SureSelect Automated Target Enrichment for Illumina Multiplexed

SureSelect Automated Target Enrichment for Illumina Multiplexed
SureSelectQXT
Automated Target
Enrichment for Illumina
Multiplexed Sequencing
Featuring Transposase-Based Library
Prep Technology
Automated using Agilent NGS Bravo
Option A
Protocol
Version B0, November 2015
SureSelect platform manufactured with Agilent
SurePrint Technology
For Research Use Only. Not for use in diagnostic
procedures.
Agilent Technologies
Notices
© Agilent Technologies, Inc. 2015
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Manual Part Number
G9681-90020
Edition
Version B0, November 2015
Printed in USA
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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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SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Safety Notices
CA U T I O N
A CAUTION notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not
correctly performed or adhered to, could result in damage to the product or loss of important data. Do not proceed
beyond a CAUTION notice until the indicated conditions are fully understood and met.
WARN I NG
A WARNING notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if
not correctly performed or adhered to, could result in personal injury or death. Do not proceed beyond a
WARNING notice until the indicated conditions are fully understood and met.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
3
In this Guide...
This guide describes an optimized protocol for Illumina
paired-end multiplexed library preparation using the
SureSelectQXT Automated Target Enrichment system.
This protocol is specifically developed and optimized 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 Agilent’s NGS Bravo Option A.
1
Before You Begin
This chapter contains information that you should read and
understand before you start an experiment.
2
Using the Agilent NGS Bravo Option A for SureSelectQXT Target
Enrichment
This chapter contains an orientation to the Agilent NGS
Bravo, an overview of the SureSelect target enrichment
protocol, and considerations for designing SureSelect
experiments for automated processing using the Agilent NGS
Bravo Option A.
3
Sample Preparation
This chapter describes the steps to prepare gDNA
sequencing libraries for target enrichment.
4
Hybridization
This chapter describes the steps to hybridize and capture
the prepared DNA library using a SureSelect or ClearSeq
Capture Library.
5
Indexing
This chapter describes the steps for post-capture
amplification and guidelines for sequencing sample
preparation.
4
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
6
Reference
This chapter contains reference information, including
component kit contents and index sequences.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
5
What’s New in Version B0
• Updates to custom sequencing primer mixtures for the
NextSeq 500 v2 platform (seeTable 61 and Table 62 on
page 114)
• Update to P5 Index details for NextSeq 500 platform runs
using BaseSpace (see Table 70 on page 124)
• Update to Qubit dsDNA Assay Kit nomenclature (Table 1
on page 12 and step 4 on page 39)
• Updates to SureCycler 8800 PCR plate compatibility
considerations (see Caution on page 34)
• Support for Agilent 4200 TapeStation (see Table 4 on
page 16)
• Correction to ordering information for Axygen 96 Deep
Well plates (see Table 4 on page 15)
• Revised ordering information for nucleic acid surface
decontamination wipes (Table 4 on page 15)
What’s New in Version A1
• Support for ClearSeq Capture Libraries, including
ClearSeq Comprehensive Cancer Libraries (see Table 3 on
page 14).
• Support for Human All Exon v6 Capture Libraries (see
Table 2 on page 13).
• Update to SBS Kit Configuration details for HiSeq 2500
Rapid Run sequencing (see Table 57 on page 111).
• Support for sequencing using NextSeq 500 v2 (see
Table 61 on page 111).
• Update to Qubit dsDNA Assay Kit ordering information
(see Table 1 on page 12).
6
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Content
1
Before You Begin
9
Procedural Notes 10
Safety Notes 11
Required Reagents 12
Optional Reagents 14
Required Equipment 15
2
Using the Agilent NGS Bravo Option A for SureSelectQXT Target Enrichment
17
About the Agilent NGS Bravo Option A 18
About the Bravo Platform 18
VWorks Automation Control Software 22
Overview of the SureSelectQXT Target Enrichment Procedure
29
Experimental Setup Considerations for Automated Runs 32
Considerations for Placement of gDNA Samples in 96-well Plates for
Automated Processing 33
Considerations for Equipment Setup 33
PCR Plate Type Considerations 34
3
Sample Preparation
37
Step 1. Prepare the genomic DNA samples and Library Prep reagents
Step 2. Fragment and adaptor-tag the genomic DNA samples 40
Step 3. Purify adaptor-tagged DNA using AMPure XP beads 45
Step 4. Amplify adaptor-ligated libraries 48
Step 5. Purify amplified DNA using AMPure XP beads 55
Step 6. Assess Library DNA quantity and quality 58
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
38
7
Contents
4
Hybridization
63
Step 1. Aliquot prepped DNA samples for hybridization 64
Step 2. Hybridize the gDNA library and SureSelect or ClearSeq Capture
Library 69
Step 3. Capture the hybridized DNA 83
Step 4. Wash the captured DNA 89
5
Indexing
93
Step 1. Amplify the captured DNA libraries to add index tags 94
Step 2. Purify the amplified indexed libraries using Agencourt AMPure XP
beads 102
Step 3. Assess indexed DNA quality 105
Step 4. Quantify each index-tagged library by QPCR (optional) 109
Step 5. Pool samples for Multiplexed Sequencing 110
Step 6. Prepare sequencing samples 111
Step 7. Set up the sequencing run and trim adaptors from the reads 115
6
Reference
119
Kit Contents 120
Nucleotide Sequences of SureSelectQXT Dual Indexes 123
Guidelines for Multiplexing with Dual-Indexed Samples 125
8
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
SureSelectQXT Automated Target Enrichment for Illumina Multiplexed
Sequencing Protocol
1
Before You Begin
Procedural Notes 10
Safety Notes 11
Required Reagents 12
Optional Reagents 14
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.
NOTE
This protocol describes automated sample processing using the Agilent NGS Bravo
Option A. For automated sample processing using the Agilent NGS Workstation Option B,
see publication G9681-90010. For non-automated sample processing procedures see
publication G9681-90000.
NOTE
Agilent cannot guarantee the SureSelect Target Enrichment kits and cannot provide
technical support for the use of non-Agilent protocols or instruments to process samples
for enrichment.
Agilent Technologies
9
1
Before You Begin
Procedural Notes
Procedural Notes
• The SureSelectQXT system requires high-quality DNA samples for
optimal performance. Use best practices for verifying DNA sample
quality before initiating the workflow. For best practice, store diluted
DNA solutions at 4°C to avoid repeated freeze-thaw cycles, which may
compromise DNA quality.
• Performance of the SureSelectQXT library preparation protocol is very
sensitive to variations in amounts of DNA sample and other reaction
components. It is important to quantify and dilute DNA samples as
described on page 39. Carefully measure volumes for all reaction
components, and combine components as described in this instruction
manual. Use best-practices for liquid handling, including regular pipette
calibration, to ensure precise volume measurement.
• Use care in handling the SureSelect QXT Enzyme Mix. After removing
the vial from storage at –20°C, keep on ice or in a cold block while in
use. Return the vial to storage at –20°C promptly after use.
• 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 Bravo to allow rapid and
efficient plate transfer.
• Use best-practices to prevent PCR product contamination of samples
throughout the workflow:
1 Assign separate pre-PCR and post-PCR pipettors, supplies, and
reagents. In particular, never use materials designated to post-PCR
segments for the pre-PCR segments of the workflow. For the pre-PCR
workflow steps, always use dedicated pre-PCR pipettors with
nuclease-free aerosol-resistant tips to pipette dedicated pre-PCR
solutions.
2 Maintain clean work areas. Clean pre-PCR surfaces that pose the
highest risk of contamination daily using a 10% bleach solution.
3 Wear powder-free gloves. Use good laboratory hygiene, including
changing gloves after contact with any potentially-contaminated
surfaces.
• Possible stopping points, where samples may be stored at –20°C, are
marked in the protocol. Do not subject the samples to multiple
freeze/thaw cycles.
10
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Before You Begin
Safety Notes
1
• 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.
• In general, follow Biosafety Level 1 (BL1) safety rules.
Safety Notes
CA U T I O N
• Wear appropriate personal protective equipment (PPE) when working in the
laboratory.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
11
1
Before You Begin
Required Reagents
Required Reagents
Required Reagents for SureSelectQXT Target Enrichment
Table 1
12
Description
Vendor and part number
SureSelect or ClearSeq Capture Library
Select one library from Table 2 or
Table 3
SureSelectQXT Reagent Kit
Illumina HiSeq or MiSeq platform (ILM), 96 Samples
Illumina NextSeq platform (NSQ), 96 Samples
Agilent
p/n G9681B
p/n G9683B
Agencourt AMPure XP Kit
5 ml
60 ml
450 ml
Beckman Coulter Genomics
p/n A63880
p/n A63881
p/n A63882
Dynabeads MyOne Streptavidin T1
2 ml
10 ml
100 ml
Life Technologies
p/n 65601
p/n 65602
p/n 65603
1X Low TE Buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA)
Life Technologies p/n 12090-015, or
equivalent
100% Ethanol, molecular biology grade
Sigma-Aldrich p/n E7023
Qubit dsDNA HS Assay Kit or
Life Technologies p/n Q32851
Qubit dsDNA BR Assay Kit
100 assays
500 assays
Life Technologies
p/n Q32850
p/n Q32853
Nuclease-free Water (not DEPC-treated)
Ambion Cat #AM9930
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Before You Begin
Required Reagents
Table 2
SureSelectXT Automation Capture Libraries
Capture Library
96 Reactions
480 Reactions
SureSelect
XT Human All Exon v6
5190-8865
5 × 5190-8865
SureSelect
XT
5190-8883
5 × 5190-8883
5190-9309
5 × 5190-9309
Human All Exon v6 + UTRs
SureSelectXT Human All Exon v6 + COSMIC
SureSelect
XT
Human All Exon v6 Plus 1
5190-8868
5 × 5190-8868
SureSelect
XT
Human All Exon v6 Plus 2
5190-8871
5 × 5190-8871
SureSelect
XT
Clinical Research Exome
5190-7344
5 × 5190-7344
5190-7789
5 × 5190-7789
SureSelectXT Focused Exome
SureSelect
XT
Focused Exome Plus 1
5190-7792
5 × 5190-7792
SureSelect
XT Focused Exome Plus 2
5190-7796
5 × 5190-7796
SureSelect
XT Human All Exon v5
5190-6210
5 × 5190-6210
SureSelectXT Human All Exon v5 + UTRs
5190-6215
5 × 5190-6215
SureSelect
XT Human All Exon v5 + lncRNA
5190-6448
5 × 5190-6448
SureSelect
XT Human All Exon v5 Plus
5190-6224
5 × 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
(reorder)
1
XT Custom 12 Mb up to 24 Mb
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
13
1
Before You Begin
Optional Reagents
Table 3
Compatible ClearSeq Automation Capture Libraries
Capture Library
96 Reactions
480 Reactions
ClearSeq Comprehensive Cancer XT
5190-8013
5 × 5190-8013
ClearSeq Comprehensive Cancer Plus XT
5190-8016
5 × 5190-8016
ClearSeq Inherited Disease XT
5190-7520
5 × 5190-7520
ClearSeq Inherited Disease Plus XT
5190-7523
5 × 5190-7523
ClearSeq DNA Kinome XT
5190-4648
5190-4650
Optional Reagents
14
Description
Vendor and part number
Agilent QPCR NGS Library Quantification Kit (Illumina GA)
Agilent p/n G4880A
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Before You Begin
Required Equipment
1
Required Equipment
Table 4
Required Equipment for SureSelectQXT Target Enrichment
Description
Vendor and part number
Agilent NGS Bravo Option A with VWorks software
version 11.3.0.1195
Contact Agilent Automation Solutions for
ordering information:
[email protected]
Robotic Pipetting Tips (Sterile, Filtered, 250 L)
Agilent p/n 19477-022
PlateLoc Thermal Microplate Sealer with Small
Hotplate
Agilent p/n G5402#226
Clear Peelable Seal plate seals (for use with the
PlateLoc Thermal Plate Sealer)
Agilent p/n 16985-001
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
When selecting plates for another thermal cycler,
see Table 8 on page 35 for the list of PCR plates
supported in 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
Nucleic acid surface decontamination wipes
DNA Away Surface Decontaminant Wipes,
Thermo Scientific p/n 7008, or equivalent
Qubit Fluorometer
Life Technologies p/n Q32857
Qubit Assay Tubes
Life Technologies p/n Q32856
Magnetic separator
DynaMag-50 magnet, Life Technologies p/n
123-02D or equivalent
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
15
1
Before You Begin
Required Equipment
Required Equipment for SureSelectQXT Target Enrichment
Table 4
Description
Vendor and part number
Vacuum concentrator
Savant SpeedVac, model DNA120, with
96-well plate rotor, model RD2MP, or
equivalent
DNA Analysis Platform and Consumables
Agilent 2100 Bioanalyzer Laptop Bundle
Agilent p/n G2943CA
Agilent 2100 Bioanalyzer Electrophoresis Set
Agilent p/n G2947CA
Agilent DNA 1000 Kit
Agilent p/n 5067-1504
Agilent High Sensitivity DNA Kit
Agilent p/n 5067-4626
Agilent 4200 TapeStation
Agilent p/n G2991AA
Agilent D1000 ScreenTape
Agilent p/n 5067-5582
Agilent D1000 Reagents
Agilent p/n 5067-5583
Agilent High Sensitivity D1000 ScreenTape
Agilent p/n 5067-5584
Agilent D1000 Reagents
Agilent p/n 5067-5585
Agilent 2200 TapeStation
Agilent p/n G2964AA or G2965AA
Agilent D1000 ScreenTape
Agilent p/n 5067-5582
Agilent D1000 Reagents
Agilent p/n 5067-5583
Agilent High Sensitivity D1000 ScreenTape
Agilent p/n 5067-5584
Agilent D1000 Reagents
Agilent p/n 5067-5585
OR
OR
Centrifuge
Eppendorf Centrifuge model 5804 or
equivalent
Plate or strip tube centrifuge
Labnet International MPS1000 Mini Plate
Spinner p/n C1000 (requires adapter, p/n
C1000-ADAPT, for use with strip tubes) or
equivalent
Pipettes (multichannel pipette and P10, P20, P200
and P1000 pipettes)
Pipetman or equivalent
Vortex mixer
Ice bucket
Powder-free gloves
Sterile, nuclease-free aerosol barrier pipette tips
16
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
SureSelectQXT Automated Target Enrichment for Illumina Multiplexed
Sequencing Protocol
2
Using the Agilent NGS Bravo Option A
for SureSelectQXT Target Enrichment
About the Agilent NGS Bravo Option A 18
Overview of the SureSelectQXT Target Enrichment Procedure 29
Experimental Setup Considerations for Automated Runs 32
This chapter contains an orientation to the Agilent NGS Bravo Option A,
an overview of the SureSelectQXT target enrichment protocol, and
considerations for designing SureSelectQXT experiments for automated
processing using the Agilent NGS Bravo Option A.
Agilent Technologies
17
2
Using the Agilent NGS Bravo Option A for SureSelectQXT Target Enrichment
About the Agilent NGS Bravo Option A
About the Agilent NGS Bravo Option A
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
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Using the Agilent NGS Bravo Option A for SureSelectQXT 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).
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
19
2
Using the Agilent NGS Bravo Option A for SureSelectQXT 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
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Using the Agilent NGS Bravo Option A for SureSelectQXT 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.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
21
2
Using the Agilent NGS Bravo Option A for SureSelectQXT Target Enrichment
VWorks Automation Control Software
VWorks Automation Control Software
VWorks software, included with your Agilent NGS Bravo Option A, allows
you to control the integrated devices using a PC. The Agilent NGS Bravo
Option A 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 SureSelectQXT automation protocols version 1.0.
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 QXT_ILM_OptA_v1.0.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.)
22
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Using the Agilent NGS Bravo Option A for SureSelectQXT Target Enrichment
VWorks Automation Control Software
2
Using the QXT_ILM_OptA_v1.0.VWForm to setup and start a run
Use the VWorks form QXT_ILM_OptA_v1.0.VWForm, shown below, to set
up and start each SureSelect automation protocol.
1 Open the form using the QXT_ILM_OptA_v1.0.VWForm shortcut on your
desktop.
2 Use the drop-down menus on the form to select the appropriate
SureSelect workflow step and other Parameters for the run.
3 Once all run parameters have been specified on the form, click Display
Initial Bravo Deck Setup.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
23
2
Using the Agilent NGS Bravo Option A for SureSelectQXT Target Enrichment
VWorks Automation Control Software
4 The form will then display the NGS Bravo deck configuration needed
for the specified run parameters.
Load the Bravo Deck with labware and reagents as specified in the
Bravo Deck Setup region of the form.
Review the temperature preset and in-run labware transfer information
shown in the Information section of the form. Set the temperature of
Bravo Deck positions as needed.
24
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Using the Agilent NGS Bravo Option A for SureSelectQXT Target Enrichment
VWorks Automation Control Software
2
5 Verify that the Current Tip State indicator on the form (shown below)
matches the configuration of unused tips in the tip box at Bravo Deck
position 2.
For a fresh tip box, containing 12 columns of tips, all positions of the
Current Tip State unused tip indicator (top portion, Box 2) should be
selected, as shown below. Clicking Reset selects all columns for position
2.
Also verify that the used tip indicator (bottom portion, Box 8) matches
the configuration of used tips in the tip box at Bravo Deck position 8.
For an empty tip box, all positions of the Current Tip State used tip
indicator (bottom portion, Box 8) should be cleared, as shown above.
Clicking Reset clears all columns for position 8.
NOTE
It is important that the Current Tip State indicator matches the configuration of tips present
at Bravo Deck positions 2 and 8 when initiating the run. Tips that are inappropriately loaded
onto the Bravo platform pipette head, or tips missing from the pipette head, will interfere
with automated processing steps.
You can use partial tip boxes for NGS Bravo Option A automation protocols, as long as
positions of available tips are accurately indicated during run setup.
6 After verifying that the NGS Bravo has been set up correctly, click Run
Selected Protocol.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
25
2
Using the Agilent NGS Bravo Option A for SureSelectQXT Target Enrichment
VWorks Automation Control Software
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 Bravo or your run setup.
1 If you encounter the G-axis error message shown below, select Ignore
and Continue, leaving device in current state.
26
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Using the Agilent NGS Bravo Option A for SureSelectQXT Target Enrichment
VWorks Automation Control Software
2
2 If you encounter the W-axis error message shown below, select Retry.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
27
2
Using the Agilent NGS Bravo Option A for SureSelectQXT Target Enrichment
VWorks Automation Control Software
Verifying the Simulation setting
VWorks software may be run in simulation mode, during which commands
entered on screen are not completed by the NGS Bravo. If NGS Bravo
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
28
If you cannot see the toolbar above the 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.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Using the Agilent NGS Bravo Option A for SureSelectQXT Target Enrichment
Overview of the SureSelectQXT Target Enrichment Procedure
2
Overview of the SureSelectQXT Target Enrichment Procedure
Figure 2 summarizes the SureSelectQXT 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, up to 96 samples can be pooled and sequenced
in a single lane using the dual index tags that are provided with
SureSelectQXT Library Prep kits.
Table 6 summarizes how the VWorks protocols are integrated into the
SureSelectQXT workflow. See Sample Preparation, Hybridization, and
Indexing chapters for complete instructions for use of the VWorks
protocols for sample processing.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
29
2
Using the Agilent NGS Bravo Option A for SureSelectQXT Target Enrichment
Overview of the SureSelectQXT Target Enrichment Procedure
Figure 2
30
Overall sequencing sample preparation workflow.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Using the Agilent NGS Bravo Option A for SureSelectQXT Target Enrichment
Overview of the SureSelectQXT Target Enrichment Procedure
Table 6
2
Overview of VWorks protocols used for SureSelectQXT Target Enrichment using NGS Bravo Option A
Workflow Step
(Protocol Chapter)
Substep
VWorks Protocols Used for Agilent NGS Bravo
Option A automation
Prepare fragmented and adaptor-tagged DNA
01 Tn_QXT_ILM_v1.0.pro
Purify DNA using AMPure XP beads
02 Cleanup_Tn_v1.0.pro
Amplify adaptor-tagged DNA
03b Pre-CapturePCR_QXT_ILM_v1.0.pro
Purify DNA using AMPure XP beads
04b Cleanup_Pre-CapturePCR_QXT_ILM_v1.0.pro
Aliquot prepped libraries for hybridization
05 Aliquot_Libraries_v1.0.pro
Hybridize prepped DNA to Capture Library
06 Hybridization_QXT_v1.0.pro
Capture DNA hybrids
07 SureSelectQXT_Capture_v1.0.pro
Wash captured DNA hybrids
08 SureSelectQXT_Wash_v1.0.pro
Add index tags by PCR
09 Post-CapturePCR_QXT_ILM_v1.0.pro
Purify DNA using AMPure XP beads
10 Cleanup_Post-CapturePCR_QXT_ILM_v1.0.pro
Sample Preparation
Hybridization
Indexing
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
31
2
Using the Agilent NGS Bravo Option A for SureSelectQXT Target Enrichment
Experimental Setup Considerations for Automated Runs
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 7
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 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.
32
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Using the Agilent NGS Bravo Option A for SureSelectQXT Target Enrichment
Considerations for Placement of gDNA Samples in 96-well Plates for Automated Processing
2
Considerations for Placement of gDNA Samples in 96-well
Plates for Automated Processing
• The NGS Bravo 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
SureSelect or ClearSeq Capture Library to each row of the plate. Plan
your experiment such that each prepared DNA library plate position
corresponds to the appropriate Capture Library.
• For post-capture amplification (see Figure 2), different SureSelect or
ClearSeq 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 53
on page 101 to determine which Capture Libraries may be amplified on
the same plate.
• Post-capture dual index assignments for the DNA samples can affect
sample placement decisions at the beginning of the workflow. For
example, all samples on the same row of the DNA sample plate must be
assigned to the same P5 indexing primer during sample indexing after
hybridization to the Capture Library (see Figure 2). It is important to
review and understand the guidelines for assignment of dual indexing
primers on page 96 while planning sample placement for the run to
ensure that the indexing design is compatible with the initial DNA
sample placement.
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 Bravo to allow rapid and
efficient plate transfer.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
33
2
Using the Agilent NGS Bravo Option A for SureSelectQXT Target Enrichment
PCR Plate Type Considerations
• Several workflow steps require that the sample plate be sealed using
the PlateLoc thermal microplate sealer and then centrifuged to collect
any dispersed liquid. To maximize efficiency, locate the centrifuge in
close proximity to the Agilent NGS Bravo.
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 QXT_ILM_OptA_v1.0.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 8.
CA U T I O N
The plates listed in Table 8 are compatible with the Agilent NGS Bravo and associated
VWorks automation protocols, designed to support use of various thermal cyclers.
Accordingly, some plates listed in Table 8 are not compatible with the recommended
SureCycler 8800 Thermal Cycler. When using the SureCycler 8800 Thermal Cycler in
the SureSelect automation workflow, use 96 Agilent semi-skirted PCR plates.
When using a different thermal cycler in the workflow, be sure to select a PCR plate
that is compatible with your thermal cycler and that is listed in Table 8.
34
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Using the Agilent NGS Bravo Option A for SureSelectQXT Target Enrichment
PCR Plate Type Considerations
Table 8
2
Ordering information for supported PCR plates
Description in VWorks menu
Vendor and part number
96 ABI PCR half-skirted plates (MicroAmp Optical
plates)
Life Technologies p/n N8010560
96 Agilent semi-skirted PCR plate
Agilent p/n 401334
96 Eppendorf Twin.tec half-skirted PCR plates
Eppendorf p/n 951020303
96 Eppendorf Twin.tec PCR plates (full-skirted)
Eppendorf p/n 951020401 or 951020619
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
35
2
36
Using the Agilent NGS Bravo Option A for SureSelectQXT Target Enrichment
PCR Plate Type Considerations
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
SureSelectQXT Automated Target Enrichment for Illumina Multiplexed
Sequencing Protocol
3
Sample Preparation
Step 1. Prepare the genomic DNA samples and Library Prep reagents 38
Step 2. Fragment and adaptor-tag the genomic DNA samples 40
Step 3. Purify adaptor-tagged DNA using AMPure XP beads 45
Step 4. Amplify adaptor-ligated libraries 48
Step 5. Purify amplified DNA using AMPure XP beads 55
Step 6. Assess Library DNA quantity and quality 58
This section contains instructions for gDNA library preparation specific to
the Illumina paired-read sequencing platform and to automated processing
using the Agilent NGS Bravo Option A.
Agilent Technologies
37
3
Sample Preparation
Step 1. Prepare the genomic DNA samples and Library Prep reagents
Step 1. Prepare the genomic DNA samples and Library Prep
reagents
It is important to have all materials prepared in advance of use in the
SureSelectQXT automated Library Prep protocol. In this step, the gDNA is
carefully quantified and dispensed into the sample plate. Additional
reagents that require modification or temperature equilibration before use
are also prepared in this step.
1 Remove the DMSO vial from the SureSelect QXT Library Prep Kit Box 2
in –20°C storage. Leave the DMSO vial at room temperature in
preparation for use on page 50.
2 Prepare reagents for the purification protocols on page 45 and page 55.
a Transfer the AMPure XP beads to room temperature. The beads
should be held at room temperature for at least 30 minutes before
use. Do not freeze the beads at any time.
b Prepare 150 ml of fresh 70% ethanol for use in the purification steps.
The 70% ethanol may be used for multiple steps done on the same
day, when stored in a sealed container.
3 Obtain the bottle of SureSelect QXT Stop Solution from SureSelect QXT
Hyb Module Box 1 (stored at room temperature). Verify that the
SureSelect QXT Stop Solution contains 25% ethanol, by referring to the
container label and the instructions below.
Before the first use of a fresh container, add 1.5 ml of ethanol to the
provided bottle containing 4.5 ml of stop solution, for a final ethanol
concentration of 25%. Seal the bottle then vortex well to mix. After
adding the ethanol, be sure to mark the label for reference by later
users.
Keep the prepared 1X SureSelect QXT Stop Solution at room
temperature, tightly sealed, until it is used on page 41.
38
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Sample Preparation
Step 1. Prepare the genomic DNA samples and Library Prep reagents
3
4 Quantify and dilute gDNA samples using two serial fluorometric assays:
a Use the Qubit dsDNA BR Assay or Qubit dsDNA HS Assay to
determine the initial concentration of each gDNA sample. Follow the
manufacturer’s instructions for the specific assay kit and the Qubit
instrument. This step is critical for successful preparation of input
DNA at the required concentration to ensure optimal fragmentation.
b Dilute each gDNA sample with nuclease-free water to a final
concentration of 100 ng/µl in a 1.5-ml LoBind tube.
c Carefully measure the DNA concentration of each of the 100 ng/µl
dilutions using a second Qubit dsDNA BR or HS Assay.
d Adjust each gDNA sample with nuclease-free water to a final
concentration of 10 ng/µl in a 1.5-ml LoBind tube.
5 Transfer 5 µl of the 10 ng/µl-DNA samples into the wells of a 96-well
Eppendorf plate, column-wise, for processing on the NGS Bravo, 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 Experimental Setup
Considerations for Automated Runs on page 32 for additional sample
placement considerations.
6 Seal the 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 remove air bubbles.
Store the sample plate on ice until it is used on page 43.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
39
3
Sample Preparation
Step 2. Fragment and adaptor-tag the genomic DNA samples
Step 2. Fragment and adaptor-tag the genomic DNA samples
In this step, automation protocol 01 Tn_QXT_ILM_v1.0.pro is used to
enzymatically fragment the gDNA and to add adaptors to ends of the
fragments in a single reaction. This step uses the SureSelectQXT Reagent
Kit components listed in Table 9 in addition to reagents prepared for use
on page 38 to page 39.
Table 9
Reagents for DNA fragmentation and adaptor-tagging
Kit Component
Storage Location
Where Used
SureSelect QXT Buffer
SureSelect QXT Library Prep Kit Box 2, –20°C page 40
SureSelect QXT Enzyme Mix ILM SureSelect QXT Library Prep Kit Box 2, –20°C page 40
Prepare the NGS Bravo
1 Clear the Bravo deck of all plates and tip boxes, then wipe it down
with a DNA Away decontamination wipe.
2 Pre-set the temperature of Bravo deck positions 4, 6, and 9 as indicated
in Table 10. See page 19 to page 21 for more information on how to do
this step.
Table 10
Bravo Deck Temperature Presets
Bravo Deck Position Temperature Preset
Preset Method
4
52°C
Inheco Multi TEC control touchscreen (CPAC 2-1)
6
4°C
Inheco Multi TEC control touchscreen (CPAC 2-2)
9
0°C
ThermoCube control panel
3 Place red PCR plate inserts at Bravo deck positions 4 and 9.
40
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Sample Preparation
Step 2. Fragment and adaptor-tag the genomic DNA samples
3
Prepare the Library Prep Master Mix and Stop Solution source plates
4 Prepare the Stop Solution source plate using an Eppendorf twin.tec
full-skirted PCR plate. Add 35 µl of 1X SureSelect QXT Stop Solution
per well, for each well to be processed. Place the source plate on Bravo
deck position 3.
5 Before use, vortex the SureSelect QXT Buffer and SureSelect QXT
Enzyme Mix ILM tubes vigorously at high speed.
These components are in liquid form when removed from –20°C storage
and should be returned to –20°C storage promptly after use.
CA U T I O N
Minor variations in volumes of the solutions combined in step 6 below may result in
DNA fragment size variation.
The SureSelect QXT Buffer and Enzyme Mix solutions are highly viscous. Thorough
mixing of the reagents is critical for optimal performance.
6 Prepare the appropriate volume of Library Prep Master Mix, according
to Table 11. Mix well by vortexing for 20 seconds and then keep on ice.
Table 11
Preparation of Library Prep Master Mix
SureSelectQXT 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 QXT Buffer
17.0 µl
216.8 µl
361.3 µl
505.8 µl
650.3 µl
939.3 µl
1878.5 µl
SureSelect QXT Enzyme
Mix ILM
2.0 µL
25.5 µl
42.5 µl
59.5 µl
76.5 µl
110.5 µl
221.0 µl
Total Volume
19 µl
242.3 µl
403.8 µl
565.3 µl
726.8 µl
1049.8 µl
2099.5 µl
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
41
3
Sample Preparation
Step 2. Fragment and adaptor-tag the genomic DNA samples
7 Prepare the Library Prep master mix source plate using a Nunc
DeepWell plate, containing the mixture from step 6. Add the volume
indicated in Table 12 to all wells of column 1 of the Nunc DeepWell
plate. Keep the master mix on ice during the aliquoting steps. The final
configuration of the master mix source plate is shown in Figure 3.
Table 12
Preparation of the Master Mix Source Plate for 01 Tn_QXT_ILM_v1.0.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
Library Prep
Master Mix
Column 1
27.9 µl
48.1 µl
68.3 µl
88.5 µl
128.8 µl
260.1 µl
(A1-H1)
Figure 3
42
Configuration of the master mix source plate for 01 Tn_QXT_ILM_v1.0.pro
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Sample Preparation
Step 2. Fragment and adaptor-tag the genomic DNA samples
3
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.
Setup and run VWorks protocol 01 Tn_QXT_ILM_v1.0.pro
10 On the SureSelect setup form, under Select Protocol to Run, select
01 Tn_QXT_ILM_v1.0.pro.
11 Select the number of columns of samples to be processed. Runs must
include 1, 2, 3, 4, 6, or 12 columns.
12 Click Display Initial Bravo Deck Setup.
13 Load the Bravo deck according to the Bravo Deck Setup region of the
form and as shown in Table 13.
Table 13
Initial Bravo deck configuration for 01 Tn_QXT_ILM_v1.0.pro
Location
Content
1
–(empty)–
2
New tip box
3
Stop Solution in Eppendorf plate
4
Empty red insert
5
–(empty)–
6
Library Prep Master Mix source plate (unsealed)
7
gDNA samples (5 µl of 10 ng/µl DNA per well) in Eppendorf plate (unsealed)
8
Empty tip box
9
Empty Eppendorf plate on red insert
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
43
3
Sample Preparation
Step 2. Fragment and adaptor-tag the genomic DNA samples
14 Verify that the Current Tip State indicator on the form matches the
configuration of unused and used tips in the tip boxes at Bravo Deck
positions 2 and 8, respectively. See page 23 for more information on
using this segment of the form during the run.
15 When setup and verification is complete, click Run Selected Protocol.
Running the 01 Tn_QXT_ILM_v1.0.pro protocol takes approximately
20 minutes. Once complete, the adaptor-tagged DNA samples are located
in the Eppendorf plate at position 6 of the Bravo deck.
44
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Sample Preparation
Step 3. Purify adaptor-tagged DNA using AMPure XP beads
3
Step 3. Purify adaptor-tagged DNA using AMPure XP beads
This step uses automation protocol 02 Cleanup_Tn_v1.0.pro.
In this step the NGS Bravo combines the adaptor-tagged samples with
AMPure XP beads and then collects and washes the bead-bound DNA. The
purified eluted DNA is transferred to an Eppendorf twin.tec plate for
further processing.
Prepare the NGS Bravo and reagents
1 Clear the Bravo deck of all plates and tip boxes, then wipe it down
with a DNA Away decontamination wipe.
2 Pre-set the temperature of Bravo deck positions 4 and 6 as indicated in
Table 14. See page 19 for more information on how to do this step.
Table 14
Bravo Deck Temperature Presets
Bravo Deck Position Temperature Preset
Preset Method
4
45°C
Inheco Multi TEC control touchscreen (CPAC 2-1)
6
20°C
Inheco Multi TEC control touchscreen (CPAC 2-2)
3 Mix the AMPure XP 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 52 µl of
homogeneous AMPure XP beads per well, for each well to be processed.
5 Prepare a Thermo Scientific Matrix reservoir containing 15 ml of
nuclease-free water.
6 Prepare a separate Thermo Scientific Matrix reservoir containing 45 ml
of freshly-prepared 70% ethanol.
Setup and Run VWorks protocol 02 Cleanup_Tn_v1.0.pro
7 On the SureSelect setup form, under Select Protocol, select
02 Cleanup_Tn_v1.0.pro.
8 Select the number of columns of samples to be processed. Runs must
include 1, 2, 3, 4, 6, or 12 columns.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
45
3
Sample Preparation
Step 3. Purify adaptor-tagged DNA using AMPure XP beads
9 Click Display Initial Bravo Deck Setup.
10 Load the Bravo deck according to the Bravo Deck Setup region of the
form and as shown in Table 15.
Table 15
Initial Bravo deck configuration for 02 Cleanup_Tn_v1.0.pro
Location
Content
1
Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)
2
New tip box
3
–(empty)–
4
–(empty)–
5
AMPure XP beads in Nunc DeepWell plate (52 µl beads per processing well)
6
Adaptor-tagged DNA samples in Eppendorf twin.tec plate
7
–(empty)–
8
Empty tip box
9
70% ethanol in Matrix reservoir
11 Verify that the Current Tip State indicator on the form matches the
configuration of unused and used tips in the tip boxes at Bravo Deck
positions 2 and 8, respectively. See page 23 for more information on
using this segment of the form during the run.
12 When setup and verification is complete, click Run Selected Protocol.
46
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Sample Preparation
Step 3. Purify adaptor-tagged DNA using AMPure XP beads
3
Running the 02 Cleanup_Tn_v1.0.pro protocol takes approximately
45 minutes. An operator must be present during the run to complete tip
box replacement and to transfer the water reservoir to the Bravo deck,
when directed by the VWorks prompt shown below.
Once the protocol is complete, the purified DNA samples are located in
the Nunc DeepWell plate at position 7 of the Bravo deck.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
47
3
Sample Preparation
Step 4. Amplify adaptor-ligated libraries
Step 4. Amplify adaptor-ligated libraries
In this step, the Agilent NGS Bravo completes the liquid handling steps for
amplification of the adaptor-ligated DNA samples using automation
protocol 03b Pre-CapturePCR_QXT_ILM_v1.0.pro. Afterward, you transfer
the PCR plate to a thermal cycler for amplification.
This step uses the SureSelectQXT Reagent Kit components listed in
Table 16.
Table 16
Reagents for precapture amplification
Kit Component
Storage Location
Where Used
Herculase II Fusion DNA Polymerase
SureSelect QXT Library Prep Kit Box 2, –20°C
page 49
Herculase II 5× Reaction Buffer
SureSelect QXT Library Prep Kit Box 2, –20°C
page 49
100 mM dNTP Mix (25 mM each dNTP)
SureSelect QXT Library Prep Kit Box 2, –20°C
page 49
SureSelect QXT Primer Mix
SureSelect QXT Hyb Module Box 2, –20°C
page 49
DMSO
Transferred to Room Temperature storage on page 38
page 49
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 NGS Bravo
1 Clear the Bravo deck of all plates and tip boxes, then wipe it down
with a DNA Away decontamination wipe.
2 Pre-set the temperature of Bravo deck positions 6 and 9 as indicated in
Table 17. See page 19 to page 21 for more information on how to do
this step.
Table 17
48
Bravo Deck Temperature Presets
Bravo Deck Position Temperature Preset
Preset Method
6
4°C
Inheco Multi TEC control touchscreen (CPAC 2-2)
9
0°C
ThermoCube control panel
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Sample Preparation
Step 4. Amplify adaptor-ligated libraries
3
3 Place a red PCR plate insert at Bravo deck position 6 and a silver deep
well plate insert at Bravo deck position 9.
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 18. Mix well using a vortex mixer and keep on ice.
Table 18
Preparation of Pre-Capture PCR Master Mix
SureSelectQXT
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
13.5 µl
172.1 µl
286.9 µl
401.6 µl
516.4 µl
745.9 µl
1491.8 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
DMSO
2.5 µL
31.9 µl
53.1 µl
74.4 µl
95.6 µl
138.1 µl
276.3 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 QXT
Primer Mix
1.0 µL
12.8 µl
21.3 µl
29.8 µl
38.3 µl
55.3 µl
110.5 µl
Herculase II
Fusion DNA
Polymerase
1.0 µL
12.8 µl
21.3 µl
29.8 µl
38.3 µl
55.3 µl
110.5 µl
Total Volume
28.5 µl
363.4 µl
605.6 µl
847.9 µl
1090.1 µl
1574.6 µl
3149.3 µl
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
49
3
Sample Preparation
Step 4. Amplify adaptor-ligated libraries
5 Using the same Nunc DeepWell master mix source plate that was used
for the 01 Tn_QXT_ILM_v1.0.pro run, add the volume of PCR Master
Mix indicated in Table 19 to all wells of column 2 of the master mix
source plate. The final configuration of the master mix source plate is
shown in Figure 4.
Table 19
Preparation of the Master Mix Source Plate for 03b Pre-CapturePCR_QXT_ILM_v1.0.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 2
41.9 µl
72.1 µl
102.4 µl
132.7 µl
193.3 µl
390.1 µl
NOTE
50
(A2-H2)
If you are using a new DeepWell plate for the pre-capture PCR source plate, leave column 1
empty and add the PCR Master Mix to column 2 of the new plate.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Sample Preparation
Step 4. Amplify adaptor-ligated libraries
Figure 4
3
Configuration of the master mix source plate for
03b Pre-CapturePCR_QXT_ILM_v1.0.pro. Column 1 was used to dispense
master mix 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
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.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
51
3
Sample Preparation
Step 4. Amplify adaptor-ligated libraries
Setup and run VWorks protocol 03b Pre-CapturePCR_QXT_ILM_v1.0.pro
8 On the SureSelect setup form, under Select Protocol to Run, select
03b Pre-CapturePCR_QXT_ILM_v1.0.pro.
9 Under Select PCR plate Labware, select the specific type of PCR plate
to be used for thermal cycling (placed at position 6 of the Bravo deck).
10 Select the number of columns of samples to be processed. Runs must
include 1, 2, 3, 4, 6, or 12 columns.
11 Click Display Initial Bravo Deck Setup.
12 Load the Bravo deck according to the Bravo Deck Setup region of the
form and as shown in Table 20.
Table 20
Initial Bravo deck configuration for 01 Tn_QXT_ILM_v1.0.pro
Location
Content
1
Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)
2
New tip box
3
–(empty)–
4
–(empty)–
5
–(empty)–
6
Empty PCR plate seated in red insert (PCR plate type must be specified on
setup form under step 2)
7
Purified adaptor-tagged DNA samples in Nunc DeepWell plate
8
Empty tip box
9
Master mix plate containing PCR Master Mix in Column 2 (unsealed), seated
in silver insert
13 Verify that the Current Tip State indicator on the form matches the
configuration of unused and used tips in the tip boxes at Bravo Deck
positions 2 and 8, respectively. See page 23 for more information on
using this segment of the form during the run.
52
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Sample Preparation
Step 4. Amplify adaptor-ligated libraries
3
14 When setup and verification is complete, click Run Selected Protocol.
15 Running the 03b Pre-CapturePCR_QXT_ILM_v1.0.pro protocol takes
approximately 10 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.
16 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.
17 Centrifuge the plate for 30 seconds to drive the well contents off the
walls and plate seal and to eliminate air bubbles.
18 Transfer the PCR plate to a thermal cycler and run the PCR
amplification program shown in Table 21.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
53
3
Sample Preparation
Step 4. Amplify adaptor-ligated libraries
Table 21
54
Pre-Capture PCR cycling program
Segment
Number of
Cycles
Temperature
Time
1
1
68°C
2 minutes
2
1
98°C
2 minutes
3
8
98°C
30 seconds
57°C
30 seconds
72°C
1 minute
4
1
72°C
5 minutes
5
1
4°C
Hold
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Sample Preparation
Step 5. Purify amplified DNA using AMPure XP beads
3
Step 5. Purify amplified DNA using AMPure XP beads
In this step, the Agilent NGS Bravo transfers AMPure XP beads and
amplified adaptor-tagged DNA to a Nunc DeepWell plate and then collects
and washes the bead-bound DNA.
This step uses protocol 04b Cleanup_Pre-CapturePCR_QXT_ILM_v1.0.pro.
Prepare the NGS Bravo and reagents
1 Clear the Bravo deck of all plates and tip boxes, then wipe it down
with a DNA Away decontamination wipe.
2 Pre-set the temperature of Bravo deck positions 4 and 6 as indicated in
Table 14. See page 19 for more information on how to do this step.
Table 22
Bravo Deck Temperature Presets
Bravo Deck Position Temperature Preset
Preset Method
4
45°C
Inheco Multi TEC control touchscreen (CPAC 2-1)
6
4°C
Inheco Multi TEC control touchscreen (CPAC 2-2)
3 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.
4 Mix the AMPure XP bead suspension well so that the reagent appears
homogeneous and consistent in color.
5 Prepare a Nunc DeepWell source plate for the beads by adding 50 µl of
homogeneous AMPure XP beads per well, for each well to be processed.
6 Prepare a Thermo Scientific Matrix reservoir containing 15 ml of
nuclease-free water.
7 Prepare a separate Thermo Scientific Matrix reservoir containing 45 ml
of freshly-prepared 70% ethanol.
8 Centrifuge the amplified DNA sample plate for 30 seconds to drive the
well contents off the walls and plate seal.
Setup and run VWorks 04b Cleanup_Pre-CapturePCR_QXT_ILM_v1.0.pro
9 On the SureSelect setup form, under Select Protocol, select
04b Cleanup_Pre-CapturePCR_QXT_ILM_v1.0.pro.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
55
3
Sample Preparation
Step 5. Purify amplified DNA using AMPure XP beads
10 Under Select PCR plate Labware, select the specific type of PCR plate
used for pre-capture amplification.
11 Select the number of columns of samples to be processed. Runs must
include 1, 2, 3, 4, 6, or 12 columns.
12 Click Display Initial Bravo Deck Setup.
13 Load the Bravo deck according to the Bravo Deck Setup region of the
form and as shown in Table 15.
Table 23
Initial Bravo deck configuration for 04b
Cleanup_Pre-CapturePCR_QXT_ILM_v1.0.pro
Location
Content
1
Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)
2
New tip box
3
–(empty)–
4
–(empty)–
5
AMPure XP beads in Nunc DeepWell plate (50 µl beads per processing well)
6
Amplified DNA libraries in unsealed PCR plate seated in red insert (PCR plate
type must be specified on setup form under step 2)
7
–(empty)–
8
Empty tip box
9
70% ethanol in Matrix reservoir
14 Verify that the Current Tip State indicator on the form matches the
configuration of unused and used tips in the tip boxes at Bravo Deck
positions 2 and 8, respectively. See page 23 for more information on
using this segment of the form during the run.
56
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Sample Preparation
Step 5. Purify amplified DNA using AMPure XP beads
3
15 When setup and verification is complete, click Run Selected Protocol.
Running the 04b Cleanup_Pre-CapturePCR_QXT_ILM_v1.0.pro protocol
takes approximately 45 minutes. An operator must be present during the
run to complete tip box replacement and to transfer the water reservoir to
the Bravo deck, when directed by the VWorks prompt shown below.
Once the protocol is complete, the purified DNA samples are located in
the Eppendorf plate at position 3 of the Bravo deck.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
57
3
Sample Preparation
Step 6. Assess Library DNA quantity and quality
Step 6. Assess Library DNA quantity and quality
Measure the concentration of each library using one of the methods
detailed below.
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 245 to 325 bp. Sample electropherograms are shown
in Figure 5. Variability of fragmentation profiles may be observed.
NOTE
A peak DNA fragment size significantly less than 245 bp may indicate too little gDNA in the
fragmentation reaction and may be associated with increased duplicates in the sequencing
data. In contrast, a peak DNA fragment size significantly greater than 325 bp may indicate
too much gDNA in the fragmentation reaction and may be associated with decreased
percent-on-target performance in sequencing results.
7 Measure the concentration of each library by integrating under the
entire peak. For accurate quantification, make sure that the
concentration falls within the linear range of the assay.
Stopping Point
58
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.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Sample Preparation
Step 6. Assess Library DNA quantity and quality
Figure 5
3
Representative sample electropherograms showing pre-capture analysis of
amplified library DNA using the Agilent 2100 Bioanalyzer and a DNA 1000 Assay.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
59
3
Sample Preparation
Step 6. Assess Library DNA quantity and quality
Option 2: Analysis using an Agilent 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 using the Agilent 4200
TapeStation or 2200 TapeStation. For more information to do this step,
see the appropriate TapeStation user manual at www.genomics.agilent.com.
1 Prepare the TapeStation samples as instructed in the instrument 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 sample buffer on a vortex
mixer for 5 seconds for accurate quantitation.
2 Load the sample plate or tube strips from step 1, the D1000
ScreenTape, and loading tips into the TapeStation as instructed in the
instrument user manual. Start the run.
3 Verify that the electropherogram shows the peak of DNA fragment size
positioned between 245 to 325 bp. Sample electropherograms are shown
in Figure 6. Variability of fragmentation profiles may be observed.
NOTE
A peak DNA fragment size significantly less than 245 bp may indicate too little gDNA in the
fragmentation reaction and may be associated with increased duplicates in the sequencing
data. In contrast, a peak DNA fragment size significantly greater than 325 bp may indicate
too much gDNA in the fragmentation reaction and may be associated with decreased
percent-on-target performance in sequencing results.
4 Measure the concentration of each library by integrating under the
entire peak.
Stopping Point
60
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.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Sample Preparation
Step 6. Assess Library DNA quantity and quality
Figure 6
3
Representative sample electropherograms showing pre-capture analysis of
amplified library DNA using the 2200 TapeStation with a D1000 ScreenTape.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
61
3
62
Sample Preparation
Step 6. Assess Library DNA quantity and quality
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
SureSelectQXT Automated Target Enrichment for Illumina Multiplexed
Sequencing Protocol
4
Hybridization
Step 1. Aliquot prepped DNA samples for hybridization 64
Step 2. Hybridize the gDNA library and SureSelect or ClearSeq Capture
Library 69
Step 3. Capture the hybridized DNA 83
This chapter describes the steps to combine the prepped library with the
blocking agents and the SureSelect or ClearSeq 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 Capture Library to prepped library is critical for successful capture.
Agilent Technologies
63
4
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.
The amount of prepared gDNA library used in the hybridization reaction
varies according to the size of the Capture Library used for hybridization
as outlined in Table 24 below. Use the maximum possible amount of each
prepped DNA, within the range listed in Table 24.
Table 24
Amount of adaptor-tagged DNA libraries used for hybridization
Capture Library Size
Amount of prepared gDNA library used in hybridization
Libraries 3.0 Mb (except ClearSeq
DNA Kinome; see below)
750 to 1500 ng DNA
Libraries 3.0 Mb and ClearSeq DNA
Kinome (3.2 Mb)
500 to 750 ng DNA
Using the DNA concentration for each sample determined on page 58 to
page 60, calculate the volume of each sample to be used for hybridization
using the appropriate formula below:
Volume (µl) = 750 ng/concentration (ng/µl)
OR
Volume (µl) = 1500 ng/concentration (ng/µl)
If the concentration of any sample is not sufficient to allow use of the
recommended amount of DNA (750 ng for ClearSeq DNA Kinome and
libraries 3.0 Mb or 1500 ng for libraries >3.0 Mb), then use the full
remaining volume of DNA sample (approximately 12 µl) for the
hybridization step.
The automation protocol 05 Aliquot_Libraries_v1.0.pro is used to prepare
a new sample plate containing the appropriate amount of each DNA
sample for hybridization. Before running the automation protocol, you
must create a table containing instructions for the NGS Bravo indicating
the volume of each sample to aliquot, as described in the steps below.
64
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Hybridization
Step 1. Aliquot prepped DNA samples for hybridization
4
1 Create a .csv (comma separated value) file with the headers shown in
Figure 7. 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) of each DNA sample to
be used in the hybridization step (see page 64 for guidelines). For all
empty wells on the plate, enter the value 0, as shown in Figure 7; do
not delete rows for empty wells.
Figure 7
NOTE
Sample spreadsheet for 1-column run.
You can find a sample spreadsheet in the directory C: > VWorks Workspace > NGS Option
A> QXT_ILM_v1.0 > Aliquot Library Input Files >
Aliquot_Libraries_full_plate_template.csv.
The Aliquot_Libraries_full_plate_template.csv file may be copied and used as a template
for creating the .csv files for each 05 Aliquot_Libraries_v1.0.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.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
65
4
Hybridization
Step 1. Aliquot prepped DNA samples for hybridization
3 Load the .csv file onto the PC containing the VWorks software into a
suitable folder, such as C: > VWorks Workspace > NGS Option A>
QXT_ILM_v1.0 > Aliquot Library Input Files.
Setup and Run VWorks protocol 05 Aliquot_Libraries_v1.0.pro
1 Clear the Bravo deck of all plates and tip boxes, then wipe it down
with a DNA Away decontamination wipe.
2 Pre-set the temperature of Bravo deck position 9 as indicated in
Table 25. See page 21 for more information on how to do this step.
Table 25
Bravo Deck Temperature Presets
Bravo Deck Position Temperature Preset
Preset Method
9
ThermoCube control panel
0°C
3 On the SureSelect setup form, under Select Protocol, select 05
Aliquot_Libraries_v1.0.pro.
4 Select the number of columns of samples to be processed. Runs must
include 1, 2, 3, 4, 6, or 12 columns.
5 Click Display Initial Bravo Deck Setup.
66
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Hybridization
Step 1. Aliquot prepped DNA samples for hybridization
4
6 Load the Bravo deck according to the Bravo Deck Setup region of the
form and as shown in Table 26.
Table 26
CA U T I O N
Initial Bravo deck configuration for 05 Aliquot_Libraries_v1.0.pro
Location
Content
1
–(empty)–
2
–(empty)–
3
–(empty)–
4
–(empty)–
5
Empty Eppendorf plate
6
Empty tip box
7
–(empty)–
8
New tip box
9
Prepped library DNA in Eppendorf plate
This protocol does not use the Current Tip State indicator function. Be sure to place a
completely full box of tips at position 8 and a completely empty tip box at position 6.
7 When verification is complete, click Run Selected Protocol.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
67
4
Hybridization
Step 1. Aliquot prepped DNA samples for hybridization
8 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 DNA sample plate is on Bravo deck position 5.
9 Remove the sample plate from the Bravo deck and use a vacuum
concentrator to dry the samples at  45°C.
10 Reconstitute each dried sample with 12 µl of nuclease-free water.
Pipette up and down along the sides of each well for optimal recovery.
11 Seal the plate using the PlateLoc Thermal Microplate Sealer, with
sealing settings of 165°C and 1.0 sec.
12 Vortex the plate for 30 seconds to ensure complete reconstitution, then
centrifuge the plate for 1 minute to drive the well contents off the walls
and plate seal.
68
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Hybridization
Step 2. Hybridize the gDNA library and SureSelect or ClearSeq Capture Library
4
Step 2. Hybridize the gDNA library and SureSelect or
ClearSeq Capture Library
In this step, automation protocol 06 Hybridization_QXT_v1.0.pro is used to
complete the liquid handling steps to set up the hybridization reactions.
Afterward, you transfer the sample plate to a thermal cycler, held at 65°C,
to allow hybridization of the DNA samples to the Capture Library.
This step uses the SureSelectQXT Reagent Kit components listed in
Table 27. Thaw each component under the conditions indicated in the
table. Vortex each reagent to mix, then spin briefly to collect the liquid.
Table 27
Reagents for Hybridization and Capture
Kit Component
Storage Location
Thawing Conditions
Where Used
SureSelect QXT Fast
Hybridization Buffer
SureSelect QXT Hyb Module
Box 2, –20°C
Warm to Room
Temperature (RT), then
keep at RT
page 75
SureSelect QXT Fast
Blocker Mix
SureSelect QXT Hyb Module
Box 2, –20°C
Thaw on ice
page 72
SureSelect RNase Block
SureSelect QXT Hyb Module
Box 2, –20°C
Thaw on ice
page 73 or
page 74
Capture Library
–80°C
Thaw on ice
page 73 or
page 74
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
69
4
Hybridization
Step 2. Hybridize the gDNA library and SureSelect or ClearSeq Capture Library
Program the thermal cycler
1 Pre-program the thermal cycler for the Hybridization workflow by
entering the thermal cycling program shown in Table 28 below.
It is critical to pre-program the thermal cycler before starting the
automation protocol for Hybridization, in order to maintain the required
sample and reagent temperatures during the workflow.
Table 28
Thermal cycler program for Hybridization*
Segment
Number
Purpose
Number of Cycles Temperature
Time
1
Denaturation
1
95°C
5 minutes
2
Blocking
1
65°C
10 minutes
3
Hold for NGS Bravo
steps†
1
65°C
Hold
4
Hybridization
60
65°C
1 minute
37°C
3 seconds
65°C
Hold
5
Hold until start of
Capture‡
1
* When setting up the thermal cycling program, use a reaction volume setting of 35 l (final volume
of hybridization reactions during cycling in Segment 4).
† Samples are transferred to the NGS Bravo during this Hold step when prompted by the VWorks
software.
‡ Samples are held at 65°C until they are processed in the Capture & Wash automation protocol that
begins on page 83.
CA U T I O N
70
The lid of the thermal cycler is hot and can cause burns. Use caution when working
near the lid.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Hybridization
Step 2. Hybridize the gDNA library and SureSelect or ClearSeq Capture Library
4
Prepare the NGS Bravo
1 Clear the Bravo deck of all plates and tip boxes, then wipe it down
with a DNA Away decontamination wipe.
2 Pre-set the temperature of Bravo deck positions 4 and 6 as indicated in
Table 29. See page 19 for more information on how to do this step.
Table 29
Bravo Deck Temperature Presets
Bravo Deck Position Temperature Preset
Preset Method
4
23°C
Inheco Multi TEC control touchscreen (CPAC 2-1)
6
23°C
Inheco Multi TEC control touchscreen (CPAC 2-2)
3 Place a red PCR plate insert at Bravo deck position 4.
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. When loading a
source plate on the silver insert, make sure the plate is seated properly
to ensure proper heat transfer.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
71
4
Hybridization
Step 2. Hybridize the gDNA library and SureSelect or ClearSeq Capture Library
Prepare the Block Master Mix
5 Prepare the appropriate volume of Block Master Mix, on ice, as
indicated in Table 30.
Table 30
Preparation of Block Master Mix
SureSelectQXT
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
2.5 µl
31.9 µl
53.1 µl
74.4 µl
95.6 µl
138.1 µl
276.3 µl
SureSelect QXT
Fast Blocker Mix
(blue cap)
5.0 µl
63.8 µl
106.3 µl
148.8 µl
191.3 µl
276.3 µl
552.5 µl
Total Volume
7.5 µl
95.6 µl
159.4 µl
223.1 µl
286.9 µl
414.4 µl
828.8 µl
Prepare one or more Capture Library Master Mixes
6 Prepare the appropriate volume of Capture Library Master Mix for each
library that will be used for hybridization as indicated in Table 31 to
Table 34. Mix thoroughly by vortexing at high speed then spin down
briefly. Keep the Capture Library Master Mix(es) on ice.
NOTE
Each row of the prepped gDNA sample plate may be hybridized to a different Capture
Library. However, 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 31 or Table 32) on page 73.
For runs that use different Capture Libraries for individual rows, prepare each master mix as
described in Step b (Table 33 or Table 34) on page 74.
72
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Hybridization
Step 2. Hybridize the gDNA library and SureSelect or ClearSeq Capture Library
4
a For runs that use a single Capture Library for all rows, prepare a
Master Mix as described in Table 31 or Table 32, according to the
size of the Capture Library.
Table 31
Preparation of Capture Library Master Mix for Capture Libraries <3 Mb, 8 rows of well
Target size <3.0 Mb
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 32
Preparation of Capture Library Master Mix for Capture Libraries 3 Mb*, 8 rows of wells
Target size >3.0 Mb
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
* Includes ClearSeq DNA Kinome XT Library (3.2 Mb)
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
73
4
Hybridization
Step 2. Hybridize the gDNA library and SureSelect or ClearSeq Capture Library
b For runs that use different Capture Libraries in individual rows,
prepare a Master Mix for each Capture Library as listed in Table 33
or Table 34, according to the library size. The volumes listed in
Table 33 and Table 34 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 33
Preparation of Capture Library Master Mix for Capture Libraries <3 Mb, single row of wells
Target size <3.0 Mb
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 34
Preparation of Capture Library Master Mix for Capture Libraries 3 Mb*, single row of wells
Target size >3.0 Mb
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
* Includes ClearSeq DNA Kinome XT Library (3.2 Mb)
74
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Hybridization
Step 2. Hybridize the gDNA library and SureSelect or ClearSeq Capture Library
4
Prepare the Hybridization Buffer master mix
7 Prepare the appropriate volume of Hybridization Buffer Master Mix, at
room temperature, as indicated in Table 35.
Table 35
Preparation of Hybridization Buffer Master Mix
SureSelectQXT
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 2.5 µl
53.1 µl
74.4 µl
95.6 µl
116.9 µl
159.4 µl
297.5 µl
SureSelect QXT Fast
Hybridization Buffer
(yellow cap)
6.0 µl
127.5 µl
178.5 µl
229.5 µl
280.5 µl
382.5 µl
714.0 µl
Total Volume
8.5 µl
180.6 µl
252.9 µl
325.1 µl
397.4 µl
541.9 µl
1011.5 µl
Prepare the master mix source plate
8 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 36 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 8.
Table 36
Preparation of the Master Mix Source Plate for 06 Hybridization_QXT_v1.0.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
Block Master Mix
Column 1
11.0 µl
19.0 µl
27.0 µl
34.9 µl
50.9 µl
102.7 µl
14.0 µl
21.4 µl
28.9 µl
36.3 µl
58.6 µl
114.4 µl
19.9 µl
29.0 µl
38.0 µl
47.0 µl
65.1 µl
123.8 µl
(A1-H1)
Capture Library
Master Mix
Column 2
(A2-H2)
Hybridization
Column 3
Buffer Master Mix (A3-H3)
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
75
4
Hybridization
Step 2. Hybridize the gDNA library and SureSelect or ClearSeq Capture Library
Figure 8
Configuration of the master mix source plate for
06 Hybridization_QXT_v1.0.pro.
9 Seal the master mix source plate using the PlateLoc Thermal Microplate
Sealer, with sealing settings of 165°C and 1.0 sec.
10 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.
Setup and run VWorks protocol 06 Hybridization_QXT_v1.0.pro
11 On the SureSelect setup form, under Select Protocol, select 06
Hybridization_QXT_v1.0.pro.
12 Under Select PCR Plate Labware, select the plate type to be used for
the hybridization step (to be loaded at Bravo deck position 4).
76
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Hybridization
Step 2. Hybridize the gDNA library and SureSelect or ClearSeq Capture Library
4
13 Select the number of columns of samples to be processed. Runs must
include 1, 2, 3, 4, 6, or 12 columns.
14 Click Display Initial Bravo Deck Setup.
15 Load the Bravo deck according to the Bravo Deck Setup region of the
form and as shown in Table 37.
Table 37
Initial Bravo deck configuration for 06 Hybridization_QXT_v1.0.pro
Location
Content
1
–(empty)–
2
New tip box
3
–(empty)–
4
Empty PCR plate, seated in red insert (PCR plate type must be specified on setup
form under step 2)
5
Empty Eppendorf twin.tec plate
6
Master Mixes in Columns 1-3 of Nunc DeepWell, seated in silver Nunc
DeepWell insert
7
–(empty)–
8
Empty tip box
9
Prepped library aliquots in Eppendorf twin.tec plate
16 Verify that the Current Tip State indicator on the form matches the
configuration of unused and used tips in the tip boxes at Bravo Deck
positions 2 and 8, respectively. See page 23 for more information on
using this segment of the form during the run.
17 When setup and verification is complete, click Run Selected Protocol.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
77
4
Hybridization
Step 2. Hybridize the gDNA library and SureSelect or ClearSeq Capture Library
Running the 06 Hybridization_QXT_v1.0.pro protocol takes approximately
30 minutes. An operator must be present during the run to complete tip
box replacement and other labware transfer steps, as directed by the
VWorks prompts detailed below.
The NGS Bravo combines the prepped gDNA in the wells of the sample
plate with the aliquotted SureSelect Block Master Mix. When this process
is complete, you will be prompted to transfer the plate to the thermal
cycler for sample denaturation and blocking prior to hybridization.
18 When prompted by VWorks as shown below, remove the PCR plate from
position 4 of the Bravo deck, leaving the red insert in place. After
removing the sample plate, click Continue.
19 Seal the sample plate using the PlateLoc Thermal Microplate Sealer,
with sealing settings of 165°C and 3.0 sec.
78
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Hybridization
Step 2. Hybridize the gDNA library and SureSelect or ClearSeq Capture Library
4
20 Transfer the sealed plate to a thermal cycler and initiate the
preprogrammed thermal cycling program described in Table 28 on
page 70. The denaturation and blocking segments of the preprogrammed
thermal cycler program are shown in Figure 9 below for reference.
Figure 9
Preprogrammed thermal cycler segments used for sample denaturation and
blocking prior to hybridization.
While the sample plate incubates on the thermal cycler, the NGS Bravo
combines aliquots of the Capture Library Master Mix and Hybridization
Buffer Master Mix.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
79
4
Hybridization
Step 2. Hybridize the gDNA library and SureSelect or ClearSeq Capture Library
CA U T I O N
You must complete step 21 to step 25 quickly, and immediately after being prompted by
the VWorks software. It is important that sample temperature remains approximately
65°C during transfers between the NGS Bravo and thermal cycler.
21 When the NGS Bravo 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.
80
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Hybridization
Step 2. Hybridize the gDNA library and SureSelect or ClearSeq Capture Library
4
22 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 NGS Bravo transfers the Capture Library-Hybridization Buffer mixture
to the wells of the PCR plate that contain the mixture of prepped gDNA
samples and blocking agents.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
81
4
Hybridization
Step 2. Hybridize the gDNA library and SureSelect or ClearSeq Capture Library
23 When prompted by VWorks as shown below, quickly remove the PCR
sample plate from Bravo deck position 4, leaving the red insert in place.
24 Seal the sample plate using the PlateLoc Thermal Microplate Sealer,
with sealing settings of 165°C and 3.0 sec.
25 Quickly transfer the plate back to the thermal cycler, held at 65°C.
Press the Play button to initiate the hybridization segment of the
pre-programmed thermal cycling program (segment 4 from Table 28 on
page 70). During this step, the prepared DNA samples are hybridized to
the SureSelect or ClearSeq Capture Library.
CA U T I O N
The thermal cycler is held at 65°C using a heated lid at 105°C. The lid of the thermal
cycler is hot and can cause burns. Use caution when working near the lid.
26 After initiating hybridization on the thermal cycler, click Continue on
the VWorks screen to finish the NGS Bravo protocol.
During the thermal cycler incubation for hybridization (approximately
1.5-hour duration), complete the reagent and NGS Bravo setup steps for
the capture automation protocol as described on page 83 to page 85.
82
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Hybridization
Step 3. Capture the hybridized DNA
4
Step 3. Capture the hybridized DNA
This step uses automation protocol 07 SureSelectQXT_Capture_v1.0.pro to
automate capture of the gDNA-Capture Library hybrids using
streptavidin-coated magnetic beads. Setup tasks for the Capture protocol
(step 1, below, through step 16 on page 86) should be completed during
the thermal cycler incubation for hybridization (approximately 1.5-hour
duration) started on page 82.
This step uses the SureSelectQXT Reagent Kit components in Table 38 in
addition to streptavidin-coated magnetic beads obtained from another
supplier (see Table 1 on page 12).
Table 38
Reagents for hybrid capture
Kit Component
Storage Location
Where Used
SureSelect Binding Buffer
SureSelect QXT Hyb Module Box 1, RT
page 84
SureSelect Wash Buffer 2
SureSelect QXT Hyb Module Box 1, RT
page 85
Prepare the NGS Bravo
1 Clear the Bravo deck of all plates and tip boxes, then wipe it down
with a DNA Away decontamination wipe.
2 Pre-set the temperature of Bravo deck position 4 as indicated in
Table 39. See page 19 for more information on how to do this step.
Table 39
Bravo Deck Temperature Presets
Bravo Deck Position Temperature Preset
Preset Method
4
Inheco Multi TEC control touchscreen (CPAC 2-1)
66°C
3 Place a red PCR plate insert at Bravo deck position 4.
4 Place the silver Nunc DeepWell plate insert at Bravo deck position 6.
This insert is required to facilitate heat transfer to DeepWell source
plate wells.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
83
4
Hybridization
Step 3. Capture the hybridized DNA
Prepare the Dynabeads streptavidin beads and Wash Buffer 2 source plates
5 Vigorously resuspend the Dynabeads MyOne Streptavidin T1 magnetic
beads on a vortex mixer. The beads settle during storage.
6 Wash the magnetic beads.
a In a conical vial, combine the components listed in Table 40. The
volumes below include the required overage.
Table 40
Magnetic bead washing mixture
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.)
7 Resuspend the beads in SureSelect Binding buffer, according to
Table 41 below.
Table 41
Preparation of magnetic beads for 07 SureSelectQXT_Capture_v1.0.pro
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
84
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Hybridization
Step 3. Capture the hybridized DNA
4
8 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.
9 Place the streptavidin bead source plate at position 5 of the Bravo deck.
10 Prepare a Nunc DeepWell source plate labeled Wash #2. For each well
to be processed, add 1150 µl of SureSelect Wash Buffer 2. Place the
Wash #2 source plate at position 6 of the Bravo deck.
Setup VWorks protocol 07 SureSelectQXT_Capture_v1.0.pro
11 On the SureSelect setup form, under Select Protocol, select
07 SureSelectQXT_Capture_v1.0.pro.
12 Under Select PCR Plate Labware, 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.
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 Bravo Deck Setup.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
85
4
Hybridization
Step 3. Capture the hybridized DNA
15 Load the Bravo deck according to the Bravo Deck Setup region of the
form and as shown in Table 42.
Table 42
Initial Bravo deck configuration for 07 SureSelectQXT_Capture_v1.0.pro
Location
Content
1
–(empty)–
2
New tip box
3
–(empty)–
4
Empty red insert
5
Prepared Dynabeads streptavidin bead DeepWell source plate
6
Wash #2 DeepWell source plate seated on silver Nunc DeepWell insert
7
–(empty)–
8
Empty tip box
9
–(empty)–
16 Verify that the Current Tip State indicator on the form matches the
configuration of unused and used tips in the tip boxes at Bravo Deck
positions 2 and 8, respectively. See page 23 for more information on
using this segment of the form during the run.
Run VWorks protocol 07 SureSelectQXT_Capture_v1.0.pro
Start the 07 SureSelectQXT_Capture_v1.0.pro protocol upon completion of
the hybridization incubation that was started on page 82, when the
thermal cycler program reaches the 65°C Hold step in Segment 5.
After verifying that the hybridization step is complete and that all NGS
Bravo setup steps for capture are complete, click Run Selected Protocol.
Leave the hybridization plate in the thermal cycler until you are prompted
to transfer the plate to the NGS Bravo.
The total duration of the 07 SureSelectQXT_Capture_v1.0.pro protocol is
approximately 35 minutes. An operator must be present to transfer the
hybridization plate from the thermal cycler when prompted by VWorks as
shown in step 17 below (<5 minutes after starting the protocol).
86
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Hybridization
Step 3. Capture the hybridized DNA
4
If the temperature of Bravo deck position 4 was not pre-set to 66°C, the
protocol will pause while position 4 reaches temperature.
CA U T I O N
It is important to complete step 17 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 NGS
Bravo is completely prepared, with deck platforms at temperature and all components
in place, before you transfer the sample plate to the Bravo deck.
17 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 protocol.
WARN I NG
Warning
Bravo deck position 4 will be hot.
Use caution when handling components that contact heated deck positions.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
87
4
Hybridization
Step 3. Capture the hybridized DNA
18 When the capture incubation period is complete you will be prompted
by VWorks as shown below. Retain the hybrid-capture bead suspension
plate at position 5 and proceed immediately to automation protocol 08
SureSelectQXT_Wash_v1.0.pro.
88
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Hybridization
Step 4. Wash the captured DNA
4
Step 4. Wash the captured DNA
This step uses automation protocol 08 SureSelectQXT_Wash_v1.0.pro to
automate washing of the captured DNA-RNA hybrids.
This step uses the SureSelectQXT Reagent Kit component in Table 38 in
addition to components retained from the previous automation protocol.
Table 43
Reagents for capture wash protocol
Kit Component
Storage Location
Where Used
SureSelect Wash Buffer 1
SureSelect QXT Hyb Module Box 1, RT
page 89
Prepare the NGS Bravo
1 Retain all plates, inserts, and tip boxes used in the previous automation
protocol on the Bravo deck, including the hybrid-capture bead
suspension plate at position 5 and the pre-warmed Wash Buffer 2
source plate at position 6.
2 Pre-set the temperature of Bravo deck positions 4 and 6 as indicated in
Table 29. See page 19 for more information on how to do this step.
Table 44
Bravo Deck Temperature Presets
Bravo Deck Position Temperature Preset
Preset Method
4
69°C
Inheco Multi TEC control touchscreen (CPAC 2-1)
6
74°C
Inheco Multi TEC control touchscreen (CPAC 2-2)
3 Place an empty square-well plate waste reservoir at position 1 of the
Bravo deck.
4 Prepare an Eppendorf source plate labeled Wash #1. For each well to
be processed, add 160 µl of SureSelect Wash Buffer 1. Place the Wash
#1 source plate at position 3 of the Bravo deck.
5 Prepare a Thermo Scientific reservoir containing 15 ml of nuclease-free
water and place the reservoir at position 9 of the Bravo deck.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
89
4
Hybridization
Step 4. Wash the captured DNA
Setup and Run VWorks protocol 08 SureSelectQXT_Wash_v1.0.pro
6 On the SureSelect setup form, under Select Protocol, select
08 SureSelectQXT_Wash_v1.0.pro.
7 Select the number of columns of samples to be processed. Runs must
include 1, 2, 3, 4, 6, or 12 columns.
8 Click Display Initial Bravo Deck Setup.
9 Verify that the Bravo deck has been set up according to the Bravo Deck
Setup region of the form and as shown in Table 45.
Table 45
Initial Bravo deck configuration for 08 SureSelectQXT_Wash_v1.0.pro
Location
Content
1
Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)
2
New tip box
3
Wash #1 Eppendorf source plate
4
Empty red insert
5
DNA-RNA hybrids captured on streptavidin beads in DeepWell plate
6
Wash #2 DeepWell source plate seated on silver Nunc DeepWell insert
(pre-heated during Capture protocol)
7
–(empty)–
8
Empty tip box
9
Nuclease-free water reservoir
10 Verify that the Current Tip State indicator on the form matches the
configuration of unused and used tips in the tip boxes at Bravo Deck
positions 2 and 8, respectively. See page 23 for more information on
using this segment of the form during the run.
11 When setup and verification is complete, click Run Selected Protocol.
90
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Hybridization
Step 4. Wash the captured DNA
4
Running the 08 SureSelectQXT_Wash_v1.0.pro protocol takes approximately
60 minutes. An operator must be present during the run to complete tip
box replacement as directed by VWorks prompts. Once complete, you will
be prompted as shown below.
12 When the wash protocol is complete, the captured, bead-bound DNA
samples are located in the Eppendorf plate at position 5 of the Bravo
deck, and you will be prompted by VWorks as shown below. Click
Continue on the VWorks screen to finish the protocol.
Remove the DNA sample plate from position 5 and seal the wells using
the PlateLoc Thermal Microplate Sealer, with sealing settings of 165°C
and 1.0 sec. Store the plate on ice until it is used on page 101. Proceed
immediately to the 09 Post-CapturePCR_QXT_ILM_v1.0.pro protocol,
starting on page 94.
NOTE
Captured DNA is retained on the streptavidin beads during the post-capture amplification
step.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
91
4
92
Hybridization
Step 4. Wash the captured DNA
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
SureSelectQXT Automated Target Enrichment for Illumina Multiplexed
Sequencing Protocol
5
Indexing
Step 1. Amplify the captured DNA libraries to add index tags 94
Step 2. Purify the amplified indexed libraries using Agencourt AMPure XP
beads 102
Step 3. Assess indexed DNA quality 105
Step 4. Quantify each index-tagged library by QPCR (optional) 109
Step 5. Pool samples for Multiplexed Sequencing 110
Step 6. Prepare sequencing samples 111
Step 7. Set up the sequencing run and trim adaptors from the reads 115
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
93
5
Indexing
Step 1. Amplify the captured DNA libraries to add index tags
Step 1. Amplify the captured DNA libraries to add index tags
In this step, the Agilent NGS Bravo completes the liquid handling steps for
PCR-based addition of dual indexing tags to the captured DNA samples
using automation protocol 09 Post-CapturePCR_QXT_ILM_v1.0.pro. After
the PCR plate is prepared by the NGS Bravo, you transfer the plate to a
thermal cycler for amplification.
This step uses the components listed in Table 46. Thaw then vortex to mix
the reagents listed below and keep on ice.
Table 46
Reagents for post-capture indexing by PCR amplification
Kit Component
Storage Location
Where Used
Herculase II Fusion DNA Polymerase
SureSelect QXT Library Prep Kit Box 2, –20°C
page 95
Herculase II 5× Reaction Buffer
SureSelect QXT Library Prep Kit Box 2, –20°C
page 95
100 mM dNTP Mix (25 mM each dNTP)
SureSelect QXT Library Prep Kit Box 2, –20°C
page 95
SureSelect QXT P7 and P5 dual indexing primers
SureSelect QXT Library Prep Kit Box 2, –20°C
page 96
Prepare the NGS Bravo
1 Clear the Bravo deck of all plates and tip boxes, then wipe it down
with a DNA Away decontamination wipe.
2 Pre-set the temperature of Bravo deck positions 6 and 9 as indicated in
Table 47. See page 19 to page 21 for more information on how to do
this step.
Table 47
Bravo Deck Temperature Presets
Bravo Deck Position Temperature Preset
Preset Method
6
4°C
Inheco Multi TEC control touchscreen (CPAC 2-2)
9
0°C
ThermoCube control panel
3 Place a red PCR plate insert at Bravo deck position 6 and a silver deep
well plate insert at Bravo deck position 9.
94
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Indexing
Step 1. Amplify the captured DNA libraries to add index tags
5
Prepare the PCR master mix
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 appropriate volume of PCR master mix, according to
Table 48. Mix well using a vortex mixer and keep on ice.
Table 48
Preparation of PCR Master Mix for 09 Post-CapturePCR_QXT_ILM_v1.0.pro,
SureSelectQXT
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 9.5 µl
121.1 µl
201.9 µl
282.6 µl
363.4 µl
524.9 µl
1049.8 µl
Herculase II 5×
Reaction Buffer
10.0 µL
127.5 µl
212.5 µl
297.5 µl
382.5 µl
552.5 µl
1105.0 µl
100 mM 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 Fusion
DNA Polymerase
1.0 µl
12.8 µl
21.3 µl
29.8 µl
38.3 µl
55.3 µl
110.5 µl
Total Volume
21.0 µl
267.8 µl
446.3 µl
624.8 µl
803.3 µl
1160.3 µl
2320.6 µl
5 Using the same Nunc DeepWell master mix source plate that was used
for the 06 Hybridization_QXT_v1.0.pro protocol, add the volume of PCR
master mix indicated in Table 49 to all wells of column 4 of the plate.
Keep the source plate on ice until it is used on page 97.
Table 49
Preparation of the Master Mix Source Plate for 09 Post-CapturePCR_QXT_ILM_v1.0.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
PCR Master Mix
Column 4
30.8 µl
53.2 µl
75.5 µl
97.8 µl
142.4 µl
287.4 µl
(A4-H4)
NOTE
If you are using a new DeepWell plate for the post-capture PCR source plate, leave
columns 1 to 3 empty and add the PCR Master Mix to column 4 of the new plate.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
95
5
Indexing
Step 1. Amplify the captured DNA libraries to add index tags
Assign and aliquot indexing primers
6 Determine the appropriate index assignments for each sample. See the
Reference section for sequences of the index portion of the P7 and P5
indexing primers used to amplify the DNA libraries in this step. (See
Table 69 and Table 70 for sequencing on HiSeq and MiSeq platforms or
see Table 69 and Table 71 for sequencing on the NextSeq platform.
Use the following guidelines for dual index assignments:
• Use a different indexing primer combination for each sample to be
sequenced in the same lane.
• All samples on the same row of the target-enriched DNA library
plate must be assigned to the same P5 indexing primer (P5 i13
through P5 i20). This design results from the automation protocol
configuration in which the P5 indexing primer is dispensed from a
single source plate column to all columns of the indexing PCR plate.
Each row of samples may be assigned to the same or different P5
primers, depending on run size and multiplexing requirements. (See
step 9, below, for details of P5 primer addition to the master mix
source plate.)
• The automation protocol configuration allows for any of the provided
P7 indexing primers (P7 i1 through P7 i12) to be assigned to any
sample position of the target-enriched DNA library plate. (See step 7
and step 8 below, for P7 primer source plate setup details.)
• For sample multiplexing, Agilent recommends maximizing index
diversity on both P7 and P5 primers as required for color balance.
For example, when 8-plexing, use eight different P7 index primers
with two P5 index primers. See Table 72 on page 125 for additional
details.
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SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Indexing
Step 1. Amplify the captured DNA libraries to add index tags
5
7 Dilute each P7 indexing primer (P7 i1 through P7 i12) to be used in the
run according to Table 50. The volumes below include the required
excess.
Table 50
Preparation of P7 indexing primer dilutions
Reagent
Volume to Index 1 Sample
Volume to Index 8 Samples
Nuclease-free water
4.0 µl
34 µl
SureSelect QXT P7 dual indexing
primer (P7 i1 to P7 i12)
1.0 µl
8.5 µl
Total Volume
5.0 µl
42.5 µl
8 In a fresh PCR plate, aliquot 5 µl of the appropriate P7 indexing primer
dilution from Table 50 to the intended sample indexing well position(s).
Keep the plate on ice.
9 Obtain the Nunc DeepWell master mix source plate containing the PCR
Master Mix in column 4 (prepared in step 5, above). Add each P5
indexing primer (P5 i13 through P5 i20) to be used in the run to the
master mix in the appropriate well of column 4. Add the volume listed
in Table 51 to each well of column 4, according to the number of
sample columns in the run. Each well of column 4 can contain the
same or different P5 indexing primers. The final configuration of the
master mix source plate is shown in Figure 10 on page 98.
Keep the source plate on ice.
Table 51
Addition of P5 indexing primers to the post-capture PCR master mix source plate
Solution added to
Source Plate
Position on
Source Plate
Volume of Primer added per Well of Nunc Deep Well Source Plate
1-Column
Runs
2-Column
Runs
3-Column
Runs
4-Column
Runs
6-Column
Runs
12-Column
Runs
SureSelect QXT
P5 dual indexing
primer(s)*
Column 4
1.5 µl
2.5 µl
3.6 µl
4.7 µl
6.8 µl
13.7 µl
(A4-H4)
* Each well of column 4 may contain the same or different P5 indexing primer. Typical 12-column runs include all eight of the
provided SureSelect QXT P5 dual indexing primers (P5 i13 through P5 i20), resulting in a different P5 primer assignment to
each row of the PCR indexing plate.
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Step 1. Amplify the captured DNA libraries to add index tags
Figure 10
Configuration of the master mix source plate for
09 Post-CapturePCR_QXT_ILM_v1.0.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 Vortex the plate to ensure complete mixing, then centrifuge the plate
for 30 seconds to drive the well contents off the walls and plate seal
and to eliminate any bubbles.
98
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Step 1. Amplify the captured DNA libraries to add index tags
5
Setup and run VWorks protocol 09 Post-CapturePCR_QXT_ILM_v1.0.pro,
12 On the SureSelect setup form, under Select Protocol to Run, select
09 Post-CapturePCR_QXT_ILM_v1.0.pro.
13 Under Select PCR plate Labware, 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 Bravo Deck Setup.
16 Load the Bravo deck according to the Bravo Deck Setup region of the
form and as shown in Table 52.
Table 52
Initial Bravo deck configuration for 01 Tn_QXT_ILM_v1.0.pro
Location
Content
1
–(empty)–
2
New tip box
3
–(empty)–
4
–(empty)–
5
Captured DNA bead suspensions in Eppendorf twin.tec plate
6
Diluted P7 indexing primers in PCR plate seated in red insert (PCR plate type
must be specified on setup form under step 2)
7
–(empty)–
8
Empty tip box
9
Master mix plate containing P5 indexing primers and PCR Master Mix in
Column 4 (unsealed), seated in silver insert
17 Verify that the Current Tip State indicator on the form matches the
configuration of unused and used tips in the tip boxes at Bravo Deck
positions 2 and 8, respectively. See page 23 for more information on
using this segment of the form during the run.
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Step 1. Amplify the captured DNA libraries to add index tags
18 When setup and verification is complete, click Run Selected Protocol.
19 Running the 09 Post-CapturePCR_QXT_ILM_v1.0.pro, protocol takes
approximately 15 minutes. Once complete, the PCR-ready samples,
containing captured DNA, PCR master mix, and indexing primers are
located in the PCR plate at position 6 of the Bravo deck.
20 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.
21 Centrifuge the plate for 30 seconds to drive the well contents off the
walls and plate seal and to eliminate air bubbles.
100
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Step 1. Amplify the captured DNA libraries to add index tags
5
22 Transfer the PCR plate to a thermal cycler and run the PCR
amplification program shown in Table 53.
Table 53
Post-Capture PCR cycling program
Segment
Number of Cycles
Temperature
Time
1
1
98°C
2 minutes
2
Capture Libraries3 Mb: 10 Cycles
98°C
30 seconds
Capture Libraries 1 to 3 Mb: 12 Cycles
58°C
30 seconds
72°C
1 minute
Capture Libraries1 Mb: 14 Cycles
3
1
72°C
5 minutes
4
1
4°C
Hold
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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 Bravo transfers AMPure XP beads to the
indexed DNA sample plate and then collects and washes the bead-bound
DNA.
This step uses protocol 10 Cleanup_Post-CapturePCR_QXT_ILM_v1.0.pro.
Prepare the NGS Bravo and reagents
1 Clear the Bravo deck of all plates and tip boxes, then wipe it down
with a DNA Away decontamination wipe.
2 Pre-set the temperature of Bravo deck positions 4 and 6 as indicated in
Table 54. See page 19 for more information on how to do this step.
Table 54
Bravo Deck Temperature Presets
Bravo Deck Position Temperature Preset
Preset Method
4
45°C
Inheco Multi TEC control touchscreen (CPAC 2-1)
6
4°C
Inheco Multi TEC control touchscreen (CPAC 2-2)
3 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.
4 Mix the AMPure XP bead suspension well so that the reagent appears
homogeneous and consistent in color.
5 Prepare a Nunc DeepWell source plate for the beads by adding 60 µl of
homogeneous AMPure XP beads per well, for each well to be processed.
6 Prepare a Thermo Scientific Matrix reservoir containing 20 ml of
nuclease-free water.
7 Prepare a separate Thermo Scientific Matrix reservoir containing 45 ml
of freshly-prepared 70% ethanol.
8 Centrifuge the amplified DNA sample plate for 30 seconds to drive the
well contents off the walls and plate seal.
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Step 2. Purify the amplified indexed libraries using Agencourt AMPure XP beads
5
Setup and run VWorks 10 Cleanup_Post-CapturePCR_QXT_ILM_v1.0.pro
9 On the SureSelect setup form, under Select Protocol, select
10 Cleanup_Post-CapturePCR_QXT_ILM_v1.0.pro.
10 Under Select PCR plate Labware, select the specific type of PCR plate
used for post-capture amplification.
11 Select the number of columns of samples to be processed. Runs must
include 1, 2, 3, 4, 6, or 12 columns.
12 Click Display Initial Bravo Deck Setup.
13 Load the Bravo deck according to the Bravo Deck Setup region of the
form and as shown in Table 55.
Table 55
Initial Bravo deck setup for 10 Cleanup_Post-CapturePCR_QXT_ILM_v1.0.pro
Location
Content
1
Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)
2
New tip box
3
Empty Eppendorf plate
4
–(empty)–
5
AMPure XP beads in Nunc DeepWell plate (60 µl beads per processing well)
6
Amplified DNA libraries in unsealed PCR plate seated in red insert (PCR plate
type must be specified on setup form under step 2)
7
–(empty)–
8
Empty tip box
9
70% ethanol in Matrix reservoir
14 Verify that the Current Tip State indicator on the form matches the
configuration of unused and used tips in the tip boxes at Bravo Deck
positions 2 and 8, respectively. See page 23 for more information on
using this segment of the form during the run.
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Step 2. Purify the amplified indexed libraries using Agencourt AMPure XP beads
15 When setup and verification is complete, click Run Selected Protocol.
Running the 10 Cleanup_Post-CapturePCR_QXT_ILM_v1.0.pro protocol
takes approximately 45 minutes. An operator must be present during the
run to complete tip box replacement and to transfer the water reservoir to
the Bravo deck, when directed by the VWorks prompt shown below.
Once the protocol is complete, the purified DNA samples are located in
the Eppendorf plate at position 3 of the Bravo deck.
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Step 3. Assess indexed DNA quality
5
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 325 and 450 bp. A sample electropherogram is
shown in Figure 11.
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.
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Step 3. Assess indexed DNA quality
Figure 11
106
Analysis of indexed DNA using the High Sensitivity DNA Assay.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Indexing
Step 3. Assess indexed DNA quality
5
Option 2: Analysis using an Agilent 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 using the Agilent 4200
TapeStation or 2200 TapeStation. For more information to do this step,
see the appropriate 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 instrument 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 TapeStation as instructed
in the instrument user manual. Start the run.
5 Verify that the electropherogram shows the peak of DNA fragment size
positioned between 325 and 450 bp. A sample electropherogram is
shown in Figure 12.
Stopping Point
If you do not continue to the next step, seal the indexed DNA sample
plate and store at 4°C overnight or at –20°C for prolonged storage.
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Indexing
Step 3. Assess indexed DNA quality
Figure 12
108
Analysis of indexed DNA using the 2200 TapeStation.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Indexing
Step 4. Quantify each index-tagged library by QPCR (optional)
5
Step 4. Quantify each index-tagged library by QPCR
(optional)
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.
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Indexing
Step 5. Pool samples for Multiplexed Sequencing
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.
 f   C f 
Volume of Index = V
--------------------------------#  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 56 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 56
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.
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Step 6. Prepare sequencing samples
5
Step 6. Prepare sequencing samples
The optimal seeding concentration for SureSelectQXT target-enriched
libraries is 8 to 12 pM on HiSeq or MiSeq instruments and 1.2 to 1.4 pM
on the NextSeq platform. Seeding concentration and cluster density may
also need to be optimized based on the DNA fragment size range for the
library and on the desired output and data quality. Follow Illumina’s
recommendation for a PhiX control in a low-concentration spike-in for
improved sequencing quality control.
Proceed to cluster amplification using the appropriate Illumina Paired-End
Cluster Generation Kit. See Table 57 for kit configurations compatible with
the recommended read length plus reads for the SureSelectQXT 8-bp dual
indexes. To do this step, refer to the manufacturer’s instructions, using
the modifications described in “Using the SureSelectQXT Read Primers with
Illumina’s Paired-End Cluster Generation Kits” on page 112.
Table 57
Illumina Kit Configuration Selection Guidelines
Platform
Run Type
Read Length*
SBS Kit Configuration
Chemistry
HiSeq 2500
Rapid Run
2 × 100 bp
200 Cycle Kit
v1 or v2
v3
HiSeq 2500
High Output
2 × 100 bp
4 x 50 Cycle Kit†
HiSeq 2500
High Output
2 × 100 bp
250 Cycle Kit
v4
v3
HiSeq 2000
All Runs
2 × 100 bp
4 x 50 Cycle Kit†
MiSeq
All Runs
2 × 100 bp
300 Cycle Kit
v2
MiSeq
All Runs
2 × 76 bp
150 Cycle Kit
v3
NextSeq 500
All Runs
2 × 100 bp
300 Cycle Kit
v2
* If your application requires a different read length, verify that you have sufficient sequencing reagents to complete Reads 1 and 2 in addition to the dual 8-bp index reads.
† A single 200-cycle kit does not include enough reagents to complete Reads 1 and 2 in addition to
the dual 8-bp index reads in this format. If preferred, the additional reads may be supported by using
one 200-cycle kit plus one 50-cycle kit.
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Step 6. Prepare sequencing samples
Using the SureSelectQXT Read Primers with Illumina’s Paired-End Cluster
Generation Kits
To sequence the SureSelectQXT libraries on Illumina’s sequencing
platforms, you need to use the following custom sequencing primers,
provided in SureSelect QXT Library Prep Kit Box 2:
• SureSelect QXT Read Primer 1
• SureSelect QXT Read Primer 2
• SureSelect QXT Index Read Primer
• SureSelect QXT Index 2 Read Primer NSQ (NextSeq platform only)
These SureSelectQXT custom sequencing primers are provided at 100 M
and must be diluted 1:200 in the corresponding Illumina primer solution,
using the platform-specific instructions below:
For the HiSeq platform, combine the primers as shown in Table 58 or
Table 59 on page 113.
For the MiSeq platform, combine the primers as shown in Table 60 on
page 113.
For the NextSeq platform, combine the primers as shown in Table 61 or
Table 62 on page 114.
NOTE
112
It is important to combine the primers precisely in the indicated ratios. Be sure to use
measured volumes of each solution; do not use volumes reported on vial labels when
preparing the mixtures. Vortex each mixture vigorously to ensure homogeneity for proper
detection of the indexes using the custom read primers.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Indexing
Step 6. Prepare sequencing samples
Table 58
5
HiSeq2000 and HiSeq 2500 High Output custom sequencing primer preparation
Sequencing
Read
Volume of SureSelectQXT Primer
Volume of Illumina TruSeq Primer
Total Volume
Read 1
5 µl SureSelect QXT Read Primer 1 (brown cap)
995 µl HP6 or HP10
1 ml*
Index
15 µl SureSelect QXT Index Read Primer (clear cap)
2985 µl HP8 or HP12
3 ml
Read 2
15 µl SureSelect QXT Read Primer 2 (black cap)
2985 µl HP7 or HP11
3 ml
* Aliquot the mixture as directed for HP6 or HP10 in Illumina’s cluster generation protocol.
Table 59
HiSeq 2500 Rapid Mode custom sequencing primer preparation
Sequencing
Read
Volume of SureSelectQXT Primer
Volume of Illumina TruSeq Primer
Total Volume
Read 1
8.8 µl SureSelect QXT Read Primer 1 (brown cap)
1741.2 µl HP10
1.75 ml*
Index
8.8 µl SureSelect QXT Index Read Primer (clear cap)
1741.2 µl HP12
1.75 ml
Read 2
8.8 µl SureSelect QXT Read Primer 2 (black cap)
1741.2 µl HP11
1.75 ml
* Aliquot the mixture as directed for HP10 in Illumina’s cluster generation protocol.
Table 60
MiSeq platform custom sequencing primer preparation
Sequencing Volume of SureSelectQXT Primer
Read
Volume of Illumina TruSeq
Primer
Total
Volume
Final
Cartridge
Position
Read 1
3 µl SureSelect QXT Read Primer 1 (brown cap)
597 µl HP10 (well 12)
0.6 ml
well 18
Index
3 µl SureSelect QXT Index Read Primer (clear cap)
597 µl HP12 (well 13)
0.6 ml
well 19
Read 2
3 µl SureSelect QXT Read Primer 2 (black cap)
597 µl HP11 (well 14)
0.6 ml
well 20
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Step 6. Prepare sequencing samples
Table 61
NextSeq 500/550 High-Output v2 Kit custom sequencing primer preparation
Sequencing Volume of SureSelectQXT Primer
Read
Volume of Illumina Primer
Total
Volume
Final
Cartridge
Position
Read 1
3.9 µl SureSelect QXT Read Primer 1 (brown cap)
1296.1 µl BP10 (from well 20)
1.3 ml
well 7
Read 2
4.2 µl SureSelect QXT Read Primer 2 (black cap)
1395.8 µl BP11 (from well 21)
1.4 ml
well 8
Index +
Index 2
6 µl SureSelect QXT Index Read Primer (clear cap)
+ 6 µl SureSelect QXT Index 2 Read Primer NSQ
(purple cap)
1988 µl BP14 (from well 22)
2 ml
well 9
Table 62
NextSeq 500/550 Mid-Output v2 Kit custom sequencing primer preparation
Sequencing Volume of SureSelectQXT Primer
Read
Volume of Illumina Primer
Total
Volume
Final
Cartridge
Position
Read 1
2.7 µl SureSelect QXT Read Primer 1 (brown cap)
897.3 µl BP10 (from well 20)
0.9 ml
well 7
Read 2
3.3 µl SureSelect QXT Read Primer 2 (black cap)
1096.7 µl BP11 (from well 21)
1.1 ml
well 8
Index +
Index 2
4.8 µl SureSelect QXT Index Read Primer (clear
cap) + 4.8 µl SureSelect QXT Index 2 Read Primer
NSQ (purple cap)
1590.4 µl BP14 (from well 22)
1.6 ml
well 9
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Step 7. Set up the sequencing run and trim adaptors from the reads
5
Step 7. Set up the sequencing run and trim adaptors from the
reads
Refer to Illumina protocols to set up custom sequencing primer runs,
using the additional guidelines outlined below.
For SureSelectQXT dual index sequence information, see tables starting on
page 123.
Before aligning reads to the reference genome, SureSelectQXT adaptor
sequences must be trimmed from the reads. You can use SureCall,
Agilent's NGS data analysis software, to perform adaptor trimming,
alignment of reads and variant calling of sequencing data generated from
either the HiSeq or the MiSeq platform. To download SureCall
free-of-charge and for additional information, including tutorials on this
software, visit the SureCall page at www.genomics.agilent.com.
SureCall is compatible with FASTQ files generated by both the HiSeq and
MiSeq platforms. To use SureCall to analyze SureSelectQXT-generated data,
you first need to define an analysis workflow. This analysis workflow
identifies the libraries as SureSelectQXT libraries and enables automated
adaptor trimming. The trimmed FASTQ files can then be used for
alignment to generate BAMs for downstream analysis.
To create the analysis workflow, refer to Figure 13 on page 116. Upon
starting SureCall, click the Analysis Workflow tab. Choose the appropriate
analysis type (single sample, paired, or trio analysis), and then click the
Import Unaligned Files button. Within the Select Unaligned Sample Files
window, specify your read 1 and read 2 files using the Add buttons. Using
the menus near the bottom of the screen, select Default SureSelect QXT
Method from the Analysis Method menu, choose the appropriate design
description from the Design menu, and select Illumina from the Platform
menu. Once done, refer to the SureCall guide for next steps on alignment
and variant calling.
If using another pipeline for alignment and downstream analysis, refer to
the platform-specific guidelines starting on page 116.
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Step 7. Set up the sequencing run and trim adaptors from the reads
Figure 13
Analysis workflow setup in SureCall.
MiSeq platform sequencing run setup and adaptor trimming guidelines
Use the Illumina Experiment Manager (IEM) software to generate a custom
primer Sample Sheet.
Set up the run to include adapter trimming using the IEM Sample Sheet
Wizard. When prompted by the wizard, select the Use Adapter Trimming
option, and specify CTGTCTCTTGATCACA as the adapter sequence. This enables
the MiSeq Reporter software to identify the adaptor sequence and trim
the adaptor from reads.
HiSeq or NextSeq 500 platform sequencing run setup and adaptor trimming
guidelines
Set up sequencing runs using the Custom setting. Since custom primers
are spiked into the standard sequencing primer tubes, no additional
specialized settings are required to accommodate the use of custom
primers in the run.
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Step 7. Set up the sequencing run and trim adaptors from the reads
5
Use the Cycles settings shown in Table 63. Cycle number settings can be
specified on the Run Configuration screen of the instrument control
software interface after choosing Custom from the index type selection
buttons.
Table 63
Run Configuration screen Cycle Number settings
Run Segment
Cycle Number
Read 1
100
Index 1 (i7)
8
Index 2 (i5)
8
Read 2
100
After the sequencing run is complete, generate demultiplexed FASTQ data
following Illumina’s instructions and then trim adaptor sequences from the
reads using Agilent’s Read Trimmer tool. This tool takes in data in FASTQ
format and removes the adaptor sequence from the ends of the sequencing
reads, generating trimmed FASTQ data as output. To download the Read
Trimmer tool free-of-charge and for additional information on this
resource, visit www.agilent.com/genomics.
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118
Indexing
Step 7. Set up the sequencing run and trim adaptors from the reads
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
SureSelectQXT Automated Target Enrichment for Illumina Multiplexed
Sequencing Protocol
6
Reference
Kit Contents 120
Nucleotide Sequences of SureSelectQXT Dual Indexes 123
Guidelines for Multiplexing with Dual-Indexed Samples 125
This chapter contains reference information, including component kit
contents and reference information for use during the downstream sample
sequencing steps.
Agilent Technologies
119
6
Reference
Kit Contents
Kit Contents
SureSelectQXT Reagent Kits contain the following component kits:
Table 64
SureSelectQXT Reagent Kit Contents
Component Kits
Storage Condition
HiSeq or
MiSeq (ILM)
96 Samples
NextSeq
(NSQ)
96 Samples
SureSelect QXT Library Prep Kit, ILM, Box 2* –20°C
5500-0121
5500-0127
SureSelect QXT Target Enrichment Kit, ILM
Hyb Module, Box #1
Room Temperature
5190-7335
5190-7335
SureSelect QXT Target Enrichment Kit, ILM
Hyb Module, Box #2
–20°C
5190-7334
5190-7334
* SureSelect QXT Library Prep Kit, ILM, Box 1 is not required for the workflow described in this manual.
The contents of each of the component kits listed in Table 64 are
described in Table 66 to Table 68 below.
120
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Reference
Kit Contents
Table 65
6
SureSelect QXT Library Prep, ILM, Box 2 Content
Kit Component
HiSeq or MiSeq (ILM) 96 Reactions
SureSelect QXT Buffer
bottle
SureSelect QXT Enzyme Mix ILM
tube with orange cap
Herculase II Fusion DNA Polymerase
tube with red cap
Herculase II 5× Reaction Buffer
tube with clear cap
100 mM dNTP Mix (25 mM each dNTP)
tube with green cap
DMSO
tube with green cap
SureSelect QXT Read Primer 1
tube with amber cap
SureSelect QXT Read Primer 2
tube with black cap
SureSelect QXT Index Read Primer
tube with clear cap
SureSelect QXT P7 dual indexing primers
P7 i1 through P7 i12 provided in 12 tubes with yellow caps (one tube per primer)
SureSelect QXT P5 dual indexing primers
P5 i13 through P5 i20 provided in 8 tubes with blue caps (one tube per primer)
Table 66
SureSelect QXT Library Prep, NSQ, Box 2 Content
Kit Component
NextSeq (NSQ) 96 Reactions
SureSelect QXT Buffer
bottle
SureSelect QXT Enzyme Mix ILM
tube with orange cap
Herculase II Fusion DNA Polymerase
tube with red cap
Herculase II 5× Reaction Buffer
tube with clear cap
100 mM dNTP Mix (25 mM each dNTP)
tube with green cap
DMSO
tube with green cap
SureSelect QXT Read Primer 1
tube with amber cap
SureSelect QXT Read Primer 2
tube with black cap
SureSelect QXT Index Read Primer
tube with clear cap
SureSelect QXT Index 2 Read Primer NSQ
tube with purple cap
SureSelect QXT P7 dual indexing primers
P7 i1 through P7 i12 provided in 12 tubes with yellow caps (one tube per primer)
SureSelect QXT P5 dual indexing primers
P5 i13 through P5 i20 provided in 8 tubes with blue caps (one tube per primer)
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
121
6
Reference
Kit Contents
Table 67
Kit Component
96 Reactions
SureSelect QXT Stop Solution
bottle
SureSelect Binding Buffer
bottle
SureSelect Wash Buffer 1
bottle
SureSelect Wash Buffer 2
bottle
Table 68
122
SureSelect QXT Hyb Module Box 1 Content
SureSelect QXT Hyb Module Box 2 Content
Kit Component
96 Reactions
SureSelect QXT Fast Hybridization Buffer
bottle
SureSelect QXT Fast Blocker Mix
tube with blue cap
SureSelect QXT Primer Mix
tube with clear cap
SureSelect RNase Block
tube with purple cap
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
Reference
Nucleotide Sequences of SureSelectQXT Dual Indexes
6
Nucleotide Sequences of SureSelectQXT Dual Indexes
The nucleotide sequence of each SureSelectQXT index is provided in the
tables below.
Note that some index number assignments of the SureSelectQXT P5 and P7
indexes differ from the index number assignments used by Illumina for
indexes of similar or identical sequence.
Each index is 8 bases in length. Refer to Illumina’s sequencing run setup
instructions for sequencing libraries using 8-base indexes.
Table 69
SureSelectQXT P7 Indexes 1 to 12
Index Number
Sequence
P7 Index 1 (P7 i1)
TAAGGCGA
P7 Index 2 (P7 i2)
CGTACTAG
P7 Index 3 (P7 i3)
AGGCAGAA
P7 Index 4 (P7 i4)
TCCTGAGC
P7 Index 5 (P7 i5)
GTAGAGGA
P7 Index 6 (P7 i6)
TAGGCATG
P7 Index 7 (P7 i7)
CTCTCTAC
P7 Index 8 (P7 i8)
CAGAGAGG
P7 Index 9 (P7 i9)
GCTACGCT
P7 Index 10 (P7 i10)
CGAGGCTG
P7 Index 11 (P7 i11)
AAGAGGCA
P7 Index 12 (P7 i12)
GGACTCCT
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
123
6
Reference
Nucleotide Sequences of SureSelectQXT Dual Indexes
Table 70
SureSelectQXT P5 Indexes 13 to 20 for HiSeq platform, MiSeq platform, or
NextSeq platform runs through BaseSpace
Index Number
Sequence
P5 Index 13 (P5 i13)
TAGATCGC
P5 Index 14 (P5 i14)
CTCTCTAT
P5 Index 15 (P5 i15)
TATCCTCT
P5 Index 16 (P5 i16)
AGAGTAGA
P5 Index 17 (P5 i17)
GTAAGGAG
P5 Index 18 (P5 i18)
ACTGCATA
P5 Index 19 (P5 i19)
AAGGAGTA
P5 Index 20 (P5 i20)
CTAAGCCT
Table 71
SureSelectQXT P5 Indexes 13 to 20 for NextSeq platform*
Index Number
Sequence
P5 Index 13 (P5 i13)
GCGATCTA
P5 Index 14 (P5 i14)
ATAGAGAG
P5 Index 15 (P5 i15)
AGAGGATA
P5 Index 16 (P5 i16)
TCTACTCT
P5 Index 17 (P5 i17)
CTCCTTAC
P5 Index 18 (P5 i18)
TATGCAGT
P5 Index 19 (P5 i19)
TACTCCTT
P5 Index 20 (P5 i20)
AGGCTTAG
* When doing NextSeq runs through BaseSpace, use the reverse complement sequences provided in Table 70.
124
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Reference
Guidelines for Multiplexing with Dual-Indexed Samples
6
Guidelines for Multiplexing with Dual-Indexed Samples
Agilent recommends following the dual index sample pooling guidelines
and shown in Table 72. These are designed to maintain color balance at
each cycle of the index reads on both ends. They also provide flexibility of
demultiplexing as single or dual indexed samples in low-plexity
experiments. One-base mismatches should also be allowed during
demultiplexing in order to maximize sequencing output per sample.
Table 72
Dual index sample pooling guidelines for 96 Reaction Kits
Plexity of Sample
Pool
Recommended SureSelectQXT P7
Recommended SureSelectQXT P5 Indexes
1-plex
Any P7 index i1 to i11
Any P5 index (i13 to i20)
2-plex
P7 i1 and P7 i2 OR
P5 i13 and P5 i14 OR
P7 i2 and P7 i4
P5 i15 and P5 i16 OR
Indexes
P5 i17 and P5 i18
3-plex
4-plex
5-plex
6- to 11-plex
P7 i1, P7 i2 and P7 i4 OR
P5 i13 and P5 i14 OR
P7 i3, P7 i4 and P7 i6 OR
P5 i15 and P5 i16 OR
P7 i5, P7 i7 and P7 i8
P5 i17 and P5 i18 (as needed)
* and P7 i4 OR
P5 i13 and P5 i14 OR
P7 i3, P7 i4, P7 i5* and P7 i6 OR
P5 i15 and P5 i16 OR
P7 i5, P7 i6*, P7 i7 and P7 i8
P5 i17 and P5 i18 (as needed)
P7 i1, P7 i2, P7 i3
P7 i1, P7 i2, P7 i3*, P7 i4 and P7 i5* OR
P5 i13 and P5 i14 OR
P7 i3, P7 i4, P7 i5*, P7 i6 and p7 i7* OR
P5 i15 and P5 i16 OR
P7 i5, P7 i6*, P7 i7, P7 i8 and p7 i9*
P5 i17 and P5 i18 (as needed)
Any combination of P7 indexes i1 to i11
using each index only once
P5 i13 and P5 i14 OR
P5 i15 and P5 i16 OR
P5 i17 and P5 i18 (as needed)
12-to 88-plex
Any combination of P7 indexes i1 to i11
(as needed)
P5 i13 and P5 i14 and any third P5 index OR
P5 i15 and P5 i16 and any third P5 index OR
P5 i17 and P5 i18 and any third P5 index (as needed)
89-to 96-plex
All twelve P7 indexes (i1 to i12)
Any P5 indexes (i13 to i20, as needed)
* The indicated indexes may be substituted with another index within range of P7 i1 to P7 i11, as long as the substitute index
differs from all others used in the sample pool.
SureSelectQXT Target Enrichment for Illumina Sequencing (NGS Bravo Option A)
125
www.agilent.com
In This Book
This guide contains
information to run the
SureSelectQXT Automated
Library Prep and Target
Enrichment protocol using
Agilent’s NGS Bravo
Option A.
Agilent Technologies, Inc. 2015
Version B0, November 2015
*G9681-90020*
p/n G9681-90020
Agilent Technologies
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