 lane single-read or
paired-end flow cells.
Pooling Preparation with Adapter Plate
A TruSeq HT or TruSeq Exome Library Prep Kit (8 rxn × 12 plex) contains a DAP or RAP
and enables preparation of up to 96 libraries with unique dual indexes.
Figure 2 DAP or RAP Dual-Indexed Layout
When less than the full set of 96 libraries are pooled and sequenced, it is important that
libraries with compatible index combinations are used in the indexed pool. Illumina
strongly recommends the following planning steps before beginning library prep:
11
1
Determine the number of libraries to pool for sequencing.
2
Make sure that the pool contains the required index combinations, as described in
Adapter Plate Pooling Guidelines on page 17. Select the DNA or RNA index adapters
based on the same guidelines.
Document # 15042173 v01
Use Illumina Experiment Manager (IEM) or BaseSpace® to:
} Create a sample sheet used during the sequencing run. This step also identifies
any unbalanced index combinations, allowing redesign before library prep starts.
} Specify the layout of all sample plates in 96-well plate format for compatibility
with the 96-well DAP or RAP. Arrange samples to pool in the same orientation as
the indexes in the DAP or RAP.
For more information, see the Illumina Experiment Manager Guide (part # 15031335)
and IEM quick reference card for the kit that you are using or BaseSpace Support.
Handling Adapter Plate
The DAP or RAP is designed for use in a TruSeq library prep HS or Plate protocol,
depending on the workflow options for the kit that you are using.
} The DAP or RAP is single-use for each well.
} Do not put a DAP or RAP through more than 4 freeze-thaw cycles.
} To maximize the use of the DAP or RAP, process more than 24 samples at a time.
These samples can then be pooled in any supported configuration.
Prepare Adapter Plate
Prepare the DAP or RAP for use as follows:
1
Thaw the plate for 10 minutes at room temperature on the benchtop. Visually inspect
the wells to make sure that they all are thawed.
2
Remove the adapter plate tape seal.
3
Centrifuge the plate at 280 × g for 1 minute to collect all the adapter to the bottom of
the well.
4
Remove the plastic cover and save the cover if you are not processing the entire
plate at one time.
5
Apply the DAP or RAP barcode label to the DAP or RAP, if a label is included your
kit.
If using only part of the DAP or RAP, use a lab pen to mark on the foil seal the
adapter wells being used. When doing so, be careful not to pierce the foil seal.
Pierce Adapter Plate Seal
Pierce the DAP or RAP foil seal as follows:
1
Place the DAP or RAP on the benchtop. The part number barcode, on the long side of
the plate, must face you and the clipped corner must be on the lower left.
Figure 3 Correct DAP or RAP Orientation
TruSeq Library Prep Pooling Guide
12
Adapter Options
3
2
Do the following:
} If using the entire plate at one time, use the bottom of a clean 96-well semiskirted
PCR plate to pierce a hole in all the well seals simultaneously. Gently but firmly
press the clean plate over the foil seal.
} If using only part of the plate, use the bottom of a clean 8-tube strip, with caps
attached, to pierce holes in the seals of the wells used for ligation. Repeat with a
new, clean 8-tube strip, with caps attached, for each row or column of adapters
used for ligation.
Do not reuse a dual-indexed adapter in a future library prep after the foil seal has
been pierced.
Pipette Adapter Plate
Pipette the adapters from the DAP or RAP into the ligation reaction as follows, while
keeping the plate in the same orientation:
1
Using a multichannel pipette, transfer the thawed adapter from the DAP or RAP well
to each well of the sample plate.
2
Change pipette tips between wells of the DAP or RAP. Changing tips is critical to
avoid cross-contamination between wells.
3
Aspirate each dual-indexed adapter by column or row depending on the adapters
being used.
4
Discard the tips after pipetting into the ligation reaction.
Adapter Plate Storage
If not all adapter wells are used in a single experiment (< 96 samples), the DAP or RAP
can be stored for future use of unused wells as follows:
1
Wipe the foil seal covering unused wells with a sterile 70% Ethanol wipe.
2
Allow the foil seal to dry.
3
Put the plastic cover that came with the DAP or RAP back on the plate.
4
Store the DAP or RAP at -25°C to -15°C.
NOTE
Do not reseal the plate with a disposable seal. A disposable seal will rip the original
foil seal when it is removed for future use.
Reagent Plate
NeoPrep index adapters are provided in a sealed reagent plate. Adapters are transferred
from the reagent plate to the NeoPrep library card before starting a library prep run.
NeoPrep libraries are pooled manually after they are unloaded from the library card.
Illumina recommends using the default index adapters in the order that they are
arranged in the reagent plate and to arrange the samples used with those index adapters
in the same order on the library card.
NeoPrep index adapters can be used to perform pooled sequencing.
} Each adapter is a unique single 6 base index adapter on the P7 strand.
} Libraries prepared with these adapters can be sequenced on the MiSeq using a
6 cycle Single Index Read and any other Illumina sequencing platform using a
7 cycle Single Index Read.
13
Document # 15042173 v01
For more information on sequencing libraries prepared using a NeoPrep kit, see the
system guide for your Illumina sequencing platform.
TruSeq Library Prep Pooling Guide
14
Adapter Options
For more information on pooling guidelines when using a NeoPrep kit, see NeoPrep
Adapter Pooling Guidelines on page 23.
Illumina sequencing platforms use a green laser to sequence G/T and a red laser to
sequence A/C. At each cycle, at least 1 of 2 nucleotides for each color channel are read to
ensure proper image registration. It is important to maintain color balance for each base
of the Index Read being sequenced, otherwise Index Read sequencing could fail due to
registration failure. Follow these low plex pooling guidelines, depending on the TruSeq
library prep kit you are using.
TruSeq Small RNA Library Prep Kit Pooling Guidelines
When using the indexed adapter tubes from a TruSeq Small RNA Library Prep Kit,
follow these pooling guidelines for single-indexed sequencing. TruSeq Small RNA
Library Prep Kit Set A, B, C, and D, each contain 12 unique indexed adapter tubes. When
designing low-plexity index pools for single-indexed sequencing, always use at least 2
unique and compatible barcodes for each index sequenced. The following table describes
possible pooling strategies for 2–4 samples generated with the adapter index tubes in
each set.
} For 5–11-plex pools, use 4-plex options with any other available adapters
} Not all color-balanced pools are listed. Check the color balance using IEM. For more
information, see the Illumina Experiment Manager Guide (part # 15031335) and the IEM
TruSeq Small RNA Quick Reference Card (document # 15037153).
Table 15 Single-Indexed Pooling Strategies for 2–4 Samples
Plexity
2
Option Set A Only
1
RPI6 and RPI12
3
1
2
3
4
1
2
3
RPI1 and RPI3
and RPI7
RPI2 and RPI4
and RPI8
2-plex option
with any other
adapter
RPI2 and RPI9
and RPI10 and
PRI11
RPI4 and RPI5
and RPI6 and
RPI7
3-plex option
with any other
adapter
Set B Only
Not
recommended
RPI16 and RPI17
and RPI18
RPI13 and RPI17
and RPI23
3-plex option
with any other
adapter
Set C Only *
Not
recommended
Not
recommended
Set D Only
RPI37 and RPI45
RPI38 and RPI44
and RPI46
RPI40 and RPI47
and RPI48
2-plex option
with any other
adapter
Not
RPI37 and RPI39
recommended and RPI42 and
RPI43
RPI37 and RPI38
and RPI45 and
RPI46
3-plex option
with any other
adapter
* Use TruSeq Small RNA Set C with another TruSeq Small RNA set. Do not use it on its
own
For more information on the single-indexed sequencing workflow, see the system guide
for your Illumina sequencing platform.
TruSeq Library Prep Pooling Guide
15
Pooling Guidelines
Pooling Guidelines
TruSeq LT Kit or ChIP Kit Adapter Tube Pooling Guidelines
When using the indexed adapter tubes from a TruSeq LT Kit or ChIP Kit, follow these
pooling guidelines for single-indexed sequencing. TruSeq LT Kit Sets A and B, each
contain 12 unique indexed adapter tubes.
When designing low-plexity index pools for single-indexed sequencing, always use at
least 2 unique and compatible barcodes for each index sequenced. The following table
describes possible pooling strategies for 2–4 samples generated with the adapter index
tubes in each set.
} For 5–11-plex pools, use 4-plex options with any other available adapters
} Not all color-balanced pools are listed. Check the color balance using IEM. For more
information, see the Illumina Experiment Manager Guide (part # 15031335) and IEM
quick reference card for the kit that you are using.
Table 16 Single-Indexed Pooling Strategies for 2–4 Samples with DNA Kits
Plexity Option Set A Only
Set B Only
2
1
AD006 and AD012
Not recommended
2
AD005 and AD019
3
1
AD002 and AD007 and AD019
AD001 and AD010 and AD020
2
AD005 and AD006 and AD015
AD003 and AD009 and AD025
3
2-plex options with any other
AD008 and AD011 and AD022
adapter
4
1
AD005 and AD006 and AD012
AD001 and AD008 and AD010
and AD019
and AD011
2
AD002 and AD004 and AD007
AD003 and AD009 and AD022
and AD016
and AD027
3
3-plex options with any other
3-plex options with any other
adapter
adapter
Table 17 Single-Indexed Pooling Strategies for 2–4 Samples with RNA and ChIP Kits
Plexity
2
3
4
Option
1
2
1
2
3
1
2
3
Set A Only
AR006 and AR012
AR005 and AR019
AR002 and AR007 and AR019
AR005 and AR006 and AR015
2-plex options with any other
adapter
AR005 and AR006 and AR012
and AR019
AR002 and AR004 and AR007
and AR016
3-plex options with any other
adapter
Set B Only
Not recommended
AR001 and AR010 and AR020
AR003 and AR009 and AR025
AR008 and AR011 and AR022
AR001 and AR008 and AR010
and AR011
AR003 and AR009 and AR022
and AR027
3-plex options with any other
adapter
For more information on the single-indexed sequencing workflow, see the system guide
for your Illumina sequencing platform.
16
Document # 15042173 v01
When using the indexed adapter tubes from a TruSeq Exome Library Prep 8 rxn × 3 plex,
8 rxn × 6 plex, and 8 rxn × 9 plex kits, follow these pooling guidelines for single-indexed
sequencing.
When designing low-plexity index pools for single-indexed sequencing, always use at
least 2 unique and compatible barcodes for each index sequenced. The following table
describes possible pooling strategies for 2–4 samples generated with the adapter index
tubes in each set.
} Not all color-balanced pools are listed. Check the color balance using IEM. For more
information, see the Illumina Experiment Manager Guide (part # 15031335) and IEM
quick reference card for the kit that you are using.
Table 18 Single-Indexed Pooling Strategies for TruSeq Exome Library Prep
8 rxn × 3 plex, 8 rxn × 6 plex, and 8 rxn × 9 plex Kits
Plexity Indexes - Option
Indexes - Option 2
Indexes - Option 3
1
2*
AD006 and AD012 AD005 and AD019
3
AD002 and AD007 AD006 and AD012 and any AD005 and AD019 and any
and AD019
other adapter
other adapter
6
AD002 and AD005 and AD006 and AD007 and AD012 and AD019
9
AD002 and AD004 and AD005 and AD006 and AD007 and AD012 and AD013
and AD015
* Not an option for 8 rxn × 3 plex kits.
When pooling:
} 3–6-plex, use indexes required for a 3-plex and select from the indexes required for a
6-plex pool
} 6–9-plex, use indexes required for a 6-plex and select from the indexes required for a
9-plex pool
For more information on the single-indexed sequencing workflow, see the system guide
for your Illumina sequencing platform.
Adapter Plate Pooling Guidelines
When using a DAP or RAP from a TruSeq HT or TruSeq Exome library prep kit (8 rxn ×
12 plex), follow these pooling guidelines. In addition, review Pooling Preparation with
Adapter Plate on page 11 and Handling Adapter Plate on page 12.
Single-Indexed Sequencing
Follow the single-indexed sequencing workflow when pooling 12 or fewer samples.
When designing low-plexity index pools, always use at least 2 unique and compatible
barcodes for each index sequenced. The following figures illustrate possible pooling
strategies for 2–12 samples generated with the DAP or RAP.
} Color balanced pools are shaded light gray with green wells.
} For 5-plex pools, dark gray wells are not used for pooled sequencing. They are
available for individual sequencing.
} For 7–11-plex pools, combine any of the 2–6-plex pools.
TruSeq Library Prep Pooling Guide
17
Pooling Guidelines
TruSeq Exome Library Prep Kit Adapter Tube Pooling Guidelines
}
Not all color-balanced pools are listed. Check the color balance using IEM. For more
information, see the Illumina Experiment Manager Guide (part # 15031335) and IEM
quick reference card for the kit that you are using.
For more information on the single-indexed sequencing workflow, see the system guide
for your Illumina sequencing platform.
Figure 4 Single-Indexed–2-plex
Figure 5 Single-Indexed–3-plex
Figure 6 Single-Indexed–4-plex
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Figure 7 Single-Indexed–5-plex
Figure 8 Single-Indexed–6-plex
Figure 9 Single-Indexed–12-plex
Dual-Indexed Sequencing
Follow the dual-indexed sequencing workflow when pooling more than 12 samples into
one pool. When designing the low-plexity index pools, always use at least 2 unique and
compatible barcodes for each index sequenced. The following figures illustrate possible
pooling strategies for 2–16 samples generated with the DAP or RAP.
} Color balanced pools are shaded light gray with green wells. The 2-plex pools are
diagonal and shaded in light or dark gray with green wells.
TruSeq Library Prep Pooling Guide
19
}
}
Odd-numbered pools display dark gray wells that are not used for pooled
sequencing. They are available for individual sequencing.
Not all color-balanced pools are listed. Check the color balance using IEM. For more
information, see the Illumina Experiment Manager Guide (part # 15031335) and IEM
quick reference card for the kit that you are using.
For more information on the dual-indexed sequencing workflow, see the system guide for
your Illumina sequencing platform.
Figure 10 Dual-Indexed–2-plex
Figure 11 Dual-Indexed–3-plex
Figure 12 Dual-Indexed–4-plex
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Pooling Guidelines
Figure 13 Dual-Indexed–5-plex
Figure 14 Dual-Indexed–6-plex
Figure 15 Dual-Indexed–7-plex
TruSeq Library Prep Pooling Guide
21
Figure 16 Dual-Indexed–8-plex, Option 1
Figure 17 Dual-Indexed–8-plex, Option 2
Figure 18 Dual-Indexed–12-plex
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Pooling Guidelines
Figure 19 Dual-Indexed–16-plex
NeoPrep Adapter Pooling Guidelines
Follow these pooling guidelines for single-indexed sequencing with NeoPrep index
adapters. The reagent plate contains 24 unique index adapters.
When designing low-plexity index pools for single-indexed sequencing, always use at
least 2 unique and compatible barcodes for each index sequenced. The following table
describes possible pooling strategies for 2–4 samples generated with the NeoPrep index
adapters.
} For 5–11-plex pools, use 4-plex options with any other available adapters
} Not all color-balanced pools are listed. Check the color balance using IEM. For more
information, see the Illumina Experiment Manager Guide (part # 15031335) and IEM
quick reference card for the kit that you are using.
Table 19 Single-Indexed Pooling Strategies for 2–4 Samples with NeoPrep DNA Kits
Plexity
Options
2
ND006 and ND012
ND005 and ND019
3
ND001 and ND010 and ND020
ND002 and ND007 and ND019
ND003 and ND009 and ND025
ND005 and ND006 and ND015
ND008 and ND011 and ND022
2-plex option with any other adapter
4
ND001 and ND008 and ND010 and ND011
ND002 and ND004 and ND007 and ND016
ND003 and ND009 and ND022 and ND027
ND005 and ND006 and ND012 and ND019
3-plex option with any other adapter
TruSeq Library Prep Pooling Guide
23
Table 20 Single-Indexed Pooling Strategies for 2–4 Samples with NeoPrep RNA Kits
Plexity
Options
2
NR006 and NR012
NR005 and NR019
3
NR001 and NR010 and NR020
NR002 and NR007 and NR019
NR003 and NR009 and NR025
NR005 and NR006 and NR015
NR008 and NR011 and NR022
2-plex option with any other adapter
4
NR001 and NR008 and NR010 and NR011
NR002 and NR004 and NR007 and NR016
NR003 and NR009 and NR022 and NR027
NR005 and NR006 and NR012 and NR019
3-plex option with any other adapter
For more information on the single-indexed sequencing workflow, see the system guide
for your Illumina sequencing platform.
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Document # 15042173 v01
For technical assistance, contact Illumina Technical Support.
Table 21 Illumina General Contact Information
Website
Email
www.illumina.com
[email protected]
Table 22 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
North America
1.800.809.4566
Italy
Australia
1.800.775.688
Netherlands
Austria
0800.296575
New Zealand
Belgium
0800.81102
Norway
Denmark
80882346
Spain
Finland
0800.918363
Sweden
France
0800.911850
Switzerland
Germany
0800.180.8994
United Kingdom
Ireland
1.800.812949
Other countries
Contact Number
800.874909
0800.0223859
0800.451.650
800.16836
900.812168
020790181
0800.563118
0800.917.0041
+44.1799.534000
Safety data sheets (SDSs)—Available on the Illumina website at
support.illumina.com/sds.html.
Product documentation—Available for download in PDF from the Illumina website. Go
to support.illumina.com, select a product, then select Documentation & Literature.
TruSeq Library Prep Pooling Guide
Technical Assistance
Technical Assistance
Illumina
San Diego, California 92122 U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com
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