TruSeq Exome Library Prep Protocol Guide (15059912 v01)

TruSeq Exome Library Prep Protocol Guide (15059912 v01)
TruSeq Exome Library Prep Kit
Protocol Guide
For Research Use Only. Not for use in diagnostic procedures.
Fragment DNA
Repair Ends and Select Library Size
Adenylate 3ʹ Ends
Ligate Adapters
Enrich DNA Fragments
Validate Libraries
Hybridize Probes
Capture Hybridized Probes
Perform Second Hybridization
Perform Second Capture
Clean Up Captured Library
Amplify Enriched Library
Clean Up Amplified Enriched Library
Validate Enriched Libraries
Acronyms
Technical Assistance
ILLUMINA PROPRIETARY
Document # 15059912 v01
November 2015
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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).
© 2015 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,
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TruSeq, TruSight, Understand Your Genome, UYG, VeraCode, verifi, VeriSeq, the pumpkin orange color, and the
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names, logos, and other trademarks are the property of their respective owners.
Preparation
1
Turn on and set up the Covaris instrument according to manufacturer guidelines.
2
[Plate] Calibrate the microplate shaker with a stroboscope and set it to 1200 rpm.
Procedure
1
Quantify gDNA using a fluorometric-based method.
2
Mix 5 ml RSB and 10 μl EDTA.
3
Normalize 100 ng gDNA with shearing buffer premix to 50 μl, and then mix
thoroughly as follows.
} [Plate] Shake at 1200 rpm for 2 minutes.
} [Tube] Pipette up and down.
4
Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
5
Transfer 50 μl DNA to Covaris tubes or plate wells.
6
Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
7
Fragment the DNA using the following Covaris settings.
Covaris Setting
Duty Factor (%)
Intensity
Peak Power (W)
Cycles/Burst
Duration (seconds)
Temperature (°C)
Water Level
Intensifier
M220
S2
S220
E220
LE220
20
—
50
200
375
20
—
—
10
5
—
200
280
7
12
—
10
—
175
200
280
7
12
—
10
—
175
200
280
7
6
Yes
30
—
450
200
360/rack; 420/tube
7
6
—
8
Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
9
Transfer 50 μl supernatant to a new midi plate or to a new 1.5 ml microcentrifuge
tube or 8-tube strip.
10 Add 100 μl SPB, and then mix thoroughly as follows.
} [Plate] Shake at 1200 rpm for 2 minutes.
} [Tube] Pipette up and down.
11 Incubate at room temperature for 5 minutes.
TruSeq Exome Library Prep Protocol Guide
3
Fragment DNA
Fragment DNA
12 Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
13 Place on a magnetic stand and wait until the liquid is clear (~8 minutes).
14 Remove and discard all supernatant.
15 Wash 2 times with 200 μl 80% EtOH.
16 Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
17 Incubate on the magnetic stand for 30 seconds.
18 Use a 20 μl pipette to remove residual EtOH.
19 Air-dry on the magnetic stand until dry (~5 minutes).
20 Add 62.5 μl RSB.
21 Remove from the magnetic stand, and then mix thoroughly as follows.
} [Plate] Shake at 1200 rpm for 2 minutes.
} [Tube] Pipette up and down.
22 Incubate at room temperature for 2 minutes.
23 Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
24 Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
25 Transfer 60 μl supernatant to a new Hard-Shell PCR plate or to a new 8-tube strip.
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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Document # 15059912 v01
Repair Ends and Select Library Size
Repair Ends and Select Library Size
Preparation
1
[Plate] Preheat the microheating system to 30°C.
2
[Tube] Save the following ERP program on the thermal cycler:
} Choose the preheat lid option and set to 100°C
} 30°C for 30 minutes
} Hold at 4°C
} Each tube contains 100 μl.
Procedure
1
Add 40 μl ERP3, and then mix thoroughly as follows.
} [Plate] Shake at 1200 rpm for 2 minutes.
} [Tube] Pipette up and down.
2
Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
3
Incubate as follows.
} [Plate] Place on the 30°C microheating system with the lid closed for 30 minutes,
and then place on ice.
} [Tube] Place on the thermal cycler and run the ERP program.
4
Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
5
Add 90 μl SPB, and then mix thoroughly as follows.
} [Plate] Shake at 1200 rpm for 2 minutes.
} [Tube] Pipette up and down.
6
Incubate at room temperature for 5 minutes.
7
Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
8
Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
9
Transfer 185 μl supernatant to a new midi plate or to a new 1.5 ml microcentrifuge
tube.
10 Add 125 μl SPB, and then mix thoroughly as follows.
} [Plate] Shake at 1200 rpm for 2 minutes.
} [Tube] Pipette up and down.
11 Incubate at room temperature for 5 minutes.
12 Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
TruSeq Exome Library Prep Protocol Guide
5
13 Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
14 Remove and discard all supernatant.
15 Wash 2 times with 200 μl 80% EtOH.
16 Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
17 Incubate on the magnetic stand for 30 seconds.
18 Use a 20 μl pipette to remove residual EtOH.
19 Air-dry on the magnetic stand until dry (~5 minutes).
20 Add 20 μl RSB.
21 Remove from the magnetic stand, and then mix thoroughly as follows.
} [Plate] Shake at 1200 rpm for 2 minutes.
} [Tube] Pipette up and down.
22 Incubate at room temperature for 2 minutes.
23 Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
24 Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
25 Transfer 17.5 μl supernatant to a new Hard-Shell PCR plate or to a new 8-tube strip.
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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Document # 15059912 v01
Adenylate 3ʹ Ends
Adenylate 3ʹ Ends
Preparation
1
[Plate] Preheat 2 microheating systems, one to 37°C and another to 70°C.
2
[Tube] Save the following ATAIL70 program on the thermal cycler:
} Choose the preheat lid option and set to 100°C
} 37°C for 30 minutes
} 70°C for 5 minutes
} 4°C for 5 minutes
} Hold at 4°C
} Each tube contains 30 μl.
Procedure
1
Add 12.5 μl ATL2, and then mix thoroughly as follows.
} [Plate] Shake at 1200 rpm for 2 minutes.
} [Tube] Pipette up and down.
2
Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
3
[Plate] Incubate as follows.
a
b
c
Place on the 37°C microheating system with the lid closed for 30 minutes.
Move to the 70°C microheating system with the lid closed for 5 minutes.
Place on ice for 5 minutes.
4
[Tube] Place on the thermal cycler and run the ATAIL70 program.
5
Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
TruSeq Exome Library Prep Protocol Guide
7
Ligate Adapters
Preparation
1
[Plate] Preheat a microheating system to 30°C.
2
[Tube] Save the following LIG program on the thermal cycler:
} Choose the preheat lid option and set to 100°C
} 30°C for 10 minutes
} Hold at 4°C
} Each tube contains 37.5 μl.
Procedure
1
Add the following reagents in the order listed.
} RSB (2.5 μl)
} LIG2 (2.5 μl)
} DNA adapters (2.5 μl)
2
Mix thoroughly as follows.
} [Plate] Shake at 1200 rpm for 2 minutes.
} [Tube] Pipette up and down.
3
Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
4
Incubate as follows.
} [Plate] Place on the 30°C microheating system with the lid closed for 10 minutes,
and then place on ice.
} [Tube] Place on the thermal cycler and run the LIG program.
5
Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
6
Add 5 μl STL, and then mix thoroughly as follows.
} [Plate] Shake at 1200 rpm for 2 minutes.
} [Tube] Pipette up and down.
7
Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
8
Perform steps 9 through 24 using the Round 1 volumes.
9
Add SPB.
SPB
Round 1
42.5 μl
Round 2
50 μl
10 Mix thoroughly as follows.
} [Plate] Shake at 1200 rpm for 2 minutes.
} [Tube] Pipette up and down.
8
Document # 15059912 v01
Ligate Adapters
11 Incubate at room temperature for 5 minutes.
12 Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
13 Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
14 Remove and discard all supernatant.
15 Wash 2 times with 200 μl 80% EtOH.
16 Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
17 Incubate on the magnetic stand for 30 seconds.
18 Use a 20 μl pipette to remove residual EtOH.
19 Air-dry on the magnetic stand until dry (~5 minutes).
20 Add RSB.
RSB
Round 1
52.5 μl
Round 2
27.5 μl
21 Mix thoroughly as follows.
} [Plate] Shake at 1200 rpm for 2 minutes.
} [Tube] Pipette up and down.
22 Incubate at room temperature for 2 minutes.
23 Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
24 Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
25 Transfer 50 μl supernatant to a new midi plate or to a new 1.5 ml microcentrifuge
tube or 8-tube strip.
26 Repeat steps 9 through 24 with the new plate or tube using the Round 2 volumes.
27 Transfer 25 μl supernatant to a new Hard-Shell PCR plate or to a new 8-tube strip.
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.
TruSeq Exome Library Prep Protocol Guide
9
Enrich DNA Fragments
Preparation
1
Save the following PCRNano program on the thermal cycler:
} Choose the preheat lid option and set to 100°C
} 95°C for 3 minutes
} 8 cycles of:
} 98°C for 20 seconds
} [Plate] 60°C for 20 seconds
} [Tube] 60°C for 15 seconds
} 72°C for 30 seconds
} 72°C for 5 minutes
} Hold at 4°C
} Each well or tube contains 50 μl.
Procedure
1
Place the plate or tube on ice and add 5 μl PPC.
2
Add 20 μl EPM, and then mix thoroughly as follows.
} [Plate] Shake at 1200 rpm for 20 seconds.
} [Tube] Pipette up and down.
3
Centrifuge briefly.
4
Place on the thermal cycler and run the PCRNano program.
5
Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
6
Add 35 μl SPB.
7
Mix thoroughly, as follows.
} [Plate] Shake at 1200 rpm for 2 minutes.
} [Tube] Pipette up and down.
8
Incubate at room temperature for 5 minutes.
9
Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
10 Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
11 Transfer 82 μl supernatant to a new Hard-Shell PCR plate or to a new 1.5 ml
microcentrifuge tube or 8-tube strip.
12 Add 82 μl SPB, and then mix thoroughly as follows.
} [Plate] Shake at 1200 rpm for 2 minutes.
} [Tube] Pipette up and down.
13 Incubate at room temperature for 5 minutes.
14 Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
10
Document # 15059912 v01
Enrich DNA Fragments
15 Remove and discard all supernatant.
16 Wash 2 times with 200 μl 80% EtOH.
17 Centrifuge briefly.
18 Incubate on the magnetic stand for 30 seconds.
19 Use a 20 μl pipette to remove residual EtOH.
20 Air-dry on the magnetic stand until dry (~5 minutes).
21 Add 17.5 μl RSB, and then mix thoroughly as follows.
} [Plate] Shake at 1200 rpm for 2 minutes.
} [Tube] Pipette up and down.
22 Incubate at room temperature for 2 minutes.
23 Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
24 Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
25 Transfer 15 μl supernatant to a new Hard-Shell PCR plate or to a new 1.5 ml
microcentrifuge tube or 8-tube strip.
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.
TruSeq Exome Library Prep Protocol Guide
11
Validate Libraries
Quantify Libraries
1
Quantify the libraries using the Qubit dsDNA HS Assay Kit.
a
b
c
d
Use 1 μl as the loading volume.
Use the dsDNA and high sensitivity settings.
Record STD1 and STD2 readings.
Measure the library concentration in duplicate and use the average.
Check Library Quality
1
12
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.
Document # 15059912 v01
Hybridize Probes
Hybridize Probes
Preparation
1
Save the TE HYB program on the thermal cycler:
} Choose the preheat lid option and set to 100°C
} 95°C for 10 minutes
} 18 cycles of 1 minute each, starting at 94°C, then decreasing 2°C per cycle
} 58°C for 90 minutes
} Hold at 58°C
} Each tube contains 100 μl.
Pool Libraries
1
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
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.
Procedure
1
Add the following reagents in the order listed to a new 8-tube strip. Pipette to mix.
} DNA library pool (40 μl)
} CT3 (50 μl)
} CEX (10 μl)
2
Centrifuge briefly.
3
Place on the thermal cycler and run the TE HYB program.
4
Keep at the 58°C holding temperature for at least 90 minutes and up to 24 hours.
TruSeq Exome Library Prep Protocol Guide
13
Capture Hybridized Probes
Preparation
1
Preheat a heat block to 50°C.
Procedure
1
Add 250 μl SMB to a new 1.5 ml microcentrifuge tube.
2
Immediately transfer the total sample volume (~100 μl) from the thermal cycler to
the 1.5 ml microcentrifuge tube containing SMB. Pipette to mix.
3
Incubate at room temperature for 25 minutes.
4
Centrifuge briefly.
5
Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
6
Remove and discard all supernatant.
7
Remove from the magnetic stand.
8
Add 200 μl SWS. Pipette to mix.
9
Place on the 50°C heat block for 30 minutes.
10 Immediately place on a magnetic stand and wait until the liquid is clear
(~2 minutes).
11 Remove and discard all supernatant.
12 Remove from the magnetic stand.
13 Repeat steps 8–12 for a total of 2 washes.
14 Mix 28.5 μl EE1 and 1.5 μl HP3, and then vortex.
15 Add 23 μl elution premix. Pipette to mix.
16 Incubate at room temperature for 2 minutes.
17 Centrifuge briefly.
18 Place on a magnetic stand and wait until the liquid is clear (~5 minutes).
19 Transfer 21 μl supernatant to a new 8-tube strip.
20 Add 4 μl ET2. Pipette to mix.
21 Centrifuge briefly.
14
Document # 15059912 v01
Procedure
1
Add the following reagents in the order listed to the 8-tube strip. Pipette to mix.
} DNA library pool (25 μl)
} RSB (15 μl)
} CT3 (50 μl)
} CEX (10 μl)
2
Centrifuge briefly.
3
Place on the thermal cycler and run the TE HYB program.
4
Keep at the 58°C holding temperature for at least 14.5 hours and up to 24 hours.
TruSeq Exome Library Prep Protocol Guide
15
Perform Second Hybridization
Perform Second Hybridization
Perform Second Capture
Preparation
1
Preheat a heat block to 50°C.
Procedure
1
Add 250 μl SMB to a new 1.5 ml microcentrifuge tube.
2
Immediately transfer the total sample volume (~100 μl) from the thermal cycler to
the 1.5 ml microcentrifuge tube containing SMB. Pipette to mix.
3
Incubate at room temperature for 25 minutes.
4
Centrifuge briefly.
5
Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
6
Remove and discard all supernatant.
7
Remove from the magnetic stand.
8
Add 200 μl SWS. Pipette to mix.
9
Place on the 50°C heat block for 30 minutes.
10 Immediately place on a magnetic stand and wait until the liquid is clear
(~2 minutes).
11 Remove and discard all supernatant.
12 Remove from the magnetic stand.
13 Repeat steps 8–12 for a total of 2 washes.
14 Mix 28.5 μl EE1 and 1.5 μl HP3, and then vortex.
15 Add 23 μl elution premix. Pipette to mix.
16 Incubate at room temperature for 2 minutes.
17 Centrifuge briefly.
18 Place on a magnetic stand and wait until the liquid is clear (~5 minutes).
19 Transfer 21 μl supernatant to a new Hard-Shell PCR plate or to a new 1.5 ml
microcentrifuge tube or 8-tube strip.
20 Add 4 μl ET2, and then mix thoroughly as follows.
} [Plate] Shake at 1200 rpm for 1 minute.
} [Tube] Pipette up and down.
21 Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
16
Document # 15059912 v01
Clean Up Captured Library
Clean Up Captured Library
Procedure
1
Add 45 μl SPB. Pipette to mix.
2
Incubate at room temperature for 5 minutes.
3
Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
4
Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
5
Remove and discard all supernatant.
6
Wash 2 times with 200 μl 80% EtOH.
7
Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
8
Incubate on the magnetic stand for 30 seconds.
9
Use a 20 μl pipette to remove residual EtOH.
10 Air-dry on the magnetic stand until dry (~5 minutes).
11 Add 27.5 μl RSB. Pipette to mix.
12 Incubate at room temperature for 2 minutes.
13 Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
14 Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
15 Transfer 25 μl supernatant to a new Hard-Shell PCR plate or to a new 8-tube strip.
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.
TruSeq Exome Library Prep Protocol Guide
17
Amplify Enriched Library
Preparation
1
Save the following AMP8 program on the thermal cycler:
} Choose the preheat lid option and set to 100°C
} 98°C for 30 seconds
} 8 cycles of:
} 98°C for 10 seconds
} [Plate] 60°C for 35 seconds
} [Tube] 60°C for 30 seconds
} 72°C for 30 seconds
} 72°C for 5 minutes
} Hold at 4°C
} Each well or tube contains 50 μl.
Procedure
1
Add 5 μl PPC.
2
Add 20 μl NEM. Pipette to mix.
3
Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
4
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.
18
Document # 15059912 v01
Clean Up Amplified Enriched Library
Clean Up Amplified Enriched Library
Procedure
1
Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
2
Add 45 μl SPB. Pipette to mix.
3
Incubate at room temperature for 5 minutes.
4
Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
5
Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
6
Remove and discard all supernatant.
7
Wash 2 times with 200 μl 80% EtOH.
8
Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
9
Incubate on the magnetic stand for 30 seconds.
10 Use a 20 μl pipette to remove residual EtOH.
11 Air-dry on the magnetic stand until dry (~5 minutes).
12 Add 22 μl RSB. Pipette to mix.
13 Incubate at room temperature for 2 minutes.
14 Centrifuge as follows.
} [Plate] Centrifuge at 280 × g for 1 minute.
} [Tube] Centrifuge briefly.
15 Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
16 Transfer 20 μl supernatant to a new Hard-Shell PCR plate or to a new 1.5 ml
microcentrifuge tube or 8-tube strip.
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.
TruSeq Exome Library Prep Protocol Guide
19
Validate Enriched Libraries
Quantify Libraries
1
Quantify the postenriched library using the Qubit dsDNA HS Assay Kit.
a
b
c
d
Use 1 μl as the loading volume.
Use the dsDNA and high sensitivity settings.
Record STD1 and STD2 readings.
Measure the library concentration in duplicate and use the average of the 2
measurements.
Assess Quality
1
20
Run 1 μl of post enriched library using a High Sensitivity DNA chip.
Document # 15059912 v01
Acronyms
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
TruSeq Exome Library Prep Protocol Guide
21
Notes
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
Italy
Australia
1.800.775.688
Netherlands
Austria
0800.296575
New Zealand
Belgium
0800.81102
Norway
Denmark
80882346
Spain
Finland
0800.918363
Sweden
France
0800.911850
Switzerland
Germany
0800.180.8994
United Kingdom
Ireland
1.800.812949
Other countries
Contact Number
800.874909
0800.0223859
0800.451.650
800.16836
900.812168
020790181
0800.563118
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 Exome Library Prep Protocol Guide
Technical Assistance
Technical Assistance
Illumina
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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