TruSight Rapid Capture Reference Guide (15043291 v01)

TruSight Rapid Capture Reference Guide (15043291 v01)
TruSight® Rapid Capture
Reference Guide
For Research Use Only. Not for use in diagnostic procedures.
ILLUMINA PROPRIETARY
Document # 15043291 v01
January 2016
Customize a short end-to-end workflow guide with the Custom Protocol Selector
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ii
Document # 15043291 v01
Revision History
Revision History
Document
Date
Document # 15043291
v01
January
2016
Part # 15043291 Rev. A
May
2013
TruSight Rapid Capture Reference Guide
Description of Change
• Changed title of this document to Reference Guide.
• Updated design of workflow diagram.
• Renamed and combined some procedures as needed to improve
continuity.
• Simplified consumables information at the beginning of each
section.
• Revised step-by-step instructions to be more succinct.
• Removed reference to obsolete Experienced User Cards and
added references to Custom Protocol Selector and new protocol
and checklist.
Initial release.
iii
Table of Contents
Revision History
Table of Contents
Chapter 1 Overview
Introduction
DNA Input Recommendations
Additional Resources
Chapter 2 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
Appendix A Supporting Information
Introduction
Acronyms
Kit Contents
Consumables and Equipment
Index Sequences
DNA Quantification
Technical Assistance
TruSight Rapid Capture Reference Guide
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Document # 15043291 v01
Chapter 1 Overview
Introduction
DNA Input Recommendations
Additional Resources
TruSight Rapid Capture Reference Guide
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Chapter 1
Overview
Overview
Introduction
This protocol explains how to prepare up to 96 indexed, paired-end libraries, followed by
enrichment using a TruSight® Content Set and reagents provided in an Illumina® TruSight
Rapid Capture kit. The goal of this protocol is to fragment and add adapter sequences onto
template DNA to generate indexed sequencing libraries that can be carried through
enrichment for targeted resequencing applications.
The TruSight Rapid Capture protocol offers:
} Excellent data quality with low input of 50 ng
} Fast, easy preparation of up to 96 enriched libraries in ~1.5 days, including ~5 hours of
hands-on time
} High throughput, automation-friendly procedures
2
Document # 15043291 v01
Using an enzymatic DNA fragmentation step allows TruSight Rapid Capture library
preparation to be more sensitive to DNA input than mechanical fragmentation methods.
Accurate quantification of the starting gDNA is essential to enrichment success.
} Quantify gDNA with a fluorometric method specific for double-stranded DNA
(dsDNA) and run samples in triplicate.
} Avoid methods that measure total nucleic acid content, such as NanoDrop or other UV
absorbance methods.
} Perform a 2-step method of gDNA normalization to minimize gDNA sample input
variability during the tagmentation step.
} After initial quantification, gDNA samples are normalized to 10 ng/µl.
} Using a similar fluorometric method, samples are quantified and normalized to a
final 5 ng/µl.
} The TruSight Rapid Capture protocol is optimized for 50 ng of total gDNA.
} A higher mass input of gDNA can result in incomplete tagmentation and larger
insert sizes, affecting enrichment performance.
} A lower mass input of gDNA or low quality gDNA in the tagmentation reaction can
generate smaller insert sizes, which can be lost during subsequent cleanup steps and
cause lower diversity.
TruSight Rapid Capture Reference Guide
3
DNA Input Recommendations
DNA Input Recommendations
Overview
Additional Resources
Visit the TruSight Rapid Capture support page on the Illumina website for documentation,
software downloads, training resources, and information about compatible Illumina
products.
4
Resource
Description
Custom Protocol Selector
http://support.illumina.com/custom-protocol-selector.html
A wizard for generating customized end-to-end
documentation that is tailored to the library prep method, run
parameters, and analysis method used for the sequencing run.
TruSight Rapid Capture Protocol
Guide (document #
1000000005002)
Provides only protocol instructions.
The protocol guide is intended for experienced users. For new
or less experienced users, see the TruSight Rapid Capture
Reference Guide.
TruSight Rapid Capture Checklist
(document # 1000000005003)
Provides a checklist of the protocol steps.
The checklist is intended for experienced users. For new or less
experienced users, see the TruSight Rapid Capture Reference
Guide.
Document # 15043291 v01
Chapter 2 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
TruSight Rapid Capture Reference Guide
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Chapter 2
Protocol
Protocol
Introduction
This chapter describes the TruSight Rapid Capture protocol.
} Follow the protocols in the order shown, using the specified volumes and incubation
parameters.
} Review Best Practices from the TruSight Rapid Capture support page on the Illumina
website.
} Include a common index in each column. A common index facilitates pipetting
operations when dispensing index adapters and pooling indexed libraries later in
the protocol.
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 Rapid Capture support page for more information.
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Document # 15043291 v01
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 Rapid Capture Reference Guide
7
Tips and Techniques
Tips and Techniques
Protocol
Library Prep Workflow
The following diagram illustrates the workflow using a TruSight Rapid Capture kit. Safe
stopping points are marked between steps.
Figure 1 TruSight Rapid Capture Workflow
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Tagment Genomic DNA
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
gDNA
TD
TDE1
SPB
ST
2
Storage
Instructions
-25°C to -15°C Thaw on ice. Invert to mix, and then centrifuge briefly.
-25°C to -15°C Thaw on ice. Invert to mix, and then centrifuge briefly.
-25°C to -15°C Thaw on ice. Invert to mix, and then centrifuge briefly. Set aside on
ice.
2°C to 8°C
Let stand for 30 minutes to bring to room temperature. Set aside at
room temperature.
15°C to 30°C
Check for precipitates. If present, vortex until all particulates are
resuspended.
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 Rapid Capture Reference Guide
9
Protocol
10
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.
Document # 15043291 v01
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
2
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.
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
b
c
Add 200 µl fresh 80% EtOH to each well.
Incubate on the magnetic stand for 30 seconds.
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 Rapid Capture Reference Guide
11
Clean Up Tagmented DNA
Clean Up Tagmented DNA
Protocol
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 # 15043291 v01
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
2
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.
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 Rapid Capture Reference Guide
13
Amplify Tagmented DNA
Amplify Tagmented DNA
Protocol
Figure 2 TruSeq Index Plate Fixture (96 libraries)
A
B
C
Columns 1–12: Index 1 (i7) adapters (orange caps)
Rows A–H: Index 2 (i5) adapters (white caps)
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.
14
Document # 15043291 v01
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
2
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.
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
b
c
Add 200 µl fresh 80% EtOH to each well.
Incubate on the magnetic stand for 30 seconds.
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 27.5 µl RSB to each well.
TruSight Rapid Capture Reference Guide
15
Clean Up Amplified DNA
Clean Up Amplified DNA
Protocol
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.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at -25°C to -15°C for up to 14 days.
16
Document # 15043291 v01
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)
CSO (Custom Selected Oligos) from the TruSight Content Set
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
CSO
EHB
RSB
2
Storage
-25°C to -15°C
-25°C to -15°C
2°C to 8°C
Instructions
Thaw at room temperature.
Thaw at room temperature.
Let stand for 30 minutes to bring to room temperature.
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.
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
drying rate.
TruSight Rapid Capture Reference Guide
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Hybridize Probes
Hybridize Probes
Protocol
} 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
18
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)
} CSO (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.
Document # 15043291 v01
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.
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.
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.
TruSight Rapid Capture Reference Guide
19
Capture Hybridized Probes
Capture Hybridized Probes
Protocol
First Wash
1
Wash 2 times as follows.
a
b
c
d
e
f
g
Add 200 µl EWS to each well.
Shake at 1800 rpm for 4 minutes.
Pipette to resuspend the bead pellet further.
Place on the 50°C microheating system with the lid closed for 30 minutes.
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
Remove and discard all supernatant from each well.
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)
} 2 N NaOH (1.5 µl)
2
Add 23.5 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.
20
Document # 15043291 v01
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)
CSO (Custom Selected Oligos) from the TruSight Content Set
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
CSO
EHB
RSB
Storage
-25°C to -15°C
-25°C to -15°C
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)
} CSO (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 Rapid Capture Reference Guide
21
Perform Second Hybridization
Perform Second Hybridization
Protocol
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
2
Prepare the following consumables.
Item
EE1
Storage
-25°C to -15°C
EWS
-25°C to -15°C
2 N NaOH
-25°C to -15°C
ET2
2°C to 8°C
SMB
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.
Preheat a microheating system with midi plate insert to 50°C.
Procedure
Second Bind
22
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.
Document # 15043291 v01
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.
Perform Second Capture
6
Second Wash
1
Wash 2 times as follows.
a
b
c
d
e
f
g
Add 200 µl EWS to each well.
Shake at 1800 rpm for 4 minutes.
Pipette to resuspend the bead pellet further.
Place on the 50°C microheating system with the lid closed for 30 minutes.
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
Remove and discard all supernatant from each well.
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)
} 2 N NaOH (1.5 µl)
2
Add 23.5 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 Rapid Capture Reference Guide
23
Protocol
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
2
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.
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
b
c
Add 200 µl fresh 80% EtOH to each well.
Incubate on the magnetic stand for 30 seconds.
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 # 15043291 v01
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 Rapid Capture Reference Guide
25
Clean Up Captured Library
14 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
Protocol
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
2
Storage
-25°C to -15°C
-25°C to -15°C
Instructions
Thaw on ice.
Thaw on ice.
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 # 15043291 v01
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
2
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.
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
b
c
Add 200 µl fresh 80% EtOH to each well.
Incubate on the magnetic stand for 30 seconds.
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 32.5 µl RSB to each well.
TruSight Rapid Capture Reference Guide
27
Clean Up Amplified Enriched Library
Clean Up Amplified Enriched Library
Protocol
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 # 15043291 v01
Perform the following procedures to check enriched library quality.
Quantify Libraries
Use the following procedure to quantify libraries using a fluorometric method.
Alternatively, you can use qPCR. For instructions, see the Sequencing Library qPCR
Quantification Guide (document # 11322363).
1
Dilute the postenriched library before quantification:
} 3-plex to 12-plex enrichments—Add 1 µl library to 29 µl RSB in a new tube or well.
Use this dilution for quantification, quality assessment, and sequencing.
} 1-plex or 2-plex enrichments—Use a 1:15 dilution.
2
Convert from ng/µl to nM using the following formula. 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)
3
x 10^6
= 57 nM
Choose whether to assess library quality:
} Proceed to Assess Quality [Optional] on page 29.
} Skip the assessment and proceed to cluster generation. For instructions, see the
system guide for your instrument.
Assess Quality [Optional]
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 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 3 Example Post Enrichment Library Distributions
TruSight Rapid Capture Reference Guide
29
Check Enriched Libraries
Check Enriched Libraries
Protocol
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.
30
Document # 15043291 v01
Appendix A Supporting Information
Introduction
Acronyms
Kit Contents
Consumables and Equipment
Index Sequences
DNA Quantification
TruSight Rapid Capture Reference Guide
32
33
34
39
41
42
31
Appendix A
Supporting Information
Supporting Information
Introduction
The protocols described in this guide assume that you have reviewed the contents of this
appendix, confirmed your kit contents, and obtained all the required consumables and
equipment.
32
Document # 15043291 v01
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 Reference Guide
33
Supporting Information
Kit Contents
Make sure that you have all reagents identified in this section before proceeding to the
library preparation procedures. TruSight Rapid Capture kits are available in the following
configurations.
Table 1 TruSight Rapid Capture Kits
Kit Name
TruSight Rapid Capture Kit (1 Index, 8 Samples)
TruSight Rapid Capture Kit (2 Indices, 8 Samples)
TruSight Rapid Capture Kit (4 Indices, 16 Samples)
TruSight Rapid Capture Kit (24 Indices, 48 Samples)
TruSight Rapid Capture Kit (24 Indices, 96 Samples)
TruSight Rapid Capture Kit (96 Indices, 288 Samples)
Catalog #
FC-140-1101
FC-140-1102
FC-140-1103
FC-140-1104
FC-140-1105
FC-140-1106
TG Catalog #*
TG-140-1101
TG-140-1102
TG-140-1103
TG-140-1104
TG-140-1105
TG-140-1106
* Consumables labeled TG include features to help reduce the frequency of revalidation. These
consumables are available with a supply agreement and require providing a binding forecast.
For more information, contact your Illumina representative.
Note regarding biomarker patents and other patents unique to specific uses of products.
Some genomic variants, including some nucleic acid sequences, and their use in specific
applications might be protected by patents. Customers are advised to determine whether
they are required to obtain licenses from the party that owns or controls such patents to use
the product for their specific application.
TruSight Rapid Capture Kit Contents
(1 Index, 8 Samples) (FC-140-1101, TG-140-1101)
Box 1
Quantity
1
4
1
1
Reagent
SPB
SMB
ET2
ST
Description
Sample Purification Beads
Streptavidin Magnetic Beads
Elute Target Buffer 2
Stop Tagment Buffer
Storage Temperature
2°C to 8°C
2°C to 8°C
2°C to 8°C
15°C to 30°C
Box 2, Store at -25°C to -15°C
Quantity
2
2
2
1
1
2
2
1
1
4
34
Reagent
TDE1
EE1
TD
RSB
NLM
EHB
EWS
HP3
PPC
NEM
Description
Tagment DNA Enzyme
Enrichment Elution Buffer 1
Tagment DNA Buffer
Resuspension Buffer
Nextera Library Amplification Mix
Enrichment Hybridization Buffer
Enrichment Wash Solution
2 N NaOH
PCR Primer Cocktail
Nextera Enrichment Amplification Mix
Document # 15043291 v01
Kit Contents
Box 3, Store at -25°C to -15°C
Quantity
1 tube
1 tube
Reagent
Index Primer, E501
Index Primer, N701
TruSight Rapid Capture Kit Contents
(2 Indexes, 8 Samples) (FC-140-1102, TG-140-1102)
Box 1
Quantity
1
2
1
1
Reagent
SPB
SMB
ET2
ST
Description
Sample Purification Beads
Streptavidin Magnetic Beads
Elute Target Buffer 2
Stop Tagment Buffer
Storage Temperature
2°C to 8°C
2°C to 8°C
2°C to 8°C
15°C to 30°C
Box 2, Store at -25°C to -15°C
Quantity
1
1
1
1
1
1
1
1
1
2
Reagent
TDE1
EE1
TD
RSB
NLM
EHB
EWS
HP3
PPC
NEM
Description
Tagment DNA Enzyme
Enrichment Elution Buffer 1
Tagment DNA Buffer
Resuspension Buffer
Nextera Library Amplification Mix
Enrichment Hybridization Buffer
Enrichment Wash Solution
2 N NaOH
PCR Primer Cocktail
Nextera Enrichment Amplification Mix
Box 3, Store at -25°C to -15°C
Quantity
1 tube
2 tubes
Reagent
Index Primer, E501
Index Primer, N701 to N702
TruSight Rapid Capture Kit Contents
(4 Indexes, 16 Samples) (FC-140-1103, TG-140-1103)
Box 1
Quantity
1
2
1
1
Reagent
SPB
SMB
ET2
ST
TruSight Rapid Capture Reference Guide
Description
Sample Purification Beads
Streptavidin Magnetic Beads
Elute Target Buffer 2
Stop Tagment Buffer
Storage Temperature
2°C to 8°C
2°C to 8°C
2°C to 8°C
15°C to 30°C
35
Supporting Information
Box 2, Store at -25°C to -15°C
Quantity
1
1
1
1
1
1
1
1
1
2
Reagent
TDE1
EE1
TD
RSB
NLM
EHB
EWS
HP3
PPC
NEM
Description
Tagment DNA Enzyme
Enrichment Elution Buffer 1
Tagment DNA Buffer
Resuspension Buffer
Nextera Library Amplification Mix
Enrichment Hybridization Buffer
Enrichment Wash Solution
2 N NaOH
PCR Primer Cocktail
Nextera Enrichment Amplification Mix
Box 3, Store at -25°C to -15°C
Quantity
1 tube
4 tubes
Reagent
Index Primer, E501
Index Primer, N701 to N704
TruSight Rapid Capture Kit Contents
(24 Indexes, 48 Samples) (FC-140-1104, TG-140-1104)
Box 1
Quantity
2
2
1
1
Acronym
SPB
SMB
ET2
ST
Reagent Name
Sample Purification Beads
Streptavidin Magnetic Beads
Elute Target Buffer 2
Stop Tagment Buffer
Storage Temperature
2°C to 8°C
2°C to 8°C
2°C to 8°C
15°C to 30°C
Box 2, Store at -25°C to -15°C
Quantity
2
1
1
1
2
1
1
1
1
2
Acronym
TDE1
EE1
TD
RSB
NLM
EHB
EWS
HP3
PPC
NEM
Reagent Name
Tagment DNA Enzyme
Enrichment Elution Buffer 1
Tagment DNA Buffer
Resuspension Buffer
Nextera Library Amplification Mix
Enrichment Hybridization Buffer
Enrichment Wash Solution
2 N NaOH
PCR Primer Cocktail
Nextera Enrichment Amplification Mix
Box 3, Store at -25°C to -15°C
Quantity
2 tubes
12 tubes
36
Reagent Name
Index Primer, E501 to E502
Index Primers, N701 to N712
Document # 15043291 v01
Box 1
Quantity
4
4
1
1
Reagent
SPB
SMB
ET2
ST
Description
Sample Purification Beads
Streptavidin Magnetic Beads
Elute Target Buffer 2
Stop Tagment Buffer
Storage Temperature
2°C to 8°C
2°C to 8°C
2°C to 8°C
15°C to 30°C
Box 2, Store at -25°C to -15°C
Quantity
4
1
2
1
4
2
2
1
1
4
Reagent
TDE1
EE1
TD
RSB
NLM
EHB
EWS
HP3
PPC
NEM
Description
Tagment DNA Enzyme
Enrichment Elution Buffer 1
Tagment DNA Buffer
Resuspension Buffer
Nextera Library Amplification Mix
Enrichment Hybridization Buffer
Enrichment Wash Solution
2 N NaOH
PCR Primer Cocktail
Nextera Enrichment Amplification Mix
Box 3, Store at -25°C to -15°C
Quantity
2 tubes
12 tubes
Reagent
Index Primer, E501 to E502
Index Primers, N701 to N712
TruSight Rapid Capture Kit Contents
(96 Indexes, 288 Samples) (FC-140-1106, TG-140-1106)
Box 1
Quantity
12
12
3
3
Reagent
SPB
SMB
ET2
ST
Description
Sample Purification Beads
Streptavidin Magnetic Beads
Elute Target Buffer 2
Stop Tagment Buffer
Storage Temperature
2°C to 8°C
2°C to 8°C
2°C to 8°C
15°C to 30°C
Box 2, Store at -25°C to -15°C
Quantity
12
3
Reagent
TDE1
EE1
TruSight Rapid Capture Reference Guide
Description
Tagment DNA Enzyme
Enrichment Elution Buffer 1
37
Kit Contents
TruSight Rapid Capture Kit Contents
(24 Indexes, 96 Samples) (FC-140-1105, TG-140-1105)
Supporting Information
Quantity
6
1
12
6
6
3
3
12
Reagent
TD
RSB
NLM
EHB
EWS
HP3
PPC
NEM
Description
Tagment DNA Buffer
Resuspension Buffer
Nextera Library Amplification Mix
Enrichment Hybridization Buffer
Enrichment Wash Solution
2 N NaOH
PCR Primer Cocktail
Nextera Enrichment Amplification Mix
Box 3, Store at -25°C to -15°C
Quantity
8 tubes
12 tubes
38
Reagent
Index Primer, E501 to E508
Index Primers, N701 to N712
Document # 15043291 v01
Make sure that you have the required user-supplied consumables and equipment before
starting the protocol.
The protocol has been optimized and validated using the items listed. Comparable
performance is not guaranteed when using alternate consumables and equipment.
Consumables
Consumable
Supplier
1.7 ml microcentrifuge tubes
General lab supplier
20 µl barrier pipette tips
General lab supplier
20 µl multichannel pipettes
General lab supplier
20 µl single channel pipettes
General lab supplier
200 µl barrier pipette tips
General lab supplier
200 µl multichannel pipettes
General lab supplier
200 µl single channel pipettes
General lab supplier
1000 µl barrier pipette tips
General lab supplier
1000 µl multichannel pipettes
General lab supplier
1000 µl single channel pipettes
General lab supplier
Adhesive seal roller
General lab supplier
96-well storage plates, round well, 0.8 ml
(midi plate)
Fisher Scientific, part # AB-0859
Hard-Shell 96-well PCR Plates
Bio-Rad, part # HSP-9601
Ethanol 200 proof (absolute)
for molecular biology (500 ml)
Sigma-Aldrich, part # E7023
Microseal 'A' film
Bio-Rad, part # MSA-5001
Microseal 'B' adhesive seals
Bio-Rad, part # MSB-1001
RNase/DNase-free 8-tube strips and caps
General lab supplier
RNase/DNase-free multichannel reagent
reservoirs, disposable
VWR, part # 89094-658
Tris-HCl 10 mM, pH 8.5
General lab supplier
PCR-grade water
General lab supplier
[Optional] Amicon Ultra-0.5 centrifugal filter
unit (0.5 ml, 30 kDa)*
Millipore, part # UFC503008
[Optional] DNA 1000 Kit
Agilent Technologies, part # 5067-1504
[Optional] High Sensitivity DNA Kit
Agilent Technologies, part # 5067-4626
TruSight Rapid Capture Reference Guide
39
Consumables and Equipment
Consumables and Equipment
Supporting Information
* Use to concentrate a pooled library. Otherwise, use a vacuum concentrator.
Equipment
Equipment
Supplier
DNA Engine Multi-Bay Thermal Cycler
See Thermal Cyclers on page 40.
Bio-Rad, part # PTC-0240G
or
PTC-0220G, with Alpha Unit,
part # ALS-1296GC
High-Speed Microplate Shaker
VWR, catalog # • 13500-890 (110 V/120 V)
or
• 14216-214 (230 V)
Magnetic stand-96
Life Technologies, part # AM10027
Microcentrifuge
General lab supplier
Microheating System-SciGene TruTemp
Heating System
Illumina, catalog #
• SC-60-503 (115 V) or
• SC-60-504 (220 V)
Microplate centrifuge
General lab supplier
Midi plate insert for microheating system
Illumina, catalog # BD-60-601
Fluorometric quantification with dsDNA
binding dye reagents
General lab supplier
Vortexer
General lab supplier
[Optional] 2100 Bioanalyzer Desktop System
Agilent Technologies, part # G2940CA
[Optional] TruSeq Index Plate Fixture Kit¹
Illumina, catalog # FC-130-1005
[Optional] Vacuum concentrator²
General lab supplier
¹ Reusable and recommended for setting up indexed adapters.
² Use to concentrate a pooled library. Alternatively, use Amicon Ultra-0.5 centrifugal filter units.
Thermal Cyclers
The following table lists the recommended settings for the recommended thermal cycler,
and other comparable models. If your lab has a thermal cycler that is not listed, validate
the thermal cycler before performing the protocol.
40
Thermal Cycler
Temp Mode
Lid Temp
Vessel Type
Bio-Rad DNA Engine
Tetrad 2
Calculated
Heated, Constant
at 100°C
Polypropylene plates
and tubes
MJ Research DNA
Engine Tetrad
Calculated
Heated
Plate
Eppendorf
Mastercycler Pro S
Gradient S,
Simulated Tube
Heated
Plate
Document # 15043291 v01
The Illumina dual-index strategy adds 2 8-base indexes, Index 1 (i7) and Index 2 (i5), to
each sample.
There are 12 different Index 1 (i7) adapters (N701–N712) and 2 different Index 2 (i5)
adapters (E501 AND E502), depending on the kit you are using. In the Index adapter name:
} N refers to Nextera
} E refers to enrichment
} 7 refers to Index 1 (i7)
} 5 refers to Index 2 (i5)
} 01–12 refers to the index number
Use the following bases for entry on your sample sheet.
Index 1 (i7)
N701
N702
N703
N704
N705
N706
N707
N708
N709
N710
N711
N712
Sequence
TAAGGCGA
CGTACTAG
AGGCAGAA
TCCTGAGC
GGACTCCT
TAGGCATG
CTCTCTAC
CAGAGAGG
GCTACGCT
CGAGGCTG
AAGAGGCA
GTAGAGGA
Index 2 (i5)*
E501
E502
Sequence
TAGATCGC
CTCTCTAT
* Although E500 series Index 2 sequences are identical to S500 series sequences used in other kits,
the Index 2 adapters are not interchangeable.
TruSight Rapid Capture Reference Guide
41
Index Sequences
Index Sequences
Supporting Information
DNA Quantification
Perform the QuantiFluor dsDNA assay to quantify dsDNA samples. The assay can
quantify small DNA volumes and measure DNA directly. Other techniques can pick up
contaminates, such as RNA and proteins. Use a spectrofluorometer for DNA-specific
quantification. Spectrophotometry can also measure RNA and yield values that are too
high.
Consumables
}
}
}
}
}
}
}
}
}
1X TE
96-well flat clear bottom black microplates (2)
96-well midi plates (2)
Aluminum foil
Conical centrifuge tube (15 ml or 50 ml)
Lambda DNA
Microseal 'B' adhesive seals
QuantiFluor dsDNA dye
RNase/DNase-free Reagent Reservoir
About Reagents
}
QuantiFluor dsDNA dye often crystallizes at room temperature. Make sure that the dye
is thawed and liquid.
Preparation
1
Remove the QuantiFluor dsDNA dye from to 2°C to 8°C and let stand at room
temperature for 60 minutes in a light-impermeable container.
Procedure
Make Lambda DNA Stock Plate
1
Dilute lambda DNA in well A1 of a new midi plate to 1 ng/µl in a final volume of
300 µl. Pipette to mix.
} Use the following formula to calculate the amount of lambda DNA to add to A1:
(300 µl) X (1 ng/µl)
(stock Lambda DNA concentration)
=
µl of stock Lambda DNA to add to
A1
} Dilute DNA in well A1 using the following formula:
(300 µl) - (µl of stock Lambda DNA in well A1) = µl of 1X TE to add to A1
2
42
Add 150 µl 1X TE to wells B, C, D, E, F, G, and H of column 1.
Document # 15043291 v01
DNA Quantification
Figure 4 Dilution of Stock Lambda DNA Standard
3
Transfer 150 µl lambda DNA from well A1 to well B1. Pipette to mix.
4
Transfer 150 µl from well B1 to well C1. Pipette to mix.
5
Repeat the transfer for wells D1, E1, F1, and G1, changing tips each time. Well H1
serves as the blank 0 ng/µl Lambda DNA.
Table 2 Concentrations of Lambda DNA
Row-Column
Concentration (ng/µl)
A1
1
B1
0.5
C1
0.25
D1
0.125
E1
0.0625
F1
0.03125
G1
0.015625
H1
0
Final Volume in Well (µl)
150
150
150
150
150
150
300
150
Figure 5 Serial Dilutions of Lambda DNA
TruSight Rapid Capture Reference Guide
43
Supporting Information
Make DNA Stock Plate
In a new midi plate, prepare the appropriate dilutions of your DNA samples using 1X TE.
Measure each sample in triplicate. Make sure that at least 50 µl of diluted sample is
prepared for quantification with the QuantiFluor dsDNA dye. Scale for replicate
measurements.
1
Dilute using 1 of the following options, depending on the sample quality or library
type:
} High-quality gDNA—Dilute 1:1000. For example: 2 µl of gDNA + 1998 µl of 1X TE.
} Pre-enriched TruSight Rapid Capture libraries—Dilute 1:200. For example: 2 µl of
library sample + 398 µl of 1X TE.
} Post-enriched TruSight Rapid Capture library dilution:
} 1-plex, 3-plex, 6-plex, and 9-plex (8 reaction kits)—Dilute 1:50. For example: 2 µl
of postenriched library + 98 µl of 1X TE.
} 12-plex—Dilute 1:100. For example: 2 µl of postenriched library + 198 µl of 1X
TE.
2
Shake at 1200 rpm for 1 minute.
3
Centrifuge at 280 × g for 1 minute
Dilute QuantiFluor dsDNA Dye
1
Prepare a 1:200 dilution of QuantiFluor dsDNA dye in 1X TE in a conical centrifuge
tube wrapped in aluminum foil.
Run each sample and standard in triplicate. For each measurement, 40 µl of diluted
QuantiFluor dye is required. Scale as appropriate.
2
Vortex to mix.
Make Lambda DNA Quant Plate
44
1
Pour the diluted QuantiFluor dsDNA dye/1X TE into a new reagent reservoir.
2
Transfer 40 µl diluted QuantiFluor dsDNA dye/1X TE into each well of columns 1–3 of
a new microplate.
3
Transfer 40 µl from each well of the lambda DNA stock plate to columns 1–3.
Document # 15043291 v01
DNA Quantification
Figure 6 Lambda DNA Quant Plate with QuantiFluor dsDNA Dye/1X TE
4
Shake at 1200 rpm for 1 minute.
5
Centrifuge at 280 × g for 1 minute
6
Protect from light until read by the spectrofluorometer.
Make DNA Quant Plate
1
Transfer 40 µl QuantiFluor dsDNA reagent/1X TE dilution to each well of the
microplate.
2
Transfer 40 µl DNA sample in the DNA stock plate to the microplate.
3
Shake at 1200 rpm for 1 minute.
4
Centrifuge at 280 × g for 1 minute
5
Protect from light until read by the spectrofluorometer.
Read Quant Plate
1
Measure fluorescence (485 nm Ex / 538 nm Em) of both the Lambda DNA quant and
DNA quant plates according to the spectrofluorometer/software recommendations.
2
Calculate the DNA concentration of your unknown samples using the fluorescence
values determined from step 1 as follows:
a
b
c
d
e
f
Calculate the average relative fluorescence units (RFU) of the Lambda DNA
standards run in triplicate on the lambda DNA quant plate.
Calculate an Adjusted RFU by subtracting the RFU of the blank Lambda DNA
standard (0 ng/µl) Row H from all unknown and standard samples.
Create a scatter plot of the lambda DNA standard curve values with the Adjusted
RFU on the Y axis and DNA concentration (ng/µl) on the X axis.
Determine the equation of the line for the lambda DNA standard curve values,
which is in the format of y = mx + b is equivalent to RFU = (slope*concentration) +
y_int.
Calculate the concentration for each unknown sample by using the RFU for each
sample for y in the equation and determining the value for x in ng/µl.
Multiply the resulting concentration by the appropriate dilution factor.
TruSight Rapid Capture Reference Guide
45
Supporting Information
g
Use the following formula to convert from ng/µl to nM.
(concentration in ng/µl)
×
106
=
concentration in nM
×
106
=
57 nM
(660 g/mol × average library
size)
For example:
15 ng/µl
(660 g/mol × 400)
46
Document # 15043291 v01
For technical assistance, contact Illumina Technical Support.
Table 3 Illumina General Contact Information
Website
Email
www.illumina.com
[email protected]
Table 4 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 Reference Guide
47
Technical Assistance
Technical Assistance
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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