TruSeq Amplicon - Cancer Panel Protocol Guide (1000000007000 v01)

TruSeq Amplicon - Cancer Panel Protocol Guide (1000000007000 v01)
TruSeq Amplicon - Cancer Panel
Protocol Guide
For Research Use Only. Not for use in diagnostic procedures.
Hybridize Oligo Pool
Remove Unbound Oligos
Extend and Ligate Bound Oligos
Amplify Libraries
Clean Up Libraries
Normalize Libraries
Pool Libraries
Acronyms
Technical Assistance
ILLUMINA PROPRIETARY
Document # 1000000007000 v01
February 2016
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This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for the
contractual use of its customer in connection with the use of the product(s) described herein and for no other purpose. This
document and its contents shall not be used or distributed for any other purpose and/or otherwise communicated, disclosed,
or reproduced in any way whatsoever without the prior written consent of Illumina. Illumina does not convey any license
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
DAMAGE TO OTHER PROPERTY.
ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S)
DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE).
© 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
Corporation for distribution by Illumina, Inc. Licensed to Promega Corporation under
U.S. Patent Nos. 5,338,671 and 5,587,287 and their corresponding foreign patents.
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
Set a 96-well heat block to 95°C.
2
Preheat an incubator to 37°C to prepare for the extension-ligation step.
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. 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 Protocol Guide
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Hybridize Oligo Pool
Hybridize Oligo Pool
Remove Unbound Oligos
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
Assemble the filter plate unit (FPU) from top to bottom as follows: lid, filter plate,
adapter collar, and midi plate.
2
Wash the wells to be used in the assay with 45 µl SW1.
Procedure
1
Make sure that the heat block has cooled to 40˚C.
2
Remove from the heat block.
3
Centrifuge at 1000 × g for 1 minute.
4
Transfer each sample to the FPU plate.
5
Cover and centrifuge at 2400 × g for 2 minutes.
6
Wash 2 times with 45 µl SW1.
7
Discard flow-through.
8
Reassemble the FPU plate.
9
Add 45 µl UB1.
10 Cover and centrifuge at 2400 × g for 2 minutes.
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Document # 1000000007000 v01
Extend and Ligate Bound Oligos
Extend and Ligate Bound Oligos
Procedure
1
Add 45 µl ELM3 to the FPU plate.
2
Incubate at 37°C for 45 minutes.
TruSeq Amplicon - Cancer Panel Protocol Guide
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Amplify Libraries
Preparation
1
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
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.
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.
5
Using a multichannel pipette, add 4 µl of each Index 2 (i5) adapter across each row.
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. Pipette to mix.
Incubate at room temperature for 5 minutes.
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Add 56 µl TDP1 to a full tube (2.8 ml) of PMM2. Invert to mix.
8
Transfer 22 µl PMM2/TDP1 mixture to the IAP plate.
9
Transfer eluted samples from the FPU plate to the IAP plate.
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.
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Document # 1000000007000 v01
Procedure
1
Centrifuge the IAP plate at 1000 × g for 1 minute.
2
Run an aliquot of library and control on 4% agarose gel (5 µl) or Bioanalyzer (1 µl).
3
Add 45 µl AMPure XP beads to the CLP plate.
4
Transfer all the supernatant from the IAP plate to 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 with 200 µl 80% EtOH.
10 Use a 20 µl pipette to remove residual EtOH.
11 Remove from the magnetic stand and air-dry for 10 minutes.
12 Add 30 µl EBT.
13 Shake at 1800 rpm for 2 minutes.
14 Incubate at room temperature for 2 minutes.
15 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
16 Transfer 20 µl supernatant from the CLP plate to the LNP plate.
17 Centrifuge at 1000 × g for 1 minute.
TruSeq Amplicon - Cancer Panel Protocol Guide
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Clean Up Libraries
Clean Up Libraries
Normalize Libraries
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.
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 tube of LNA1.
4
Add 45 µl LNA1/LNB1 to the LNP plate.
5
Shake at 1800 rpm for 30 minutes.
6
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
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Remove and discard all supernatant.
8
Remove from the magnetic stand.
9
Wash 2 times with 45 µl LNW1.
10 Use a 20 µl pipette to remove residual LNW1.
11 Remove from the magnetic stand.
12 Add 30 µl fresh 0.1 N NaOH.
13 Shake at 1800 rpm for 5 minutes.
14 Place the LNP plate on a magnetic stand and wait until the liquid is clear (~2
minutes).
15 Add 30 µl LNS1 to the SGP plate.
16 Transfer 30 µl supernatant from the LNP plate to the SGP plate.
17 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.
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Document # 1000000007000 v01
Pool Libraries
Pool Libraries
Procedure
1
Centrifuge at 1000 × g for 1 minute.
2
Transfer 5 µl of each library to an 8-tube strip, column by column.
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.
TruSeq Amplicon - Cancer Panel Protocol Guide
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Acronyms
Acronym
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
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Definition
Pooled Amplicon Library
PCR Master Mix 2
SGP
Storage Plate
SW1
Stringent Wash 1
TDP1
TruSeq DNA Polymerase 1
UB1
Universal Buffer 1
Document # 1000000007000 v01
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 Protocol Guide
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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