TruSight Myeloid Sequencing Panel Protocol Guide (1000000005004 v00)

TruSight Myeloid Sequencing Panel Protocol Guide (1000000005004 v00)
TruSight Myeloid Sequencing 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 # 1000000005004 v00
January 2016
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Hybridize Oligo Pool
Hybridize Oligo Pool
Preparation
1
Set a 96-well heat block to 95°C.
2
Preheat an incubator to 37°C.
3
Label a new 96-well PCR plate HYP.
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.
3
Add 5 µl TSO to each well containing gDNA.
4
Centrifuge at 1000 × g for 1 minute.
5
Add 35 µl OHS2. Pipette to mix.
6
Centrifuge at 1000 × g for 1 minute.
7
Place on the preheated heat block and incubate for 1 minute.
8
With the plate on the heat block, reset the temperature to 40°C and continue
incubating for 80 minutes.
TruSight Myeloid Sequencing Panel Protocol Guide
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Remove Unbound Oligos
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 5 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 5 minutes.
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Document # 1000000005004 v00
Extend and Ligate Bound Oligos
Extend and Ligate Bound Oligos
Procedure
1
Add 45 µl ELM4 to the FPU plate.
2
Incubate at 37°C for 45 minutes.
TruSight Myeloid Sequencing Panel Protocol Guide
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Amplify Libraries
Preparation
1
Prepare fresh 50 mM NaOH from 10 N NaOH.
2
Label a new PCR plate IAP.
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 IAP plate on a TruSeq Index Plate Fixture.
4
Use a multichannel pipette to add 4 µl of each Index 1 (i7) adapter to each row.
Replace the cap on each i7 adapter tube with a new orange cap.
5
Use a multichannel pipette to add 4 µl of each Index 2 (i5) adapter to each column.
Replace the cap on each i5 adapter tube with a new white cap.
6
Add 56 µl TDP1 to 2.8 ml PMM2.
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Invert to mix.
8
When incubation is complete, remove the FPU plate from the incubator and remove
the seal.
9
Cover and centrifuge at 2400 × g for 5 minutes.
10 Use a multichannel pipette to add 25 µl 50 mM NaOH.
11 Incubate at room temperature for 5 minutes.
12 Transfer 22 µl PMM2/TDP1 master mix to the IAP plate.
13 Transfer samples eluted from the FPU plate to the IAP plate as follows.
a
b
c
Pipette to mix the NaOH in the first column.
Transfer 20 µl from the FPU plate to the IAP plate. Pipette to mix.
Discard the waste collection midi plate.
14 Centrifuge at 1000 × g for 1 minute.
15 Transfer to the post-amplification area.
16 Determine the required number (X) of PCR cycles using the following table:
Table 1 50–99 ng
Number of Amplicons in TSO
< 96 amplicons
97–384 amplicons
385–768 amplicons
769–1536 amplicons
Number of PCR Cycles (X)
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17 Perform PCR on a thermal cycler using the following program:
} 95°C for 3 minutes
} X cycles of:
} 95°C for 30 seconds
} 66°C for 30 seconds
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Document # 1000000005004 v00
Amplify Libraries
} 72°C for 60 seconds
} 72°C for 5 minutes
} Hold at 10°C
SAFE STOPPING POINT
If you are stopping, leave the plate on the thermal cycler at 2°C to 8°C overnight.
TruSight Myeloid Sequencing Panel Protocol Guide
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Clean Up Libraries
Preparation
1
Prepare fresh 80% EtOH from absolute ethanol.
2
Label 2 new midi plates CLP and LNP.
Procedure
1
Centrifuge the IAP plate at 1000 × g for 1 minute.
2
Run an aliquot of the libraries 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.
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Incubate at room temperature for 10 minutes.
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Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
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Remove and discard all supernatant from each well.
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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.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at 2°C to 8°C for up to 3 days. Alternatively,
store at -25°C to -15°C for up to 7 days.
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Document # 1000000005004 v00
Normalize Libraries
Normalize Libraries
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
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
Add 4.4 µl LNA1 per library 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 the LNA1/LNB1 mix to a trough.
5
Add 45 µl LNA1/LNB1 to the LNP plate.
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Shake at 1800 rpm for 30 minutes.
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Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
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Remove and discard all supernatant.
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Remove from the magnetic stand.
10 Wash 2 times with 45 µl LNW1.
11 Remove residual LNW1.
12 Remove from the magnetic stand.
13 Add 30 µl fresh 0.1 N NaOH.
14 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 the SGP plate.
17 Transfer 30 µl supernatant from the LNP plate to the SGP plate.
18 Centrifuge at 1000 × g for 1 minute.
TruSight Myeloid Sequencing Panel Protocol Guide
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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 # 1000000005004 v00
Preparation
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If the SGP plate was stored frozen, thaw at room temperature and then centrifuge at
1000 × g for 1 minute. Pipette to mix.
Procedure
1
Transfer 5 µl of each library from the SGP plate to an 8-tube strip, column by
column.
2
Seal the plate and store at -25°C to -15°C.
3
Transfer the contents of the 8-tube strip to the PAL tube.
4
Denature and dilute pooled libraries to the loading concentration for the instrument
you are using. See the denature and dilute libraries guide for your instrument.
TruSight Myeloid Sequencing Panel Protocol Guide
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Pool Libraries
Pool Libraries
Acronyms
Acronym
ACD1
Amplicon Control DNA 1
ACP1
Amplicon Control Oligo Pool 1
TSO
TruSight Oligos
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
Index 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
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Definition
Pooled Amplicon Library
Document # 1000000005004 v00
For technical assistance, contact Illumina Technical Support.
Table 2 Illumina General Contact Information
Website
Email
www.illumina.com
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Table 3 Illumina Customer Support Telephone Numbers
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Contact Number
Region
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1.800.809.4566
Japan
Australia
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Austria
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Denmark
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France
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Germany
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Hong Kong
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Ireland
1.800.812949
Other countries
Italy
800.874909
Safety data sheets (SDSs)—Available on the Illumina website at
support.illumina.com/sds.html.
TruSight Myeloid Sequencing Panel Protocol Guide
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
Technical Assistance
Technical Assistance
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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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