SureSelect Automated Target Enrichment for Illumina Paired

SureSelect Automated Target Enrichment for Illumina Paired
SureSelectXT Automated
Target Enrichment for
Illumina Paired-End
Multiplexed Sequencing
Automated using Agilent NGS
Workstation Option B
Protocol
Version G.2, April 2015
SureSelect platform manufactured with Agilent
SurePrint Technology
Research Use Only. Not for use in Diagnostic
Procedures.
Agilent Technologies
Notices
© Agilent Technologies, Inc. 2015
Warranty
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Manual Part Number
G7550-90000
Edition
Version G.2, April 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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Notice to Purchaser
Research Use Only. Not for use in diagnostic
procedures.
SureSelect capture libraries and reagents
must be used within one year of receipt.
2
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SureSelect XT Automated Library Prep and Capture System
In this Guide...
This guide describes an optimized protocol for Illumina
paired-end multiplexed library preparation using the Agilent
SureSelectXT 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 Agilent NGS
Workstation Option B.
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 SureSelect target enrichment
protocol, and considerations for designing SureSelect
experiments for automated processing using the Agilent NGS
Workstation.
3
Sample Preparation (3 µg DNA Samples)
This chapter describes the steps to prepare the DNA samples
for target enrichment when starting with 3 g of gDNA.
4
Sample Preparation (200 ng DNA Samples)
This chapter describes the steps to prepare the DNA samples
for target enrichment when starting with 200 ng of gDNA.
SureSelect XT Automated Library Prep and Capture System
3
5
Hybridization
This chapter describes the steps to hybridize and capture
samples.
6
Indexing
This chapter describes the steps to amplify, purify, and
assess quality of the sample libraries. Samples are pooled by
mass prior to sequencing.
7
Reference
This chapter contains reference information.
4
SureSelect XT Automated Library Prep and Capture System
What’s New in Version G.2
• Support for the OneSeq Capture Libraries (see Table 3 on
page 14 and Table 75 on page 141).
• Support for ClearSeq Capture Libraries, including
ClearSeq Comprehensive Cancer XT Libraries (see Table 2
on page 13).
• Correction to ordering information for Axygen 96 Deep
Well Plates (see Table 4 on page 15).
• Updates to workflow diagram (see Figure 2 on page 29).
• Updates to sequencing data guidelines (see page 133).
What’s New in Version G.1
• Support for kits with either 8-bp indexes A01 to H12
(revised index configuration, typically received December
2014 or later) or 6-bp indexes 1 to 16 (original index
configuration, typically received before December 2014).
Kits with 8-bp indexes include 96 indexing primers
provided in a blue plate format. For indexing protocol
details, see page 134. For kit content details see
page 154. For nucleotide sequences of the 8-bp indexes,
see Table 86 on page 158. User guide version G.1
includes updates to the 8-bp index sequences in
Table 86 on page 158. Do not use version G.0 for 8-bp
index sequence information.
Kits with 6-bp indexes include 16 indexing primers
provided in clear-capped tubes. For indexing protocol
details, see page 134. For kit content details see
page 159. For nucleotide sequences of the 6-bp indexes,
see Table 91 on page 162.
• Updates to sequencing sample and run setup guidelines
(see page 152).
• Removal of SureSelect Elution Buffer and SureSelect
Neutralization Buffer from SureSelect Target Enrichment
Box 1 (p/n 5190-8646, provided with kits with revised
index configuration; see Table 83 on page 156).
SureSelect XT Automated Library Prep and Capture System
5
6
SureSelect XT Automated Library Prep and Capture System
Content
1
Before You Begin
9
Procedural Notes 10
Safety Notes 11
Required Reagents 12
Required Equipment 15
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
17
22
Overview of the SureSelect Target Enrichment Procedure
28
Experimental Setup Considerations for Automated Runs 31
Considerations for Placement of gDNA Samples in 96-well Plates for
Automated Processing 32
Considerations for Equipment Setup 32
PCR Plate Type Considerations 33
3
Sample Preparation (3 µg DNA Samples)
35
Step 1. Shear DNA 36
Step 2. Purify sheared DNA using AMPure XP beads 39
Step 3. Assess sample quality (optional) 44
Step 4. Modify DNA ends for target enrichment 47
Step 5. Amplify adaptor-ligated libraries 55
Step 6. Purify amplified DNA using AMPure XP beads 63
Step 7. Assess Library DNA quantity and quality 66
4
Sample Preparation (200 ng DNA Samples)
Step 1. Shear DNA 72
Step 2. Assess sample quality (optional)
SureSelect XT Automated Library Prep and Capture System
71
75
7
Contents
Step 3. Modify DNA ends for target enrichment 78
Step 4. Amplify adaptor-ligated libraries 87
Step 5. Purify amplified DNA using AMPure XP beads
Step 6. Assess Library DNA quantity and quality 98
5
Hybridization
95
103
Step 1. Aliquot prepped DNA samples for hybridization 104
Step 2. Hybridize DNA Samples to the Capture Library 108
Step 3. Capture the hybridized DNA 123
6
Indexing
131
Step 1. Amplify the captured libraries to add index tags 132
Step 2. Purify the amplified indexed libraries using Agencourt AMPure XP
beads 142
Step 3. Assess indexed DNA quality 146
Step 4. Quantify each index-tagged library by QPCR 150
Step 5. Pool samples for Multiplexed Sequencing 151
Guidelines for sequencing sample preparation and run setup 152
7
Reference
153
Reference Information for Kits with Revised Index Configuration (8-bp indexes with
indexing primers in blue plate) 154
Kit Contents 154
Nucleotide Sequences of SureSelectXT 8-bp Indexes 158
Reference Information for Kits with Original Index Configuration (6-bp indexes with
indexing primers in 16 tubes) 159
Kit Contents 159
Nucleotide Sequences of SureSelectXT 6-bp Indexes 162
8
SureSelect XT Automated Library Prep and Capture System
SureSelectXT Automated Target Enrichment for Illumina Paired-End
Multiplexed Sequencing Protocol
1
Before You Begin
Procedural Notes 10
Safety Notes 11
Required Reagents 12
Required Equipment 15
Make sure you read and understand the information in this chapter and
have the necessary equipment and reagents listed before you start an
experiment.
CA U T I O N
This Protocol supports the SureSelect Target Enrichment workflow with on-bead
post-capture PCR, using version 1.5.1 (v1.5.1) VWorks SureSelect automation
protocols.
If your VWorks SureSelect setup form displays earlier versions of the automation
protocols, please contact [email protected] for assistance.
NOTE
This protocol describes automated sample processing using the Agilent NGS Workstation.
For non-automated sample processing procedures for Agilent's SureSelectXT Target
Enrichment Kit for Illumina Multiplex Sequencing, see publication G7530-90000.
NOTE
This protocol differs from other SureSelect protocols at several steps. Pay close attention
to the primers used for each amplification step and the blocking agents used during
hybridization.
Agilent Technologies
9
1
Before You Begin
Procedural Notes
Procedural Notes
• This User Guide includes protocols for library preparation using either
3 g DNA samples (see Chapter 3 on page 35) or 200 ng DNA samples
(see Chapter 4 on page 71). Make sure that you are following the
appropriate protocol for your DNA input amount. After the prepared
libraries are amplified, both DNA input options use the same protocol
for hybridization and post-capture processing.
• 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 3 on page 41.
• 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 stock solutions and 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 frozen reagent stock solutions for use:
1 Thaw the aliquot as rapidly as possible without heating above room
temperature.
2 Mix briefly on a vortex mixer, then spin in a centrifuge for 5 to
10 seconds to drive the contents off of walls and lid.
3 Store on ice or in a cold block until use.
• In general, follow Biosafety Level 1 (BL1) safety rules.
10
SureSelect XT Automated Library Prep and Capture System
Before You Begin
Safety Notes
1
Safety Notes
CA U T I O N
• Wear appropriate personal protective equipment (PPE) when working in the
laboratory.
SureSelect XT Automated Library Prep and Capture System
11
1
Before You Begin
Required Reagents
Required Reagents
Table 1
Required Reagents
Description
Vendor and part number
SureSelect, ClearSeq or OneSeq Capture Library*
Select the appropriate library from
Table 2 or Table 3
SureSelectXT Automation Reagent Kit*†
HiSeq platform (HSQ), 96 reactions
HiSeq platform (HSQ), 480 reactions
MiSeq platform (MSQ), 96 reactions
MiSeq platform (MSQ), 480 reactions
Agilent p/n G9641B
Agilent p/n G9641C
Agilent p/n G9642B
Agilent p/n G9642C
Herculase II Fusion DNA Polymerase, 400 reactions
(includes dNTP mix and 5x Buffer)
Agilent p/n 600679
QPCR NGS Library Quantification Kit (Illumina GA)
Agilent p/n G4880A
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 12090-015, or
equivalent
Agencourt AMPure XP Kit
60 mL
450 mL
Beckman Coulter Genomics
p/n A63881
p/n A63882
Quant-iT dsDNA BR Assay Kit, for use with the Qubit
fluorometer
100 assays, 2-1000 ng
500 assays, 2-1000 ng
Life Technologies
Cat #Q32850
Cat #Q32853
Dynabeads MyOne Streptavidin T1
2 mL
10 mL
100 mL
Life Technologies
Cat #65601
Cat #65602
Cat #65603
100% Ethanol, molecular biology grade
Sigma-Aldrich p/n E7023
* SureSelect, ClearSeq, and OneSeq reagents must be used within one year of receipt.
† Each 96-reaction kit contains sufficient reagents for 96 reactions used in runs that include at least
3 columns of samples per run.
12
SureSelect XT Automated Library Prep and Capture System
Before You Begin
Required Reagents
Table 2
SureSelectXT Automation Capture Libraries
Capture Library
SureSelect
XT Clinical Research Exome
SureSelectXT Focused Exome
96 Reactions
480 Reactions
5190-7344
–
5190-7789
–
SureSelect
XT Focused Exome Plus 1
5190-7792
–
SureSelect
XT Focused Exome Plus 2
5190-7796
–
SureSelect
XT Human All Exon v5
5190-6210
–
SureSelectXT Human All Exon v5 + UTRs
5190-6215
–
SureSelect
XT Human All Exon v5 + lncRNA
5190-6448
–
SureSelect
XT Human All Exon v5 Plus
5190-6224
–
SureSelect
XT Human All Exon v4
5190-4633
5190-4635
SureSelectXT Human All Exon v4 + UTRs
5190-4638
5190-4640
SureSelect
XT Mouse All Exon
5190-4643
5190-4645
SureSelect
XT Custom 1 kb up to 499 kb
5190-4808
5190-4810
(5190-4813)
(5190-4815)
(reorder)
XT Custom 0.5 Mb up to 2.9 Mb
5190-4818
5190-4820
(reorder)
(5190-4823)
(5190-4825)
SureSelectXT Custom 3 Mb up to 5.9 Mb
5190-4828
5190-4830
(reorder)
(5190-4833)
(5190-4835)
5190-4838
5190-4840
(5190-4843)
(5190-4845)
5190-4898
5190-4900
(5190-4903)
(5190-4905)
SureSelect
SureSelect
XT Custom 6 Mb up to 11.9 Mb
(reorder)
SureSelect
1
XT Custom 12 Mb up to 24 Mb
(reorder)
SureSelect XT Automated Library Prep and Capture System
13
1
Before You Begin
Required Reagents
Table 3
14
Compatible ClearSeq and OneSeq Automation Capture Libraries
Capture Library
96 Reactions
480 Reactions
ClearSeq Comprehensive Cancer XT
5190-8013
–
ClearSeq Comprehensive Cancer Plus XT
5190-8016
–
ClearSeq Inherited Disease XT
5190-7520
–
ClearSeq Inherited Disease Plus XT
5190-7523
–
ClearSeq DNA Kinome XT
5190-4648
OneSeq Constitutional Research Panel
5190-8704
–
OneSeq Hi Res CNV Backbone + Custom 1–499 kb
5190-8888
–
OneSeq Hi Res CNV Backbone + Custom 0.5 –2.9 Mb
5190-8891
–
OneSeq Hi Res CNV Backbone + Custom 3–5.9 Mb
5190-8894
–
OneSeq Hi Res CNV Backbone + Custom 6–11.9 Mb
5190-8897
–
5190-4650
SureSelect XT Automated Library Prep and Capture System
Before You Begin
Required Equipment
1
Required Equipment
Table 4
Required Equipment
Description
Vendor and part number
Agilent NGS Workstation Option B, with VWorks
software version 11.3.0.1195
Agilent p/n G5522A
Contact Agilent Automation Solutions for
more information:
[email protected]
Robotic Pipetting Tips (Sterile, Filtered, 250 L)
Agilent p/n 19477-022
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 Table 9 on page 33 for a list of supported PCR
plates for automation protocols
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 mL, Square Well
(waste reservoirs; working volume 2.2 mL)
Axygen p/n P-2ML-SQ-C
E & K Scientific p/n EK-2440
DNA LoBind Tubes, 1.5-mL PCR clean, 250 pieces
Eppendorf p/n 022431021 or equivalent
Qubit Fluorometer
Life Technologies p/n Q32857
Qubit assay tubes
Life Technologies p/n Q32856
Covaris Sample Preparation System, S-series of
E-series model
Covaris
Covaris sample holders
96 microTUBE plate (E-series only)
Covaris p/n 520078
microTUBE for individual sample processing
Covaris p/n 520045
SureSelect XT Automated Library Prep and Capture System
15
1
Before You Begin
Required Equipment
Table 4
Required Equipment (continued)
Description
Vendor and part number
DNA Analysis Platform and Consumables
Agilent 2100 Bioanalyzer Laptop Bundle
Agilent p/n G2943CA
Agilent 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
Agilent 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
High Sensitivity D1000 Reagents
Agilent p/n 5067-5585
OR
Centrifuge
Eppendorf Centrifuge model 5804 or
equivalent
P10, P20, P200 and P1000 pipettes
Pipetman P10, P20, P200, P1000 or
equivalent
Vacuum concentrator
Savant SpeedVac, model DNA120, with
96-well plate rotor, model RD2MP, or
equivalent
Magnetic separator
DynaMag-50 magnet, Life Technologies p/n
123-02D or equivalent
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
NucleoClean Decontamination Wipes
Millipore p/n 3097
Ice bucket
Powder-free gloves
Sterile, nuclease-free aerosol barrier pipette tips
Vortex mixer
Timer
16
SureSelect XT Automated Library Prep and Capture System
SureSelectXT Automated Target Enrichment for Illumina Paired-End
Multiplexed Sequencing Protocol
2
Using the Agilent NGS Workstation for
SureSelect Target Enrichment
About the Agilent NGS Workstation 18
Overview of the SureSelect Target Enrichment Procedure 28
Experimental Setup Considerations for Automated Runs 31
This chapter contains an orientation to the Agilent NGS Workstation, an
overview of the SureSelectXT 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
SureSelect XT 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 5 for designations of the heat block-containing Bravo deck positions
on the Multi TEC control device.
Table 5
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).
SureSelect XT 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
SureSelect XT 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 then initiates temperature control of Bravo deck position
9 at the displayed set point.
SureSelect XT Automated Library Prep and Capture System
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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,
including SureSelectXT automation protocols version 1.5.1.
If you have questions about VWorks version compatibility, please contact
[email protected]
Logging in to the VWorks software
1 Double-click the VWorks icon or the XT_ILM_v1.5.1.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
SureSelect XT Automated Library Prep and Capture System
Using the Agilent NGS Workstation for SureSelect Target Enrichment
VWorks Automation Control Software
2
Using the SureSelectXT_ILM_v1.5.1.VWForm to setup and start a run
Use the VWorks form SureSelectXT_ILM_v1.5.1.VWForm, shown below, to
set up and start each SureSelect automation protocol or runset.
1 Open the form using the XT_ILM_v1.5.1.VWForm shortcut on your
desktop.
2 Use the form drop-down menus to select the appropriate SureSelect
workflow step 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.
SureSelect XT 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
SureSelect XT 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.
SureSelect XT 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
SureSelect XT 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 SureSelect_XT_Illumina VWorks form, click the
Full Screen button 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.
SureSelect XT Automated Library Prep and Capture System
27
2
Using the Agilent NGS Workstation for SureSelect Target Enrichment
Overview of the SureSelect Target Enrichment Procedure
Overview of the SureSelect Target Enrichment Procedure
Figure 2 summarizes the SureSelect target enrichment workflow for
samples to be sequenced using the Illumina paired-read sequencing
platform. For each sample to be sequenced, individual library
preparations, hybridizations, and captures are performed. The samples are
then tagged by PCR with an index sequence. Depending on the target size
of the SureSelect capture, multiple samples can be pooled and sequenced
in a single lane using the Illumina-specified multiplex index tags that are
provided with SureSelect Library Prep kits.
The SureSelectXT automated target enrichment system is compatible with
gDNA samples containing either 3 g or 200 ng DNA, with minor
differences in the VWorks protocols used during the Sample Preparation
segment of the workflow for the two DNA input options. Both DNA input
options use identical automation protocols for the Hybridization and
Indexing segments of the workflow.
When starting with 3 g gDNA samples, see Table 6 for a summary of the
VWorks protocols used during the workflow. Then, see Sample Preparation
(3 µg DNA Samples), Hybridization, and Indexing chapters for complete
instructions for use of the VWorks protocols for sample processing.
When starting with 200 ng gDNA samples, see Table 7 for a summary of
the VWorks protocols used during the workflow. Then, see Sample
Preparation (200 ng DNA Samples), Hybridization, and Indexing chapters
for complete instructions for use of the VWorks protocols for sample
processing.
28
SureSelect XT Automated Library Prep and Capture System
Using the Agilent NGS Workstation for SureSelect Target Enrichment
Overview of the SureSelect Target Enrichment Procedure
Figure 2
2
Overall sequencing sample preparation workflow.
SureSelect XT Automated Library Prep and Capture System
29
2
Using the Agilent NGS Workstation for SureSelect Target Enrichment
Overview of the SureSelect Target Enrichment Procedure
Table 6
Overview of VWorks protocols and runsets used for 3 g gDNA samples
Workflow Step
(Protocol Chapter)
Substep
VWorks Protocols Used for Agilent NGS Workstation
automation
Purify DNA using AMPure XP beads
AMPureXP_XT_ILM_v1.5.1.pro:Shearing-3 µg only
Prepare adaptor-ligated DNA
LibraryPrep_XT_ILM_v1.5.1.rst
Amplify adaptor-ligated DNA
Pre-CapturePCR_XT_ILM_3µg_v1.5.1.pro
Purify DNA using AMPure XP beads
AMPureXP_XT_ILM_v1.5.1.pro:Pre-Capture PCR
Aliquot 750-ng of prepped libraries for
hybridization
Aliquot_Libraries_v1.5.1.pro
Hybridize prepped DNA to Capture
Library
Hybridization_v1.5.1.pro
Capture and wash DNA hybrids
SureSelectCapture&Wash_v1.5.1.rst
Add index tags by PCR
Post-CaptureIndexing_XT_ILM_v1.5.1.pro
Purify DNA using AMPure XP beads
AMPureXP_XT_ILM_v1.5.1.pro:Post-Capture PCR
Sample Preparation
Hybridization
Indexing
Table 7
Overview of VWorks protocols and runsets used for 200 ng gDNA samples
Workflow Step
(Protocol Chapter)
Substep
VWorks Protocols Used for Agilent NGS Workstation
automation
Prepare adaptor-ligated DNA
LibraryPrep_XT_ILM_v1.5.1.rst
Sample Preparation Amplify adaptor-ligated DNA
Hybridization
Indexing
30
Pre-CapturePCR_XT_ILM_200ng_v1.5.1.pro
Purify DNA using AMPure XP beads
AMPureXP_XT_ILM_v1.5.1.pro:Pre-Capture PCR
Aliquot 750-ng of prepped libraries for
hybridization
Aliquot_Libraries_v1.5.1.pro
Hybridize prepped DNA to Capture
Library
Hybridization_v1.5.1.pro
Capture and wash DNA hybrids
SureSelectCapture&Wash_v1.5.1.rst
Add index tags by PCR
Post-CaptureIndexing_XT_ILM_v1.5.1.pro
Purify DNA using AMPure XP beads
AMPureXP_XT_ILM_v1.5.1.pro:Post-Capture PCR
SureSelect XT 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
Agilent SureSelect Automated Library Prep and Capture System runs may
include 1, 2, 3, 4, 6, or 12 columns (equivalent to 8, 16, 24, 32, 48, or 96
wells) of gDNA samples to be enriched for sequencing on the Illumina
platform. Plan your experiments using complete columns of samples.
Table 8
Columns to Samples Equivalency
Number of Columns Processed
Total Number of Samples Processed
1
8
2
16
3
24
4
32
6
48
12
96
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 reactions configured as 4 runs of 3 columns of samples per run.
SureSelect XT Automated Library Prep and Capture System
31
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.
• At the hybridization step (see Figure 2), you can add a different
Capture Library to each row of the plate. Plan your experiment such
that each prepared DNA library corresponds to the appropriate Capture
Library row in the sample plate.
• For sample indexing after hybridization to the SureSelect library (see
Figure 2), you will need to prepare a separate plate containing the
indexing primers. Assign the wells to be indexed with their respective
indexing primers during experimental design.
• 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 75 on page 141 to
determine which Capture Libraries may be amplified on the same plate.
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 using
the PlateLoc thermal microplate sealer included with the Agilent NGS
Workstation, and then centrifuged to collect any dispersed liquid. To
maximize efficiency, locate the centrifuge in close proximity to the
Agilent NGS Workstation.
32
SureSelect XT Automated Library Prep and Capture System
Using the Agilent NGS Workstation for SureSelect Target Enrichment
PCR Plate Type Considerations
2
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 SureSelectXT_ILM_v1.5.1.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 9.
Table 9
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
SureSelect XT Automated Library Prep and Capture System
33
2
34
Using the Agilent NGS Workstation for SureSelect Target Enrichment
PCR Plate Type Considerations
SureSelect XT Automated Library Prep and Capture System
SureSelectXT Automated Target Enrichment for Illumina Paired-End
Multiplexed Sequencing Protocol
3
Sample Preparation (3 µg DNA
Samples)
Step 1. Shear DNA 36
Step 2. Purify sheared DNA using AMPure XP beads 39
Step 3. Assess sample quality (optional) 44
Step 4. Modify DNA ends for target enrichment 47
Step 5. Amplify adaptor-ligated libraries 55
Step 6. Purify amplified DNA using AMPure XP beads 63
Step 7. Assess Library DNA quantity and quality 66
This section contains instructions for the preparation of gDNA libraries
from samples containing 3 g of DNA. For lower input (200 ng) DNA
samples, see the protocol on page 71.
This section contains instructions for gDNA library preparation specific to
the Illumina paired-read sequencing platform and to automated processing
using the Agilent NGS Workstation. For each sample to be sequenced,
individual library preparations, hybridizations, and captures are performed
in separate wells of a 96-well plate. The samples are then tagged by PCR
with an index sequence. Depending on the target size of the SureSelect
capture, multiple samples can be pooled and sequenced in a single lane
using the Illumina-specified index tags that are provided with
SureSelectXT target enrichment kits.
Agilent Technologies
35
3
Sample Preparation (3 µg DNA Samples)
Step 1. Shear DNA
Step 1. Shear DNA
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. Make sure the gDNA is of high quality (non-degraded,
A260/A280 is 1.8 to 2.0).
Follow the instructions for the instrument.
2 Dilute 3 µg of high-quality gDNA with 1X Low TE Buffer in a 1.5-mL
LoBind tube to a total volume of 130 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 at least 30 minutes, 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
When using a Covaris E-series instrument to prepare multiple gDNA samples in the same
experiment, you can also use the 96 microTube plate (see Table 4 on page 15) for the DNA
shearing step.
5 Use a tapered pipette tip to slowly transfer the 130-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 10 or Table 11, depending on the Covaris instrument
SonoLab software version used.
36
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (3 µg DNA Samples)
Step 1. Shear DNA
3
The target peak size is 150 to 200 bp.
Table 10
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 11
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.
SureSelect XT Automated Library Prep and Capture System
37
3
Sample Preparation (3 µg DNA Samples)
Step 1. Shear DNA
9 Transfer the sheared DNA into the wells of a 96-well Eppendorf 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
38
If you do not continue to the next step, store the sample plate at 4°C
overnight or at –20°C for prolonged storage.
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (3 µg DNA Samples)
Step 2. Purify sheared DNA using AMPure XP beads
3
Step 2. Purify sheared DNA using AMPure XP beads
In this step, the Agilent NGS Workstation transfers AMPure XP beads and
gDNA samples 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 Gently wipe down the Labware MiniHub, Bravo deck, and BenchCel
with a NucleoClean decontamination wipe.
3 Turn on the ThermoCube, 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 Let the AMPure XP beads come to room temperature for at least
30 minutes. Do not freeze the beads at any time.
5 Mix the bead suspension well so that the reagent appears homogeneous
and consistent in color.
6 Prepare a Nunc DeepWell source plate for the beads by adding 185 µL
of homogeneous AMPure XP beads per well, for each well to be
processed.
7 Prepare a Thermo Scientific reservoir containing 15 mL of nuclease-free
water.
8 Prepare a separate Thermo Scientific reservoir containing 45 mL of
freshly-prepared 70% ethanol.
SureSelect XT Automated Library Prep and Capture System
39
3
Sample Preparation (3 µg DNA Samples)
Step 2. Purify sheared DNA using AMPure XP beads
9 Load the Labware MiniHub according to Table 12, using the plate
orientations shown in Figure 3.
Table 12
40
Initial MiniHub configuration for AMPureXP_XT_ILM_v1.5.1.pro:Shearing-3 µg
only
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
Plate
Empty
Empty
Shelf 2
Empty
Nuclease-free
water reservoir
from step 7
AMPure XP beads
in Nunc DeepWell
plate from step 6
Empty
Shelf 1 (Bottom)
Empty
70% ethanol
reservoir from
step 8
Empty
Empty Tip Box
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (3 µg DNA Samples)
Step 2. Purify sheared DNA using AMPure XP beads
Figure 3
3
Agilent Labware MiniHub plate orientation. For Thermo Scientific reservoirs,
place the notched corner facing the center of the hub.
10 Load the Bravo deck according to Table 13.
Table 13
Initial Bravo deck configuration for AMPureXP_XT_ILM_v1.5.1.pro:Shearing-3
µg only
Location
Content
1
Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)
9
Sheared gDNA samples in unsealed PCR plate seated on red insert (PCR
plate type must be specified on setup form under step 2)
SureSelect XT Automated Library Prep and Capture System
41
3
Sample Preparation (3 µg DNA Samples)
Step 2. Purify sheared DNA using AMPure XP beads
11 Load the BenchCel Microplate Handling Workstation according to
Table 14.
Table 14
Initial BenchCel configuration for AMPureXP_XT_ILM_v1.5.1.pro:Shearing-3 µg
only
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
Run VWorks protocol AMPureXP_XT_ILM_v1.5.1.pro:Shearing-3 g only
12 Open the SureSelect setup form using the XT_ILM_v1.5.1.VWForm
shortcut on your desktop.
13 Log in to the VWorks software.
14 On the setup form, under Select Protocol to Run, select
AMPureXP_XT_ILM_v1.5.1.pro:Shearing-3 g only.
NOTE
AMPureXP purification protocols are used during multiple steps of the SureSelect
automation workflow. Be sure to select the correct workflow step when initiating the
automation protocol.
15 Under Select PCR plate labware for Thermal Cycling, select the
specific type of PCR plate containing the sheared gDNA samples at
position 9.
16 Select the number of columns of samples to be processed. Runs must
include 1, 2, 3, 4, 6, or 12 columns.
17 Click Display Initial Workstation Setup.
42
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (3 µg DNA Samples)
Step 2. Purify sheared DNA using AMPure XP beads
3
18 Verify that the NGS workstation has been set up as displayed in the
Workstation Setup region of the form.
19 When verification is complete, click Run Selected Protocol.
NOTE
If workstation devices do not respond when you start the run, but activity is recorded in the
Log, verify that VWorks is not running in Simulation mode. See page 27 for more
information.
Running the AMPureXP purification protocol takes approximately
45 minutes. Once complete, the purified DNA samples are located in the
Eppendorf plate at position 7 of the Bravo deck.
SureSelect XT Automated Library Prep and Capture System
43
3
Sample Preparation (3 µg DNA Samples)
Step 3. Assess sample quality (optional)
Step 3. Assess sample quality (optional)
Analysis of the purified sheared DNA samples prior to library preparation
is optional. If you elect to include this step, follow the instructions below.
Option 1: Analysis using the 2100 Bioanalyzer and DNA 1000 Assay
Use a Bioanalyzer DNA 1000 chip and reagent kit. For more information,
see the Agilent DNA 1000 Kit Guide at www.genomics.agilent.com.
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 the peak of DNA fragment size
positioned between 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 plate and store at 4°C
overnight or at –20°C for prolonged storage.
Figure 4
44
Analysis of sheared DNA using a DNA 1000 Bioanalyzer assay.
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (3 µg DNA Samples)
Step 3. Assess sample quality (optional)
3
Option 2: Analysis using the 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). For more information to do this step, see the Agilent
2200 TapeStation User Manual at www.genomics.agilent.com.
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 the peak of DNA fragment size
positioned between 150 to 200 bp. A sample electropherogram is shown
in Figure 5.
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.
SureSelect XT Automated Library Prep and Capture System
45
3
Sample Preparation (3 µg DNA Samples)
Step 3. Assess sample quality (optional)
Figure 5
46
Analysis of sheared DNA using the 2200 TapeStation.
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (3 µg DNA Samples)
Step 4. Modify DNA ends for target enrichment
3
Step 4. Modify DNA ends for target enrichment
In this step, the Agilent NGS Workstation completes the DNA end
modification steps required for SureSelect target enrichment, including GA
end-repair, A-tailing, and adaptor ligation. After the appropriate
modification steps, the Agilent NGS Workstation purifies the prepared
DNA using AMPure XP beads.
Before starting the run, you need to prepare master mixes (with overage)
for each step, without the DNA sample. Master mixes for runs that include
1, 2, 3, 4, 6, and 12 columns (including overage) are shown in each table.
Prepare each master mix on ice.
Prepare the workstation
1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes.
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 Turn on the ThermoCube, 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.
SureSelect XT Automated Library Prep and Capture System
47
3
Sample Preparation (3 µg DNA Samples)
Step 4. Modify DNA ends for target enrichment
Prepare the SureSelect DNA end-repair master mix
4 Prepare the appropriate volume of end-repair master mix, according to
Table 15. Mix well using a vortex mixer and keep on ice.
Table 15
Preparation of End-Repair Master Mix
SureSelect XT
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
Nuclease-free
water
35.2 µL
448.8 µL
748.0 µL
1047.2 µL
1346.4 µL
1944.8 µL
3889.6 µL
10X End-Repair
Buffer
10.0 µL
127.5 µL
212.5 µL
297.5 µL
382.5 µL
552.5 µL
1105.0 µL
dNTP mix
1.6 µL
20.4 µL
34.0 µL
47.6 µL
61.2 µL
88.4 µL
176.8 µL
T4 DNA
Polymerase
1.0 µL
12.8 µL
21.3 µL
29.8 µL
38.3 µL
55.3 µL
110.5 µL
Klenow DNA
Polymerase
2.0 µL
25.5 µL
42.5 µL
59.5 µL
76.5 µL
110.5 µL
221.0 µL
T4 Polynucleotide
Kinase
2.2 µL
28.1 µL
46.8 µL
65.5 µL
84.2 µL
121.6 µL
243.1 µL
Total Volume
52 µL
663 µL
1105 µL
1547 µL
1989 µL
2873 µL
5746 µL
48
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (3 µg DNA Samples)
Step 4. Modify DNA ends for target enrichment
3
Prepare the A-tailing master mix
5 Prepare the appropriate volume of A-tailing master mix, according to
Table 16. Mix well using a vortex mixer and keep on ice.
Table 16
Preparation of A-Tailing Master Mix
SureSelectXT
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
Nuclease-free
water
11.0 µL
187.0 µL
280.5 µL
374.0 µL
467.5 µL
654.5 µL
1262.3 µL
10x Klenow
Polymerase Buffer
5.0 µL
85.0 µL
127.5 µL
170.0 µL
212.5 µL
297.5 µL
573.8 µL
dATP
1.0 µL
17.0 µL
25.5 µL
34.0 µL
42.5 µL
59.5 µL
114.8 µL
Exo (–) Klenow
3.0 µL
51.0 µL
76.5 µL
102.0 µL
127.5 µL
178.5 µL
344.3 µL
Total Volume
20 µL
340 µL
510 µL
680 µL
850 µL
1190 µL
2295 µL
Prepare the adaptor ligation master mix
6 Prepare the appropriate volume of adaptor ligation master mix,
according to Table 17. Mix well using a vortex mixer and keep on ice.
Table 17
Preparation of Adaptor Ligation Master Mix (use only for the 3 µg DNA input workflow)
SureSelectXT
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
Nuclease-free
water
15.5 µL
197.6 µL
329.4 µL
461.1 µL
592.9 µL
856.4 µL
1712.8 µL
5X T4 DNA Ligase
Buffer
10.0 µL
127.5 µL
212.5 µL
297.5 µL
382.5 µL
552.5 µL
1105.0 µL
SureSelect
Adaptor Oligo Mix
10.0 µL
127.5 µL
212.5 µL
297.5 µL
382.5 µL
552.5 µL
1105.0 µL
T4 DNA Ligase
1.5 µL
19.1 µL
31.9 µL
44.6 µL
57.4 µL
82.9 µL
165.8 µL
Total Volume
37.0 µL
471.8 µL
786.3 µL
1100.8 µL
1415.3 µL
2044.3 µL
4088.5 µL
SureSelect XT Automated Library Prep and Capture System
49
3
Sample Preparation (3 µg DNA Samples)
Step 4. Modify DNA ends for target enrichment
Prepare the master mix source plate
7 In a Nunc DeepWell plate, prepare the master mix source plate
containing the master mixes prepared in steps 3 to 5. Add the volumes
indicated in Table 18 of each master mix to all wells of the indicated
column of the Nunc DeepWell plate. Keep the master mixes on ice
during the aliquoting steps. The final configuration of the master mix
source plate is shown in Figure 6.
Table 18
Preparation of the Master Mix Source Plate for LibraryPrep_XT_ILM_v1.5.1.rst
Master Mix
Solution
Position on
Source Plate
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
End Repair
Master Mix
Column 1
76.4 µL
131.6 µL
186.9 µL
242.1 µL
352.6 µL
711.8 µL
A-Tailing Master
Mix
Column 2
40.0 µL
61.3 µL
82.5 µL
103.8 µL
146.3µL
284.4 µL
Adaptor Ligation
Master Mix
Column 3
54.3 µL
93.7 µL
133.0 µL
172.3 µL
250.9 µL
506.4 µL
50
(A1-H1)
(A2-H2)
(A3-H3)
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (3 µg DNA Samples)
Step 4. Modify DNA ends for target enrichment
Figure 6
3
Configuration of the master mix source plate for
LibraryPrep_XT_ILM_v1.5.1.rst
8 Seal the master mix source plate using the PlateLoc Thermal Microplate
Sealer, with sealing settings of 165°C and 1.0 sec.
9 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.
SureSelect XT Automated Library Prep and Capture System
51
3
Sample Preparation (3 µg DNA Samples)
Step 4. Modify DNA ends for target enrichment
Prepare the purification reagents
10 Verify that the AMPure XP bead suspension is at room temperature. Do
not freeze the beads at any time.
11 Mix the bead suspension well so that the reagent appears homogeneous
and consistent in color.
12 Prepare a separate Nunc DeepWell source plate for the beads by adding
370 µL of homogeneous AMPure XP beads per well, for each well to be
processed.
13 Prepare a Thermo Scientific reservoir containing 20 mL of nuclease-free
water.
14 Prepare a separate Thermo Scientific reservoir containing 150 mL of
freshly-prepared 70% ethanol.
Load the Agilent NGS Workstation
15 Load the Labware MiniHub according to Table 19, using the plate
orientations shown in Figure 3.
Table 19
52
Initial MiniHub configuration for LibraryPrep_XT_ILM_v1.5.1.rst
Vertical Shelf
Position
Cassette 1
Cassette 2
Cassette 3
Cassette 4
Shelf 5 (Top)
Empty Nunc
DeepWell plate
Empty Nunc
DeepWell plate
Empty Nunc
DeepWell plate
Empty
Shelf 4
Empty
Empty Eppendorf
plate
Empty Eppendorf
plate
Empty
Shelf 3
Empty
Empty
Empty
Empty
Eppendorf plate
Shelf 2
Empty tip box
Nuclease-free
water reservoir
from step 13
AMPure XP beads Empty
in Nunc DeepWell
plate from step 12
Shelf 1 (Bottom)
New tip box
70% ethanol
reservoir from
step 14
Empty
Empty tip box
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (3 µg DNA Samples)
Step 4. Modify DNA ends for target enrichment
3
16 Load the Bravo deck according to Table 20.
Table 20
Initial Bravo deck configuration for LibraryPrep_XT_ILM_v1.5.1.rst
Location
Content
1
Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)
6
Empty Eppendorf plate
7
Eppendorf plate containing purified gDNA samples
9
DNA End Modification Master Mix Source Plate, unsealed and seated on
silver Nunc DeepWell insert
17 Load the BenchCel Microplate Handling Workstation according to
Table 21.
Table 21
Initial BenchCel configuration for LibraryPrep_XT_ILM_v1.5.1.rst
No. of Columns
Processed
Rack 1
Rack 2
Rack 3
Rack 4
1
2 Tip boxes
Empty
Empty
Empty
2
4 Tip boxes
Empty
Empty
Empty
3
5 Tip boxes
Empty
Empty
Empty
4
7 Tip boxes
Empty
Empty
Empty
6
10 Tip boxes
Empty
Empty
Empty
12
11 Tip boxes
8 Tip boxes
Empty
Empty
Run VWorks runset LibraryPrep_XT_ILM_v1.5.1.rst
18 On the SureSelect setup form, under Select Protocol to Run, select
LibraryPrep_XT_ILM_v1.5.1.rst.
19 Select the number of columns of samples to be processed. Runs must
include 1, 2, 3, 4, 6, or 12 columns.
SureSelect XT Automated Library Prep and Capture System
53
3
Sample Preparation (3 µg DNA Samples)
Step 4. Modify DNA ends for target enrichment
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.
Running the LibraryPrep_XT_ILM_v1.5.1.rst runset takes approximately
3.5 hours. Once complete, the purified, adaptor-ligated DNA samples are
located in the Eppendorf plate at position 7 of the Bravo deck.
Stopping Point
54
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.
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (3 µg DNA Samples)
Step 5. Amplify adaptor-ligated libraries
3
Step 5. Amplify adaptor-ligated libraries
In this step, the Agilent NGS Workstation completes the liquid handling
steps for amplification of the adaptor-ligated DNA samples. Afterward, you
transfer the PCR plate to a thermal cycler for amplification.
In this protocol, one half of the adaptor-ligated 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 Turn on the ThermoCube, 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 Leave tip boxes on shelves 1 and 2 in cassette 1 of the Labware
MiniHub from the previous LibraryPrep_XT_ILM_v1.5.1.rst run.
Otherwise, clear the remaining positions of the MiniHub and BenchCel
of plates and tip boxes.
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.
SureSelect XT Automated Library Prep and Capture System
55
3
Sample Preparation (3 µg DNA Samples)
Step 5. Amplify adaptor-ligated libraries
Prepare the pre-capture PCR master mix and master mix source plate
4 Prepare the appropriate volume of pre-capture PCR Master Mix,
according to Table 22. Mix well using a vortex mixer and keep on ice.
Table 22
Preparation of Pre-Capture PCR Master Mix (use only for the 3 µg DNA input workflow)
SureSelectXT
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
Nuclease-free
water
21.0 µL
267.8 µL
446.3 µL
624.8 µL
803.3 µL
1160.3 µL
2320.5 µL
Herculase II 5X
Reaction Buffer*
10.0 µL
127.5 µL
212.5 µL
297.5 µL
382.5 µL
552.5 µL
1105 µL
dNTP mix*
0.5 µL
6.4 µL
10.6 µL
14.9 µL
19.1 µL
27.6 µL
55.3 µL
SureSelect Primer†
1.25 µL
15.9 µL
26.6 µL
37.2 µL
47.8 µL
69.1 µL
138.1 µL
SureSelect
Indexing
Pre-Capture PCR
(Reverse) Primer‡
1.25 µL
15.9 µL
26.6 µL
37.2 µL
47.8 µL
69.1 µL
138.1 µL
Herculase II
Polymerase
1.0 µL
12.8 µL
21.3 µL
29.8 µL
38.3 µL
55.3 µL
110.5 µL
Total Volume
35 µL
446.3 µL
743.8 µL
1041.3 µL
1338.8 µL
1933.8 µL
3867.5 µL
(Forward)
* Included with the Herculase II Fusion DNA Polymerase. Do not use the buffer or dNTP mix from any other kit.
† Included in SureSelect XT Library Prep Kit ILM.
‡ Included in SureSelect XT Automation ILM Module Box 2. Ensure that the correct primer is selected from Box 2 at this step
(do not use the SureSelect Indexing Post-Capture PCR (Forward) Primer).
56
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (3 µg DNA Samples)
Step 5. Amplify adaptor-ligated libraries
3
5 Using the same Nunc DeepWell master mix source plate that was used
for the LibraryPrep_XT_ILM_v1.5.1.rst run, add the volume of PCR
Master Mix indicated in Table 23 to all wells of column 4 of the master
mix source plate. The final configuration of the master mix source plate
is shown in Figure 7.
Table 23
Preparation of the Master Mix Source Plate for Pre-CapturePCR_XT_ILM_3µg_v1.5.1.pro
Master Mix
Solution
Position on
Source Plate
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
Pre-Capture PCR
Master Mix
Column 4
51.4 µL
88.6 µL
125.8 µL
163.0 µL
237.3 µL
479.1 µL
NOTE
(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 adaptor-ligated DNA sample), leave columns 1 to 3
empty and add the PCR Master Mix to column 4 of the new plate.
SureSelect XT Automated Library Prep and Capture System
57
3
Sample Preparation (3 µg DNA Samples)
Step 5. Amplify adaptor-ligated libraries
Figure 7
Configuration of the master mix source plate for
Pre-CapturePCR_XT_ILM_3µg_v1.5.1.pro. Columns 1-3 were used to dispense
master mixes during the previous protocol.
6 Seal the master mix source plate using the PlateLoc Thermal Microplate
Sealer, with sealing settings of 165°C and 1.0 sec.
7 Centrifuge the plate for 30 seconds to drive the well contents off the
walls and plate seal and to eliminate any bubbles.
NOTE
58
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.
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (3 µg DNA Samples)
Step 5. Amplify adaptor-ligated libraries
3
Load the Agilent NGS Workstation
8 Load the Labware MiniHub according to Table 24, using the plate
orientations shown in Figure 3.
Table 24
Initial MiniHub configuration for Pre-CapturePCR_XT_ILM_3µg_v1.5.1.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
* The waste tip box (Cassette 1, Shelf 2) and clean tip box (Cassette 1, Shelf 1) are retained from the
LibraryPrep_XT_ILM_v1.5.1.rst run and reused here.
NOTE
If you are using a new box of tips on shelf 1 of cassette 1 (for example, when amplifying the
second half of the adaptor-ligated DNA sample), first remove the tips from columns 1 to 3 of
the tip box. Any tips present in columns 1 to 3 of the tip box may be inappropriately loaded
onto the Bravo platform pipette heads and may interfere with automated processing steps.
9 Load the Bravo deck according to Table 25.
Table 25
Initial Bravo deck configuration for Pre-CapturePCR_XT_ILM_3µg_v1.5.1.pro
Location
Content
6
Empty PCR plate seated in red insert (PCR plate type must be
specified on setup form under step 2)
7
Adaptor-ligated DNA samples in Eppendorf plate
9
Master mix plate containing PCR Master Mix in Column 4 (unsealed)
SureSelect XT Automated Library Prep and Capture System
59
3
Sample Preparation (3 µg DNA Samples)
Step 5. Amplify adaptor-ligated libraries
10 Load the BenchCel Microplate Handling Workstation according to
Table 26.
Table 26
Initial BenchCel configuration for Pre-CapturePCR_XT_ILM_3µg_v1.5.1.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
Run VWorks protocol Pre-CapturePCR_XT_ILM_3g_v1.5.1.pro
11 On the SureSelect setup form, under Select Protocol to Run, select
Pre-CapturePCR_XT_ILM_3g_v1.5.1.pro.
12 Under Select PCR plate labware for Thermal Cycling, select the
specific type of PCR plate used at position 6 of the Bravo deck.
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.
60
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (3 µg DNA Samples)
Step 5. Amplify adaptor-ligated libraries
3
16 When verification is complete, click Run Selected Protocol.
Running the Pre-CapturePCR_XT_ILM_3g_v1.5.1.pro protocol takes
approximately 15 minutes. Once complete, the PCR-ready samples,
containing prepped 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 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.
SureSelect XT Automated Library Prep and Capture System
61
3
Sample Preparation (3 µg DNA Samples)
Step 5. Amplify adaptor-ligated libraries
19 Transfer the PCR plate to a thermal cycler and run the PCR
amplification program shown in Table 27.
Table 27
NOTE
62
Pre-Capture PCR cycling program (use only for the 3 µg DNA input workflow)
Segment
Number of
Cycles
Temperature
Time
1
1
98°C
2 minutes
2
4 to 6
98°C
30 seconds
65°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.
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (3 µg DNA Samples)
Step 6. Purify amplified DNA using AMPure XP beads
3
Step 6. 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.) Do not freeze the beads at any time.
3 Mix the bead suspension well so that the reagent appears homogeneous
and consistent in color.
4 Prepare a Nunc DeepWell source plate for the beads by adding 95 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 28, using the plate
orientations shown in Figure 3.
Table 28
Initial MiniHub configuration for AMPureXP_XT_ILM_v1.5.1.pro:Pre-Capture
PCR
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
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
SureSelect XT Automated Library Prep and Capture System
63
3
Sample Preparation (3 µg DNA Samples)
Step 6. Purify amplified DNA using AMPure XP beads
8 Load the Bravo deck according to Table 29.
Table 29
Initial Bravo deck configuration for AMPureXP_XT_ILM_v1.5.1.pro:Pre-Capture
PCR
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)
9 Load the BenchCel Microplate Handling Workstation according to
Table 30.
Table 30
Initial BenchCel configuration for AMPureXP_XT_ILM_v1.5.1.pro:Pre-Capture
PCR
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
Run VWorks protocol AMPureXP_XT_ILM_v1.5.1.pro:Pre-Capture PCR
10 On the SureSelect setup form, under Select Protocol to Run, select
AMPureXP_XT_ILM_v1.5.1.pro:Pre-Capture PCR.
NOTE
64
AMPureXP purification protocols are used during multiple steps of the SureSelect
automation workflow. Be sure to select the correct workflow step when initiating the
automation protocol.
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (3 µg DNA Samples)
Step 6. Purify amplified DNA using AMPure XP beads
3
11 Under Select PCR plate labware for Thermal Cycling, select the
specific type of PCR plate containing the amplified libraries at position
9.
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 purified DNA samples are in the Eppendorf plate located on Bravo
deck position 7.
SureSelect XT Automated Library Prep and Capture System
65
3
Sample Preparation (3 µg DNA Samples)
Step 7. Assess Library DNA quantity and quality
Step 7. Assess Library DNA quantity and quality
The hybridization protocol in the following section requires 750 ng of each
amplified DNA library. Measure the concentration of each library using
one of the methods detailed below. Once DNA concentration for each
sample is determined, calculate the volume of the library to be used for
hybridization using the following formula:
Volume (µL) = 750 ng/concentration (ng/µL)
Option 1: Analysis using the Agilent 2100 Bioanalyzer and DNA 1000 Assay
Use a Bioanalyzer DNA 1000 chip and reagent kit to analyze the amplified
libraries. For more information to do this step, see the Agilent DNA 1000
Kit Guide at www.genomics.agilent.com.
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 the peak of DNA fragment size
positioned between 225 to 275 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
66
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.
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (3 µg DNA Samples)
Step 7. Assess Library DNA quantity and quality
Figure 8
3
Analysis of amplified library DNA using a DNA 1000 assay.
SureSelect XT Automated Library Prep and Capture System
67
3
Sample Preparation (3 µg DNA Samples)
Step 7. 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 libraries. For more information to do
this step, see the Agilent 2200 TapeStation User Manual at
www.genomics.agilent.com.
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 1 µL of each amplified library 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 the peak of DNA fragment size
positioned between 225 to 275 bp. A sample electropherogram is shown
in Figure 9.
Stopping Point
68
If you do not continue to the next step, seal the library DNA sample plate
and store at 4°C overnight or at –20°C for prolonged storage.
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (3 µg DNA Samples)
Step 7. Assess Library DNA quantity and quality
Figure 9
3
Analysis of amplified library DNA using the 2200 TapeStation.
SureSelect XT Automated Library Prep and Capture System
69
3
70
Sample Preparation (3 µg DNA Samples)
Step 7. Assess Library DNA quantity and quality
SureSelect XT Automated Library Prep and Capture System
SureSelectXT Automated Target Enrichment for Illumina Paired-End
Multiplexed Sequencing Protocol
4
Sample Preparation (200 ng DNA
Samples)
Step 1. Shear DNA 72
Step 2. Assess sample quality (optional) 75
Step 3. Modify DNA ends for target enrichment 78
Step 4. Amplify adaptor-ligated libraries 87
Step 5. Purify amplified DNA using AMPure XP beads 95
Step 6. Assess Library DNA quantity and quality 98
This section contains instructions for the preparation of gDNA libraries
from samples containing 200 ng of DNA. For higher input (3 g) DNA
samples, see the protocol on page 35.
This section contains instructions for gDNA library preparation specific to
the Illumina paired-read sequencing platform and to automated processing
using the Agilent NGS Workstation. For each sample to be sequenced,
individual library preparations, hybridizations, and captures are performed
in separate wells of a 96-well plate. The samples are then tagged by PCR
with an index sequence. Depending on the target size of the SureSelect
capture, multiple samples can be pooled and sequenced in a single lane
using the Illumina-specified index tags that are provided with
SureSelectXT target enrichment kits.
Agilent Technologies
71
4
Sample Preparation (200 ng DNA Samples)
Step 1. Shear DNA
Step 1. Shear DNA
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. Make sure the gDNA is of high quality (non-degraded,
A260/A280 is 1.8 to 2.0).
Follow the instructions for the instrument.
2 Dilute 200 ng 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 at least 30 minutes, 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
When using a Covaris E-series instrument to prepare multiple gDNA samples in the same
experiment, you can also use the 96 microTube plate (see Table 4 on page 15) for the DNA
shearing step.
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 31 or Table 32, depending on the Covaris instrument
SonoLab software version used.
72
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (200 ng DNA Samples)
Step 1. Shear DNA
4
The target peak size is 150 to 200 bp.
Table 31
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 32
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.
SureSelect XT Automated Library Prep and Capture System
73
4
Sample Preparation (200 ng DNA Samples)
Step 1. Shear DNA
9 Transfer the sheared DNA into the wells of a 96-well Eppendorf 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
If you do not continue to the next step, store the sample plate at 4°C
overnight or at –20°C for prolonged storage.
CA U T I O N
The Sample Preparation protocol for 200 ng gDNA samples does not include the
post-shear purification step that is included in the Sample Preparation protocol for 3 g
gDNA samples.
If you wish to analyze the sheared DNA fragment size prior to library preparation, use
the optional protocol on page 75. Otherwise, proceed directly to “Step 3. Modify DNA
ends for target enrichment” on page 78.
74
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (200 ng DNA Samples)
Step 2. Assess sample quality (optional)
4
Step 2. Assess sample quality (optional)
Analysis of the sheared DNA samples prior to library preparation is
optional. If you elect to include this step, follow the instructions below.
Option 1: Analysis using the 2100 Bioanalyzer and High Sensitivity DNA Assay
Use the Bioanalyzer High Sensitivity DNA Assay to analyze the 200-ng
sheared DNA samples. See the High Sensitivity DNA Kit Guide at
www.genomics.agilent.com for more information on doing this step.
1 Set up the 2100 Bioanalyzer as instructed in the reagent kit guide.
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 chip, samples and ladder as instructed in the reagent kit
guide, using 1 µL of each sample for the analysis.
4 Load the prepared chip into the 2100 Bioanalyzer and start the run
within five minutes after preparation.
5 Verify that the electropherogram shows the peak of DNA fragment size
positioned between 120 to 150 bp. A sample electropherogram is shown
in Figure 10.
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 10
Analysis of sheared DNA using a High Sensitivity DNA Bioanalyzer assay.
SureSelect XT Automated Library Prep and Capture System
75
4
Sample Preparation (200 ng DNA Samples)
Step 2. Assess sample quality (optional)
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 200-ng sheared DNA samples. For more
information to do this step, see the Agilent 2200 TapeStation User
Manual at www.genomics.agilent.com.
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 2 µL of each indexed 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 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 the peak of DNA fragment size
positioned between 120 to 150 bp. A sample electropherogram is shown
in Figure 11.
Stopping Point
76
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.
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (200 ng DNA Samples)
Step 2. Assess sample quality (optional)
Figure 11
4
Analysis of sheared DNA using the 2200 TapeStation.
SureSelect XT Automated Library Prep and Capture System
77
4
Sample Preparation (200 ng DNA Samples)
Step 3. Modify DNA ends for target enrichment
Step 3. Modify DNA ends for target enrichment
In this step, the Agilent NGS Workstation completes the DNA end
modification steps required for SureSelect target enrichment, including GA
end-repair, A-tailing, and adaptor ligation. After the appropriate
modification steps, the Agilent NGS Workstation purifies the prepared
DNA using AMPure XP beads.
Before starting the run, you need to prepare master mixes (with overage)
for each step, without the DNA sample. Master mixes for runs that include
1, 2, 3, 4, 6, and 12 columns (including overage) are shown in each table.
Prepare each master mix on ice.
CA U T I O N
The Library Prep automation protocol for 200 ng gDNA samples differs from the
3 g gDNA protocol in the amount of SureSelect Adaptor Oligo Mix used in the adaptor
ligation master mix. Be sure to use the master mix preparation table provided on
page 80 for 200 ng DNA samples.
Prepare the workstation
1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes.
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 Turn on the ThermoCube, 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.
78
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (200 ng DNA Samples)
Step 3. Modify DNA ends for target enrichment
4
Prepare the SureSelect DNA end-repair master mix
4 Prepare the appropriate volume of end-repair master mix, according to
Table 33. Mix well using a vortex mixer and keep on ice.
Table 33
Preparation of End-Repair Master Mix
SureSelect XT
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
Nuclease-free
water
35.2 µL
448.8 µL
748.0 µL
1047.2 µL
1346.4 µL
1944.8 µL
3889.6 µL
10X End-Repair
Buffer
10.0 µL
127.5 µL
212.5 µL
297.5 µL
382.5 µL
552.5 µL
1105.0 µL
dNTP mix
1.6 µL
20.4 µL
34.0 µL
47.6 µL
61.2 µL
88.4 µL
176.8 µL
T4 DNA
polymerase
1.0 µL
12.8 µL
21.3 µL
29.8 µL
38.3 µL
55.3 µL
110.5 µL
Klenow DNA
polymerase
2.0 µL
25.5 µL
42.5 µL
59.5 µL
76.5 µL
110.5 µL
221.0 µL
T4 Polynucleotide
Kinase
2.2 µL
28.1 µL
46.8 µL
65.5 µL
84.2 µL
121.6 µL
243.1 µL
Total Volume
52 µL
663 µL
1105 µL
1547 µL
1989 µL
2873 µL
5746 µL
SureSelect XT Automated Library Prep and Capture System
79
4
Sample Preparation (200 ng DNA Samples)
Step 3. Modify DNA ends for target enrichment
Prepare the A-tailing master mix
5 Prepare the appropriate volume of A-tailing master mix, according to
Table 34. Mix well using a vortex mixer and keep on ice.
Table 34
Preparation of A-Tailing Master Mix
SureSelect XT
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
Nuclease-free
water
11.0 µL
187.0 µL
280.5 µL
374.0 µL
467.5 µL
654.5 µL
1262.3 µL
10x Klenow
Polymerase
Buffer
5.0 µL
85.0 µL
127.5 µL
170.0 µL
212.5 µL
297.5 µL
573.8 µL
dATP
1.0 µL
17.0 µL
25.5 µL
34.0 µL
42.5 µL
59.5 µL
114.8 µL
Exo (–) Klenow
3.0 µL
51.0 µL
76.5 µL
102.0 µL
127.5 µL
178.5 µL
344.3 µL
Total Volume
20 µL
340 µL
510 µL
680 µL
850 µL
1190 µL
2295 µL
Prepare the adaptor ligation master mix
6 Prepare the appropriate volume of adaptor ligation master mix,
according to Table 35. Mix well using a vortex mixer and keep on ice.
Table 35
Preparation of Adaptor Ligation Master Mix (use only for the 200 ng DNA input workflow)
SureSelect XT
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
Nuclease-free
water
24.5 µL
312.4 µL
520.6 µL
728.9 µL
937.1 µL
1353.6 µL
2707.3 µL
5X T4 DNA Ligase
Buffer
10.0 µL
127.5 µL
212.5 µL
297.5 µL
382.5 µL
552.5 µL
1105.0 µL
SureSelect
1.0 µL
Adaptor Oligo Mix*
12.8 µL
21.3 µL
29.8 µL
38.3 µL
55.3 µL
110.5 µL
T4 DNA Ligase
1.5 µL
19.1 µL
31.9 µL
44.6 µL
57.4 µL
82.9 µL
165.8 µL
Total Volume
37.0 µL
471.8 µL
786.3 µL
1100.8 µL
1415.3 µL
2044.3 µL
4088.5 µL
* Previously labeled as InPE Adaptor Oligo Mix.
80
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (200 ng DNA Samples)
Step 3. Modify DNA ends for target enrichment
4
Prepare the master mix source plate
7 In a Nunc DeepWell plate, prepare the master mix source plate
containing the master mixes prepared in steps 3 to 5. Add the volumes
indicated in Table 36 of each master mix to all wells of the indicated
column of the Nunc DeepWell plate. Keep the master mixes on ice
during the aliquoting steps. The final configuration of the master mix
source plate is shown in Figure 12.
Table 36
Preparation of the Master Mix Source Plate for LibraryPrep_XT_ILM_v1.5.1.rst
Master Mix
Solution
Position on
Source Plate
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
End Repair
Master Mix
Column 1
76.4 µL
131.6 µL
186.9 µL
242.1 µL
352.6 µL
711.8 µL
A-Tailing Master
Mix
Column 2
40.0 µL
61.3 µL
82.5 µL
103.8 µL
146.3µL
284.4 µL
Adaptor Ligation
Master Mix
Column 3
54.3 µL
93.7 µL
133.0 µL
172.3 µL
250.9 µL
506.4 µL
(A1-H1)
(A2-H2)
(A3-H3)
SureSelect XT Automated Library Prep and Capture System
81
4
Sample Preparation (200 ng DNA Samples)
Step 3. Modify DNA ends for target enrichment
Figure 12
Configuration of the master mix source plate for
LibraryPrep_XT_ILM_v1.5.1.rst
8 Seal the master mix source plate using the PlateLoc Thermal Microplate
Sealer, with sealing settings of 165°C and 1.0 sec.
9 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
82
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.
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (200 ng DNA Samples)
Step 3. Modify DNA ends for target enrichment
4
Prepare the purification reagents
10 Verify that the AMPure XP bead suspension is at room temperature. Do
not freeze the beads at any time.
11 Mix the bead suspension well so that the reagent appears homogeneous
and consistent in color.
12 Prepare a separate Nunc DeepWell source plate for the beads by adding
370 µL of homogeneous AMPure XP beads per well, for each well to be
processed.
13 Prepare a Thermo Scientific reservoir containing 20 mL of nuclease-free
water.
14 Prepare a separate Thermo Scientific reservoir containing 150 mL of
freshly-prepared 70% ethanol.
Load the Agilent NGS Workstation
15 Load the Labware MiniHub according to Table 37, using the plate
orientations shown in Figure 13.
Table 37
Initial MiniHub configuration for LibraryPrep_XT_ILM_v1.5.1.rst
Vertical Shelf
Position
Cassette 1
Cassette 2
Cassette 3
Cassette 4
Shelf 5 (Top)
Empty Nunc
DeepWell plate
Empty Nunc
DeepWell plate
Empty Nunc
DeepWell plate
Empty
Shelf 4
Empty
Empty Eppendorf
plate
Empty Eppendorf
plate
Empty
Shelf 3
Empty
Empty
Empty
Empty
Eppendorf plate
Shelf 2
Empty tip box
Nuclease-free
water reservoir
from step 13
AMPure XP beads Empty
in Nunc DeepWell
plate from step 12
Shelf 1 (Bottom)
New tip box
70% ethanol
reservoir from
step 14
Empty
SureSelect XT Automated Library Prep and Capture System
Empty tip box
83
4
Sample Preparation (200 ng DNA Samples)
Step 3. Modify DNA ends for target enrichment
Figure 13
Agilent Labware MiniHub plate orientation. For Thermo Scientific reservoirs,
place the notched corner facing the center of the hub.
16 Load the Bravo deck according to Table 38.
Table 38
84
Initial Bravo deck configuration for LibraryPrep_XT_ILM_v1.5.1.rst
Location
Content
1
Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)
6
Empty Eppendorf plate
7
Eppendorf plate containing sheared gDNA samples (unsealed)
9
DNA End Modification Master Mix Source Plate (unsealed) seated on silver
Nunc DeepWell insert
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (200 ng DNA Samples)
Step 3. Modify DNA ends for target enrichment
4
17 Load the BenchCel Microplate Handling Workstation according to
Table 39.
Table 39
Initial BenchCel configuration for LibraryPrep_XT_ILM_v1.5.1.rst
No. of Columns
Processed
Rack 1
Rack 2
Rack 3
Rack 4
1
2 Tip boxes
Empty
Empty
Empty
2
4 Tip boxes
Empty
Empty
Empty
3
5 Tip boxes
Empty
Empty
Empty
4
7 Tip boxes
Empty
Empty
Empty
6
10 Tip boxes
Empty
Empty
Empty
12
11 Tip boxes
8 Tip boxes
Empty
Empty
Run VWorks runset LibraryPrep_XT_ILM_v1.5.1.rst
18 On the SureSelect setup form, under Select Protocol to Run, select
LibraryPrep_XT_ILM_v1.5.1.rst.
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.
SureSelect XT Automated Library Prep and Capture System
85
4
Sample Preparation (200 ng DNA Samples)
Step 3. Modify DNA ends for target enrichment
22 When verification is complete, click Run Selected Protocol.
23 When ready to begin the run, click OK in the following window.
Running the LibraryPrep_XT_ILM_v1.5.1.rst runset takes approximately
3.5 hours. Once complete, the purified, adaptor-ligated DNA samples are
located in the Eppendorf plate at position 7 of the Bravo deck.
Stopping Point
86
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.
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (200 ng DNA Samples)
Step 4. Amplify adaptor-ligated libraries
4
Step 4. Amplify adaptor-ligated libraries
In this step, the Agilent NGS Workstation completes the liquid handling
steps for amplification of the adaptor-ligated DNA samples. Afterward, you
transfer the PCR plate to a thermal cycler for amplification.
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 Turn on the ThermoCube, 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 Leave tip boxes on shelves 1 and 2 in cassette 1 of the Labware
MiniHub from the previous LibraryPrep_XT_ILM_v1.5.1.rst run.
Otherwise, clear the remaining positions of the MiniHub and BenchCel
of plates and tip boxes.
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.
SureSelect XT Automated Library Prep and Capture System
87
4
Sample Preparation (200 ng DNA Samples)
Step 4. Amplify adaptor-ligated libraries
Prepare the pre-capture PCR master mix and master mix source plate
4 Prepare the appropriate volume of pre-capture PCR Master Mix,
according to Table 40. Mix well using a vortex mixer and keep on ice.
Table 40
Preparation of Pre-Capture PCR Master Mix (use only for the 200 ng DNA input workflow)
SureSelectXT
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
Nuclease-free
water
6.0 µL
76.5 µL
127.5 µL
178.5 µL
229.5 µL
331.5 µL
663.0 µL
Herculase II 5X
Reaction Buffer*
10.0 µL
127.5 µL
212.5 µL
297.5 µL
382.5 µL
552.5 µL
1105 µL
dNTP mix*
0.5 µL
6.4 µL
10.6 µL
14.9 µL
19.1 µL
27.6 µL
55.3 µL
SureSelect Primer†
1.25 µL
15.9 µL
26.6 µL
37.2 µL
47.8 µL
69.1 µL
138.1 µL
SureSelect
Indexing
Pre-Capture PCR
(Reverse) Primer‡
1.25 µL
15.9 µL
26.6 µL
37.2 µL
47.8 µL
69.1 µL
138.1 µL
Herculase II
Polymerase
1.0 µL
12.8 µL
21.3 µL
29.8 µL
38.3 µL
55.3 µL
110.5 µL
Total Volume
20 µL
255 µL
425 µL
595 µL
765 µL
1105 µL
2210 µL
(Forward)
* Included with the Herculase II Fusion DNA Polymerase. Do not use the buffer or dNTP mix from any other kit.
† Included in SureSelect XT Library Prep Kit ILM.
‡ Included in SureSelect XT Automation ILM Module Box 2. Ensure that the correct primer is selected from Box 2 at this step
(do not use the SureSelect Indexing Post-Capture PCR (Forward) Primer).
88
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (200 ng DNA Samples)
Step 4. Amplify adaptor-ligated libraries
4
5 Using the same Nunc DeepWell master mix source plate that was used
for the LibraryPrep_XT_ILM_v1.5.1.rst run, add the volume of PCR
Master Mix indicated in Table 41 to all wells of column 4 of the master
mix source plate. The final configuration of the master mix source plate
is shown in Figure 14.
Table 41
Preparation of the Master Mix Source Plate for Pre-CapturePCR_XT_ILM_200ng_v1.5.1.pro
Master Mix
Solution
Position on
Source Plate
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
Pre-Capture PCR
Master Mix
Column 4
29.4 µL
50.6 µL
71.9 µL
93.1 µL
135.6 µL
273.8 µL
NOTE
(A4-H4)
If you are using a new DeepWell plate for the pre-capture PCR source plate, leave columns
1 to 3 empty and add the PCR Master Mix to column 4 of the new plate.
SureSelect XT Automated Library Prep and Capture System
89
4
Sample Preparation (200 ng DNA Samples)
Step 4. Amplify adaptor-ligated libraries
Figure 14
Configuration of the master mix source plate for
Pre-CapturePCR_XT_ILM_200ng_v1.5.1.pro. Columns 1-3 were used to dispense master mixes during the previous protocol.
6 Seal the master mix source plate using the PlateLoc Thermal Microplate
Sealer, with sealing settings of 165°C and 1.0 sec.
7 Centrifuge the plate for 30 seconds to drive the well contents off the
walls and plate seal and to eliminate any bubbles.
NOTE
90
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.
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (200 ng DNA Samples)
Step 4. Amplify adaptor-ligated libraries
4
Load the Agilent NGS Workstation
8 Load the Labware MiniHub according to Table 42, using the plate
orientations shown in Figure 13.
Table 42
Initial MiniHub configuration for Pre-CapturePCR_XT_ILM_200ng_v1.5.1.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
* The waste tip box (Cassette 1, Shelf 2) and clean tip box (Cassette 1, Shelf 1) are retained from the
LibraryPrep_XT_ILM_v1.5.1.rst run and reused here.
NOTE
If you are using a new box of tips on shelf 1 of cassette 1, first remove the tips from
columns 1 to 3 of the tip box. Any tips present in columns 1 to 3 of the tip box may be
inappropriately loaded onto the Bravo platform pipette heads and may interfere with
automated processing steps.
9 Load the Bravo deck according to Table 43.
Table 43
Initial Bravo deck configuration for Pre-CapturePCR_XT_ILM_200ng_v1.5.1.pro
Location
Content
6
Empty PCR plate seated on red insert (PCR plate type must be
specified on setup form under step 2)
7
Adaptor-ligated DNA samples in Eppendorf plate
9
Master mix plate containing PCR Master Mix in Column 4 (unsealed)
SureSelect XT Automated Library Prep and Capture System
91
4
Sample Preparation (200 ng DNA Samples)
Step 4. Amplify adaptor-ligated libraries
10 Load the BenchCel Microplate Handling Workstation according to
Table 44.
Table 44
Initial BenchCel configuration for Pre-CapturePCR_XT_ILM_200ng_v1.5.1.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
Run VWorks protocol Pre-CapturePCR_XT_ILM_200ng_v1.5.1.pro
11 On the SureSelect setup form, under Select Protocol to Run, select
Pre-CapturePCR_XT_ILM_200ng_v1.5.1.pro.
12 Under Select PCR plate labware for Thermal Cycling, select the
specific type of PCR plate used at position 6 of the Bravo deck.
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.
92
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (200 ng DNA Samples)
Step 4. Amplify adaptor-ligated libraries
4
16 When verification is complete, click Run Selected Protocol.
Running the Pre-CapturePCR_XT_ILM_200ng_v1.5.1.pro protocol takes
approximately 15 minutes. Once complete, the PCR-ready samples,
containing prepped DNA and PCR master mix, are located in the PCR
plate at position 6 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.
19 Transfer the PCR plate to a thermal cycler and run the PCR
amplification program shown in Table 45.
SureSelect XT Automated Library Prep and Capture System
93
4
Sample Preparation (200 ng DNA Samples)
Step 4. Amplify adaptor-ligated libraries
Table 45
94
Pre-Capture PCR cycling program (use only for the 200 ng DNA input workflow)
Segment
Number of
Cycles
Temperature
Time
1
1
98°C
2 minutes
2
10
98°C
30 seconds
65°C
30 seconds
72°C
1 minute
3
1
72°C
10 minutes
4
1
4°C
Hold
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (200 ng DNA Samples)
Step 5. Purify amplified DNA using AMPure XP beads
4
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.) Do not freeze the beads at any time.
3 Mix the bead suspension well so that the reagent appears homogeneous
and consistent in color.
4 Prepare a Nunc DeepWell source plate for the beads by adding 95 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 46, using the plate
orientations shown in Figure 13.
Table 46
Initial MiniHub configuration for AMPureXP_XT_ILM_v1.5.1.pro:Pre-Capture
PCR
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
Plate
Empty
Empty
Shelf 2
Empty
Nuclease-free water AMPure XP beads
reservoir from step 5 in Nunc DeepWell
plate from step 4
Empty
Shelf 1 (Bottom)
Empty
70% ethanol
Empty
reservoir from step 6
Empty tip box
SureSelect XT Automated Library Prep and Capture System
95
4
Sample Preparation (200 ng DNA Samples)
Step 5. Purify amplified DNA using AMPure XP beads
8 Load the Bravo deck according to Table 47.
Table 47
Initial Bravo deck configuration for AMPureXP_XT_ILM_v1.5.1.pro:Pre-Capture
PCR
Location
Content
1
Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)
9
Amplified DNA libraries in PCR plate seated in red insert (PCR plate type must
be specified on setup form under step 2)
9 Load the BenchCel Microplate Handling Workstation according to
Table 48.
Table 48
Initial BenchCel configuration for AMPureXP_XT_ILM_v1.5.1.pro:Pre-Capture
PCR
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
Run VWorks protocol AMPureXP_XT_ILM_v1.5.1.pro:Pre-Capture PCR
10 On the SureSelect setup form, under Select Protocol to Run, select
AMPureXP_XT_ILM_v1.5.1.pro:Pre-Capture PCR.
NOTE
96
AMPureXP purification protocols are used during multiple steps of the SureSelect
automation workflow. Be sure to select the correct workflow step when initiating the
automation protocol.
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (200 ng DNA Samples)
Step 5. Purify amplified DNA using AMPure XP beads
4
11 Under Select PCR plate labware for Thermal Cycling, select the
specific type of PCR plate containing the amplified libraries at position
9.
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 purified DNA samples are in the Eppendorf plate located on Bravo
deck position 7.
SureSelect XT Automated Library Prep and Capture System
97
4
Sample Preparation (200 ng DNA Samples)
Step 6. Assess Library DNA quantity and quality
Step 6. Assess Library DNA quantity and quality
The hybridization protocol in the following section requires 750 ng of each
amplified DNA library. Measure the concentration of each library using
one of the methods detailed below. Once DNA concentration for each
sample is determined, calculate the volume of the library to be used for
hybridization using the following formula:
Volume (µL) = 750 ng/concentration (ng/µL)
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 at
www.genomics.agilent.com.
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 the peak of DNA fragment size
positioned between 225 to 275 bp. A sample electropherogram is shown
in Figure 15.
7 Determine the concentration of the library (ng/µL) by integrating under
the peak.
Stopping Point
98
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.
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (200 ng DNA Samples)
Step 6. Assess Library DNA quantity and quality
Figure 15
4
Analysis of amplified library DNA using a DNA 1000 assay.
SureSelect XT Automated Library Prep and Capture System
99
4
Sample Preparation (200 ng DNA Samples)
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 libraries. For more information to do
this step, see the Agilent 2200 TapeStation User Manual at
www.genomics.agilent.com.
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 1 µL of each amplified library 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 the peak of DNA fragment size
positioned between 225 to 275 bp. A sample electropherogram is shown
in Figure 16.
Stopping Point
100
If you do not continue to the next step, seal the library DNA sample plate
and store at 4°C overnight or at –20°C for prolonged storage.
SureSelect XT Automated Library Prep and Capture System
Sample Preparation (200 ng DNA Samples)
Step 6. Assess Library DNA quantity and quality
Figure 16
4
Analysis of amplified library DNA using the 2200 TapeStation.
SureSelect XT Automated Library Prep and Capture System
101
4
102
Sample Preparation (200 ng DNA Samples)
Step 6. Assess Library DNA quantity and quality
SureSelect XT Automated Library Prep and Capture System
SureSelectXT Automated Target Enrichment for Illumina Paired-End
Multiplexed Sequencing Protocol
5
Hybridization
Step 1. Aliquot prepped DNA samples for hybridization 104
Step 2. Hybridize DNA Samples to the Capture Library 108
Step 3. Capture the hybridized DNA 123
This chapter describes the steps to combine the prepped library with the
blocking agents and the SureSelect capture library. Each DNA library
sample must be hybridized and captured individually prior to addition of
the indexing tag by PCR.
CA U T I O N
The ratio of SureSelect capture library to prepped library is critical for successful
capture.
Agilent Technologies
103
5
Hybridization
Step 1. Aliquot prepped DNA samples for hybridization
Step 1. Aliquot prepped DNA samples for hybridization
For each sample library prepared, do one hybridization and capture. Do
not pool samples at this stage.
Each hybridization reaction will contain 750 ng of the prepped gDNA
sample. Before starting the hybridization step, you must create a table
containing instructions for the Agilent NGS Workstation indicating the
volume of each sample required for a 750-ng aliquot.
1 Create a .csv (comma separated value) file with the headers shown in
Figure 17. The header text must not contain spaces. The table may be
created using a spreadsheet application, such as Microsoft Excel
software, and then saved in .csv format. The file must include rows for
all 96 wells of the plate.
2 Enter the information requested in the header for each DNA sample.
• In the SourceBC field, enter the sample plate description or barcode.
The SourceBC field contents must be identical for all rows.
• In the SourceWell and DestinationWell fields, enter each well position
for the plate. SourceWell and DestinationWell field contents must be
identical for a given sample.
• In the Volume field, enter the volume (in µL) equivalent to 750 ng
DNA for each sample. These values are determined from the
concentration values obtained from Bioanalyzer or TapeStation traces
in the previous section. For all empty wells on the plate, enter the
value 0, as shown in Figure 17; do not delete rows for empty wells.
Figure 17
104
Sample spreadsheet for 750-ng sample aliquot for 1-column run.
SureSelect XT Automated Library Prep and Capture System
Hybridization
Step 1. Aliquot prepped DNA samples for hybridization
NOTE
5
You can find a sample spreadsheet in the directory C: > VWorks Workspace > NGS Option
B > XT Illumina_1.5.1> Aliquot Library Input Files >
750ng_transfer_full_plate_template_xlsx.
The 750ng_transfer_full_plate_template.xlsx file may be copied and used as a template for
creating the .csv files for each Aliquot_Libraries_v1.5.1.pro run. If you are using the sample
file as a template for runs with fewer than 12 columns, be sure to retain rows for all 96
wells, and populate the Volume column with 0 for unused wells.
3 Load the .csv file onto the PC containing the VWorks software into a
suitable folder, such as C: > VWorks Workspace > NGS Option B > XT
Illumina_1.5.1 > Aliquot Library Input Files.
4 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.
5 Load the Bravo deck according to Table 49.
Table 49
Initial Bravo deck configuration for Aliquot_Libraries_v1.5.1.pro
Location
Content
5
Empty Eppendorf plate
6
Empty tip box
8
New tip box
9
Prepped library DNA in Eppendorf plate
6 On the SureSelect setup form, under Select Protocol to Run, select
Aliquot_Libraries_v1.5.1.pro.
7 Click Display Initial Workstation Setup.
SureSelect XT Automated Library Prep and Capture System
105
5
Hybridization
Step 1. Aliquot prepped DNA samples for hybridization
8 Verify that the NGS workstation has been set up as displayed in the
Workstation Setup region of the form.
9 When verification is complete, click Run Selected Protocol.
10 When prompted by the dialog below, browse to the .csv file created for
the source plate of the current run, and then click OK to start the run.
The library aliquoting protocol takes approximately 1 hour for 96 samples.
When complete, the 750-ng samples are in the PCR plate located on Bravo
deck position 5.
11 Remove the 750-ng sample plate from the Bravo deck and use a
vacuum concentrator to dry the sample at  45°C.
12 Reconstitute each dried sample with 3.4 µL of nuclease-free water to
bring the final concentration to 221 ng/µL. Pipette up and down along
the sides of each well for optimal recovery.
13 Seal the plate using the PlateLoc Thermal Microplate Sealer, with
sealing settings of 165°C and 1.0 sec.
106
SureSelect XT Automated Library Prep and Capture System
Hybridization
Step 1. Aliquot prepped DNA samples for hybridization
5
14 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.
SureSelect XT Automated Library Prep and Capture System
107
5
Hybridization
Step 2. Hybridize DNA Samples to the Capture Library
Step 2. Hybridize DNA Samples to the Capture Library
In this step, the Agilent NGS Workstation completes the liquid handling
steps to prepare for hybridization. Afterward, you transfer the sample
plate to a thermal cycler, held at 65°C, to allow hybridization of the
prepared DNA samples to one or more Capture Libraries.
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 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 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 Hybridization protocol.
108
SureSelect XT Automated Library Prep and Capture System
Hybridization
Step 2. Hybridize DNA Samples to the Capture Library
5
Prepare the SureSelect Block master mix
5 Prepare the appropriate volume of SureSelect Block master mix, on ice,
as indicated in Table 50.
Table 50
Preparation of SureSelect Block Master Mix
SureSelectXT
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
Nuclease-free
water
6.0 µL
76.5 µL
127.5 µL
178.5 µL
229.5 µL
331.5 µL
663.0 µL
SureSelect
Indexing Block 1
(green cap)
2.5 µL
31.9 µL
53.1 µL
74.4 µL
95.6 µL
138.1 µL
276.3 µL
SureSelect Block 2
(blue cap)
2.5 µL
31.9 µL
53.1 µL
74.4 µL
95.6 µL
138.1 µL
276.3 µL
SureSelect ILM
Indexing Block 3
(brown cap)
0.6 µL
7.7 µL
12.8 µL
17.9 µL
23.0 µL
33.2 µL
66.3 µL
Total Volume
11.6 µL
147.9 µL
246.5 µL
345.1 µL
443.7 µL
640.9 µL
1281.9 µL
Prepare one or more Capture Library master mixes
6 Prepare the appropriate volume of SureSelect capture library master
mix for each of the capture libraries that will be used for hybridization
as indicated in Table 51 to Table 54. Mix the components by pipetting.
Keep the master mixes on ice during preparation and aliquoting.
NOTE
Each row of the prepped gDNA sample 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 51 or Table 52) below.
For runs that use different capture libraries for individual rows, prepare each master mix as
described in Step b (Table 53 or Table 54) below.
SureSelect XT Automated Library Prep and Capture System
109
5
Hybridization
Step 2. Hybridize DNA Samples to the Capture Library
a For runs that use a single capture library for all rows, prepare the
Capture Library Master Mix as listed in Table 51 or Table 52, based
on the Mb target size of your design.
Table 51
Preparation of Capture Library Master Mix for target sizes <3.0 Mb, 8 rows of wells
Target size <3.0 Mb
SureSelectXT
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
Nuclease-free
water
4.5 µL
76.5 µL
114.8 µL
153.0 µL
191.3 µL
306.0 µL
592.9 µL
RNase Block
(purple cap)
0.5 µL
8.5 µL
12.8 µL
17.0 µL
21.3 µL
34.0 µL
65.9 µL
Capture Library
2.0 µL
34.0 µL
51.0 µL
68.0 µL
85.0 µL
136.0 µL
263.5 µL
Total Volume
7.0 µL
119.0 µL
178.6 µL
238.0 µL
297.6 µL
476.0 µL
922.3 µL
Table 52
Preparation of Capture Library Master Mix for target sizes >3.0 Mb, 8 rows of wells
Target size >3.0 Mb
SureSelectXT
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
Nuclease-free
water
1.5 µL
25.5 µL
38.3 µL
51.0 µL
63.8 µL
102.0 µL
197.6 µL
RNase Block
(purple cap)
0.5 µL
8.5 µL
12.8 µL
17.0 µL
21.3 µL
34.0 µL
65.9 µL
Capture Library
5.0 µL
85.0 µL
127.5 µL
170.0 µL
212.5 µL
340.0 µL
658.8 µL
Total Volume
7.0 µL
119.0 µL
178.6 µL
238.0 µL
297.6 µL
476.0 µL
922.3 µL
110
SureSelect XT Automated Library Prep and Capture System
Hybridization
Step 2. Hybridize DNA Samples to the Capture Library
5
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 53 or Table 54, based on the Mb target size of your
design. The volumes listed in Table 53 and Table 54 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 53
Preparation of Capture Library Master Mix for target sizes <3.0 Mb, single row of wells
Target size <3.0 Mb
SureSelectXT
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
Nuclease-free
water
4.5 µL
9.0 µL
13.8 µL
18.6 µL
23.3 µL
37.7 µL
73.5 µL
RNase Block
(purple cap)
0.5 µL
1.0 µL
1.5 µL
2.1 µL
2.6 µL
4.2 µL
8.2 µL
Capture Library
2.0 µL
4.0 µL
6.1 µL
8.3 µL
10.4 µL
16.8 µL
32.7 µL
Total Volume
7.0 µL
14.0 µL
21.4 µL
28.9 µL
36.3 µL
58.6 µL
114.4 µL
Table 54
Preparation of Capture Library Master Mix for target sizes >3.0 Mb, single row of wells
Target size >3.0 Mb
SureSelectXT
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
Nuclease-free
water
1.5 µL
3.0 µL
4.6 µL
6.2 µL
7.8 µL
12.6 µL
24.5 µL
RNase Block
(purple cap)
0.5 µL
1.0 µL
1.5 µL
2.1 µL
2.6 µL
4.2 µL
8.2 µL
Capture Library
5.0 µL
10.0 µL
15.3 µL
20.6 µL
25.9 µL
41.9 µL
81.7 µL
Total Volume
7.0 µL
14.0 µL
21.4 µL
28.9 µL
36.3 µL
58.6 µL
114.4 µL
SureSelect XT Automated Library Prep and Capture System
111
5
Hybridization
Step 2. Hybridize DNA Samples to the Capture Library
Prepare the Hybridization Buffer master mix
7 Prepare the appropriate volume of Hybridization Buffer Master Mix, at
room temperature, as indicated in Table 55.
Table 55
Preparation of Hybridization Buffer Master Mix
SureSelectXT Reagent 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 Hyb 1
(orange cap or bottle)
140.9 µL
197.3 µL
250.0 µL
310.1 µL
422.8 µL
789.3 µL
SureSelect Hyb 2 (red
cap)
5.6 µL
7.9 µL
10.0 µL
12.4 µL
16.9 µL
31.6 µL
SureSelect Hyb 3
(yellow cap or bottle)
56.4 µL
78.9 µL
100.0 µL
124.0 µL
169.1 µL
315.7 µL
SureSelect Hyb 4
(black cap or bottle)
73.3 µL
102.6 µL
130.0 µL
161.2 µL
219.9 µL
410.4 µL
Total Volume
276.2 µL
386.7 µL
490.0 µL
607.7 µL
828.7 µL
1547 µL
8 If precipitate forms, warm the hybridization buffer at 65°C for
5 minutes.
112
SureSelect XT Automated Library Prep and Capture System
Hybridization
Step 2. Hybridize DNA Samples to the Capture Library
5
Prepare the master mix source plate
9 In a Nunc DeepWell plate, prepare the master mix source plate
containing the master mixes prepared in step 5 to step 7 at room
temperature. Add the volumes indicated in Table 56 of each master mix
to each well of the indicated column of the Nunc DeepWell plate. 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 18.
Table 56
Preparation of the Master Mix Source Plate for Hybridization_v1.5.1.pro
Master Mix
Solution
Block Master Mix
Position on
Source Plate
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
Column 1
17.0 µL
29.4 µL
41.7 µL
54.0 µL
78.7 µL
158.8 µL
14.0 µL
21.4 µL
28.9 µL
36.3 µL
58.6 µL
114.4 µL
30.5 µL
44.3 µL
57.2 µL
71.9 µL
99.5 µL
189.3 µL
(A1-H1)
Capture Library
Master Mix
Column 2
(A2-H2)
Hybridization
Column 3
Buffer Master Mix (A3-H3)
SureSelect XT Automated Library Prep and Capture System
113
5
Hybridization
Step 2. Hybridize DNA Samples to the Capture Library
Figure 18
Configuration of the master mix source plate for Hybridization_v1.5.1.pro.
10 Seal the master mix source 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 eliminate any bubbles. Keep the master mix
plate at room temperature.
114
SureSelect XT Automated Library Prep and Capture System
Hybridization
Step 2. Hybridize DNA Samples to the Capture Library
5
Load the Agilent NGS Workstation
12 Load the Labware MiniHub according to Table 57, using the plate
orientations shown in Figure 3.
Table 57
Initial MiniHub configuration for Hybridization_v1.5.1.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
13 Load the Bravo deck according to Table 58.
Table 58
Initial Bravo deck configuration for Hybridization_v1.5.1.pro
Location
Content
4
Empty PCR plate seated in red insert (PCR plate type must be
specified on setup form under step 2)
5
Empty Eppendorf plate
6
Hybridization Master Mix source plate (unsealed) seated on silver
Nunc DeepWell insert
8
Empty tip box
9
750-ng aliquots of prepped gDNA (reconstituted at 221 ng/µL), in
Eppendorf plate (unsealed)
SureSelect XT Automated Library Prep and Capture System
115
5
Hybridization
Step 2. Hybridize DNA Samples to the Capture Library
14 Load the BenchCel Microplate Handling Workstation according to
Table 59.
Table 59
Initial BenchCel configuration for Hybridization_v1.5.1.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
Run VWorks protocol Hybridization_v1.5.1.pro
15 On the SureSelect setup form, under Select Protocol to Run, select
Hybridization_v1.5.1.pro.
16 Under Select PCR plate labware for Thermal Cycling, select the
specific type of PCR plate used at position 4 of the Bravo deck.
17 Select the number of columns of samples to be processed. Runs must
include 1, 2, 3, 4, 6, or 12 columns.
18 Click Display Initial Workstation Setup.
19 Verify that the NGS workstation has been set up as displayed in the
Workstation Setup region of the form.
116
SureSelect XT Automated Library Prep and Capture System
Hybridization
Step 2. Hybridize DNA Samples to the Capture Library
5
20 When verification is complete, click Run Selected Protocol.
The Agilent NGS Workstation transfers SureSelect Block Master Mix to the
prepped gDNA-containing wells of the sample plate. When this process is
complete, you will be prompted to transfer the plate to the thermal cycler
for sample denaturation prior to hybridization.
21 When prompted by VWorks as shown below, remove the PCR plate from
position 4 of the Bravo deck, 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.
SureSelect XT Automated Library Prep and Capture System
117
5
Hybridization
Step 2. Hybridize DNA Samples to the Capture Library
23 Transfer the sealed plate to a thermal cycler and run the following
program shown in Table 60. After transferring the plate, click Continue
on the VWorks screen.
Table 60
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 Master Mix.
118
SureSelect XT Automated Library Prep and Capture System
Hybridization
Step 2. Hybridize DNA Samples to the Capture Library
CA U T I O N
5
You must complete step 24 to step 28 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.
24 When the workstation has finished aliquoting the Capture Library and
Hybridization Buffer master mixes, 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.
SureSelect XT Automated Library Prep and Capture System
119
5
Hybridization
Step 2. Hybridize DNA Samples to the Capture Library
25 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
prepped gDNA samples and blocking agents.
120
SureSelect XT Automated Library Prep and Capture System
Hybridization
Step 2. Hybridize DNA Samples to the Capture Library
5
26 When prompted by VWorks as shown below, quickly remove the PCR
sample plate from Bravo deck position 4, leaving the red insert in place.
27 Seal the sample plate using the PlateLoc Thermal Microplate Sealer,
with sealing settings of 165°C and 3.0 sec.
28 Quickly transfer the plate back to the thermal cycler, held at 65°C.
After transferring the plate, click Continue on the VWorks screen.
29 To finish the VWorks protocol, click Continue in the Unused Tips and
Empty Tip box dialogs, and click Yes in the Protocol Complete dialog.
SureSelect XT Automated Library Prep and Capture System
121
5
Hybridization
Step 2. Hybridize DNA Samples to the Capture Library
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.
30 Incubate the hybridization mixture in the thermal cycler for 16 or
24 hours at 65°C with a heated lid at 105°C.
NOTE
122
If you are using the SureCycler 8800 thermal cycler for this step, be sure to set up the
incubation using a compression mat (see Table 4 on page 15 for ordering information).
SureSelect XT Automated Library Prep and Capture System
Hybridization
Step 3. Capture the hybridized DNA
5
Step 3. Capture the hybridized DNA
In this step, the gDNA-capture library hybrids are captured using
streptavidin-coated magnetic beads. This step is run immediately after the
16 or 24-hour hybridization period.
This step is automated by the NGS workstation using the
SureSelectCapture&Wash_v1.5.1.rst runset, with a total duration of
approximately 3 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 61
Operator action
Approximate time after run start
Transfer hybridization reactions from
thermal cycler to NGS workstation
<5 minutes
Remove PCR plate from red aluminum
insert
5-10 minutes
SureSelect XT Automated Library Prep and Capture System
123
5
Hybridization
Step 3. Capture the hybridized DNA
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. Bravo deck position 4
corresponds to CPAC 2, position 1 on the Multi TEC control
touchscreen.
Prepare the Dynabeads streptavidin beads
4 Vigorously resuspend the Dynabeads MyOne Streptavidin T1 magnetic
beads on a vortex mixer. The beads settle during storage.
5 Wash the magnetic beads.
a In a conical vial, combine the components listed in Table 62. The
volumes below include the required overage.
Table 62
Components required for magnetic bead washing procedure
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
Dynabeads
MyOne
Streptavidin T1
bead suspension
50 µL
425 µL
825 µL
1225 µL
1.65 mL
2.5 mL
5.0 mL
SureSelect
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
4.125 mL
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 Binding Buffer.)
124
SureSelect XT Automated Library Prep and Capture System
Hybridization
Step 3. Capture the hybridized DNA
5
6 Resuspend the beads in SureSelect Binding buffer, according to
Table 63 below.
Table 63
Preparation of magnetic beads for SureSelect Capture&Wash_v1.5.1.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
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 streptavidin 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.
Prepare capture and wash solution source plates
9 Prepare a Thermo Scientific reservoir containing 15 mL of nuclease-free
water.
10 Prepare an Eppendorf source plate labeled Wash #1. For each well to
be processed, add 160 µL of SureSelect Wash Buffer 1.
11 Prepare a Nunc DeepWell source plate labeled Wash #2. For each well
to be processed, add 1150 µL of SureSelect Wash Buffer 2.
12 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 Capture&Wash runset.
13 Place the Wash #2 source plate on the insert at position 6 of the Bravo
deck. Make sure the plate is seated properly on the silver DeepWell
insert.
SureSelect XT Automated Library Prep and Capture System
125
5
Hybridization
Step 3. Capture the hybridized DNA
Load the Agilent NGS Workstation
14 Load the Labware MiniHub according to Table 64, using the plate
orientations shown in Figure 3.
Table 64
Initial MiniHub configuration for SureSelect Capture&Wash_v1.5.1.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
plate
Empty
Wash #1
Eppendorf source
plate
Empty
Shelf 2
Empty
Nuclease-free
water reservoir
Empty
Empty
Shelf 1 (Bottom)
Empty
Empty
Empty
Empty tip box
15 Load the Bravo deck according to Table 65 (positions 5 and 6 should
already be loaded).
Table 65
126
Initial Bravo deck configuration for SureSelectCapture&Wash_v1.5.1.rst
Location
Content
1
Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)
4
Empty red insert
5
Dynabeads streptavidin bead DeepWell source plate
6
Wash #2 DeepWell source plate seated on silver Nunc DeepWell insert
SureSelect XT Automated Library Prep and Capture System
Hybridization
Step 3. Capture the hybridized DNA
5
16 Load the BenchCel Microplate Handling Workstation according to
Table 66.
Table 66
Initial BenchCel configuration for SureSelectCapture&Wash_v1.5.1.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
10 Tip boxes
3 Tip boxes
Empty
Empty
Run VWorks runset SureSelectCapture&Wash_v1.5.1.rst
17 On the SureSelect setup form, under Select Protocol to Run, select
SureSelectCapture&Wash_v1.5.1.rst.
18 Under Select PCR plate labware for Thermal Cycling, select the
specific type of PCR plate used for hybridization. This plate will be
transferred from the thermal cycler to Bravo deck position 4 when
prompted by VWorks.
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.
SureSelect XT Automated Library Prep and Capture System
127
5
Hybridization
Step 3. Capture the hybridized DNA
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.
128
SureSelect XT Automated Library Prep and Capture System
Hybridization
Step 3. Capture the hybridized DNA
CA U T I O N
5
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
insert. Click Continue to resume the runset.
WARN I NG
Warning
Bravo deck position 4 will be hot.
Use caution when handling components that contact heated deck positions.
SureSelect XT Automated Library Prep and Capture System
129
5
Hybridization
Step 3. Capture the hybridized DNA
25 When prompted by VWorks as shown below, remove the PCR plate from
position 4 of the Bravo deck, leaving the red aluminum insert in place.
When finished, click Continue to resume the runset.
The remainder of the SureSelectCapture&Wash_v1.5.1.rst runset takes
approximately 2 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
130
Captured DNA is retained on the streptavidin beads during the post-capture amplification
step.
SureSelect XT Automated Library Prep and Capture System
SureSelectXT Automated Target Enrichment for Illumina Paired-End
Multiplexed Sequencing Protocol
6
Indexing
Step 1. Amplify the captured libraries to add index tags 132
Step 2. Purify the amplified indexed libraries using Agencourt AMPure XP
beads 142
Step 3. Assess indexed DNA quality 146
Step 4. Quantify each index-tagged library by QPCR 150
Step 5. Pool samples for Multiplexed Sequencing 151
Guidelines for sequencing sample preparation and run setup 152
This chapter describes the steps to add index tags by amplification, purify,
assess quality and quantity of the libraries, and pool indexed samples for
multiplexed sequencing.
Agilent Technologies
131
6
Indexing
Step 1. Amplify the captured libraries to add index tags
Step 1. Amplify the captured libraries to add index tags
In this step, the Agilent NGS Workstation completes the liquid handling
steps for PCR-based addition of indexing tags to 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.
The size of your Capture Library determines the amplification cycle
number used for indexing. Plan your experiments for amplification of
samples prepared using Capture Libraries of similar sizes on the same
plate. See Table 75 on page 141 for cycle number recommendations.
CA U T I O N
This chapter includes instructions for kits containing two different sets of indexing
primers. Verify that you are referencing the information appropriate for your kit
version before you proceed.
Kits with revised index configuration (typically received December, 2014 or later)
include 96 different primers with 8-bp indexes A01 through H12 supplied in Library Prep
Kit p/n 5500-0133 in a blue plate format. Refer to Table 86 on page 158 for the
nucleotide sequences of the 8-bp indexes.
Kits with original index configuration (typically received before December, 2014)
include 16 different primers with 6-bp indexes 1–16 supplied in Library Prep Kit p/n
5500-0075 in format of clear-capped tubes. Refer to Table 91 on page 162 for the
nucleotide sequences of the 6-bp indexes.
The 8-bp index primers are provided at a lower concentration than the 6-bp index
primers. Make sure you are adding the amount appropriate for your primer type when
completing step 4 on page 134.
132
SureSelect XT Automated Library Prep and Capture System
Indexing
Step 1. Amplify the captured libraries to add index tags
6
Assign indexes to DNA samples
Select the appropriate indexing primer for each sample.
Use a different index primer for each sample to be sequenced in the same
lane. The number of samples that may be combined per lane depends on
the sequencing platform performance and the Capture Library size.
As a guideline, Agilent recommends analyzing 100X amount of sequencing
data compared to the Capture Library size for each sample. Specific
examples of sequence data requirement recommendations are provided in
Table 67. Calculate the number of indexes that can be combined per lane
based on these guidelines.
Table 67
Sequencing data requirement guidelines
Capture Library Size
Recommended Amount of Sequencing Data per Sample*
1 kb up to 0.5 Mb
0.1 to 50 Mb
0.5 Mb up to 2.9 Mb
50 to 290 Mb
3 Mb up to 5.9 Mb
300 to 590 Mb
6 Mb up to 11.9 Mb
600 to 1190 Mb
12 Mb up to 24 Mb
1.2 to 2.4 Gb
Human All Exon v5
4 Gb
Human All Exon v5 + UTRs
6 Gb
Human All Exon 50 Mb
5 Gb
Human DNA Kinome
320 Mb
Mouse All Exon
5 Gb
* Agilent recommends analyzing 100X amount of sequencing data compared to the Capture Library size for each sample. Pool samples according to your expected sequencing output.
SureSelect XT Automated Library Prep and Capture System
133
6
Indexing
Step 1. Amplify the captured libraries to add index tags
Prepare the workstation
1 Turn on the ThermoCube, 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 plates and tip boxes.
3 Pre-set the temperature of Bravo deck positions 4 and 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 4
corresponds to CPAC 2, position 1 and Bravo deck position 6
corresponds to CPAC 2, position 2 on the Multi TEC control
touchscreen.
Prepare indexing primers and PCR master mix
CA U T I O N
Do not use amplification enzymes other than Herculase II Fusion DNA Polymerase.
Other enzymes have not been validated.
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.
4 Prepare the indexing primers in the amplification protocol PCR plate.
In each well of the PCR plate, combine the specific indexing primer
assigned to the sample well with water, using the amounts of each
reagent shown in Table 68. Keep the plate on ice.
Table 68
134
Preparation of PCR plate with indexing primers
Reagent
8-bp Indexes A01–H12
(obtained from blue plate)
6-bp Indexes 1–16 (obtained
from clear-capped tubes)
Nuclease-free water
4.0 µL
8.0 µL
Indexing PCR primer (reverse)
5.0 µL
1.0 µL
Total Volume
9.0 µL
9.0 µL
SureSelect XT Automated Library Prep and Capture System
Indexing
Step 1. Amplify the captured libraries to add index tags
6
5 Prepare the appropriate volume of PCR master mix, according to
Table 69. Mix well using a vortex mixer and keep on ice.
Table 69
Preparation of PCR Master Mix for Post-CaptureIndexing_XT_ILM_v1.5.1.pro
SureSelectXT
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
Nuclease-free
water
14.5 µL
184.9 µL
308.1 µL
431.4 µL
554.6 µL
801.1 µL
1602.3 µL
Herculase II 5X
Reaction Buffer*
10.0 µL
127.5 µL
212.5 µL
297.5 µL
382.5 µL
552.5 µL
1105.0 µL
SureSelect
Indexing
Post-Capture PCR
(Forward) Primer†
1.0 µL
12.8 µL
21.3 µL
29.8 µL
38.3 µL
55.3 µL
110.5 µL
dNTP mix*
0.5 µL
6.4 µL
10.6 µL
14.9 µL
19.1 µL
27.6 µL
55.3 µL
Herculase II
polymerase
1.0 µL
12.8 µL
21.3 µL
29.8 µL
38.3 µL
55.3 µL
110.5 µL
Total Volume
27.0 µL
344.3 µL
573.8 µL
803.3 µL
1032.8 µL
1491.8 µL
2983.5 µL
* Included with the Herculase II Fusion DNA Polymerase. Do not use the buffer or dNTP mix from any other kit.
† Included in SureSelect XT Automation ILM Module Box 2.
SureSelect XT Automated Library Prep and Capture System
135
6
Indexing
Step 1. Amplify the captured libraries to add index tags
6 Using the same Nunc DeepWell master mix source plate that was used
for the Hybridization_v1.5.1.pro protocol, add the volume of PCR master
mix indicated in Table 70 to all wells of column 4 of the master mix
source plate. The final configuration of the master mix source plate is
shown in Figure 19.
Table 70
Preparation of the Master Mix Source Plate for Post-CaptureIndexing_XT_ILM_v1.5.1.pro
Master Mix
Solution
PCR Master Mix
Position on
Source Plate
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
Column 4
39.7 µL
68.3 µL
97.0 µL
125.7 µL
183.1 µL
369.6 µL
(A4-H4)
NOTE
136
If you are using a new DeepWell plate for the post-capture PCR source plate (for example,
when amplifying the second half of the captured DNA sample), leave columns 1 to 3 empty
and add the PCR Master Mix to column 4 of the new plate.
SureSelect XT Automated Library Prep and Capture System
Indexing
Step 1. Amplify the captured libraries to add index tags
Figure 19
6
Configuration of the master mix source plate for Post-CaptureIndexing_
XT_ILM_v1.5.1.pro. Columns 1–3 were used to dispense master mixes for the
Hybridization_v1.5.1.pro protocol.
7 Seal the master mix source plate using the PlateLoc Thermal Microplate
Sealer, with sealing settings of 165°C and 1.0 sec.
8 Centrifuge the plate for 30 seconds to drive the well contents off the
walls and plate seal and to eliminate any bubbles.
SureSelect XT Automated Library Prep and Capture System
137
6
Indexing
Step 1. Amplify the captured libraries to add index tags
Load the Agilent NGS Workstation
9 Load the Labware MiniHub according to Table 71, using the plate
orientations shown in Figure 3.
Table 71
Initial MiniHub configuration for Post-CaptureIndexing_XT_ILM_v1.5.1.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
10 Load the Bravo deck according to Table 72.
Table 72
138
Initial Bravo deck configuration for Post-CaptureIndexing_XT_ILM_v1.5.1.pro
Location
Content
4
Captured DNA bead suspensions in Eppendorf twin.tec plate (unsealed)
6
Diluted indexing primers in PCR plate seated in red insert (PCR plate type must
be specified on setup form under step 2)
9
Master mix plate containing PCR Master Mix in Column 4 (unsealed) seated on
silver Nunc DeepWell insert
SureSelect XT Automated Library Prep and Capture System
Indexing
Step 1. Amplify the captured libraries to add index tags
6
11 Load the BenchCel Microplate Handling Workstation according to
Table 73.
Table 73
Initial BenchCel configuration for Post-CaptureIndexing_XT_ILM_v1.5.1.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
Run VWorks protocol Post-CaptureIndexing_XT_ILM_v1.5.1.pro
12 On the SureSelect setup form, under Select Protocol to Run, select
Post-CaptureIndexing_XT_ILM_v1.5.1.pro.
13 Under Select PCR plate labware for Thermal Cycling, select the
specific type of PCR plate used at position 6 of the Bravo deck.
14 Select the number of columns of samples to be processed. Runs must
include 1, 2, 3, 4, 6, or 12 columns.
15 Click Display Initial Workstation Setup.
16 Verify that the NGS workstation has been set up as displayed in the
Workstation Setup region of the form.
SureSelect XT Automated Library Prep and Capture System
139
6
Indexing
Step 1. Amplify the captured libraries to add index tags
17 When verification is complete, click Run Selected Protocol.
Running the Post-CaptureIndexing_XT_ILM_v1.5.1.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 bead-bound captured DNA samples, which may be stored for
future use at 4°C overnight, or at –20°C for longer-term storage, is located
at position 4 of the Bravo deck.
18 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.
19 Centrifuge the plate for 30 seconds to drive the well contents off the
walls and plate seal and to eliminate air bubbles.
140
SureSelect XT Automated Library Prep and Capture System
Indexing
Step 1. Amplify the captured libraries to add index tags
6
20 Transfer the PCR plate to a thermal cycler and run the PCR
amplification program shown in Table 74 using the cycle number
specified in Table 75.
Table 74
Segment
Number of Cycles
Temperature
Time
1
1
98°C
2 minutes
2
10 to 16 Cycles
98°C
30 seconds
see Table 75 for recommendations
based on Capture Library size
57°C
30 seconds
72°C
1 minute
3
1
72°C
10 minutes
4
1
4°C
Hold
Table 75
NOTE
Post-Capture PCR cycling program
Recommended cycle number based on Capture Library size
Size of Capture Library
Cycles
<0.5 Mb
16 cycles
0.5 to 1.49 Mb
14 cycles
> 1.5 Mb
12 cycles
All Exon and Exome libraries
10 to 12 cycles
OneSeq libraries (all designs)
10 cycles
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.
SureSelect XT Automated Library Prep and Capture System
141
6
Indexing
Step 2. Purify the amplified indexed libraries using Agencourt AMPure XP beads
Step 2. Purify the amplified indexed libraries using Agencourt
AMPure XP beads
In this step, the Agilent NGS Workstation transfers AMPure XP beads to
the indexed DNA sample 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 Gently wipe down the Labware MiniHub, Bravo decks, and BenchCel
with a Nucleoclean decontamination wipe.
3 Turn on the ThermoCube, 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 Let the AMPure XP beads come to room temperature for at least
30 minutes. Do not freeze the beads at any time.
5 Mix the bead suspension well so that the reagent appears homogeneous
and consistent in color.
6 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.
7 Prepare a Thermo Scientific reservoir containing 15 mL of nuclease-free
water.
8 Prepare a separate Thermo Scientific reservoir containing 45 mL of
freshly-prepared 70% ethanol.
142
SureSelect XT Automated Library Prep and Capture System
Indexing
Step 2. Purify the amplified indexed libraries using Agencourt AMPure XP beads
6
9 Load the Labware MiniHub according to Table 76, using the plate
orientations shown in Figure 3.
Table 76
Initial MiniHub configuration for AMPureXP_XT_ILM_v1.5.1.pro:Post-Capture
PCR
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
Plate
Empty
Empty
Shelf 2
Empty
Nuclease-free
water reservoir
from step 7
AMPure XP beads
in Nunc DeepWell
plate from step 6
Empty
Shelf 1 (Bottom)
Empty
70% ethanol
reservoir from
step 8
Empty
Empty tip box
10 Load the Bravo deck according to Table 77.
Table 77
Initial Bravo deck configuration for
AMPureXP_XT_ILM_v1.5.1.pro:Post-Capture PCR
Location
Content
1
Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)
9
Indexed library samples in PCR plate seated in red insert (PCR plate type must be
specified on setup form under step 2)
SureSelect XT Automated Library Prep and Capture System
143
6
Indexing
Step 2. Purify the amplified indexed libraries using Agencourt AMPure XP beads
11 Load the BenchCel Microplate Handling Workstation according to
Table 78.
Table 78
Initial BenchCel configuration for AMPureXP_XT_ILM_v1.5.1.pro:Post-Capture
PCR
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
Run VWorks protocol AMPureXP_XT_ILM_v1.5.1.pro:Post-Capture PCR
12 On the SureSelect setup form, under Select Protocol to Run, select
AMPureXP_XT_ILM_v1.5.1.pro:Post-Capture PCR.
NOTE
AMPureXP purification protocols are used during multiple steps of the SureSelect
automation workflow. Be sure to select the correct workflow step when initiating the
automation protocol.
13 Under Select PCR plate labware for Thermal Cycling, select the
specific type of PCR plate containing the indexed libraries at position 9.
14 Select the number of columns of samples to be processed. Runs must
include 1, 2, 3, 4, 6, or 12 columns.
15 Click Display Initial Workstation Setup.
144
SureSelect XT Automated Library Prep and Capture System
Indexing
Step 2. Purify the amplified indexed libraries using Agencourt AMPure XP beads
6
16 Verify that the NGS workstation has been set up as displayed in the
Workstation Setup region of the form.
17 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.
SureSelect XT Automated Library Prep and Capture System
145
6
Indexing
Step 3. Assess indexed DNA quality
Step 3. Assess indexed DNA quality
Option 1: Analysis using the 2100 Bioanalyzer and High Sensitivity DNA Assay
1 Set up the 2100 Bioanalyzer as instructed in the High Sensitivity DNA
Kit Guide at www.genomics.agilent.com.
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 each sample for the analysis.
NOTE
For some samples, Bioanalyzer results are improved by diluting 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 the peak of DNA fragment size
positioned between 250 to 350 bp. A sample electropherogram is shown
in Figure 20.
Stopping Point
146
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.
SureSelect XT Automated Library Prep and Capture System
Indexing
Step 3. Assess indexed DNA quality
Figure 20
6
Analysis of indexed DNA using the High Sensitivity DNA Assay.
SureSelect XT Automated Library Prep and Capture System
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6
Indexing
Step 3. Assess indexed DNA quality
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 indexed DNA. For more information to do
this step, see the Agilent 2200 TapeStation User Manual at
www.genomics.agilent.com.
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 indexed 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 the peak of DNA fragment size
positioned between 250 to 350 bp. A sample electropherogram is shown
in Figure 21.
Stopping Point
148
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.
SureSelect XT Automated Library Prep and Capture System
Indexing
Step 3. Assess indexed DNA quality
Figure 21
6
Analysis of indexed DNA using the 2200 TapeStation.
SureSelect XT Automated Library Prep and Capture System
149
6
Indexing
Step 4. Quantify each index-tagged library by QPCR
Step 4. Quantify each index-tagged library by QPCR
Refer to the protocol that is included with the Agilent QPCR NGS Library
Quantification Kit (p/n G4880A) for more details to do this step.
1 Use the Agilent QPCR NGS Library Quantification Kit (for Illumina) to
determine the concentration of each index-tagged captured library.
2 Prepare a standard curve using the quantification standard included in
the kit, according to the instructions provided in the user guide.
3 Dilute each index-tagged captured library such that it falls within the
range of the standard curve.
Typically this corresponds to approximately a 1:1000 to 1:10,000
dilution of the captured DNA.
4 Prepare the QPCR master mix with Illumina adaptor-specific PCR
primers according to instructions provided in the kit.
5 Add an aliquot of the master mix to PCR tubes and add template.
6 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.
7 Use the standard curve to determine the concentration of each
unknown index-tagged library, in nM.
The concentration will be used to accurately pool samples for
multiplexed sequencing.
NOTE
150
In most cases, the cycle numbers in Table 75 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.
SureSelect XT Automated Library Prep and Capture System
Indexing
Step 5. Pool samples for Multiplexed Sequencing
6
Step 5. Pool samples for Multiplexed Sequencing
1 Combine the libraries such that each index-tagged sample is present in
equimolar amounts in the pool. For each library, use the formula below
to determine the amount of indexed sample to use.
V  f   C f 
Volume of Index = -------------------------------#  C i
where V(f) is the final desired volume of the pool,
C(f) is the desired final concentration of all the DNA in the pool
# is the number of indexes, and
C(i) is the initial concentration of each indexed sample.
Table 79 shows an example of the amount of 4 index-tagged samples
(of different concentrations) and Low TE needed for a final volume of
20 µL at 10 nM.
Table 79
Example of index volume calculation for a total volume of 20 µL
Component
V(f)
C(i)
C(f)
#
Volume to use (µL)
Sample 1
20 µL
20 nM
10 nM
4
2.5
Sample 2
20 µL
10 nM
10 nM
4
5
Sample 3
20 µL
17 nM
10 nM
4
2.9
Sample 4
20 µL
25 nM
10 nM
4
2
Low TE
7.6
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.
3 If you store the library before sequencing, add Tween 20 to 0.1% v/v
and store at -20°C short term.
SureSelect XT Automated Library Prep and Capture System
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6
Indexing
Guidelines for sequencing sample preparation and run setup
Guidelines for sequencing sample preparation and run setup
Use the appropriate Illumina Paired-End Cluster Generation Kit to do
cluster amplification.
Refer to the instructions that are included with the Illumina Paired-End
Cluster Generation Kit. The optimal seeding concentration for SureSelectXT
libraries is 6 to 8 pM, depending on the desired output and data quality.
Sequencing run setup guidelines for 8-bp indexes
For libraries prepared using kits with 8-bp indexes, sequencing runs must
be set up to perform an 8-bp index read. For the HiSeq platform, use the
Cycles settings shown in Table 80. 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.
For complete 8-bp index sequence information, see Table 86 on page 158.
Table 80
HiSeq platform Run Configuration screen Cycle Number settings*
Run Segment
Cycle Number
Read 1
100
Index 1 (i7)
9
Index 2 (i5)
0
Read 2
100
* Settings apply to v3.0 SBS chemistry.
152
SureSelect XT Automated Library Prep and Capture System
SureSelectXT Automated Target Enrichment for Illumina Paired-End
Multiplexed Sequencing Protocol
7
Reference
Reference Information for Kits with Revised Index Configuration (8-bp
indexes with indexing primers in blue plate) 154
Reference Information for Kits with Original Index Configuration (6-bp
indexes with indexing primers in 16 tubes) 159
This chapter contains reference information, including component kit
contents and index sequences.
Agilent Technologies
153
7
Reference
Reference Information for Kits with Revised Index Configuration (8-bp indexes with 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 8-bp indexes supplied in Library Prep Kit p/n 5500-0133
(typically received December, 2014 or later). The second section covers kits with 6-bp
indexes supplied in Library Prep Kit 5500-0075 (typically received before December,
2014). Verify that you are referencing the information appropriate for your kit version
before you proceed.
Reference Information for Kits with Revised Index Configuration
(8-bp indexes with indexing primers in blue plate)
Use the reference information in this section if your kit includes
Library Prep Kit p/n 5500-0133. If your kit does not include this
component kit, see page 159 for kit content and indexing primer
information.
Kit Contents
Each SureSelectXT Automation Reagent Kit contains the following
component kits:
Table 81
SureSelectXT Automation Reagent Kit Contents-Revised Index Configuration
Product
Storage Condition
96 Reactions
480 Reactions
SureSelect XT Library Prep Kit ILM
–20°C
5500-0133
5 x 5500-0133
SureSelect Target Enrichment Box 1
Room Temperature
5190-8646
5 x 5190-8646
SureSelect XT Automation ILM Module Box 2
–20°C
5190-3730
5190-3732
NOTE
154
SureSelect capture libraries and reagents must be used within one year of receipt.
SureSelect XT Automated Library Prep and Capture System
Reference
Kit Contents
7
The contents of each of the component kits listed in Table 81 are
described in the tables below.
Table 82
SureSelect XT Library Prep Kit ILM Content-Revised Index Configuration
Kit Component
Format
10X End Repair Buffer
tube with clear cap
10X Klenow Polymerase Buffer
tube with blue cap
5X T4 DNA Ligase Buffer
tube with green cap
T4 DNA Ligase
tube with red cap
Exo(–) Klenow
tube with red cap
T4 DNA Polymerase
tube with purple cap
Klenow DNA Polymerase
tube with yellow cap
T4 Polynucleotide Kinase
tube with orange cap
dATP
tube with green cap
dNTP Mix
tube with green cap
SureSelect Adaptor Oligo Mix
tube with brown cap
SureSelect Primer (forward primer)
tube with brown cap
SureSelectXT Indexes, 8 bp reverse
primers*
SureSelect 8bp Indexes A01 through H12, provided in blue
96-well plate†
* See Table 86 on page 158 for index sequences.
† See Table 85 on page 157 for a plate map.
SureSelect XT Automated Library Prep and Capture System
155
7
Reference
Kit Contents
Table 83
Kit Component
Format
SureSelect Hyb 1
tube with orange cap
SureSelect Hyb 2
tube with red cap
SureSelect Hyb 4
tube with black cap
SureSelect Binding Buffer
bottle
SureSelect Wash Buffer 1
bottle
SureSelect Wash Buffer 2
bottle
Table 84
156
SureSelect Target Enrichment-Box 1 Content
SureSelect XT Automation ILM Module Box 2 Content
Kit Component
96 Reactions
480 Reactions
SureSelect Hyb 3
tube with yellow cap
bottle
SureSelect Indexing Block 1
tube with green cap
tube with green cap
SureSelect Block 2
tube with blue cap
tube with blue cap
SureSelect ILM Indexing Block 3
tube with brown cap
tube with brown cap
SureSelect RNase Block
tube with purple cap
tube with purple cap
SureSelect Indexing Pre-Capture PCR (Reverse)
Primer
tube with clear cap
tube with clear cap
SureSelect Indexing Post-Capture PCR (Forward)
Primer
tube with orange cap
tube with orange cap
SureSelect XT Automated Library Prep and Capture System
Reference
Kit Contents
Table 85
7
Plate map for SureSelect 8bp Indexes A01 through H12, provided in blue plate in Library Prep kit p/n 5500-0133
1
2
3
4
5
6
7
8
9
10
11
12
A
A01
A02
A03
A04
A05
A06
A07
A08
A09
A10
A11
A12
B
B01
B02
B03
B04
B05
B06
B07
B08
B09
B10
B11
B12
C
C01
C02
C03
C04
C05
C06
C07
C08
C09
C10
C11
C12
D
D01
D02
D03
D04
D05
D06
D07
D08
D09
D10
D11
D12
E
E01
E02
E03
E04
E05
E06
E07
E08
E09
E10
E11
E12
F
F01
F02
F03
F04
F05
F06
F07
F08
F09
F10
F11
F12
G
G01
G02
G03
G04
G05
G06
G07
G08
G09
G10
G11
G12
H
H01
H02
H03
H04
H05
H06
H07
H08
H09
H10
H11
H12
SureSelect XT Automated Library Prep and Capture System
157
7
Reference
Nucleotide Sequences of SureSelectXT 8-bp Indexes
Nucleotide Sequences of SureSelectXT 8-bp Indexes
Each index is 8 nt in length. See page 152 for sequencing run setup
requirements for sequencing libraries using 8-bp indexes.
Table 86
SureSelectXT 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
158
SureSelect XT Automated Library Prep and Capture System
Reference
7
Reference Information for Kits with Original Index Configuration (6-bp indexes with indexing primers in
16 tubes)
Reference Information for Kits with Original Index Configuration
(6-bp indexes with indexing primers in 16 tubes)
Use the reference information in this section if your kit includes
Library Prep Kit p/n 5500-0075. If your kit does not include this
component kit, see page 154 for kit content and indexing primer
information.
Kit Contents
Each SureSelectXT Automation Reagent Kit contains the following
component kits:
Table 87
SureSelect XT Automation Reagent Kit Contents-Original Index Configuration
Product
Storage Condition
96 Reactions
480 Reactions
SureSelect XT Library Prep Kit ILM
–20°C
5500-0075
5 x 5500-0075
SureSelect Target Enrichment Box 1
Room Temperature
5190-4394
5190-4395
SureSelect XT Automation ILM Module Box 2
–20°C
5190-3730
5190-3732
NOTE
SureSelect capture libraries and reagents must be used within one year of receipt.
SureSelect XT Automated Library Prep and Capture System
159
7
Reference
Kit Contents
The contents of each of the component kits listed in Table 87 are
described in the tables below.
Table 88
SureSelect XT Library Prep Kit ILM Content-Original Index Configuration
Kit Component
Format
10X End Repair Buffer
tube with clear cap
10X Klenow Polymerase Buffer
tube with blue cap
5X T4 DNA Ligase Buffer
tube with green cap
T4 DNA Ligase
tube with red cap
Exo(–) Klenow
tube with red cap
T4 DNA Polymerase
tube with purple cap
Klenow DNA Polymerase
tube with yellow cap
T4 Polynucleotide Kinase
tube with orange cap
dATP
tube with green cap
dNTP Mix
tube with green cap
SureSelect Adaptor Oligo Mix
tube with brown cap
SureSelect Primer (forward primer)
tube with brown cap
PCR Primer Index 1 through Index 16 (reverse primers
containing 6-bp index sequences)*
16 tubes with clear caps
* See Table 91 on page 162 for index sequences.
160
SureSelect XT Automated Library Prep and Capture System
Reference
Kit Contents
Table 89
7
SureSelect Target Enrichment Box 1 Content
Kit Component
96 Reactions
480 Reactions
SureSelect Hyb 1
tube with orange cap
bottle
SureSelect Hyb 2
tube with red cap
tube with red cap
SureSelect Hyb 4
tube with black cap
bottle
SureSelect Binding Buffer
bottle
bottle
SureSelect Wash Buffer 1
bottle
bottle
SureSelect Wash Buffer 2
bottle
bottle
SureSelect Elution Buffer*
bottle
bottle
SureSelect Neutralization Buffer*
bottle
bottle
* The provided SureSelect Elution Buffer and Neutralization Buffer are not used in the workflow described in this manual.
Table 90
SureSelect XT Automation ILM Module Box 2 Content
Kit Component
96 Reactions
480 Reactions
SureSelect Hyb 3
tube with yellow cap
bottle
SureSelect Indexing Block 1
tube with green cap
tube with green cap
SureSelect Block 2
tube with blue cap
tube with blue cap
SureSelect ILM Indexing Block 3
tube with brown cap
tube with brown cap
SureSelect RNase Block
tube with purple cap
tube with purple cap
SureSelect Indexing Pre-Capture PCR (Reverse) Primer
tube with clear cap
tube with clear cap
SureSelect Indexing Post-Capture PCR (Forward) Primer
tube with orange cap
tube with orange cap
SureSelect XT Automated Library Prep and Capture System
161
7
Reference
Nucleotide Sequences of SureSelectXT 6-bp Indexes
Nucleotide Sequences of SureSelectXT 6-bp Indexes
Refer to the sequence information in Table 91 only if your kit includes
Library Prep kit p/n 5500-0075, with indexing primers provided in 16
clear-capped tubes (original kit configuration).
Table 91
162
SureSelectXT Indexes 1-16
Index Number
Sequence
1
ATCACG
2
CGATGT
3
TTAGGC
4
TGACCA
5
ACAGTG
6
GCCAAT
7
CAGATC
8
ACTTGA
9
GATCAG
10
TAGCTT
11
GGCTAC
12
CTTGTA
13
AAACAT
14
CAAAAG
15
GAAACC
16
AAAGCA
SureSelect XT Automated Library Prep and Capture System
www.agilent.com
In This Book
This guide contains
information to run the
SureSelectXT Automated
Target Enrichment for
Illumina Paired-End
Multiplexed Sequencing
protocol using a
SureSelectXT Automated
Reagent Kit (HSQ or
MSQ) and automation
protocols provided with
the Agilent NGS
Workstation Option B.
Agilent Technologies, Inc. 2015
Version G.2, April 2015
*G7550-90000*
G7550-90000
Agilent Technologies
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