TruSight Myeloid Sequencing Panel Reference Guide (15054779 v01)

TruSight Myeloid Sequencing Panel Reference Guide (15054779 v01)
TruSight® Myeloid
Sequencing Panel
Reference Guide
For Research Use Only. Not for use in diagnostic procedures.
ILLUMINA PROPRIETARY
Document # 15054779 v01
January 2016
Customize a short end-to-end workflow guide with the Custom Protocol Selector
support.illumina.com/custom-protocol-selector.html
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.
ii
Document # 15054779 v01
Revision History
Document
Date
Document # 15054779
v01
January
2016
Part # 15054779 Rev. B
November
2014
Part # 15054779 Rev. A
May 2014
Description of Change
• Changed the title of this document to TruSight Myeloid
Sequencing Panel Reference Guide, and updated the kit name
to TruSight Myeloid Sequencing Panel throughout.
• Updated design of workflow diagram.
• Added a safe stopping point before library normalization.
• 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 guide and checklist.
• Clarified minimum batch size. The TruSight Myeloid
Sequencing Panel does not provide enough reagents to process
fewer than 8 samples at a time.
• Clarified that unused volume is already included in calculation
when preparing fewer than 96 samples and calculating volumes
of TDP1 and PMM2.
Initial release.
TruSight Myeloid Sequencing Panel Reference Guide
iii
iv
Document # 15054779 v01
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
Hybridize Oligo Pool
Remove Unbound Oligos
Extend and Ligate Bound Oligos
Amplify Libraries
Clean Up Libraries
Normalize Libraries
Pool Libraries
Appendix A Supporting Information
Introduction
Acronyms
Kit Contents
Consumables and Equipment
Technical Assistance
TruSight Myeloid Sequencing Panel Reference Guide
iii
v
1
2
3
4
5
6
7
8
9
11
14
15
18
21
24
25
26
27
28
30
33
v
vi
Document # 15054779 v01
Chapter 1 Overview
Introduction
DNA Input Recommendations
Additional Resources
TruSight Myeloid Sequencing Panel Reference Guide
2
3
4
1
Chapter 1
Overview
Overview
Introduction
This protocol explains how to prepare up to 96 indexed, pooled libraries using the
Illumina® TruSight® Myeloid Sequencing Panel. The goal of this protocol is to hybridize a
custom oligo pool to targeted regions of unfragmented gDNA. After extension and ligation,
a polymerase chain reaction (PCR) amplifies the library template and adds indexes and
sequence adapters to generate DNA libraries ready for clustering and sequencing.
The TruSight Myeloid protocol offers:
} A targeted approach for quickly and efficiently assessing various genetic variants.
} Multiplexing capability to generate up to 1536 amplicons in 1 reaction and sequence
up to 96 libraries per sequencing run.
} Fast and easy workflow to prepare up to 96 pooled libraries in 1 plate with
approximately 3 hours of hands-on time.
} Variant calling and analysis across all libraries using automated, on-instrument
analysis software.
} Fully integrated DNA-to-data solution from assay, sequencing, and automated data
analysis to offline software for reviewing results.
2
Document # 15054779 v01
Quantify the starting genomic DNA (gDNA) using a fluorescence-based quantification
method, such as PicoGreen.
} Fluorescence-based methods employ dye specific to double-stranded DNA (dsDNA)
and accurately quantify dsDNA, even in the presence of many common contaminants.
} Avoid UV spectrometer methods based on 260 OD readings, which can overestimate
DNA concentrations due to the presence of RNA and other common contaminants.
The TruSight Myeloid protocol has been optimized for 50 ng of total gDNA.
Input (≥ 15
µl)
High-quality gDNA 150, 175, 250, and 425 bp 50 ng
DNA Type
Amplicon Size
TruSight Myeloid Sequencing Panel Reference Guide
A260/A1280
1.8–2.0
FFPE DNA
Not
supported
3
DNA Input Recommendations
DNA Input Recommendations
Overview
Additional Resources
Visit the TruSight Myeloid Sequencing Panel 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 Myeloid Sequencing
Panel Protocol Guide (document #
1000000005004)
Provides only protocol instructions.
The protocol guide is intended for experienced users. For new
or less experienced users, see the TruSight Myeloid
Sequencing Panel Reference Guide.
TruSight Myeloid Sequencing
Panel Checklist (document #
1000000005005)
Provides a checklist of the protocol steps.
The checklist is intended for experienced users. For new or less
experienced users, see the TruSight Myeloid Sequencing Panel
Reference Guide.
Document # 15054779 v01
Chapter 2 Protocol
Introduction
Tips and Techniques
Library Prep Workflow
Hybridize Oligo Pool
Remove Unbound Oligos
Extend and Ligate Bound Oligos
Amplify Libraries
Clean Up Libraries
Normalize Libraries
Pool Libraries
TruSight Myeloid Sequencing Panel Reference Guide
6
7
8
9
11
14
15
18
21
24
5
Chapter 2
Protocol
Protocol
Introduction
This chapter describes the TruSight Myeloid Sequencing Panel protocol.
} Follow the protocols in the order shown, using the specified volumes and incubation
parameters.
} Review Best Practices from the TruSight Myeloid Sequencing Panel 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 Myeloid Sequencing Panel support page for more information.
6
Document # 15054779 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 Myeloid Sequencing Panel Reference Guide
7
Tips and Techniques
Tips and Techniques
Protocol
Library Prep Workflow
The following diagram illustrates the workflow using the TruSight Myeloid Sequencing
Panel. Safe stopping points are marked between steps.
Figure 1 TruSight Myeloid Sequencing Panel Workflow
8
Document # 15054779 v01
This step hybridizes a custom oligo pool that contains upstream and downstream oligos
specific to your targeted regions of interest. Perform replicates to increase confidence in
somatic variant calls.
Consumables
}
}
}
}
}
}
}
TSO (TruSight Oligos)
OHS2 (Oligo Hybridization for Sequencing 2)
ACD1 (Amplicon Control DNA 1)
HYP (Hybridization Plate) barcode label
gDNA (50 ng per sample)
96-well PCR plate, skirted
Foil adhesive seals (2)
WARNING
This set of reagents contains formamide, an aliphatic amide that is a probable
reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact,
and eye contact. Wear protective equipment, including eye protection, gloves, and
laboratory coat. Handle used reagents as chemical waste and discard in accordance with
the governmental safety standards for your region. For environmental, health, and safety
information, see the SDS for this kit at support.illumina.com/sds.html.
About Reagents
}
}
OHS2
} Aspirate and dispense slowly due to the viscosity of the reagent.
} Before each use, vortex thoroughly and then centrifuge briefly. Make sure that all
precipitates have dissolved.
} When mixing, mix thoroughly.
} Do not mix with TSO for storage purposes. TSO is unstable when combined with
other reagents.
ACD1
} Although the control oligo pool ACP1 is included with the kit, using it for the
TruSight Myeloid Sequencing Panel protocol is not necessary. Use TSO with ACD1
as a positive control.
} Include ACD1 and TSO in your assay to establish a baseline and monitor overall
performance. Use of these controls enables Illumina Technical Support to
troubleshoot if you need assistance.
Preparation
1
Prepare the following consumables:
Item
TSO
OHS2
ACD1
gDNA
Storage
-25°C to -15°C
-25°C to -15°C
-25°C to -15°C
-25°C to -15°C
2
Set a 96-well heat block to 95°C.
3
Preheat an incubator to 37°C.
TruSight Myeloid Sequencing Panel Reference Guide
Instructions
Thaw at room temperature.
Thaw at room temperature.
Thaw at room temperature.
Thaw at room temperature.
9
Hybridize Oligo Pool
Hybridize Oligo Pool
Protocol
4
Label a new 96-well PCR plate HYP.
Procedure
1
Add 5 µl ACD1 and 5 µl TE or water to 1 well of the HYP plate.
2
Add 10 µl gDNA to each remaining well.
For more diluted samples (eg, < 25 ng/µl), you can use up to 15 µl gDNA.
Table 1 Example Setup for High-Quality gDNA
Input (ng)
Volume (µl)
50
10
50
up to 15
10
DNA Concentration (ng/µl)
5
≥ 3.3
3
Add 5 µl TSO to each well containing gDNA.
4
Centrifuge at 1000 × g for 1 minute.
5
Add 35 µl OHS2 to each well. Pipette to mix.
6
Centrifuge at 1000 × g for 1 minute.
7
Place on the preheated heat block and incubate for 1 minute.
8
With the plate on the heat block, reset the temperature to 40°C and continue incubating
for 80 minutes.
Document # 15054779 v01
This step uses a filter to remove unbound oligos from gDNA. Two wash steps using SW1
ensure complete removal of unbound oligos. A third wash step using UB1 removes
residual SW1 and prepares samples for extension and ligation.
Consumables
}
}
}
}
}
}
}
ELM4 (Extension Ligation Mix 4)
SW1 (Stringent Wash 1)
UB1 (Universal Buffer 1)
FPU (Filter Plate Unit) barcode label
Filter plate with lid
Adapter collar, reusable
96-well midi plate
WARNING
This set of reagents contains formamide, an aliphatic amide that is a probable
reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact,
and eye contact. Wear protective equipment, including eye protection, gloves, and
laboratory coat. Handle used reagents as chemical waste and discard in accordance with
the governmental safety standards for your region. For environmental, health, and safety
information, see the SDS for this kit at support.illumina.com/sds.html.
WARNING
This set of reagents contains ß-mercaptoethanol. Perform the following procedure in a hood
or well-ventilated area.
About Reagents
}
Incomplete drainage of SW1 compromises target enrichment specificity.
Preparation
1
Prepare the following consumables:
Item
ELM4
Storage
-25°C to -15°C
SW1
UB1
2°C to 8°C
2°C to 8°C
Instructions
Let stand to bring to room temperature in preparation for a later
procedure.
Set aside at room temperature.
Set aside at room temperature.
TruSight Myeloid Sequencing Panel Reference Guide
11
Remove Unbound Oligos
Remove Unbound Oligos
Protocol
2
Assemble the filter plate unit (FPU) from top to bottom.
Figure 2 FPU Assembly
A
B
C
D
Lid
Filter plate
Adapter collar
Midi plate
3
Label the completed assembly FPU.
4
Wash the wells to be used in the assay as follows. Use new wells only.
a
b
c
5
Add 45 µl SW1 to each well.
Cover the FPU plate.
Centrifuge at 2400 × g for 5 minutes.
If a significant amount (> 15 µl/well) of residual buffer remains in multiple wells (≥ 10
wells/plate), switch to a new filter plate.
Procedure
1
Make sure that the heat block has cooled to 40˚C and the HYP plate seal is secure.
2
Remove from the heat block.
3
Centrifuge at 1000 × g for 1 minute.
4
Transfer each sample to the corresponding well of the FPU plate.
5
Cover and centrifuge at 2400 × g for 5 minutes.
6
Wash 2 times as follows.
a
b
c
7
12
Add 45 µl SW1 to each sample well.
Cover and centrifuge at 2400 × g for 5 minutes.
If SW1 does not drain completely, centrifuge again for up to 10 minutes.
Discard flow-through.
Document # 15054779 v01
Reassemble the FPU plate for continued use.
9
Add 45 µl UB1 to each sample well.
Remove Unbound Oligos
8
10 Cover and centrifuge at 2400 × g for 5 minutes.
11 If UB1 does not drain completely, centrifuge again for up to 10 minutes.
TruSight Myeloid Sequencing Panel Reference Guide
13
Protocol
Extend and Ligate Bound Oligos
This step connects the hybridized upstream and downstream oligos. A DNA polymerase
extends from the upstream oligo through the targeted region, followed by ligation to the 5'
end of the downstream oligo using a DNA ligase. The result is the formation of products
containing the targeted regions of interest flanked by sequences required for amplification.
Consumables
}
}
ELM4 (Extension-Ligation Mix 4)
Foil adhesive seal
Procedure
14
1
Add 45 µl ELM4 to each sample well of the FPU plate.
2
Incubate at 37°C for 45 minutes.
3
During incubation, proceed to the next step.
Document # 15054779 v01
This step amplifies the extension-ligation products and adds Index 1 (i7) adapters, Index 2
(i5) adapters, and sequences required for cluster formation.
Consumables
}
}
}
}
}
}
}
}
PMM2 (PCR Master Mix 2)
Index i5 adapters (A5XX)
Index i7 adapters (A7XX)
TDP1 (TruSeq DNA Polymerase 1)
IAP (Indexed Amplification Plate) barcode label
Microseal 'A' adhesive film
50 mM NaOH (3.5 ml for 96 samples)
96-well PCR plate, skirted
About Reagents
}
}
Always use a fresh dilution of NaOH.
Do not store combined PMM2 and TDP1.
Preparation
1
Prepare the following consumables:
Item
PMM2
Index adapters (i5 and i7)
Storage
-25°C to -15°C
-25°C to -15°C
2
Prepare fresh 50 mM NaOH from 10 N NaOH.
3
Label a new PCR plate IAP.
Instructions
Thaw at room temperature.
Thaw at room temperature.
Procedure
1
Arrange the Index 1 (i7) adapters in columns 1–12 of the TruSeq Index Plate Fixture.
2
Arrange the Index 2 (i5) adapters in rows A–H of the TruSeq Index Plate Fixture.
3
Place the IAP plate on a TruSeq Index Plate Fixture.
TruSight Myeloid Sequencing Panel Reference Guide
15
Amplify Libraries
Amplify Libraries
Protocol
Figure 3 TruSeq Index Plate Fixture
A
B
C
Columns 1–12: Index 1 (i7) adapters (orange caps)
Rows A–H: Index 2 (i5) adapters (white caps)
IAP plate
4
Use a multichannel pipette to add 4 µl of each Index 1 (i7) adapter to each row.
Replace the cap on each i7 adapter tube with a new orange cap.
5
Use a multichannel pipette to add 4 µl of each Index 2 (i5) adapter to each column.
Replace the cap on each i5 adapter tube with a new white cap.
6
Add 56 µl TDP1 to 2.8 ml PMM2.
7
Invert to mix.
8
When incubation is complete, remove the FPU plate from the incubator and remove the
seal.
9
Cover and centrifuge at 2400 × g for 5 minutes.
10 Use a multichannel pipette to add 25 µl 50 mM NaOH to each well. Pipette to mix.
11 Incubate at room temperature for 5 minutes.
12 During incubation, transfer 22 µl PMM2/TDP1 master mix to each well of the IAP
plate.
13 Transfer samples eluted from the FPU plate to the IAP plate as follows.
a
b
c
Pipette to mix the NaOH in the first column.
Transfer 20 µl from the FPU plate to the corresponding column of the IAP plate.
Pipette to mix.
Discard the waste collection midi plate.
14 Centrifuge the IAP plate at 1000 × g for 1 minute.
15 Transfer to the post-amplification area.
16 Determine the required number (X) of PCR cycles using the following table:
16
Document # 15054779 v01
Amplify Libraries
Table 2 50–99 ng
Number of Amplicons in TSO
< 96 amplicons
97–384 amplicons
385–768 amplicons
769–1536 amplicons
Number of PCR Cycles (X)
33
28
27
26
17 Perform PCR on a thermal cycler using the following program:
} 95°C for 3 minutes
} X cycles of:
} 95°C for 30 seconds
} 66°C for 30 seconds
} 72°C for 60 seconds
} 72°C for 5 minutes
} Hold at 10°C
SAFE STOPPING POINT
If you are stopping, leave the plate on the thermal cycler at 2°C to 8°C overnight.
TruSight Myeloid Sequencing Panel Reference Guide
17
Protocol
Clean Up Libraries
This step uses AMPure XP beads to purify the PCR products from other reaction
components.
Consumables and Equipment
}
}
}
}
}
}
EBT (Elution Buffer with Tris)
AMPure XP beads
Barcode labels
} CLP (Clean-up Plate)
} LNP (Library Normalization Plate)
Freshly prepared 80% ethanol (EtOH) (40 ml per 96 samples)
96-well midi plates (2)
Microseal 'B' adhesive film
About Reagents
}
Always prepare fresh 80% EtOH for wash steps.
Preparation
1
Prepare the following consumables.
Reagent
AMPure XP beads
Storage
Instructions
2°C to 8°C Let stand for 30 minutes to bring to room temperature.
2
Prepare fresh 80% EtOH from absolute ethanol.
3
Label 2 new midi plates CLP and LNP.
Procedure
1
Centrifuge the IAP plate at 1000 × g for 1 minute.
2
Run an aliquot of the libraries on 4% agarose gel (5 µl) or Bioanalyzer (1 µl).
Expected PCR product size for 250 bp amplicons is ~350 bp.
The following table provides the expected PCR product sizes.
Amplicon Size (bp)
150
175
250
425
PCR Product Size (bp)
~280
~310
~350
~570
Figure 4 Agarose Gel Example
18
Document # 15054779 v01
Clean Up Libraries
A
B
Expected PCR product for 250 bp amplicons (~350 bp)
Primers
Figure 5 Bioanalyzer Example
A
B
C
Marker
Expected PCR product for 250 bp amplicons (~350 bp)
Marker
3
Add 45 µl AMPure XP beads to each well of the CLP plate.
4
Transfer all the supernatant from each well of the IAP plate to the corresponding well
of the CLP plate.
5
Shake at 1800 rpm for 2 minutes.
6
Incubate at room temperature for 10 minutes.
7
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
TruSight Myeloid Sequencing Panel Reference Guide
19
Protocol
8
Remove and discard all supernatant from each well.
9
Wash 2 times as follows.
a
b
c
Add 200 µl of 80% EtOH to each sample 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 Remove from the magnetic stand and air-dry for 10 minutes.
12 Add 30 µl EBT to each well.
13 Shake at 1800 rpm for 2 minutes.
14 Make sure that all beads are resuspended. If necessary, pipette to mix and repeat the
shaking step.
15 Incubate at room temperature for 2 minutes.
16 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
17 Transfer 20 µl supernatant from each well of the CLP plate to the corresponding well of
the LNP plate.
18 Centrifuge at 1000 × g for 1 minute.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at 2°C to 8°C for up to 3 days. Alternatively,
store at -25°C to -15°C for up to 7 days.
20
Document # 15054779 v01
This step normalizes the quantity of each library for balanced representation in pooled
libraries. Only samples containing DNA require processing through the subsequent steps.
Consumables and Equipment
}
}
}
}
}
}
}
}
}
}
LNA1 (Library Normalization Additives 1)
LNB1 (Library Normalization Beads 1)
LNW1 (Library Normalization Wash 1)
LNS2 (Library Normalization Storage buffer 2)
SGP (Storage Plate) barcode label
0.1 N NaOH (freshly prepared)
96-well PCR plate, skirted
15 ml conical tube
Microseal 'B' adhesive seals
Magnetic stand-96 (use with midi 96-well storage plates)
WARNING
This set of reagents contains formamide, an aliphatic amide that is a probable
reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact,
and eye contact. Wear protective equipment, including eye protection, gloves, and
laboratory coat. Handle used reagents as chemical waste and discard in accordance with
the governmental safety standards for your region. For environmental, health, and safety
information, see the SDS for this kit at support.illumina.com/sds.html.
WARNING
This set of reagents contains ß-mercaptoethanol. Perform the following procedure in a hood
or well-ventilated area.
About Reagents
}
}
}
}
}
Use a P1000 pipette to transfer LNB1 to LNA1.
When mixing, mix thoroughly.
Mix only the amounts of LNA1 and LNB1 required for the current experiment.
Store remaining LNA1 and LNB1 separately at their respective temperatures.
Make sure that LNB1 is resuspended before use. Homogeneous resuspension is
essential for consistent cluster density on the flow cell.
TruSight Myeloid Sequencing Panel Reference Guide
21
Normalize Libraries
Normalize Libraries
Protocol
Preparation
1
Prepare the following consumables.
Reagent
LNA1
Storage
-25°C to -15°C
LNB1
2°C to 8°C
LNW1
2°C to 8°C
LNS2
15°C to 30°C
Instructions
Thaw at room temperature. Let stand for 30 minutes to
bring to room temperature.
Vortex to mix. Make sure that all precipitate has
dissolved.
Let stand for 30 minutes to bring to room temperature.
Vortex for at least 1 minute. Invert intermittently to
resuspend. Make sure that the bottom of the tube is free
of pellets.
Thaw at room temperature. Let stand for 30 minutes to
bring to room temperature.
If frozen, thaw at room temperature for 20 minutes.
Vortex to mix.
2
Prepare fresh 0.1 N NaOH.
3
Label a new 96-well plate SGP.
Procedure
1
Add 4.4 µl LNA1 per library to a new 15 ml conical tube.
2
Use a P1000 pipette to resuspend LNB1.
3
Transfer 800 µl LNB1 to the 15 ml conical tube of LNA1. Invert to mix.
The LNB1/NA1 mix is sufficient for 96 libraries.
4
Add the LNA1/LNB1 mix to a trough.
5
Add 45 µl LNA1/LNB1 to each well of the LNP plate.
6
Shake at 1800 rpm for 30 minutes.
Durations other than 30 minutes can affect library representation and cluster density.
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 as follows.
a
b
c
d
Add 45 µl LNW1 to each library well.
Shake at 1800 rpm for 5 minutes.
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
Remove and discard all supernatant.
11 Remove residual LNW1 from each well.
12 Remove from the magnetic stand.
13 Add 30 µl fresh 0.1 N NaOH to each well.
14 Shake at 1800 rpm for 5 minutes.
15 If the libraries are not resuspended, pipette to mix, and then shake at 1800 rpm for 5
minutes.
22
Document # 15054779 v01
17 Add 30 µl LNS2 to each well of the SGP plate.
18 Transfer 30 µl supernatant from each well of the LNP plate to the corresponding well
of the SGP plate.
19 Centrifuge at 1000 × 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 30 days.
TruSight Myeloid Sequencing Panel Reference Guide
23
Normalize Libraries
16 Place the LNP plate on a magnetic stand and wait until the liquid is clear (~2 minutes).
Protocol
Pool Libraries
Pooling libraries combines equal volumes of normalized libraries in a single tube. After
pooling, dilute and heat-denature the library pool before loading libraries for the
sequencing run.
Consumables
}
}
}
}
HT1 (Hybridization Buffer)
PAL (Pooled Amplicon Library) barcode label
Eppendorf tubes, screw top (2)
PCR 8-tube strip
About Reagents
}
Store the PAL tube at -25°C to -15°C for later use.
Preparation
1
If the SGP plate was stored frozen, prepare as follows.
a
b
c
Thaw at room temperature.
Centrifuge at 1000 × g for 1 minute.
Pipette to mix.
2
To prepare for the sequencing run, begin thawing reagents according to the instructions
for your instrument.
3
Label a new Eppendorf tube PAL.
Procedure
24
1
Transfer 5 µl of each library from the SGP plate to an 8-tube strip, column by column.
2
Seal the plate and store at -25°C to -15°C.
3
Transfer the contents of the 8-tube strip to the PAL tube. Pipette to mix.
4
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.
Document # 15054779 v01
Appendix A Supporting Information
Introduction
Acronyms
Kit Contents
Consumables and Equipment
TruSight Myeloid Sequencing Panel Reference Guide
26
27
28
30
25
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.
26
Document # 15054779 v01
Acronyms
Acronyms
Acronym
Definition
ACD1
Amplicon Control DNA 1
ACP1
Amplicon Control Oligo Pool 1
TSO
TruSight Oligos
CLP
Clean-up Plate
EBT
Elution Buffer with Tris
ELM4
Extension Ligation Mix 4
FPU
Filter Plate Unit
HT1
Hybridization Buffer
HYP
Hybridization Plate
IAP
Index Amplification Plate
LNA1
Library Normalization Additives 1
LNB1
Library Normalization Beads 1
LNP
Library Normalization Plate
LNS2
Library Normalization Storage Buffer 2
LNW1
Library Normalization Wash 1
OHS2
Oligo Hybridization for Sequencing Reagent 2
PAL
Pooled Amplicon Library
TruSight Myeloid Sequencing Panel Reference Guide
27
Supporting Information
Kit Contents
Make sure that you have all reagents identified in this section before proceeding to the
library preparation procedures. TruSight Myeloid kits are available in the following
configurations.
Kit Name
TruSight Myeloid
TruSeq Custom Amplicon Index Kit
Catalog #
FC-130-1010
FC-130-1003
TruSight Myeloid Sequencing Panel Kit Contents
(96 Samples) (FC-130-1010)
Box 1
Quantity
1
1
1
Reagent
ACD1
ACP1
OHS2
1
1
1
1
1
5 each
ELM4
PMM2
TDP1
SW1
UB1
--
Description
Amplicon Control DNA 1
Amplicon Control Oligo Pool 1
Oligo Hybridization
for Sequencing Reagent 2
Extension Ligation Mix 4
PCR Master Mix 2
TruSeq DNA Polymerase 1
Stringent Wash 1
Universal Buffer 1
Barcode labels for FPU, HYP,
and IAP
Storage Temperature
-25°C to -15°C
-25°C to -15°C
-25°C to -15°C
Area
Pre-amp
Pre-amp
Pre-amp
-25°C to -15°C
-25°C to -15°C
-25°C to -15°C
2°C to 8°C
2°C to 8°C
Room temperature
Pre-amp
Pre-amp
Pre-amp
Pre-amp
Pre-amp
Pre-amp
WARNING
This set of reagents contains formamide, an aliphatic amide that is a probable
reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact,
and eye contact. Wear protective equipment, including eye protection, gloves, and
laboratory coat. Handle used reagents as chemical waste and discard in accordance with
the governmental safety standards for your region. For environmental, health, and safety
information, see the SDS for this kit at support.illumina.com/sds.html.
Box 2
Quantity
1
1
Reagent
-LNB1
Description
Filter plate with lid
Library Normalization
Beads 1
Quantity
1
1
Reagent
HT1
LNA1
1
LNW1
Description
Hybridization Buffer
Library Normalization
Additives 1
Library Normalization
Wash 1
Storage Temperature
Room temperature
2°C to 8°C
Area
Pre-amp
Post-amp
Box 3
28
Storage Temperature
-25°C to -15°C
-25°C to -15°C
Area
Post-amp
Post-amp
2°C to 8°C
Post-amp
Document # 15054779 v01
Reagent
LNS2
1
5 each
EBT
--
Description
Library Normalization
Storage Buffer 2
Elution Buffer with Tris
Barcode labels for CLP,
DAL, LNP, PAL, and
SGP
Storage Temperature
Room temperature
Area
Post-amp
Room temperature
Room temperature
Post-amp
Post-amp
TruSight Myeloid Custom Amplicon Oligos Box, Store at -25°C to 15°C
Quantity
1
Reagent
TSO
Description
TruSight Oligo Tube
Area
Pre-amp
TruSeq Custom Amplicon Index Kit Contents
(96 Indexes, 384 Samples)(FC-130-1003)
TruSeq Custom Amplicon Index Kit, Store at -25°C to -15°C
Quantity
8
12
Description
i5 Index Primers, A501 to A508
i7 Index Primers, A701 to A712
Area
Pre-amp
Pre-amp
Index Adapter Replacement Caps, Store at Room Temperature
Quantity
48
32
Reagent Name
i5 Index Tube Caps, White
i7 Index Tube Caps, Orange
Area
Pre-amp
Pre-amp
Additional Components
Illumina Consumable
Catalog #
TruSeq Custom Amplicon Filter Plate¹
TruSeq Index Plate Fixture and Collar
Kit²
FC-130-1006
FC-130-1007
Storage
Temperature
Room temperature
Room temperature
Area
Pre-amp
Pre-amp
¹ Highly recommended
² Required and reusable
TruSight Myeloid Sequencing Panel Reference Guide
29
Kit Contents
Quantity
1
Supporting Information
Consumables and Equipment
Make sure that you have the required user-supplied consumables and equipment before
starting the protocol.
Consumables
Consumable
Supplier
10 N NaOH (prepare from tablets or use a
standard solution)
General lab supplier
96-well skirted PCR plates, 0.2 ml,
polypropylene
Bio-Rad, Part # MSP-9601
96-well storage plates, 0.8 ml (midi plates)
Fisher Scientific, Part # AB-0859
Fisher Scientific, Part # AB-0765
Agencourt AMPure XP, 60 ml kit
Beckman Coulter, Part # A63881/A63880
Foil seals
Beckman Coulter, Part # 538619
Conical tubes, 15 ml
General lab supplier
Eppendorf microcentrifuge tubes, screw top
General lab supplier
Ethanol, 200 proof for molecular biology
General lab supplier
Microseal 'A' adhesive seals
Bio-Rad, Part # MSA-5001
Microseal 'B' adhesive seals
Bio-Rad, Part # MSB-1001
PCR 8-tube strips
General lab supplier
Solution basin, PVC, non-sterile (trough)
Labcor, Part# 730-001
Agarose gel (2% for 250 bp and 425 bp
amplicons, or 4% for 150 bp, 175 bp, and 250
bp amplicons)
General Lab Supplier
DNA 1000 Kit for Bioanalyzer
Agilent 5067–1504 (for 300 samples)
DNA molecular weight markers
General Lab Supplier
Ice bucket
General Lab Supplier
Pre-PCR Equipment
30
Equipment
Supplier
37° incubator
Forced Air Oven, VWR International or
comparable
Heat block, 96-well
SciGene, Hybex Microsample Incubator for
PCR plate
Tabletop centrifuge
General lab supplier
Document # 15054779 v01
Consumables and Equipment
Post-PCR Equipment
Equipment
Supplier
Magnetic stand-96
Invitrogen DynaMag™-96 Side Skirted
Post-PCR plate shaker
Q Instruments BioShake iQ high-speed
thermoshaker, part # 1808-0506, or
Q Instruments BioShake XP high-speed lab
shake, part # 1808-0505
Tabletop centrifuge
General lab supplier
Gel electrophoresis supplies and apparatus
General lab supplier
Heat block for 1.5 ml centrifuge tubes
General lab supplier
Bioanalyzer System
Agilent Technologies
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.
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
TruSight Myeloid Sequencing Panel Reference Guide
31
32
Document # 15054779 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 Myeloid Sequencing Panel Reference Guide
33
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
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

Download PDF

advertisement