TruSight Cardio Sequencing Kit Reference Guide (15063774 v01)

TruSight Cardio Sequencing Kit Reference Guide (15063774 v01)

  Chapter 2  Protocol

Protocol

Introduction

Tips and Techniques

Library Prep Workflow

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

17

19

21

22

9

11

13

15

24

26

27

29

6

7

8

TruSight Cardio Sequencing Kit Reference Guide

5

Introduction

This chapter describes the TruSight Cardio protocol.

}

Follow the protocols in the order shown, using the specified volumes and incubation parameters.

} Review Best Practices from the TruSight Cardio support page on the Illumina website.

Prepare for Pooling

If you plan to pool libraries, record information about your samples before beginning library prep. Different methods are available depending on the sequencing instrument you are using. See the TruSight Cardio Sequencing Kit support page for more information.

6

Document # 15063774 v01

Tips and Techniques

Unless a safe stopping point is specified in the protocol, proceed immediately to the next step.

Avoiding Cross-Contamination

} When adding or transferring samples, change tips between each sample.

} When adding adapters or primers, change tips between each row and each column.

}

Remove unused index adapter tubes from the working area.

Sealing the Plate

}

Always seal the 96-well plate before the following steps in the protocol:

}

Shaking steps

}

Vortexing steps

} Centrifuge steps

}

Thermal cycling steps

}

Apply the adhesive seal to cover the plate and seal with a rubber roller.

}

Microseal 'B' adhesive seals are effective at -40°C to 110°C, and suitable for skirted or semiskirted PCR plates. Use Microseal 'B' for shaking, centrifuging, and long-term storage.

}

Microseal 'A' adhesive film is effective for thermal cycling and easy to cut when using fewer than 96 wells.

Plate Transfers

}

When transferring volumes between plates, transfer the specified volume from each well of a plate to the corresponding well of the other plate.

Centrifugation

}

Centrifuge at any step in the procedure to consolidate liquid or beads in the bottom of the well, and to prevent sample loss.

Handling Beads

}

Pipette bead suspension slowly.

}

When mixing, mix thoroughly.

}

If beads are aspirated into the pipette tips, dispense back to the plate on the magnetic stand and wait until the liquid is clear (~2 minutes).

} When washing beads:

}

Use the appropriate magnet for the plate.

}

Dispense liquid so that beads on the side of the wells are wetted.

} Keep the plate on the magnet until the instructions specify to remove it.

} Do not agitate the plate while on the magnetic stand. Do not disturb the bead pellet.

TruSight Cardio Sequencing Kit Reference Guide

7

Library Prep Workflow

The following diagram illustrates the workflow using a TruSight Cardio Sequencing Kit.

Safe stopping points are marked between steps.

Figure 1 TruSight Cardio Sequencing Kit Workflow

8

Document # 15063774 v01

Tagment Genomic DNA

This step uses the Nextera transposome to tagment gDNA, which is a process that fragments DNA and then tags the DNA with adapter sequences in a single step.

Consumables

} SPB (Sample Purification Beads)

} ST (Stop Tagment Buffer)

}

TD (Tagment DNA Buffer)

}

TDE1 (Tagment DNA Enzyme)

} gDNA (50 ng per sample)

} Tris-HCl 10 mM, pH 8.5

}

96-well midi plate (1)

}

Microseal 'B' adhesive seals

Preparation

1 Prepare the following consumables.

Item Storage Instructions

gDNA -25°C to -15°C Thaw on ice. Invert to mix, and then centrifuge briefly.

TD -25°C to -15°C Thaw on ice. Invert to mix, and then centrifuge briefly.

TDE1 -25°C to -15°C Thaw on ice. Invert to mix, and then centrifuge briefly. Set aside on ice.

SPB 2°C to 8°C Let stand for 30 minutes to bring to room temperature. Set aside at room temperature.

ST 15°C to 30°C Check for precipitates. If present, vortex until all particulates are resuspended.

2 Preheat a microheating system with midi plate insert to 58°C.

Procedure

Quantify and Normalize gDNA

1 Quantify gDNA using a fluorometric method, such as QuantiFluor or Qubit.

2 Normalize gDNA in Tris-HCl 10 mM, pH 8.5 to 10 ng/µl.

3 Requantify the normalized gDNA using the same fluorometric quantification method.

4 Dilute the normalized gDNA in Tris-HCl 10 mM, pH 8.5 to a final volume of 10 µl at

5 ng/µl (50 ng total).

Tagment DNA

1 Add the following items in the order listed to each well of a new midi plate.

}

Normalized gDNA (10 µl)

}

TD (25 µl)

} TDE1 (15 µl)

2 Shake at 1800 rpm for 1 minute.

3 Centrifuge at 280 × g for 1 minute.

TruSight Cardio Sequencing Kit Reference Guide

9

4 Place on the 58°C microheating system with the lid closed for 10 minutes.

5 Add 15 µl ST to each well.

6 Shake at 1800 rpm for 1 minute.

7 Centrifuge at 280 × g for 1 minute.

8 Incubate at room temperature for 4 minutes.

10

Document # 15063774 v01

Clean Up Tagmented DNA

This step uses SPB (Sample Purification Beads) to purify the tagmented DNA from the

Nextera transposome. The cleanup step removes the Nextera transposome that can otherwise bind to DNA ends and interfere with downstream processes.

Consumables

} RSB (Resuspension Buffer)

} SPB (Sample Purification Beads)

}

Freshly prepared 80% ethanol (EtOH)

}

96-well Hard-Shell 0.3 ml PCR plate

} Microseal 'B' adhesive seals

About Reagents

}

Vortex SPB before each use.

} Vortex SPB frequently to make sure that beads are evenly distributed.

}

Aspirate and dispense SPB slowly due to the viscosity of the solution.

Preparation

1 Prepare the following consumables:

Item

RSB

SPB

Storage

2°C to 8°C

2°C to 8°C

Instructions

Let stand for 30 minutes to bring to room temperature.

Let stand for 30 minutes to bring to room temperature.

2 Prepare fresh 80% EtOH.

Procedure

1 Add 65 µl SPB to each well.

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 from each well.

7 Wash 2 times as follows.

a Add 200 µl fresh 80% EtOH to each well.

b Incubate on the magnetic stand for 30 seconds.

c Remove and discard all supernatant from each well.

8 Use a 20 µl pipette to remove residual EtOH from each well.

9 Air-dry on the magnetic stand for 10 minutes.

10 Remove from the magnetic stand.

11 Add 22.5 µl RSB to each well.

12 Shake at 1800 rpm for 1 minute.

TruSight Cardio Sequencing Kit Reference Guide

11

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 the corresponding well of a new Hard-Shell PCR plate.

12

Document # 15063774 v01

Amplify Tagmented DNA

This step amplifies purified tagmented DNA and adds index adapters using a 10-cycle

PCR program. This PCR step adds Index 1 (i7) adapters, Index 2 (i5) adapters, and sequencing adapters required for cluster amplification.

Consumables

} Index 1 (i7) adapters and orange tube caps

} Index 2 (i5) adapters and white tube caps

}

NLM (Library Amp Mix)

}

1.7 ml microcentrifuge tubes (1 per index adapter tube)

} Microseal 'A' film

} Microseal 'B' adhesive seal

}

[Optional] TruSeq Index Plate Fixture Kit

NOTE

Use Microseal 'A' when sealing the plate before placing on the thermal cycler. Use Microseal

'B' for other steps that require a sealed plate.

Preparation

1 Prepare the following consumables.

Item

Index adapters

(i5 and i7)

NLM

Storage Instructions

-25°C to -15°C Only remove adapters being used. Thaw at room temperature for 20 minutes.

Vortex each tube to mix. Centrifuge briefly using a 1.7 ml

Eppendorf tube.

-25°C to -15°C Thaw on ice.

2 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.

TruSight Cardio Sequencing Kit Reference Guide

13

Figure 2 TruSeq Index Plate Fixture (96 libraries)

14

A Columns 1–12: Index 1 (i7) adapters (orange caps)

B Rows A–H: Index 2 (i5) adapters (white caps)

C 96-well plate

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 to each well.

7 Shake at 1200 rpm for 1 minute.

8 Centrifuge at 280 × g for 1 minute.

9 Place on the preprogrammed 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.

Document # 15063774 v01

Clean Up Amplified DNA

This step uses SPB (Sample Purification Beads) to purify the DNA library and remove unwanted products.

Consumables

} RSB (Resuspension Buffer)

} SPB (Sample Purification Beads)

}

Freshly prepared 80% ethanol (EtOH)

}

96-well Hard-Shell 0.3 ml PCR plate

} 96-well midi plate

} Microseal 'B' adhesive seals

About Reagents

} Vortex SPB before each use.

}

Vortex SPB frequently to make sure that beads are evenly distributed.

}

Aspirate and dispense SPB slowly due to the viscosity of the solution.

Preparation

1 Prepare the following consumables.

Item

RSB

SPB

Storage Instructions

2°C to 8°C Let stand for 30 minutes to bring to room temperature.

2°C to 8°C Let stand for 30 minutes to bring to room temperature.

2 Prepare fresh 80% EtOH.

Procedure

1 Centrifuge at 280 × g for 1 minute.

2 Transfer 50 µl supernatant to the corresponding well of a new midi plate.

3 Add 90 µl SPB to each well.

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 from each well.

9 Wash 2 times as follows.

a Add 200 µl fresh 80% EtOH to each well.

b Incubate on the magnetic stand for 30 seconds.

c Remove and discard all supernatant from each well.

10 Use a 20 µl pipette to remove residual EtOH from each well.

11 Air-dry on the magnetic stand for 10 minutes.

12 Add 27 µl RSB to each well.

TruSight Cardio Sequencing Kit Reference Guide

15

16

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 the corresponding well of a new Hard-Shell PCR plate.

18 Quantify the library using a fluorometric method, such as QuantiFluor or Qubit.

19 [Optional] Run 1 µl of the library on an Agilent Technologies 2100 Bioanalyzer using a

DNA 1000 chip.

Expect a distribution of DNA fragments with a size range from ~300 bp to ~1 kbp.

A sharp peak is not necessary, but most of the fragments must fall within the desired range. Traces can vary from library to library. The following traces show examples of possible distributions, but are not inclusive of successful libraries.

Figure 3 Example of Post-PCR, Pre-Enriched Library Distribution

NOTE

The sample peak must not be significantly shifted compared to the example shown in

Figure 3 , but traces can differ depending on sample quality. A larger peak distribution

(> 350 bp) can indicate > 50 ng gDNA input going into tagmentation, which can cause lower on-target reads. A smaller sample peak distribution (< 225 bp) can indicate < 50 ng gDNA or low quality gDNA, which can cause reduced library diversity or elevated duplicates.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at -25°C to -15°C for up to 14 days.

Document # 15063774 v01

Hybridize Probes

This step combines DNA libraries containing unique indexes into a single pool, and then binds targeted regions of the DNA with capture probes.

Consumables

} EHB (Enrichment Hybridization Buffer)

} TCO (TruSight Cardio Oligos)

}

RSB (Resuspension Buffer)

}

96-well Hard-Shell 0.3 ml PCR plate

} Microseal 'B' adhesive seal

} [Optional] Amicon Ultra-0.5 centrifugal filter unit (0.5 ml, 30 kDa) (1 per pooled sample)

About Reagents

} Before using EHB, vortex to resuspend the solution. Make sure that no crystal structures are present. If crystals and cloudiness are observed, vortex until the solution is clear.

Preparation

1 Prepare the following consumables.

Item Storage

TCO -25°C to -15°C

EHB -25°C to -15°C

RSB 2°C to 8°C

Instructions

Thaw at room temperature.

Thaw at room temperature.

Let stand for 30 minutes to bring to room temperature.

2 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.

Up to 12 DNA libraries can be pooled before adding capture probes.

Library Pool

Complexity

Total DNA Library

Mass (ng)

Library Pool

Complexity

Total DNA Library

Mass (ng)

1-plex

2-plex

3-plex

4-plex

5-plex

6-plex

500

1000

1500

2000

2500

3000

7-plex

8-plex

9-plex

10-plex

11-plex

12-plex

3500

4000

4500

5000

5500

6000

}

If the total volume is > 40 µl, use a vacuum concentrator or Amicon Ultra-0.5

centrifugal filter unit (0.5 ml, 30 kDa) to concentrate the pooled sample to 40 µl.

} If you are using a vacuum concentrator, use a no heat setting and a medium

TruSight Cardio Sequencing Kit Reference Guide

17

drying rate.

}

If you are using an Amicon Ultra-0.5 centrifugal filter unit (0.5 ml, 30 kDa), rinsing the device before use is not required. Most volume filters through in

5 minutes. Up to 30 minutes might be needed, depending on the starting volume.

} 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 each well of a new Hard-Shell PCR plate.

}

DNA library sample or pool (40 µl)

} EHB (50 µl)

}

TCO (10 µl)

2 Shake at 1200 rpm for 1 minute.

3 Centrifuge at 280 × g for 1 minute.

4 Place on the preprogrammed thermal cycler and run the NRC HYB program. Each well contains 100 µl.

5 Keep at the 58°C holding temperature for at least 90 minutes and up to 24 hours.

18

Document # 15063774 v01

Capture Hybridized Probes

This step uses SMB (Streptavidin Magnetic Beads) to capture probes hybridized to the targeted regions of interest. Two heated washes remove nonspecific binding from the beads.

The enriched library is then eluted from the beads and prepared for a second round of hybridization.

Consumables

} EE1 (Enrichment Elution Buffer 1)

} ET2 (Elute Target Buffer 2)

}

EWS (Enrichment Wash Solution)

}

HP3 (2 N NaOH)

} SMB (Streptavidin Magnetic Beads)

} 96-well Hard-Shell 0.3 ml PCR plate

}

96-well midi plate

}

1.7 ml microcentrifuge tube

} Microseal 'B' adhesive seals

About Reagents

}

EWS can be cloudy after reaching room temperature.

} Vortex EWS before use.

}

Make sure that you use SMB (2 ml tube) and not SPB (15 ml tube) for this procedure.

}

Invert and vortex SMB to mix before use.

}

Discard elution premix after use.

Preparation

1 Prepare the following consumables.

Item Storage

EE1 -25°C to -15°C

EWS -25°C to -15°C

HP3

ET2

SMB

-25°C to -15°C

2°C to 8°C

2°C to 8°C

Instructions

Thaw at room temperature.

Return to storage after use.

Thaw at room temperature.

Return to storage after use.

Thaw at room temperature.

Return to storage after use.

Let stand at room temperature.

Return to storage after use.

Let stand for 30 minutes to bring to room temperature.

Return to storage after use.

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

Procedure

First Bind

1 Centrifuge at 280 × g for 1 minute.

2 Transfer all volumes to the corresponding well of a new midi plate.

3 Add 250 µl SMB to each well.

TruSight Cardio Sequencing Kit Reference Guide

19

20

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 from each well.

9 Remove from the magnetic stand.

First Wash

1 Wash 2 times as follows.

a Add 200 µl EWS to each well.

b Shake at 1800 rpm for 4 minutes.

c Pipette to resuspend the bead pellet further.

d Place on the 50°C microheating system with the lid closed for 30 minutes.

e Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

f Remove and discard all supernatant from each well.

g Remove from the magnetic stand.

First Elution

1 Create elution premix by combining the following volumes per sample in a 1.7 ml microcentrifuge tube, and then vortex.

}

EE1 (28.5 µl)

}

HP3 (1.5 µl)

2 Add 23 µl elution premix to each well.

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 the corresponding well of a new Hard-Shell PCR plate.

8 Add 4 µl ET2 to each well.

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.

Document # 15063774 v01

Perform Second Hybridization

This step binds targeted regions of the enriched DNA with capture probes a second time.

This second hybridization ensures high specificity of the captured regions.

Consumables

} EHB (Enrichment Hybridization Buffer)

} TCO (TruSight Cardio Oligos)

}

RSB (Resuspension Buffer)

}

Microseal 'B' adhesive seals

About Reagents

}

Before using EHB, vortex to resuspend the solution. Make sure that no crystal structures are present. If crystals and cloudiness are observed, vortex until the solution is clear.

Preparation

1 Prepare the following consumables.

Item Storage

TCO -25°C to -15°C

EHB -25°C to -15°C

RSB 2°C to 8°C

Instructions

Thaw at room temperature.

Thaw at room temperature.

Let stand for 30 minutes to bring to room temperature.

Procedure

1 Add the following reagents in the order listed to each sample well.

} RSB (15 µl)

} EHB (50 µl)

}

TCO (10 µl)

2 Shake at 1200 rpm for 1 minute.

3 Centrifuge at 280 × g for 1 minute.

4 Place on the preprogrammed thermal cycler and run the NRC HYB program. Each well contains 100 µl.

5 Keep at the 58°C holding temperature for at least 14.5 hours and up to 24 hours.

TruSight Cardio Sequencing Kit Reference Guide

21

Perform Second Capture

This step uses SMB (Streptavidin Magnetic Beads) to capture probes hybridized to the targeted regions of interest. Two heated washes remove nonspecific binding from the beads.

The enriched library is then eluted from the beads and prepared for sequencing.

Consumables

} EE1 (Enrichment Elution Buffer 1)

}

ET2 (Elute Target Buffer 2)

}

EWS (Enrichment Wash Solution)

} HP3 (2 N NaOH)

} SMB (Streptavidin Magnetic Beads)

}

96-well midi plates (2)

}

1.7 ml microcentrifuge tube

} Microseal 'B' adhesive seals

About Reagents

}

EWS can be cloudy after reaching room temperature.

} Vortex EWS before use.

}

Invert SMB to mix before use.

}

Discard elution premix after use.

Preparation

1 Prepare the following consumables.

Item Storage

EE1 -25°C to -15°C

EWS -25°C to -15°C

HP3

ET2

SMB

-25°C to -15°C

2°C to 8°C

2°C to 8°C

Instructions

Thaw at room temperature.

Return to storage after use.

Thaw at room temperature.

Return to storage after use.

Thaw at room temperature.

Return to storage after use.

Let stand at room temperature.

Return to storage after use.

Let stand for 30 minutes to bring to room temperature.

Return to storage after use.

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

Procedure

Second Bind

1 Centrifuge at 280 × g for 1 minute.

2 Transfer supernatant to the corresponding well of a new midi plate.

3 Add 250 µl SMB to each well.

4 Shake at 1200 rpm for 5 minutes.

5 Incubate at room temperature for 25 minutes.

22

Document # 15063774 v01

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 from each well.

9 Remove from the magnetic stand.

Second Wash

1 Wash 2 times as follows.

a Add 200 µl EWS to each well.

b Shake at 1800 rpm for 4 minutes.

c Pipette to resuspend the bead pellet further.

d Place on the 50°C microheating system with the lid closed for 30 minutes.

e Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

f Remove and discard all supernatant from each well.

g Remove from the magnetic stand.

Second Elution

1 Create elution premix by combining the following volumes per sample in a 1.7 ml microcentrifuge tube, and then vortex:

} EE1 (28.5 µl)

}

HP3 (1.5 µl)

2 Add 23 µl elution premix to each well.

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 the corresponding well of a new midi plate.

8 Add 4 µl ET2 to each well.

9 Shake at 1800 rpm for 1 minute.

10 Centrifuge at 280 × g for 1 minute.

TruSight Cardio Sequencing Kit Reference Guide

23

Clean Up Captured Library

This step uses SPB (Sample Purification Beads) to purify the captured library before PCR amplification.

Consumables

} RSB (Resuspension Buffer)

}

SPB (Sample Purification Beads)

}

Freshly prepared 80% ethanol (EtOH)

} 96-well Hard-Shell 0.3 ml PCR plate

} Microseal 'B' adhesive seals

About Reagents

} Vortex SPB before each use.

}

Vortex SPB frequently to make sure that beads are evenly distributed.

}

Aspirate and dispense SPB slowly due to the viscosity of the solution.

Preparation

1 Prepare the following consumables.

Item

RSB

SPB

Storage Instructions

2°C to 8°C Let stand for 30 minutes to bring to room temperature.

2°C to 8°C Let stand for 30 minutes to bring to room temperature.

2 Prepare fresh 80% EtOH.

Procedure

1 Add 45 µl SPB to each well.

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 from each well.

7 Wash 2 times as follows.

a Add 200 µl fresh 80% EtOH to each well.

b Incubate on the magnetic stand for 30 seconds.

c Remove and discard all supernatant from each well.

8 Use a 20 µl pipette to remove residual EtOH from each well.

9 Air-dry on the magnetic stand for 10 minutes.

10 Add 27.5 µl RSB to each well.

11 Shake at 1800 rpm for 1 minute.

12 Incubate at room temperature for 2 minutes.

13 Centrifuge at 280 × g for 1 minute.

24

Document # 15063774 v01

14 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

15 Transfer 25 µl supernatant to the corresponding well of 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 Cardio Sequencing Kit Reference Guide

25

Amplify Enriched Library

This step uses a 12-cycle PCR program to amplify the enriched library.

Consumables

} NEM (Enrichment Amp Mix)

}

PPC (PCR Primer Cocktail)

}

Microseal 'A' film

} Microseal 'B' adhesive seal

NOTE

Use Microseal 'A' when sealing the plate before placing on the thermal cycler. Use Microseal

'B' for other steps that require a sealed plate.

Preparation

1 Prepare the following consumables.

Item

NEM

PPC

Storage

-25°C to -15°C

-25°C to -15°C

Instructions

Thaw on ice.

Thaw on ice.

2 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 to each well.

2 Add 20 µl NEM to each well.

3 Shake at 1200 rpm for 1 minute.

4 Centrifuge at 280 × g for 1 minute.

5 Place on the preprogrammed thermal cycler and run the NEM AMP12 program. Each well contains 50 µl.

SAFE STOPPING POINT

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

26

Document # 15063774 v01

Clean Up Amplified Enriched Library

This step uses SPB (Sample Purification Beads) to purify the enriched library and remove unwanted products.

Consumables

} RSB (Resuspension Buffer)

} SPB (Sample Purification Beads)

}

Freshly prepared 80% ethanol (EtOH)

}

96-well Hard-Shell 0.3 ml PCR plate

} 96-well midi plate

} Microseal 'B' adhesive seals

About Reagents

} Vortex SPB before each use.

}

Vortex SPB frequently to make sure that beads are evenly distributed.

}

Aspirate and dispense SPB slowly due to the viscosity of the solution.

Preparation

1 Prepare the following consumables.

Item

RSB

SPB

Storage Instructions

2°C to 8°C Let stand for 30 minutes to bring to room temperature.

2°C to 8°C Let stand for 30 minutes to bring to room temperature.

2 Prepare fresh 80% EtOH.

Procedure

1 Centrifuge at 280 × g for 1 minute.

2 Transfer 50 µl to the corresponding well of a new midi plate.

3 Add 90 µl SPB to each well.

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 from each well.

9 Wash 2 times as follows.

a Add 200 µl fresh 80% EtOH to each well.

b Incubate on the magnetic stand for 30 seconds.

c Remove and discard all supernatant from each well.

10 Use a 20 µl pipette to remove residual EtOH from each well.

11 Air-dry on the magnetic stand for 10 minutes.

12 Add 32 µl RSB to each well.

TruSight Cardio Sequencing Kit Reference Guide

27

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 the corresponding well of 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.

28

Document # 15063774 v01

Check Enriched Libraries

Perform the following procedures to check enriched library quality.

Quantify Libraries

Accurately quantify DNA libraries to ensure optimum cluster densities on the flow cell.

Use a fluorometric dsDNA assay to quantify dsDNA libraries. Other techniques can introduce contamination such as RNA and proteins. Use a spectrofluorometer for DNAspecific quantification. Spectrophotometry can also measure RNA and yield values that are too high.

1 If the library concentration is higher than the supported quantified range of the quantification method, dilute the library with RSB.

2 Quantify using a fluorometric method.

Use the following formula to convert from ng/µl to nM. Assume a 400 bp library size or calculate based on the average size of the enriched library.

(concentration in ng/µl)

(660 g/mol * average library size) x 10^6 = concentration in nM

For example:

(15 ng/µl)

(660 g/mol * 400) x 10^6 = 57 nM

Assess Quality

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 diluted post enriched library on an Agilent Technologies 2100 Bioanalyzer using a High Sensitivity DNA chip.

Expect a distribution of DNA fragments with a size range from ~200 bp to ~1 kbp.

Depending on the level of indexing, insert size distribution can vary slightly. However, the sample peak must not be significantly shifted compared to the following example.

Figure 4 Example Post Enrichment Library Distributions

TruSight Cardio Sequencing Kit Reference Guide

29

NOTE

The blue lines indicate the boundaries that were manually created to determine average library size. In the first example, a second minor peak at ~2000 bp is visible. Do not include minor peaks in the determination of average library size. The presence of these larger fragments does not affect downstream clustering and sequencing of your enriched library.

3 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.

30

Document # 15063774 v01

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