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Agilent SureSelect Target Enrichment System Sequencer Protocol Manual V2 PDF
Summary of Content for Agilent SureSelect Target Enrichment System Sequencer Protocol Manual V2 PDF
SureSelectXT Target Enrichment System for the Illumina Platform
Protocol Version D1, July 2021
SureSelect platform manufactured with Agilent SurePrint Technology
For Research Use Only. Not for use in diagnostic procedures.
Agilent Technologies
Notices Agilent Technologies, Inc. 2010-2021
No part of this manual may be reproduced in any form or by any means (including elec- tronic storage and retrieval or translation into a foreign language) without prior agree- ment and written consent from Agilent Technologies, Inc. as governed by United States and international copyright laws.
Manual Part Number G7530-90000
Edition Version D1, July 2021
Printed in USA
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Warranty The material contained in this document is provided as is, and is subject to being changed, with- out notice, in future editions. Fur- ther, to the maximum extent permitted by applicable law, Agi- lent disclaims all warranties, either express or implied, with regard to this manual and any information contained herein, including but not limited to the implied warranties of merchant- ability and fitness for a particular purpose. Agilent shall not be lia- ble for errors or for incidental or consequential damages in con- nection with the furnishing, use, or performance of this document or of any information contained herein. Should Agilent and the user have a separate written agreement with warranty terms covering the material in this doc- ument that conflict with these terms, the warranty terms in the separate agreement shall control.
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CAUTION
A CAUTION notice denotes a haz- ard. It calls attention to an operat- ing procedure, practice, or the like that, if not correctly performed or adhered to, could result in damage to the product or loss of important data. Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met.
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2 SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
In this Guide...
SureSelectXT Target Enrichment Sys
This guide provides an optimized protocol for Illumina paired- end multiplexed library preparation using the SureSelectXT Library Prep and Capture System.
1
This chapter contains information (such as procedural notes, safety information, required reagents and equipment) that you should read and understand before you start an experiment.
2
This chapter describes the steps to prepare libraries for target enrichment from 3- g gDNA samples.
3
This chapter describes the steps to prepare libraries for target enrichment from 200- ng gDNA samples.
4
This chapter describes the steps to hybridize and capture the prepared library DNA.
5
This chapter describes the steps to amplify, purify, and assess quality and quantity of the sample libraries. Samples are pooled by mass prior to sequencing.
6
This chapter contains recommended protocol modifications for FFPE- derived DNA samples.
7
This chapter contains reference information, including component kit contents and index sequences.
tem for Illumina Multiplexed Sequencing 3
Whats New in Version D1
4 Sur
Support for SureSelect XT Human All Exon V8 Probe (see Table 3 on page 17)
Minor updates to instructions in theHybridization and Capture chapter on page 63 to page 71
Updates to downstream sequencing platform and kit support information (see Table 41 on page 86 and footnote to Table 2 on page 16)
Whats New in Version D0
produced using an updated manufacturing process beginning August, 2020 (see Table 4 on page 18). Custom probes produced using the legacy manufacturing process are also fully supported by the protocols in this document. Probe information was reorganized (see Table 3 on page 17 through Table 5 on page 18), and probe nomenclature throughout document was updated.
Updates to thermal cycler and plasticware recommendations (see Table 6 on page 19, Caution on page 19 and on page 65, Procedural Notes on page 14, and Table 55 on page 101).
Updates to ordering information for Dynabeads MyOne Streptavidin T1 beads, 1X Low TE Buffer, and AMPure XP Kits (Table 2 on page 16) and for Eppendorf ThermoMixer C and Qubit Fluorometer (Table 6 on page 19).
Updates to optional materials in Table 7 on page 21, including removal of ethylene glycol supplier information (see page 24 and page 44 for related updates to DNA shearing set up instructions).
Updates to Agilent TapeStation 4200/4150 ordering information (Table 6 on page 20) and sample mixing information (for example, see Caution on page 61).
Support for 5200 Fragment Analyzer (see footnote to Table 6 on page 20).
eSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
SureSelectXT Target Enrichment Sys
Correction to Downstream Sequencing Modifications" on page 93 to indicate that FFPE sample adjustments based on DIN score.
Updates to Technical Support contact information (see page 2)
tem for Illumina Multiplexed Sequencing 5
6 Sur
Content
1 Before You Begin 11
Overview of the Workflow 12
Procedural Notes 14
Safety Notes 15
Required Reagents 16
Required Equipment 19
Optional Reagents and Equipment 21
2 Sample Preparation (3 g DNA Samples) 23
Step 1. Shear the DNA 24 Step 2. Purify the sample using AMPure XP beads 26 Step 3. Assess quality (optional) 28 Step 4. Repair the ends 30 Step 5. Purify the sample using AMPure XP beads 31 Step 6. dA-tail the 3' end of the DNA fragments 32 Step 7. Purify the sample using AMPure XP beads 33 Step 8. Ligate the paired-end adaptor 34 Step 9. Purify the sample using AMPure XP beads 35 Step 10. Amplify the adaptor-ligated library 36 Step 11. Purify the amplified library with AMPure XP beads 39 Step 12. Assess quality and quantity 40
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing 7
Contents
3 Sample Preparation (200 ng DNA Samples) 43
Step 1. Shear the DNA 44 Step 2. Assess quality (optional) 47 Step 3. Repair the ends 48 Step 4. Purify the sample using AMPure XP beads 49 Step 5. dA-tail the 3' end of the DNA fragments 51 Step 6. Purify the sample using AMPure XP beads 52 Step 7. Ligate the paired-end adaptor 53 Step 8. Purify the sample using AMPure XP beads 55 Step 9. Amplify the adaptor-ligated library 56 Step 10. Purify the amplified library with AMPure XP beads 59 Step 11. Assess quality and quantity 60
4 Hybridization and Capture 63
Step 1. Hybridize DNA samples to the probe 64 Step 2. Prepare streptavidin-coated magnetic beads 70 Step 3. Capture the hybridized DNA using streptavidin-coated beads 71
5 Indexing and Sample Processing for Multiplexed Sequencing 73
Step 1. Amplify the captured libraries with indexing primers 74 Step 2. Purify the amplified captured libraries using AMPure XP beads 77 Step 3. Assess indexed library DNA quantity and quality 79 Step 4. Quantify each index-tagged library by QPCR (optional) 83 Step 5. Pool samples for multiplexed sequencing 84 Step 6. Prepare sequencing samples 86
6 Appendix: Using FFPE-derived DNA Samples 89
Modifications for all FFPE DNA samples 90
Modifications for samples assessed using the Agilent NGS FFPE QC Kit 91
Modifications for samples assessed using Agilents Genomic DNA ScreenTape 93
8 SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Contents
7 Reference 95
Kit Contents 96
Nucleotide Sequences of SureSelectXT Indexes A01 to H12 100
Alternative Capture Equipment Combinations 101
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing 9
Contents
10 SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
SureSelectXT Target Enrichment System for the Illumina Platform Protocol
1 Before You Begin
Overview of the Workflow 12
Procedural Notes 14
Safety Notes 15
Required Reagents 16
Required Equipment 19
Optional Reagents and Equipment 21
Make sure you have the most current protocol. Go to agilent.com and search for G7530- 90000.
Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an experiment.
This protocol differs from the Illumina Multiplexed Paired-End sequencing manual and other SureSelect protocols at several steps. Pay close attention to the primers used for each amplification step and the blocking agents used during hybridization.
Agilent guarantees performance and provides technical support for the SureSelect reagents required for this workflow only when used as directed in this Protocol.
NOTE
NOTE
11Agilent Technologies
1 Before You Begin Overview of the Workflow
Overview of the Workflow
12
The SureSelectXT target enrichment workflow is summarized in Figure 1. The estimated time requirements for each step are summarized in Table 1.
Figure 1 Overall target-enriched sequencing sample preparation workflow.
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Before You Begin 1 Overview of the Workflow
SureSelectXT Target
Table 1 Estimated time requirements (up to 16 sample run size)
Step Time
Library Preparation 5 hours
Hybridization and Capture 16 or 24 hours
Post-capture amplification 1 hour
QC using Bioanalyzer or TapeStation and sample pooling 1.5 hours
Enrichment System for Illumina Multiplexed Sequencing 13
1 Before You Begin Procedural Notes
Procedural Notes
14
This protocol includes sample processing steps in PCR plates or strip tubes, with certain protocol steps using liquid volumes exceeding 0.2 ml. Before you begin, you must verify that the wells of the plasticware selected for use can accommodate at least 0.31 ml for processing of 3- g DNA samples or can accommodate at least 0.28 ml for processing of 200- ng DNA samples. If needed, samples may be transferred to 1.5- ml tubes for the high- volume protocol steps, with possible impacts on sample throughput and yield.
To prevent contamination of reagents by nucleases, always wear powder- free laboratory gloves and use dedicated solutions and pipettors with nuclease- free aerosol- resistant tips.
Use best- practices to prevent PCR product contamination of samples throughout the workflow:
1 Assign separate pre- PCR and post- PCR work areas and use dedicated equipment, supplies, and reagents in each area. In particular, never use materials designated to post- PCR work areas for pre- PCR segments of the workflow.
2 Maintain clean work areas. Clean pre- PCR surfaces that pose the highest risk of contamination daily using a 10% bleach solution.
3 Always use dedicated pre- PCR pipettors with nuclease- free aerosol- resistant tips to pipette dedicated pre- PCR solutions.
4 Wear powder- free gloves. Use good laboratory hygiene, including changing gloves after contact with any potentially- contaminated surfaces.
Do not mix stock solutions of gDNA on a vortex mixer. Instead, gently tap the tube with your finger to mix the sample.
When preparing frozen reagent stock solutions for use:
1 Thaw the aliquot as rapidly as possible without heating above room temperature.
2 Mix briefly on a vortex mixer, then spin in a centrifuge for 5 to 10 seconds to drive the contents off of walls and lid.
3 Store on ice or in a cold block until use.
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Before You Begin 1 Safety Notes
SureSelectXT Target
For each protocol step that requires removal of tube cap strips, reseal the tubes with a fresh strip of domed caps. Cap deformation may result from exposure of the cap strips to the heated lid of the thermal cycler and from other procedural steps. Reuse of strip caps can cause sample loss, sample contamination, or imprecision in sample temperatures during thermal cycler incubation steps.
In general, follow Biosafety Level 1 (BSL1) safety rules.
Possible stopping points, where samples may be stored at 20C, are marked in the protocol. Do not subject the samples to multiple freeze/thaw cycles.
Safety Notes
Wear appropriate personal protective equipment (PPE) when working in the laboratory.
CAUTION
Enrichment System for Illumina Multiplexed Sequencing 15
1 Before You Begin Required Reagents
Required Reagents
16
Table 2 Required Reagents
Description Vendor and part number
SureSelect, ClearSeq or OneSeq Probe Select the appropriate probe from Table 3, Table 4, or Table 5
SureSelectXT Reagent Kit, Illumina (ILM) platforms*
HiSeq platform, 16 reactions HiSeq platform, 96 reactions HiSeq platform, 480 reactions MiSeq platform, 16 reaction MiSeq platform, 96 reactions MiSeq platform, 480 reactions
Agilent
p/n G9611A p/n G9611B p/n G9611C p/n G9612A p/n G9612B p/n G9612C
AMPure XP Kit 5 mL 60 mL 450 mL
Beckman Coulter Genomics p/n A63880 p/n A63881 p/n A63882
Herculase II Fusion DNA Polymerase (includes dNTPs and 5 Buffer) 200 Reactions (processes 100 XT libraries) 400 Reactions
Agilent p/n 600677 p/n 600679
Dynabeads MyOne Streptavidin T1 2 mL 10 mL 50 mL
Thermo Fisher Scientific p/n 65601 p/n 65602 p/n 65604D
1X Low TE Buffer (10 mM Tris-HCl, pH 7.5-8.0, 0.1 mM EDTA) Thermo Fisher Scientific p/n 12090-015, or equivalent
100% Ethanol, molecular biology grade Sigma-Aldrich p/n E7023
Qubit dsDNA HS Assay Kit or
Qubit BR dsDNA Assay Kit 100 assays 500 assays
Thermo Fisher Scientific p/n Q32851
Thermo Fisher Scientific p/n Q32850 p/n Q32853
Nuclease-free Water (not DEPC-treated) Thermo Fisher Scientific p/n AM9930
* HiSeq and MiSeq Reagent Kits are also compatible with the NextSeq 500 and NovaSeq 6000 platforms.
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Before You Begin 1 Required Reagents
SureSelectXT Target
Table 3 Compatible Pre-Designed Probes
Probe 16 Reactions 96 Reactions
SureSelect XT Human All Exon V8 5191-6879 5191-6891
SureSelect XT Human All Exon V7 5191-4004 5191-4005
SureSelect XT Human All Exon V6 5190-8863 5190-8864
SureSelect XT Human All Exon V6 + UTRs 5190-8881 5190-8882
SureSelect XT Human All Exon V6 + COSMIC 5190-9307 5190-9308
SureSelect XT Clinical Research Exome V2 5190-9491 5190-9492
SureSelect XT Focused Exome 5190-7787 5190-7788
SureSelect XT Mouse All Exon 5190-4641 5190-4642
SureSelect XT Human X-Chromosome 5190-4651 5190-4652
ClearSeq Comprehensive Cancer XT 5190-8011 5190-8012
ClearSeq Inherited Disease XT 5190-7518 5190-7519
Pre-designed probes customized with additional Plus custom content
SureSelect XT Human All Exon V7 Plus 1
Please visit the SureDesign website to design the customized Plus content and obtain ordering information. Contact the SureSelect support team (see page 2) or
your local representative if you need assistance.
SureSelect XT Human All Exon V7 Plus 2
SureSelect XT Human All Exon V6 Plus 1
SureSelect XT Human All Exon V6 Plus 2
SureSelect XT Clinical Research Exome V2 Plus 1
SureSelect XT Clinical Research Exome V2 Plus 2
SureSelect XT Focused Exome Plus 1
SureSelect XT Focused Exome Plus 2
ClearSeq Comprehensive Cancer Plus XT
ClearSeq Inherited Disease Plus XT
Enrichment System for Illumina Multiplexed Sequencing 17
1 Before You Begin Required Reagents
18
Table 4 Compatible Custom Probes*
* Custom probes designed August 2020 or later are produced using an updated manufacturing process; design size Tier is shown on labeling for these products. Custom probes designed and ordered prior to August 2020 may be reordered, with these probes produced using the legacy manufacturing process; design-size Tier is not shown on labeling for the legacy-process products. Custom probes of both categories use the same optimized target enrichment protocols detailed in this publication.
Probe 16 Reactions 96 Reactions
SureSelect Custom Tier1 1499 kb Please visit the SureDesign website to design Custom SureSelect probes and obtain ordering
information. Contact the SureSelect support team (see page 2) or your local representative if you
need assistance. Custom probes are also available in 480 Reaction size.
SureSelect Custom Tier2 0.5 2.9 Mb
SureSelect Custom Tier3 3 5.9 Mb
SureSelect Custom Tier4 6 11.9 Mb
SureSelect Custom Tier5 1224 Mb
Table 5 Compatible OneSeq CNV Probes
Probe 16 Reactions 96 Reactions
OneSeq 300kb CNV Backbone + Human All Exon V7 5191-4022 5191-4023
OneSeq 1Mb CNV Backbone + Human All Exon V7 5191-4025 5191-4026
OneSeq Constitutional Research Panel 5190-8702 5190-8703
Pre-designed probes customized with additional Plus custom content
OneSeq 1Mb CNV Backbone + Custom 1499 kb
Please visit the SureDesign website to design the customized Plus content and obtain ordering
information. Contact the SureSelect support team (see page 2) or your
local representative if you need assistance.
OneSeq 1Mb CNV Backbone + Custom 0.52.9 Mb
OneSeq 1Mb CNV Backbone + Custom 35.9 Mb
OneSeq 1Mb CNV Backbone + Custom 611.9 Mb
OneSeq 1Mb CNV Backbone + Custom 1224 Mb
OneSeq Hi Res CNV Backbone + Custom 1499 kb
OneSeq Hi Res CNV Backbone + Custom 0.5 2.9 Mb
OneSeq Hi Res CNV Backbone + Custom 35.9 Mb
OneSeq Hi Res CNV Backbone + Custom 611.9 Mb
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Before You Begin 1 Required Equipment
Required Equipment
CAUTION Sample volumes exceed 0.2 ml in certain steps of this protocol. Make sure that the plasticware used with the selected thermal cycler holds 0.31 ml per well when processing 3-g DNA samples or 0.28 ml per well when processing 200-ng DNA samples. Samples may be transferred to 1.5-ml tubes for processing where required, with possible impacts on sample throughput and yield.
SureSelectXT Target
R
Equipment
Description Vendor and part number
Thermal Cycler with 96-well, 0.2 ml block Various suppliers
Plasticware compatible with the selected thermal cycler:
96-well plates or 8-well strip tubes Tube cap strips, domed
Consult the thermal cycler manufacturers recommendations
Qubit Fluorometer Thermo Fisher Scientific p/n Q33238
Qubit Assay Tubes Thermo Fisher Scientific p/n Q32856
Covaris Sample Preparation System, S-series or E-series model
Covaris
Covaris sample holders
96 microTUBE plate (E-series only)
microTUBE for individual sample processing
Covaris p/n 520078
Covaris p/n 520045
DNA LoBind Tubes, 1.5-mL PCR clean, 250 pieces Eppendorf p/n 022431021 or equivalent
Microcentrifuge Eppendorf microcentrifuge, model 5417C or equivalent
Plate or strip tube centrifuge Labnet International MPS1000 Mini Plate Spinner, p/n C1000 (requires adapter, p/n C1000-ADAPT, for use with strip tubes) or equivalent
96-well plate mixer Eppendorf ThermoMixer C, p/n 5382000023 and Eppendorf SmartBlock 96 PCR, p/n 5306000006, or equivalent
Enrichment System for Illumina Multiplexed Sequencing 19
1 Before You Begin Required Equipment
DNA Analysis Platform and Consumables*
Agilent 2100 Bioanalyzer Instrument
Agilent 2100 Expert SW Laptop Bundle (optional)
DNA 1000 Kit
High Sensitivity DNA Kit
OR
Agilent 4200/4150 TapeStation
96-well sample plates
96-well plate foil seals
8-well tube strips
8-well tube strip caps
D1000 ScreenTape
D1000 Reagents
High Sensitivity D1000 ScreenTape
High Sensitivity D1000 Reagents
Agilent p/n G2939BA
Agilent p/n G2953CA
Agilent p/n 5067-1504
Agilent p/n 5067-4626
Agilent p/n G2991AA/G2992AA
Agilent p/n 5042-8502
Agilent p/n 5067-5154
Agilent p/n 401428
Agilent p/n 401425
Agilent p/n 5067-5582
Agilent p/n 5067-5583
Agilent p/n 5067-5584
Agilent p/n 5067-5585
Magnetic separator Thermo Fisher Scientific p/n 12331D or equivalent
Vacuum concentrator Savant SpeedVac, model DNA120, or equivalent
Multichannel pipette Rainin Pipet-Lite Multi Pipette or equivalent
P10, P20, P200 and P1000 pipettes Rainin Pipet-Lite Pipettes or equivalent
Sterile, nuclease-free aerosol barrier pipette tips general laboratory supplier
Vortex mixer general laboratory supplier
Ice bucket general laboratory supplier
Powder-free gloves general laboratory supplier
* DNA samples may also be analyzed using the Agilent 5200 Fragment Analyzer, p/n M5310AA, and associated NGS Fragment Kits (DNF-473-0500 and DNF-474-0500). Implement any sample dilution instructions provided in protocols in this document, and then follow the assay instructions provided for each NGS Fragment Kit.
Select a magnetic separator configured to collect magnetic particles on one side of each well. Do not use a magnetic sep- arator configured to collect the particles in a ring formation.
Table 6 Required Equipment
Description Vendor and part number
20 SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Before You Begin 1 Optional Reagents and Equipment
Optional Reagents and Equipment
SureSelectXT Target
Table 7 Optional materials for processing of all samples
Description Vendor and part number
Tween 20 Sigma-Aldrich p/n P9416-50ML
PlateLoc Thermal Microplate Sealer with Small Hotplate and Peelable Aluminum Seal for PlateLoc Sealer
Please contact the SureSelect support team (see page 2) or your local representative for ordering information
MicroAmp Clear Adhesive Film Thermo Fisher Scientific p/n 4311971
Agilent QPCR NGS Library Quantification Kit (Illumina GA) Agilent p/n G4880A
Mx3005P Real-Time PCR System Agilent p/n 401449 or equivalent
Mx3000P/Mx3005P 96-well tube plates Agilent p/n 410088 or equivalent
Mx3000P/Mx3005P optical strip caps Agilent p/n 401425 or equivalent
Table 8 Optional materials for processing of FFPE samples
Description Vendor and part number
Agilent NGS FFPE QC Kit
16 reactions 96 reactions
Agilent
p/n G9700A p/n G9700B
TapeStation Genomic DNA Analysis Consumables:
Genomic DNA ScreenTape
Genomic DNA Reagents
Agilent p/n 5067-5365
Agilent p/n 5067-5366
Enrichment System for Illumina Multiplexed Sequencing 21
1 Before You Begin Optional Reagents and Equipment
22
SureSelectXT Target Enrichment System for the Illumina Platform Protocol
2 Sample Preparation (3 g DNA Samples)
Step 1. Shear the DNA 24
Step 2. Purify the sample using AMPure XP beads 26
Step 3. Assess quality (optional) 28
Step 4. Repair the ends 30
Step 5. Purify the sample using AMPure XP beads 31
Step 6. dA-tail the 3' end of the DNA fragments 32
Step 7. Purify the sample using AMPure XP beads 33
Step 8. Ligate the paired-end adaptor 34
Step 9. Purify the sample using AMPure XP beads 35
Step 10. Amplify the adaptor-ligated library 36
Step 11. Purify the amplified library with AMPure XP beads 39
Step 12. Assess quality and quantity 40
CAUTION This section contains instructions for the preparation of gDNA libraries from 3 g DNA samples. For 200 ng DNA samples and FFPE-derived DNA samples, see the library preparation protocol on page 43.
The sample preparation protocol is used to prepare DNA libraries for sequencing using the Illumina paired- read platform. For each sample to be sequenced, an individual indexed library is prepared. For an overview of the SureSelectXT target enrichment workflow, see Figure 1 on page 12.
The steps in this section differ from the Illumina protocol in the use of the Covaris system for gDNA shearing, smaller target shear size, elimination of size selection by gel purification, implementation of AMPure XP beads for all purification steps, and primers used for PCR. Refer to the Illumina protocol Preparing Samples for Multiplexed Paired- End Sequencing (p/n1005361) or the appropriate Illumina protocol for more information.
23Agilent Technologies
2 Sample Preparation (3 g DNA Samples) Step 1. Shear the DNA
Step 1. Shear the DNA Make sure genomic DNA samples are of high quality with an OD 260/280 ratio ranging from 1.8 to 2.0. Use the Qubit system to quantify genomic DNA before library preparation.
This protocol has been optimized using a Covaris model E220 instrument and 130-l Covaris microTUBE for shearing 130-l DNA samples to a target DNA fragment size of 150 to 200 bp. To shear using a different Covaris instrument model/sample holder, or if your NGS workflow requires a different DNA fragment size, consult the manufacturers literature for recommended shearing conditions.
NOTE
24
1 Set up the Covaris E- series or S- series instrument.
a Check that the water in the Covaris tank is filled with fresh deionized water to the appropriate fill line level according to the manufacturers recommendations for the specific instrument model and sample tube or plate in use.
b Check that the water covers the visible glass part of the tube.
c On the instrument control panel, push the Degas button. Degas the instrument for least 30 minutes before use, or according to the manufacturers recommendations.
d Set the chiller temperature to between 2C to 5C to ensure that the temperature reading in the water bath displays 5C. Consult the manufacturers recommendations for addition of coolant fluids to prevent freezing.
2 Put a Covaris microTUBE into the loading and unloading station.
Keep the cap on the tube.
You can use the 96 microTUBE plate (see Table 6 on page 19) for the DNA shearing step when preparing multiple gDNA samples in the same experiment.
NOTE
3 Use the Qubit dsDNA Assay to determine the concentration of your gDNA sample.
Follow the instructions for the instrument.
4 Dilute 3 g of high- quality gDNA with 1X Low TE Buffer (10 mM Tris- HCl, pH 8.0, 0.1 mM EDTA) in a 1.5- mL LoBind tube to a total volume of 130 L.
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Sample Preparation (3 g DNA Samples) 2 Step 1. Shear the DNA
SureSelectXT Target
5 Use a tapered pipette tip to slowly transfer the 130- L DNA sample through the pre- split septum.
Be careful not to introduce a bubble into the bottom of the tube.
6 Secure the microTUBE in the tube holder and shear the DNA with the settings in Table 9.
The target DNA fragment size is 150 to 200 bp.
7 Put the Covaris microTUBE back into the loading and unloading station.
8 While keeping the snap- cap on, insert a pipette tip through the pre- split septum, then slowly remove the sheared DNA.
9 Transfer each sheared DNA sample (approximately 130 L) to a separate well of a 96- well plate or strip tube.
Table 9 Shear settings for Covaris instruments (SonoLab software v7 or later)
Setting Value
Duty Factor 10%
Peak Incident Power (PIP) 175
Cycles per Burst 200
Treatment Time 360 seconds
Bath Temperature 4 to 8 C
Enrichment System for Illumina Multiplexed Sequencing 25
2 Sample Preparation (3 g DNA Samples) Step 2. Purify the sample using AMPure XP beads
Step 2. Purify the sample using AMPure XP beads Instructions in this manual are for sample processing in 96-well PCR plates. When processing a small number of samples, you can instead use strip tubes or individual tubes that are compatible with the thermal cycler and magnetic separation device used in the protocol.
The total liquid volume is 0.31 ml in step 4 through step 7 of the protocol below. Before you begin, make sure that wells hold this volume. Samples may be transferred to 1.5-ml tubes for this step, if needed.
NOTE
26
1 Let the AMPure XP beads come to room temperature for at least 30 minutes. Do not freeze the beads at any time.
2 Prepare 400 L of 70% ethanol per sample, plus excess, for use in step 8.
The freshly-prepared 70% ethanol may be used for subsequent purification steps run on the same day. The complete Library Preparation protocol requires 2 mL of fresh 70% ethanol per sample.
NOTE
3 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color.
4 Add 180 L of homogeneous AMPure XP beads to each sheared DNA sample (approximately 130 L) in the PCR plate. Pipette up and down 10 times to mix.
5 Incubate samples for 5 minutes at room temperature.
6 Put the plate into a magnetic separation device. Wait for the solution to clear (approximately 3 to 5 minutes).
7 Keep the plate in the magnetic stand. Carefully remove and discard the cleared solution from each well. Do not touch the beads while removing the solution.
NOTE If some magnetic beads remain suspended in solution after 5 minutes, carefully remove and discard 100 l of cleared solution from near the bottom of the wells, and continue incubating the plate in the magnetic stand for an additional 3 minutes. After the remaining suspension has cleared, remove and discard the remaining cleared solution (approximately 210 l) from the wells.
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Sample Preparation (3 g DNA Samples) 2 Step 2. Purify the sample using AMPure XP beads
SureSelectXT Target
8 Continue to keep the plate in the magnetic stand while you dispense 200 L of 70% ethanol in each sample well.
Use fresh 70% ethanol for optimal results.
9 Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol.
10 Repeat step 8 to step 9 once.
11 Seal the wells with strip caps, then briefly spin the plate to collect the residual ethanol. Return the plate to the magnetic stand for 30 seconds. Remove the residual ethanol with a P20 pipette.
12 Dry the samples by placing the unsealed plate on the thermal cycler, set to hold samples at 37C, for 3 to 5 minutes or until the residual ethanol completely evaporates.
Do not dry the bead pellet to the point that the pellet appears cracked during any of the bead drying steps in the protocol. Elution efficiency is significantly decreased when the bead pellet is excessively dried.
NOTE
13 Add 50 L nuclease- free water to each sample well.
14 Seal the wells with strip caps, then mix well on a vortex mixer and briefly spin the plate to collect the liquid.
15 Incubate for 2 minutes at room temperature.
16 Put the plate in the magnetic stand and leave for 2 to 3 minutes, until the solution is clear.
17 Remove the cleared supernatant (approximately 48 L) to a fresh PCR plate well. You can discard the beads at this time.
Stopping Point
Enrichment System for Illumina Multiplexed Sequencing 27
2 Sample Preparation (3 g DNA Samples) Step 3. Assess quality (optional)
Step 3. Assess quality (optional)
28
Analysis of the purified sheared DNA samples prior to library preparation is optional. If you elect to include this step, follow the instructions below.
Option 1: Analysis using the 2100 Bioanalyzer and DNA 1000 Assay
Use a Bioanalyzer DNA 1000 chip and reagent kit and perform the assay according to the Agilent DNA 1000 Kit Guide.
1 Set up the 2100 Bioanalyzer instrument as instructed in the reagent kit guide.
2 Prepare the chip, samples and ladder as instructed in the reagent kit guide, using 1 L of each sample for the analysis. Load the prepared chip into the instrument and start the run within five minutes after preparation.
3 Check that the electropherogram shows a DNA fragment size peak between 150200 bp. A sample electropherogram is shown in Figure 2.
Figure 2 Analysis of sheared DNA using a DNA 1000 Bioanalyzer assay.
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Sample Preparation (3 g DNA Samples) 2 Step 3. Assess quality (optional)
SureSelectXT Target
Option 2: Analysis using an Agilent TapeStation and D1000 ScreenTape
Use a D1000 ScreenTape and associated reagent kit for analysis of the 3 g sheared DNA samples. Perform the assay according to the Agilent D1000 Assay Quick Guide.
1 Prepare the TapeStation samples as instructed in the reagent kit guide. Use 1 L of each DNA sample diluted with 3 L of D1000 sample buffer for the analysis.
CAUTION For accurate quantitation, make sure to thoroughly mix the combined DNA and sample buffer by vortexing the assay plate or tube strip for 1 minute on the IKA MS3 vortex mixer provided with the 4200/4150 TapeStation system before loading the samples.
2 Load the sample plate or tube strips from step 1, the D1000 ScreenTape, and loading tips into the TapeStation as instructed in the reagent kit guide. Start the run.
3 Verify that the electropherogram shows a DNA fragment size peak between 150200 bp. A sample electropherogram is shown in Figure 3.
Figure 3 Analysis of sheared DNA using a D1000 ScreenTape.
Enrichment System for Illumina Multiplexed Sequencing 29
2 Sample Preparation (3 g DNA Samples) Step 4. Repair the ends
Step 4. Repair the ends
30
Use the SureSelect XT Library Prep Kit ILM for this step.
To process multiple samples, prepare master mixes with overage at each step, without the DNA sample. Master mixes for preparation of 16 samples (including excess) are shown in each table as an example.
Hold samples on ice while setting up this step.
1 Prepare the appropriate volume of End Repair master mix, as described in Table 10, on ice. Mix well on a vortex mixer.
Table 10 Preparation of End Repair master mix
2 Add 52 L of the master mix to each PCR plate sample well containing purified, sheared DNA. Mix by pipetting up and down.
3 Incubate the plate in the thermal cycler and run the program in Table 11. Do not use a heated lid.
Table 11 End-Repair Thermal Cycler Program
Reagent Volume for 1 reaction Volume for 16 reactions (includes excess)
Nuclease-free water 35.2 L 580.8 L
10 End Repair Buffer (clear cap) 10 L 165 L
dNTP Mix (green cap) 1.6 L 26.4 L
T4 DNA Polymerase (purple cap) 1 L 16.5 L
Klenow DNA Polymerase (yellow cap) 2 L 33 L
T4 Polynucleotide Kinase (orange cap) 2.2 L 36.3 L
Total 52 L 858 L
Step Temperature Time
Step 1 20C 30 minutes
Step 2 4C Hold
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Sample Preparation (3 g DNA Samples) 2 Step 5. Purify the sample using AMPure XP beads
Step 5. Purify the sample using AMPure XP beads
SureSelectXT Target
1 Let the AMPure XP beads come to room temperature for at least 30 minutes. Do not freeze the beads at any time.
2 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color.
3 Add 180 L of homogeneous AMPure XP beads to each 100- L end- repaired DNA sample in the PCR plate. Pipette up and down 10 times to mix.
4 Incubate samples for 5 minutes at room temperature.
5 Put the plate into a magnetic separation device. Wait for the solution to clear (approximately 3 to 5 minutes).
6 Keep the plate in the magnetic stand. Carefully remove and discard the cleared solution from each well. Do not touch the beads while removing the solution.
7 Continue to keep the plate in the magnetic stand while you dispense 200 L of freshly- prepared 70% ethanol in each sample well.
8 Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol.
9 Repeat step 7 to step 8 step once.
10 Seal the wells with strip caps, then briefly spin the plate to collect the residual ethanol. Return the plate to the magnetic stand for 30 seconds. Remove the residual ethanol with a P20 pipette.
11 Dry the samples by placing the unsealed plate on the thermal cycler, set to hold samples at 37C, for 3 to 5 minutes or until the residual ethanol completely evaporates.
12 Add 32 L nuclease- free water to each sample well.
13 Seal the wells with strip caps, then mix well on a vortex mixer and briefly spin the plate to collect the liquid.
14 Incubate for 2 minutes at room temperature.
15 Put the plate in the magnetic stand and leave for 2 to 3 minutes, until the solution is clear.
16 Remove the cleared supernatant (approximately 30 L) to a fresh PCR plate well. You can discard the beads at this time.
Stopping Point
Enrichment System for Illumina Multiplexed Sequencing 31
2 Sample Preparation (3 g DNA Samples) Step 6. dA-tail the 3' end of the DNA fragments
Step 6. dA-tail the 3' end of the DNA fragments
32
Use the SureSelect XT Library Prep Kit ILM for this step.
Hold samples on ice while setting up this step.
1 Prepare the appropriate volume of dA- Tailing master mix, as described in Table 12, on ice. Mix well on a vortex mixer.
Table 12 Preparation of dA-Tailing master mix
2 Add 20 L of the dA- Tailing master mix to each end- repaired, purified DNA sample (approximately 30 L).
3 Mix well by pipetting up and down.
4 Incubate the plate in the thermal cycler and run the program in Table 13. Do not use a heated lid.
Table 13 dA-Tailing Thermal Cycler Program
Reagent Volume for 1 reaction Volume for 16 reactions (includes excess)
Nuclease-free water 11 L 181.5 L
10 Klenow Polymerase Buffer (blue cap) 5 L 82.5 L
dATP (green cap) 1 L 16.5 L
Exo() Klenow (red cap) 3 L 49.5 L
Total 20 L 330 L
Step Temperature Time
Step 1 37C 30 minutes
Step 2 4C Hold
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Sample Preparation (3 g DNA Samples) 2 Step 7. Purify the sample using AMPure XP beads
Step 7. Purify the sample using AMPure XP beads
SureSelectXT Target
1 Let the AMPure XP beads come to room temperature for at least 30 minutes. Do not freeze the beads at any time.
2 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color.
3 Add 90 L of homogeneous AMPure XP beads to each 50- L dA- tailed DNA sample in the PCR plate. Pipette up and down 10 times to mix.
4 Incubate samples for 5 minutes at room temperature.
5 Put the plate into a magnetic separation device. Wait for the solution to clear (approximately 3 to 5 minutes).
6 Keep the plate in the magnetic stand. Carefully remove and discard the cleared solution from each well. Do not touch the beads while removing the solution.
7 Continue to keep the plate in the magnetic stand while you dispense 200 L of freshly- prepared 70% ethanol in each sample well.
8 Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol.
9 Repeat step 7 to step 8 step once.
10 Seal the wells with strip caps, then briefly spin the plate to collect the residual ethanol. Return the plate to the magnetic stand for 30 seconds. Remove the residual ethanol with a P20 pipette.
11 Dry the samples by placing the unsealed plate on the thermal cycler, set to hold samples at 37C, for 1 to 2 minutes or until the residual ethanol completely evaporates.
12 Add 15 L nuclease- free water to each sample well.
13 Seal the wells with strip caps, then mix well on a vortex mixer and briefly spin the plate to collect the liquid.
14 Incubate for 2 minutes at room temperature.
15 Put the plate in the magnetic stand and leave for 2 to 3 minutes, until the solution is clear.
16 Remove 13 L of the cleared supernatant to a fresh PCR plate well. You can discard the beads at this time.
17 Proceed immediately to the next step, Step 8. Ligate the paired- end adaptor.
Enrichment System for Illumina Multiplexed Sequencing 33
2 Sample Preparation (3 g DNA Samples) Step 8. Ligate the paired-end adaptor
Step 8. Ligate the paired-end adaptor
34
Use the SureSelect XT Library Prep Kit ILM for this step.
Hold samples on ice while setting up this step.
1 Prepare the appropriate volume of Ligation master mix, as described in Table 14, on ice. Mix well on a vortex mixer.
Table 14 Preparation of Ligation master mix
2 Add 37 L of the Ligation master mix to each dA- tailed, purified DNA sample (13 L) in the PCR plate wells.
3 Mix well by pipetting up and down.
4 Incubate the plate in the thermal cycler and run the program in Table 15. Do not use a heated lid.
Table 15 Ligation Thermal Cycler Program
Reagent Volume for 1 reaction Volume for 16 reactions (includes excess)
Nuclease-free water 15.5 L 255.75 L
5 T4 DNA Ligase Buffer (green cap) 10 L 165 L
SureSelect Adaptor Oligo Mix (brown cap) 10 L 165 L
T4 DNA Ligase (red cap) 1.5 L 24.75 L
Total 37 L 610.5 L
Step Temperature Time
Step 1 20C 15 minutes
Step 2 4C Hold
Stopping Point
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Sample Preparation (3 g DNA Samples) 2 Step 9. Purify the sample using AMPure XP beads
Step 9. Purify the sample using AMPure XP beads
SureSelectXT Target
1 Let the AMPure XP beads come to room temperature for at least 30 minutes. Do not freeze the beads at any time.
2 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color.
3 Add 90 L of homogeneous AMPure XP beads to each adaptor- ligated DNA sample in the PCR plate (50 L). Pipette up and down to mix.
4 Incubate samples for 5 minutes at room temperature.
5 Put the plate into a magnetic separation device. Wait for the solution to clear (approximately 3 to 5 minutes).
6 Keep the plate in the magnetic stand. Carefully remove and discard the cleared solution from each well. Do not touch the beads while removing the solution.
7 Continue to keep the plate in the magnetic stand while you dispense 200 L of freshly- prepared 70% ethanol in each sample well.
8 Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol.
9 Repeat step 7 and step 8 step once.
10 Seal the wells with strip caps, then briefly spin the plate to collect the residual ethanol. Return the plate to the magnetic stand for 30 seconds. Remove the residual ethanol with a P20 pipette.
11 Dry the samples by placing the unsealed plate on the thermal cycler, set to hold samples at 37C, for 1 to 2 minutes or until the residual ethanol completely evaporates.
12 Add 32 L nuclease- free water to each sample well.
13 Seal the wells with strip caps, then mix well on a vortex mixer and briefly spin the plate to collect the liquid.
14 Incubate for 2 minutes at room temperature.
15 Put the plate in the magnetic stand and leave for 2 to 3 minutes, until the solution is clear.
16 Remove the cleared supernatant (approximately 32 L) to a fresh PCR plate well. You can discard the beads at this time.
Stopping Point
Enrichment System for Illumina Multiplexed Sequencing 35
2 Sample Preparation (3 g DNA Samples) Step 10. Amplify the adaptor-ligated library
Step 10. Amplify the adaptor-ligated library
36
This step uses the components listed in Table 16. Thaw the reagents listed below and keep on ice.
This protocol uses half of the adaptor- ligated library for amplification. The remainder can be saved at 20C for future use, if needed.
Table 16 Reagents for pre-capture PCR amplification
Component Storage Location
SureSelect Primer SureSelect XT Library Prep Kit ILM, 20C
SureSelect ILM Index Pre-Capture PCR Reverse Primer
SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2, 20C
Herculase II Fusion DNA Polymerase Herculase II Fusion DNA Polymerase kit, 20C
5 Herculase II Reaction Buffer Herculase II Fusion DNA Polymerase kit*, 20C
* Do not use the PCR Reaction Buffer or dNTP mix from any other kit.
100 mM dNTP Mix Herculase II Fusion DNA Polymerase kit*, 20C
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Sample Preparation (3 g DNA Samples) 2 Step 10. Amplify the adaptor-ligated library
CAUTION To avoid cross-contaminating libraries, set up PCR reactions (all components except the library DNA) in a dedicated clean area or PCR hood with UV sterilization and positive air flow.
SureSelectXT Target
1 Prepare the appropriate volume of pre- capture PCR reaction mix, as described in Table 17, on ice. Mix well on a vortex mixer.
2 Combine 35 L of the PCR reaction mixture prepared in Table 17 and 15 L of each purified DNA library sample from step 16 on page 35. Add a single DNA library sample to each well of the plate or strip tube.
Mix by pipetting.
Table 17 Preparation of SureSelect Pre-Capture PCR Reaction Mix
Reagent Volume for 1 reaction
Volume for 16 reactions (includes excess)
Nuclease-free water 21 L 346.5 L
SureSelect Primer (brown cap) 1.25 L 20.6 L
SureSelect ILM Index Pre-Capture PCR Reverse Primer (clear cap)
1.25 L 20.6 L
5 Herculase II Reaction Buffer (clear cap) 10 L 165 L
100 mM dNTP Mix (green cap) 0.5 L 8.25 L
Herculase II Fusion DNA Polymerase (red cap) 1 L 16.5 L
Total 35 L 577.5 L
Enrichment System for Illumina Multiplexed Sequencing 37
2 Sample Preparation (3 g DNA Samples) Step 10. Amplify the adaptor-ligated library
38
3 Run the program in Table 18 in a thermal cycler.
. Table 18 Pre-Capture PCR Thermal Cycler Program
Segment Number of Cycles
Temperature Time
1 1 98C 2 minutes
2 46 98C 30 seconds
65C 30 seconds
72C 1 minute
3 1 72C 10 minutes
4 1 4C Hold
Different library preparations can produce slightly different results, based on varying DNA quality. In most cases, five cycles will produce an adequate yield for subsequent capture without introducing bias or non-specific products. If yield is too low, or too high (where non-specific high molecular weight products are observed), adjust the number of cycles accordingly to amplify the remaining library template.
NOTE
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Sample Preparation (3 g DNA Samples) 2 Step 11. Purify the amplified library with AMPure XP beads
Step 11. Purify the amplified library with AMPure XP beads
SureSelectXT Target
1 Let the AMPure XP beads come to room temperature for at least 30 minutes. Do not freeze the beads at any time.
2 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color.
3 Add 90 L of homogeneous AMPure XP beads to each 50- L amplified DNA sample in the PCR plate. Pipette up and down to mix.
4 Incubate samples for 5 minutes at room temperature.
5 Put the plate into a magnetic separation device. Wait for the solution to clear (approximately 3 to 5 minutes).
6 Keep the plate in the magnetic stand. Carefully remove and discard the cleared solution from each well. Do not touch the beads while removing the solution.
7 Continue to keep the plate in the magnetic stand while you dispense 200 L of freshly- prepared 70% ethanol in each sample well.
8 Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol.
9 Repeat step 7 and step 8 step once.
10 Seal the wells with strip caps, then briefly spin the plate to collect the residual ethanol. Return the plate to the magnetic stand for 30 seconds. Remove the residual ethanol with a P20 pipette.
11 Dry the samples by placing the unsealed plate on the thermal cycler, set to hold samples at 37C, for 1 to 2 minutes or until the residual ethanol completely evaporates.
12 Add 30 L nuclease- free water to each sample well.
13 Seal the wells, then mix well on a vortex mixer and briefly spin the plate in a centrifuge or mini- plate spinner to collect the liquid.
14 Incubate for 2 minutes at room temperature.
15 Put the plate in the magnetic stand and leave for 2 to 3 minutes, until the solution is clear.
16 Remove the cleared supernatant (approximately 30 L) to a fresh PCR plate well. You can discard the beads at this time.
Stopping Point
Enrichment System for Illumina Multiplexed Sequencing 39
2 Sample Preparation (3 g DNA Samples) Step 12. Assess quality and quantity
Step 12. Assess quality and quantity
40
Quality assessment can be done with either the 2100 Bioanalyzer instrument or Agilent TapeStation instrument.
Option 1: Analysis using the 2100 Bioanalyzer and DNA 1000 Assay
Use a Bioanalyzer DNA 1000 chip and reagent kit and perform the assay according to the Agilent DNA 1000 Kit Guide.
1 Set up the 2100 Bioanalyzer instrument as instructed in the reagent kit guide.
2 Prepare the chip, samples and ladder as instructed in the reagent kit guide, using 1 L of each sample for the analysis. Load the prepared chip into the instrument and start the run within five minutes after preparation.
3 Verify that the electropherogram shows a distribution with a DNA fragment size peak of approximately 225 to 275 bp. Determine the concentration of the library DNA by integrating under the peak. A sample electropherogram is shown in Figure 4.
Figure 4 Analysis of amplified library DNA using a DNA 1000 Bioanalyzer assay.
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Sample Preparation (3 g DNA Samples) 2 Step 12. Assess quality and quantity
SureSelectXT Target
Option 2: Analysis using an Agilent TapeStation and D1000 ScreenTape
Use a D1000 ScreenTape and associated reagent kit. Perform the assay according to the Agilent D1000 Assay Quick Guide.
1 Prepare the TapeStation samples as instructed as instructed in the reagent kit guide. Use 1 L of each DNA sample diluted with 3 L of D1000 sample buffer for the analysis.
CAUTION For accurate quantitation, make sure to thoroughly mix the combined DNA and sample buffer by vortexing the assay plate or tube strip for 1 minute on the IKA MS3 vortex mixer provided with the 4200/4150 TapeStation system before loading the samples.
2 Load the sample plate or tube strips from step 1, the D1000 ScreenTape, and loading tips into the TapeStation as instructed in the reagent kit guide. Start the run.
3 Verify that the electropherogram shows a distribution with a DNA fragment size peak of approximately 225 to 275 bp. Determine the concentration of the library DNA by integrating under the peak. A sample electropherogram is shown in Figure 5.
Figure 5 Analysis of amplified library DNA using a D1000 ScreenTape.
Enrichment System for Illumina Multiplexed Sequencing 41
2 Sample Preparation (3 g DNA Samples) Step 12. Assess quality and quantity
42
SureSelectXT Target Enrichment System for the Illumina Platform Protocol
3 Sample Preparation (200 ng DNA Samples)
Step 1. Shear the DNA 44
Step 2. Assess quality (optional) 47
Step 3. Repair the ends 48
Step 4. Purify the sample using AMPure XP beads 49
Step 5. dA-tail the 3' end of the DNA fragments 51
Step 6. Purify the sample using AMPure XP beads 52
Step 7. Ligate the paired-end adaptor 53
Step 8. Purify the sample using AMPure XP beads 55
Step 9. Amplify the adaptor-ligated library 56
Step 10. Purify the amplified library with AMPure XP beads 59
Step 11. Assess quality and quantity 60
CAUTION This section contains instructions for the preparation of gDNA libraries from 200 ng DNA samples. For higher input (3 g) DNA samples, see the library preparation protocol on page 23.
The sample preparation protocol is used to prepare DNA libraries for sequencing using the Illumina paired- read platform. For each sample to be sequenced, an individual indexed library is prepared. For an overview of the SureSelectXT target enrichment workflow, see Figure 1 on page 12.
The steps in this section differ from the Illumina protocol in the use of the Covaris system for gDNA shearing, smaller target shear size, elimination of size selection by gel purification, implementation of AMPure XP beads for all purification steps, and primers used for PCR. Refer to the Illumina protocol Preparing Samples for Multiplexed Paired- End Sequencing (p/n1005361) or the appropriate Illumina protocol for more information.
43Agilent Technologies
3 Sample Preparation (200 ng DNA Samples) Step 1. Shear the DNA
Step 1. Shear the DNA Make sure genomic DNA samples are of high quality with an OD 260/280 ratio ranging from 1.8 to 2.0. Use the Qubit system to quantify genomic DNA before library preparation.
For FFPE-derived DNA samples, review the protocol modifications detailed in the Appendix on page 89 before starting the library preparation protocol.
This protocol has been optimized using a Covaris model E220 instrument and 130-l Covaris microTUBE for shearing 50-l DNA samples to a target DNA fragment size of 150 to 200 bp. To shear using a different Covaris instrument model/sample holder (e.g. 50-l microTUBE), or if your NGS workflow requires a different DNA fragment size, consult the manufacturers literature for recommended shearing conditions.
NOTE
44
For each DNA sample to be sequenced, prepare 1 library.
1 Set up the Covaris E- series or S- series instrument. Refer to the Covaris instrument user guide for details.
a Check that the water in the Covaris tank is filled with fresh deionized water to the appropriate fill line level according to the manufacturers recommendations for the specific instrument model and sample tube or plate in use.
b Check that the water covers the visible glass part of the tube.
c On the instrument control panel, push the Degas button. Degas the instrument for least 30 minutes before use, or according to the manufacturers recommendations.
d Set the chiller temperature to between 2C to 5C to ensure that the temperature reading in the water bath displays 5C. Consult the manufacturers recommendations for addition of coolant fluids to prevent freezing.
2 Put a Covaris microTUBE into the loading and unloading station.
Keep the cap on the tube.
You can use the 96 microTUBE plate (see Table 6 on page 19) for the DNA shearing step when preparing multiple gDNA samples in the same experiment.
NOTE
3 Use the Qubit dsDNA Assay to determine the concentration of your gDNA sample.
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Sample Preparation (200 ng DNA Samples) 3 Step 1. Shear the DNA
SureSelectXT Target
Follow the instructions for the instrument.
For FFPE-derived DNA samples with significantly degraded DNA, use the concentration of amplifiable DNA as determined by qPCR and use the maximum amount of DNA available in the range of 100200 ng. See Chapter 6 for more information on when protocol modifications are appropriate for FFPE samples.
NOTE
4 Dilute 200 ng of high- quality gDNA with 1X Low TE Buffer (10 mM Tris- HCl, pH 8.0, 0.1 mM EDTA) in a 1.5- mL LoBind tube to a total volume of 50 L.
5 Use a tapered pipette tip to slowly transfer the 50- L DNA sample through the pre- split septum.
Be careful not to introduce a bubble into the bottom of the tube.
6 Secure the microTUBE in the tube holder and shear the DNA with the settings in Table 19.
The target DNA fragment size is 150 to 200 bp.
For FFPE-derived DNA samples, reduce the duration of shearing from 6 minutes to 4 minutes. See Chapter 6 for a complete list of modifications recommended for FFPE samples.
NOTE
7 Put the Covaris microTUBE back into the loading and unloading station.
8 While keeping the snap- cap on, insert a pipette tip through the pre- split septum, then slowly remove the sheared DNA.
Table 19 Shear settings for Covaris instruments (SonoLab software v7 or later)
Setting Value
Duty Factor 10%
Peak Incident Power (PIP) 175
Cycles per Burst 200
Treatment Time 360 seconds*
* For more complete shearing, the 360-second treatment time may be completed in two rounds of 180 seconds each. After completing the first round of shearing for 180 seconds, spin the microTUBE briefly to collect the liquid, then shear the DNA for an additional 180 seconds.
Bath Temperature 4 to 8 C
Enrichment System for Illumina Multiplexed Sequencing 45
3 Sample Preparation (200 ng DNA Samples) Step 1. Shear the DNA
46
9 Transfer each sheared DNA sample (approximately 50 L) to a separate well of a 96- well plate or strip tube.
Instructions in this manual are for sample processing in 96-well PCR plates. When processing a small number of samples, you can instead use strip tubes or individual tubes that are compatible with the thermal cycler and magnetic separation device used in the protocol.
NOTE
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Sample Preparation (200 ng DNA Samples) 3 Step 2. Assess quality (optional)
Step 2. Assess quality (optional)
SureSelectXT Target
This step is optional.
Quality assessment can be done with the 2100 Bioanalyzer instrument.
For analysis of 200 ng sheared DNA samples, Use a High Sensitivity DNA chip and reagent kit. Perform the assay according to the High Sensitivity DNA Kit Guide.
1 Set up the 2100 Bioanalyzer instrument as instructed in the reagent kit guide.
2 Prepare the chip, samples and ladder as instructed in the reagent kit guide, using 1 L of each sample for the analysis. Load the prepared chip into the instrument and start the run within five minutes after preparation.
3 Check that the electropherogram shows a DNA fragment size peak at approximately 150 bp. A sample electropherogram is shown in Figure 6.
Figure 6 Analysis of sheared DNA using a High Sensitivity DNA Bioanalyzer assay.
Enrichment System for Illumina Multiplexed Sequencing 47
3 Sample Preparation (200 ng DNA Samples) Step 3. Repair the ends
Step 3. Repair the ends
48
Use the SureSelect XT Library Prep Kit ILM for this step.
To process multiple samples, prepare master mixes with overage at each step, without the DNA sample. Master mixes for preparation of 16 samples (including excess) are shown in each table as an example.
Hold samples on ice while setting up this step.
1 Prepare the appropriate volume of End Repair master mix, as described in Table 20, on ice. Mix well on a vortex mixer.
Table 20 Preparation of End Repair master mix
2 Add 52 L of the master mix to each PCR plate well containing the sheared DNA samples (approximately 4850 L). Mix by pipetting up and down.
3 Incubate the plate in the thermal cycler and run the program in Table 21. Do not use a heated lid.
Table 21 End-Repair Thermal Cycler Program
Reagent Volume for 1 reaction Volume for 16 reactions (includes excess)
Nuclease-free water 35.2 L 580.8 L
10 End Repair Buffer (clear cap) 10 L 165 L
dNTP Mix (green cap) 1.6 L 26.4 L
T4 DNA Polymerase (purple cap) 1 L 16.5 L
Klenow DNA Polymerase (yellow cap) 2 L 33 L
T4 Polynucleotide Kinase (orange cap) 2.2 L 36.3 L
Total 52 L 858 L
Step Temperature Time
Step 1 20C 30 minutes
Step 2 4C Hold
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Sample Preparation (200 ng DNA Samples) 3 Step 4. Purify the sample using AMPure XP beads
Step 4. Purify the sample using AMPure XP beads
SureSelectXT Target
1 Let the AMPure XP beads come to room temperature for at least 30 minutes. Do not freeze the beads at any time.
2 Prepare 400 L of 70% ethanol per sample, plus excess, for use in step 8.
The freshly-prepared 70% ethanol may be used for subsequent purification steps run on the same day. The complete Library Preparation protocol requires 1.6 mL of fresh 70% ethanol per sample.
NOTE
3 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color.
4 Add 180 L of homogeneous AMPure XP beads to each end- repaired DNA sample (approximately 100 L) in the PCR plate. Pipette up and down 10 times to mix.
5 Incubate samples for 5 minutes at room temperature.
6 Put the plate into a magnetic separation device. Wait for the solution to clear (approximately 3 to 5 minutes).
7 Keep the plate in the magnetic stand. Carefully remove and discard the cleared solution from each well. Do not touch the beads while removing the solution.
NOTE If some magnetic beads remain suspended in solution after 5 minutes, carefully remove and discard 100 l of cleared solution from near the bottom of the wells, and continue incubating the plate in the magnetic stand for an additional 3 minutes. After the remaining suspension has cleared, remove and discard the remaining cleared solution (approximately 180 l) from the wells.
8 Continue to keep the plate in the magnetic stand while you dispense 200 L of 70% ethanol in each sample well.
Use fresh 70% ethanol for optimal results.
9 Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol.
10 Repeat step 8 to step 9 once.
Enrichment System for Illumina Multiplexed Sequencing 49
3 Sample Preparation (200 ng DNA Samples) Step 4. Purify the sample using AMPure XP beads
50
11 Seal the wells with strip caps, then briefly spin the plate to collect the residual ethanol. Return the plate to the magnetic stand for 30 seconds. Remove the residual ethanol with a P20 pipette.
12 Dry the samples by placing the unsealed plate on the thermal cycler, set to hold samples at 37C, for 3 to 5 minutes or until the residual ethanol completely evaporates.
Do not dry the bead pellet to the point that the pellet appears cracked during any of the bead drying steps in the protocol. Elution efficiency is significantly decreased when the bead pellet is excessively dried.
NOTE
13 Add 32 L nuclease- free water to each sample well.
14 Seal the wells with strip caps, then mix well on a vortex mixer and briefly spin the plate to collect the liquid.
15 Incubate for 2 minutes at room temperature.
16 Put the plate in the magnetic stand and leave for 2 to 3 minutes, until the solution is clear.
17 Remove the cleared supernatant (approximately 30 L) to a fresh PCR plate well. You can discard the beads at this time.
Stopping Point
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Sample Preparation (200 ng DNA Samples) 3 Step 5. dA-tail the 3' end of the DNA fragments
Step 5. dA-tail the 3' end of the DNA fragments
SureSelectXT Target
Use the SureSelect XT Library Prep Kit ILM for this step.
Hold samples on ice while setting up this step.
1 Prepare the appropriate volume of dA- Tailing master mix, as described in Table 22, on ice. Mix well on a vortex mixer.
Table 22 Preparation of dA-Tailing master mix
2 Add 20 L of the dA- Tailing master mix to each end- repaired, purified DNA sample (approximately 30 L).
3 Mix well by pipetting up and down.
4 Incubate the plate in the thermal cycler and run the program in Table 23. Do not use a heated lid.
Table 23 dA-Tailing Thermal Cycler Program
Reagent Volume for 1 reaction Volume for 16 reactions (includes excess)
Nuclease-free water 11 L 181.5 L
10 Klenow Polymerase Buffer (blue cap) 5 L 82.5 L
dATP (green cap) 1 L 16.5 L
Exo() Klenow (red cap) 3 L 49.5 L
Total 20 L 330 L
Step Temperature Time
Step 1 37C 30 minutes
Step 2 4C Hold
Enrichment System for Illumina Multiplexed Sequencing 51
3 Sample Preparation (200 ng DNA Samples) Step 6. Purify the sample using AMPure XP beads
Step 6. Purify the sample using AMPure XP beads
52
1 Let the AMPure XP beads come to room temperature for at least 30 minutes. Do not freeze the beads at any time.
2 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color.
3 Add 90 L of homogeneous AMPure XP beads to each 50- L dA- tailed DNA sample in the PCR plate. Pipette up and down 10 times to mix.
4 Incubate samples for 5 minutes at room temperature.
5 Put the plate into a magnetic separation device. Wait for the solution to clear (approximately 3 to 5 minutes).
6 Keep the plate in the magnetic stand. Carefully remove and discard the cleared solution from each well. Do not touch the beads while removing the solution.
7 Continue to keep the plate in the magnetic stand while you dispense 200 L of freshly- prepared 70% ethanol in each sample well.
8 Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol.
9 Repeat step 7 to step 8 step once.
10 Seal the wells with strip caps, then briefly spin the plate to collect the residual ethanol. Return the plate to the magnetic stand for 30 seconds. Remove the residual ethanol with a P20 pipette.
11 Dry the samples by placing the unsealed plate on the thermal cycler, set to hold samples at 37C, for 1 to 2 minutes or until the residual ethanol completely evaporates.
12 Add 15 L nuclease- free water to each sample well.
13 Seal the wells with strip caps, then mix well on a vortex mixer and briefly spin the plate to collect the liquid.
14 Incubate for 2 minutes at room temperature.
15 Put the plate in the magnetic stand and leave for 2 to 3 minutes, until the solution is clear.
16 Remove 13 L of the cleared supernatant to a fresh PCR plate well. You can discard the beads at this time.
17 Proceed immediately to the next step, Step 7. Ligate the paired- end adaptor.
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Sample Preparation (200 ng DNA Samples) 3 Step 7. Ligate the paired-end adaptor
Step 7. Ligate the paired-end adaptor
SureSelectXT Target
Use the SureSelect XT Library Prep Kit ILM for this step.
Hold samples on ice while setting up this step.
1 Dilute the SureSelect Adaptor Oligo Mix ten- fold in nuclease- free water immediately before use. For example, for a 16- reaction run, combine 17 L SureSelect Adaptor Oligo Mix (brown cap) with 153 L of nuclease- free water.
Use the diluted oligo mix when preparing the Ligation master mix in the next step.
For FFPE-derived DNA samples, skip this dilution step and use the undiluted SureSelect Adaptor Oligo Mix in step 2 below. See Chapter 6 for a complete list of modifications recommended for FFPE samples.
NOTE
2 Prepare the appropriate volume of Ligation master mix, as described in Table 24, on ice. Mix well on a vortex mixer.
Table 24 Preparation of Ligation master mix
3 Add 37 L of the Ligation master mix to each dA- tailed, purified DNA sample (13 L) in the PCR plate wells.
4 Mix well by pipetting up and down.
Reagent Volume for 1 reaction
Volume for 16 reactions (includes excess)
Nuclease-free water 15.5 L 255.75 L
5 T4 DNA Ligase Buffer (green cap) 10 L 165 L
Diluted SureSelect Adaptor Oligo Mix from step 1 10 L 165 L
T4 DNA Ligase (red cap) 1.5 L 24.75 L
Total 37 L 610.5 L
Enrichment System for Illumina Multiplexed Sequencing 53
3 Sample Preparation (200 ng DNA Samples) Step 7. Ligate the paired-end adaptor
54
5 Incubate the plate in the thermal cycler and run the program in Table 25. Do not use a heated lid.
Table 25 Ligation Thermal Cycler Program
Step Temperature Time
Step 1 20C 15 minutes
Step 2 4C Hold
Stopping Point
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Sample Preparation (200 ng DNA Samples) 3 Step 8. Purify the sample using AMPure XP beads
Step 8. Purify the sample using AMPure XP beads
SureSelectXT Target
1 Let the AMPure XP beads come to room temperature for at least 30 minutes. Do not freeze the beads at any time.
2 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color.
3 Add 90 L of homogeneous AMPure XP beads to each adaptor- ligated DNA sample in the PCR plate (50 L). Pipette up and down to mix.
4 Incubate samples for 5 minutes at room temperature.
5 Put the plate into a magnetic separation device. Wait for the solution to clear (approximately 3 to 5 minutes).
6 Keep the plate in the magnetic stand. Carefully remove and discard the cleared solution from each well. Do not touch the beads while removing the solution.
7 Continue to keep the plate in the magnetic stand while you dispense 200 L of freshly- prepared 70% ethanol in each sample well.
8 Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol.
9 Repeat step 7 and step 8 step once.
10 Seal the wells with strip caps, then briefly spin the plate to collect the residual ethanol. Return the plate to the magnetic stand for 30 seconds. Remove the residual ethanol with a P20 pipette.
11 Dry the samples by placing the unsealed plate on the thermal cycler, set to hold samples at 37C, for 1 to 2 minutes or until the residual ethanol completely evaporates.
12 Add 32 L nuclease- free water to each sample well.
13 Seal the wells with strip caps, then mix well on a vortex mixer and briefly spin the plate to collect the liquid.
14 Incubate for 2 minutes at room temperature.
15 Put the plate in the magnetic stand and leave for 2 to 3 minutes, until the solution is clear.
16 Remove the cleared supernatant (approximately 30 L) to a fresh PCR plate well. You can discard the beads at this time.
Stopping Point
Enrichment System for Illumina Multiplexed Sequencing 55
3 Sample Preparation (200 ng DNA Samples) Step 9. Amplify the adaptor-ligated library
Step 9. Amplify the adaptor-ligated library
56
This step uses the components listed in Table 26. Thaw the reagents listed below and keep on ice.
Table 26 Reagents for pre-capture PCR amplification
Component Storage Location
SureSelect Primer (brown cap) SureSelect XT Library Prep Kit ILM, 20C
SureSelect ILM Index Pre-Capture PCR Reverse Primer (clear cap)
SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2, 20C
Herculase II Fusion DNA Polymerase (red cap) Herculase II Fusion DNA Polymerase kit, 20C
5 Herculase II Reaction Buffer (clear cap) Herculase II Fusion DNA Polymerase kit*, 20C
* Do not use the PCR Reaction Buffer or dNTP mix from any other kit.
100 mM dNTP Mix (green cap) Herculase II Fusion DNA Polymerase kit*, 20C
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Sample Preparation (200 ng DNA Samples) 3 Step 9. Amplify the adaptor-ligated library
CAUTION To avoid cross-contaminating libraries, set up PCR reactions (all components except the library DNA) in a dedicated clean area or PCR hood with UV sterilization and positive air flow.
SureSelectXT Target
1 Prepare the appropriate volume of pre- capture PCR reaction mix, as described in Table 27, on ice. Mix well on a vortex mixer.
2 Add 20 L of the PCR reaction mixture prepared in Table 27 to each purified DNA library sample (30 L) in the PCR plate wells.
Mix by pipetting.
Table 27 Preparation of SureSelect Pre-Capture PCR Reaction Mix
Reagent Volume for 1 reaction
Volume for 16 reactions (includes excess)
Nuclease-free water 6 L 99.0 L
SureSelect Primer (brown cap) 1.25 L 20.6 L
SureSelect ILM Index Pre-Capture PCR Reverse Primer (clear cap)
1.25 L 20.6 L
5 Herculase II Reaction Buffer (clear cap) 10 L 165 L
100 mM dNTP Mix (green cap) 0.5 L 8.3 L
Herculase II Fusion DNA Polymerase (red cap) 1 L 16.5 L
Total 20 L 330 L
Enrichment System for Illumina Multiplexed Sequencing 57
3 Sample Preparation (200 ng DNA Samples) Step 9. Amplify the adaptor-ligated library
58
3 Run the program in Table 28 in a thermal cycler.
. Table 28 Pre-Capture PCR Thermal Cycler Program
Segment Number of Cycles
Temperature Time
1 1 98C 2 minutes
2 10 98C 30 seconds
65C 30 seconds
72C 1 minute
3 1 72C 10 minutes
4 1 4C Hold
NOTE FFPE-derived DNA samples may require a different cycle number for amplification, depending on DNA integrity. See page 91 or page 93 for DNA integrity score-based cycle number recommendations.
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Sample Preparation (200 ng DNA Samples) 3 Step 10. Purify the amplified library with AMPure XP beads
Step 10. Purify the amplified library with AMPure XP beads
SureSelectXT Target
1 Let the AMPure XP beads come to room temperature for at least 30 minutes. Do not freeze the beads at any time.
2 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color.
3 Add 90 L of homogeneous AMPure XP beads to each 50- L amplified DNA sample in the PCR plate. Pipette up and down to mix.
4 Incubate samples for 5 minutes at room temperature.
5 Put the plate into a magnetic separation device. Wait for the solution to clear (approximately 3 to 5 minutes).
6 Keep the plate in the magnetic stand. Carefully remove and discard the cleared solution from each well. Do not touch the beads while removing the solution.
7 Continue to keep the plate in the magnetic stand while you dispense 200 L of freshly- prepared 70% ethanol in each sample well.
8 Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol.
9 Repeat step 7 and step 8 step once.
10 Seal the wells with strip caps, then briefly spin the plate to collect the residual ethanol. Return the plate to the magnetic stand for 30 seconds. Remove the residual ethanol with a P20 pipette.
11 Dry the samples by placing the unsealed plate on the thermal cycler, set to hold samples at 37C, for 1 to 2 minutes or until the residual ethanol completely evaporates.
12 Add 30 L nuclease- free water to each sample well.
13 Seal the wells, then mix well on a vortex mixer and briefly spin the plate in a centrifuge or mini- plate spinner to collect the liquid.
14 Incubate for 2 minutes at room temperature.
15 Put the plate in the magnetic stand and leave for 2 to 3 minutes, until the solution is clear.
16 Remove the cleared supernatant (approximately 30 L) to a fresh PCR plate well. You can discard the beads at this time.
Stopping Point
Enrichment System for Illumina Multiplexed Sequencing 59
3 Sample Preparation (200 ng DNA Samples) Step 11. Assess quality and quantity
Step 11. Assess quality and quantity
60
Sample analysis can be done with either the 2100 Bioanalyzer instrument or an Agilent TapeStation instrument.
Option 1: Analysis using the 2100 Bioanalyzer and DNA 1000 Assay
Use a Bioanalyzer DNA 1000 chip and reagent kit and perform the assay according to the Agilent DNA 1000 Kit Guide.
1 Set up the 2100 Bioanalyzer instrument as instructed in the reagent kit guide.
2 Prepare the chip, samples and ladder as instructed in the reagent kit guide, using 1 L of each sample for the analysis. Load the prepared chip into the instrument and start the run within five minutes after preparation.
3 Verify that the electropherogram shows a distribution with a DNA fragment size peak of approximately 225 to 275 bp. Determine the concentration of the library DNA by integrating under the peak. A sample electropherogram is shown in Figure 7.
Figure 7 Analysis of amplified library DNA using a DNA 1000 Bioanalyzer assay.
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Sample Preparation (200 ng DNA Samples) 3 Step 11. Assess quality and quantity
SureSelectXT Target
Option 2: Analysis using an Agilent TapeStation and D1000 ScreenTape
Use a D1000 ScreenTape and associated reagent kit. Perform the assay according to the Agilent D1000 Assay Quick Guide.
1 Prepare the TapeStation samples as instructed as instructed in the reagent kit guide. Use 1 L of each DNA sample diluted with 3 L of D1000 sample buffer for the analysis.
CAUTION For accurate quantitation, make sure to thoroughly mix the combined DNA and sample buffer by vortexing the assay plate or tube strip for 1 minute on the IKA MS3 vortex mixer provided with the 4200/4150 TapeStation system before loading the samples.
2 Load the sample plate or tube strips from step 1, the D1000 ScreenTape, and loading tips into the TapeStation as instructed in the reagent kit guide. Start the run.
3 Verify that the electropherogram shows a distribution with a DNA fragment size peak of approximately 225 to 275 bp. Determine the concentration of the library DNA by integrating under the peak. A sample electropherogram is shown in Figure 8.
Figure 8 Analysis of amplified library DNA using a D1000 ScreenTape.
Stopping Point
Enrichment System for Illumina Multiplexed Sequencing 61
3 Sample Preparation (200 ng DNA Samples) Step 11. Assess quality and quantity
62
SureSelectXT Target Enrichment System for the Illumina Platform Protocol
4 Hybridization and Capture
Step 1. Hybridize DNA samples to the probe 64
Step 2. Prepare streptavidin-coated magnetic beads 70
Step 3. Capture the hybridized DNA using streptavidin-coated beads 71
This chapter describes the steps to hybridize the prepared gDNA libraries with a target- specific probe. After hybridization, the targeted molecules are captured on streptavidin beads.
Each DNA library sample is hybridized and captured individually prior to addition of the indexing tag by PCR.
CAUTION The ratio of probe to gDNA library is critical for successful capture.
63Agilent Technologies
4 Hybridization and Capture Step 1. Hybridize DNA samples to the probe
Step 1. Hybridize DNA samples to the probe
64
In this step, the prepared gDNA libraries are hybridized to a target- specific probe.
This step uses the components listed in Table 29. Thaw each component under the conditions indicated in the table. Vortex each reagent to mix, then spin tubes briefly to collect the liquid.
Table 29 Reagents for Hybridization
Kit Component Storage Location Thawing Conditions Where Used
SureSelect Hyb 1 SureSelect Target Enrichment-Box 1, RT page 66
SureSelect Hyb 2 SureSelect Target Enrichment-Box 1, RT page 66
SureSelect Hyb 3 SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2, 20C
Warm to Room Temperature (RT)
page 66
SureSelect Hyb 4 SureSelect Target Enrichment-Box 1, RT page 66
SureSelect Indexing Block 1 SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2, 20C
Thaw on ice page 66
SureSelect Block 2 SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2, 20C
Thaw on ice page 66
SureSelect Indexing Block 3 SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2, 20C
Thaw on ice page 66
SureSelect RNase Block SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2, 20C
Thaw on ice page 67
Probe 80C Thaw on ice page 68
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Hybridization and Capture 4 Step 1. Hybridize DNA samples to the probe
SureSelectXT Target
For each sample library prepared, do one hybridization and capture. Do not pool samples at this stage.
The hybridization reaction requires 750 ng of prepared DNA in a volume of 3.4 L (initial concentration of 221 ng/L).
NOTE For FFPE-derived DNA samples, add the maximum amount of DNA available in range of 500750 ng DNA. See Chapter 6 for a complete list of modifications recommended for FFPE samples.
1 For prepped libraries with DNA concentrations above 221 ng/L, prepare 3.4 L of a 221 ng/L dilution of each library.
2 For prepped libraries with DNA concentrations below 221 ng/L, use a vacuum concentrator to concentrate the samples at 45C.
a Add the entire 30- L volume of prepped library to an Eppendorf tube. Poke one or more holes in the lid with a narrow gauge needle.
You can also break off the cap, cover with parafilm, and poke a hole in the parafilm.
b Dehydrate using a vacuum concentrator on low heat (less than 45C).
c Reconstitute with nuclease- free water to a final concentration of 221 ng/L. Pipette up and down along the sides of the tube for optimal recovery.
d Mix well on a vortex mixer and spin in a centrifuge for 1 minute.
3 Transfer each 3.4- L gDNA library sample (750 ng) to a separate well of a 96- well plate or strip tube. Seal the wells and keep on ice.
CAUTION You must avoid evaporation from the small volumes of the capture during the 16 or 24 hour incubation.
Before you do the first experiment, make sure the plasticware and sealing method (strip caps or sealing tape), are appropriate for the thermal cycler. To test evaporation under the conditions used for hybridization, incubate 27 L of water at 65C for 24 hours. Include water in each well that you might use, including center and edge wells. Check that no more than 4 L is lost to evaporation.
For a partial list of tested options showing minimal evaporation, refer to Alternative Capture Equipment Combinations on page 101.
Enrichment System for Illumina Multiplexed Sequencing 65
4 Hybridization and Capture Step 1. Hybridize DNA samples to the probe
66
4 Prepare the Hybridization Buffer by mixing the components in Table 30 at room temperature.
If a precipitate forms, warm the Hybridization Buffer at 65C for 5 minutes.
Keep the prepared Hybridization Buffer at room temperature until it is used in step 9.
Table 30 Preparation of Hybridization Buffer
5 Prepare the SureSelect Block Mix by mixing the components in Table 31. Keep the mixture on ice until it is used in step 6.
Table 31 Preparation of SureSelect Block Mix
Reagent Volume for 1 reaction*
* Prepare Hybridization Buffer for at least 5 reaction equivalents per run to allow accurate pipetting volumes.
Volume for 16 reactions (includes excess)
SureSelect Hyb 1 (orange cap) 6.63 L 116 L
SureSelect Hyb 2 (red cap) 0.27 L 4.7 L
SureSelect Hyb 3 (yellow cap or bottle) 2.65 L 46.4 L
SureSelect Hyb 4 (black cap) 3.45 L 60.4 L
Total 13 L 227.5
Reagent Volume for 1 reaction Volume for 16 reactions (includes excess)
SureSelect Indexing Block 1 (green cap) 2.5 L 42.5 L
SureSelect Block 2 (blue cap) 2.5 L 42.5 L
SureSelect Indexing Block 3 (brown cap) 0.6 L 10.2 L
Total 5.6 L 95.2 L
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Hybridization and Capture 4 Step 1. Hybridize DNA samples to the probe
SureSelectXT Target
CAUTION For each protocol step that requires removal of tube cap strips, make sure to reseal the tubes with a fresh strip of caps. Reuse of strip caps can cause sample loss, sample contamination, or imprecision in sample temperatures during incubations.
6 To each gDNA library sample well prepared in step 3 on page 65, add 5.6 L of the SureSelect Block Mix prepared in Table 31. Pipette up and down to mix.
7 Cap the wells, then transfer the sealed plate or strip tube to the thermal cycler and run the following program shown in Table 32.
Use a heated lid, set at 105C, to hold the temperature at 65C.
Make sure that the DNA + Block Mix samples are held at 65C for at least 5 minutes before adding the remaining hybridization reaction components in step 10 below.
Table 32 Thermal cycler program for DNA + Block Mix prior to hybridization
Step Temperature Time
Step 1 95C 5 minutes
Step 2 65C Hold (at least 5 minutes)
CAUTION The lid of the thermal cycler is hot and can cause burns. Use caution when working near the lid.
8 Prepare the appropriate dilution of SureSelect RNase Block, based on the design size of your probe, according to Table 33. Prepare the amount required for the number of hybridization reactions in the run, plus excess.
Table 33 Preparation of RNase Block dilution
Keep the mixture on ice until it is used in step 9.
Probe Size RNase Block dilution (parts RNase Block:water)
Volume of dilute RNase Block Required per hybridization reaction
3.0 Mb 25% (1:3) 2 L
<3.0 Mb 10% (1:9) 5 L
Enrichment System for Illumina Multiplexed Sequencing 67
4 Hybridization and Capture Step 1. Hybridize DNA samples to the probe
68
NOTE Prepare the mixture described in step 9, below, near the end of the 65C hold step of >5 minute duration described in Table 32. Keep the mixture at room temperature briefly, until adding the mixture to sample wells in step 10. Do not keep solutions containing the probe at room temperature for extended periods.
9 Prepare the Probe Hybridization Mix appropriate for your probe design size. Use Table 34 for probes3 Mb or Table 35 for probes<3 Mb.
Mix well by vortexing at high speed for 5 seconds then spin down briefly. Keep the mixture at room temperature briefly, until use in step 10.
Table 34 Preparation of Probe Hybridization Mix for probes 3 Mb
Table 35 Preparation of Probe Hybridization Mix for probes<3 Mb
Reagent Volume for 1 reaction Volume for 16 reactions (includes excess)
Hybridization Buffer mixture from step 4 13 L 221 L
25% RNase Block solution from step 8 2 L 34 L
Probe (with design 3 Mb) 5 L 85 L
Total 20 L 340 L
Reagent Volume for 1 reaction Volume for 16 reactions (includes excess)
Hybridization Buffer mixture from step 4 13 L 221 L
10% RNase Block solution from step 8 5 L 85 L
Probe (with design <3 Mb) 2 L 34 L
Total 20 L 340 L
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Hybridization and Capture 4 Step 1. Hybridize DNA samples to the probe
SureSelectXT Target
10 Maintain the gDNA library + Block Mix plate or strip tube at 65C while you add 20 L of the Probe Hybridization Mix from step 9 to each sample well. Mix well by pipetting up and down 8 to 10 times.
The hybridization reaction wells now contain approximately 27 to 29 L, depending on the degree of evaporation during the thermal cycler incubation.
11 Seal the wells with strip caps or using the PlateLoc Thermal Microplate Sealer. Make sure that all wells are completely sealed.
CAUTION Wells must be adequately sealed to minimize evaporation, or your results can be negatively impacted.
When using the SureCycler 8800 thermal cycler and sealing with strip caps, make sure to use domed strip caps or to place a compression mat over the PCR plate or strip tubes in the thermal cycler.
12 Incubate the hybridization mixture for 16 or 24 hours at 65C with a heated lid at 105C.
Enrichment System for Illumina Multiplexed Sequencing 69
4 Hybridization and Capture Step 2. Prepare streptavidin-coated magnetic beads
Step 2. Prepare streptavidin-coated magnetic beads
70
The hybrid capture protocol uses reagents provided in SureSelect Target Enrichment Box 1 (stored at room temperature) in addition to the streptavidin- coated magnetic beads obtained from another supplier (see Table 2 on page 16).
1 Prewarm SureSelect Wash Buffer 2 at 65C in a circulating water bath or heat block for use in Step 3. Capture the hybridized DNA using streptavidin- coated beads on page 71.
2 Vigorously resuspend the Dynabeads MyOne Streptavidin T1 magnetic beads on a vortex mixer. The magnetic beads settle during storage.
3 For each hybridization sample, add 50 L of the resuspended beads to wells of a fresh PCR plate or strip tube.
4 Wash the beads:
a Add 200 L of SureSelect Binding Buffer.
b Mix by pipetting up and down until beads are fully resuspended.
c Put the plate or strip tube into a magnetic separator device.
d Wait until the solution is clear, then remove and discard the supernatant.
e Repeat step a through step d two more times for a total of 3 washes.
5 Resuspend the beads in 200 L of SureSelect Binding Buffer.
If you are equipped for higher-volume magnetic bead captures, the streptavidin beads may be batch-washed in an Eppendorf tube or conical vial. Start the batch-washing procedure using excess bead solution. After resuspending the washed beads in the appropriate volume of SureSelect Binding Buffer, aliquot 200 l of the washed beads to plate or strip tube wells to be used for hybridization capture.
NOTE
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Hybridization and Capture 4 Step 3. Capture the hybridized DNA using streptavidin-coated beads
Step 3. Capture the hybridized DNA using streptavidin-coated beads
SureSelectXT Target
1 Estimate and record the volume of hybridization solution that remains after the 16 or 24 hour incubation.
2 Maintain the hybridization plate or strip tube at 65C while you use a multichannel pipette to transfer the entire volume (approximately 25 to 29 L) of each hybridization mixture to the plate or strip tube wells containing 200 L of washed streptavidin beads.
Mix well by slowly pipetting up and down until beads are fully resuspended.
Excessive evaporation, such as when less than 20 L remains after hybridization, can indicate suboptimal capture performance. See Table 55 on page 101 for tips to minimize evaporation.
NOTE
3 Cap the wells, then incubate the capture plate or strip tube on a 96- well plate mixer, mixing vigorously (14001800 rpm) for 30 minutes at room temperature.
Make sure the samples are properly mixing in the wells.
4 Briefly spin the plate or strip tube in a centrifuge or mini- plate spinner.
5 Put the plate or strip tube in a magnetic separator to collect the beads. Wait until the solution is clear, then remove and discard all of the supernatant.
6 Resuspend the beads in 200 L of SureSelect Wash Buffer 1. Mix by pipetting up and down until beads are fully resuspended.
7 Incubate the samples for 15 minutes at room temperature.
8 Briefly spin in a centrifuge or mini- plate spinner.
9 Put the plate or strip tube in the magnetic separator. Wait for the solution to clear, then remove and discard all of the supernatant.
Enrichment System for Illumina Multiplexed Sequencing 71
4 Hybridization and Capture Step 3. Capture the hybridized DNA using streptavidin-coated beads
72
CAUTION It is important to maintain bead suspensions at 65C during the washing procedure below to ensure specificity of capture.
Make sure that the SureSelect Wash Buffer 2 is pre-warmed to 65C before use.
Do not use a tissue incubator, or other devices with significant temperature fluctuations, for the incubation steps.
10 Wash the beads with SureSelect Wash Buffer 2:
a Resuspend the beads in 200 L of 65C prewarmed Wash Buffer 2. Pipette up and down until beads are fully resuspended.
b Cap the wells, then incubate the sample plate or strip tube for 10 minutes at 65C on the thermal cycler.
c Put the plate or strip tube in the magnetic separator. Wait for the solution to clear, then remove and discard the supernatant.
d Repeat step a through step c for a total of 3 washes.
Make sure all of the wash buffer has been removed during the final wash.
11 Add 30 L of nuclease- free water to each sample well. Pipette up and down to resuspend the beads.
Keep the samples on ice until they are used on page 75.
Captured DNA is retained on the streptavidin beads during the post-capture amplification step.
NOTE
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
SureSelectXT Target Enrichment System for the Illumina Platform Protocol
5 Indexing and Sample Processing for Multiplexed Sequencing
Step 1. Amplify the captured libraries with indexing primers 74
Step 2. Purify the amplified captured libraries using AMPure XP beads 77
Step 3. Assess indexed library DNA quantity and quality 79
Step 4. Quantify each index-tagged library by QPCR (optional) 83
Step 5. Pool samples for multiplexed sequencing 84
Step 6. Prepare sequencing samples 86
This chapter describes the steps to add index tags by amplification, and to purify and assess quality and quantity of the captured libraries. Sample pooling instructions are provided to prepare the indexed samples for multiplexed sequencing.
73Agilent Technologies
5 Indexing and Sample Processing for Multiplexed Sequencing Step 1. Amplify the captured libraries with indexing primers
Step 1. Amplify the captured libraries with indexing primers
74
In this step, the SureSelect- enriched DNA libraries are PCR amplified in PCR reactions that include the appropriate indexing primer for each sample.
This step uses the components listed in Table 36. Thaw then vortex to mix the reagents listed below and keep on ice.
Prepare one indexing amplification reaction for each DNA library.
Table 36 Reagents for post-capture indexing by PCR amplification
Kit Component Storage Location
5 Herculase II Reaction Buffer Herculase II Fusion DNA Polymerase kit*, 20C
100 mM dNTP Mix (25 mM each dNTP) Herculase II Fusion DNA Polymerase kit*, 20C
* Do not use the PCR Reaction Buffer or dNTP mix from any other kit.
Herculase II Fusion DNA Polymerase Herculase II Fusion DNA Polymerase kit, 20C
SureSelect ILM Indexing Post Capture Forward PCR Primer
SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2, 20C
SureSelect 8 bp Indexes (reverse primers) SureSelect XT Library Prep Kit ILM, 20C
NOTE When processing FFPE-derived DNA samples, some details of this step should be modified. See Table 44 on page 90 for more information.
CAUTION To avoid cross-contaminating libraries, set up PCR mixes in a dedicated clean area or PCR hood with UV sterilization and positive air flow.
1 Determine the appropriate index assignments for each sample. See Table 54 in the Reference chapter for sequences of the index portion of the SureSelect 8 bp Indexes A01 through H12 indexing primers used to amplify the DNA libraries in this step.
Use a different indexing primer for each sample to be sequenced in the same lane.
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Indexing and Sample Processing for Multiplexed Sequencing 5 Step 1. Amplify the captured libraries with indexing primers
SureSelectXT Target
2 Prepare the appropriate volume of PCR reaction mix, as described in Table 37, on ice. Mix well on a vortex mixer.
Table 37 Preparation of post-capture PCR Reaction mix
3 Add 31 L of the PCR reaction mix prepared in Table 37 to each sample well of a fresh PCR plate or strip tube.
4 Add 5 L of the appropriate indexing primer (SureSelect 8 bp Indexes A01 through H02, provided in white- capped tubes or A01 through H12, provided in blue plate) to each well. Add only one of the 16 or 96 possible indexing primers to each reaction well.
5 Add the DNA library samples to the PCR reactions:
a Obtain the PCR plate or strip tube containing 30 L of bead- bound target- enriched DNA samples from ice (prepared on page 72).
b Pipette each DNA sample up and down until the bead suspension is homogeneous, then transfer 14 L of the sample to the appropriate well of the PCR plate or strip tube containing PCR reaction mix and indexing primer.
c Mix the PCR reactions well by pipetting.
d Store the remaining library- bound beads at 20C for future use, if needed.
Reagent Volume for 1 reaction
Volume for 16 reactions (includes excess)
Nuclease-free water 18.5 L 314.5 L
5 Herculase II Reaction Buffer (clear cap) 10 L 170 L
Herculase II Fusion DNA Polymerase (red cap) 1 L 17 L
100 mM dNTP Mix (green cap) 0.5 L 8.5 L
SureSelect ILM Indexing Post Capture Forward PCR Primer (orange cap)
1 L 17 L
Total 31 L 527 L
Enrichment System for Illumina Multiplexed Sequencing 75
5 Indexing and Sample Processing for Multiplexed Sequencing Step 1. Amplify the captured libraries with indexing primers
76
6 Transfer the PCR plate or strip tube to a thermal cycler and run the PCR amplification program shown in Table 38.
Table 38 Post-Capture PCR cycling program
7 When the PCR amplification program is complete, spin the plate or strip tube briefly.
Segment Number of Cycles Temperature Time
1 1 98C 2 minutes
2 10 to 16 Cycles
See Table 39 for recommendations for specific Probes
98C 30 seconds
57C 30 seconds
72C 1 minute
3 1 72C 10 minutes
4 1 4C Hold
Table 39 Post-capture PCR cycle number recommendations
Probe Design Size/Description Cycles
1 kb up to 0.5 Mb 16 cycles
0.5 Mb up to 1.49 Mb 14 cycles
>1.5 Mb 12 cycles
All Exon and Exome libraries 10 to 12 cycles
OneSeq Constitutional Research Panel 10 cycles
OneSeq Hi Res CNV Backbone-based custom designs 10 cycles
OneSeq 1Mb CNV Backbone-based custom designs 10 to 12 cycles
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Indexing and Sample Processing for Multiplexed Sequencing 5 Step 2. Purify the amplified captured libraries using AMPure XP beads
Step 2. Purify the amplified captured libraries using AMPure XP beads
SureSelectXT Target
1 Let the AMPure XP beads come to room temperature for at least 30 minutes. Do not freeze the beads at any time.
2 Prepare 400 L of fresh 70% ethanol per sample, plus excess, for use in step 9.
3 Mix the AMPure XP bead suspension well so that the suspension appears homogeneous and consistent in color.
4 Add 90 L of the homogeneous AMPure XP bead suspension to each 50- L amplified DNA sample bead suspension in the PCR plate or strip tube.
5 Mix thoroughly by pipetting up and down.
Check that the beads are in a homogeneous suspension in the sample wells. Each well should have a uniform color with no layers of beads or clear liquid present.
6 Incubate samples for 5 minutes at room temperature.
7 Put the plate or strip tube on the magnetic stand at room temperature. Wait for the solution to clear (approximately 3 to 5 minutes).
8 While keeping the plate or tubes in the magnetic stand, carefully remove and discard the cleared solution from each well. Do not disturb the beads while removing the solution.
9 Continue to keep the plate or tubes in the magnetic stand while you dispense 200 L of freshly prepared 70% ethanol in each sample well.
10 Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol.
11 Repeat step 9 and step 10 once for a total of two washes. Make sure to remove all of the ethanol at each wash step.
12 Seal the wells with strip caps, then briefly spin to collect the residual ethanol. Return the plate or strip tube to the magnetic stand for 30 seconds. Remove the residual ethanol with a P20 pipette.
13 Dry the samples by placing the unsealed plate or strip tube on the thermal cycler, set to hold samples at 37C, for 1 to 2 minutes or until the residual ethanol completely evaporates.
14 Add 30 L of nuclease- free water to each sample well.
Enrichment System for Illumina Multiplexed Sequencing 77
5 Indexing and Sample Processing for Multiplexed Sequencing Step 2. Purify the amplified captured libraries using AMPure XP beads
78
15 Seal the sample wells, then mix well on a vortex mixer and briefly spin to collect the liquid.
16 Incubate for 2 minutes at room temperature.
17 Put the plate or strip tube in the magnetic stand and leave for 2 minutes or until the solution is clear.
18 Remove the cleared supernatant (approximately 30 L) to a fresh well. You can discard the beads at this time.
Stopping Point
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Indexing and Sample Processing for Multiplexed Sequencing 5 Step 3. Assess indexed library DNA quantity and quality
Step 3. Assess indexed library DNA quantity and quality
SureSelectXT Target
Option 1: Analysis using the Agilent 2100 Bioanalyzer and High Sensitivity DNA Assay
Use the Bioanalyzer High Sensitivity DNA Assay to analyze the amplified indexed DNA. Perform the assay according to the High Sensitivity DNA Kit Guide.
1 Set up the 2100 Bioanalyzer as instructed in the reagent kit guide.
2 Prepare the chip, samples and ladder as instructed in the reagent kit guide, using 1 L of each sample for the analysis.
3 Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation.
4 Verify that the electropherogram shows the peak of DNA fragment size positioned between 250 and 350 bp. A sample electropherogram is shown in Figure 9.
5 Measure the concentration of each library by integrating under the entire peak. For accurate quantification, make sure that the concentration falls within the linear range of the assay.
If you wish to more- precisely quantify the target enriched samples prior to pooling, proceed to Step 4. Quantify each index- tagged library by QPCR (optional) on page 83.
Otherwise, proceed to Step 5. Pool samples for multiplexed sequencing on page 84.
Stopping Point
Enrichment System for Illumina Multiplexed Sequencing 79
5 Indexing and Sample Processing for Multiplexed Sequencing Step 3. Assess indexed library DNA quantity and quality
80
Figure 9 Post-capture analysis of amplified indexed library DNA using the 2100 Bioanalyzer and a High Sensitivity DNA Assay.
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Indexing and Sample Processing for Multiplexed Sequencing 5 Step 3. Assess indexed library DNA quantity and quality
SureSelectXT Target
Option 2: Analysis using an Agilent TapeStation and High Sensitivity D1000 ScreenTape
Use a High Sensitivity D1000 ScreenTape and associated reagent kit. Perform the assay according to the Agilent High Sensitivity D1000 Assay Quick Guide.
1 Prepare the TapeStation samples as instructed in the reagent kit guide. Use 2 L of each indexed DNA sample diluted with 2 L of High Sensitivity D1000 sample buffer for the analysis.
CAUTION For accurate quantitation, make sure to thoroughly mix the combined DNA and sample buffer by vortexing the assay plate or tube strip for 1 minute on the IKA MS3 vortex mixer provided with the 4200/4150 TapeStation system before loading the samples.
2 Load the sample plate or tube strips from step 1, the High Sensitivity D1000 ScreenTape, and loading tips into the TapeStation instrument as instructed in the reagent kit guide. Start the run.
3 Verify that the electropherogram shows the peak of DNA fragment size positioned between 250 and 350 bp. A sample electropherogram is shown in Figure 10.
4 Measure the concentration of each library by integrating under the entire peak.
If you wish to more- precisely quantify the target enriched samples prior to pooling, proceed to Step 4. Quantify each index- tagged library by QPCR (optional) on page 83.
Otherwise, proceed to Step 5. Pool samples for multiplexed sequencing on page 84.
Stopping Point
Enrichment System for Illumina Multiplexed Sequencing 81
5 Indexing and Sample Processing for Multiplexed Sequencing Step 3. Assess indexed library DNA quantity and quality
82
Figure 10 Post-capture analysis of amplified indexed library DNA using a High Sensitivity D1000 ScreenTape.
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Indexing and Sample Processing for Multiplexed Sequencing 5 Step 4. Quantify each index-tagged library by QPCR (optional)
Step 4. Quantify each index-tagged library by QPCR (optional)
SureSelectXT Target
You can use the Agilent QPCR NGS Library Quantification Kit (for Illumina) to accurately determine the concentration of each index- tagged captured library. Refer to the protocol that is included with the Agilent QPCR NGS Library Quantification Kit (p/n G4880A) for more details to do this step.
1 Prepare a standard curve using the quantification standard included in the kit, according to the instructions provided in the user guide.
2 Dilute each index- tagged captured library such that it falls within the range of the standard curve.
Typically this corresponds to approximately a 1:1000 to 1:10,000 dilution of the captured DNA.
3 Prepare the QPCR master mix with Illumina adaptor- specific PCR primers according to instructions provided in the kit.
4 Add an aliquot of the master mix to PCR tubes and add template.
5 On a QPCR system, such as the Mx3005p, run the thermal profile outlined in the QPCR NGS Library Quantification kit user guide. Use the SYBR Green instrument setting.
6 Use the standard curve to determine the concentration of each unknown index- tagged library, in nM.
Enrichment System for Illumina Multiplexed Sequencing 83
5 Indexing and Sample Processing for Multiplexed Sequencing Step 5. Pool samples for multiplexed sequencing
Step 5. Pool samples for multiplexed sequencing
84
The number of indexed libraries that may be multiplexed in a single sequencing lane is determined by the output specifications of the platform used, together with the amount of sequencing data required for your research design. Calculate the number of indexes that can be combined per lane, according to the capacity of your platform and the amount of sequencing data required per sample.
1 Combine the libraries such that each index- tagged sample is present in equimolar amounts in the pool. For each library, use the formula below to determine the amount of indexed sample to use.
Volume of Index V f C f # C i
--------------------------------=
where V(f) is the final desired volume of the pool,
C(f) is the desired final concentration of all the DNA in the pool
# is the number of indexes, and
C(i) is the initial concentration of each indexed sample.
Table 40 shows an example of the amount of 4 index- tagged samples (of different concentrations) and Low TE needed for a final volume of 20 L at 10 nM.
2 Adjust the final volume of the pooled library to the desired final concentration.
Table 40 Example of indexed sample volume calculation for total volume of 20 L
Component V(f) C(i) C(f) # Volume to use (L)
Sample 1 20 L 20 nM 10 nM 4 2.5
Sample 2 20 L 10 nM 10 nM 4 5
Sample 3 20 L 17 nM 10 nM 4 2.9
Sample 4 20 L 25 nM 10 nM 4 2
Low TE 7.6
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Indexing and Sample Processing for Multiplexed Sequencing 5 Step 5. Pool samples for multiplexed sequencing
SureSelectXT Target
If the final volume of the combined index- tagged samples is less than the desired final volume, V(f), add Low TE to bring the volume to the desired level.
If the final volume of the combined index- tagged samples is greater than the final desired volume, V(f), lyophilize and reconstitute to the desired volume.
3 If you store the library before sequencing, add Tween 20 to 0.1% v/v and store at - 20C short term.
Enrichment System for Illumina Multiplexed Sequencing 85
5 Indexing and Sample Processing for Multiplexed Sequencing Step 6. Prepare sequencing samples
Step 6. Prepare sequencing samples The sequencing workflow for FFPE-derived DNA libraries should employ the modifications discussed in the Appendix, starting on page 89. Modifications include the requirement for adapter trimming and increased sequencing depth for lower-integrity DNA samples.
NOTE
86
Proceed to cluster amplification using the appropriate Illumina Paired- End Cluster Generation Kit. See Table 41 for kit configurations compatible with the recommended read length.
The optimal seeding concentration for SureSelectXT target- enriched libraries varies according to sequencing platform, run type, and Illumina kit version. See Table 41 for guidelines. Seeding concentration and cluster density may also need to be optimized based on the DNA fragment size range for the library and on the desired output and data quality. Begin optimization using a seeding concentration in the middle of the range listed in Table 41.
Follow Illuminas recommendation for a PhiX control in a low- concentration spike- in for improved sequencing quality control.
Table 41 Illumina Kit Configuration and Seeding Concentration Guidelines
Platform Run Type Read Length SBS Kit Configuration Chemistry Seeding Concentration
HiSeq 2500 Rapid Run 2 100 bp 200 Cycle Kit v2 910 pM
HiSeq 2500 High Output 2 100 bp 250 Cycle Kit v4 1214 pM
MiSeq All Runs 2 100 bp 300 Cycle Kit v2 910 pM
MiSeq All Runs 2 75 bp 150 Cycle Kit v3 1216 pM
NextSeq 500/550 All Runs 2 100 bp 300 Cycle Kit v2.5 1.21.5 pM
HiSeq 3000/4000 All Runs 2 100 bp 300 Cycle Kit v1 300400 pM
NovaSeq 6000 Standard Workflow Runs 2 100 bp 300 Cycle Kit v1.0 or v1.5 300600 pM
NovaSeq 6000 Xp Workflow Runs 2 100 bp 300 Cycle Kit v1.0 or v1.5 200400 pM
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Indexing and Sample Processing for Multiplexed Sequencing 5 Step 6. Prepare sequencing samples
SureSelectXT Target
Sequencing run setup guidelines for 8-bp indexes
Sequencing runs must be set up to perform an 8- bp index read. For complete index sequence information, see the Table 54 on page 100.
For the HiSeq platform, Cycles settings can be specified on the Run Configuration screen of the instrument control software interface after choosing Custom from the Index Type selection buttons. Use the Cycles settings shown in Table 42.
For the NextSeq and NovaSeq platforms, the Read Length settings shown in Table 42 can be specified on the Run Setup screen of the instrument control software interface. In the Custom Primers section of the NextSeq or NovaSeq platform Run Setup screen, clear (do not select) the checkboxes for all primers (Read 1, Read 2, Index 1 and Index 2)
For the MiSeq platform, use the Illumina Experiment Manager (IEM) software to generate a Sample Sheet that includes the run parameters specified in Table 43.
Table 42 Cycle Number settings for HiSeq/NextSeq/NovaSeq platforms
Run Segment Cycles/Read Length
Read 1 100
Index 1 (i7) 8
Index 2 (i5) 0
Read 2 100
Table 43 Run parameters for MiSeq platform Sample Sheet
Parameter Entry
Workflow GenerateFASTQ
Cycles for Read 1 100 for v2 chemistry
75 for v3 chemistry
Cycles for Read 2 100 for v2 chemistry
75 for v3 chemistry
Index 1 (i7) Sequence (enter in Data Section for each sample)
Type the 8-nt index sequence for each individual sample (see Table 54 on page 100).
Enrichment System for Illumina Multiplexed Sequencing 87
5 Indexing and Sample Processing for Multiplexed Sequencing Step 6. Prepare sequencing samples
88
SureSelectXT Target Enrichment System for the Illumina Platform Protocol
6 Appendix: Using FFPE-derived DNA Samples
Modifications for all FFPE DNA samples 90
Modifications for samples assessed using the Agilent NGS FFPE QC Kit 91
Modifications for samples assessed using Agilents Genomic DNA ScreenTape 93
FFPE- derived DNA samples may be used in the Library Preparation protocol for 200 ng samples and subsequent Target Enrichment protocol after making the minor protocol modifications detailed in this chapter.
Protocol modifications that should be applied to all FFPE samples are detailed on page 90.
Additional protocol modifications may be appropriate, depending on the integrity of the FFPE sample DNA. DNA integrity may be assessed using the Agilent NGS FFPE QC Kit or using the Agilent TapeStation instrument and Genomic DNA ScreenTape.
The Agilent NGS FFPE QC Kit provides a qPCR- based assay for DNA sample integrity determination. Results include the precise quantity of amplifiable DNA in the sample and a Cq DNA integrity score. Protocol modifications based on Cq scores for individual samples are detailed on page 91.
TThe Agilent TapeStation instrument, combined with the Genomic DNA ScreenTape assay, provides an automated electrophoresis method for determination of a DNA Integrity Number (DIN) score. Protocol modifications based on DIN scores for individual samples are detailed on page 93.
89Agilent Technologies
6 Appendix: Using FFPE-derived DNA Samples Modifications for all FFPE DNA samples
Modifications for all FFPE DNA samples
SureSelectXT Protocol Modifications
90
Protocol modifications that should be applied to all FFPE samples are detailed in Table 44.
Downstream Sequencing Modifications
Table 44 SureSelectXT protocol modifications for all FFPE samples
Workflow Step and page Parameter Condition for non-FFPE Samples
Condition for FFPE Samples
Library Preparation using 200 ng DNA, page 45
Duration of DNA Shearing 6 minutes 4 minutes
Library Preparation using 200 ng DNA, page 53
Dilution of SureSelect Adaptor Oligo Mix for Ligation reaction
Use ten-fold dilution of SureSelect Adaptor Oligo Mix
Use undiluted SureSelect Adaptor Oligo Mix
Hybridization, page 65 Amount of prepared library added to Hybridization
750 ng 500750 ng (use maximum available in range)
Post-capture PCR, page 75
Amount of captured DNA bead suspension added to PCR
14 l 30 l (decrease the amount of water added to post-capture PCR by 16 l to compensate for greater volume of captured DNA)
For all FFPE sample- derived libraries, set up the sequencing run to include adapter trimming.
To do this step, use the IEM Sample Sheet Wizard. When prompted by the wizard, select the Use Adapter Trimming and Use Adapter Trimming Read 2 options. This enables the MiSeq Reporter software to identify the adaptor sequence and trim the adaptor from reads.
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Appendix: Using FFPE-derived DNA Samples 6 Modifications for samples assessed using the Agilent NGS FFPE QC Kit
Modifications for samples assessed using the Agilent NGS FFPE QC Kit
SureSelectXT Target
Before applying protocol modifications in this section, use the Agilent NGS FFPE QC Kit to determine the Cq DNA integrity score and the quantity of amplifiable DNA for each FFPE DNA sample. For the complete Agilent NGS FFPE QC Kit protocol, go to agilent.com and search for document part number G9700- 90000.
SureSelectXT Protocol Modifications
Protocol modifications that should be applied to FFPE samples based on the Cq score determined for each sample are detailed in Table 45.
Downstream Sequencing Modifications
Table 45 SureSelectXT protocol modifications based on Cq DNA integrity score
Protocol Step and Parameter
non-FFPE Samples
FFPE Samples
Cq1* Cq between 1 and 4 Cq>4
DNA input for Library Preparation, page 44
200 ng, based on Qubit Assay
200 ng, based on Qubit Assay
100 to 200 ng of amplifiable DNA, based on qPCR quantification
100 to 200 ng of amplifiable DNA, based on qPCR quantification
Pre-capture PCR cycle number, page 58
10 cycles 10 cycles 10 cycles 13 cycles
* FFPE samples with Cq scores 1 should be treated like non-FFPE samples at these steps. For samples of this type, make sure to use the DNA concentration determined by the Qubit Assay, instead of the concentration determined by qPCR, to cal- culate the volume required for 200 ng DNA.
After determining the amount of sequencing output required for intact DNA samples to meet the goals of your project, use the guidelines in Table 46 to determine the amount of extra sequencing output required for FFPE DNA samples, based on the Cq DNA integrity score.
Enrichment System for Illumina Multiplexed Sequencing 91
6 Appendix: Using FFPE-derived DNA Samples Downstream Sequencing Modifications
92
For example, if your workflow demands 100 Mb output for intact DNA samples to achieve the required coverage, an FFPE sample with Cq score of 1 requires 150200 Mb of sequencing output to achieve the same coverage.
Table 46 Recommended sequencing augmentation for FFPE-derived DNA samples
Cq value Recommended fold increase for FFPE-derived sample
<0.5 No extra sequencing output
between 0.5 and 2 Increase sequencing allocation by 1.5 to 2
between 2 and 3.5 Increase sequencing allocation by 3
between 3.5 and 5 Increase sequencing allocation by 4 to 5
>5 Increase sequencing allocation by 6 to 10
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Appendix: Using FFPE-derived DNA Samples 6 Modifications for samples assessed using Agilents Genomic DNA ScreenTape
Modifications for samples assessed using Agilents Genomic DNA ScreenTape
SureSelectXT Target
Before applying protocol modifications in this section, use the Agilent TapeStation instrument and Genomic DNA ScreenTape to determine the DNA Integrity Number (DIN) score for each sample. For more information on how to obtain DIN numbers using the TapeStation system, go to agilent.com and search for document part number 5991- 5442.
Use the DIN score to determine whether additional SureSelectXT protocol or downstream sequencing modifications are appropriate for each sample.
SureSelectXT Protocol Modifications
Protocol modifications that should be applied to FFPE samples based on DIN score are detailed in Table 47.
Downstream Sequencing Modifications
Table 47 SureSelectXT protocol modifications based on DIN score
Protocol Step and Parameter
non-FFPE Samples
FFPE Samples
DIN 5 DIN between 2 and 5 DIN <2
DNA input for Library Preparation, page 44
200 ng, based on Qubit Assay
200 ng, based on Qubit Assay
100 to 200 ng of amplifiable DNA, based on qPCR quantification*
100 to 200 ng of amplifiable DNA, based on qPCR quantification
Pre-capture PCR cycle number, page 58
10 cycles 10 cycles 10 cycles 13 cycles
* Use the Agilent NGS FFPE QC Kit for qPCR-based sample quantification. See Table 8 on page 21 for ordering information.
After determining the amount of sequencing output required for intact DNA samples to meet the goals of your project, use the guidelines in Table 48 below to determine the amount of extra sequencing output required for FFPE DNA samples, based on the DNA integrity (DIN) score.
Enrichment System for Illumina Multiplexed Sequencing 93
6 Appendix: Using FFPE-derived DNA Samples Downstream Sequencing Modifications
94
For example, if your workflow demands 100 Mb output for intact DNA samples to achieve the required coverage, an FFPE sample with DIN score of 4 requires approximately 200 Mb of sequencing output to achieve the same coverage.
Table 48 Recommended sequencing augmentation for FFPE-derived DNA samples
DIN value Recommended fold increase for FFPE-derived sample
8 No extra sequencing output
between 5 and 8 Increase sequencing allocation by 1.5
between 3 and 5 Increase sequencing allocation by 2
between 1.5 and 3 Increase sequencing allocation by 3 to 5
<1.5 Increase sequencing allocation by 6 to 10
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
SureSelectXT Target Enrichment System for the Illumina Platform Protocol
7 Reference
Kit Contents 96
Nucleotide Sequences of SureSelectXT Indexes A01 to H12 100
Alternative Capture Equipment Combinations 101
This chapter contains reference information, including component kit contents and index sequences.
95Agilent Technologies
7 Reference Kit Contents
Kit Contents
96
Each SureSelectXT Reagent Kit contains the following component kits:
Table 49 Component Kits
Product Storage Condition 16 Reactions 96 Reactions 480 Reactions
SureSelect XT Library Prep Kit ILM 20C 5500-0132 5500-0133 5 x 5500-0133
SureSelect Target Enrichment Box 1 Room Temperature 5190-8645 5190-8646 5 x 5190-8646
SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2
20C 5190-4455 5190-4456 5190-4457
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Reference 7 Kit Contents
SureSelectXT Target
The contents of each of the component kits listed in Table 49 are described in the tables below.
Table 50 SureSelect XT Library Prep Kit ILM Content
Kit Component 16 Reactions 96 or 480 Reactions
10X End Repair Buffer tube with clear cap tube with clear cap
10X Klenow Polymerase Buffer tube with blue cap tube with blue cap
5X T4 DNA Ligase Buffer tube with green cap tube with green cap
T4 DNA Ligase tube with red cap tube with red cap
Exo(-) Klenow tube with red cap tube with red cap
T4 DNA Polymerase tube with purple cap tube with purple cap
Klenow DNA Polymerase tube with yellow cap tube with yellow cap
T4 Polynucleotide Kinase tube with orange cap tube with orange cap
dATP tube with green cap tube with green cap
dNTP Mix tube with green cap tube with green cap
SureSelect Adaptor Oligo Mix tube with brown cap tube with brown cap
SureSelect Primer (forward primer) tube with brown cap tube with brown cap
SureSelectXT 8 bp Index reverse primers* SureSelect 8bp Indexes A01 through H02, provided in 16 white-capped tubes
SureSelect 8bp Indexes A01 through H12, provided in blue 96-well plate
* See Table 54 on page 100 for index sequences.
See Table 53 on page 99 for a plate map.
Enrichment System for Illumina Multiplexed Sequencing 97
7 Reference Kit Contents
98
Table 51 SureSelect Target Enrichment Box 1 Content
Kit Component 16 Reactions 96 or 480 Reactions
SureSelect Hyb 1 tube with orange cap tube with orange cap
SureSelect Hyb 2 tube with red cap tube with red cap
SureSelect Hyb 4 tube with black cap tube with black cap
SureSelect Binding Buffer bottle bottle
SureSelect Wash Buffer 1 bottle bottle
SureSelect Wash Buffer 2 bottle bottle
Table 52 SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2 Content
Kit Component 16 Reactions 96 Reactions 480 Reactions
SureSelect Hyb 3 tube with yellow cap tube with yellow cap bottle
SureSelect Indexing Block 1 tube with green cap tube with green cap tube with green cap
SureSelect Block 2 tube with blue cap tube with blue cap tube with blue cap
SureSelect Indexing Block 3 tube with brown cap tube with brown cap tube with brown cap
SureSelect RNase Block tube with purple cap tube with purple cap tube with purple cap
SureSelect ILM Index Pre Capture PCR Reverse Primer
tube with clear cap tube with clear cap tube with clear cap
SureSelect ILM Indexing Post Capture Forward PCR Primer
tube with orange cap tube with orange cap tube with orange cap
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Reference 7 Kit Contents
Table 53 Plate map for SureSelect 8bp Indexes A01 through H12, provided in blue plate in Library Prep kit p/n 5500-0133
1 2 3 4 5 6 7 8 9 10 11 12
A A01 A02 A03 A04 A05 A06 A07 A08 A09 A10 A11 A12
B B01 B02 B03 B04 B05 B06 B07 B08 B09 B10 B11 B12
C C01 C02 C03 C04 C05 C06 C07 C08 C09 C10 C11 C12
D D01 D02 D03 D04 D05 D06 D07 D08 D09 D10 D11 D12
E E01 E02 E03 E04 E05 E06 E07 E08 E09 E10 E11 E12
F F01 F02 F03 F04 F05 F06 F07 F08 F09 F10 F11 F12
G G01 G02 G03 G04 G05 G06 G07 G08 G09 G10 G11 G12
H H01 H02 H03 H04 H05 H06 H07 H08 H09 H10 H11 H12
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing 99
7 Reference Nucleotide Sequences of SureSelectXT Indexes A01 to H12
Nucleotide Sequences of SureSelectXT Indexes A01 to H12
100
Each index is 8 nt in length. See page 87 for sequencing run setup requirements for sequencing libraries using 8- bp indexes.
XT
Index Sequence Index Sequence Index Sequence Index Sequence
A01 ATGCCTAA A04 AACTCACC A07 ACGTATCA A10 AATGTTGC
B01 GAATCTGA B04 GCTAACGA B07 GTCTGTCA B10 TGAAGAGA
C01 AACGTGAT C04 CAGATCTG C07 CTAAGGTC C10 AGATCGCA
D01 CACTTCGA D04 ATCCTGTA D07 CGACACAC D10 AAGAGATC
E01 GCCAAGAC E04 CTGTAGCC E07 CCGTGAGA E10 CAACCACA
F01 GACTAGTA F04 GCTCGGTA F07 GTGTTCTA F10 TGGAACAA
G01 ATTGGCTC G04 ACACGACC G07 CAATGGAA G10 CCTCTATC
H01 GATGAATC H04 AGTCACTA H07 AGCACCTC H10 ACAGATTC
A02 AGCAGGAA A05 AACGCTTA A08 CAGCGTTA A11 CCAGTTCA
B02 GAGCTGAA B05 GGAGAACA B08 TAGGATGA B11 TGGCTTCA
C02 AAACATCG C05 CATCAAGT C08 AGTGGTCA C11 CGACTGGA
D02 GAGTTAGC D05 AAGGTACA D08 ACAGCAGA D11 CAAGACTA
E02 CGAACTTA E05 CGCTGATC E08 CATACCAA E11 CCTCCTGA
F02 GATAGACA F05 GGTGCGAA F08 TATCAGCA F11 TGGTGGTA
G02 AAGGACAC G05 CCTAATCC G08 ATAGCGAC G11 AACAACCA
H02 GACAGTGC H05 CTGAGCCA H08 ACGCTCGA H11 AATCCGTC
A03 ATCATTCC A06 AGCCATGC A09 CTCAATGA A12 CAAGGAGC
B03 GCCACATA B06 GTACGCAA B09 TCCGTCTA B12 TTCACGCA
C03 ACCACTGT C06 AGTACAAG C09 AGGCTAAC C12 CACCTTAC
D03 CTGGCATA D06 ACATTGGC D09 CCATCCTC D12 AAGACGGA
E03 ACCTCCAA E06 ATTGAGGA E09 AGATGTAC E12 ACACAGAA
F03 GCGAGTAA F06 GTCGTAGA F09 TCTTCACA F12 GAACAGGC
G03 ACTATGCA G06 AGAGTCAA G09 CCGAAGTA G12 AACCGAGA
H03 CGGATTGC H06 CCGACAAC H09 CGCATACA H12 ACAAGCTA
SureSelectXT Target Enrichment System for Illumina Multiplexed Sequencing
Reference 7 Alternative Capture Equipment Combinations
Alternative Capture Equipment Combinations
SureSelectXT Target
Table 55 below lists combinations of thermal cyclers, lid temperatures, plates or strip tubes and sealing methods that have shown minimal evaporation when used for the Hybridization protocol on page 64. Note that minimal evaporation is required to ensure optimal capture results.
Table 55 Hybridization equipment options that show minimal evaporation
PCR Machine Plate/Strips Cover Comments
Agilent SureCycler 8800 Thermal Cycler, p/n G8800A
96-well plates, p/n 410088
8-well strip tubes, p/n 410092
Domed cap strips, p/n 410096
Heated lid
Agilent Mx3005P Real-Time PCR System
Mx3005P Strip Tubes (Agilent p/n 401428)
Mx3005P Optical Strip Caps (Agilent p/n 401425)
Heated lid
Agilent Mx3005P Real-Time PCR System
Thermo Fisher Scientific ABI MicroAmp Optical 96-well plates (p/n N8010560)
MicroAmp clear adhesive film (p/n 4306311)
Heated lid; use ABI compression pad (4312639); use two layers of film
ABI GeneAmp 9700 Thermo Fisher Scientific ABI MicroAmp Optical 96-well plates (p/n N8010560)
MicroAmp caps (p/n N8010535)
Heated lid
ABI Veriti (p/n 4375786) Thermo Fisher Scientific ABI MicroAmp Optical 96-well plates (p/n N8010560)
MicroAmp clear adhesive film (p/n 4306311)
Heated lid; use ABI compression pad (4312639); use two layers of film
Eppendorf Mastercycler Eppendorf 8-Tube PCR Tubes Attached caps Lid heating set to 75C
BioRad (MJ Research) PTC-200 Mx4000 Strip Tubes (Agilent p/n 410022)
Mx4000 Optical Caps (Agilent p/n 401024)
Heated lid
BioRad (MJ Research) PTC-200 Mx4000 Strip Tubes (Agilent p/n 410022)
Mx3005P Optical Strip Caps (Agilent p/n 401425)
Heated lid
BioRad (MJ Research) PTC-200 Mx3005P 96-well plate (Agilent p/n 410088)
Mx3005P Optical Strip Caps (Agilent p/n 401425)
Heated lid
Enrichment System for Illumina Multiplexed Sequencing 101
www.agilent.com
In This Book
This guide contains information to run the SureSelectXT target enrichment protocol.
Agilent Technologies, Inc. 2010-2021
Version D1, July 2021
*G7530-90000 * p/n G7530-90000
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