TruSight One Sequencing Panel Protocol Guide (1000000006694 v00)

TruSight One Sequencing Panel Protocol Guide (1000000006694 v00)

TruSight One Sequencing Panel

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

9

10

11

7

8

5

6

3

4

12

13

14

15

17

ILLUMINA PROPRIETARY

Document # 1000000006694 v00

January 2016

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,

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.

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 (5 μl)

}

PCR-grade water (10 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 One Sequencing Panel Protocol Guide

3

Clean Up Tagmented DNA

Preparation

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.

4

Document # 1000000006694 v00

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.

5 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 One Sequencing Panel Protocol Guide

5

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.

6

Document # 1000000006694 v00

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)

} TOO (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 One Sequencing Panel Protocol Guide

7

Capture Hybridized Probes

Preparation

1 Preheat a microheating system with midi plate insert to 50°C.

Procedure

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.

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 HP3, 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 Hard-Shell PCR plate.

18 Add 4 μl ET2.

19 Shake at 1200 rpm for 1 minute.

20 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.

8

Document # 1000000006694 v00

Perform Second Hybridization

Procedure

1 Add the following reagents in the order listed.

}

RSB (15 μl)

} EHB (50 μl)

}

TOO (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 One Sequencing Panel Protocol Guide

9

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 HP3, 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.

10

Document # 1000000006694 v00

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 One Sequencing Panel Protocol Guide

11

Amplify Enriched Library

Preparation

1 Save the following NEM AMP10 program on the thermal cycler:

}

Choose the preheat lid option and set to 100°C

}

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 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 AMP10 program.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at 2°C to 8°C for up to 2 days.

12

Document # 1000000006694 v00

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 One Sequencing Panel Protocol Guide

13

Check Enriched Libraries

Quantify Libraries

1 Quantify the enriched library using a fluorometric method.

Assess Quality

[Optional]

1 Run 1 μl post enriched library using a High Sensitivity DNA chip.

14

Document # 1000000006694 v00

Acronyms

PPC

RSB

SMB

SPB

ST

TD

TDE1

TOO

NEM

NEW1

NEW2

NIL

NLA

NLC

NLM

NLT

Acronym

EE1

EHB

ET2

EWS

HP3

NEC1

NEC2

NEH1

NEH2

NEL

Definition

Enrichment Elution Buffer 1

Enrichment Hybridization Buffer

Elute Target Buffer 2

Enrichment Wash Solution

2N NaOH

Nextera Enriched Clean Up Plate 1

Nextera Enriched Clean Up Plate 2

Nextera Enrichment Hyb Plate 1

Nextera Enrichment Hyb Plate 2

Nextera Enrichment Library Plate

Enrichment Amp Mix

Nextera Enrichment Wash Plate 1

Nextera Enrichment Wash Plate 2

Nextera Index Library Plate

Nextera Library Amplification Plate

Nextera Library Clean Up Plate

Library Amp Mix

Nextera Library Tagment Plate

PCR Primer Cocktail

Resuspension Buffer

Streptavidin Magnetic Beads

Sample Purification Beads

Stop Tagment Buffer

Tagment DNA Buffer

Tagment DNA Enzyme TDE

TruSight One Oligos

TruSight One Sequencing Panel Protocol Guide

15

Notes

Technical Assistance

For technical assistance, contact Illumina Technical Support.

Table 1 Illumina General Contact Information

Website

www.illumina.com

Email

[email protected]

Table 2 Illumina Customer Support Telephone Numbers

Region

North America

Australia

Austria

Belgium

China

Denmark

Finland

France

Germany

Hong Kong

Ireland

Italy

Contact Number

1.800.809.4566

1.800.775.688

0800.296575

0800.81102

400.635.9898

80882346

0800.918363

0800.911850

0800.180.8994

800960230

1.800.812949

800.874909

Region

Japan

Netherlands

New Zealand

Norway

Singapore

Spain

Sweden

Switzerland

Taiwan

United Kingdom

Other countries

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 One Sequencing Panel Protocol Guide

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

Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project