TruSeq Custom Amplicon v1.5 Reference Guide (15027983 v02)

TruSeq Custom Amplicon v1.5 Reference Guide (15027983 v02)
TruSeq® Custom Amplicon v1.5
Reference Guide
For Research Use Only. Not for use in diagnostic procedures.
ILLUMINA PROPRIETARY
Document # 15027983 v02
February 2016
Customize a short end-to-end workflow guide with the Custom Protocol Selector
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© 2016 Illumina, Inc. All rights reserved.
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Epicentre, ForenSeq, Genetic Energy, GenomeStudio, GoldenGate, HiScan, HiSeq, HiSeq X, Infinium, iScan, iSelect,
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Patent pending for methods performed by components in this kit.
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ii
Document # 15027983 v02
Revision History
Document
Date
Description of Change
Document # 15027983
v02
February
2016
The following changes were made to the user guide:
• Corrected the About Reagents section in the Hybridize
Oligo Pool step.
• Removed the About Reagents section in Remove
Unbound Oligos.
• Removed the beta-mercaptoethanol warning in Remove
Unbound Oligos because it does not apply to the
reagents listed.
• Revised instructions in Normalize Libraries to remove
"per library" wording.
Document # 15027983
v01
January
2016
The following changes were made to the user guide:
• Removed use of plate name (eg, IMP plate), except for
first and last instance in each procedure.
• Revised step-by-step instructions to be more succinct.
• Removed box and tube part numbers from Kit
Contents.
• Removed instrument-specific instructions. Information
is now available in the denature and dilute libraries
guide and system guide for your sequencing
instrument.
Part # 15027983 Rev. C
August
2013
The following changes were made to the user guide:
• Reorganized Getting Started content and moved some
topics to the Supporting Information Appendix.
• Introduction of reagents OHS2, ELM4, and LNS2.
• Revised DNA input and volume recommendations for
high-quality genomic DNA. See DNA Input
Recommendations.
• Revised instructions for hybridization using OHS2.
• Revised PCR cycling conditions for DNA input <100 ng.
See PCR Amplification.
• Revised instructions for preparation of DAL and sample
pooling recommendations in Library Pooling and
MiSeq Sample Loading to support CAT Reagent Kit v3.
TruSeq Custom Amplicon v1.5 Reference Guide
iii
Document
Date
Part # 15027983 Rev. B
September
2012
Part # 15027983 Rev. A
October
2011
iv
Description of Change
The following changes were made to the user guide:
• Revised DNA input recommendations for high-quality
genomic DNA and recommendations for the use of
FFPE DNA. See DNA Input Recommendations.
• Updated filter plate pre-wash and wash
recommendations for removal of unbound oligos. See
Removal of Unbound Oligos.
• Revised instructions for removal of LNW1 supernatant
after library normalization. See Library Normalization.
• Revised instructions to specify required workflows
when using the Illumina Experiment Manager. See
Sample Sheet Preparation.
• Overview of the TSCA process, from design to data
analysis, including a new section for the Amplicon
Viewer. See Process Overview.
• Expanded assay conditions to support new amplicon
sizes, FFPE DNA, and various target multiplexing
levels. See PCR Amplification and PCR Clean-Up.
Initial release.
Document # 15027983 v02
Table of Contents
Revision History
Table of Contents
Chapter 1 Overview
Introduction
DNA Input Recommendations
Additional Resources
Chapter 2 Protocol
Protocol Introduction
Tips and Techniques
TruSeq Custom Amplicon Workflow
Prepare for Pooling
Hybridize Oligo Pool
Remove Unbound Oligos
Extend and Ligate Bound Oligos
Amplify Libraries
Clean Up Libraries
Normalize Libraries
Pool Libraries
Appendix A Supporting Information
Introduction
Acronyms
Kit Contents
Consumables and Equipment
Index Sequences
Technical Assistance
TruSeq Custom Amplicon v1.5 Reference Guide
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Document # 15027983 v02
Chapter 1 Overview
Introduction
DNA Input Recommendations
Additional Resources
TruSeq Custom Amplicon v1.5 Reference Guide
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Chapter 1
Overview
Overview
Introduction
This protocol explains how to prepare up to 96 uniquely indexed paired-end libraries of
genomic DNA (gDNA) using the Illumina® TruSeq® Custom Amplicon Library Prep Kit.
The kit supports sequencing targeted regions of the genome spanning upwards of 600 kb
with up to 1536 amplicons in a single multiplex reaction. This highly targeted approach
enables a wide range of applications for discovering, validating, and screening genetic
variants in a rapid and efficient manner.
The TruSeq Custom Amplicon library prep protocol offers:
} Multiplexing capability to amplify up to 1536 amplicons in a single reaction and
sequence up to 96 samples in a single sequencing run.
} Fast and simple workflow to generate up to 1536 amplicons across 96 samples within
a single plate with less than 3 hours hands on time.
} Streamlined 96-well based workflow amenable to automation.
The TruSeq Custom Amplicon library prep supports:
} Qualified FFPE samples.
} Customized design to create and manage projects using the Illumina online
DesignStudio software for a range of amplicon sizes and reference genomes.
} Automated data analysis to perform variant calling and analysis across all samples
using simple on-instrument, automated analysis software.
} The convenience of a fully integrated DNA-to-data solution including online probe
design and ordering, assay, sequencing, automated data analysis, and offline software
for reviewing results.
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Document # 15027983 v02
High-quality genomic DNA
Supported
Amplicon Size
(bp)
150, 175, 250, 425
FFPE genomic DNA
150 and 175
Type of DNA
Input (Up to
15 µl)
50 ng minimum
250 ng
recommended
250 ng
minimum
A260/A280
FFPE
DNA QC
1.8–2.0
Not
required
1.8–2.0
Illumina
FFPE QC
Kit (WG321-1001)
Δ Cq ≤ 2.0
Input DNA Quantification
Quantify the starting genomic material using a fluorescence-based quantification method,
such as a Qubit dsDNA Assay Kit or PicoGreen, rather than a UV-spectrometer-based
method. Fluorescence-based methods, which employ a double-stranded DNA (dsDNA)
specific dye, specifically and accurately quantify dsDNA even in the presence of many
common contaminants. In contrast, UV spectrometer methods based on 260 OD readings
are prone to overestimating DNA concentrations due to the presence of RNA and other
contaminants commonly found in genomic DNA (gDNA) preparations.
Assessing DNA Quality
To determine FFPE DNA quality and input amounts for the TruSeq Custom Amplicon
library prep, use the TruSeq FFPE DNA Library Prep QC Kit (catalog # WG-321-1001).
In addition, the ratio of absorbance at 260 nm to absorbance at 280 nm is used as an
indication of sample purity. This protocol is optimized for DNA with absorbance ratio
values of 1.8–2.0.
TruSeq Custom Amplicon v1.5 Reference Guide
3
DNA Input Recommendations
DNA Input Recommendations
Overview
Additional Resources
Visit theTruSeq Custom Amplicon Kit kit support page on the Illumina website for
documentation, software downloads, training resources, and information about compatible
Illumina products.
The following documentation is available for download from the Illumina website.
Resource
Custom Protocol Selector
Description
http://support.illumina.com/custom-protocol-selector.html
A wizard for generating customized end-to-end documentation
that is tailored to the library prep method, run parameters, and
analysis method used for the sequencing run.
4
TruSeq Custom Amplicon v1.5
Protocol Guide (document #
1000000005006)
Provides instructions for the experienced user.
TruSeq Custom Amplicon v1.5
Checklist (document #
1000000005007)
Provides a checklist of steps for the experienced user.
TruSeq Custom Amplicon v1.5
Consumables & Equipment List
(document # 1000000006989)
Provides an interactive checklist of user-provided
consumables and equipment.
TruSeq FFPE DNA Library Prep
QC Reference Guide (document #
1000000002136)
Provides instructions on how to determine the fragmentation
status and the amplification potential of FFPE-extracted gDNA
samples using the TruSeq FFPE DNA QC Kit.
Document # 15027983 v02
Chapter 2 Protocol
Protocol Introduction
Tips and Techniques
TruSeq Custom Amplicon Workflow
Prepare for Pooling
Hybridize Oligo Pool
Remove Unbound Oligos
Extend and Ligate Bound Oligos
Amplify Libraries
Clean Up Libraries
Normalize Libraries
Pool Libraries
TruSeq Custom Amplicon v1.5 Reference Guide
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Chapter 2
Protocol
Protocol
Protocol Introduction
This section describes the TruSeq Custom Amplicon Kit protocol.
} Prepare at least 8 samples at a time for the 16-sample kit. Prepare at least 16 samples
at a time for the 96-sample kit.
} Follow the protocol in the order described using the specified parameters.
} Before proceeding, confirm your kit contents and make sure that you have the required
consumables and equipment. This protocol requires different magnetic stands for prePCR and post-PCR procedures.
6
Document # 15027983 v02
Unless a safe stopping point is specified in the protocol, proceed immediately to the next
step.
Avoiding Cross-Contamination
}
}
}
When adding or transferring samples, change tips between each sample.
When adding adapters or primers, change tips between each row and each column.
Remove unused index adapter tubes from the working area.
Sealing the Plate
}
}
}
}
Always seal the 96-well plate before the following steps in the protocol:
} Shaking steps
} Vortexing steps
} Centrifuge steps
} Thermal cycling steps
Apply the adhesive seal to cover the plate and seal with a rubber roller.
Microseal 'B' adhesive seals are effective at -40°C to 110°C, and suitable for skirted or
semiskirted PCR plates. Use Microseal 'B' for shaking, centrifuging, and long-term
storage.
Microseal 'A' adhesive film is effective for thermal cycling and easy to cut when using
fewer than 96 wells.
Plate Transfers
}
When transferring volumes between plates, transfer the specified volume from each
well of a plate to the corresponding well of the other plate.
Centrifugation
}
Centrifuge at any step in the procedure to consolidate liquid or beads in the bottom of
the well, and to prevent sample loss.
Handling Beads
}
}
}
}
Pipette bead suspension slowly.
When mixing, mix thoroughly.
If beads are aspirated into the pipette tips, dispense back to the plate on the magnetic
stand and wait until the liquid is clear (~2 minutes).
When washing beads:
} Use the appropriate magnet for the plate.
} Dispense liquid so that beads on the side of the wells are wetted.
} Keep the plate on the magnet until the instructions specify to remove it.
} Do not agitate the plate while on the magnetic stand. Do not disturb the bead pellet.
TruSeq Custom Amplicon v1.5 Reference Guide
7
Tips and Techniques
Tips and Techniques
Protocol
TruSeq Custom Amplicon Workflow
The following figure illustrates the TruSeq Custom Amplicon workflow. Safe stopping
points are marked between steps.
Figure 1 TruSeq Custom Amplicon Workflow
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Document # 15027983 v02
If you plan to pool libraries, record information about your samples before beginning
library prep. Different methods are available depending on the sequencing instrument you
are using. See the TruSeq Custom Amplicon Kit support page for more information.
TruSeq Custom Amplicon v1.5 Reference Guide
9
Prepare for Pooling
Prepare for Pooling
Protocol
Hybridize Oligo Pool
This process hybridizes a custom oligo pool that contains upstream and downstream
oligos specific to your targeted regions of interest. Perform replicates to increase confidence
in somatic variant calls.
Consumables
}
}
}
}
}
}
}
}
CAT (Custom Amplicon Oligo Tube)
OHS2 (Oligo Hybridization for Sequencing 2)
ACD1 (Amplicon Control DNA)
ACP1 (Control Oligo Pool)
HYP (Hybridization Plate) barcode label
Diluted high-quality or FFPE gDNA
96-well PCR plate
Adhesive aluminum foil seal
WARNING
This set of reagents contains formamide, an aliphatic amide that is a probable
reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact,
and eye contact. Wear protective equipment, including eye protection, gloves, and
laboratory coat. Handle used reagents as chemical waste and discard in accordance with
the governmental safety standards for your region. For environmental, health, and safety
information, see the SDS for this kit at support.illumina.com/sds.html.
About Reagents
}
}
OHS2
} Aspirate and dispense slowly due to the viscosity of the reagent.
} Before each use, vortex thoroughly and then centrifuge briefly. Make sure that all
precipitates have dissolved.
} When mixing, mix thoroughly.
ACD1 and ACP1
} Include ACD1 and ACP1 in every batch of samples being prepared. Use of these
controls enables Illumina Technical Support to troubleshoot if you need assistance. If
these controls are excluded from your assay, assistance will not be provided.
Preparation
1
Prepare the following consumables.
Reagent
gDNA
CAT
ACD1
ACP1
OHS2
2
10
Storage
Instructions
-25°C to -15°C Let stand for 30 minutes to bring to room temperature.
Flick to mix, and then centrifuge briefly. Do not vortex.
-25°C to -15°C Thaw at room temperature.
Vortex to mix, and then centrifuge briefly.
-25°C to -15°C Thaw at room temperature.
Vortex to mix, and then centrifuge briefly.
-25°C to -15°C Thaw at room temperature.
Vortex to mix, and then centrifuge briefly.
-25°C to -15°C Thaw at room temperature.
Vortex vigorously to mix. Inspect in front of a light. Make sure
that all precipitates have dissolved.
Set a 96-well heat block to 95°C.
Document # 15027983 v02
Preheat an incubator to 37°C to prepare for the extension-ligation step.
4
Apply the HYP barcode label to a new 96-well PCR plate.
Hybridize Oligo Pool
3
Procedure
1
Add 5 µl ACD1 and 5 µl TE or water to 1 well of the HYP plate.
2
Add 10 µl gDNA to each remaining well.
For more diluted samples (eg, < 25 ng/µl), you can use up to 15 µl gDNA.
Table 1 Example Setup for High-Quality gDNA
Input (ng)
Volume (µl)
250
10
250
up to 15
50
10
50
up to 15
DNA Concentration (ng/µl)
25
≥ 16.7
5
≥ 3.3
Table 2 Example Setup for FFPE gDNA
Input (ng)
Volume (µl)
250
10
250
up to 15
DNA Concentration (ng/µl)
25
≥ 16.7
3
Add 5 µl ACP1 to the well containing ACD1.
4
Add 5 µl CAT to each well containing gDNA.
5
Centrifuge at 1000 × g for 1 minute.
6
Add 35 µl OHS2 to each well. Pipette to mix.
7
Centrifuge at 1000 × g for 1 minute.
8
Place on the preheated heat block and incubate for 1 minute.
9
With the plate on the heat block, reset the temperature to 40°C and continue incubating
for 80 minutes.
TruSeq Custom Amplicon v1.5 Reference Guide
11
Protocol
Remove Unbound Oligos
This process removes unbound oligos from genomic DNA using a size-selection filter. Two
wash steps using SW1 ensure complete removal of unbound oligos. A third wash step
using UB1 removes residual SW1 and prepares samples for the next step.
Consumables and Equipment
}
}
}
}
}
}
ELM4 (Extension-ligation Mix 4)
SW1 (Stringent Wash 1)
UB1 (Universal Buffer 1)
Filter plate with lid
Adapter roller
Midi plate
WARNING
This set of reagents contains formamide, an aliphatic amide that is a probable
reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact,
and eye contact. Wear protective equipment, including eye protection, gloves, and
laboratory coat. Handle used reagents as chemical waste and discard in accordance with
the governmental safety standards for your region. For environmental, health, and safety
information, see the SDS for this kit at support.illumina.com/sds.html.
Preparation
1
12
Prepare the following consumables:
Item
ELM4
Storage
-25°C to -15°C
SW1
UB1
2°C to 8°C
2°C to 8°C
Instructions
Let stand to bring to room temperature in preparation for a later
procedure.
Set aside at room temperature.
Set aside at room temperature.
Document # 15027983 v02
Remove Unbound Oligos
2
Assemble the filter plate unit (FPU) from top to bottom.
Figure 2 FPU Assembly
A
B
C
D
Lid
Filter plate
Adapter collar
Midi plate
3
Label the completed assembly FPU.
4
Wash the wells to be used in the assay as follows. Use new wells only.
a
b
c
5
Add 45 µl SW1 to each well.
Cover the FPU plate.
Centrifuge at 2400 × g for 10 minutes.
If a significant amount (> 15 µl/well) of residual buffer remains in multiple wells (≥ 10
wells/plate), switch to a new filter plate.
Procedure
1
Make sure that the heat block has cooled to 40˚C and the HYP plate seal is secure.
2
Remove from the heat block.
3
Centrifuge at 1000 × g for 1 minute.
4
Transfer each sample to the corresponding well of the FPU plate.
5
Cover and centrifuge at 2400 × g for 2 minutes.
6
Wash 2 times as follows.
a
b
c
7
Add 45 µl SW1 to each sample well.
Cover and centrifuge at 2400 × g for 2 minutes.
If SW1 does not drain completely, centrifuge again for up to 10 minutes.
Discard flow-through.
TruSeq Custom Amplicon v1.5 Reference Guide
13
Protocol
8
Reassemble the FPU plate for continued use.
9
Add 45 µl UB1 to each sample well.
10 Cover and centrifuge at 2400 × g for 2 minutes.
11 If UB1 does not drain completely, centrifuge again for up to 10 minutes.
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Document # 15027983 v02
This step connects the hybridized upstream and downstream oligos. A DNA polymerase
extends from the upstream oligo through the targeted region, followed by ligation to the 5'
end of the downstream oligo using a DNA ligase. The result is the formation of products
containing the targeted regions of interest flanked by sequences required for amplification.
Consumables
}
}
ELM4 (Extension-Ligation Mix 4)
Foil adhesive seal
Procedure
1
Add 45 µl ELM4 to each sample well of the FPU plate.
2
Incubate at 37°C for 45 minutes.
3
During incubation, proceed to the next step.
TruSeq Custom Amplicon v1.5 Reference Guide
15
Extend and Ligate Bound Oligos
Extend and Ligate Bound Oligos
Protocol
Amplify Libraries
This step amplifies the extension-ligation products and adds index 1 (i7) adapters, index 2
(i5) adapters, and sequences required for cluster formation.
Consumables
}
}
}
}
}
}
}
}
PMM2 (PCR Master Mix 2)
Index i5 adapters (A5XX)
Index i7 adapters (A7XX)
TDP1 (TruSeq DNA Polymerase 1)
96-well skirted PCR plate
IAP (Indexed Amplification Plate) barcode label
Microseal 'A' adhesive film
Microseal 'B' adhesive seal
About Reagents
}
PMM2/TDP1
} Combine PMM1 and TDP1 immediately before use. Do not combine and store the
combined PMM1/TDP1 mixture.
} When mixing, mix thoroughly.
Preparation
1
16
Prepare the following consumables.
Reagent
PMM2
Storage
-25° to -15° C
Index i5
adapters
(A5XX)
Index i7
adapters
(A7XX)
-25° to -15° C
-25° to -15° C
Instructions
Thaw at room temperature for 20 minutes. Vortex to mix,
and then centrifuge briefly.
Let stand for 20 minutes to bring to room temperature.
Vortex to mix, and then centrifuge briefly using a 1.7 ml
Eppendorf tube.
Let stand for 20 minutes to bring to room temperature.
Vortex to mix, and then centrifuge briefly using a 1.7 ml
Eppendorf tube.
2
Prepare fresh 50 mM NaOH.
3
Save the following PCR program on a thermal cycler using the appropriate number of
PCR cycles listed under PCR Cycle Number Guidelines.
} 95°C for 3 minutes
} X cycles of:
} 95°C for 30 seconds
} 66°C for 30 seconds
} 72°C for 60 seconds
} 72°C for 5 minutes
} Hold at 10°C
4
Apply the IAP label to a new 96-well PCR plate.
Document # 15027983 v02
Amplify Libraries
PCR Cycle Number Guidelines
Table 3 50–99 ng
Plexity
< 96 amplicons
97–384 amplicons
385–768 amplicons
769–1536 amplicons
150/175 bp
32
28
26
25
Number of PCR Cycles (X)
250 bp
33
28
27
26
425 bp
33
29
28
27
150/175 bp
29
25
23
22
Number of PCR Cycles (X)
250 bp
30
25
24
23
425 bp
30
26
25
24
Table 4 100–250 ng
Amplicon Size
< 96 amplicons
97–384 amplicons
385–768 amplicons
769–1536 amplicons
Procedure
1
Arrange the Index 1 (i7) adapters in columns 1–12 of the TruSeq Index Plate Fixture.
2
Arrange the Index 2 (i5) adapters in rows A–H of the TruSeq Index Plate Fixture.
Figure 3 TruSeq Index Plate Fixture
A
B
C
Rows A–H: Index 2 (i5) adapters (white caps)
Columns 1–12: Index (i7) adapters (orange caps)
IAP plate
3
Place the plate on a TruSeq Index Plate Fixture.
4
Using a multichannel pipette, add 4 µl of each Index 1 (i7) adapter down each column.
Replace the cap on each i7 adapter tube with a new orange cap.
TruSeq Custom Amplicon v1.5 Reference Guide
17
Protocol
5
Using a multichannel pipette, add 4 µl of each Index 2 (i5) adapter across each row.
Replace the cap on each i5 adapter tube with a new white cap.
6
Remove the FPU plate from the incubator and do the following.
a
b
c
d
Replace the aluminum foil seal with the filter plate lid.
Centrifuge at 2400 × g for 2 minutes.
Add 25 µl 50 mM NaOH to each well. Pipette to mix.
Incubate at room temperature for 5 minutes.
7
Add 56 µl TDP1 to a full tube (2.8 ml) of PMM2. Invert to mix.
8
Transfer 22 µl PMM2/TDP1 mixture to each well of the IAP plate.
9
Transfer eluted samples from the FPU plate to the IAP plate as follows.
a
b
c
d
Using fine tips, pipette to mix the NaOH in the first column of the FPU plate.
Transfer 20 µl NaOH to the corresponding column of the IAP plate. Pipette to mix.
Transfer remaining columns from the FPU to the IAP plate.
Discard the waste collection midi plate.
NOTE
Set aside the metal adapter collar for future use. If you partially used an FPU plate, mark
the used wells and store the FPU plate and lid in a sealed plastic bag.
10 Centrifuge at 1000 × g for 1 minute.
11 Transfer the IAP plate to the post-amplification area.
12 Place on the preprogrammed thermal cycler and run the PCR program.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at 2°C to 8°C for up to 2 days. Alternatively,
leave on the thermal cycler overnight.
18
Document # 15027983 v02
Clean Up Libraries
Clean Up Libraries
This step uses AMPure XP beads to purify the PCR products from other reaction
components.
Consumables and Equipment
}
}
}
}
}
}
}
EBT (Elution Buffer with Tris)
AMPure XP beads
Barcode labels
} CLP (Cleanup Plate)
} LNP (Library Normalization Plate)
96-well midi plates (2)
Microseal 'B' adhesive seals
Freshly prepared 80% ethanol (EtOH)
Magnetic stand-96
Preparation
1
Prepare the following consumables.
Reagent
AMPure XP
beads
Storage
2°C to
8°C
Instructions
Let stand for 30 minutes to bring to room temperature.
2
Prepare 40 ml (for 96 samples) fresh 80% ethanol from 100% ethanol.
3
Apply the CLP barcode label to a new midi plate.
4
Apply the LNP barcode label to a new midi plate.
Procedure
1
Centrifuge the IAP plate at 1000 × g for 1 minute.
2
Run an aliquot of the library and control on 4% agarose gel (5 µl) or Bioanalyzer (1 µl).
The following table provides the expected PCR product sizes.
Amplicon Size (bp)
150
175
250
425
TruSeq Custom Amplicon v1.5 Reference Guide
PCR Product Size (bp)
~280
~310
~350
~570
19
Protocol
Figure 4 Agarose Gel Example
A
B
Expected PCR product for 250 bp amplicons (~350 bp)
Primers
Figure 5 Bioanalyzer Example
A
B
C
Marker
Expected PCR product for 250 bp amplicons (~350 bp)
Marker
NOTE
Assess library quality by gel electrophoresis or Bioanalyzer for oligo pools being used
for the first time. You do not need to assess the quality of every sample in the
experiment.
20
Document # 15027983 v02
3
Add the appropriate volume of AMPure XP beads indicated in Table 5 to each well of
the CLP plate.
Table 5 Bead Volume
Amplicon Size (bp)
150
175
250
425
AMPure XP beads (µl)
60
60
45
35
4
Transfer all the supernatant from each well of the IAP plate to the corresponding well
of the CLP plate.
5
Shake at 1800 rpm for 2 minutes.
6
Incubate at room temperature for 10 minutes.
7
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
8
Remove and discard all supernatant from each well.
9
Wash 2 times as follows.
a
b
c
Add 200 µl of 80% EtOH to each sample well.
Incubate on the magnetic stand for 30 seconds.
Remove and discard all supernatant from each well.
10 Use a 20 µl pipette to remove residual EtOH from each well.
11 Remove from the magnetic stand and air-dry for 10 minutes.
12 Add 30 µl EBT to each well.
13 Shake at 1800 rpm for 2 minutes.
14 Make sure that all beads are resuspended. If necessary, pipette to mix and repeat the
shaking step.
15 Incubate at room temperature for 2 minutes.
16 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
17 Transfer 20 µl supernatant from each well of the CLP plate to the corresponding well of
the LNP plate.
18 Centrifuge at 1000 × g for 1 minute.
TruSeq Custom Amplicon v1.5 Reference Guide
21
Clean Up Libraries
To enable Illumina Technical Support to troubleshoot if you need assistance, assess the
quality of the control reaction generated with the ACD1 and ACP1. Process the control
reaction using the same conditions as CAT.
Protocol
Normalize Libraries
This step normalizes the quantity of each library for balanced representation in pooled
libraries. Only samples containing DNA require processing through the subsequent steps.
Consumables and Equipment
}
}
}
}
}
}
}
}
}
}
LNA1 (Library Normalization Additives 1)
LNB1 (Library Normalization Beads 1)
LNW1 (Library Normalization Wash 1)
LNS2 (Library Normalization Storage buffer 2)
SGP (Storage Plate) barcode label
0.1 N NaOH (freshly prepared)
96-well PCR plate, skirted
15 ml conical tube
Microseal 'B' adhesive seals
Magnetic stand-96 (use with midi 96-well storage plates)
WARNING
This set of reagents contains formamide, an aliphatic amide that is a probable
reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact,
and eye contact. Wear protective equipment, including eye protection, gloves, and
laboratory coat. Handle used reagents as chemical waste and discard in accordance with
the governmental safety standards for your region. For environmental, health, and safety
information, see the SDS for this kit at support.illumina.com/sds.html.
WARNING
This set of reagents contains ß-mercaptoethanol. Perform the following procedure in a
hood or well-ventilated area.
About Reagents
}
}
}
}
}
22
Use a P1000 pipette to transfer LNB1 to LNA1.
When mixing, mix thoroughly.
Mix only the amounts of LNA1 and LNB1 required for the current experiment.
Store remaining LNA1 and LNB1 separately at their respective temperatures.
Make sure that LNB1 is resuspended before use. Homogeneous resuspension is
essential for consistent cluster density on the flow cell.
Document # 15027983 v02
1
Prepare the following consumables.
Reagent
LNA1
Storage
-25°C to -15°C
LNB1
2°C to 8°C
LNW1
2°C to 8°C
LNS2
15°C to 30°C
Instructions
Thaw at room temperature. Let stand for 30 minutes to
bring to room temperature.
Vortex to mix. Make sure that all precipitate has
dissolved.
Let stand for 30 minutes to bring to room temperature.
Vortex for at least 1 minute. Invert intermittently to
resuspend. Make sure that the bottom of the tube is free
of pellets.
Thaw at room temperature. Let stand for 30 minutes to
bring to room temperature.
If frozen, thaw at room temperature for 20 minutes.
Vortex to mix.
2
Prepare fresh 0.1 N NaOH.
3
Label a new 96-well plate SGP.
Procedure
1
For 96 samples, add 4.4 ml LNA1 to a new 15 ml conical tube.
2
Use a P1000 pipette to resuspend LNB1.
3
Transfer 800 µl LNB1 to the 15 ml conical tube of LNA1. Invert to mix.
4
Add 45 µl LNA1/LNB1 to each well of the LNP plate.
5
Shake at 1800 rpm for 30 minutes.
Durations other than 30 minutes can affect library representation and cluster density.
6
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
7
Remove and discard all supernatant.
8
Remove from the magnetic stand.
9
Wash 2 times as follows.
a
b
c
d
Add 45 µl LNW1 to each library well.
Shake at 1800 rpm for 5 minutes.
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
Remove and discard all supernatant.
10 Use a 20 µl pipette to remove residual LNW1 from each well.
11 Remove from the magnetic stand.
12 Add 30 µl fresh 0.1 N NaOH to each well.
13 Shake at 1800 rpm for 5 minutes.
14 If the libraries are not resuspended, pipette to mix, and then shake at 1800 rpm for 5
minutes.
15 Place the LNP plate on a magnetic stand and wait until the liquid is clear (~2 minutes).
16 Add 30 µl LNS2 to each well of the SGP plate.
TruSeq Custom Amplicon v1.5 Reference Guide
23
Normalize Libraries
Preparation
Protocol
17 Transfer 30 µl supernatant from each well of the LNP plate to the corresponding well
of the SGP plate.
18 Centrifuge at 1000 × g for 1 minute.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at -25°C to -15°C for up to 30 days.
24
Document # 15027983 v02
Pooling libraries combines equal volumes of normalized libraries in a single tube. After
pooling, dilute and denature the library pool before loading libraries for the sequencing run.
Consumables
}
}
}
PAL (Pooled Amplicon Library) barcode label
LoBind microcentrifuge tube
RNase/DNase-free 8-tube strips and caps
About Reagents
}
Store the PAL tube at -25°C to -15°C for later use.
Preparation
1
If the SGP plate was stored frozen, prepare as follows.
a
b
c
Thaw at room temperature.
Centrifuge at 1000 × g for 1 minute.
Pipette to mix.
2
To prepare for the sequencing run, begin thawing reagents according to the instructions
for your instrument.
3
Label a new Eppendorf tube PAL.
Procedure
1
Centrifuge at 1000 × g for 1 minute.
2
Transfer 5 µl of each library to an 8-tube strip, column by column. Seal the plate and
store at -25°C to -15°C.
3
Transfer the contents of the 8-tube strip to the PAL tube. Pipette to mix.
TruSeq Custom Amplicon v1.5 Reference Guide
25
Pool Libraries
Pool Libraries
26
Document # 15027983 v02
Appendix A Supporting Information
Introduction
Acronyms
Kit Contents
Consumables and Equipment
Index Sequences
TruSeq Custom Amplicon v1.5 Reference Guide
28
29
30
32
34
27
Appendix A
Supporting Information
Supporting Information
Introduction
The protocols described in this guide assume that you have reviewed the contents of this
appendix, confirmed your kit contents, and obtained all the required consumables and
equipment.
28
Document # 15027983 v02
Acronyms
Acronyms
Acronym
Definition
ACD1
Amplicon Control DNA 1
ACP1
Amplicon Control Oligo Pool 1
CAT
Custom Amplicon Oligo Tube
CLP
Clean-up Plate
EBT
Elution Buffer with Tris
ELM4
Extension Ligation Mix 4
FPU
Filter Plate Unit
HT1
Hybridization Buffer
HYP
Hybridization Plate
IAP
Indexed Amplification Plate
LNA1
Library Normalization Additives 1
LNB1
Library Normalization Beads 1
LNP
Library Normalization Plate
LNS2
Library Normalization Storage Buffer 2
LNW1
Library Normalization Wash 1
OHS2
Oligo Hybridization for Sequencing Reagent 2
PAL
PMM2
Pooled Amplicon Library
PCR Master Mix 2
SGP
Storage Plate
SW1
Stringent Wash 1
TDP1
TruSeq DNA Polymerase 1
UB1
Universal Buffer 1
TruSeq Custom Amplicon v1.5 Reference Guide
29
Supporting Information
Kit Contents
Make sure that you have all the reagents identified in this section before proceeding to the
library preparation procedures. A TruSeq Custom Amplicon Kit Kit and a TruSeq Custom
Amplicon Index Kit are required.
TruSeq Custom Amplicon Kit Contents (FC-130-1001)
Box 1—Store in the Pre-PCR Area
This box also contains the HYP, FPU, and IAP barcode labels.
Quantity
1
1
1
1
1
1
1
1
Reagent
ACD1
ACP1
OHS2
ELM4
PMM2
TDP1
SW1
UB1
Description
Amplicon Control DNA 1
Amplicon Control Oligo Pool 1
Oligo Hybridization for Sequencing Reagent 2
Extension Ligation Mix 4
PCR Master Mix 2
TruSeq DNA Polymerase 1
Stringent Wash 1
Universal Buffer 1
Storage Temperature
-25°C to -15°C
-25°C to -15°C
-25°C to -15°C
-25°C to -15°C
-25°C to -15°C
-25°C to -15°C
2°C to 8°C
2°C to 8°C
WARNING
This set of reagents contains formamide, an aliphatic amide that is a probable
reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact,
and eye contact. Wear protective equipment, including eye protection, gloves, and
laboratory coat. Handle used reagents as chemical waste and discard in accordance with
the governmental safety standards for your region. For environmental, health, and safety
information, see the SDS for this kit at support.illumina.com/sds.html.
Box 2—Store in the Pre-PCR Area
This box is shipped at room temperature. As soon as you receive your kit, remove LNB1
from box 2 and store at 2°C to 8°C in the post-amplification area. Keep the filter plate in the
pre-PCR area at room temperature.
Quantity
Reagent
1
LNB1
Description
Filter plate with lid
Library Normalization Beads 1
Storage Temperature
Room temperature
2°C to 8°C
Box 3—Store in the Post-PCR Area
This box also contains the CLP, LNP, SGP, PAL, DAL barcode labels.
Quantity
1
1
1
1
1
30
Reagent
HT1
LNA1
LNW1
LNS2
EBT
Description
Hybridization Buffer
Library Normalization Additives 1
Library Normalization Wash 1
Library Normalization Storage Buffer 2
Elution Buffer with Tris
Storage Temperature
-25°C to -15°C
-25°C to -15°C
2°C to 8°C
Room temperature
Room temperature
Document # 15027983 v02
Quantity
Reagent
CAT
Description
Custom Amplicon oligo Tube
Storage Temperature
-25°C to -15°C
TruSeq Custom Amplicon Index Kit Contents (FC-130-1003)
Box 1—Store in Pre-PCR Area
Quantity
8 tubes
12 tubes
Reagent Name
i5 Index Primers, A501 to A508
i7 Index Primers, A701 to A712
Storage Temperature
-25°C to -15°C
-25°C to -15°C
Box 2—Store in Pre-PCR Area
Quantity
1 box
1 box
Reagent Name
i5 Index Tube Caps, White
i7 Index Tube Caps, Orange
Storage Temperature
Room temperature
Room temperature
Additional Components
Consumable
TruSeq Index Plate Fixture Kit
(Recommended)
TruSeq Custom Amplicon Filter Plate
(Required)
TruSeq Index Plate Fixture and Collar
Kit (Recommended)
Illumina FFPE QC Kit (Recommended
for FFPE DNA samples)
TruSeq Custom Amplicon v1.5 Reference Guide
Catalog #
FC-130-1005
Storage Temperature
Room temperature
Area
Pre-PCR
FC-130-1006
Room temperature
Pre-PCR
FC-130-1007
Room temperature
Pre-PCR
WG-321-1001
-25°C to -15°C
Pre-PCR
31
Kit Contents
Box 4—Store in the Pre-PCR Area
Supporting Information
Consumables and Equipment
Make sure that you have the required user-supplied consumables and equipment before
starting the protocol.
NOTE
• Use a dedicated set of consumables and equipment for pre-PCR and post-PCR
procedures.
• The TruSeq Custom Amplicon library prep protocol requires different magnetic stands
for pre-PCR and post-PCR procedures.
Consumables
32
Consumable
Supplier
10 N NaOH (prepare from tablets or use a
standard solution)
General lab supplier
96-well skirted PCR plates, 0.2 ml,
polypropylene
Bio-Rad, Part # MSP-9601
96-well storage plates, 0.8 ml (midi plates)
Fisher Scientific, Part # AB-0859
Fisher Scientific, Part # AB-0765
Agencourt AMPure XP, 60 ml kit
Beckman Coulter, Part # A63881/A63880
Foil seals
Beckman Coulter, Part # 538619
Conical tubes, 15 ml
General lab supplier
Eppendorf microcentrifuge tubes, screw top
General lab supplier
Ethanol, 200 proof for molecular biology
General lab supplier
Microseal 'A' adhesive seals
Bio-Rad, Part # MSA-5001
Microseal 'B' adhesive seals
Bio-Rad, Part # MSB-1001
PCR 8-tube strips
General lab supplier
Solution basin, PVC, non-sterile (trough)
Labcor, Part# 730-001
Agarose gel (2% for 250 bp and 425 bp
amplicons, or 4% for 150 bp, 175 bp, and 250
bp amplicons)
General Lab Supplier
DNA 1000 Kit for Bioanalyzer
Agilent 5067–1504 (for 300 samples)
DNA molecular weight markers
General Lab Supplier
Ice bucket
General Lab Supplier
Document # 15027983 v02
Equipment
Supplier
37°C incubator
Forced Air Oven, VWR International or
comparable
Heat block, 96-well
Scigene, Hybex Microsample Incubator for
PCR plate
Tabletop centrifuge
General lab supplier
[Optional] Heat sealer
Agilent PlateLoc Thermal Microplate Sealer
Post-PCR Equipment
Equipment
Supplier
Magnetic stand-96
Invitrogen DynaMag™-96 Side Skirted
Post-PCR plate shaker
Q Instruments BioShake iQ high-speed
thermoshaker, part # 1808-0506, or
Q Instruments BioShake XP high-speed lab
shake, part # 1808-0505
Tabletop centrifuge
General lab supplier
Gel electrophoresis supplies and apparatus
General lab supplier
Heat block for 1.5 ml centrifuge tubes
General lab supplier
[Optional] Bioanalyzer System
Agilent Technologies
Thermal Cyclers
Use the following recommended settings for selected thermal cycler models. Before
performing library prep, validate any thermal cyclers not listed.
Thermal Cycler
Temp Mode
Lid Temp
Vessel Type
Bio-Rad DNA Engine
Tetrad 2
Calculated
Heated, constant
at 100°C
Polypropylene plates
and tubes
MJ Research DNA
Engine Tetrad
Calculated
Heated
Plate
Eppendorf
Mastercycler Pro S
Gradient S,
Simulated Tube
Heated
Plate
TruSeq Custom Amplicon v1.5 Reference Guide
33
Consumables and Equipment
Pre-PCR Equipment
Supporting Information
Index Sequences
The TruSeq Custom Amplicon Index Kit contains the following indexed adapter sequences.
34
i7 Index Adapter
Sequence
A701
ATCACGAC
A702
ACAGTGGT
A703
CAGATCCA
A704
ACAAACGG
A705
ACCCAGCA
A706
AACCCCTC
A707
CCCAACCT
A708
CACCACAC
A709
GAAACCCA
A710
TGTGACCA
A711
AGGGTCAA
A712
AGGAGTGG
i5 Index Adapter
Sequence
A501
TGAACCTT
A502
TGCTAAGT
A503
TGTTCTCT
A504
TAAGACAC
A505
CTAATCGA
A506
CTAGAACA
A507
TAAGTTCC
A508
TAGACCTA
Document # 15027983 v02
For technical assistance, contact Illumina Technical Support.
Table 6 Illumina General Contact Information
Website
Email
www.illumina.com
[email protected]
Table 7 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
North America
1.800.809.4566
Japan
Australia
1.800.775.688
Netherlands
Austria
0800.296575
New Zealand
Belgium
0800.81102
Norway
China
400.635.9898
Singapore
Denmark
80882346
Spain
Finland
0800.918363
Sweden
France
0800.911850
Switzerland
Germany
0800.180.8994
Taiwan
Hong Kong
800960230
United Kingdom
Ireland
1.800.812949
Other countries
Italy
800.874909
Contact Number
0800.111.5011
0800.0223859
0800.451.650
800.16836
1.800.579.2745
900.812168
020790181
0800.563118
00806651752
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 Custom Amplicon v1.5 Reference Guide
35
Technical Assistance
Technical Assistance
Illumina
5200 Illumina Way
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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