TruSight Rapid Capture Protocol Guide (1000000005002 v00)

TruSight Rapid Capture Protocol Guide (1000000005002 v00)
TruSight Rapid Capture
Protocol Guide
For Research Use Only. Not for use in diagnostic procedures.
Tagment Genomic DNA
Clean Up Tagmented DNA
Amplify Tagmented DNA
Clean Up Amplified DNA
Hybridize Probes
Capture Hybridized Probes
Perform Second Hybridization
Perform Second Capture
Clean Up Captured Library
Amplify Enriched Library
Clean Up Amplified Enriched Library
Check Enriched Libraries
Acronyms
Technical Assistance
ILLUMINA PROPRIETARY
Document # 1000000005002 v00
January 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
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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
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DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE).
© 2016 Illumina, Inc. All rights reserved.
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Tagment Genomic DNA
Tagment Genomic DNA
Preparation
1
Preheat a microheating system with midi plate insert to 58°C.
Procedure
1
Quantify gDNA using a fluorometric method.
2
Dilute gDNA in Tris-HCl 10 mM, pH 8.5 to a final volume of 10 μl at 5 ng/μl.
3
Add the following items in the order listed to a new midi plate.
} Normalized gDNA (10 μl)
} TD (25 μl)
} TDE1 (15 μl)
4
Shake at 1800 rpm for 1 minute.
5
Centrifuge at 280 × g for 1 minute.
6
Place on the 58°C microheating system with the lid closed for 10 minutes.
7
Add 15 μl ST.
8
Shake at 1800 rpm for 1 minute.
9
Centrifuge at 280 × g for 1 minute.
10 Incubate at room temperature for 4 minutes.
TruSight Rapid Capture Protocol Guide
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Clean Up Tagmented DNA
Procedure
1
Add 65 μl SPB.
2
Shake at 1800 rpm for 1 minute.
3
Incubate at room temperature for 8 minutes.
4
Centrifuge at 280 × g for 1 minute.
5
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
6
Remove and discard all supernatant.
7
Wash 2 times with 200 μl 80% EtOH.
8
Use a 20 μl pipette to remove residual EtOH.
9
Air-dry on the magnetic stand for 10 minutes.
10 Remove from the magnetic stand.
11 Add 22.5 μl RSB.
12 Shake at 1800 rpm for 1 minute.
13 Incubate at room temperature for 2 minutes.
14 Centrifuge at 280 × g for 1 minute.
15 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
16 Transfer 20 μl supernatant to a new Hard-Shell PCR plate.
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Document # 1000000005002 v00
Amplify Tagmented DNA
Amplify Tagmented DNA
Preparation
1
Save the following NLM AMP program on the thermal cycler:
} Choose the preheat lid option and set to 100°C
} 72°C for 3 minutes
} 98°C for 30 seconds
} 10 cycles of:
} 98°C for 10 seconds
} 60°C for 30 seconds
} 72°C for 30 seconds
} 72°C for 5 minutes
} Hold at 10°C
Procedure
1
Arrange Index 1 (i7) adapters in columns 1–12 of the TruSeq Index Plate Fixture.
2
Arrange Index 2 (i5) adapters in rows A–H of the TruSeq Index Plate Fixture.
3
Place the plate on the TruSeq Index Plate Fixture.
4
Using a multichannel pipette, add 5 μl of each Index 1 (i7) adapter down each
column. Replace the cap on each i7 adapter tube with a new orange cap.
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Using a multichannel pipette, add 5 μl of each Index 2 (i5) adapter across each row.
Replace the cap on each i5 adapter tube with a new white cap.
6
Add 20 μl NLM.
7
Shake at 1200 rpm for 1 minute.
8
Centrifuge at 280 × g for 1 minute.
9
Place on the thermal cycler and run the NLM AMP 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.
TruSight Rapid Capture Protocol Guide
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Clean Up Amplified DNA
Procedure
1
Centrifuge at 280 × g for 1 minute.
2
Transfer 50 μl supernatant to a new midi plate.
3
Add 90 μl SPB.
4
Shake at 1800 rpm for 1 minute.
5
Incubate at room temperature for 10 minutes.
6
Centrifuge at 280 × g for 1 minute.
7
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
8
Remove and discard all supernatant.
9
Wash 2 times with 200 μl 80% EtOH.
10 Use a 20 μl pipette to remove residual EtOH.
11 Air-dry on the magnetic stand for 10 minutes.
12 Add 27.5 μl RSB.
13 Shake at 1800 rpm for 1 minute.
14 Incubate at room temperature for 2 minutes.
15 Centrifuge at 280 × g for 1 minute.
16 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
17 Transfer 25 μl supernatant to a new Hard-Shell PCR plate.
18 Quantify the library using a fluorometric method.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at -25°C to -15°C for up to 14 days.
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Hybridize Probes
Hybridize Probes
Preparation
1
Save the NRC 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
} Hold at 58°C
Pool Libraries
1
Combine 500 ng of each DNA library. Make sure that each library has a unique
index.
} 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 items in the order listed to a new Hard-Shell PCR plate.
} DNA library sample or pool (40 μl)
} EHB (50 μl)
} CSO (10 μl)
2
Shake at 1200 rpm for 1 minute.
3
Centrifuge at 280 × g for 1 minute.
4
Place on the thermal cycler and run the NRC HYB program.
5
Keep at the 58°C holding temperature for at least 90 minutes and up to 24 hours.
TruSight Rapid Capture Protocol Guide
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Capture Hybridized Probes
First Bind
1
Centrifuge at 280 × g for 1 minute.
2
Transfer all volumes to a new plate.
3
Add 250 μl SMB.
4
Shake at 1200 rpm for 5 minutes.
5
Incubate at room temperature for 25 minutes.
6
Centrifuge at 280 × g for 1 minute.
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Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
8
Remove and discard all supernatant.
9
Remove from the magnetic stand.
First Wash
1
Wash 2 times with 200 μl EWS.
First Elution
1
Mix 28.5 μl EE1 and 1.5 μl 2 N NaOH, and then vortex.
2
Add 23.5 μl elution premix.
3
Shake at 1800 rpm for 2 minutes.
4
Incubate at room temperature for 2 minutes.
5
Centrifuge at 280 × g for 1 minute.
6
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
7
Transfer 21 μl supernatant to a new Hard-Shell PCR plate.
8
Add 4 μl ET2.
9
Shake at 1200 rpm for 1 minute.
10 Centrifuge at 280 × 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 7 days.
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Document # 1000000005002 v00
Perform Second Hybridization
Perform Second Hybridization
Procedure
1
Add the following reagents in the order listed.
} RSB (15 μl)
} EHB (50 μl)
} CSO (10 μl)
2
Shake at 1200 rpm for 1 minute.
3
Centrifuge at 280 × g for 1 minute.
4
Place on the thermal cycler and run the NRC HYB program.
5
Keep at the 58°C holding temperature for at least 14.5 hours and up to 24 hours.
TruSight Rapid Capture Protocol Guide
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Perform Second Capture
Preparation
1
Preheat a microheating system with midi plate insert to 50°C.
Procedure
1
Centrifuge at 280 × g for 1 minute.
2
Transfer supernatant to a new midi plate.
3
Add 250 μl SMB.
4
Shake at 1200 rpm for 5 minutes.
5
Incubate at room temperature for 25 minutes.
6
Centrifuge at 280 × g for 1 minute.
7
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
8
Remove and discard all supernatant.
9
Remove from the magnetic stand.
10 Wash 2 times with 200 μl EWS.
11 Mix 28.5 μl EE1 and 1.5 μl 2 N NaOH, and then vortex.
12 Add 23.5 μl elution premix.
13 Shake at 1800 rpm for 2 minutes.
14 Incubate at room temperature for 2 minutes.
15 Centrifuge at 280 × g for 1 minute.
16 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
17 Transfer 21 μl supernatant to a new midi plate.
18 Add 4 μl ET2.
19 Shake at 1800 rpm for 1 minute.
20 Centrifuge at 280 × g for 1 minute.
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Document # 1000000005002 v00
Clean Up Captured Library
Clean Up Captured Library
Procedure
1
Add 45 μl SPB.
2
Shake at 1800 rpm for 1 minute.
3
Incubate at room temperature for 10 minutes.
4
Centrifuge at 280 × g for 1 minute.
5
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
6
Remove and discard all supernatant.
7
Wash 2 times with 200 μl 80% EtOH.
8
Use a 20 μl pipette to remove residual EtOH.
9
Air-dry on the magnetic stand for 10 minutes.
10 Add 27.5 μl RSB.
11 Shake at 1800 rpm for 1 minute.
12 Incubate at room temperature for 2 minutes.
13 Centrifuge at 280 × g for 1 minute.
14 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
15 Transfer 25 μl supernatant to a new Hard-Shell PCR plate.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at -25°C to -15°C for up to 7 days.
TruSight Rapid Capture Protocol Guide
11
Amplify Enriched Library
Preparation
1
Save the following NEM AMP12 program on the thermal cycler:
} Choose the preheat lid option and set to 100°C
} 98°C for 30 seconds
} 12 cycles of:
} 98°C for 10 seconds
} 60°C for 30 seconds
} 72°C for 30 seconds
} 72°C for 5 minutes
} Hold at 10°C
Procedure
1
Add 5 μl PPC.
2
Add 20 μl NEM.
3
Shake at 1200 rpm for 1 minute.
4
Centrifuge at 280 × g for 1 minute.
5
Place on the thermal cycler and run the NEM AMP12 program.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at 2°C to 8°C for up to 2 days.
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Document # 1000000005002 v00
Clean Up Amplified Enriched Library
Clean Up Amplified Enriched Library
Procedure
1
Centrifuge at 280 × g for 1 minute.
2
Transfer 50 μl to a new midi plate.
3
Add 90 μl SPB.
4
Shake at 1800 rpm for 1 minute.
5
Incubate at room temperature for 10 minutes.
6
Centrifuge at 280 × g for 1 minute.
7
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
8
Remove and discard all supernatant.
9
Wash 2 times with 200 μl 80% EtOH.
10 Use a 20 μl pipette to remove residual EtOH.
11 Air-dry on the magnetic stand for 10 minutes.
12 Add 32.5 μl RSB.
13 Shake at 1800 rpm for 1 minute.
14 Incubate at room temperature for 2 minutes.
15 Centrifuge at 280 × g for 1 minute.
16 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
17 Transfer 30 μl supernatant to a new Hard-Shell PCR plate.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at -25°C to -15°C for up to 7 days.
TruSight Rapid Capture Protocol Guide
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Check Enriched Libraries
Quantify Libraries
1
Quantify the enriched library using a fluorometric method.
Assess Quality [Optional]
14
1
If the library concentration is higher than the supported quantitative range for the
High Sensitivity DNA chip, dilute the library 1:10 with RSB.
2
Run 1 μl post enriched library using a High Sensitivity DNA chip.
Document # 1000000005002 v00
Acronyms
Acronyms
Acronym
Definition
EE1
Enrichment Elution Buffer 1
EHB
Enrichment Hybridization Buffer
ET2
Elute Target Buffer 2
EWS
Enrichment Wash Solution
NEC1
Nextera Enriched Clean Up Plate 1
NEC2
Nextera Enriched Clean Up Plate 2
NEH1
Nextera Enrichment Hyb Plate 1
NEH2
Nextera Enrichment Hyb Plate 2
NEL
Nextera Enrichment Library Plate
NEM
Enrichment Amp Mix
NEW1
Nextera Enrichment Wash Plate 1
NEW2
Nextera Enrichment Wash Plate 2
NIL
Nextera Index Library Plate
NLA
Nextera Library Amplification Plate
NLC
Nextera Library Clean Up Plate
NLM
Library Amp Mix
NLT
Nextera Library Tagment Plate
PPC
PCR Primer Cocktail
RCO
Rapid Capture Oligos
RSB
Resuspension Buffer
SMB
Streptavidin Magnetic Beads
SPB
Sample Purification Beads
ST
Stop Tagment Buffer
TD
Tagment DNA Buffer
TDE1
Tagment DNA Enzyme TDE
TruSight Rapid Capture Protocol Guide
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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
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.
TruSight Rapid Capture Protocol Guide
Technical Assistance
Technical Assistance
*12345678*
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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