TruSeq® Amplicon - Cancer Panel
Reference Guide
For Research Use Only. Not for use in diagnostic procedures.
ILLUMINA PROPRIETARY
Document # 15031875 v02
February 2016
Customize a short end-to-end workflow guide with the Custom Protocol Selector
support.illumina.com/custom-protocol-selector.html
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under its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this document.
The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order
to ensure the proper and safe use of the product(s) described herein. All of the contents of this document must be fully read
and understood prior to using such product(s).
FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN
MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND
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ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S)
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© 2016 Illumina, Inc. All rights reserved.
Illumina, 24sure, BaseSpace, BeadArray, BlueFish, BlueFuse, BlueGnome, cBot, CSPro, CytoChip, DesignStudio,
Epicentre, ForenSeq, Genetic Energy, GenomeStudio, GoldenGate, HiScan, HiSeq, HiSeq X, Infinium, iScan, iSelect,
MiniSeq, MiSeq, MiSeqDx, MiSeq FGx, NeoPrep, NextBio, Nextera, NextSeq, Powered by Illumina, SureMDA,
TruGenome, TruSeq, TruSight, Understand Your Genome, UYG, VeraCode, verifi, VeriSeq, the pumpkin orange color,
and the streaming bases design are trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/or other countries. All
other names, logos, and other trademarks are the property of their respective owners.
Patent pending for methods performed by components in this kit.
For Research Use Only– not for any clinical or therapeutic use in humans or animals.
This product includes GoTaq® Hot Start Polymerase manufactured by Promega
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U.S. Patent Nos. 5,338,671 and 5,587,287 and their corresponding foreign patents.
ii
Document # 15031875 v02
Revision History
Document
Date
Description of Change
Document # 15031875
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.
• Corrected the Consumables list in Remove Unbound
Oligos.
• Added the number of minutes to centrifuge during the
wash step in Remove Unbound Oligos.
• Corrected the Consumables list and Preparation section
in Normalize Libraries to reference LNS1.
• Revised instructions in Normalize Libraries to remove
"per library" wording.
Document # 15031875
v01
January
2016
The following changes were made to the user guide:
• Reorganized Getting Started content and moved some
topics to the Supporting Information Appendix.
• 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.
TruSeq Amplicon - Cancer Panel Reference Guide
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Document # 15031875 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 Amplicon - Cancer Panel 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
Technical Assistance
TruSeq Amplicon - Cancer Panel Reference Guide
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Document # 15031875 v02
Chapter 1 Overview
Introduction
DNA Input Recommendations
Additional Resources
TruSeq Amplicon - Cancer Panel Reference Guide
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Chapter 1
Overview
Overview
Introduction
The TruSeq Amplicon - Cancer Panel is a highly multiplexed targeted resequencing assay
for detecting somatic mutations within important cancer-related genes, including BRAF,
KRAS, and EGFR. The streamlined protocol includes an integrated quality control assay for
DNA from formalin-fixed, paraffin-embedded (FFPE) samples.
TruSeq Amplicon - Cancer Panel provides predesigned, optimized oligonucleotide probes
for sequencing mutational hotspots in > 35 kilobases (kb) of target genomic sequence. 48
genes are targeted with 212 amplicons in a highly multiplexed, single-tube reaction. This
highly targeted approach enables a wide range of applications for discovering, validating,
and screening genetic variants in a rapid and efficient manner.
How Does the Assay Work?
One pair of oligos is designed for each amplicon. Hybridization of these oligos to
unfragmented genomic DNA occurs in a 96-well plate, followed by extension and ligation
to form DNA templates consisting of the regions of interest flanked by universal primer
sequences. Using index adapters provided with the kit, libraries are indexed, PCR
amplified, and then pooled into a single tube before sequencing.
A
B
C
D
2
Hybridization of Cancer Panel oligonucleotide probes
Extension and ligation
Addition of indices and sequencing adapters by PCR
Final amplicon ready for sequencing
Document # 15031875 v02
A260/A280
Type of DNA
Input
FFPE DNA QC
High quality
genomic DNA
150 ng (minimum)
250 ng (recommended)
1.8–2.0
Not required
FFPE genomic DNA
250 ng (minimum)
1.8–2.0
Illumina FFPE QC Kit
(WG-321-1001)
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 Amplicon - Cancer
Panel 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 Amplicon - Cancer Panel Reference Guide
3
DNA Input Recommendations
DNA Input Recommendations
Overview
Additional Resources
Visit theTruSeq Amplicon - Cancer Panel 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.
TruSeq Amplicon - Cancer Panel
Protocol Guide (document #
1000000007000)
Provides instructions for the experienced user.
TruSeq Amplicon - Cancer Panel
Checklist (document #
1000000006993)
Provides a checklist of steps for the experienced user.
TruSeq Amplicon - Cancer Panel
Consumables & Equipment List
(document # 1000000006997)
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.
Required Manifest File
The Manifest file corresponding to your Amplicon Fixed Panel 1 Tube (AFP1) is available
from www.illumina.com/truseq_amplicon_cancer_panel_manifest.
4
Document # 15031875 v02
Chapter 2 Protocol
Protocol Introduction
Tips and Techniques
TruSeq Amplicon - Cancer Panel 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 Amplicon - Cancer Panel Reference Guide
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Chapter 2
Protocol
Protocol
Protocol Introduction
This section describes the TruSeq Amplicon - Cancer Panel Kit protocol.
} 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 # 15031875 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 Amplicon - Cancer Panel Reference Guide
7
Tips and Techniques
Tips and Techniques
Protocol
TruSeq Amplicon - Cancer Panel Workflow
The following diagram illustrates the workflow using the TruSeq Amplicon - Cancer Panel
Kit. Safe stopping points are marked between steps.
Figure 1 TruSeq Amplicon - Cancer Panel Workflow
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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 Amplicon - Cancer Panel Kit support page for more information.
TruSeq Amplicon - Cancer Panel 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
}
}
}
}
}
}
}
}
AFP1 (Amplicon Fixed Panel 1)
OHS1 (Oligo Hybridization for Sequencing 1)
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
}
}
OHS1
} 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
AFP1
ACD1
ACP1
OHS1
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 # 15031875 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 to 1 well of the HYP plate.
2
Add 5 µl gDNA to each remaining well.
3
Add 5 µl ACP1 to the well containing ACD1.
4
Add 5 µl AFP1 to each well containing gDNA.
5
Centrifuge at 1000 × g for 1 minute.
6
Add 40 µl OHS1 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 Amplicon - Cancer Panel 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
}
}
}
}
}
}
ELM3 (Extension-Ligation Mix 3)
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
ELM3
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 # 15031875 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 2 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 Amplicon - Cancer Panel 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.
14
Document # 15031875 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
}
}
ELM3 (Extension-Ligation Mix 3)
Foil adhesive seal
Procedure
1
Add 45 µl ELM3 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 Amplicon - Cancer Panel 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
Microseal 'A' adhesive film
Microseal 'B' adhesive seal
Preparation
1
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.
} 95°C for 3 minutes
} 27 cycles of:
} 95°C for 30 seconds
} 62°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.
Procedure
16
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.
Document # 15031875 v02
Amplify Libraries
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.
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.
TruSeq Amplicon - Cancer Panel Reference Guide
17
Protocol
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
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Clean Up Libraries
Clean Up Libraries
This step uses AMPure XP beads to purify the PCR products from other reaction
components.
Consumables and Equipment
}
}
}
}
}
}
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).
Expect the PCR product sizes to be around 350 bp for the control (ACP1) and 310 bp
for the cancer panel (AFP1).
TruSeq Amplicon - Cancer Panel Reference Guide
19
Protocol
Figure 4 Example Agarose Gel: Control (ACP1)
A
B
Expected Control (ACP1) Panel PCR Product (~350bp)
Primers
Figure 5 Example Agarose Gel: Cancer Panel (AFP1)
20
Document # 15031875 v02
Clean Up Libraries
A
B
Expected Cancer Panel (AFP1) PCR Product (~310bp)
Primers
Figure 6 Bioanalyzer Example
A
B
C
Marker
Expected Cancer Panel (AFP1) PCR Product (~310bp)
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.
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 AFP1.
3
Add 45 µl AMPure XP beads to each well of the CLP plate.
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.
TruSeq Amplicon - Cancer Panel Reference Guide
21
Protocol
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.
22
Document # 15031875 v02
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)
LNS1 (Library Normalization Storage buffer 1)
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
}
}
}
}
}
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.
TruSeq Amplicon - Cancer Panel Reference Guide
23
Normalize Libraries
Normalize Libraries
Protocol
Preparation
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
LNS1
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 LNS1 to each well of the SGP plate.
24
Document # 15031875 v02
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.
TruSeq Amplicon - Cancer Panel Reference Guide
25
Normalize Libraries
17 Transfer 30 µl supernatant from each well of the LNP plate to the corresponding well
of the SGP plate.
Protocol
Pool Libraries
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
26
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.
4
Denature and dilute pooled libraries to the loading concentration for the sequencing
instrument you are using. See the denature and dilute libraries guide for your
instrument.
Document # 15031875 v02
Appendix A Supporting Information
Introduction
Acronyms
Kit Contents
Consumables and Equipment
TruSeq Amplicon - Cancer Panel Reference Guide
28
29
30
32
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 # 15031875 v02
Acronyms
Acronyms
Acronym
Definition
ACD1
Amplicon Control DNA 1
ACP1
Amplicon Control Oligo Pool 1
AFP1
Amplicon Fixed Panel 1
CLP
Clean-up Plate
EBT
Elution Buffer with Tris
ELM3
Extension Ligation Mix 3
FPU
Filter Plate Unit
HT1
Hybridization Buffer
HYP
Hybridization Plate
IAP
Indexed Amplification Plate
LNA1
Library Normalization Additives 1
LNB1
Library Normalization Beads 1
LNS1
Library Normalization Storage Buffer 1
LNW1
Library Normalization Wash 1
LNP
Library Normalization Plate
OHS1
Oligo Hybridization for Sequencing Reagent 1
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 Amplicon - Cancer Panel 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 Amplicon - Cancer Panel Kit Kit and a Indexing
Kit are required.
TruSeq Amplicon - Cancer Panel Kit Contents (FC-130-1008)
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
OHS1
ELM3
PMM2
TDP1
SW1
UB1
Description
Amplicon Control DNA 1
Amplicon Control Oligo Pool 1
Oligo Hybridization for Sequencing Reagent 1
Extension Ligation Mix 3
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-amplification area at room temperature.
Quantity
1
1
Reagent
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
LNS1
EBT
Description
Hybridization Buffer
Library Normalization Additives 1
Library Normalization Wash 1
Library Normalization Storage Buffer 1
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 # 15031875 v02
Quantity
Reagent
AFP1
Description
Amplicon Fixed Panel 1
Storage Temperature
-25°C to -15°C
Indexing 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 Amplicon - Cancer Panel 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 Amplicon - Cancer Panel 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 # 15031875 v02
Consumables and Equipment
Pre-PCR Equipment
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
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
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 Amplicon - Cancer Panel Reference Guide
33
34
Document # 15031875 v02
For technical assistance, contact Illumina Technical Support.
Table 1 Illumina General Contact Information
Website
Email
www.illumina.com
[email protected]
Table 2 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 Amplicon - Cancer Panel 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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