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SureSelectXT2
Automated Library Prep
and Capture System
For Illumina Paired-End
Multiplexed Sequencing
Automated using Agilent NGS
Workstation Option B
Protocol
Version B1, June 2015
SureSelect platform manufactured with Agilent
SurePrint Technology
For Research Use Only. Not for Use in Diagnostic
Procedures.
Agilent Technologies
Notices
© Agilent Technologies, Inc. 2015
Warranty
Notice to Purchaser
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into a foreign language) without prior agreement and written consent from Agilent
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No. 1 283 875 B1, owned by Bio-Rad Laboratories, Inc. Purchase of this product conveys to the buyer the non-transferable right
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and components of the product in PCR (but
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including all Applied Research Fields
(including but not limited to forensics, animal testing, and food testing).
Manual Part Number
G9450-90000
Edition
Version B1, June 2015
Printed in USA
Agilent Technologies, Inc.
5301 Stevens Creek Blvd
Santa Clara, CA 95051 USA
Acknowledgement
Oligonucleotide sequences © 2006, 2008,
and 2011 Illumina, Inc. All rights reserved.
Only for use with the Illumina sequencer
systems and associated assays.
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2
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SureSelectXT2 Automated Library Prep and Capture System
Safety Notices
CA U T I O N
A CAUTION notice denotes a hazard. It calls attention to an operating 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.
WARN I NG
A WARNING notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if
not correctly performed or adhered to, could result in personal injury or death. Do not proceed beyond a
WARNING notice until the indicated conditions are fully understood and met.
SureSelectXT2 Automated Library Prep and Capture System
3
In this Guide...
This guide describes an optimized protocol for Illumina
paired-end multiplexed library preparation using the
SureSelectXT2 Automated Library Prep and Capture System.
This protocol is specifically developed and optimized to
capture the genomic regions of interest using Agilent’s
SureSelect system to enrich targeted regions of the genome
from repetitive sequences and sequences unrelated to the
research focus prior to sample sequencing. Sample
processing steps are automated using the NGS Workstation.
1
Before You Begin
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
Using the Agilent NGS Workstation for SureSelect Target
Enrichment
This chapter contains an orientation to the Agilent NGS
Workstation, an overview of the SureSelectXT2 target
enrichment protocol, and considerations for designing
SureSelectXT2 experiments for automated processing using
the Agilent NGS Workstation.
3
Sample Preparation
This chapter describes the steps to prepare index-tagged
DNA samples for target enrichment.
4
Hybridization
This chapter describes the steps to pool indexed libraries
and then hybridize and capture the pooled DNA.
4
SureSelectXT2 Automated Library Prep and Capture System
5
Post-Capture Sample Processing for Multiplexed Sequencing
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
Reference
This chapter contains reference information, including
component kit contents and index sequences.
SureSelectXT2 Automated Library Prep and Capture System
5
What’s New in Version B.1
• Support for ClearSeq Capture Libraries, including
ClearSeq Comprehensive Cancer Libraries (see Table 3 on
page 13).
• Support for Human All Exon v6 Capture Libraries (see
Table 2 on page 12).
• Update to name of end-repair reagent from SureSelect
End Repair Oligo Mix to SureSelect End Repair
Nucleotide Mix (see Table 65 on page 129).
• Update to name of indexing adaptor reagent from
SureSelectXT2 Pre-Capture Index to SureSelect
Pre-Capture Indexed Adaptor (see Table 65 on
page 129).
• Updates to post-capture pooling and sequencing setup
guidelines, including support for the NextSeq 500
platform (see page 121 to page 122).
What’s New in Version B.0
• Support for kits supplied with either of two indexing
primer configurations.
Kits with revised index configuration (typically received
February 2015 or later) include indexing primers A01
through H12 provided in a blue plate. For kit content
details see page 128. For nucleotide sequences of the
8-bp indexes in this revised configuration, see Table 69
on page 131.
Kits with original index configuration (typically received
before February 2015), include indexing primers 1–96
provided in clear-capped tubes. For kit content details
see page 132. For nucleotide sequences of the 8-bp
indexes in this original configuration, see Table 75 on
page 134 through Table 80 on page 139.
• Support for revised Library Prep kit configuration, now
including End Repair Enzyme Mix and End Repair Oligo
Mix (both replacing End Repair Master Mix). For
instructions for use of the revised kit components, see
page 44. See Table 65 on page 129 for updated kit
contents.
6
SureSelectXT2 Automated Library Prep and Capture System
Content
1
Before You Begin
9
Procedural Notes 10
Safety Notes 10
Required Reagents 11
Required Equipment 14
Optional Reagents 16
Optional Equipment 16
2
Using the Agilent NGS Workstation for SureSelect Target Enrichment
About the Agilent NGS Workstation 18
About the Bravo Platform 18
VWorks Automation Control Software
Overview of the Workflow
17
22
28
Experimental Setup Considerations for Automated Runs 31
Considerations for Placement of gDNA Samples in 96-well Plates for
Automated Processing 34
Considerations for Indexed DNA Sample Placement for Automated
Hybridization and Post-Hybridization Processing 34
Considerations for Equipment Setup 35
PCR Plate Type Considerations 36
3
Sample Preparation
37
Step 1. Shear DNA 38
Step 2. Assess sample quality and DNA fragment size
Step 3. Prepare indexed gDNA library samples 43
Step 4. Amplify the indexed libraries 51
Step 5. Purify amplified DNA using AMPure XP beads
Step 6. Assess Library DNA quantity and quality 61
SureSelectXT2 Automated Library Prep and Capture System
41
58
7
Contents
4
Hybridization
63
Step 1. Pool indexed DNA samples for hybridization 64
Step 2. Hybridize the gDNA library and Capture Library 72
Hybridization Option A: Master Mixes in Columns
(Hybridization_MMCol_v2.0.pro) 73
Hybridization Option B: Master Mixes in Rows
(Hybridization_MMRow_v2.0.pro) 86
Step 3. Capture the hybridized DNA 97
5
Post-Capture Sample Processing for Multiplexed Sequencing
105
Step 1. Amplify the captured libraries 106
Step 2. Purify the amplified captured libraries using AMPure XP beads
Step 3. Assess quantity and quality of the amplified captured library
pools 117
Step 4. Prepare samples for multiplexed sequencing 121
Step 5. Optional: Quantify captured library pools by QPCR 123
Step 6. Optional: Pool captured libraries for sequencing 124
6
Reference
113
127
Reference Information for Kits with Revised Index Configuration (indexing primers
in blue plate) 128
Kit Contents 128
Nucleotide Sequences of SureSelectXT2 Indexes A01 to H12 131
Reference Information for Kits with Original Index Configuration (indexing primers
in clear-capped tubes) 132
Kit Contents 132
Nucleotide Sequences of SureSelect Pre-Capture Indexes-Original Index
Configuration 134
8
SureSelectXT2 Automated Library Prep and Capture System
SureSelectXT2 Automated Library Prep and Capture System Protocol
1
Before You Begin
Procedural Notes 10
Safety Notes 10
Required Reagents 11
Required Equipment 14
Optional Reagents 16
Optional Equipment 16
Make sure you read and understand the information in this chapter and
have the necessary equipment and reagents listed before you start an
experiment.
NOTE
Agilent cannot guarantee the SureSelect Target Enrichment kits and cannot provide
technical support for the use of non-Agilent protocols or instruments to process samples
for enrichment.
Agilent Technologies
9
1
Before You Begin
Procedural Notes
Procedural Notes
• Certain protocol steps require the rapid transfer of sample plates
between the Bravo deck and a thermal cycler. Locate your thermal
cycler in close proximity to the Agilent NGS Workstation to allow rapid
and efficient plate transfer.
• Prepare and load the Agilent NGS Workstation as detailed in each of
the protocol steps before initiating each automated protocol run. When
loading plates in the workstation’s Labware MiniHub, always place
plates in the orientation shown in Figure 6 on page 48.
• 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.
• Maintain a clean work area.
• Do not mix reactions containing gDNA on a vortex mixer. Instead,
gently tap the tube with your finger to mix the sample.
• Avoid repeated freeze-thaw cycles of stock and diluted gDNA solutions.
Possible stopping points, where gDNA samples may be stored overnight
at 4°C, are marked in the protocol. When storing samples for >24 hours,
store the samples at –20°C, but do not subject the samples to multiple
freeze/thaw cycles.
• When preparing 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 vials used during an experiment on ice or in a cold block.
4 If reagents will be used for multiple experiments, aliquot to multiple
vials to minimize freeze/thaw cycles for each vial.
• In general, follow Biosafety Level 1 (BL1) safety rules.
Safety Notes
CA U T I O N
10
• Wear appropriate personal protective equipment (PPE) when working in the
laboratory.
SureSelectXT2 Automated Library Prep and Capture System
Before You Begin
Required Reagents
1
Required Reagents
Table 1
Required Reagents for SureSelectXT2 Automated Target Enrichment
Description
Vendor and part number
SureSelect or ClearSeq Capture Library*
Select one library from Table 2 or
Table 3
SureSelectXT2 Automation Reagent Kit*†
Agilent
HiSeq platform (HSQ), 96 Samples
HiSeq platform (HSQ), 480 Samples
p/n G9661B
p/n G9661C
MiSeq platform (MSQ), 96 Samples
MiSeq platform (MSQ), 480 Samples
p/n G9662B
p/n G9662C
Nuclease-free Water (not DEPC-treated)
Ambion Cat #AM9930
1X Low TE Buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA)
Life Technologies p/n 4389764
Agencourt AMPure XP Kit
60 mL
450 mL
Beckman Coulter Genomics
p/n A63881
p/n A63882
Dynabeads MyOne Streptavidin T1
2 mL
10 mL
100 mL
Life Technologies
Cat #65601
Cat #65602
Cat #65603
Quant-iT dsDNA BR Assay Kit, for use with the Qubit
fluorometer
100 assays, 2-1000 ng
500 assays, 2-1000 ng
100% Ethanol, molecular biology grade
Life Technologies p/n Q32850
Life Technologies p/n Q32853
Sigma-Aldrich p/n E7023
* SureSelect reagents and Capture Libraries must be used within one year of receipt.
† HiSeq and MiSeq Reagent Kits are also compatible with the NextSeq 500 platform.
SureSelectXT2 Automated Library Prep and Capture System
11
1
Before You Begin
Required Reagents
Table 2
SureSelectXT2 Automation Capture Libraries
Capture Library
96 Reactions
480 Reactions
SureSelect
XT2 Human All Exon v6*
5190-8874
5 × 5190-8874
SureSelect
XT2
Human All Exon v6 + UTRs*
5190-9306
5 × 5190-9306
SureSelect
XT2
Human All Exon v6 + COSMIC*
5190-9312
5 × 5190-9312
SureSelect
XT2
Human All Exon v6 Plus 1*
5190-8877
5 × 5190-8877
SureSelect
XT2
Human All Exon v6 Plus 2*
5190-8880
5 × 5190-8880
SureSelect
XT2
Clinical Research Exome*
5190-7347
5 × 5190-7347
SureSelect
XT2
Focused Exome*
5190-7799
5 × 5190-7799
SureSelect
XT2
Focused Exome Plus 1*
5190-7807
5 × 5190-7807
SureSelect
XT2 Focused Exome Plus 2*
5190-7810
5 × 5190-7810
SureSelect
XT2 Human All Exon v5*
5190-6218
5 × 5190-6218
SureSelect
XT2 Human All Exon v5 + UTRs*
5190-6223
5 × 5190-6223
SureSelect
XT2 Human All Exon v5 + lncRNA*
5190-6454
5 × 5190-6454
SureSelect
XT2 Human All Exon v5 Plus*
5190-6225
5 × 5190-6225
SureSelect
XT2 Human All Exon v4 *
5190-4668
5190-4670
SureSelect
XT2 Human All Exon v4+ UTRs*
5190-4673
5190-4675
SureSelect
XT2 Mouse All Exon*
5190-4683
5190-4685
SureSelect
XT2 Custom 1 kb up to 499 kb†
5190-4848
5190-4850
(5190-4853)
(5190-4855)
5190-4858
5190-4860
(5190-4863)
(5190-4865)
5190-4868
5190-4870
(5190-4873)
(5190-4875)
5190-4878
5190-4880
(5190-4883)
(5190-4885)
5190-4888
5190-4890
(5190-4893)
(5190-4895)
(reorder)
SureSelect
XT2 Custom 0.5 Mb up to 2.9 Mb†
(reorder)
SureSelect
XT2 Custom 3 Mb up to 5.9 Mb†
(reorder)
SureSelect
XT2 Custom 6 Mb up to 11.9 Mb†
(reorder)
SureSelect
(reorder)
XT2 Custom 12 Mb up to 24 Mb†
* Eight gDNA samples are enriched in one capture reaction after sample pooling. Capture Libraries
are provided for the number of capture reactions needed to enrich the indicated number of samples.
† Sixteen gDNA samples are enriched in one capture reaction after sample pooling. Capture Libraries
are provided for the number of capture reactions needed to enrich the indicated number of samples.
12
SureSelectXT2 Automated Library Prep and Capture System
Before You Begin
Required Reagents
Table 3
1
Compatible ClearSeq Automation Capture Libraries
Capture Library
ClearSeq Comprehensive Cancer XT2
*
ClearSeq Comprehensive Cancer Plus XT2*
†
ClearSeq Inherited Disease XT2
ClearSeq Inherited Disease Plus XT2
†
ClearSeq DNA Kinome XT2*
96 Reactions
480 Reactions
5190-8019
5 × 5190-8019
5190-8022
5 × 5190-8022
5190-7526
5 × 5190-7526
5190-7529
5 × 5190-7529
5190-4678
5190-4680
* Sixteen gDNA samples are enriched in one capture reaction after sample pooling. Capture Libraries
are provided for the number of capture reactions needed to enrich the indicated number of samples.
† Eight gDNA samples are enriched in one capture reaction after sample pooling. Capture Libraries
are provided for the number of capture reactions needed to enrich the indicated number of samples.
SureSelectXT2 Automated Library Prep and Capture System
13
1
Before You Begin
Required Equipment
Required Equipment
Table 4
Required Equipment for SureSelectXT2 Automated Target Enrichment
Description
Vendor and part number
Agilent NGS Workstation Option B, with VWorks
software version 11.3.0.1195
Contact Agilent Automation Solutions for
ordering information:
[email protected]
Robotic Pipetting Tips (Sterile, Filtered, 250 L)
Agilent p/n 19477-022
Eppendorf twin.tec full-skirted 96-well PCR plates
Eppendorf p/n 951020401 or 951020619
Thermo Scientific Reservoirs
Thermo Scientific p/n 1064156
Nunc DeepWell Plates, sterile, 1.3-mL well volume
Thermo Scientific p/n 260251
Axygen 96 Deep Well Plate, 2.2 mL, Square Well
(waste reservoirs)
Axygen p/n P-2ML-SQ-C
E & K Scientific p/n EK-2440
Thermal cycler and accessories
SureCycler 8800 Thermal Cycler (Agilent p/n
G8810A), 96 well plate module (Agilent p/n
G8810A) and compression mats (Agilent p/n
410187) or equivalent
PCR plates compatible with selected Thermal
Cycler, e.g. Agilent semi-skirted PCR plate for the
SureCycler 8800 Thermal Cycler
Agilent p/n 401334
See page 36 for a list of supported PCR plates for
automation protocols
Covaris Sample Preparation System, E-series or
S-series
Covaris
Covaris sample holders
14
96 microTUBE plate (E-series only)
Covaris p/n 520078
microTUBE for individual sample processing
Covaris p/n 520045
DNA LoBind Tubes, 1.5-mL PCR clean, 250 pieces
Eppendorf p/n 022431021 or equivalent
Centrifuge
Eppendorf Centrifuge model 5804 or
equivalent
Qubit Fluorometer
Life Technologies p/n Q32857
Qubit assay tubes
Life Technologies p/n Q32856
SureSelectXT2 Automated Library Prep and Capture System
Before You Begin
Required Equipment
Table 4
1
Required Equipment for SureSelectXT2 Automated Target Enrichment
Description
Vendor and part number
P10, P20, P200 and P1000 pipettes
Pipetman P10, P20, P200, P1000 or
equivalent
Magnetic separator
DynaMag-50 magnet, Life Technologies p/n
123-02D or equivalent
DNA Analysis Platform and Consumables
2100 Bioanalyzer Laptop Bundle
Agilent p/n G2943CA
2100 Bioanalyzer Electrophoresis Set
Agilent p/n G2947CA
DNA 1000 Kit
Agilent p/n 5067-1504
High Sensitivity DNA Kit
Agilent p/n 5067-4626
2200 TapeStation
Agilent p/n G2964AA or G2965AA
D1000 ScreenTape
Agilent p/n 5067-5582
D1000 Reagents
Agilent p/n 5067-5583
High Sensitivity D1000 ScreenTape
Agilent p/n 5067-5584
D1000 Reagents
Agilent p/n 5067-5585
OR
NucleoClean Decontamination Wipes
Millipore p/n 3097
Vacuum concentrator
Savant SpeedVac, model DNA120, with
96-well plate rotor, model RD2MP, or
equivalent
Ice bucket
Powder-free gloves
Sterile, nuclease-free aerosol barrier pipette tips
Vortex mixer
SureSelectXT2 Automated Library Prep and Capture System
15
1
Before You Begin
Optional Reagents
Optional Reagents
Table 5
Reagents for Optional Quantitation Methods
Description
Vendor and part number
QPCR NGS Library Quantification Kit (Illumina)
Agilent p/n G4880A
Optional Equipment
Table 6
16
Equipment for Optional Quantitation Methods
Description
Vendor and part number
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
SureSelectXT2 Automated Library Prep and Capture System
SureSelectXT2 Automated Library Prep and Capture System Protocol
2
Using the Agilent NGS Workstation for
SureSelect Target Enrichment
About the Agilent NGS Workstation 18
Overview of the Workflow 28
Experimental Setup Considerations for Automated Runs 31
This chapter contains an orientation to the Agilent NGS Workstation, an
overview of the SureSelectXT2 target enrichment protocol, and
considerations for designing SureSelect experiments for automated
processing using the Agilent NGS Workstation.
Agilent Technologies
17
2
Using the Agilent NGS Workstation for SureSelect Target Enrichment
About the Agilent NGS Workstation
About the Agilent NGS Workstation
About the Bravo Platform
The Bravo platform is a versatile liquid handler with a nine plate-location
platform deck, suitable for handling 96-well, 384-well, and 1536-well
plates. The Bravo platform is controlled by the VWorks Automation
Control software. Fitted with a choice of seven interchangeable fixed-tip
or disposable-tip pipette heads, it accurately dispenses fluids from 0.1 µL
to 250 µL.
CA U T I O N
Before you begin, make sure that you have read and understand operating,
maintenance and safety instructions for using your Bravo platform. Refer to the Bravo
Platform User Guide (G5409-90006) and the VWorks Software User Guide
(G5415-90063).
Bravo Platform Deck
The protocols in the following sections include instructions for placing
plates and reagent reservoirs on specific Bravo deck locations. Use
Figure 1 to familiarize yourself with the location numbering convention on
the Bravo platform deck.
Figure 1
18
Bravo platform deck
SureSelectXT2 Automated Library Prep and Capture System
Using the Agilent NGS Workstation for SureSelect Target Enrichment
About the Bravo Platform
2
Setting the Temperature of Bravo Deck Heat Blocks
Bravo deck positions 4 and 6 are equipped with Inheco heat blocks, used
to incubate sample plates at defined temperatures during the run. Runs
that include high- (85°C) or low- (4°C) temperature incubation steps may
be expedited by pre-setting the temperature of the affected block before
starting the run.
Bravo deck heat block temperatures may be changed using the Inheco
Multi TEC Control device touchscreen as described in the steps below. See
Table 7 for designations of the heat block-containing Bravo deck positions
on the Multi TEC control device.
Table 7
Inheco Multi TEC Control touchscreen designations
Bravo Deck Position
Designation on Inheco Multi TEC Control Screen
4
CPAC 2 1
6
CPAC 2 2
1 Using the arrow buttons, select the appropriate block (CPAC 2 block 1
or CPAC 2 block 2).
SureSelectXT2 Automated Library Prep and Capture System
19
2
Using the Agilent NGS Workstation for SureSelect Target Enrichment
About the Bravo Platform
2 To set the temperature of the selected block, press the SET button.
3 Using the numeral pad, enter the desired temperature. The entered
temperature appears in the top, left rectangle. Once the correct
temperature is displayed, press the rectangle to enter the temperature.
4 Press the Temp button until the new temperature is displayed on the
SET button and until the Temp button is darkened, indicating that the
selected heat block is heating or cooling to the new temperature setting.
The current temperature of the block is indicated in the center of the
display.
20
SureSelectXT2 Automated Library Prep and Capture System
Using the Agilent NGS Workstation for SureSelect Target Enrichment
About the Bravo Platform
2
Setting the Temperature of Bravo Deck Position 9 Using the ThermoCube Device
Bravo deck position 9 is equipped with a ThermoCube thermoelectric
temperature control system, used to incubate components at a defined
temperature during the run. During protocols that require temperature
control at position 9, you will be instructed to start and set the
temperature of the ThermoCube device before starting the run.
ThermoCube temperature settings are modified using the control panel
(LCD display screen and four input buttons) on the front panel of the
device using the following steps.
1 Turn on the ThermoCube and wait for the LCD screen to display
TEMP.
2 Press the UP or DOWN button to change SET TEMP 1 to the required
set point.
3 Press the START button.
The ThermoCube will then initates temperature control of Bravo deck
position 9 at the displayed set point.
SureSelectXT2 Automated Library Prep and Capture System
21
2
Using the Agilent NGS Workstation for SureSelect Target Enrichment
VWorks Automation Control Software
VWorks Automation Control Software
VWorks software, included with your Agilent NGS Workstation, allows you
to control the robot and integrated devices using a PC. The Agilent NGS
Workstation is preloaded with VWorks software containing all of the
necessary SureSelect system liquid handling protocols. General
instructions for starting up the VWorks software and the included
protocols is provided below. Each time a specific VWorks protocol is used
in the SureSelect procedure, any settings required for that protocol are
included in the relevant section of this manual.
NOTE
The instructions in this manual are compatible with VWorks software version 11.3.0.1195.
If you have questions about VWorks version compatibility, please contact
[email protected]
Logging in to the VWorks software
1 Double-click the XT2_ILM.VWForm shortcut on the Windows desktop to
start the VWorks software.
2 If User Authentication dialog is not visible, click Log in on the VWorks
window toolbar.
3 In the User Authentication dialog, type your VWorks user name and
password, and click OK. (If no user account is set up, contact the
administrator.)
VWorks protocol and runset files
VWorks software uses two file types for automation runs, .pro (protocol)
files and .rst (runset) files. Runset files are used for automated procedures
in which the workstation uses more than one automation protocol during
the run.
22
SureSelectXT2 Automated Library Prep and Capture System
Using the Agilent NGS Workstation for SureSelect Target Enrichment
VWorks Automation Control Software
2
Using the XT2_ILM.VWForm.VWForm to setup and start a run
Use the VWorks form XT2_ILM.VWForm.VWForm, shown below, to set up
and start each SureSelect automation protocol or runset.
1 Open the form using the XT2_ILM.VWForm.VWForm shortcut on your
desktop.
2 Use the drop-down menus on the form to select the appropriate
SureSelectXT2 workflow step, PCR plate labware description, and
number of columns of samples for the run.
3 Once all run parameters have been specified on the form, click Display
Initial Workstation Setup.
SureSelectXT2 Automated Library Prep and Capture System
23
2
Using the Agilent NGS Workstation for SureSelect Target Enrichment
VWorks Automation Control Software
4 The Workstation Setup region of the form will then display the required
placement of reaction components and labware in the NGS Workstation
for the specified run parameters.
5 After verifying that the NGS Workstation has been set up correctly,
click Run Selected Protocol.
24
SureSelectXT2 Automated Library Prep and Capture System
Using the Agilent NGS Workstation for SureSelect Target Enrichment
VWorks Automation Control Software
2
Error messages encountered at start of run
After starting the run, you may see the error messages displayed below.
When encountered, make the indicated selections and proceed with the
run. Encountering either or both of these error messages is not indicative
of a problem with the NGS workstation or your run setup.
1 If you encounter the G-axis error message shown below, select Ignore
and Continue, leaving device in current state.
SureSelectXT2 Automated Library Prep and Capture System
25
2
Using the Agilent NGS Workstation for SureSelect Target Enrichment
VWorks Automation Control Software
2 If you encounter the W-axis error message shown below, select Retry.
26
SureSelectXT2 Automated Library Prep and Capture System
Using the Agilent NGS Workstation for SureSelect Target Enrichment
VWorks Automation Control Software
2
Verifying the Simulation setting
VWorks software may be run in simulation mode, during which commands
entered on screen are not completed by the NGS workstation. If
workstation devices do not respond when you start a run, verify the
simulation mode status in VWorks using the following steps.
1 Verify that Simulation is off is displayed on the status indicator
(accessible by clicking View > Control Toolbar).
2 If the indicator displays Simulation is on, click the status indicator
button to turn off the simulation mode.
NOTE
If you cannot see the toolbar above the VWorks form, click Full Screen on/off to exit full
screen mode. If the toolbar is still not visible, right-click on the form and then select Control
Toolbar from the menu.
Finishing a protocol or runset
The window below appears when each run is complete. Click Yes to
release the BenchCel racks to allow removal of components used in the
current run in preparation for the next .pro or .rst run.
SureSelectXT2 Automated Library Prep and Capture System
27
2
Using the Agilent NGS Workstation for SureSelect Target Enrichment
Overview of the Workflow
Overview of the Workflow
Figure 2 summarizes the SureSelectXT2 pre-capture indexing and target
enrichment workflow. For each sample to be sequenced, an individual
library indexing reaction is performed. Indexed libraries are then pooled
for hybridization and capture steps, using a pooling strategy appropriate
for the size of the Capture Library and the sequencing design.
Table 8 summarizes how the VWorks protocols are integrated into the
SureSelectXT2 workflow. See the Sample Preparation, Hybridization, and
Post-Capture Sample Processing for Multiplexed Sequencing chapters for
complete instructions for use of the VWorks protocols for sample
processing.
For greater flexibility, two versions of the Hybridization automation
protocol are available (see Table 8). The Hybridization_MMCol_v2.0.pro
protocol is used optimally when processing full plates of hybridization
samples and may be set up using different Capture Libraries in each row,
allowing enrichment with up to 8 different libraries in a run. The
Hybridization_MMRow_v2.0.pro protocol is designed for optimal reagent
usage when processing plates containing 6 columns of samples and may
be set up using different Capture Libraries in each column, allowing
enrichment with up to 12 different libraries in a run.
28
SureSelectXT2 Automated Library Prep and Capture System
Using the Agilent NGS Workstation for SureSelect Target Enrichment
Overview of the Workflow
Figure 2
2
Overall sequencing sample preparation workflow.
SureSelectXT2 Automated Library Prep and Capture System
29
2
Using the Agilent NGS Workstation for SureSelect Target Enrichment
Overview of the Workflow
Table 8
Overview of VWorks protocols and runsets used during the workflow
Workflow Step
(Protocol Chapter)
Sample Preparation
Hybridization
Sample Processing for
Multiplexed Sequencing
30
Substep
VWorks Protocols Used for Agilent NGS Workstation
automation
Prepare indexing adaptor-ligated
DNA
LibraryPrep_XT_Illumina_v2.0.rst
Amplify indexed DNA
Pre-CapturePCR_XT_Illumina_v2.0.pro
Purify indexed DNA amplicons
using AMPure XP beads
SPRI_XT_Illumina_v2.0.pro:Pre-Capture PCR Cleanup
Prepare indexed DNA pools for
hybridization
PreCapture_Pooling_v1.0.pro (initiated using
XT2_Pooling.VWForm)
Hybridize pooled indexed DNA to
Capture Library
Hybridization_MMCol_v2.0.pro OR
Capture and wash DNA hybrids
SureSelectCapture&Wash_v2.0.rst
PCR amplify captured DNA
Post-CaptureOnBeadPCR_XT_Illumina_v2.0.pro
Purify captured DNA amplicons
using AMPure XP beads
SPRI_XT_Illumina_v2.0.pro:Post-CaptureOnBeadPCR
Cleanup
Hybridization_MMRow_v2.0.pro
SureSelectXT2 Automated Library Prep and Capture System
Using the Agilent NGS Workstation for SureSelect Target Enrichment
Experimental Setup Considerations for Automated Runs
2
Experimental Setup Considerations for Automated Runs
SureSelectXT2 automated Library Prep runs may include 1, 2, 3, 4, 6, or 12
columns (equivalent to 8, 16, 24, 32, 48, or 96 wells) of gDNA samples.
Plan your experiments using complete columns of samples.
The number of columns or samples that may be processed using the
supplied reagents (see Table 1) will depend on the experimental design.
For greatest efficiency of reagent use, plan experiments using at least
3 columns per run. Each 96-reaction kit contains sufficient reagents for
96 Library Prep reactions configured as 4 runs of 3 columns of samples
per run.
Prior to hybridization, indexed library samples are pooled in sets of
8 samples or in sets of 16 samples, based on the type of Capture Library
to be used for hybridization(see Table 27 on page 65). Thus one Library
Prep run corresponds to 0.5 to 12 hybridization wells, depending on the
number of columns processed (see Table 9). Hybridization runs are set up
using 1, 2, 3, 4, 6, or 12 columns (equivalent to 8, 16, 24, 32, 48, or 96
wells). Accordingly, it is typically beneficial to consolidate the indexed
library pools from multiple Library Prep runs to prepare full columns of
samples for Hybridization runs and downstream workflow steps.
Table 9
Hybridization reaction numbers derived from each Library Prep run size
Number of Columns
Processed in Library
Prep Protocol
Total Libraries
Prepared
1
Number of Hyb Reactions
Pools containing
8 indexed libraries
Pools containing
16 indexed libraries
8
1
0.5
2
16
2
1
3
24
3
1.5
4
32
4
2
6
48
6
3
12
96
12
6
SureSelectXT2 Automated Library Prep and Capture System
31
2
Using the Agilent NGS Workstation for SureSelect Target Enrichment
Experimental Setup Considerations for Automated Runs
Optimal reagent usage is obtained using Hybridization runs that include 3,
6, or 12 columns. Hybridization runs of this size result from processing
indexed library samples from multiple 96-well plates in the same
Hybridization run. To determine the number of Library Prep reaction
plates required for various Hybridization run sizes, see Table 10 for All
Exon captures and see Table 11 for all other captures. Sample numbers
required for optimal 3, 6, and 12 column runs are highlighted in gray.
For greatest efficiency of reagent use, plan experiments using at least
3 columns per run. Each 2x96-reaction kit contains sufficient reagents for
hybridization reactions configured as 1 run of 3 columns of samples per
run.
Table 10
Sample number conversion using Exome libraries (8-sample pools)
Number of 96-Well
Plates Processed
though Library Prep
Total Indexed
Libraries Prepared
Number of
Hybridization
Reactions
Columns of Samples
in Hybridization
Protocol
1
96
12
1.5*
2
192
24
3
3
288
36
4.5*
4
384
48
6
5
480
60
7.5*
6
576
72
9†
7
672
84
10.5*
8
768
96
12
* Not a valid run size. Hybridization runs should include 1, 2, 3, 4, 6, or 12 complete columns of
samples.
† When planning a run using 6 plates of gDNA samples to generate 9 columns of Hybridization
samples, split the Hybridization samples into one 6-column plate and one 3-column plate.
32
SureSelectXT2 Automated Library Prep and Capture System
Using the Agilent NGS Workstation for SureSelect Target Enrichment
Experimental Setup Considerations for Automated Runs
Table 11
2
Sample number conversion using 16-sample pools
Number of 96-Well
Plates Processed
though Library Prep
Total Indexed
Libraries Prepared
Number of
Hybridization
Reactions
Columns of Samples in
Hybridization Protocol
1
96
6
0.75*
2
192
12
1.5*
3
288
18
2.25*
4
384
24
3
5
480
30
3.75*
6
576
36
4.5*
7
672
42
5.25*
8
768
48
6
9
864
54
6.75*
10
960
60
7.5*
11
1056
66
8.25*
12
1152
72
9†
13
1248
78
9.75*
14
1344
84
10.5*
15
1440
90
11.25*
16
1536
96
12
* Not a valid run size. Hybridization runs should include 1, 2, 3, 4, 6, or 12 complete columns of samples.
† When planning a run using 12 plates of gDNA samples to generate 9 columns of Hybridization
samples, split the Hybridization samples into one 6-column plate and one 3-column plate.
SureSelectXT2 Automated Library Prep and Capture System
33
2
Using the Agilent NGS Workstation for SureSelect Target Enrichment
Considerations for Placement of gDNA Samples in 96-well Plates for Automated Processing
Considerations for Placement of gDNA Samples in 96-well
Plates for Automated Processing
• The Agilent NGS Workstation processes samples column-wise beginning
at column 1. gDNA samples should be loaded into 96-well plates
column-wise, in well order A1 to H1, then A2 to H2, ending with A12
to H12. When processing partial runs with <12 sample columns, do not
leave empty columns between sample columns; always load the plate
using the left-most column that is available.
• Samples are indexed during the LibraryPrep_XT_Illumina_v2.0.rst
runset using indexing adaptors supplied in the corresponding well on a
separate plate. Assign the gDNA sample wells to be indexed with their
respective indexing primers during experimental design.
Considerations for Indexed DNA Sample Placement for
Automated Hybridization and Post-Hybridization Processing
Indexed DNA samples are pooled before the hybridization step (see
Figure 2) and captured DNA samples may be pooled again when preparing
samples for sequencing. It is important to develop a pooling strategy that
is compatible with the specific Capture Library sizes and sequencing goals
of the experiment using the following considerations:
• At the hybridization step (see Figure 2), you can add a different
SureSelect or ClearSeq Capture Library to different rows or columns of
the plate. See page 28 for guidelines on selecting the appropriate
hybridization run configuration. Plan your experiment such that each
indexed DNA library is placed in a pool in the row or column of the
sample plate that corresponds to the appropriate Capture Library for
hybridization.
• For post-capture amplification (see Figure 2), different Capture
Libraries can require different amplification cycle numbers, based on
sizes of the captured targets. It is most efficient to process similar-sized
Capture Libraries on the same plate. See Table 57 on page 112 to
determine which Capture Libraries may be amplified on the same plate.
34
SureSelectXT2 Automated Library Prep and Capture System
Using the Agilent NGS Workstation for SureSelect Target Enrichment
Considerations for Equipment Setup
2
• After the SureSelectXT2 capture process, DNA samples enriched using
small Capture Libraries are typically pooled a second time before
sequencing. See page 121 for post-capture secondary sample pooling
guidelines. When using such a secondary pooling strategy, develop a
pre-capture indexed library pooling strategy that is compatible with
post-capture pooling and sequencing designs.
Considerations for Equipment Setup
• Some workflow steps require the rapid transfer of sample plates
between the Bravo deck and a thermal cycler. Locate your thermal
cycler in close proximity to the Agilent NGS Workstation to allow rapid
and efficient plate transfer.
• Several workflow steps require that the sample plate be sealed, then
centrifuged to collect any dispersed liquid, before being transfered
between instruments. To maximize efficiency, locate the PlateLoc
thermal microplate sealer and the centrifuge in close proximity to the
Agilent NGS Workstation and thermal cycler.
SureSelectXT2 Automated Library Prep and Capture System
35
2
Using the Agilent NGS Workstation for SureSelect Target Enrichment
PCR Plate Type Considerations
PCR Plate Type Considerations
Automation protocols include several liquid-handling steps in which
reagents are dispensed to PCR plates in preparation for transfer to a
thermal cycler. For these steps you must specify the PCR plate type to be
used on the SureSelect_RNA_ILM.VWForm to allow correct configuration
of the liquid handling components for the PCR plate type. Before you
begin the automation protocol, make sure that you are using a supported
PCR plate type. The PCR plate type to be used in the protocol is specified
using the menu below. Vendor and part number information is provided
for the supported plate types in Table 12.
Table 12
36
Ordering information for supported PCR plates
Description in VWorks menu
Vendor and part number
96 ABI PCR half-skirted plates (MicroAmp Optical
plates)
Life Technologies p/n N8010560
96 Agilent semi-skirted PCR plate
Agilent p/n 401334
96 Eppendorf Twin.tec half-skirted PCR plates
Eppendorf p/n 951020303
96 Eppendorf Twin.tec PCR plates (full-skirted)
Eppendorf p/n 951020401 or 951020619
SureSelectXT2 Automated Library Prep and Capture System
SureSelectXT2 Automated Library Prep and Capture System Protocol
3
Sample Preparation
Step 1. Shear DNA 38
Step 2. Assess sample quality and DNA fragment size 41
Step 3. Prepare indexed gDNA library samples 43
Step 4. Amplify the indexed libraries 51
Step 5. Purify amplified DNA using AMPure XP beads 58
Step 6. Assess Library DNA quantity and quality 61
This section contains instructions for indexed gDNA library preparation
specific to the Illumina multiplexed, paired end sequencing platform and
to automated processing using the Agilent NGS Workstation. For each
sample to be sequenced, an individual indexed library is prepared. See the
Reference chapter, starting on page 127 for sequences of the index portion
of the indexing adaptors ligated to gDNA libraries in this section.
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 (SPRI 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.
Agilent Technologies
37
3
Sample Preparation
Step 1. Shear DNA
Step 1. Shear DNA
Before you begin, you can use the SureSelect gDNA Extraction Kit to
extract genomic DNA. Refer to the gDNA Extraction Kit Protocol
(p/n 5012-8701).
NOTE
Make sure genomic DNA samples are of high quality with an OD 260/280 ratio ranging
from 1.8 to 2.0.
For each DNA sample to be sequenced, prepare 1 library.
1 Use the Qubit dsDNA BR Assay to determine the concentration of your
gDNA sample.
Follow the instructions for the instrument.
2 Dilute 1 µg of high-quality gDNA with 1X Low TE Buffer in a 1.5-mL
LoBind tube to a total volume of 50 µL.
3 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
manufacturer’s 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 2 hours before use, or according to the
manufacturer’s recommendations.
d Set the chiller temperature to between 2°C to 5°C to ensure that the
temperature reading in the water bath displays 5°C.
e Optional. Supplement the circulated water chiller with ethylene
glycol to 20% volume to prevent freezing.
Refer to the Covaris instrument user guide for more details.
4 Put a Covaris microTube into the loading and unloading station.
Keep the cap on the tube.
NOTE
38
You can use the 96 microTube plate (see Table 4 on page 14) for the DNA shearing step
when preparing multiple gDNA samples in the same experiment.
SureSelectXT2 Automated Library Prep and Capture System
Sample Preparation
Step 1. Shear DNA
3
5 Use a tapered pipette tip to slowly transfer the 50 µL DNA sample
through the pre-split septa.
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 13 or Table 14, depending on the Covaris instrument
SonoLab software version used.
The target DNA fragment size is 150 to 200 bp.
Table 13
Shear settings for Covaris instruments using SonoLab software version 7 or
newer
Setting
Value
Duty Factor
10%
Peak Incident Power (PIP)
175
Cycles per Burst
200
Treatment Time
360 seconds
Bath Temperature
4° to 8° C
Table 14
Shear settings for Covaris instruments using SonoLab software prior to
version 7
Setting
Value
Duty Cycle
10%
Intensity
5
Cycles per Burst
200
Time
6 cycles of 60 seconds each
Set Mode
Frequency sweeping
Temperature
4° to 7° C
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 septa, then slowly remove the sheared DNA.
SureSelectXT2 Automated Library Prep and Capture System
39
3
Sample Preparation
Step 1. Shear DNA
9 Transfer 50 µL of each sheared DNA sample to a separate well of a
96-well Eppendorf twin.tec plate, column-wise for processing on the
Agilent NGS Workstation, in well order A1 to H1, then A2 to H2, ending
with A12 to H12.
NOTE
SureSelect Automated Library Prep and Capture System runs may include
1, 2, 3, 4, 6, or 12 columns of the plate. See Using the Agilent NGS
Workstation for SureSelect Target Enrichment for additional sample
placement considerations.
10 Seal the plate using the PlateLoc Thermal Microplate Sealer, with
sealing settings of 165°C and 1.0 sec.
11 Centrifuge the plate for 30 seconds to drive the well contents off the
walls and plate seal and to remove air bubbles.
Stopping Point
40
If you do not continue to the next step, store the sample plate at 4°C
overnight or at –20°C for prolonged storage.
SureSelectXT2 Automated Library Prep and Capture System
Sample Preparation
Step 2. Assess sample quality and DNA fragment size
3
Step 2. Assess sample quality and DNA fragment size
Option 1: Analysis using the Agilent 2100 Bioanalyzer and DNA 1000 Assay
Use a Bioanalyzer DNA 1000 chip and reagent kit. For more information
to do this step, see the Agilent DNA 1000 Kit Guide.
1 Set up the 2100 Bioanalyzer as instructed in the reagent kit guide.
2 Seal the sample plate using the PlateLoc Thermal Microplate Sealer,
with sealing settings of 165°C and 1.0 sec.
3 Vortex the plate to mix samples in each well, then centrifuge the plate
for 30 seconds to drive the well contents off the walls and plate seal.
4 Prepare the chip, samples and ladder as instructed in the reagent kit
guide, using 1 µL of each sample for the analysis.
5 Load the prepared chip into the 2100 Bioanalyzer and start the run
within five minutes after preparation.
6 Verify that the electropherogram shows an average DNA fragment size
of 150 to 200 bp. A sample electropherogram is shown in Figure 3.
Stopping Point
If you do not continue to the next step, seal the plate and store at 4°C
overnight or at –20°C for prolonged storage.
Figure 3
Analysis of sheared DNA using a DNA 1000 Bioanalyzer assay.
SureSelectXT2 Automated Library Prep and Capture System
41
3
Sample Preparation
Step 2. Assess sample quality and DNA fragment size
Option 2: Analysis using the Agilent 2200 TapeStation and D1000 ScreenTape
You can use Agilent’s 2200 TapeStation for rapid analysis of multiple
samples. Use a D1000 ScreenTape (p/n 5067-5582) and associated reagent
kit (p/n 5067-5583) to analyze the sheared DNA. For more information to
do this step, see the Agilent 2200 TapeStation User Manual.
1 Seal the sheared DNA sample plate using the PlateLoc Thermal
Microplate Sealer, with sealing settings of 165°C and 1.0 sec.
2 Vortex the plate to mix samples in each well, then centrifuge the plate
for 30 seconds to drive the well contents off the walls and plate seal.
3 Prepare the TapeStation samples as instructed in the Agilent 2200
TapeStation User Manual. Use 1 µL of each sheared DNA sample
diluted with 3 µL of D1000 sample buffer for the analysis.
CA U T I O N
Make sure that you thoroughly mix the combined DNA and D1000 sample buffer on a
vortex mixer for 5 seconds for accurate quantitation.
4 Load the sample plate or tube strips from step 3, the D1000
ScreenTape, and loading tips into the 2200 TapeStation as instructed in
the Agilent 2200 TapeStation User Manual. Start the run.
5 Verify that the electropherogram shows an average DNA fragment size
of 150 to 200 bp. A sample electropherogram is shown in Figure 4.
Stopping Point
If you do not continue to the next step, seal the sheared DNA sample
plate and store at 4°C overnight or at –20°C for prolonged storage.
Figure 4
42
Analysis of sheared DNA using the 2200 TapeStation.
SureSelectXT2 Automated Library Prep and Capture System
Sample Preparation
Step 3. Prepare indexed gDNA library samples
3
Step 3. Prepare indexed gDNA library samples
In this step, the Agilent NGS Workstation completes the DNA end
modification steps required for SureSelectXT2 pre-capture indexing,
including end-repair, A-tailing, and indexing adaptor ligation. Where
required, the Agilent NGS Workstation purifies the prepared DNA using
AMPure XP beads as part of the Library Prep runset.
Prepare the workstation
1 Turn on the chiller, set to 0°C, at position 9 of the Bravo deck. Be sure
that the chiller reservoir contains at least 300 mL of 25% ethanol.
2 Pre-set the temperature of Bravo deck position 6 to 4°C using the
Inheco Multi TEC control touchscreen, as described in Setting the
Temperature of Bravo Deck Heat Blocks. Bravo deck position 6
corresponds to CPAC 2, position 2 on the Multi TEC control
touchscreen.
3 Clear the Labware MiniHub and BenchCel of all plates and tip boxes.
SureSelectXT2 Automated Library Prep and Capture System
43
3
Sample Preparation
Step 3. Prepare indexed gDNA library samples
Prepare the Library Prep master mix source plate
4 In a Nunc DeepWell plate, prepare the master mix source plate by
adding the volumes indicated in Table 15 of each reagent to all wells of
the indicated column of the plate.
As indicated in the shaded portions of Table 15, Column 1 and Column
3 are prepared to contain mixtures of two reagents. Keep the reagents
and source plate on ice during the aliquoting steps.
Table 15
Preparation of the Master Mix Source Plate for LibraryPrep_XT_Illumina_v2.0.rst
Reagent Solution
SureSelect End
Repair Enzyme
Mix
Position on
Source Plate
SureSelect
Ligation Master
Mix
Nuclease-free
water
1-Column
Runs
2-Column
Runs
3-Column
Runs
4-Column
Runs
6-Column
Runs
12-Column
Runs
60 µL*
100 µL†
140 µL‡
180 µL**
260 µL††
520 µL‡‡
15 µL*
25 µL†
35 µL‡
45 µL**
65 µL††
130 µL‡‡
30 µL
50 µL
70 µL
90 µL
130 µL
260 µL
7.5 µL
15.0 µL
20.0 µL
27.5 µL
40 µL
75 µL
3.8 µL
7.5 µL
10.0 µL
13.8 µL
20 µL
37.5 µL
Column 1
(A1-H1)
SureSelect End
Repair Nucleotide
Mix***
SureSelect
dA-Tailing Master
Mix
Volume added per Well of Nunc Deep Well Source Plate
Column 2
(A2-H2)
Column 3
(A3-H3)
* For kits that include SureSelect End Repair Master Mix, add 75 µL of the pre-combined master mix for 1-column runs.
† For kits that include SureSelect End Repair Master Mix, add 125 µL of the pre-combined master mix for 2-column runs.
‡ For kits that include SureSelect End Repair Master Mix, add 175 µL of the pre-combined master mix for 3-column runs.
** For kits that include SureSelect End Repair Master Mix, add 225 µL of the pre-combined master mix for 4-column runs.
†† For kits that include SureSelect End Repair Master Mix, add 325 µL of the pre-combined master mix for 6-column runs.
‡‡ For kits that include SureSelect End Repair Master Mix, add 650 µL of the pre-combined master mix for 12-column runs.
***May also be labeled as SureSelect End Repair Oligo Mix.
44
SureSelectXT2 Automated Library Prep and Capture System
Sample Preparation
Step 3. Prepare indexed gDNA library samples
3
5 Seal the master mix source plate using the PlateLoc Thermal Microplate
Sealer, with sealing settings of 165°C and 1.0 sec.
6 Vortex the plate for 5 seconds to ensure homogeneity of the mixtures.
7 Centrifuge the plate for 30 seconds to drive the well contents off the
walls and plate seal and to eliminate any bubbles. Keep the master mix
source plate on ice.
The final configuration of the master mix source plate is shown in
Figure 5.
NOTE
The presence of bubbles in source plate solutions may cause inaccurate volume transfer by
the Bravo liquid handling platform. Ensure that the source plate is sealed and centrifuged
prior to use in a run.
Figure 5
Configuration of the master mix source plate for
LibraryPrep_XT_Illumina_v2.0.rst
SureSelectXT2 Automated Library Prep and Capture System
45
3
Sample Preparation
Step 3. Prepare indexed gDNA library samples
Prepare the Pre-capture Indexed Adaptors source plate
8 Select the appropriate index for each sample. Nucleotide sequence
information for the index portion of each indexed adaptor is provided
in the Reference chapter, starting on page 127.
Using an Eppendorf Twin.tec plate, prepare the indexed adaptors
source plate by combining 5 µL of each SureSelect Pre-capture Indexed
Adaptor solution with 2.5 µL of nuclease-free water. Each pre-capture
index dilution is made in a separate well of the source plate,
corresponding to the well position of the sample to be indexed.
Prepare the purification reagents
9 Verify that the AMPure XP bead suspension is at room temperature.
10 Mix the bead suspension well so that the reagent appears homogeneous
and consistent in color. Do not freeze.
11 Prepare a separate Nunc DeepWell source plate for the beads by adding
250 µL of homogeneous AMPure XP beads per well, for each well to be
processed.
12 Prepare a Thermo Scientific reservoir containing 20 mL of nuclease-free
water.
13 Prepare a separate Thermo Scientific reservoir containing 100 mL of
freshly-prepared 70% ethanol.
46
SureSelectXT2 Automated Library Prep and Capture System
Sample Preparation
Step 3. Prepare indexed gDNA library samples
3
Load the Agilent NGS Workstation
14 Load the Labware MiniHub according to Table 16, using the plate
orientations shown in Figure 6.
Table 16
Initial MiniHub configuration for LibraryPrep_XT_Illumina_v2.0.rst
Vertical Shelf
Position
Cassette 1
Cassette 2
Cassette 3
Cassette 4
Shelf 5 (Top)
Empty
Empty Nunc
DeepWell plate
Empty Nunc
DeepWell plate
Indexing
Adaptors in
Eppendorf
twin.tec plate
Shelf 4
Empty
Empty Eppendorf
twin.tec plate
Empty Eppendorf
twin.tec plate
Empty
Shelf 3
Empty
Empty
Empty Eppendorf
twin.tec plate
Empty
Shelf 2
Empty tip box
Nuclease-free
water reservoir
from step 12
AMPure XP beads Empty
in Nunc DeepWell
plate from step 11
Shelf 1 (Bottom)
New tip box
70% ethanol
reservoir from
step 13
Empty
SureSelectXT2 Automated Library Prep and Capture System
Empty tip box
47
3
Sample Preparation
Step 3. Prepare indexed gDNA library samples
Figure 6
Labware MiniHub plate orientation. For Thermo Scientific reservoirs, place the
notched corner facing the center of the hub.
15 Load the BenchCel Microplate Handling Workstation according to
Table 17.
Table 17
48
Initial BenchCel configuration for LibraryPrep_XT_Illumina_v2.0.rst
No. of Columns
Processed
Rack 1
Rack 2
Rack 3
Rack 4
1
2 Tip boxes
Empty
Empty
Empty
2
3 Tip boxes
Empty
Empty
Empty
3
4 Tip boxes
Empty
Empty
Empty
4
5 Tip boxes
Empty
Empty
Empty
6
7 Tip boxes
Empty
Empty
Empty
12
11 Tip boxes
3 Tip boxes
Empty
Empty
SureSelectXT2 Automated Library Prep and Capture System
Sample Preparation
Step 3. Prepare indexed gDNA library samples
3
16 Load the Bravo deck according to Table 18.
Table 18
Initial Bravo deck configuration for LibraryPrep_XT_Illumina_v2.0.rst
Location
Content
1
Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)
6
Empty Eppendorf twin.tec plate
7
Eppendorf twin.tec plate containing sheared gDNA samples, oriented with
well A1 in the upper-left
9
Library Prep Master Mix Source Plate (unsealed) seated on silver insert
Run VWorks runset LibraryPrep_XT_Illumina_v2.0.rst
NOTE
For this runset, you are not required to select PCR Plate labware under step 2 on the setup
form.
17 On the SureSelect setup form, under Select Protocol to Run, select
LibraryPrep_XT_Illumina_v2.0.rst.
18 Select the number of columns of samples to be processed. Runs must
include 1, 2, 3, 4, 6, or 12 columns.
19 Click Display Initial Workstation Setup.
20 Verify that the NGS workstation has been set up as displayed in the
Workstation Setup region of the form.
SureSelectXT2 Automated Library Prep and Capture System
49
3
Sample Preparation
Step 3. Prepare indexed gDNA library samples
21 When verification is complete, click Run Selected Protocol.
22 When ready to begin the run, click OK in the following window.
Running the LibraryPrep_XT_Illumina_v2.0.rst runset takes approximately
3 hours. Once complete, the purified, indexing adaptor-ligated DNA
samples are located in the Eppendorf twin.tec plate at position 7 of the
Bravo deck.
Stopping Point
50
If you do not continue to the next step, seal the plate and store at 4°C
overnight or at –20°C for prolonged storage.
SureSelectXT2 Automated Library Prep and Capture System
Sample Preparation
Step 4. Amplify the indexed libraries
3
Step 4. Amplify the indexed libraries
In this step, the Agilent NGS Workstation completes the liquid handling
steps for amplification of the indexing adaptor-ligated DNA samples.
Afterward, you transfer the PCR plate to a thermal cycler for
amplification.
In this protocol, one half of the DNA sample is removed from the
Eppendorf sample plate for amplification. The remainder can be saved at
4°C for future use or amplification troubleshooting, if needed. Store the
samples at –20°C for long-term storage.
CA U T I O N
To avoid cross-contaminating libraries, set up PCR master mixes in a dedicated clean
area or PCR hood with UV sterilization and positive air flow.
Prepare the workstation
1 Leave tip boxes on shelves 1 and 2 in cassette 1 of the Labware
MiniHub from the previous LibraryPrep_XT_Illumina_v2.0.rst run.
Otherwise, clear the remaining positions of the MiniHub and BenchCel
of plates and tip boxes.
2 Turn on the chiller, set to 0°C, at position 9 of the Bravo deck. Be sure
that the chiller reservoir contains at least 300 mL of 25% ethanol.
3 Pre-set the temperature of Bravo deck position 6 to 4°C using the
Inheco Multi TEC control touchscreen, as described in Setting the
Temperature of Bravo Deck Heat Blocks. Bravo deck position 6
corresponds to CPAC 2, position 2 on the Multi TEC control
touchscreen.
SureSelectXT2 Automated Library Prep and Capture System
51
3
Sample Preparation
Step 4. Amplify the indexed libraries
Prepare the pre-capture PCR master mix and the master mix source plate
4 Prepare the Pre-capture PCR Master Mix by combining SureSelect
Herculase II Master Mix and the XT2 Primer Mix in column 4 of the
master mix source plate. Add the volumes of both reagents shown in
Table 19 to each well of column 4 of the master mix source plate.
Use the same Nunc DeepWell master mix source plate that was used for
the LibraryPrep_XT_Illumina_v2.0.rst run. The final configuration of the
master mix source plate is shown in Figure 7.
Table 19
Preparation of the Master Mix Source Plate for Pre-CapturePCR_XT_Illumina_v2.0.pro
SureSelectXT2
Reagent
Position on
Source Plate
SureSelect
Herculase II
Master Mix
Column 4
XT2 Primer Mix
NOTE
52
Volume of Reagents added per Well of Nunc Deep Well Source Plate
1-Column
Runs
2-Column
Runs
3-Column
Runs
4-Column
Runs
6-Column
Runs
12-Column
Runs
37.5 µL
62.5 µL
87.5 µL
112.5 µL
162.5 µL
325 µL
1.5 µL
2.5 µL
3.5 µL
4.5 µL
6.5 µL
13.0 µL
(A4-H4)
If you are using a new DeepWell plate for the pre-capture PCR source plate (for example,
when amplifying the second half of the indexing adaptor-ligated DNA sample), leave
columns 1 to 3 empty and add the PCR Master Mix to column 4 of the new plate.
SureSelectXT2 Automated Library Prep and Capture System
Sample Preparation
Step 4. Amplify the indexed libraries
Figure 7
3
Configuration of the master mix source plate for
Pre-CapturePCR_XT_Illumina_v2.0.pro. Columns 1-3 were used to dispense
master mixes during the previous protocol.
5 Seal the master mix source plate using the PlateLoc Thermal Microplate
Sealer, with sealing settings of 165°C and 1.0 sec.
6 Vortex the plate for 5 seconds to ensure homogeneity of the PCR
Master Mix.
7 Centrifuge the plate for 30 seconds to drive the well contents off the
walls and plate seal and to eliminate any bubbles. Keep the master mix
source plate on ice.
NOTE
The presence of bubbles in source plate solutions may cause inaccurate volume transfer by
the Bravo liquid handling platform. Ensure that the source plate is sealed and centrifuged
prior to use in a run.
SureSelectXT2 Automated Library Prep and Capture System
53
3
Sample Preparation
Step 4. Amplify the indexed libraries
Load the Agilent NGS Workstation
8 Load the Labware MiniHub according to Table 20, using the plate
orientations shown in Figure 6.
The waste tip box (Cassette 1, Shelf 2) and clean tip box (Cassette 1,
Shelf 1) are retained from the LibraryPrep_XT_Illumina_v2.0.rst run
and reused here. If you are using a new box of tips on shelf 1 of
cassette 1 (for example, when amplifying the second half of the indexed
DNA sample), first remove the tips from columns 1 to 3 of the tip box.
CA U T I O N
Any tips present in columns 1 to 3 of the clean tip box (Cassette 1, Shelf 1) may be
inappropriately loaded onto the Bravo platform pipette heads and may interfere with
automated processing steps.
Table 20
Initial MiniHub configuration for Pre-CapturePCR_XT_Illumina_v2.0.pro
Vertical
Shelf
Position
Cassette 1
Cassette 2
Cassette 3
Cassette 4
Shelf 5
(Top)
Empty
Empty
Empty
Empty
Shelf 4
Empty
Empty
Empty
Empty
Shelf 3
Empty
Empty
Empty
Empty
Shelf 2
Waste tip box*
Empty
Empty
Empty
Shelf 1
(Bottom)
Clean tip box*
Empty
Empty
Empty tip box
* Retained from the LibraryPrep_XT_Illumina_v2.0.rst run and reused here.
54
SureSelectXT2 Automated Library Prep and Capture System
Sample Preparation
Step 4. Amplify the indexed libraries
3
9 Load the BenchCel Microplate Handling Workstation according to
Table 21.
Table 21
Initial BenchCel configuration for Pre-CapturePCR_XT_Illumina_v2.0.pro
No. of Columns
Processed
Rack 1
Rack 2
Rack 3
Rack 4
1
1 Tip box
Empty
Empty
Empty
2
1 Tip box
Empty
Empty
Empty
3
1 Tip box
Empty
Empty
Empty
4
1 Tip box
Empty
Empty
Empty
6
1 Tip box
Empty
Empty
Empty
12
1 Tip box
Empty
Empty
Empty
10 Load the Bravo deck according to Table 22.
Table 22
Location
6
Initial Bravo deck configuration for Pre-CapturePCR_XT_Illumina_v2.0.pro
Content
Empty PCR plate seated on red insert
(PCR plate type must be specified on setup form under step 2)
7
Indexing adaptor-ligated DNA samples, in Eppendorf twin.tec plate
9
Master mix plate (unsealed) containing Pre-Capture PCR Master Mix in
Column 4 seated on silver insert
Run VWorks protocol Pre-CapturePCR_XT_Illumina_v2.0.pro
11 On the SureSelect setup form, under Select Protocol to Run, select
Pre-CapturePCR_XT_Illumina_v2.0.pro.
12 Under Select PCR plate labware for Thermal Cycling, select the
specific type of PCR plate that was loaded on Bravo deck position 6.
13 Select the number of columns of samples to be processed. Runs must
include 1, 2, 3, 4, 6, or 12 columns.
SureSelectXT2 Automated Library Prep and Capture System
55
3
Sample Preparation
Step 4. Amplify the indexed libraries
14 Click Display Initial Workstation Setup.
15 Verify that the NGS workstation has been set up as displayed in the
Workstation Setup region of the form.
16 When verification is complete, click Run Selected Protocol.
Running the Pre-CapturePCR_XT_Illumina_v2.0.pro protocol takes
approximately 15 minutes. Once complete, the PCR-ready samples,
containing indexed DNA and PCR master mix, are located in the PCR
plate at position 6 of the Bravo deck. The Eppendorf plate containing the
remaining prepped DNA samples, which may be stored for future use at
4°C overnight, or at –20°C for long-term storage, is located at position 7
of the Bravo deck.
17 Remove the PCR plate from position 6 of the Bravo deck and seal the
plate using the PlateLoc Thermal Microplate Sealer, with sealing settings
of 165°C and 3.0 seconds.
18 Centrifuge the plate for 30 seconds to drive the well contents off the
walls and plate seal and to eliminate air bubbles.
19 Transfer the PCR plate to a thermal cycler and run the PCR
amplification program shown in Table 23.
The volume of each PCR amplification reaction is 50 µL.
56
SureSelectXT2 Automated Library Prep and Capture System
Sample Preparation
Step 4. Amplify the indexed libraries
Table 23
NOTE
3
Pre-Capture PCR cycling program
Segment
Number of
Cycles
Temperature
Time
1
1
98°C
2 minutes
2
5
98°C
30 seconds
60°C
30 seconds
72°C
1 minute
3
1
72°C
10 minutes
4
1
4°C
Hold
Different library preparations can produce slightly different results, based on varying DNA
quality. In most cases, 5 cycles will produce an adequate yield for subsequent capture
without introducing bias or non-specific products. If yield is too low or non-specific high
molecular weight products are observed, adjust the number of cycles accordingly with the
remaining library template.
SureSelectXT2 Automated Library Prep and Capture System
57
3
Sample Preparation
Step 5. Purify amplified DNA using AMPure XP beads
Step 5. Purify amplified DNA using AMPure XP beads
In this step, the Agilent NGS Workstation transfers AMPure XP beads and
amplified adaptor-ligated DNA to a Nunc DeepWell plate and then collects
and washes the bead-bound DNA.
Prepare the workstation and reagents
1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes.
2 Verify that the AMPure XP bead suspension is at room temperature. (If
necessary, allow the bead solution to come to room temperature for at
least 30 minutes.)
3 Mix the bead suspension well so that the reagent appears homogeneous
and consistent in color. Do not freeze.
4 Prepare a Nunc DeepWell source plate for the beads by adding 65 µL of
homogeneous AMPure XP beads per well, for each well to be processed.
5 Prepare a Thermo Scientific reservoir containing 15 mL of nuclease-free
water.
6 Prepare a separate Thermo Scientific reservoir containing 45 mL of
freshly-prepared 70% ethanol.
7 Load the Labware MiniHub according to Table 24, using the plate
orientations shown in Figure 6.
Table 24
58
MiniHub configuration for SPRI_XT_Illumina_v2.0.pro:Pre-Capture PCR Cleanup
Vertical Shelf
Position
Cassette 1
Cassette 2
Cassette 3
Cassette 4
Shelf 5 (Top)
Empty Nunc
DeepWell plate
Empty
Empty
Empty
Shelf 4
Empty
Empty
Empty
Empty
Shelf 3
Empty
Empty Eppendorf
twin.tec plate
Empty
Empty
Shelf 2
Empty
Nuclease-free
water reservoir
from step 5
AMPure XP beads
in Nunc DeepWell
plate from step 4
Empty
Shelf 1 (Bottom)
Empty
70% ethanol
reservoir from
step 6
Empty
Empty tip box
SureSelectXT2 Automated Library Prep and Capture System
Sample Preparation
Step 5. Purify amplified DNA using AMPure XP beads
3
8 Load the BenchCel Microplate Handling Workstation according to
Table 25.
Table 25
BenchCel configuration for SPRI_XT_Illumina_v2.0.pro:Pre-Capture PCR Cleanup
No. of Columns
Processed
Rack 1
Rack 2
Rack 3
Rack 4
1
1 Tip box
Empty
Empty
Empty
2
1 Tip box
Empty
Empty
Empty
3
2 Tip boxes
Empty
Empty
Empty
4
2 Tip boxes
Empty
Empty
Empty
6
3 Tip boxes
Empty
Empty
Empty
12
6 Tip boxes
Empty
Empty
Empty
9 Load the Bravo deck according to Table 26.
Table 26
Bravo deck configuration for SPRI_XT_Illumina_v2.0.pro:Pre-Capture PCR Cleanup
Location
Content
1
Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)
9
Amplified DNA libraries in unsealed PCR plate seated in red insert
(PCR plate type must be specified on setup form under step 2)
Run VWorks protocol SPRI_XT_Illumina_v2.0.pro:Pre-Capture PCR Cleanup
10 On the SureSelect setup form, under Select Protocol to Run, select
SPRI_XT_Illumina_v2.0.pro:Pre-Capture PCR Cleanup.
11 Under Select PCR plate labware for Thermal Cycling, select the
specific type of PCR plate that was loaded on Bravo deck position 9.
SureSelectXT2 Automated Library Prep and Capture System
59
3
Sample Preparation
Step 5. Purify amplified DNA using AMPure XP beads
12 Select the number of columns of samples to be processed. Runs must
include 1, 2, 3, 4, 6, or 12 columns.
13 Click Display Initial Workstation Setup.
14 Verify that the NGS workstation has been set up as displayed in the
Workstation Setup region of the form.
15 When verification is complete, click Run Selected Protocol.
The purification protocol takes approximately 45 minutes. When complete,
the amplified DNA samples are in the Eppendorf plate located on Bravo
deck position 7.
60
SureSelectXT2 Automated Library Prep and Capture System
Sample Preparation
Step 6. Assess Library DNA quantity and quality
3
Step 6. Assess Library DNA quantity and quality
Option 1: Analysis using the Agilent 2100 Bioanalyzer and DNA 1000 Assay
Use a Bioanalyzer DNA 1000 chip and reagent kit. For more information
to do this step, see the Agilent DNA 1000 Kit Guide.
1 Set up the 2100 Bioanalyzer as instructed in the reagent kit guide.
2 Seal the sample plate using the PlateLoc Thermal Microplate Sealer,
with sealing settings of 165°C and 1.0 sec.
3 Vortex the plate to mix samples in each well, then centrifuge the plate
for 30 seconds to drive the well contents off the walls and plate seal.
4 Prepare the chip, samples and ladder as instructed in the reagent kit
guide, using 1 µL of each sample for the analysis.
5 Load the prepared chip into the 2100 Bioanalyzer and start the run
within five minutes after preparation.
6 Verify that the electropherogram shows an average DNA amplicon size
of 250 to 300 bp. A sample electropherogram is shown in Figure 8.
7 Determine the concentration of the library (ng/µL) by integrating under
the peak.
Stopping Point
If you do not continue to the next step, seal the plate and store at 4°C
overnight or at –20°C for prolonged storage.
Figure 8
Analysis of amplified prepped library DNA using a DNA 1000 assay.
SureSelectXT2 Automated Library Prep and Capture System
61
3
Sample Preparation
Step 6. Assess Library DNA quantity and quality
Option 2: Analysis using the Agilent 2200 TapeStation and D1000 ScreenTape
Use a D1000 ScreenTape (p/n 5067-5582) and associated reagent kit (p/n
5067-5583) to analyze the amplified prepped DNA. For more information
to do this step, see the Agilent 2200 TapeStation User Manual.
1 Seal the DNA library sample plate using the PlateLoc Thermal
Microplate Sealer, with sealing settings of 165°C and 1.0 sec.
2 Vortex the plate to mix samples in each well, then centrifuge the plate
for 30 seconds to drive the well contents off the walls and plate seal.
3 Prepare the TapeStation samples as instructed in the Agilent 2200
TapeStation User Manual. Use 1 µL of each DNA sample diluted with
3 µL of D1000 sample buffer for the analysis.
CA U T I O N
Make sure that you thoroughly mix the combined DNA and D1000 sample buffer on a
vortex mixer for 5 seconds for accurate quantitation.
4 Load the sample plate or tube strips from step 3, the D1000
ScreenTape, and loading tips into the 2200 TapeStation as instructed in
the Agilent 2200 TapeStation User Manual. Start the run.
5 Verify that the electropherogram shows an average DNA amplicon size
of 250 to 300 bp. A sample electropherogram is shown in Figure 9.
6 Determine the DNA concentration (ng/µL) by integrating under the
peak.
Stopping Point
If you do not continue to the next step, seal the sheared DNA sample
plate and store at 4°C overnight or at –20°C for prolonged storage.
Figure 9
62
Analysis of amplified DNA using the 2200 TapeStation.
SureSelectXT2 Automated Library Prep and Capture System
SureSelectXT2 Automated Library Prep and Capture System Protocol
4
Hybridization
Step 1. Pool indexed DNA samples for hybridization 64
Step 2. Hybridize the gDNA library and Capture Library 72
Step 3. Capture the hybridized DNA 97
This chapter describes the steps to pool indexed gDNA libraries and then
hybridize the pooled gDNA libraries with a SureSelect or ClearSeq Capture
Library. Pools of 8 or 16 indexed samples are hybridized to the
appropriate Capture Library and the targeted molecules are captured for
sequencing. See Table 27 for the recommended number of indexes per
gDNA library pool for different types of Capture Libraries.
The size of your SureSelect or ClearSeq Capture Library determines the
post-capture amplification cycle number. See Table 57 for cycle number
recommendations for different Capture Library sizes. Plan your
experiments for capture using similar-sized Capture Libraries on the same
plate to facilitate post-capture amplification.
CA U T I O N
The ratio of Capture Library to indexed gDNA library is critical for successful capture.
CA U T I O N
You must avoid evaporation from the small volumes of the capture during the 24 hour
or greater incubation.
If you want to use a duration of hybridization >24 hours, first test the conditions.
Incubate 60 µL of SureSelect XT2 Hybridization Buffer (without DNA) at 65°C for
24 hours (or longer, if applicable) as a test. Include buffer in each well that you might
use, including those in the center and those on the edges. Check that you do not get
extensive evaporation. Evaporation should not exceed 6 to 8 µL.
Agilent Technologies
63
4
Hybridization
Step 1. Pool indexed DNA samples for hybridization
Step 1. Pool indexed DNA samples for hybridization
In this step, the workstation pools the prepped indexed gDNA samples,
before hybridization to the SureSelect or ClearSeq Capture Library. This
workflow step is set up using the VWorks Form XT2_Pooling.VWForm
shown below.
64
SureSelectXT2 Automated Library Prep and Capture System
Hybridization
Step 1. Pool indexed DNA samples for hybridization
4
Plan pooling run parameters
The Hybridization reaction requires 1500 ng indexed gDNA, made up of a
pool containing equal amounts of 8 or 16 individual libraries. See Table 27
for the recommended pool composition based on your SureSelect or
ClearSeq Capture Library.
Where possible, indexed DNA pools are prepared containing a total DNA
amount of 1500 ng. For some indexed DNA pools, the initial library pool
will contain >1500 ng DNA, as detailed below, with 1500 ng of the pooled
DNA added to the Hybridization reaction at a later step.
Table 27
Pre-capture pooling of indexed DNA libraries
Capture Library
Number of indexed
gDNA libraries per pool
Amount of each indexed
gDNA library in pool
SureSelect Custom Capture Libraries
16
93.75 ng
ClearSeq Comprehensive Cancer
16
93.75 ng
ClearSeq DNA Kinome
16
93.75 ng
SureSelect Human or Mouse All-Exon
8
187.5 ng
SureSelect Clinical Research Exome
8
187.5 ng
SureSelect Focused Exome
8
187.5 ng
ClearSeq Inherited Disease
8
187.5 ng
Before setting up the pooling run, you must determine the total amount of
DNA to pool, and the appropriate daughter plate type, based on the
starting concentrations of the DNA samples to be pooled.
Accurate normalization of pools requires a minimum pipetting volume of
2 µL for each sample. Maximum DNA concentration values for a 1500 ng
pool containing >2 µL of each sample are shown in Table 27, above. When
higher-concentration DNA samples are included in the pooling run, the
DNA pool amount must be adjusted as described below.
1 Check the DNA concentration of each sample in the set of source plates
to be pooled to a single daughter plate to determine the appropriate
amount of DNA per pool.
a If all samples contain DNA at concentrations below the maximum
DNA concentration shown in Table 27 (<94 ng/µL for All Exon and
SureSelectXT2 Automated Library Prep and Capture System
65
4
Hybridization
Step 1. Pool indexed DNA samples for hybridization
Inherited Disease captures or at <47 ng/µL for DNA Kinome and
custom captures), then prepare 1500 ng DNA pools.
b If at least one of the samples is above the maximum DNA
concentration shown in Table 27 (>94 ng/µL for All Exon and
Inherited Disease captures or at >47 ng/µL for DNA Kinome and
custom captures), then you need to calculate the appropriate DNA
pool amount. First, identify the most concentrated DNA sample and
calculate the amount of DNA contained in 2 µL of that sample. This
becomes the amount of each DNA sample used for pooling in the
run. For example, if the highest DNA sample concentration is 100
ng/µL, then the final DNA pool will contain 200 ng of each indexed
DNA. Next, determine the total amount of DNA per pool, based on
the Capture Library size. Continuing with the same example, an All
Exon capture pool would contain 8 × 200 ng, or 1600 ng DNA.
2 Determine the appropriate daughter plate type, based on DNA pool
volumes. First, calculate the volume of each indexed DNA sample to be
pooled, using the concentration values for each sample and the amount
of each DNA sample per pool from step 1 above. Next, calculate the
expected total pool volume for each indexed DNA pool included on the
daughter plate.
a If the volume for all pools in the run is <180 µL, then use an
Eppendorf twin.tec plate as the daughter (destination) plate for the
pooling protocol. This plate will be used directly as indexed DNA
pool source plate in the Hybridization protocol.
b If the volume for any pool in the run is >180 µL, then use a Nunc
DeepWell plate as the daughter (destination) plate for the pooling
protocol. After pool volumes are standardized (see page 71) the
indexed DNA pools must be transferred to an Eppendorf twin.tec
plate for the Hybridization protocol.
Plan daughter indexed DNA pool sample plate configuration
The indexed gDNA samples should be pooled into the daughter plate using
a pooled sample configuration appropriate for the subsequent
Hybridization run. Use the following plate configuration considerations for
pooling gDNA samples for automated hybridization and capture runs:
• When using a single Capture Library for all wells on the plate, fill the
plate column-wise in well order A1 to H1, then A2 to H2, ending with
A12 to H12.
66
SureSelectXT2 Automated Library Prep and Capture System
Hybridization
Step 1. Pool indexed DNA samples for hybridization
4
• When using multiple Capture Libraries, configure the plate such that all
gDNA library pools to be hybridized to a particular Capture Library are
positioned in appropriate rows or columns. When using the
Hybridization_MMCol_v2.0.pro protocol, place samples to be enriched
using the same library in the same row. When using the
Hybridization_MMRow_v2.0.pro protocol, place samples to be enriched
using the same library in the same column.
• Each 96-reaction library preparation run produces 6 or 12 gDNA pools.
For greatest efficiency of reagent use, gDNA pools from multiple library
preparation runs may be placed on the same daughter plate for
hybridization.
Prepare .csv files for pooling and normalization
Before starting the sample pooling automation protocol, you must create
comma-separated value (.csv) files containing instructions including the
specific wells to be pooled and the concentration of each sample. From
this data, the workstation calculates the volume of each sample required
to prepare each concentration-normalized pool for the Hybridization step.
See Figure 10 for required .csv file content. Pooling and normalization .csv
file templates are provided in the following directory:
C: > VWorks Workspace > NGS Option B > XT_Illumina_2.0 > Pooling and
Normalization Templates
Select the appropriate set of templates from the directory based on the
intended pool composition (8 or 16 prepared samples) and on the number
of source plates to be consolidated in the run to prepare the single
hybridization sample plate. For example, for 8-library pools, use the
template Pool8_01_SourcePlate.csv for the first DNA source plate,
continuing with additional Pool8_0X_SourcePlate.csv files for additional
DNA source plates.
1 Copy and rename the appropriate set of .csv file templates for the run.
Make sure to retain the header text used in the template files, without
introducing spaces or other new characters.
If processing a partial plate of prepped gDNA samples, delete the rows
corresponding to the WellIDs of the empty wells on the plate.
SureSelectXT2 Automated Library Prep and Capture System
67
4
Hybridization
Step 1. Pool indexed DNA samples for hybridization
Figure 10
Sample pooling and normalization .csv file content
2 In each .csv file, edit the information for each DNA sample (Well ID) as
follows:
• In the PreCap Amplified pond concentrations field, enter the
concentration (in ng/µL) determined on page 61 for each indexed
DNA sample.
• In the Target WellID field, enter the well position of the pool in
which the indexed DNA sample should be included for the
Hybridization plate. See the guidelines on page 65 for Hybridization
sample pool placement considerations.
Set up and run the PreCapture_Pooling_v1.0.pro automation protocol
1 Turn on the chiller, set to 0°C, at position 9 of the Bravo deck. Be sure
that the chiller reservoir contains at least 300 mL of 25% ethanol.
2 Clear the Labware MiniHub and BenchCel of all plates and tip boxes.
3 To set up the PreCapture_Pooling_v1.0.pro automation protocol, open
the VWorks Form XT2_Pooling.VWForm using the shortcut on your
desktop.
68
SureSelectXT2 Automated Library Prep and Capture System
Hybridization
Step 1. Pool indexed DNA samples for hybridization
4
4 In the Form, enter the run information highlighted below:
• Under Controls, specify whether the indexed DNA source plates will
be loaded in the MiniHub and will be sealed at start of run
(recommended).
• From Number of Indexes to Pool menu, select 8 or 16 (see Table 27
for guidelines).
• From Pooled DNA Quantity menu, enter the required total amount
of DNA in the pool (typically 1500 ng). See page 65 for guidelines.
• In Plate ID/Barcode field, enter the name or barcode of the
daughter Hybridization sample plate.
• From Number of Source Plates menu, select the number of indexed
DNA source plates to be provided for sample pooling. If >8 plates
will be used to create a single Hybridization sample plate, run the
pooling and normalization protocol in sets of 8 source plates.
• Under Concentration File, use the browse button to specify the
location of each .csv file that provides sample position and
concentration data for each plate.
SureSelectXT2 Automated Library Prep and Capture System
69
4
Hybridization
Step 1. Pool indexed DNA samples for hybridization
5 When finished entering run parameters in the Form, click Show Setup.
6 Load sample plates and labware as displayed in the Workstation Setup
region of the form (example shown below is for pooling run for two
source plates):
• Load each indexed DNA source plates onto its assigned shelf on the
MiniHub.
• Load the appropriate type of destination (daughter) plate on Bravo
deck position 5. See step 2 on page 66 to determine plate type
needed.
• Load an empty tip box on Bravo deck position 6.
• Load the indicated number of tip boxes in the BenchCel stacker.
70
SureSelectXT2 Automated Library Prep and Capture System
Hybridization
Step 1. Pool indexed DNA samples for hybridization
4
7 When verification is complete, click Run Protocol.
CA U T I O N
When more than one indexed DNA source plate is used in the run, a workstation
operator must be present during the run to remove and replace plate seals during the
run, in response to NGS Workstation prompts.
Running the PreCapture_Pooling_v1.0.pro protocol takes approximately one
hour per indexed DNA source plate. Once complete, the Hybridization
sample plate, containing indexed DNA pools, is located at position 5 of the
Bravo deck.
Adjust final concentration of pooled DNA
8 Remove the Hybridization sample plate from Bravo deck position 5.
9 Use a vacuum concentrator, held at  45°C, to reduce the volume in
each well to 1–2 µL.
10 Add sufficient nuclease-free water to each concentrated gDNA pool to
bring the final DNA concentration to 214.3 ng/µL. For example, for
1500 ng pools, bring the final volume in each well to 7 µL, for a final
concentration of 214.3 ng/µL.
11 Seal the plate using the PlateLoc Thermal Microplate Sealer, with
sealing settings of 165°C and 1.0 sec.
12 Vortex the plate for 30 seconds to ensure complete reconstitution, then
centrifuge the plate for 1 minute to drive the well contents off the walls
and plate seal.
13 If indexed DNA pool samples are in a Nunc DeepWell plate, carefully
transfer the samples to an Eppendorf twin.tec plate for use in the
following Hybridization protocol.
SureSelectXT2 Automated Library Prep and Capture System
71
4
Hybridization
Step 2. Hybridize the gDNA library and Capture Library
Step 2. Hybridize the gDNA library and Capture Library
In this step, the Agilent NGS Workstation completes the liquid handling
steps in preparation for hybridization of the indexed DNA pools to one or
more SureSelect or ClearSeq Capture Libraries. Afterward, you transfer
the sample plate to a thermal cycler, held at 65°C, to allow hybridization
of the indexed DNA to the Capture Library.
Two versions of the Hybridization automation protocol are available. See
Table 28 for a summary of suggested usage and to locate the instructions
for each protocol option in this manual.
For runs in which all samples will be hybridized to the same Capture
Library, hybridization protocol selection is based on run size, where
12-column runs should use the Hybridization_MMCol_v2.0.pro protocol
and 1-6 column runs should use the Hybridization_MMRow_v2.0.pro
protocol.
For runs that include hybridization to multiple Capture Libraries,
hybridization protocol selection is based on the following considerations:
1 Number of Capture Libraries
2 Appropriate positioning (rows vs. columns) of the Capture Libraries
with respect to the DNA sample plate configuration:
• In Hybridization_MMCol_v2.0.pro, master mixes are organized in the
source plate by column (see Figure 11) and each row of the DNA
sample plate may be hybridized to a different Capture library
• In Hybridization_MMRow_v2.0.pro, master mixes are organized in the
source plate by row (see Figure 12), and each column of the DNA
sample plate may be hybridized to a different Capture library
Table 28
Comparison of Hybridization protocol options
Protocol Name
Optimal Hybridization Run Size
Number of Different Capture
Libraries Allowed in Run
Instructions Start
Hybridization_MMCol_v2.0.pro
12-column runs
8
page 73
Hybridization_MMRow_v2.0.pro
6-column runs
12
page 86
72
SureSelectXT2 Automated Library Prep and Capture System
Hybridization
Hybridization Option A: Master Mixes in Columns (Hybridization_MMCol_v2.0.pro)
4
Hybridization Option A: Master Mixes in Columns
(Hybridization_MMCol_v2.0.pro)
Prepare the workstation
1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes.
2 Gently wipe down the Labware MiniHub, Bravo decks, and BenchCel
with a NucleoClean decontamination wipe.
Prepare one or more Capture Library Master Mixes
3 Prepare the appropriate volume of SureSelect or ClearSeq Capture
Library Master Mix for each of the Capture Libraries that will be used
for hybridization as indicated in Table 29 to Table 32. Mix the
components by pipetting. Keep the master mixes on ice during
preparation and aliquoting.
NOTE
Each row of the indexed gDNA pool plate may be hybridized to a different Capture Library.
However, Capture Libraries of different sizes require different post-capture amplification
cycles. Plan experiments such that similar-sized libraries are hybridized on the same plate.
For runs that use a single Capture Library for all rows of the plate, prepare the master mix
as described in Step a (Table 29 or Table 30) below.
For runs that use different Capture Libraries for individual rows, prepare each master mix as
described in Step b (Table 31 or Table 32) below.
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Hybridization
Hybridization Option A: Master Mixes in Columns (Hybridization_MMCol_v2.0.pro)
a For runs that use a single Capture Library for all rows, prepare
the Capture Library Master Mix as listed in Table 29 or Table 30,
based on the Mb target size of your design.
Table 29
Preparation of Capture Library Master Mix for target sizes <3.0 Mb; same
Capture Library for all 8 rows of wells
Target size <3.0 Mb
SureSelectXT2 Reagent
Volume for 1 Well
Volume for 12 Columns of Wells
Nuclease-free water
7.0 µL
808.5 µL
SureSelect RNase Block (purple cap)
0.5 µL
57.8 µL
SureSelect or ClearSeq Capture Library
2.0 µL
231.0 µL
Total Volume
9.5 µL
1097.3 µL
Table 30
Preparation of Capture Library Master Mix for target sizes >3.0 Mb; same
Capture Library for all 8 rows of wells
Target size >3.0 Mb
74
SureSelectXT2 Reagent
Volume for 1 Well
Volume for 12 Columns of Wells
Nuclease-free water
4.0 µL
462.0 µL
SureSelect RNase Block (purple cap)
0.5 µL
57.8 µL
SureSelect or ClearSeq Capture Library
5.0 µL
577.5 µL
Total Volume
9.5 µL
1097.3 µL
SureSelectXT2 Automated Library Prep and Capture System
Hybridization
Hybridization Option A: Master Mixes in Columns (Hybridization_MMCol_v2.0.pro)
4
b For runs that use different Capture Libraries in individual rows,
prepare a Capture Library Master Mix for each Capture Library as
listed in Table 31 or Table 32, based on the Mb target size of your
design. The volumes listed in Table 31 and Table 32 are for a single
row of sample wells. If a given Capture Library will be hybridized in
multiple rows, multiply each of the values below by the number of
rows assigned to that Capture Library.
Table 31
Preparation of Capture Library Master Mix for target sizes <3.0 Mb, single row of wells
Target size <3.0 Mb
SureSelectXT2
Reagent
Volume for
1 Well
Volume for
1 Column
Volume for
2 Columns
Volume for
3 Columns
Volume for
4 Columns
Volume for
6 Columns
Volume for
12 Columns
Nuclease-free
water
7.0 µL
14.0 µL
21.2 µL
28.4 µL
35.7 µL
53.7 µL
100.6 µL
SureSelect RNase 0.5 µL
Block (purple cap)
1.0 µL
1.5 µL
2.0 µL
2.5 µL
3.8 µL
7.2 µL
Capture Library
2.0 µL
4.0 µL
6.1 µL
8.1 µL
10.2 µL
15.3 µL
28.8 µL
Total Volume
9.5 µL
19.0 µL
28.8 µL
38.6 µL
48.4 µL
72.9 µL
136.6 µL
Table 32
Preparation of Capture Library Master Mix for target sizes >3.0 Mb, single row of wells
Target size >3.0 Mb
SureSelectXT2
Reagent
Volume for
1 Well
Volume for
1 Column
Volume for
2 Columns
Volume for
3 Columns
Volume for
4 Columns
Volume for
6 Columns
Volume for
12 Columns
Nuclease-free
water
4.0 µL
8.0 µL
12.1 µL
16.3 µL
20.4 µL
30.7 µL
57.5 µL
SureSelect RNase 0.5 µL
Block (purple cap)
1.0 µL
1.5 µL
2.0 µL
2.5 µL
3.8 µL
7.2 µL
Capture Library
5.0 µL
10.0 µL
15.2 µL
20.3 µL
25.5 µL
38.4 µL
71.9 µL
Total Volume
9.5 µL
19.0 µL
28.8 µL
38.6 µL
48.4 µL
72.9 µL
136.6 µL
SureSelectXT2 Automated Library Prep and Capture System
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4
Hybridization
Hybridization Option A: Master Mixes in Columns (Hybridization_MMCol_v2.0.pro)
Prepare the master mix source plate
4 In a Nunc DeepWell plate, prepare the hybridization master mix source
plate at room temperature. Add the volumes indicated in Table 33 to all
wells of the indicated column of the Nunc DeepWell plate. As indicated
in the shaded portion of Table 33, Blocking Mix and nuclease-free
water are combined in the wells of Column 1.
When using multiple Capture Libraries in a run, add each Capture
Library Master Mix to the appropriate row(s) of the Nunc DeepWell
plate.
The final configuration of the master mix source plate is shown in
Figure 11.
Table 33
Preparation of the Master Mix Source Plate for Hybridization_MMCol_v2.0.pro
Master Mix
Solution
Position on
Source Plate
SureSelect XT2
Blocking Mix
Column 1
Volume of Master Mix added per Well of Nunc Deep Well Source Plate
1-Column
Runs
2-Column
Runs
3-Column
Runs
4-Column
Runs
6-Column
Runs
12-Column
Runs
13.5 µL
22.5 µL
31.5 µL
40.5 µL
58.5 µL
117.0 µL
Nuclease-free
water
(A1-H1)
3.75 µL
6.25 µL
8.75 µL
11.25 µL
16.25 µL
32.5 µL
Capture Library
Master Mix
Column 2
18.4 µL
28.2 µL
38.0 µL
47.8 µL
72.3 µL
136.0 µL
SureSelect XT2
Hybridization
Buffer
Column 3
55.5 µL
92.5 µL
129.5 µL
166.5 µL
240.5 µL
481 µL
76
(A2-H2)
(A3-H3)
SureSelectXT2 Automated Library Prep and Capture System
Hybridization
Hybridization Option A: Master Mixes in Columns (Hybridization_MMCol_v2.0.pro)
Figure 11
4
Configuration of the master mix source plate for Hybridization_MMCol_v2.0.pro. Each well in column 2 may contain the same or different Capture Libraries.
5 Seal the master mix source plate using the PlateLoc Thermal Microplate
Sealer, with sealing settings of 165°C and 1.0 sec.
6 Vortex the plate for 5 seconds to ensure homogeneity of the Block
Master Mix dilution.
7 Centrifuge the plate for 30 seconds to drive the well contents off the
walls and plate seal and to eliminate any bubbles. Keep the master mix
plate at room temperature.
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Hybridization
Hybridization Option A: Master Mixes in Columns (Hybridization_MMCol_v2.0.pro)
Load the Agilent NGS Workstation
8 Load the Labware MiniHub according to Table 34, using the plate
orientations shown in Figure 6.
Table 34
Initial MiniHub configuration for Hybridization_MMCol_v2.0.pro
Vertical Shelf
Position
Cassette 1
Cassette 2
Cassette 3
Cassette 4
Shelf 5 (Top)
Empty
Empty
Empty
Empty
Shelf 4
Empty
Empty
Empty
Empty
Shelf 3
Empty
Empty
Empty
Empty
Shelf 2
Empty
Empty
Empty
Empty tip box
Shelf 1 (Bottom)
Empty
Empty
Empty
Empty
9 Load the BenchCel Microplate Handling Workstation according to
Table 35.
Table 35
78
Initial BenchCel configuration for Hybridization_MMCol_v2.0.pro
No. of Columns
Processed
Rack 1
Rack 2
Rack 3
Rack 4
1
2 Tip boxes
Empty
Empty
Empty
2
2 Tip boxes
Empty
Empty
Empty
3
2 Tip boxes
Empty
Empty
Empty
4
2 Tip boxes
Empty
Empty
Empty
6
3 Tip boxes
Empty
Empty
Empty
12
4 Tip boxes
Empty
Empty
Empty
SureSelectXT2 Automated Library Prep and Capture System
Hybridization
Hybridization Option A: Master Mixes in Columns (Hybridization_MMCol_v2.0.pro)
4
10 Load the Bravo deck according to Table 36.
Table 36
Location
4
Initial Bravo deck configuration for Hybridization_MMCol_v2.0.pro
Content
Empty PCR plate seated on red insert
(PCR plate type must be specified on setup form under step 2)
5
Empty Eppendorf twin.tec plate
6
Hybridization Master Mix source plate (unsealed) seated on silver
insert (Master Mixes in Columns 1-3)
8
Empty tip box
9
Indexed DNA pools in Eppendorf twin.tec plate (unsealed)
Run VWorks protocol Hybridization_MMCol_v2.0.pro
11 On the SureSelect setup form, under Select Protocol to Run, select
Hybridization_MMCol_v2.0.pro.
12 Under Select PCR plate labware for Thermal Cycling, select the
specific type of PCR plate that was loaded on Bravo deck position 4.
13 Select the number of columns of samples to be processed. Runs must
include 1, 2, 3, 4, 6, or 12 columns.
14 Click Display Initial Workstation Setup.
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4
Hybridization
Hybridization Option A: Master Mixes in Columns (Hybridization_MMCol_v2.0.pro)
15 Verify that the NGS workstation has been set up as displayed in the
Workstation Setup region of the form.
16 When verification is complete, click Run Selected Protocol.
The Agilent NGS Workstation transfers Blocking Mix and indexed gDNA
pools to the PCR plate. When this process is complete, you will be
prompted to transfer the plate to the thermal cycler for sample
denaturation prior to hybridization.
80
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Hybridization Option A: Master Mixes in Columns (Hybridization_MMCol_v2.0.pro)
4
17 When prompted by VWorks as shown below, remove the PCR plate from
position 4 of the Bravo deck, leaving the red insert in place.
18 Seal the sample plate using the PlateLoc Thermal Microplate Sealer,
with sealing settings of 165°C and 3.0 sec.
19 Transfer the sealed plate to a thermal cycler and run the following
program shown in Table 37. After transferring the plate, click Continue
on the VWorks screen.
Table 37
Thermal cycler program used for sample denaturation prior to hybridization
Step
Temperature
Time
Step 1
95°C
5 minutes
Step 2
65°C
Hold
While the sample plate incubates on the thermal cycler, the Agilent NGS
Workstation combines aliquots of the Capture Library master mix and
Hybridization Buffer.
SureSelectXT2 Automated Library Prep and Capture System
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Hybridization
Hybridization Option A: Master Mixes in Columns (Hybridization_MMCol_v2.0.pro)
CA U T I O N
You must complete step 20 to step 24 quickly, and immediately after being prompted by
the VWorks software. It is important that sample temperature remains approximately
65°C during transfers between the Agilent NGS Workstation and thermal cycler.
20 When the workstation has finished aliquoting the Capture Library
master mixes and Hybridization Buffer, you will be prompted by VWorks
as shown below. When the thermal cycler reaches the 65°C hold step,
click Continue. Leave the sample plate in the thermal cycler until you
are notified to move it.
82
SureSelectXT2 Automated Library Prep and Capture System
Hybridization
Hybridization Option A: Master Mixes in Columns (Hybridization_MMCol_v2.0.pro)
4
21 When prompted by VWorks as shown below, quickly remove the sample
plate from the thermal cycler, unseal the plate carefully to avoid
splashing, and transfer the plate to position 4 of the Bravo deck, seated
in the red insert. Click Continue.
WARN I NG
Warning
Bravo deck position 4 will be hot.
Use caution when handling components that contact heated deck positions.
The Agilent NGS Workstation transfers the capture library-hybridization
buffer mixture to the wells of the PCR plate, containing the mixture of
indexed gDNA pools and blocking agents.
SureSelectXT2 Automated Library Prep and Capture System
83
4
Hybridization
Hybridization Option A: Master Mixes in Columns (Hybridization_MMCol_v2.0.pro)
22 When prompted by VWorks as shown below, quickly remove the PCR
sample plate from Bravo deck position 4, leaving the red insert in place.
23 Seal the sample plate using the PlateLoc Thermal Microplate Sealer,
with sealing settings of 165°C and 3.0 sec.
24 Quickly transfer the plate back to the thermal cycler, held at 65°C.
Place a compression mat over the PCR plate in the thermal cycler. After
transferring the plate, click Continue on the VWorks screen.
25 To finish the VWorks protocol, click Continue in the Unused Tips and
Empty Tip box dialogs, and then click Yes in the Protocol Complete
dialog.
CA U T I O N
The temperature of the plate in the thermal cycler should be held at 65°C using a
heated lid at 105°C. The lid of the thermal cycler is hot and can cause burns. Use
caution when working near the lid.
26 Incubate the hybridization mixture in the thermal cycler for 24 hours at
65°C with a heated lid at 105°C.
84
SureSelectXT2 Automated Library Prep and Capture System
Hybridization
Hybridization Option A: Master Mixes in Columns (Hybridization_MMCol_v2.0.pro)
4
Samples may be hybridized for up to 72 hours, but you must verify that
the extended hybridization does not cause extensive evaporation in the
sample wells.
When hybridization is complete, proceed to “Step 3. Capture the
hybridized DNA” on page 97.
SureSelectXT2 Automated Library Prep and Capture System
85
4
Hybridization
Hybridization Option B: Master Mixes in Rows (Hybridization_MMRow_v2.0.pro)
Hybridization Option B: Master Mixes in Rows
(Hybridization_MMRow_v2.0.pro)
Prepare the workstation
1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes.
2 Gently wipe down the Labware MiniHub, Bravo decks, and BenchCel
with a NucleoClean decontamination wipe.
Prepare one or more Capture Library Master Mixes
3 Prepare the appropriate volume of Capture Library Master Mix for each
of the Capture Libraries that will be used for hybridization as indicated
in Table 38 to Table 41. Mix the components by pipetting. Keep the
master mixes on ice during preparation and aliquoting.
NOTE
Each column of the indexed gDNA pool plate may be hybridized to a different Capture
Library. However, Capture Libraries of different sizes require different post-capture
amplification cycles. Plan experiments such that similar-sized libraries are hybridized on
the same plate.
For runs that use a single Capture Library for all columns of the plate, prepare the master
mix as described in Step a (Table 38 or Table 39) below.
For runs that use different Capture Libraries for individual columns, prepare each master
mix as described in Step b (Table 40 or Table 41) below.
86
SureSelectXT2 Automated Library Prep and Capture System
Hybridization
Hybridization Option B: Master Mixes in Rows (Hybridization_MMRow_v2.0.pro)
4
a For runs that use a single Capture Library for all columns, prepare
the Capture Library Master Mix as listed in Table 38 or Table 39,
based on the Mb target size of your design.
Table 38
Preparation of Capture Library Master Mix for target sizes <3.0 Mb; same Capture Library for all
columns
Target size <3.0 Mb
SureSelectXT2 Reagent
Volume for
1 Well
Volume for
1 Column
Volume for
2 Columns
Volume for
3 Columns
Volume for
4 Columns
Volume for
6 Columns
Nuclease-free water
7.0 µL
68.3 µL
143.3 µL
211.6 µL
279.8 µL
416.3 µL
SureSelect RNase Block
(purple cap)
0.5 µL
4.9 µL
10.2 µL
15.1 µL
20.0 µL
29.7 µL
Capture Library
2.0 µL
19.5 µL
41.0 µL
60.5 µL
80.0 µL
119.0 µL
Total Volume
9.5 µL
92.7 µL
194.5 µL
287.1 µL
379.8 µL
565.0 µL
Table 39
Preparation of Capture Library Master Mix for target sizes >3.0 Mb; same Capture Library for all
columns
Target size >3.0 Mb
SureSelectXT2 Reagent
Volume for
1 Well
Volume for
1 Column
Volume for
2 Columns
Volume for
3 Columns
Volume for
4 Columns
Volume for
6 Columns
Nuclease-free water
4.0 µL
39.0 µL
81.9 µL
120.9 µL
159.9 µL
237.9 µL
SureSelect RNase Block
(purple cap)
0.5 µL
4.9 µL
10.2 µL
15.1 µL
20.0 µL
29.7 µL
Capture Library
5.0 µL
48.8 µL
102.4 µL
151.1 µL
199.9 µL
297.4 µL
Total Volume
9.5 µL
92.7 µL
194.5 µL
287.1 µL
379.8 µL
565.0 µL
SureSelectXT2 Automated Library Prep and Capture System
87
4
Hybridization
Hybridization Option B: Master Mixes in Rows (Hybridization_MMRow_v2.0.pro)
b For runs that use different Capture Libraries in individual
columns, prepare a Capture Library Master Mix for each Capture
Library as listed in Table 40 or Table 41, based on the Mb target size
of your design. The volumes listed in Table 40 and Table 41 are for a
single column of sample wells. If a given Capture Library will be
hybridized in multiple columns, multiply each of the values below by
the number of columns assigned to that Capture Library.
Table 40
Preparation of Capture Library Master Mix for target sizes <3.0 Mb, single
column of wells
Target size <3.0 Mb
SureSelectXT2 Reagent
Volume for 1 Well
Volume for 1 Column
Nuclease-free water
7.0 µL
68.3 µL
SureSelect RNase Block (purple cap)
0.5 µL
4.9 µL
SureSelect or ClearSeq Capture Library
2.0 µL
19.5 µL
Total Volume
9.5 µL
92.7 µL
Table 41
Preparation of Capture Library Master Mix for target sizes >3.0 Mb, single
column of wells
Target size >3.0 Mb
88
SureSelectXT2 Reagent
Volume for 1 Well
Volume for 1 Column
Nuclease-free water
4.0 µL
39.0 µL
SureSelect RNase Block (purple cap)
0.5 µL
4.9 µL
SureSelect or ClearSeq Capture Library
5.0 µL
48.8 µL
Total Volume
9.5 µL
92.7 µL
SureSelectXT2 Automated Library Prep and Capture System
Hybridization
Hybridization Option B: Master Mixes in Rows (Hybridization_MMRow_v2.0.pro)
4
Prepare the master mix source plate
4 In a Nunc DeepWell plate, prepare the hybridization master mix source
plate at room temperature. Add the volumes indicated in Table 42 to
the appropriate number of wells of the indicated row of the Nunc
DeepWell plate. Fill the number of wells that corresponds to the
number of DNA-sample columns in the run (1, 2, 3, 4, 6, or 12).
As indicated in the shaded portion of Table 42, Blocking Mix and
nuclease-free water are combined in the wells of Row A.
When using multiple Capture Libraries in a run, add each Capture
Library Master Mix to the appropriate column(s) of the Nunc DeepWell
plate.
The final configuration of the master mix source plate is shown in
Figure 12.
Table 42
Preparation of the Master Mix Source Plate for Hybridization_MMRow_v2.0.pro
Master Mix Solution
Position on Source
Plate
Volume of Master
Mix added per Well
SureSelect XT2 Blocking Mix
Row A
81.0 µL
Nuclease-free water
(A1-AX)
22.5 µL
Capture Library Master Mix
Row B
92.7 µL
(B1-BX)
SureSelect XT2 Hybridization Buffer
Row C
314.5 µL
(C1-CX)
SureSelectXT2 Automated Library Prep and Capture System
89
4
Hybridization
Hybridization Option B: Master Mixes in Rows (Hybridization_MMRow_v2.0.pro)
Figure 12
Configuration of the master mix source plate for Hybridization_MMRow_v2.0.pro. Rows A–C may contain 1, 2, 3, 4, 6, or 12 wells of reagents,
depending on run size (example shown is for 6-column run size). Each well in
row B may contain the same or different Capture Libraries.
5 Seal the master mix source plate using the PlateLoc Thermal Microplate
Sealer, with sealing settings of 165°C and 1.0 sec.
6 Vortex the plate for 5 seconds to ensure homogeneity of the Block
Master Mix dilution.
7 Centrifuge the plate for 30 seconds to drive the well contents off the
walls and plate seal and to eliminate any bubbles. Keep the master mix
plate at room temperature.
90
SureSelectXT2 Automated Library Prep and Capture System
Hybridization
Hybridization Option B: Master Mixes in Rows (Hybridization_MMRow_v2.0.pro)
4
Load the Agilent NGS Workstation
8 Load the BenchCel Microplate Handling Workstation according to
Table 43.
Table 43
Initial BenchCel configuration for Hybridization_MMRow_v2.0.pro
No. of Columns
Processed
Rack 1
Rack 2
Rack 3
Rack 4
1
1 Tip box
Empty
Empty
Empty
2
1 Tip box
Empty
Empty
Empty
3
2 Tip boxes
Empty
Empty
Empty
4
2 Tip boxes
Empty
Empty
Empty
6
3 Tip boxes
Empty
Empty
Empty
12
5 Tip boxes
Empty
Empty
Empty
9 Load the Bravo deck according to Table 44.
Table 44
Initial Bravo deck configuration for Hybridization_MMRow_v2.0.pro
Location
Content
1
Empty tip box
4
Empty PCR plate seated on red insert
(PCR plate type must be specified on setup form under step 2)
5
Empty Eppendorf twin.tec plate
6
Hybridization Master Mix source plate (unsealed) seated on silver
insert (Master Mixes in Rows A-C)
9
Indexed DNA pools in Eppendorf twin.tec plate
Run VWorks protocol Hybridization_MMRow_v2.0.pro
10 On the SureSelect setup form, under Select Protocol to Run, select
Hybridization_MMRow_v2.0.pro.
11 Under Select PCR plate labware for Thermal Cycling, select the
specific type of PCR plate that was loaded on Bravo deck position 4.
SureSelectXT2 Automated Library Prep and Capture System
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Hybridization
Hybridization Option B: Master Mixes in Rows (Hybridization_MMRow_v2.0.pro)
12 Select the number of columns of samples to be processed. Runs must
include 1, 2, 3, 4, 6, or 12 columns.
13 Click Display Initial Workstation Setup.
14 Verify that the NGS workstation has been set up as displayed in the
Workstation Setup region of the form.
15 When verification is complete, click Run Selected Protocol.
The Agilent NGS Workstation transfers Blocking Mix and indexed gDNA
pools to the PCR plate. When this process is complete, you will be
prompted to transfer the plate to the thermal cycler for sample
denaturation prior to hybridization.
92
SureSelectXT2 Automated Library Prep and Capture System
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Hybridization Option B: Master Mixes in Rows (Hybridization_MMRow_v2.0.pro)
4
16 When prompted by VWorks as shown below, remove the PCR plate from
position 4 of the Bravo deck, leaving the red insert in place.
17 Seal the sample plate using the PlateLoc Thermal Microplate Sealer,
with sealing settings of 165°C and 3.0 sec.
18 Transfer the sealed plate to a thermal cycler and run the following
program shown in Table 37. After transferring the plate, click Continue
on the VWorks screen.
Table 45
Thermal cycler program used for sample denaturation prior to hybridization
Step
Temperature
Time
Step 1
95°C
5 minutes
Step 2
65°C
Hold
While the sample plate incubates on the thermal cycler, the Agilent NGS
Workstation combines aliquots of the Capture Library master mix and
Hybridization Buffer.
SureSelectXT2 Automated Library Prep and Capture System
93
4
Hybridization
Hybridization Option B: Master Mixes in Rows (Hybridization_MMRow_v2.0.pro)
CA U T I O N
You must complete step 19 to step 23 quickly, and immediately after being prompted by
the VWorks software. It is important that sample temperature remains approximately
65°C during transfers between the Agilent NGS Workstation and thermal cycler.
19 When the workstation has finished aliquoting the Capture Library
master mixes and Hybridization Buffer, you will be prompted by VWorks
as shown below. When the thermal cycler reaches the 65°C hold step,
click Continue. Leave the sample plate in the thermal cycler until you
are notified to move it.
94
SureSelectXT2 Automated Library Prep and Capture System
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Hybridization Option B: Master Mixes in Rows (Hybridization_MMRow_v2.0.pro)
4
20 When prompted by VWorks as shown below, quickly remove the sample
plate from the thermal cycler, unseal the plate carefully to avoid
splashing, and transfer the plate to position 4 of the Bravo deck, seated
in the red insert. Click Continue.
WARN I NG
Warning
Bravo deck position 4 will be hot.
Use caution when handling components that contact heated deck positions.
The Agilent NGS Workstation transfers the capture library-hybridization
buffer mixture to the wells of the PCR plate, containing the mixture of
indexed gDNA pools and blocking agents.
SureSelectXT2 Automated Library Prep and Capture System
95
4
Hybridization
Hybridization Option B: Master Mixes in Rows (Hybridization_MMRow_v2.0.pro)
21 When prompted by VWorks as shown below, quickly remove the PCR
sample plate from Bravo deck position 4, leaving the red insert in place.
22 Seal the sample plate using the PlateLoc Thermal Microplate Sealer,
with sealing settings of 165°C and 3.0 sec.
23 Quickly transfer the plate back to the thermal cycler, held at 65°C.
Place a compression mat over the PCR plate in the thermal cycler. After
transferring the plate, click Continue on the VWorks screen.
24 To finish the VWorks protocol, click Continue in the Unused Tips and
Empty Tip box dialogs, and then click Yes in the Protocol Complete
dialog.
CA U T I O N
The temperature of the plate in the thermal cycler should be held at 65°C using a
heated lid at 105°C. The lid of the thermal cycler is hot and can cause burns. Use
caution when working near the lid.
25 Incubate the hybridization mixture in the thermal cycler for 24 hours at
65°C with a heated lid at 105°C.
Samples may be hybridized for up to 72 hours, but you must verify that
the extended hybridization does not cause extensive evaporation in the
sample wells.
96
SureSelectXT2 Automated Library Prep and Capture System
Hybridization
Step 3. Capture the hybridized DNA
4
Step 3. Capture the hybridized DNA
In this step, the indexed gDNA-Capture Library hybrids are captured
using streptavidin-coated magnetic beads. This step is run immediately
after the 24-hour hybridization period.
This step is automated by the NGS workstation using the
SureSelectCapture&Wash_v2.0.rst runset, with a total duration of
approximately 2 hours. A workstation operator must be present to
complete two actions during the runset, at the time points in the table
below. The times provided are approximate; each action is completed in
response to a VWorks prompt at the appropriate time in the runset.
Table 46
Operator action
Approximate time after run start
Transfer hybridization reaction plate
<5 minutes
from thermal cycler to NGS workstation
Remove hybridization plate from
position 4 after reactions transferred to
capture plate
5-10 minutes
Prepare the workstation
1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes.
2 Gently wipe down the Labware MiniHub, Bravo decks, and BenchCel
with a NucleoClean decontamination wipe.
3 Pre-set the temperature of Bravo deck position 4 to 66°C using the
Inheco Multi TEC control touchscreen, as described in Setting the
Temperature of Bravo Deck Heat Blocks. On the Multi TEC control
touchscreen, Bravo deck position 4 corresponds to CPAC 2, position 1.
Prepare the streptavidin-coated beads
4 Vigorously resuspend the Dynal MyOne Streptavidin T1 magnetic beads
on a vortex mixer. Dynal beads settle during storage.
SureSelectXT2 Automated Library Prep and Capture System
97
4
Hybridization
Step 3. Capture the hybridized DNA
5 Wash the magnetic beads.
a In a conical vial, combine the components listed in Table 47. The
volumes below include the required overage.
Table 47
Components required for magnetic bead washing procedure
Reagent
Volume for
1 Well
Volume for
1 Column
Volume for
2 Columns
Volume for
3 Columns
Volume for
4 Columns
Volume for
6 Columns
Volume for
12 Columns
Streptavidin bead
suspension
50 µL
425 µL
825 µL
1225 µL
1.65 mL
2.5 mL
5.0 mL
SureSelect XT2
Binding Buffer
0.2 mL
1.7 mL
3.3 mL
4.9 mL
6.6 mL
10 mL
20 mL
Total Volume
0.25 mL
2.125 mL
4.7125mL
6.125 mL
8.25 mL
12.5 mL
25 mL
b Mix the beads on a vortex mixer for 5 seconds.
c Put the vial into a magnetic device, such as the Dynal magnetic
separator.
d Remove and discard the supernatant.
e Repeat step a through step d for a total of 3 washes. (Retain the
beads after each wash and combine with a fresh aliquot of the
indicated volume of SureSelect XT2 Binding Buffer.)
6 Resuspend the beads in SureSelect XT2 Binding Buffer, according to
Table 48 below.
Table 48
Preparation of magnetic beads for SureSelect Capture&Wash_v2.0.rst
Reagent
Volume for
1 Library
Volume for
1 Column
Volume for
2 Columns
Volume for
3 Columns
Volume for
4 Columns
Volume for
6 Columns
Volume for
12 Columns
SureSelect XT2
Binding Buffer
0.2 mL
1.7 mL
3.3 mL
4.9 mL
6.6 mL
10 mL
20 mL
7 Prepare a Nunc DeepWell source plate for the washed bead suspension.
For each well to be processed, add 200 µL of the homogeneous bead
suspension to the Nunc DeepWell plate.
8 Place the streptavidin bead source plate at position 5 of the Bravo deck.
98
SureSelectXT2 Automated Library Prep and Capture System
Hybridization
Step 3. Capture the hybridized DNA
4
Prepare capture and wash solution source plates
9 Prepare an Eppendorf twin.tec source plate labeled Wash #1. For each
well to be processed, add 160 µL of SureSelect XT2 Wash 1.
10 Prepare a Nunc DeepWell source plate labeled Wash #2. For each well
to be processed, add 1150 µL of SureSelect XT2 Wash 2.
11 Place the silver Nunc DeepWell plate insert on position 6 of the Bravo
deck. This insert is required to facilitate heat transfer to DeepWell
source plate wells during the runset.
12 Place the Wash #2 source plate on the silver insert at position 6 of the
Bravo deck. Make sure the plate is seated properly on the silver
DeepWell insert.
13 Prepare a Thermo Scientific reservoir containing 20 mL of nuclease-free
water.
Load the Agilent NGS Workstation
14 Load the Labware MiniHub according to Table 49, using the plate
orientations shown in Figure 6.
Table 49
Initial MiniHub configuration for SureSelect Capture&Wash_v2.0.rst
Vertical Shelf
Position
Cassette 1
Cassette 2
Cassette 3
Cassette 4
Shelf 5 (Top)
Empty
Empty
Empty
Empty
Shelf 4
Empty
Empty
Empty
Empty
Shelf 3
Empty Eppendorf
twin.tec plate
Empty
Wash #1
Eppendorf
twin.tec source
plate
Empty
Shelf 2
Empty
Nuclease-free
water reservoir
from step 13
Empty
Empty
Shelf 1 (Bottom)
Empty
Empty
Empty
Empty tip box
SureSelectXT2 Automated Library Prep and Capture System
99
4
Hybridization
Step 3. Capture the hybridized DNA
15 Load the BenchCel Microplate Handling Workstation according to
Table 50.
Table 50
Initial BenchCel configuration for SureSelectCapture&Wash_v2.0.rst
No. of Columns
Processed
Rack 1
Rack 2
Rack 3
Rack 4
1
1 Tip box
Empty
Empty
Empty
2
2 Tip boxes
Empty
Empty
Empty
3
3 Tip boxes
Empty
Empty
Empty
4
4 Tip boxes
Empty
Empty
Empty
6
6 Tip boxes
Empty
Empty
Empty
12
10 Tip boxes
2 Tip boxes
Empty
Empty
16 Load the Bravo deck according to Table 51 (positions 5 and 6 should
already be loaded).
Table 51
Initial Bravo deck configuration for SureSelectCapture&Wash_v2.0.rst
Location
Content
1
Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)
4
Empty red aluminum insert
(PCR plate type used for Hybridization protocol must be specified on setup form
under step 2)
5
Streptavidin beads DeepWell source plate
6
Wash #2 DeepWell source plate seated on silver insert
Run VWorks runset SureSelectCapture&Wash_v2.0.rst
17 On the SureSelect setup form, under Select Protocol to Run, select
SureSelectCapture&Wash_v2.0.rst.
18 Under Select PCR plate labware for Thermal Cycling, select the
specific type of PCR plate used to incubate the hybridization reactions
in the thermal cycler. This plate will be transferred from the thermal
cycler to the NGS workstation in step 24 below.
100
SureSelectXT2 Automated Library Prep and Capture System
Hybridization
Step 3. Capture the hybridized DNA
4
19 Select the number of columns of samples to be processed. Runs must
include 1, 2, 3, 4, 6, or 12 columns.
20 Click Display Initial Workstation Setup.
21 Verify that the NGS workstation has been set up as displayed in the
Workstation Setup region of the form.
22 When verification is complete, click Run Selected Protocol.
23 When ready to begin the run, click OK in the following window. If the
temperature of Bravo deck position 4 was not pre-set to 66°C, the
runset will pause while position 4 reaches temperature.
SureSelectXT2 Automated Library Prep and Capture System
101
4
Hybridization
Step 3. Capture the hybridized DNA
CA U T I O N
It is important to complete step 24 quickly and carefully. Transfer the sample plate to
the Bravo platform quickly to retain the 65°C sample temperature. Unseal the plate
without tilting or jerking the plate to avoid sample splashing. Make sure that the
Agilent NGS Workstation is completely prepared, with deck platforms at temperature
and all components in place, before you transfer the sample plate to the Bravo deck.
24 When prompted by VWorks as shown below, quickly remove the PCR
plate, containing the hybridization reactions held at 65°C, from the
thermal cycler. Unseal the plate carefully to avoid splashing, and quickly
transfer the plate to position 4 of the Bravo deck, seated in the red
aluminum insert. Click Continue to resume the runset.
WARN I NG
Warning
102
Bravo deck position 4 will be hot.
Use caution when handling components that contact heated deck positions.
SureSelectXT2 Automated Library Prep and Capture System
Hybridization
Step 3. Capture the hybridized DNA
4
25 When the hybridization samples have been transferred from the PCR
plate to the capture plate wells, you will be prompted by VWorks as
shown below. Remove the PCR plate from position 4 of the Bravo deck,
leaving the red insert in place. When finished, click Continue to resume
the runset.
The remainder of the SureSelectCapture&Wash_v2.0.rst runset takes
approximately 1.5 hours. Once the runset is complete, the captured,
bead-bound DNA samples are located in the Eppendorf plate at position 9
of the Bravo deck.
When the runset is complete, seal the plate using the PlateLoc Thermal
Microplate Sealer, with sealing settings of 165°C and 1.0 sec and store the
plate on ice while setting up the next automation protocol.
NOTE
Captured DNA is retained on the streptavidin beads during the post-capture amplification
steps.
SureSelectXT2 Automated Library Prep and Capture System
103
4
104
Hybridization
Step 3. Capture the hybridized DNA
SureSelectXT2 Automated Library Prep and Capture System
SureSelectXT2 Automated Library Prep and Capture System Protocol
5
Post-Capture Sample Processing for
Multiplexed Sequencing
Step 1. Amplify the captured libraries 106
Step 2. Purify the amplified captured libraries using AMPure XP
beads 113
Step 3. Assess quantity and quality of the amplified captured library
pools 117
Step 4. Prepare samples for multiplexed sequencing 121
Step 5. Optional: Quantify captured library pools by QPCR 123
Step 6. Optional: Pool captured libraries for sequencing 124
This chapter describes the steps to amplify, purify, and assess quality and
quantity of the captured libraries. Post-capture dilution and optional
pooling instructions are provided to prepare the indexed samples for
multiplexed sequencing.
Agilent Technologies
105
5
Post-Capture Sample Processing for Multiplexed Sequencing
Step 1. Amplify the captured libraries
Step 1. Amplify the captured libraries
In this step, the Agilent NGS Workstation completes the liquid handling
steps for PCR amplification of the SureSelect-enriched DNA samples. After
the PCR plate is prepared by the Agilent NGS Workstation, you transfer
the plate to a thermal cycler for amplification.
Plan your experiments for amplification of libraries captured using
Capture Libraries of similar sizes on the same plate. See Table 57 for
cycle number recommendations for different Capture Library size ranges.
Prepare the workstation
1 Clear the Labware MiniHub and BenchCel of plates and tip boxes.
2 Gently wipe down the Labware MiniHub, Bravo decks, and BenchCel
with a Nucleoclean decontamination wipe.
3 Turn on the chiller, set to 0°C, at position 9 of the Bravo deck. Be sure
that the chiller reservoir contains at least 300 mL of 25% ethanol.
4 Pre-set the temperature of Bravo deck position 6 to 4°C using the
Inheco Multi TEC control touchscreen, as described in Setting the
Temperature of Bravo Deck Heat Blocks. Bravo deck position 6
corresponds to CPAC 2, position 2 on the Multi TEC control
touchscreen.
Prepare the Post-capture PCR master mix source plate
CA U T I O N
To avoid cross-contaminating libraries, set up PCR master mixes in a dedicated clean
area or PCR hood with UV sterilization and positive air flow.
The post-capture PCR master mix source plate must be a Nunc DeepWell
plate, with the PCR master mix for the run supplied in column 4.
If the Hybridization protocol was run with master mixes configured by
column (Hybridization_MMCol_v2.0.pro), reuse the Nunc DeepWell master
mix source plate used for the Hybridization run. The final configuration of
the master mix source plate for this scenario is shown in Figure 13.
If the Hybridization protocol was run with master mixes configured by
row (Hybridization_MMRow_v2.0.pro), use a new Nunc DeepWell plate.
106
SureSelectXT2 Automated Library Prep and Capture System
Post-Capture Sample Processing for Multiplexed Sequencing
Step 1. Amplify the captured libraries
5
5 Prepare the Post-capture PCR Master Mix by combining SureSelect
Herculase II Master Mix and the XT2 Primer Mix in column 4 of the
master mix source plate. Add the volumes of both reagents shown in
Table 52 to each well of column 4 of the master mix source plate.
Table 52
Preparation of the Master Mix Source Plate for Post-CaptureOnBeadPCR_XT_Illumina_v2.0.pro
SureSelectXT2
Reagent
Position on
Source Plate
Volume of Reagents added per Well of Nunc Deep Well Source Plate
1-Column
Runs
2-Column
Runs
3-Column
Runs
4-Column
Runs
6-Column
Runs
12-Column
Runs
SureSelect
Herculase II
Master Mix
Column 4
37.5 µL
62.5 µL
87.5 µL
112.5 µL
162.5 µL
325 µL
1.5 µL
2.5 µL
3.5 µL
4.5 µL
6.5 µL
13.0 µL
XT2 Primer Mix
(A4-H4)
6 Seal the source plate using the PlateLoc Thermal Microplate Sealer,
with sealing settings of 165°C and 1.0 sec.
7 Vortex the plate for 5 seconds then centrifuge the plate for 30 seconds
to drive the well contents off the walls and plate seal and to eliminate
any bubbles. Keep the master mix source plate on ice.
SureSelectXT2 Automated Library Prep and Capture System
107
5
Post-Capture Sample Processing for Multiplexed Sequencing
Step 1. Amplify the captured libraries
Figure 13
Configuration of the master mix source plate for
Post-CaptureOnBeadPCR_XT_Illumina_v2.0.pro. Columns 1-3 may have been used
to dispense master mixes for the Hybridization_MMCol_v2.0.pro protocol, or
may be empty.
Load the Agilent NGS Workstation
8 Load the Labware MiniHub according to Table 53, using the plate
orientations shown in Figure 6.
NOTE
108
Load a new tip box in Cassete 1, Shelf 1 for the
Post-CaptureOnBeadPCR_XT_Illumina_v2.0.pro protocol. Do not retain a partially-filled tip
box from previous runs.
SureSelectXT2 Automated Library Prep and Capture System
Post-Capture Sample Processing for Multiplexed Sequencing
Step 1. Amplify the captured libraries
Table 53
5
Initial MiniHub configuration for Post-CaptureOnBeadPCR_XT_Illumina_v2.0.pro
Vertical
Shelf
Position
Cassette 1
Cassette 2
Cassette 3
Cassette 4
Shelf 5
(Top)
Empty
Empty
Empty
Empty
Shelf 4
Empty
Empty
Empty
Empty
Shelf 3
Empty
Empty
Empty
Empty
Shelf 2
Empty tip box
Empty
Empty
Empty
Shelf 1
(Bottom)
New tip box
Empty
Empty
Empty tip box
9 Load the BenchCel Microplate Handling Workstation according to
Table 54.
Table 54
Initial BenchCel configuration for Post-CaptureOnBeadPCR_XT_Illumina_v2.0.pro
No. of Columns
Processed
Rack 1
Rack 2
Rack 3
Rack 4
1
1 Tip box
Empty
Empty
Empty
2
1 Tip box
Empty
Empty
Empty
3
2 Tip boxes
Empty
Empty
Empty
4
2 Tip boxes
Empty
Empty
Empty
6
3 Tip boxes
Empty
Empty
Empty
12
6 Tip boxes
Empty
Empty
Empty
SureSelectXT2 Automated Library Prep and Capture System
109
5
Post-Capture Sample Processing for Multiplexed Sequencing
Step 1. Amplify the captured libraries
10 Load the Bravo deck according to Table 55.
Table 55
Initial Bravo deck configuration for Post-CaptureOnBeadPCR_XT_Illumina_v2.0.pro
Location
Content
5
Eppendorf twin.tec plate (unsealed) containing captured, bead-bound DNA
samples
6
Empty PCR plate seated in red insert
(PCR plate type must be specified on setup form under step 2)
9
Master mix plate (unsealed) containing PCR Master Mix in Column 4 seated in
silver insert
Run VWorks protocol Post-CaptureOnBeadPCR_XT_Illumina_v2.0.pro
11 On the SureSelect setup form, under Select Protocol to Run, select
Post-CaptureOnBeadPCR_XT_Illumina_v2.0.pro.
12 Under Select PCR plate labware for Thermal Cycling, select the
specific type of PCR plate that was loaded on Bravo deck position 6.
13 Select the number of columns of samples to be processed. Runs must
include 1, 2, 3, 4, 6, or 12 columns.
14 Click Display Initial Workstation Setup.
15 Verify that the NGS workstation has been set up as displayed in the
Workstation Setup region of the form.
110
SureSelectXT2 Automated Library Prep and Capture System
Post-Capture Sample Processing for Multiplexed Sequencing
Step 1. Amplify the captured libraries
5
16 When verification is complete, click Run Selected Protocol.
Running the Post-CaptureOnBeadPCR_XT_Illumina_v2.0.pro protocol takes
approximately 15 minutes. Once complete, the PCR-ready samples,
containing captured DNA and PCR master mix, are located in the PCR
plate at position 6 of the Bravo deck. The Eppendorf plate containing the
remaining captured DNA samples, which may be stored for future use at
–20°C, is located at position 5 of the Bravo deck.
17 When you see the following prompt, remove the PCR plate from position
6 of the Bravo deck and seal the plate using the PlateLoc Thermal
Microplate Sealer, with sealing settings of 165°C and 3.0 seconds.
18 Centrifuge the plate for 30 seconds to drive the well contents off the
walls and plate seal and to eliminate air bubbles.
SureSelectXT2 Automated Library Prep and Capture System
111
5
Post-Capture Sample Processing for Multiplexed Sequencing
Step 1. Amplify the captured libraries
19 Transfer the PCR plate to a thermal cycler and run the PCR
amplification program shown in Table 56 using the cycle number
specified in Table 57.
The volume of each PCR amplification reaction is 50 µL.
Table 56
Post-Capture PCR cycling program
Segment
Number of
Cycles
Temperature
Time
1
1
98°C
2 minutes
2
8-14
98°C
30 seconds
see Table 57 60°C
30 seconds
72°C
1 minute
3
1
72°C
10 minutes
4
1
4°C
Hold
Table 57
Recommended cycle number based on Capture Library size
Size of Capture Library
Cycles
<0.5 Mb
12 to 14 cycles
0.5 to 1.49 Mb
9 to 11 cycles
> 1.5 Mb (including All Exon and Exome libraries) 8 to 10 cycles
NOTE
112
Amplify the captured DNA using a minimal number of PCR cycles. If yield is too low or
non-specific high molecular weight products are observed, adjust the number of cycles
accordingly with the remaining captured DNA template.
SureSelectXT2 Automated Library Prep and Capture System
Post-Capture Sample Processing for Multiplexed Sequencing
Step 2. Purify the amplified captured libraries using AMPure XP beads
5
Step 2. Purify the amplified captured libraries using AMPure
XP beads
In this step, the Agilent NGS Workstation transfers AMPure XP beads to
the amplified captured DNA and then collects and washes the bead-bound
enriched DNA amplicons.
Prepare the workstation and reagents
1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes.
2 Gently wipe down the Labware MiniHub, Bravo decks, and BenchCel
with a Nucleoclean decontamination wipe.
3 Let the AMPure XP beads come to room temperature for at least
30 minutes.
4 Mix the bead suspension well so that the reagent appears homogeneous
and consistent in color. Do not freeze.
5 Prepare a Nunc DeepWell source plate containing AMPure XP beads.
For each well to be processed, add 95 µL of homogeneous AMPure XP
beads per well to the Nunc DeepWell plate.
6 Prepare a Thermo Scientific reservoir containing 15 mL of nuclease-free
water.
7 Prepare a separate Thermo Scientific reservoir containing 45 mL of
freshly-prepared 70% ethanol.
SureSelectXT2 Automated Library Prep and Capture System
113
5
Post-Capture Sample Processing for Multiplexed Sequencing
Step 2. Purify the amplified captured libraries using AMPure XP beads
8 Load the Labware MiniHub according to Table 58, using the plate
orientations shown in Figure 6.
Table 58
Initial MiniHub configuration for DNA cleanup using
SPRI_XT_Illumina_v2.0.pro:Post-CaptureOnBeadPCR Cleanup
Vertical Shelf
Position
Cassette 1
Cassette 2
Cassette 3
Cassette 4
Shelf 5 (Top)
Empty Nunc
DeepWell plate
Empty
Empty
Empty
Shelf 4
Empty
Empty
Empty
Empty
Shelf 3
Empty
Empty Eppendorf
twin.tec Plate
Empty
Empty
Shelf 2
Empty
Nuclease-free
water reservoir
from step 6
AMPure XP beads
in Nunc DeepWell
plate from step 5
Empty
Shelf 1 (Bottom)
Empty
70% ethanol
reservoir from
step 7
Empty
Empty tip box
9 Load the BenchCel Microplate Handling Workstation according to
Table 59.
Table 59
114
Initial BenchCel configuration for DNA cleanup using
SPRI_XT_Illumina_v2.0.pro:Post-CaptureOnBeadPCR Cleanup
No. of Columns
Processed
Rack 1
Rack 2
Rack 3
Rack 4
1
1 Tip box
Empty
Empty
Empty
2
1 Tip box
Empty
Empty
Empty
3
2 Tip boxes
Empty
Empty
Empty
4
2 Tip boxes
Empty
Empty
Empty
6
3 Tip boxes
Empty
Empty
Empty
12
6 Tip boxes
Empty
Empty
Empty
SureSelectXT2 Automated Library Prep and Capture System
Post-Capture Sample Processing for Multiplexed Sequencing
Step 2. Purify the amplified captured libraries using AMPure XP beads
5
10 Load the Bravo deck according to Table 60.
Table 60
Initial Bravo deck configuration for DNA cleanup using
SPRI_XT_Illumina_v2.0.pro:Post-CaptureOnBeadPCR Cleanup
Location
Content
1
Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)
9
Amplified, captured library pools in unsealed PCR plate seated in red insert
(PCR plate type must be specified on setup form under step 2)
Run VWorks protocol SPRI_XT_Illumina_v2.0.pro:Post-CaptureOnBeadPCR
Cleanup
11 On the SureSelect setup form, under Select Protocol to Run, select
SPRI_XT_Illumina_v2.0.pro:Post-CaptureOnBeadPCR Cleanup.
12 Under Select PCR plate labware for Thermal Cycling, select the
specific type of PCR plate that was loaded on Bravo deck position 9.
13 Select the number of columns of samples to be processed. Runs must
include 1, 2, 3, 4, 6, or 12 columns.
14 Click Display Initial Workstation Setup.
15 Verify that the NGS workstation has been set up as displayed in the
Workstation Setup region of the form.
16 When verification is complete, click Run Selected Protocol.
SureSelectXT2 Automated Library Prep and Capture System
115
5
Post-Capture Sample Processing for Multiplexed Sequencing
Step 2. Purify the amplified captured libraries using AMPure XP beads
The purification protocol takes approximately 45 minutes. When complete,
the amplified DNA samples are in the Eppendorf plate located on Bravo
deck position 7.
116
SureSelectXT2 Automated Library Prep and Capture System
Post-Capture Sample Processing for Multiplexed Sequencing
Step 3. Assess quantity and quality of the amplified captured library pools
5
Step 3. Assess quantity and quality of the amplified captured
library pools
Option 1: Analysis using the Agilent 2100 Bioanalyzer and High Sensitivity DNA
Assay
1 Set up the 2100 Bioanalyzer as instructed in the High Sensitivity DNA
Assay kit guide.
NOTE
Version B.02.07 or higher of the Agilent 2100 Expert Software is required for High
Sensitivity DNA Assay Kit runs.
2 Seal the sample plate using the PlateLoc Thermal Microplate Sealer,
with sealing settings of 165°C and 1.0 sec.
3 Vortex the plate to mix samples in each well, then centrifuge the plate
for 30 seconds to drive the well contents off the walls and plate seal.
4 Prepare the chip, samples and ladder as instructed in the reagent kit
guide, using 1 µL of a ten-fold dilution of each sample for the analysis.
NOTE
Dilute 1 µL of the sample in 9 µL of 10 mM Tris, 1 mM EDTA prior to analysis. Be sure to mix
well by vortexing at 2000 rpm on the IKA vortex supplied with the Bioanalyzer before
analyzing the diluted samples.
5 Load the prepared chip into the 2100 Bioanalyzer and start the run
within five minutes after preparation.
6 Verify that the electropherogram shows an average DNA amplicon size
of approximately 250 to 300 bp. A sample electropherogram is shown in
Figure 14.
7 Determine the concentration of each amplified captured library pool by
integration under the peak in the electropherogram.
If the yield is too low or non-specific peaks are observed in the
electropherogram, repeat the PCR with more or fewer cycles. The goal is
to minimize cycles, while you produce enough library for application to
the flow cell.
Stopping Point
If you do not continue to the next step, seal the plate and store at 4°C
overnight or at –20°C for prolonged storage.
SureSelectXT2 Automated Library Prep and Capture System
117
5
Post-Capture Sample Processing for Multiplexed Sequencing
Step 3. Assess quantity and quality of the amplified captured library pools
Figure 14
118
Analysis of amplified captured DNA using the 2100 Bioanalyzer and the High
Sensitivity DNA Assay.
SureSelectXT2 Automated Library Prep and Capture System
Post-Capture Sample Processing for Multiplexed Sequencing
Step 3. Assess quantity and quality of the amplified captured library pools
5
Option 2: Analysis using the Agilent 2200 TapeStation and High Sensitivity D1000
ScreenTape
Use a High Sensitivity D1000 ScreenTape (p/n 5067-5584) and reagent kit
(p/n 5067-5585) to analyze the amplified captured DNA. For more
information to do this step, see the Agilent 2200 TapeStation User
Manual.
1 Seal the DNA sample plate using the PlateLoc Thermal Microplate
Sealer, with sealing settings of 165°C and 1.0 sec.
2 Vortex the plate to mix samples in each well, then centrifuge the plate
for 30 seconds to drive the well contents off the walls and plate seal.
3 Prepare the TapeStation samples as instructed in the Agilent 2200
TapeStation User Manual. Use 2 µL of each DNA sample diluted with
2 µL of High Sensitivity D1000 sample buffer for the analysis.
CA U T I O N
Make sure that you thoroughly mix the combined DNA and High Sensitivity D1000
sample buffer on a vortex mixer for 5 seconds for accurate quantitation.
4 Load the sample plate or tube strips from step 3, the High Sensitivity
D1000 ScreenTape, and loading tips into the 2200 TapeStation as
instructed in the Agilent 2200 TapeStation User Manual. Start the run.
5 Verify that the electropherogram shows an average DNA amplicon size
of 250 to 300 bp. A sample electropherogram is shown in Figure 15.
6 Determine the concentration of each amplified captured library pool by
integration under the peak in the electropherogram.
If the yield is too low or non-specific peaks are observed in the
electropherogram, repeat the PCR with more or fewer cycles. The goal is
to minimize cycles, while you produce enough library for application to
the flow cell.
Stopping Point
If you do not continue to the next step, seal the indexed DNA sample
plate and store at 4°C overnight or at –20°C for prolonged storage.
SureSelectXT2 Automated Library Prep and Capture System
119
5
Post-Capture Sample Processing for Multiplexed Sequencing
Step 3. Assess quantity and quality of the amplified captured library pools
Figure 15
120
Analysis of amplified captured DNA using the 2200 TapeStation.
SureSelectXT2 Automated Library Prep and Capture System
Post-Capture Sample Processing for Multiplexed Sequencing
Step 4. Prepare samples for multiplexed sequencing
5
Step 4. Prepare samples for multiplexed sequencing
The final SureSelectXT2-enriched samples contain pools of either 8 or 16
indexed libraries, based on the Capture Library used and resulting
pre-capture pooling strategy. When appropriate for your sequencing
platform, the 8-plex or 16-plex samples may be further multiplexed by
post-capture pooling.
Determine whether to do post-capture pooling by calculating the number
of indexes that can be combined per lane, according to the capacity of
your platform, together with the amount of sequencing required to achieve
the needed coverage for your specific Capture Library for each indexed
sample.
If doing post-capture pooling, use the guidelines provided in “Step 6.
Optional: Pool captured libraries for sequencing” on page 124. Prior to
post-capture pooling, the DNA concentration of each sample may be
accurately determined as described in “Step 5. Optional: Quantify captured
library pools by QPCR” on page 123.
If samples will not be further combined in post-capture pools, proceed to
cluster amplification using the Illumina Paired-End Cluster Generation Kit.
Refer to the manufacturer’s instructions for this step.
The optimal seeding concentration for SureSelectXT2 target-enriched
libraries is 6 to 8 pM on HiSeq or MiSeq instruments and 1.2 to 1.3 pM
on the NextSeq platform. 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. Follow Illumina’s
recommendation for a PhiX control in a low-concentration spike-in for
improved sequencing quality control.
Sequencing run setup guidelines
Sequencing runs must be set up to perform an 8-bp index read. For
complete 8-bp index sequence information, see the Reference chapter
starting on page 127.
SureSelectXT2 Automated Library Prep and Capture System
121
5
Post-Capture Sample Processing for Multiplexed Sequencing
Step 4. Prepare samples for multiplexed sequencing
For the HiSeq 2500 and NextSeq 500 (v1) platforms, use the Cycles
settings shown in Table 61. Cycle number settings can be specified on the
Run Configuration screen of the instrument control software interface
after choosing Custom from the index type selection buttons..
Table 61
Cycle Number settings for HiSeq and NextSeq platforms
Run Segment
Cycle Number
Read 1
100
Index 1 (i7)
9
Index 2 (i5)
0
Read 2
100
For the MiSeq platform, use the Illumina Experiment Manager (IEM)
software to generate a Sample Sheet that includes the run parameters
specified in Table 62.
Table 62
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)
122
Type the 8-nt index sequence for each individual sample (see the
index sequence tables in the Reference chapter starting on
page 127).
SureSelectXT2 Automated Library Prep and Capture System
Post-Capture Sample Processing for Multiplexed Sequencing
Step 5. Optional: Quantify captured library pools by QPCR
5
Step 5. Optional: Quantify captured library pools by QPCR
For accurate determination of the DNA concentration in each captured
library pool, use the QPCR NGS Library Quantification Kit (for Illumina).
Refer to the protocol that is included with the 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 captured library pool 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 captured library pool, in nM.
The concentration will be used to accurately pool samples for
multiplexed sequencing.
NOTE
In most cases, the cycle numbers in Table 57 will produce an adequate yield for sequencing
without introducing bias or non-specific products. If yield is too low or non-specific
products are observed, adjust the number of cycles accordingly with the remaining
captured DNA template.
SureSelectXT2 Automated Library Prep and Capture System
123
5
Post-Capture Sample Processing for Multiplexed Sequencing
Step 6. Optional: Pool captured libraries for sequencing
Step 6. Optional: Pool captured libraries for sequencing
See page 121 for post-capture pooling considerations, based on your
SureSelect or ClearSeq Capture Library size and sequencing design.
Pooling instructions are provided below.
1 Combine the capture pools such that each index-tagged sample is
present in equimolar amounts in the final sequencing sample pool. For
each final pool, use the formula below to determine the amount of each
capture pool to use.
 f   C f 
Volume of capture pool = V
--------------------------------- where
#  C i
where V(f) is the final desired volume of the sequencing sample pool,
C(f) is the desired final concentration of all the DNA in the pool
# is the number of capture pool samples to be combined, and
C(i) is the initial concentration of each capture pool sample.
2 Adjust the final volume of the pooled library to the desired final
concentration.
• 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.
Table 63 shows an example of the amount of 2 capture pool samples (of
different concentrations) and Low TE needed for a final volume of
20 µL at 10 nM final DNA concentration.
124
SureSelectXT2 Automated Library Prep and Capture System
Post-Capture Sample Processing for Multiplexed Sequencing
Step 6. Optional: Pool captured libraries for sequencing
Table 63
5
Example of capture pool volume calculations for a 20-µL final sequencing sample
pool containing 10 nM DNA
Component
V(f)
C(i)
C(f)
#
Volume to use (µL)
Sample 1
20 µL
20 nM
10 nM
6
5.0
Sample 2
20 µL
15 nM
10 nM
6
6.7
Low TE
8.3
3 If you store the library before sequencing, add Tween 20 to 0.1% v/v
and store at –20°C short term.
Proceed to cluster amplification using the Illumina Paired-End Cluster
Generation Kit; refer to the manufacturer’s instructions for this step. The
optimal seeding concentration for cluster amplification from SureSelectXT2
DNA libraries is approximately 6 to 8 pM.
NOTE
The optimal seeding concentration may vary, depending on the method used for library
quantification and fragment size distribution.
See page 121 for sequencing run setup guidelines for SureSelectXT2
libraries.
SureSelectXT2 Automated Library Prep and Capture System
125
5
126
Post-Capture Sample Processing for Multiplexed Sequencing
Step 6. Optional: Pool captured libraries for sequencing
SureSelectXT2 Automated Library Prep and Capture System
SureSelectXT2 Automated Library Prep and Capture System Protocol
6
Reference
Reference Information for Kits with Revised Index Configuration (indexing
primers in blue plate) 128
Reference Information for Kits with Original Index Configuration (indexing
primers in clear-capped tubes) 132
This chapter contains reference information, including component kit
contents and index sequences.
Agilent Technologies
127
6
Reference
Reference Information for Kits with Revised Index Configuration (indexing primers in blue plate)
CA U T I O N
This chapter contains two sets of index sequence and kit content information. The first
section covers kits with indexing primers supplied in a blue plate format in Library Prep
Kit p/n 5500-0131 (typically received February, 2015 or later). The second section
covers kits with indexing primers supplied in tube format, in p/n 5190-3936 and
p/n 5190-3937 (typically received before February, 2015). Verify that you are
referencing the information appropriate for your kit version before you proceed.
Reference Information for Kits with Revised Index Configuration
(indexing primers in blue plate)
Use the reference information in this section if your kit includes Library Prep Kit p/n
5500-0131. If your kit does not include this component kit, see page 132 for kit content and
indexing primer information.
Kit Contents
SureSelectXT2 Automation Reagent Kits contain the following components:
Table 64
SureSelectXT2 Automation Reagent Kit Content-Revised Index Configuration
Component Kits*
Storage Condition
G9661B
(96 Samples)†
G9661C
(480 Samples)‡
SureSelect XT2 Library Prep Kit, ILM
–20°C
5500-0131
5 x 5500-0131
SureSelect XT2 Pre-Capture Box 1
Room Temperature 5190-4076
5190-4077
SureSelect XT2 Pre-Capture Automation-ILM Module Box 2
–20°C
5190-4463
5190-4462
* See Table 65 through Table 67 for a list of reagents included in each component kit.
† Kits contain reagents to prepare indexed libraries from 96 gDNA samples and to enrich the samples in 6 or 12 hybridization
and capture reactions (as appropriate for the specific Capture Library size and sample pooling format).
‡ Kits contain reagents to prepare indexed libraries from 480 gDNA samples and to enrich the samples in 30 or 60 hybridization
and capture reactions (as appropriate for the specific Capture Library size and sample pooling format).
NOTE
128
SureSelect reagents and Capture Libraries must be used within one year of receipt.
SureSelectXT2 Automated Library Prep and Capture System
Reference
Kit Contents
6
The contents of each of the component kits listed in Table 64 are
described in the tables below.
Table 65
SureSelect XT2 Library Prep Kit, ILM Content-Revised Configuration
Kit Component
Format
SureSelect End Repair Enzyme Mix
bottle
SureSelect End Repair Nucleotide Mix*
tube with green cap
SureSelect dA-Tailing Master Mix
bottle
SureSelect Ligation Master Mix
tube with purple cap
SureSelect Herculase II Master Mix
bottle
XT2 Primer Mix
tube with clear cap
SureSelect Pre-Capture Indexed Adaptors†
Indexes A01 through H12,
provided in blue 96-well plate‡
* May also be labeled as SureSelect End Repair Oligo Mix.
† See Table 69 on page 131 for index sequences.
‡ See Table 68 on page 130 for a plate map.
Table 66
SureSelect XT2 Pre-Capture Box 1 Content
Kit Component
Format
SureSelect XT2 Binding Buffer
bottle
SureSelect XT2 Wash 1
bottle
SureSelect XT2 Wash 2
bottle
Table 67
SureSelect XT2 Pre-Capture Automation-ILM Module Box 2 Content
Kit Component
96 Sample Kit
480 Sample Kit
SureSelect XT2 Blocking Mix
tube with blue cap
tube with blue cap
SureSelect XT2 Hybridization Buffer
tube with yellow cap
bottle
SureSelect RNase Block
tube with purple cap
tube with purple cap
SureSelectXT2 Automated Library Prep and Capture System
129
6
Reference
Kit Contents
Table 68
Plate map for indexed adaptors containing indexes A01 through H12 (blue plate in Library Prep kit p/n 5500-0131)
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
130
SureSelectXT2 Automated Library Prep and Capture System
Reference
Nucleotide Sequences of SureSelectXT2 Indexes A01 to H12
6
Nucleotide Sequences of SureSelectXT2 Indexes A01 to H12
Each index is 8 nt in length. See page 121 for sequencing run setup
information using 8-bp indexes.
Table 69
SureSelectXT2 Indexes, for indexing primers provided in blue 96-well plate
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
SureSelectXT2 Automated Library Prep and Capture System
131
6
Reference
Reference Information for Kits with Original Index Configuration (indexing primers in clear-capped tubes)
Reference Information for Kits with Original Index Configuration
(indexing primers in clear-capped tubes)
Use the reference information in this section if your kit includes Pre-Capture Indexes in
tube format in p/n 5190-3936 and p/n 5190-3937. If your kit does not include these
component kits, see page 128 for kit content and indexing primer information.
Kit Contents
SureSelectXT2 Automation Reagent Kits contain the following component
kits:
Table 70
SureSelectXT2 Automation Reagent Kit Content-Original Index Configuration
Component Kits*
Storage Condition
G9661B
(96 Samples)†
G9661C
(480 Samples)‡
SureSelect XT2 Library Prep Kit, ILM
–20°C
5500-0103
5 x 5500-0103
SureSelect XT2 Pre-Capture Indexes, ILM**
–20°C
5190-3936 and
5 x 5190-3936 and
5190-3937
5 x 5190-3937
SureSelect XT2 Pre-Capture Box 1
Room Temperature 5190-4076
5190-4077
SureSelect XT2 Pre-Capture Automation-ILM Module Box 2
–20°C
5190-4463
5190-4462
* See Table 71 through Table 74 for a list of reagents included in each component kit.
† Kits contain reagents to prepare indexed libraries from 96 gDNA samples and to enrich the samples in 6 or 12 hybridization
and capture reactions (as appropriate for the specific Capture Library size and sample pooling format).
‡ Kits contain reagents to prepare indexed libraries from 480 gDNA samples and to enrich the samples in 30 or 60 hybridization
and capture reactions (as appropriate for the specific Capture Library size and sample pooling format).
** See Table 75 on page 134 through Table 80 on page 139 for index sequence information.
NOTE
132
SureSelect reagents and Capture Libraries must be used within one year of receipt.
SureSelectXT2 Automated Library Prep and Capture System
Reference
Kit Contents
6
The contents of each of the component kits listed in Table 70 are
described in the tables below.
Table 71
SureSelect XT2 Library Prep Kit, ILM Content-Original Index Configuration
Kit Component
Format
SureSelect End-Repair Master Mix
bottle
SureSelect dA-Tailing Master Mix
bottle
SureSelect Ligation Master Mix
tube with purple cap
SureSelect Herculase II Master Mix
bottle
XT2 Primer Mix
tube with clear cap
Table 72
SureSelect XT2 Pre-Capture Indexes Content-Original Index Configuration
Kit Component
Format
Indexes 1-48
48 clear-capped tubes, supplied in component kit 5190-3936
Indexes 49-96
48 clear-capped tubes, supplied in component kit 5190-3937
Table 73
SureSelect XT2 Pre-Capture Box 1 Content
Kit Component
Format
SureSelect XT2 Binding Buffer
bottle
SureSelect XT2 Wash 1
bottle
SureSelect XT2 Wash 2
bottle
Table 74
SureSelect XT2 Pre-Capture Automation-ILM Module Box 2 Content
Kit Component
96 Sample Kit
480 Sample Kit
SureSelect XT2 Blocking Mix
tube with blue cap
tube with blue cap
SureSelect XT2 Hybridization Buffer
tube with yellow cap
bottle
SureSelect RNase Block
tube with purple cap
tube with purple cap
SureSelectXT2 Automated Library Prep and Capture System
133
6
Reference
Nucleotide Sequences of SureSelect Pre-Capture Indexes-Original Index Configuration
Nucleotide Sequences of SureSelect Pre-Capture
Indexes-Original Index Configuration
The nucleotide sequence of each SureSelectXT2 Pre-Capture Index provided
with the original kit configuration is provided in the tables below.
Refer to the sequence information below only if your kit includes p/n
5190-3936 and p/n 5190-3937, with indexing primers provided in 96
individual clear-capped tubes.
Each index is 8 nt in length. Each index is 8 nt in length. See page 121
for sequencing run setup information using 8-bp indexes.
Table 75
134
SureSelectXT2 Pre-capture Indexes 1-16
Index Number
Sequence
1
AACGTGAT
2
AAACATCG
3
ATGCCTAA
4
AGTGGTCA
5
ACCACTGT
6
ACATTGGC
7
CAGATCTG
8
CATCAAGT
9
CGCTGATC
10
ACAAGCTA
11
CTGTAGCC
12
AGTACAAG
13
AACAACCA
14
AACCGAGA
15
AACGCTTA
16
AAGACGGA
SureSelectXT2 Automated Library Prep and Capture System
Reference
Nucleotide Sequences of SureSelect Pre-Capture Indexes-Original Index Configuration
Table 76
6
SureSelectXT2 Pre-capture Indexes 17-32
Index Number
Sequence
17
AAGGTACA
18
ACACAGAA
19
ACAGCAGA
20
ACCTCCAA
21
ACGCTCGA
22
ACGTATCA
23
ACTATGCA
24
AGAGTCAA
25
AGATCGCA
26
AGCAGGAA
27
AGTCACTA
28
ATCCTGTA
29
ATTGAGGA
30
CAACCACA
31
CAAGACTA
32
CAATGGAA
SureSelectXT2 Automated Library Prep and Capture System
135
6
Reference
Nucleotide Sequences of SureSelect Pre-Capture Indexes-Original Index Configuration
Table 77
136
SureSelectXT2 Pre-capture Indexes 33-48
Index Number
Sequence
33
CACTTCGA
34
CAGCGTTA
35
CATACCAA
36
CCAGTTCA
37
CCGAAGTA
38
CCGTGAGA
39
CCTCCTGA
40
CGAACTTA
41
CGACTGGA
42
CGCATACA
43
CTCAATGA
44
CTGAGCCA
45
CTGGCATA
46
GAATCTGA
47
GACTAGTA
48
GAGCTGAA
SureSelectXT2 Automated Library Prep and Capture System
Reference
Nucleotide Sequences of SureSelect Pre-Capture Indexes-Original Index Configuration
Table 78
6
SureSelectXT2 Pre-capture Indexes 49-64
Index Number
Sequence
49
GATAGACA
50
GCCACATA
51
GCGAGTAA
52
GCTAACGA
53
GCTCGGTA
54
GGAGAACA
55
GGTGCGAA
56
GTACGCAA
57
GTCGTAGA
58
GTCTGTCA
59
GTGTTCTA
60
TAGGATGA
61
TATCAGCA
62
TCCGTCTA
63
TCTTCACA
64
TGAAGAGA
SureSelectXT2 Automated Library Prep and Capture System
137
6
Reference
Nucleotide Sequences of SureSelect Pre-Capture Indexes-Original Index Configuration
Table 79
138
SureSelectXT2 Pre-capture Indexes 65-80
Index Number
Sequence
65
TGGAACAA
66
TGGCTTCA
67
TGGTGGTA
68
TTCACGCA
69
AACTCACC
70
AAGAGATC
71
AAGGACAC
72
AATCCGTC
73
AATGTTGC
74
ACACGACC
75
ACAGATTC
76
AGATGTAC
77
AGCACCTC
78
AGCCATGC
79
AGGCTAAC
80
ATAGCGAC
SureSelectXT2 Automated Library Prep and Capture System
Reference
Nucleotide Sequences of SureSelect Pre-Capture Indexes-Original Index Configuration
Table 80
6
SureSelectXT2 Pre-capture Indexes 81-96
Index Number
Sequence
81
ATCATTCC
82
ATTGGCTC
83
CAAGGAGC
84
CACCTTAC
85
CCATCCTC
86
CCGACAAC
87
CCTAATCC
88
CCTCTATC
89
CGACACAC
90
CGGATTGC
91
CTAAGGTC
92
GAACAGGC
93
GACAGTGC
94
GAGTTAGC
95
GATGAATC
96
GCCAAGAC
SureSelectXT2 Automated Library Prep and Capture System
139
www.agilent.com
In This Book
This guide contains
information to run the
SureSelectXT2 Automated
Library Prep and Capture
System protocol using the
automation protocols
provided with the Agilent
NGS Workstation Option
B.
Agilent Technologies, Inc. 2015
Version B1, June 2015
*G9450-90000*
G9450-90000
Agilent Technologies
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