TruSeq Exome Library Prep Checklist (15059914 v01)

TruSeq Exome Library Prep Checklist (15059914 v01)
For Research Use Only. Not for
use in diagnostic procedures.
TruSeq Exome Library Prep
Fragment DNA
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Mix 5 ml RSB and 10 μl EDTA.
Normalize 100 ng gDNA with premix to 50 μl
and mix.
Centrifuge.
Transfer 50 μl DNA to Covaris tubes or plate
wells.
Centrifuge.
Fragment the DNA using the following settings.
Setting
Duty Factor
Intensity
Peak Power
Cycles/Burst
Duration
M220
S2
S220
E220
LE220
20
—
50
200
375
10
5
—
200
280
10
—
175
200
280
10
—
175
200
280
Temp.
Water Level
Intensifier
20
—
—
7
12
—
7
12
—
7
6
Yes
30
—
450
200
360/rack
420/tube
7
6
—
□22 Centrifuge.
□23 Place on a magnetic stand until liquid is clear.
□24 Transfer 60 μl supernatant.
SAFE STOPPING POINT
If you are stopping, seal the plate or cap the tube
and store at -25°C to -15°C for up to 7 days.
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□7 Centrifuge.
□8 Transfer 50 μl supernatant.
□9 Add 100 μl SPB and mix.
□10 Incubate at room temperature for 5 minutes.
□11 Centrifuge.
□12 Place on a magnetic stand until liquid is clear.
□13 Remove and discard all supernatant.
□14 Wash 2 times with 200 μl 80% EtOH.
□15 Centrifuge.
□16 Incubate on the magnetic stand for 30 seconds.
□17 Use a 20 μl pipette to remove residual EtOH.
□18 Air-dry until dry.
□19 Add 62.5 μl RSB.
□20 Remove from the magnetic stand and mix.
□21 Incubate at room temperature for 2 minutes.
Document # 15059914 v01
Repair Ends and Select Library Size
Add 40 μl ERP3 and mix.
Centrifuge.
Incubate as follows.
} [Plate] Place on the 30°C microheating system
for 30 minutes, and then place on ice.
} [Tube] Place on the thermal cycler and run the
ERP program.
Centrifuge.
Add 90 μl SPB and mix.
Incubate at room temperature for 5 minutes.
Centrifuge.
Place on a magnetic stand until liquid is clear.
Transfer 185 μl supernatant.
Add 125 μl SPB and mix.
Incubate at room temperature for 5 minutes.
Centrifuge.
Place on a magnetic stand until liquid is clear.
Remove and discard all supernatant.
Wash 2 times with 200 μl 80% EtOH.
Centrifuge.
Incubate on the magnetic stand for 30 seconds.
Use a 20 μl pipette to remove residual EtOH.
Air-dry until dry.
Add 20 μl RSB.
Remove from the magnetic stand and mix.
Incubate at room temperature for 2 minutes.
Centrifuge.
Place on a magnetic stand until liquid is clear.
Transfer 17.5 μl supernatant.
SAFE STOPPING POINT
If you are stopping, seal the plate or cap the tube
and store at -25°C to -15°C for up to 7 days.
November 2015
ILLUMINA PROPRIETARY
Page 1 of 6
For Research Use Only. Not for
use in diagnostic procedures.
TruSeq Exome Library Prep
Adenylate 3ʹ Ends
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Add 12.5 μl ATL2 and mix.
Centrifuge.
[Plate] Incubate as follows.
a Place on the 37°C microheating system for
30 minutes.
b Move to the 70°C microheating system for
5 minutes.
c Place on ice for 5 minutes.
[Tube] Place on the thermal cycler and run the
ATAIL70 program.
Centrifuge.
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Ligate Adapters
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Add the following.
} RSB (2.5 μl)
} LIG2 (2.5 μl)
} DNA adapters (2.5 μl)
Mix thoroughly.
Centrifuge.
Incubate as follows.
} [Plate] Place on the 30°C microheating system
for 10 minutes, and then place on ice.
} [Tube] Place on the thermal cycler and run the
LIG program.
Centrifuge.
Add 5 μl STL and mix.
Centrifuge.
Perform steps 9 through 24 using the Round 1
volumes.
Add SPB.
SPB
Round 1
42.5 μl
□21 Mix thoroughly.
□22 Incubate at room temperature for 2 minutes.
□23 Centrifuge.
□24 Place on a magnetic stand until liquid is clear.
□25 Transfer 50 μl supernatant to a new plate or to a
new tube.
□26 Repeat steps 9 through 24 with the new plate or
tube using the Round 2 volumes.
□27 Transfer 25 μl supernatant.
SAFE STOPPING POINT
If you are stopping, seal the plate or cap the tube
and store at -25°C to -15°C for up to 7 days.
Round 2
50 μl
□10 Mix thoroughly.
□11 Incubate at room temperature for 5 minutes.
□12 Centrifuge.
□13 Place on a magnetic stand until liquid is clear.
□14 Remove and discard all supernatant.
□15 Wash 2 times with 200 μl 80% EtOH.
□16 Centrifuge.
□17 Incubate on the magnetic stand for 30 seconds.
□18 Use a 20 μl pipette to remove residual EtOH.
□19 Air-dry until dry.
□20 Add RSB.
RSB
Page 2 of 6
Round 1
52.5 μl
Round 2
27.5 μl
November 2015
ILLUMINA PROPRIETARY
Document # 15059914 v01
For Research Use Only. Not for
use in diagnostic procedures.
TruSeq Exome Library Prep
Enrich DNA Fragments
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Place on ice and add 5 μl PPC.
Add 20 μl EPM and mix.
Centrifuge.
Place on the thermal cycler and run the
PCRNano program.
Centrifuge.
Add 35 μl SPB.
Mix thoroughly.
Incubate at room temperature for 5 minutes.
Centrifuge.
Place on a magnetic stand until liquid is clear.
Transfer 82 μl supernatant.
Add 82 μl SPB and mix.
Incubate at room temperature for 5 minutes.
Place on a magnetic stand until liquid is clear.
Remove and discard all supernatant.
Wash 2 times with 200 μl 80% EtOH.
Centrifuge.
Incubate on the magnetic stand for 30 seconds.
Use a 20 μl pipette to remove residual EtOH.
Air-dry until dry.
Add 17.5 μl RSB and mix.
Incubate at room temperature for 2 minutes.
Centrifuge.
Place on a magnetic stand until liquid is clear.
Transfer 15 μl supernatant.
Validate Libraries
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Quantify the libraries using the Qubit dsDNA HS
Assay Kit.
a Use 1 μl as the loading volume.
b Use the dsDNA and high sensitivity
settings.
c Record STD1 and STD2 readings.
d Measure the library concentration in
duplicate and use the average.
Check the library size distribution:
} If using a High Sensitivity DNA chip:
} Dilute the DNA library 1:10 with RSB.
} Run 1 μl diluted DNA library.
} If using a DNA 1000 chip, run 1 μl undiluted
DNA library.
Hybridize Probes
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Combine the following amount of each DNA
library, making sure that each library has a
unique index.
Plexity
3-plex
6-plex
9-plex
12-plex
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Each Library
250 ng
200 ng
150 ng
100 ng
Total Pool
750 ng
1200 ng
1350 ng
1200 ng
} If the total volume is > 40 μl, concentrate the
pooled sample to 40 μl.
} If the total volume is < 40 μl, increase the
volume to 40 μl with RSB.
Add the following to a new tube. Pipette to mix.
} DNA library pool (40 μl)
} CT3 (50 μl)
} CEX (10 μl)
Centrifuge.
Place on the thermal cycler and run the TE HYB
program.
Keep at the 58°C holding temperature for at least
90 minutes and up to 24 hours.
SAFE STOPPING POINT
If you are stopping, seal the plate or cap the tube
and store at -25°C to -15°C for up to 7 days.
Document # 15059914 v01
November 2015
ILLUMINA PROPRIETARY
Page 3 of 6
For Research Use Only. Not for
use in diagnostic procedures.
TruSeq Exome Library Prep
Capture Hybridized Probes
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Add 250 μl SMB to a new tube.
Immediately transfer the sample to the tube
containing SMB. Pipette to mix.
Incubate at room temperature for 25 minutes.
Centrifuge.
Place on a magnetic stand until liquid is clear.
Remove and discard all supernatant.
Remove from the magnetic stand.
Add 200 μl SWS. Pipette to mix.
Place on the 50°C heat block for 30 minutes.
Place on a magnetic stand until liquid is clear.
Remove and discard all supernatant.
Remove from the magnetic stand.
Repeat steps 8–12 for a total of 2 washes.
Mix 28.5 μl EE1 and 1.5 μl HP3, and then vortex.
Add 23 μl elution premix. Pipette to mix.
Incubate at room temperature for 2 minutes.
Centrifuge.
Place on a magnetic stand until liquid is clear.
Transfer 21 μl supernatant.
Add 4 μl ET2. Pipette to mix.
Centrifuge.
Page 4 of 6
Perform Second Hybridization
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Add the following to the tube. Pipette to mix.
} DNA library pool (25 μl)
} RSB (15 μl)
} CT3 (50 μl)
} CEX (10 μl)
Centrifuge.
Place on the thermal cycler and run the TE HYB
program.
Keep at the 58°C holding temperature for at least
14.5 hours and up to 24 hours.
November 2015
ILLUMINA PROPRIETARY
Perform Second Capture
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Add 250 μl SMB to a new tube.
Immediately transfer the sample to the tube
containing SMB. Pipette to mix.
Incubate at room temperature for 25 minutes.
Centrifuge.
Place on a magnetic stand until liquid is clear.
Remove and discard all supernatant.
Remove from the magnetic stand.
Add 200 μl SWS. Pipette to mix.
Place on the 50°C heat block for 30 minutes.
Place on a magnetic stand until liquid is clear.
Remove and discard all supernatant.
Remove from the magnetic stand.
Repeat steps 8–12 for a total of 2 washes.
Mix 28.5 μl EE1 and 1.5 μl HP3, and then vortex.
Add 23 μl elution premix. Pipette to mix.
Incubate at room temperature for 2 minutes.
Centrifuge.
Place on a magnetic stand until liquid is clear.
Transfer 21 μl supernatant.
Add 4 μl ET2 and mix.
Centrifuge.
Document # 15059914 v01
For Research Use Only. Not for
use in diagnostic procedures.
TruSeq Exome Library Prep
Clean Up Captured Library
□1 Add 45 μl SPB. Pipette to mix.
□2 Incubate at room temperature for 5 minutes.
□3 Centrifuge.
□4 Place on a magnetic stand until liquid is clear.
□5 Remove and discard all supernatant.
□6 Wash 2 times with 200 μl 80% EtOH.
□7 Centrifuge.
□8 Incubate on the magnetic stand for 30 seconds.
□9 Use a 20 μl pipette to remove residual EtOH.
□10 Air-dry until dry.
□11 Add 27.5 μl RSB. Pipette to mix.
□12 Incubate at room temperature for 2 minutes.
□13 Centrifuge.
□14 Place on a magnetic stand until liquid is clear.
□15 Transfer 25 μl supernatant.
Amplify Enriched Library
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Add 5 μl PPC.
Add 20 μl NEM. Pipette to mix.
Centrifuge.
Place on the thermal cycler and run the AMP8
program.
SAFE STOPPING POINT
If you are stopping, seal the plate or cap the tube
and store at 2°C to 8°C for up to 2 days.
Alternatively, leave on the thermal cycler overnight.
SAFE STOPPING POINT
If you are stopping, seal the plate or cap the tube
and store at -25°C to -15°C for up to 7 days.
Clean Up Amplified Enriched
Library
□1 Centrifuge.
□2 Add 45 μl SPB. Pipette to mix.
□3 Incubate at room temperature for 5 minutes.
□4 Centrifuge.
□5 Place on a magnetic stand until liquid is clear.
□6 Remove and discard all supernatant.
□7 Wash 2 times with 200 μl 80% EtOH.
□8 Centrifuge.
□9 Incubate on the magnetic stand for 30 seconds.
□10 Use a 20 μl pipette to remove residual EtOH.
□11 Air-dry until dry.
□12 Add 22 μl RSB. Pipette to mix.
□13 Incubate at room temperature for 2 minutes.
□14 Centrifuge.
□15 Place on a magnetic stand until liquid is clear.
□16 Transfer 20 μl supernatant.
SAFE STOPPING POINT
If you are stopping, seal the plate or cap the tube
and store at -25°C to -15°C for up to 7 days.
Document # 15059914 v01
November 2015
ILLUMINA PROPRIETARY
Page 5 of 6
TruSeq Exome Library Prep
Validate Enriched Libraries
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Quantify using the Qubit dsDNA HS Assay Kit.
a Use 1 μl as the loading volume.
b Use the dsDNA and high sensitivity
settings.
c Record STD1 and STD2 readings.
d Measure the library concentration
Run 1 μl using a High Sensitivity DNA chip.
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Page 6 of 6
For Research Use Only. Not for
use in diagnostic procedures.
Acronyms
Acronym
Definition
ATL2
A Tailing Mix
CEX
Coding Exome Oligos
CT3
Capture Target Buffer 3
DAP
DNA Adapter Plate
EE1
Enrichment Elution Buffer 1
EPM
Enhanced PCR Mix
ERP
End Repair Mix
ET2
Elute Target Buffer 2
HP3
2N NaOH
LIG
Ligation Mix
NEM
Enrichment Amplification Mix
PPC
PCR Primer Cocktail
RSB
Resuspension Buffer
SMB
Streptavidin Magnetic Beads
SPB
Sample Purification Beads
STL
Stop Ligation Buffer
SWS
Streptavidin Wash Solution
November 2015
ILLUMINA PROPRIETARY
Document # 15059914 v01
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