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SureSelect Automated Library Prep and Capture SystemSureSelectXT Automated Target Enrichment for SOLiD 5500 Paired-End Multiplexed Sequencing
ProtocolVersion A, October 2012
SureSelect platform manufactured with Agilent SurePrint Technology
Research Use Only. Not for use in Diagnostic Procedures.
Agilent Technologies
Notices© Agilent Technologies, Inc. 2012
No part of this manual may be reproduced in any form or by any means (including elec-tronic storage and retrieval or translation into a foreign language) without prior agree-ment and written consent from Agilent Technologies, Inc. as governed by United States and international copyright laws.
Manual Part NumberG7550-90002
EditionVersion A, October 2012
Printed in USA
Agilent Technologies, Inc. 5301 Stevens Creek Blvd
WarrantyThe material contained in this document is provided “as is,” and is subject to being changed, with-out notice, in future editions. Fur-ther, to the maximum extent permitted by applicable law, Agi-lent disclaims all warranties, either express or implied, with regard to this manual and any information contained herein, including but not limited to the implied warranties of merchant-ability and fitness for a particular purpose. Agilent shall not be lia-ble for errors or for incidental or consequential damages in con-nection with the furnishing, use, or performance of this document or of any information contained herein. Should Agilent and the user have a separate written agreement with warranty terms covering the material in this doc-ument that conflict with these terms, the warranty terms in the separate agreement shall control.
Technology Licenses The hardware and/or software described in this document are furnished under a license and may be used or copied only in accor-dance with the terms of such license.
Restricted Rights LegendU.S. Government Restricted Rights. Soft-ware and technical data rights granted to the federal government include only those rights customarily provided to end user cus-tomers. Agilent provides this customary commercial license in Software and techni-cal data pursuant to FAR 12.211 (Technical Data) and 12.212 (Computer Software) and, for the Department of Defense, DFARS 252.227-7015 (Technical Data - Commercial Items) and DFARS 227.7202-3 (Rights in Commercial Computer Software or Com-puter Software Documentation).
Safety Notices
CAUTION
A CAUTION notice denotes a haz-ard. It calls attention to an operat-ing procedure, practice, or the like that, if not correctly performed or adhered to, could result in damage to the product or loss of important data. Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met.
WARNING
A WARNING notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly per-formed or adhered to, could result in personal injury or death. Do not proceed beyond a WARNING notice until the indicated condi-tions are fully understood and met.
Santa Clara, CA 95051 USA
AcknowledgementOligonucleotide sequences © 2006 and 2008 Applied Biosystems, a division of Life Technologies, Inc. All rights reserved. Only for use with the Applied Biosystems SOLiD System Sequencing and associated assays.
Technical SupportFor technical product support, contact your local Agilent Support Services representa-tive. For Agilent’s worldwide sales and sup-port center telephone numbers, go to www.agilent.com/chem/contactus
or send an email to: [email protected]
Notice to PurchaserResearch Use Only. Not for use in diagnostic procedures.
SureSelect capture libraries and reagents must be used within one year of receipt.
2 SureSelect Automated Library Prep and Capture System
In this Guide...
SureSelect Automated Library Prep
This guide describes an optimized protocol for SOLiD 5500 paired-end multiplexed library preparation using the Agilent SureSelect Automated Library Prep and Capture System.
This protocol is specifically developed and optimized to capture the genomic regions of interest using Agilent’s SureSelect system to enrich targeted regions of the genome from repetitive sequences and sequences unrelated to the research focus prior to sample sequencing. Sample processing steps are automated using the SureSelect Automated Library Prep and Capture System.
1
Before You BeginThis chapter contains information (such as procedural notes, safety information, required reagents and equipment) that you should read and understand before you start an experiment.
2
Using the Agilent NGS Workstation for SureSelect Target EnrichmentThis chapter contains an orientation to the Agilent NGS Workstation, an overview of the SureSelect target enrichment protocol, and considerations for designing SureSelect experiments for automated processing using the Agilent NGS Workstation.
3
Sample PreparationThis chapter describes the steps to prepare the DNA samples for target enrichment.
4
HybridizationThis chapter describes the steps to hybridize and capture the DNA samples.
and Capture System 3
5
4
Barcoding
This chapter describes the steps to amplify, purify, and assess quality of the sample libraries. Samples are pooled by mass prior to sequencing.
6
ReferenceThis chapter contains reference information.
SureSelect Automated Library Prep and Capture System
Content
1 Before You Begin 7
Procedural Notes 8Safety Notes 8Required Reagents 9Required Equipment 11
2 Using the Agilent NGS Workstation for SureSelect Target Enrichment 13
About the Agilent NGS Workstation 14
About the Bravo Platform 14VWorks Automation Control Software 18
Overview of the SureSelect Target Enrichment Procedure 24
Experimental Setup Considerations for Automated Runs 27
Considerations for Placement of gDNA Samples in 96-well Plates for Automated Processing 28
Considerations for Equipment Setup 28
3 Sample Preparation 29
Step 1. Shear DNA 30Step 2. Size-select and purify sheared DNA using AMPure XP beads 33Step 3. Assess sheared DNA fragment size 39Step 4. Modify DNA ends for target enrichment 42Step 5. Amplify adaptor-ligated libraries 52Step 6. Purify amplified DNA using AMPure XP beads 61Step 7. Assess prepared library DNA quantity and quality 66
4 Hybridization 71
Step 1. Aliquot prepped DNA samples for hybridization 72Step 2. Hybridize the gDNA library and SureSelect Capture Library 76Step 3. Capture the hybridized DNA 92
SureSelect Automated Library Prep and Capture System 5
Contents
5 Barcoding 101
Step 1. Amplify the captured libraries to add barcode tags 102Step 2. Purify the amplified barcoded libraries using Agencourt AMPure XP
beads 114Step 3. Remove primer-dimers by purification using Agencourt AMPure XP
beads 119Step 4. Assess barcoded DNA quality 124Step 5. Assess the quantity of each barcode-tagged library by QPCR 128Step 6. Pool samples for multiplexed sequencing 129
6 Reference 131
Kit Contents 132Nucleotide Sequences of SureSelect Barcodes 135
6 SureSelect Automated Library Prep and Capture System
SureSelect Automated Library Prep and Capture System Protocol
1Before You Begin
Procedural Notes 8
Safety Notes 8
Required Reagents 9
Required Equipment 11
Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an experiment.
This protocol differs from other SureSelect protocols at several steps. Pay close attention to the primers used for each amplification step and the blocking agents used during hybridization.
Agilent 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.
NOTE
NOTE
7Agilent Technologies
1 Before You Begin Procedural Notes
Procedural Notes
8
• Certain protocol steps require the rapid transfer of sample plates between the Bravo deck and a thermal cycler. Locate your thermal cycler in close proximity to the Agilent NGS Workstation to allow rapid and efficient plate transfer.
• Prepare and load the Agilent NGS Workstation as detailed in each of the protocol steps before initiating each automated protocol run. When loading plates in the workstation’s Labware MiniHub, always place plates in the orientation shown in Figure 3 on page 35.
• To prevent contamination of reagents by nucleases, always wear powder-free laboratory gloves and use dedicated solutions and pipettors with nuclease-free aerosol-resistant tips.
• Maintain a clean work area.
• Do not mix stock solutions and reactions containing gDNA on a vortex mixer. Instead, gently tap the tube with your finger to mix the sample.
• Avoid repeated freeze-thaw cycles of stock and diluted gDNA solutions. Possible stopping points, where gDNA samples may be stored overnight at 4°C, are marked in the protocol. When storing samples for >24 hours, store the samples at –20°C, but do not subject the samples to multiple freeze/thaw cycles.
• When preparing frozen reagent stock solutions for use:
1 Thaw the aliquot as rapidly as possible without heating above room temperature.
2 Mix briefly on a vortex mixer, then spin in a centrifuge for 5 to 10 seconds to drive the contents off of walls and lid.
3 Store on ice or in a cold block until use.
• In general, follow Biosafety Level 1 (BL1) safety rules.
Safety Notes
• Wear appropriate personal protective equipment (PPE) when working in the laboratory.
CAUTION
SureSelect Automated Library Prep and Capture System
Before You Begin 1 Required Reagents
Required Reagents
SureSelect Automat
Table 1 Required Reagents
Description Vendor and part number
SureSelectXT Capture Library*
* SureSelect capture libraries and reagents must be used within one year of receipt.
Select one library from Table 2 or Table 3
SureSelectXT Reagent Kit*-5500, 96 Reactions*†
† Each 96-reaction kit contains sufficient reagents for 96 reactions used in runs that include at least 3 columns of samples per run.
G9615B
Herculase II Fusion DNA Polymerase, 400 reactions (includes dNTP mix and 5x Buffer)
Agilent p/n 600679
QPCR NGS Library Quantification Kit (SOLiD) Agilent p/n G4881A
Nuclease-free Water (not DEPC-treated) Ambion Cat #AM9930
1X Low TE Buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA) Life Technologies p/n 4389764
Agencourt AMPure XP Kit 60 mL 450 mL
Beckman Coulter Genomics p/n A63881 p/n A63882
Quant-iT dsDNA BR Assay Kit, for use with the Qubit fluorometer
100 assays, 2-1000 ng 500 assays, 2-1000 ng
Life Technologies Cat #Q32850 Cat #Q32853
Dynabeads MyOne Streptavidin T1 2 mL 10 mL 100 mL
Life Technologies Cat #65601 Cat #65602 Cat #65603
100% Ethanol, molecular biology grade Sigma-Aldrich p/n E7023
ed Library Prep and Capture System 9
1 Before You Begin Required Reagents
10
Table 2 Agilent SureSelectXT Automation Custom Capture Libraries
Custom Capture Library Size 96 Reactions 96 Reactions for Reorder
480 Reactions 480 Reactions for Reorder
1 kb up to 499 kb 5190-4808 5190-4813 5190-4810 5190-4815
0.5 Mb up to 2.9 Mb 5190-4818 5190-4823 5190-4820 5190-4825
3 Mb up to 5.9 Mb 5190-4828 5190-4833 5190-4830 5190-4835
6 Mb up to 11.9 Mb 5190-4838 5190-4843 5190-4840 5190-4845
12 Mb up to 24 Mb 5190-4898 5190-4903 5190-4900 5190-4905
Table 3 Agilent SureSelectXT Automation Catalog Capture Libraries
Catalog Capture Library 96 Reactions 480 Reactions
Human All Exon v4 5190-4633 5190-4635
Human All Exon v4 + UTRs 5190-4638 5190-4640
Human All Exon 50 Mb 5190-4628 5190-4630
Human DNA Kinome 5190-4648 5190-4650
Mouse All Exon 5190-4643 5190-4645
SureSelect Automated Library Prep and Capture System
Before You Begin 1 Required Equipment
Required Equipment
SureSelect Automat
Table 4 Required Equipment
Description Vendor and part number
Agilent NGS Workstation for the SureSelect Automated Library Prep and Capture System, with VWorks software version 11.0.1.1032 or later.
Agilent p/n G5522A; contact Agilent Automation Solutions for ordering information:
Robotic Pipetting Tips (Sterile, Filtered, 250 L) Agilent p/n 19477-022
Eppendorf twin.tec full-skirted 96-well PCR plates Eppendorf p/n 951020401 or 951020619
Thermo Scientific Reservoirs Thermo Scientific p/n 1064156
Nunc DeepWell Plates, sterile, 1.3-mL well volume Thermo Scientific p/n 260251
Axygen 96 Deep Well Plate, 2.2 mL, Square Well (waste reservoirs)
Axygen p/n P-2ML-SQ-C E & K Scientific p/n EK-2440
MicroAmp Optical 96-well plates, half-skirted Life Technologies p/n N801-0560
MicroAmp 96-well base (black adapter for MicroAmp plates)
Life Technologies p/n N801-0531
DNA Analysis Platform and Consumables
2100 Bioanalyzer Laptop Bundle
2100 Bioanalyzer Electrophoresis Set
DNA 1000 Kit
High Sensitivity DNA Kit
OR
2200 TapeStation
D1K ScreenTape
D1K Reagents
High Sensitivity D1K ScreenTape
D1K Reagents
Agilent p/n G2943CA
Agilent p/n G2947CA
Agilent p/n 5067-1504
Agilent p/n 5067-4626
Agilent p/n G2964AA or G2965AA
Agilent p/n 5067-5361
Agilent p/n 5067-5362
Agilent p/n 5067-5363
Agilent p/n 5067-5364
Thermal cycler Life Technologies Veriti Thermal Cycler
Qubit Fluorometer Life Technologies p/n Q32857
Qubit assay tubes Life Technologies p/n Q32856
ed Library Prep and Capture System 11
1 Before You Begin Required Equipment
12
Covaris Sample Preparation System, S-series or E-series model
Covaris
Covaris sample holders
96 microTUBE plate (E-series only)
microTUBE for individual sample processing
Covaris p/n 520078
Covaris p/n 520045
DNA LoBind Tubes, 1.5-mL PCR clean, 250 pieces Eppendorf p/n 022431021 or equivalent
Centrifuge Eppendorf Centrifuge model 5804 or equivalent
P10, P20, P200 and P1000 pipettes Pipetman P10, P20, P200, P1000 or equivalent
Vacuum concentrator Savant SpeedVac, model DNA120, with 96-well plate rotor, model RD2MP, or equivalent
Magnetic separator DynaMag-50 magnet, Life Technologies p/n 123-02D or equivalent
Mx3005P Real-Time PCR System Agilent p/n 401449 or equivalent
Mx3000P/Mx3005P 96-well tube plates Agilent p/n 410088 or equivalent
Mx3000P/Mx3005P optical strip caps Agilent p/n 401425 or equivalent
NucleoClean Decontamination Wipes Millipore p/n 3097
Ice bucket
Powder-free gloves
Sterile, nuclease-free aerosol barrier pipette tips
Vortex mixer
Timer
Table 4 Required Equipment (continued)
Description Vendor and part number
SureSelect Automated Library Prep and Capture System
SureSelect Automated Library Prep and Capture System Protocol
2Using the Agilent NGS Workstation for SureSelect Target Enrichment
About the Agilent NGS Workstation 14
Overview of the SureSelect Target Enrichment Procedure 24
Experimental Setup Considerations for Automated Runs 27
This chapter contains an orientation to the Agilent NGS Workstation, an overview of the SureSelectXT target enrichment protocol, and considerations for designing SureSelect experiments for automated processing using the Agilent NGS Workstation.
13Agilent Technologies
2 Using the Agilent NGS Workstation for SureSelect Target Enrichment About the Agilent NGS Workstation
About the Agilent NGS Workstation
About the Bravo Platform
14
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.
CAUTION 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 (G5409-90004) and the VWorks Software User Guide (G5415-90002).
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 Bravo platform deck
SureSelect Automated Library Prep and Capture System
Using the Agilent NGS Workstation for SureSelect Target Enrichment 2 About the Bravo Platform
SureSelect Automat
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.
1 Using the arrow buttons, select the appropriate block (CPAC 2 block 1 or CPAC 2 block 2).
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
ed Library Prep and Capture System 15
2 Using the Agilent NGS Workstation for SureSelect Target Enrichment About the Bravo Platform
16
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.
SureSelect Automated Library Prep and Capture System
Using the Agilent NGS Workstation for SureSelect Target Enrichment 2 About the Bravo Platform
SureSelect Automat
Setting the Temperature of Bravo Deck Position 9 Using the ThermoCube Device
Bravo deck position 9 is equipped with a ThermoCube thermoelectric temperature control system, used to incubate components at a defined temperature during the run. During protocols that require temperature control at position 9, you will be instructed to start and set the temperature of the ThermoCube device before starting the run.
ThermoCube temperature settings are modified using the control panel (LCD display screen and four input buttons) on the front panel of the device using the following steps.
1 Turn on the ThermoCube and wait for the LCD screen to display TEMP.
2 Press the UP or DOWN button to change SET TEMP 1 to the required set point.
3 Press the START button.
The ThermoCube will then initates temperature control of Bravo deck position 9 at the displayed set point.
ed Library Prep and Capture System 17
2 Using the Agilent NGS Workstation for SureSelect Target Enrichment VWorks Automation Control Software
VWorks Automation Control Software
18
VWorks software, included with your Agilent NGS Workstation, allows you to control the robot and integrated devices using a PC. The Agilent NGS Workstation is preloaded with VWorks software containing all of the necessary SureSelect system liquid handling protocols. General instructions for starting up the VWorks software and the included protocols is provided below. Each time a specific VWorks protocol is used in the SureSelect procedure, any settings required for that protocol are included in the relevant section of this manual.
NOTE The instructions in this manual are compatible with VWorks software version 11.0.1.1032 or later.
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 SureSelect_XT_SOLiD_5500.VWForm shortcut on the Windows desktop to start the VWorks software.
2 If User Authentication dialog is not visible, click Log in on the VWorks window toolbar.
3 In the User Authentication dialog, type your VWorks user name and password, and click OK. (If no user account is set up, contact the administrator.)
VWorks protocol and runset files
VWorks software uses two file types for automation runs, .pro (protocol) files and .rst (runset) files. Runset files are used for automated procedures in which the workstation uses more than one automation protocol during the run.
SureSelect Automated Library Prep and Capture System
Using the Agilent NGS Workstation for SureSelect Target Enrichment 2 VWorks Automation Control Software
SureSelect Automat
Using the SureSelect_XT_SOLiD_5500.VWForm to setup and start a run
Use the VWorks form SureSelect_XT_SOLiD_5500.VWForm, shown below, to set up and start each SureSelect automation protocol or runset.
1 Open the form using the SureSelect_XT_SOLiD_5500.VWForm shortcut on your desktop.
2 Use the drop-down menus on the form to select the appropriate SureSelect workflow steps, plate type, and number of columns of samples for the run.
3 Once all run parameters have been specified on the form, click Display Initial Workstation Setup.
ed Library Prep and Capture System 19
2 Using the Agilent NGS Workstation for SureSelect Target Enrichment VWorks Automation Control Software
20
4 The Workstation Setup region of the form will then display the required placement of reaction components and labware in the NGS Workstation for the specified run parameters.
5 After verifying that the NGS Workstation has been set up correctly, click Run Selected Protocol.
SureSelect Automated Library Prep and Capture System
Using the Agilent NGS Workstation for SureSelect Target Enrichment 2 VWorks Automation Control Software
SureSelect Automat
Error messages encountered at start of run
After starting the run, you may see the error messages displayed below. When encountered, make the indicated selections and proceed with the run. Encountering either or both of these error messages is not indicative of a problem with the NGS workstation or your run setup.
1 If you encounter the G-axis error message shown below, select Ignore and Continue, leaving device in current state.
ed Library Prep and Capture System 21
2 Using the Agilent NGS Workstation for SureSelect Target Enrichment VWorks Automation Control Software
22
2 If you encounter the W-axis error message shown below, select Retry.
SureSelect Automated Library Prep and Capture System
Using the Agilent NGS Workstation for SureSelect Target Enrichment 2 VWorks Automation Control Software
SureSelect Automat
Verifying the Simulation setting
VWorks software may be run in simulation mode, during which commands entered on screen are not completed by the NGS workstation. If workstation devices do not respond when you start a run, verify the simulation mode status in VWorks using the following steps.
1 Verify that Simulation is off is displayed on the status indicator (accessible by clicking View > Control Toolbar).
2 If the indicator displays Simulation is on, click the status indicator button to turn off the simulation mode.
If you cannot see the toolbar above the SureSelect_XT_SOLiD_5500 VWorks form, click Full Screen on/off to exit full screen mode. If the toolbar is still not visible, right-click on the form and then select Control Toolbar from the menu.
NOTE
Finishing a protocol or runset
The window below appears when each run is complete. Click Yes to release the BenchCel racks to allow removal of components used in the current run in preparation for the next .pro or .rst run.
ed Library Prep and Capture System 23
2 Using the Agilent NGS Workstation for SureSelect Target Enrichment Overview of the SureSelect Target Enrichment Procedure
Overview of the SureSelect Target Enrichment Procedure
24
Figure 2 summarizes the SureSelect target enrichment workflow. For each sample to be sequenced, individual library preparations, hybridizations, and captures are performed. The samples are then tagged by PCR with an barcode sequence. Depending on the target size of the SureSelect capture, up to 96 samples can be pooled and sequenced in a single lane.
Table 6 summarizes how the VWorks protocols are integrated into the SureSelect workflow. See Sample Preparation, Hybridization, and Barcoding chapters for complete instructions for use of the VWorks protocols for sample processing.
SureSelect Automated Library Prep and Capture System
Using the Agilent NGS Workstation for SureSelect Target Enrichment 2 Overview of the SureSelect Target Enrichment Procedure
SureSelect Automat
Figure 2 Overall sequencing sample preparation workflow.
ed Library Prep and Capture System 25
2 Using the Agilent NGS Workstation for SureSelect Target Enrichment Overview of the SureSelect Target Enrichment Procedure
Table 6 Overview of VWorks protocols and runsets used during the workflow
Workflow Step (Protocol Chapter)
Substep VWorks Protocols Used for Agilent NGS Workstation automation
Sample Preparation Size-select and purify sheared DNA using AMPure XP beads
AMPureXP_XT_SOLiD_5500_DoubleSPRI_v1.0.pro
Prepare adaptor-ligated DNA LibraryPrep_XT_SOLiD_5500_v1.0.rst
Amplify adaptor-ligated DNA Pre-CapturePCR_XT_SOLiD_5500_v1.0.pro
Purify DNA using AMPure XP beads
AMPureXP_XT_SOLiD_5500_v1.5.pro
Hybridization Aliquot 500 ng of prepped libraries for hybridization
Aliquot_Libraries_v1.0.pro
Hybridize prepped DNA to Capture Library
Hybridization_v1.0.pro
Capture and wash DNA hybrids SureSelectCapture&Wash_v1.5.rst
Barcoding Add barcode tags by PCR Post-CaptureBarcoding_XT_SOLiD_5500_v1.5.pro
Purify DNA using AMPure XP beads
AMPureXP_XT_SOLiD_5500_v1.5.pro
26 SureSelect Automated Library Prep and Capture System
Using the Agilent NGS Workstation for SureSelect Target Enrichment 2 Experimental Setup Considerations for Automated Runs
Experimental Setup Considerations for Automated Runs
SureSelect Automat
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 SOLiD 5500 platform. Plan your experiments using complete columns of samples.
The number of columns or samples that may be processed using the supplied reagents (see Table 1) will depend on the experimental design. For greatest efficiency of reagent use, plan experiments using at least 3 columns per run. Each 96-reaction kit contains sufficient reagents for 96 reactions configured as 4 runs of 3 columns of samples per run.
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
ed Library Prep and Capture System 27
2 Using the Agilent NGS Workstation for SureSelect Target Enrichment Considerations for Placement of gDNA Samples in 96-well Plates for Automated Processing
Considerations for Placement of gDNA Samples in 96-well Plates for Automated Processing
28
• The Agilent NGS Workstation processes samples column-wise beginning at column 1. gDNA samples should be loaded into 96-well plates column-wise, in well order A1 to H1, then A2 to H2, ending with A12 to H12. When processing partial runs with <12 sample columns, do not leave empty columns between sample columns; always load the plate using the left-most column that is available.
• At the hybridization step (see Figure 2), you can add a different SureSelect Capture Library to each row of the plate. Plan your experiment such that each prepared DNA library corresponds to the appropriate SureSelect Capture Library.
• For sample barcoding after hybridization to the SureSelect library (see Figure 2), you will need to prepare a separate plate containing the barcoding primers. Assign the wells to be barcoded with the appropriate primers during experimental design.
• For post-capture amplification (see Figure 2), different SureSelect 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 55 on page 113 to determine which Capture Libraries may be amplified on the same plate.
Considerations for Equipment Setup
• Some workflow steps require the rapid transfer of sample plates between the Bravo deck and a thermal cycler. Locate your thermal cycler in close proximity to the Agilent NGS Workstation to allow rapid and efficient plate transfer.
• Several workflow steps require that the sample plate be sealed using the PlateLoc thermal microplate sealer on the Agilent NGS Workstation, and then centrifuged to collect any dispersed liquid. To maximize efficiency, locate the centrifuge in close proximity to the Agilent NGS Workstation.
SureSelect Automated Library Prep and Capture System
SureSelect Automated Library Prep and Capture System Protocol
3Sample Preparation
Step 1. Shear DNA 30
Step 2. Size-select and purify sheared DNA using AMPure XP beads 33
Step 3. Assess sheared DNA fragment size 39
Step 4. Modify DNA ends for target enrichment 42
Step 5. Amplify adaptor-ligated libraries 52
Step 6. Purify amplified DNA using AMPure XP beads 61
Step 7. Assess prepared library DNA quantity and quality 66
This section contains instructions for gDNA library preparation specific to the Life Technologies SOLiD 5500 sequencing platform and to automated processing using the Agilent NGS Workstation.
29Agilent Technologies
3 Sample Preparation Step 1. Shear DNA
Step 1. Shear DNA
30
For each DNA sample to be sequenced, prepare 1 library.
1 Use the Qubit dsDNA BR Assay to determine the concentration of your gDNA sample. Make sure the gDNA is of high quality (non-degraded, A260/A280 is 1.8 to 2.0).
Follow the instructions for the instrument.
2 Dilute 3 µg of high-quality gDNA with 1X Low TE Buffer in a 1.5-mL LoBind tube to a total volume of 130 µL.
3 Set up the Covaris E-Series or S-Series instrument.
a Check that the water in the Covaris tank is filled with fresh deionized water to the appropriate fill line level according to the manufacturer’s recommendations for the specific instrument model and sample tube or plate in use.
b Check that the water covers the visible glass part of the tube.
c On the instrument control panel, push the Degas button. Degas the instrument for at least 30 minutes, or according to the manufacturer’s recommendations.
d Set the chiller temperature to between 2°C to 5°C to ensure that the temperature reading in the water bath displays 5°C.
e Optional. Supplement the circulated water chiller with ethylene glycol to 20% volume to prevent freezing.
Refer to the Covaris instrument user guide for more details.
4 Put a Covaris microTube into the loading and unloading station.
Keep the cap on the tube.
When using a Covaris E-series instrument to prepare multiple gDNA samples in the same experiment, you can also use the 96 microTube plate (see Table 4 on page 11) for the DNA shearing step.
NOTE
5 Use a tapered pipette tip to slowly transfer the 130 µL DNA sample through the pre-split septa.
Be careful not to introduce a bubble into the bottom of the tube.
6 Secure the microTube in the tube holder and shear the DNA with the settings in Table 8 or Table 9, depending on the Covaris instrument SonoLab software version used.
SureSelect Automated Library Prep and Capture System
Sample Preparation 3 Step 1. Shear DNA
SureSelect Automat
The target sheared DNA fragment size is 150 bp.
7 Put the Covaris microTube back into the loading and unloading station.
8 While keeping the snap-cap on, insert a pipette tip through the pre-split septa, then slowly remove the sheared DNA.
Table 8 Shear settings for Covaris instruments using SonoLab software version 7 or newer
Setting Value
Duty Factor 10%
Peak Incident Power (PIP) 175
Cycles per Burst 100
Treatment Time 360 seconds
Bath Temperature 4° to 8° C
Table 9 Shear settings for Covaris instruments using SonoLab software prior to version 7
Setting Value
Duty Cycle 10%
Intensity 5
Cycles per Burst 100
Time 6 cycles of 60 seconds each
Set Mode Frequency sweeping
Temperature 4° to 7° C
ed Library Prep and Capture System 31
3 Sample Preparation Step 1. Shear DNA
32
9 Transfer the sheared DNA into the wells of a 96-well Eppendorf plate, column-wise for processing on the Agilent NGS Workstation, in well order A1 to H1, then A2 to H2, ending with A12 to H12.
SureSelect Automated Library Prep and Capture System runs may include 1, 2, 3, 4, 6, or 12 columns of the plate. See Using the Agilent NGS Workstation for SureSelect Target Enrichment for additional sample placement considerations.
NOTE
10 Seal the plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165°C and 1.0 sec.
11 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to remove air bubbles.
Stopping Point
If you do not continue to the next step, store the sample plate at 4°C overnight or at –20°C for prolonged storage.SureSelect Automated Library Prep and Capture System
Sample Preparation 3 Step 2. Size-select and purify sheared DNA using AMPure XP beads
Step 2. Size-select and purify sheared DNA using AMPure XP beads
SureSelect Automat
In this step, the Agilent NGS Workstation runs a double-SPRI bead purification protocol to size-select and purify the sheared DNA using AMPure XP beads.
Prepare the workstation and reagents
1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes.
2 Gently wipe down the Labware MiniHub, Bravo deck, and BenchCel with a NucleoClean decontamination wipe.
3 Let the AMPure XP beads come to room temperature for at least 30 minutes.
4 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color. Do not freeze.
5 Prepare a Nunc DeepWell source plate for the beads by adding 240 µL of homogenous AMPure XP beads per well, for each well to be processed.
6 Prepare a Thermo Scientific reservoir containing 15 mL of nuclease-free water.
7 Prepare a separate Thermo Scientific reservoir containing 45 mL of freshly-prepared 70% ethanol.
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3 Sample Preparation Step 2. Size-select and purify sheared DNA using AMPure XP beads
34
8 Load the Labware MiniHub according to Table 10, using the plate orientations shown in Figure 3.
Table 10 Initial MiniHub configuration for AMPureXP_XT_SOLiD_5500_DoubleSPRI_v1.0.pro
Vertical Shelf Position
Cassette 1 Cassette 2 Cassette 3 Cassette 4
Shelf 5 (Top) Empty Nunc DeepWell plate
Empty Nunc DeepWell plate
Empty Empty
Shelf 4 Empty Empty Empty Empty
Shelf 3 Empty Empty Eppendorf Plate
Empty Empty
Shelf 2 Empty Nuclease-free water reservoir from step 6
AMPure XP beads in Nunc DeepWell plate from step 5
Empty
Shelf 1 (Bottom) Empty 70% ethanol reservoir from step 7
Empty Empty
SureSelect Automated Library Prep and Capture System
Sample Preparation 3 Step 2. Size-select and purify sheared DNA using AMPure XP beads
SureSelect Automat
Figure 3 Agilent Labware MiniHub plate orientation. For Thermo Scientific reservoirs, place the notched corner facing the center of the hub.
9 Load the BenchCel Microplate Handling Workstation according to Table 11.
Table 11 Initial BenchCel configuration for AMPureXP_XT_SOLiD_5500_DoubleSPRI_v1.0.pro
No. of Columns Processed
Rack 1 Rack 2 Rack 3 Rack 4
1 1 Tip box Empty Empty Empty
2 2 Tip boxes Empty Empty Empty
3 2 Tip boxes Empty Empty Empty
4 3 Tip boxes Empty Empty Empty
6 4 Tip boxes Empty Empty Empty
12 7 Tip boxes Empty Empty Empty
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3 Sample Preparation Step 2. Size-select and purify sheared DNA using AMPure XP beads
36
10 Load the Bravo deck according to Table 12.
Table 12 Initial Bravo deck configuration for AMPureXP_XT_SOLiD_5500_DoubleSPRI_v1.0.pro
Run VWorks protocol AMPureXP_XT_SOLiD_5500_DoubleSPRI_v1.0.pro
11 Open the SureSelect setup form using the SureSelect_XT_SOLiD_5500.VWForm shortcut on your desktop.
12 Log in to the VWorks software.
Location Content
1 Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)
8 Empty tip box
9 Eppendorf plate containing sheared gDNA samples, oriented with well A1 in the upper-left
SureSelect Automated Library Prep and Capture System
Sample Preparation 3 Step 2. Size-select and purify sheared DNA using AMPure XP beads
SureSelect Automat
13 On the setup form, under Select Protocol to Run, select AMPureXP_XT_SOLiD_5500_DoubleSPRI_v1.0.pro.
14 Under Select additional Parameters, select the step Size Selection (Double SPRI) and the plate type 96 Eppendorf Twin.tec PCR.
Several different AMPureXP bead purification protocol variants are available in the VWorks menu for use during different specific steps of the SureSelect automation workflow. Be sure to select the correct protocol name, workflow step, and plate type when initiating the automation protocol.
The AMPureXP_XT_SOLiD_5500_DoubleSPRI_v1.0.pro protocol is used only for size-selection and purification of sheared DNA prior to the Library Prep protocol.
NOTE
ed Library Prep and Capture System 37
3 Sample Preparation Step 2. Size-select and purify sheared DNA using AMPure XP beads
38
15 Select the number of columns of samples to be processed. Runs must include 1, 2, 3, 4, 6, or 12 columns.
16 Click Display Initial Workstation Setup.
17 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form.
18 When verification is complete, click Run Selected Protocol.
NOTE If workstation devices do not respond when you start the run, but activity is recorded in the Log, verify that VWorks is not running in Simulation mode. See page 23 for more information.
Running the AMPureXP_XT_SOLiD_5500_DoubleSPRI_v1.0.pro protocol takes approximately one hour. Once complete, the purified DNA samples are located in the Eppendorf plate at position 7 of the Bravo deck.
SureSelect Automated Library Prep and Capture System
Sample Preparation 3 Step 3. Assess sheared DNA fragment size
Step 3. Assess sheared DNA fragment size
SureSelect Automat
Option 1: Analysis using the 2100 Bioanalyzer System and DNA 1000 Assay
Use a Bioanalyzer DNA 1000 chip and reagent kit. For more information to do this step, see the Agilent DNA 1000 Kit Guide.
1 Set up the 2100 Bioanalyzer system 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 instrument and start the run within five minutes after preparation.
6 Verify that the electropherogram shows an average DNA fragment size of approximately 150 bp. A sample electropherogram is shown in Figure 4.
Stopping Point
If you do not continue to the next step, seal the plate and store at 4°C overnight or at –20°C for prolonged storage.Figure 4 Analysis of sheared DNA using a DNA 1000 Bioanalyzer assay.
ed Library Prep and Capture System 39
3 Sample Preparation Step 3. Assess sheared DNA fragment size
40
Option 2: Analysis using the Agilent 2200 TapeStation and D1K ScreenTape
You can use Agilent’s 2200 TapeStation for rapid analysis of multiple samples. Use a D1K ScreenTape (p/n 5067-5361) and associated reagent kit (p/n 5067-5362) to analyze the sheared DNA. For more information to do this step, see the Agilent 2200 TapeStation User Manual.
1 Seal the sheared DNA sample plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165°C and 1.0 sec.
2 Vortex the plate to mix samples in each well, then centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal.
3 Prepare the TapeStation samples as instructed in the Agilent 2200 TapeStation User Manual. Use 1 µL of each sheared DNA sample diluted with 3 µL of D1K sample buffer for the analysis.
CAUTION Make sure that you thoroughly mix the combined DNA and D1K sample buffer on a vortex mixer for 5 seconds for accurate quantitation.
4 Load the sample plate or tube strips from step 3, the D1K ScreenTape, and loading tips into the 2200 TapeStation as instructed in the Agilent 2200 TapeStation User Manual. Start the run.
5 Verify that the electropherogram shows an average DNA fragment size of approximately 150 bp. A sample electropherogram is shown in Figure 5.
Stopping Point
If you do not continue to the next step, seal the sheared DNA sample plate and store at 4°C overnight or at –20°C for prolonged storage.SureSelect Automated Library Prep and Capture System
Sample Preparation 3 Step 3. Assess sheared DNA fragment size
SureSelect Automat
Figure 5 Analysis of sheared DNA using the 2200 TapeStation with a D1K ScreenTape.
ed Library Prep and Capture System 41
3 Sample Preparation Step 4. Modify DNA ends for target enrichment
Step 4. Modify DNA ends for target enrichment
42
In this step, the Agilent NGS Workstation completes the DNA end modification steps required for SureSelect target enrichment, including SOLiD end-repair, A-tailing, and adaptor ligation. After each modification step, the Agilent NGS Workstation purifies the prepared DNA using AMPure XP beads.
Before starting the run, you need to prepare master mixes (with overage) for each step, without the cDNA sample. Master mixes for runs that include 1, 2, 3, 4, 6, and 12 columns (including overage) are shown in each table.
Prepare each master mix on ice.
Prepare the workstation
1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes.
2 Pre-set the temperature of Bravo deck position 4 to 20°C using the Inheco Multi TEC control touchscreen, as described in Setting the Temperature of Bravo Deck Heat Blocks. Bravo deck position 4 corresponds to CPAC 2, position 1 on the Multi TEC control touchscreen.
3 Pre-set the temperature of Bravo deck position 6 to 4°C using the Inheco Multi TEC control touchscreen. Bravo deck position 6 corresponds to CPAC 2, position 2 on the Multi TEC control touchscreen.
4 Turn on the chiller, set to 0°C, at position 9 of the Bravo deck. Be sure that the chiller reservoir contains at least 300 mL of 25% ethanol.
SureSelect Automated Library Prep and Capture System
Sample Preparation 3 Step 4. Modify DNA ends for target enrichment
SureSelect Automat
Prepare the SureSelect DNA end-repair master mix
5 Prepare the appropriate volume of end-repair master mix, according to Table 13. Mix well using a vortex mixer and keep on ice.
Table 13 Preparation of End-Repair Master Mix
SureSelect XT Reagent
Volume for 1 Library
Volume for 1 Column
Volume for 2 Columns
Volume for 3 Columns
Volume for 4 Columns
Volume for 6 Columns
Volume for 12 Columns
Nuclease-free water
35.2 µL 448.8 µL 748 µL 1047 µL 1346 µL 1945 µL 3890 µL
10X End-Repair Buffer
10.0 µL 127.5 µL 212.5 µL 297.5 µL 382.5 µL 552.5 µL 1105 µL
dNTP mix 1.6 µL 20.4 µL 34.0 µL 47.6 µL 61.2 µL 88.4 µL 176.8 µL
T4 DNA polymerase
1.0 µL 12.8 µL 21.3 µL 29.8 µL 38.3 µL 55.3 µL 110.5 µL
Klenow DNA polymerase
2.0 µL 25.5 µL 42.5 µL 59.5 µL 76.5 µL 110.5 µL 221 µL
T4 Polynucleotide Kinase
2.2 µL 28.1 µL 46.8 µL 65.5 µL 84.2 µL 121.6 µL 243.1 µL
Total Volume 52 µL 663.1 µL 1105.1 µL 1546.9 µL 1988.7 µL 2873.3 µL 5746.4 µL
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3 Sample Preparation Step 4. Modify DNA ends for target enrichment
44
Prepare the A-tailing master mix
6 Prepare the appropriate volume of A-tailing master mix, according to Table 14. Mix well using a vortex mixer and keep on ice.
Table 14 Preparation of A-Tailing Master Mix
SureSelectXT Reagent
Volume for 1 Library
Volume for 1 Column
Volume for 2 Columns
Volume for 3 Columns
Volume for 4 Columns
Volume for 6 Columns
Volume for 12 Columns
Nuclease-free water
11.0 µL 187.0 µL 280.5 µL 374.0 µL 467.5 µL 654.5 µL 1262 µL
10x Klenow DNA Polymerase Buffer
5.0 µL 85.0 µL 127.5 µL 170.0 µL 212.5 µL 297.5 µL 573.8 µL
dATP 1.0 µL 17.0 µL 25.5 µL 34.0 µL 42.5 µL 59.5 µL 114.8 µL
Exo (-) Klenow DNA Polymerase
3.0 µL 51.0 µL 76.5 µL 102.0 µL 127.5 µL 178.5 µL 344.3 µL
Total Volume 20 µL 340 µL 510 µL 680 µL 850 µL 1190 µL 2294.9 µL
SureSelect Automated Library Prep and Capture System
Sample Preparation 3 Step 4. Modify DNA ends for target enrichment
SureSelect Automat
Prepare the adaptor ligation master mix
7 Prepare the appropriate volume of adaptor ligation master mix, according to Table 15. Mix well using a vortex mixer and keep on ice.
Table 15 Preparation of Adaptor Ligation Master Mix
SureSelect XT Reagent
Volume for 1 Library
Volume for 1 Column
Volume for 2 Columns
Volume for 3 Columns
Volume for 4 Columns
Volume for 6 Columns
Volume for 12 Columns
Nuclease-free water
16.5 µL 210.4 µL 351 µL 491 µL 631 µL 912 µL 1823 µL
5X T4 DNA Ligase Buffer
10.0 µL 127.5 µL 212.5 µL 297.5 µL 382.5 µL 552.5 µL 1105 µL
SureSelect LTI5500 P1 primer
4.5 µL 57.4 µL 95.6 µL 133.9 µL 172.1 µL 248.6 µL 497.3 µL
SureSelect LTI5500 IA primer
4.5 µL 57.4 µL 95.6 µL 133.9 µL 172.1 µL 248.6 µL 497.3 µL
T4 DNA Ligase 1.5 µL 19.1 µL 31.9 µL 44.6 µL 57.4 µL 82.9 µL 165.8 µL
Total Volume 37.0 µL 471.8 µL 786.6 µL 1100.9 µL 1415.1 µL 2044.6 µL 4088.4 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 steps 3 to 5. Add the volumes indicated in Table 16 of each master mix to all wells of the indicated column of the Nunc DeepWell plate. Keep the master mixes on ice
ed Library Prep and Capture System 45
3 Sample Preparation Step 4. Modify DNA ends for target enrichment
46
during the aliquoting steps. The final configuration of the master mix source plate is shown in Figure 6.
Table 16 Preparation of the Master Mix Source Plate for LibraryPrep_XT_SOLiD_5500_v1.0.rst
Master Mix Solution
Position on Source Plate
Volume of Master Mix added per Well of Nunc Deep Well Source Plate
1-Column Runs
2-Column Runs
3-Column Runs
4-Column Runs
6-Column Runs
12-Column Runs
End Repair Master Mix
Column 1
(A1-H1)
76.4 µL 131.6 µL 186.9 µL 242.1 µL 352.6 µL 711.8 µL
A-Tailing Master Mix
Column 2
(A2-H2)
40 µL 61.3 µL 82.5 µL 103.8 µL 146.3 µL 284.4 µL
Adaptor Ligation Master Mix
Column 3
(A3-H3)
54.3 µL 93.7 µL 133 µL 172.3 µL 250.9 µL 506.4 µL
SureSelect Automated Library Prep and Capture System
Sample Preparation 3 Step 4. Modify DNA ends for target enrichment
SureSelect Automat
Figure 6 Configuration of the master mix source plate for LibraryPrep_XT_SOLiD_5500_v1.0.rst
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 source plate on ice.
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.
NOTE
ed Library Prep and Capture System 47
3 Sample Preparation Step 4. Modify DNA ends for target enrichment
48
Prepare the purification reagents
11 Verify that the AMPure XP bead suspension is at room temperature.
12 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color. Do not freeze.
13 Prepare a separate Nunc DeepWell source plate for the beads by adding 370 µL of homogenous AMPure XP beads per well, for each well to be processed.
14 Prepare a Thermo Scientific reservoir containing 20 mL of nuclease-free water.
15 Prepare a separate Thermo Scientific reservoir containing 150 mL of freshly-prepared 70% ethanol.
Load the Agilent NGS Workstation
16 Load the Labware MiniHub according to Table 17, using the plate orientations shown in Figure 3.
Table 17 Initial MiniHub configuration for LibraryPrep_XT_SOLiD_5500_v1.0.rst
Vertical Shelf Position
Cassette 1 Cassette 2 Cassette 3 Cassette 4
Shelf 5 (Top) Empty Nunc DeepWell plate
Empty Nunc DeepWell plate
Empty Nunc DeepWell plate
Empty
Shelf 4 Empty Empty Eppendorf plate
Empty Eppendorf plate
Empty
Shelf 3 Empty Empty Empty Empty Eppendorf plate
Shelf 2 Empty tip box Nuclease-free water reservoir from step 14
AMPure XP beads in Nunc DeepWell plate from step 13
Empty
Shelf 1 (Bottom) New tip box 70% ethanol reservoir from step 15
Empty Empty tip box
SureSelect Automated Library Prep and Capture System
Sample Preparation 3 Step 4. Modify DNA ends for target enrichment
SureSelect Automat
17 Load the BenchCel Microplate Handling Workstation according to Table 18.
Table 18 Initial BenchCel configuration for LibraryPrep_XT_SOLiD_5500_v1.0.rst
18 Load the Bravo deck according to Table 19.
Table 19 Initial Bravo deck configuration for LibraryPrep_XT_SOLiD_5500_v1.0.rst
No. of Columns Processed
Rack 1 Rack 2 Rack 3 Rack 4
1 2 Tip boxes Empty Empty Empty
2 4 Tip boxes Empty Empty Empty
3 5 Tip boxes Empty Empty Empty
4 7 Tip boxes Empty Empty Empty
6 10 Tip boxes Empty Empty Empty
12 11 Tip boxes 8 Tip boxes Empty Empty
Location Content
1 Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)
6 Empty Eppendorf plate, oriented with well A1 in the upper-left
7 Size-slected gDNA samples, in Eppendorf plate oriented with well A1 in the upper-left
9 DNA End Modification Master Mix Source Plate seated on silver insert
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3 Sample Preparation Step 4. Modify DNA ends for target enrichment
50
Run VWorks runset LibraryPrep_XT_SOLiD_5500_v1.0.rst
19 On the SureSelect setup form, under Select Protocol to Run, select LibraryPrep_XT_SOLiD_5500_v1.0.rst.
20 Select the number of columns of samples to be processed. Runs must include 1, 2, 3, 4, 6, or 12 columns.
21 Click Display Initial Workstation Setup.
SureSelect Automated Library Prep and Capture System
Sample Preparation 3 Step 4. Modify DNA ends for target enrichment
SureSelect Automat
22 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form.
23 When verification is complete, click Run Selected Protocol.
24 When ready to begin the run, click OK in the following window.
Running the LibraryPrep_XT_SOLiD_5500_v1.0.rst runset takes approximately 4 hours. Once complete, the purified, adaptor-ligated DNA samples are located in the Eppendorf plate at position 7 of the Bravo deck.
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.ed Library Prep and Capture System 51
3 Sample Preparation Step 5. Amplify adaptor-ligated libraries
Step 5. Amplify adaptor-ligated libraries
52
In this step, the Agilent NGS Workstation completes the liquid handling steps for amplification of the adaptor-ligated DNA samples. Afterward, you transfer the PCR plate to a thermal cycler for amplification.
In this protocol, one half of the adaptor-ligated DNA sample is removed from the Eppendorf sample plate for amplification. The remainder can be saved at 4°C for future use or amplification troubleshooting, if needed. Store the samples at –20°C for long-term storage.
CAUTION To avoid cross-contaminating libraries, set up PCR master mixes in a dedicated clean area or PCR hood with UV sterilization and positive air flow.
Prepare the workstation
1 Turn on the chiller, set to 0°C, at position 9 of the Bravo deck. Be sure that the chiller reservoir contains at least 300 mL of 25% ethanol.
2 Leave tip boxes on shelves 1 and 2 in casette 1 of the Labware MiniHub from the previous LibraryPrep_XT_SOLiD_5500_v1.0.rst run. Otherwise, clear the remaining positions of the MiniHub and BenchCel of plates and tip boxes.
3 Pre-set the temperature of Bravo deck position 6 to 4°C using the Inheco Multi TEC control touchscreen, as described in Setting the Temperature of Bravo Deck Heat Blocks. Bravo deck position 6 corresponds to CPAC 2, position 2 on the Multi TEC control touchscreen.
SureSelect Automated Library Prep and Capture System
Sample Preparation 3 Step 5. Amplify adaptor-ligated libraries
SureSelect Automat
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 20. Mix well using a vortex mixer and keep on ice.
Table 20 Preparation of Pre-Capture PCR Master Mix
SureSelectXT Reagent
Volume for 1 Library
Volume for 1 Column
Volume for 2 Columns
Volume for 3 Columns
Volume for 4 Columns
Volume for 6 Columns
Volume for 12 Columns
Nuclease-free water 20.5 µL 261.4 µL 436 µL 610 µL 784 µL 1133 µL 2265 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
SureSelect LTI Precapture Primer
4.0 µL 51.0 µL 85.0 µL 119.0 µL 153.0 µL 221 µL 442 µL
dNTP mix* 0.5 µL 6.4 µL 10.6 µL 14.9 µL 19.1 µL 27.6 µL 55.2 µL
Herculase II Fusion DNA Polymerase
1.0 µL 12.8 µL 21.3 µL 29.8 µL 38.3 µL 55.2 µL 110.5 µL
Total Volume 36.0 µL 459.1 µL 765.4 µL 1071.2 µL 1376.9 µL 1989.3 µL 3977.7 µL
* Included with the Herculase II Fusion DNA Polymerase. Do not use the buffer or dNTP mix from any other kit.
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3 Sample Preparation Step 5. Amplify adaptor-ligated libraries
54
5 Using the same Nunc DeepWell master mix source plate that was used for the LibraryPrep_XT_SOLiD_5500_v1.0.rst run, add the volume of PCR Master Mix indicated in Table 21 to all wells of column 4 of the master mix source plate. The final configuration of the master mix source plate is shown in Figure 7.
Table 21 Preparation of the Master Mix Source Plate for Pre-CapturePCR_XT_SOLiD_5500_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 4
(A4-H4)
52.9 µL 91.1 µL 129.4 µL 167.6 µL 244.1 µL 493 µL
NOTE If you are using a new DeepWell plate for the pre-capture PCR source plate (for example, when amplifying the second half of the adaptor-ligated DNA sample), leave columns 1 to 3 empty and add the PCR Master Mix to column 4 of the new plate.
SureSelect Automated Library Prep and Capture System
Sample Preparation 3 Step 5. Amplify adaptor-ligated libraries
SureSelect Automat
Figure 7 Configuration of the master mix source plate for Pre-CapturePCR_XT_SOLiD_5500_v1.0.pro. Columns 1-3 were used to dis-pense master mixes during the previous protocol.
6 Seal the master mix source plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165°C and 1.0 sec.
7 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate any bubbles.
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.
NOTE
ed Library Prep and Capture System 55
3 Sample Preparation Step 5. Amplify adaptor-ligated libraries
56
Load the Agilent NGS Workstation
8 Load the Labware MiniHub according to Table 22, using the plate orientations shown in Figure 3.
Table 22 Initial MiniHub configuration for Pre-CapturePCR_XT_SOLiD_5500_v1.0.pro
Vertical Shelf Position
Cassette 1 Cassette 2 Cassette 3 Cassette 4
Shelf 5 (Top)
Empty Empty Empty Empty ABI MicroAmp plate seated in black adapter
Shelf 4 Empty Empty Empty Empty
Shelf 3 Empty Empty Empty Empty
Shelf 2 Waste tip box*
* The waste tip box (Cassette 1, Shelf 2) and clean tip box (Cassette 1, Shelf 1) are retained from the LibraryPrep_XT_SOLiD_5500_v1.0.rst run and reused here.
Empty Empty Empty
Shelf 1 (Bottom)
Clean tip box* Empty Empty Empty tip box
NOTE If you are using a new box of tips on shelf 1 of cassette 1 (for example, when amplifying the second half of the adaptor-ligated DNA sample), first remove the tips from columns 1 to 3 of the tip box. Any tips present in columns 1 to 3 of the tip box may be inappropriately loaded onto the Bravo platform pipette heads and may interfere with automated processing steps.
SureSelect Automated Library Prep and Capture System
Sample Preparation 3 Step 5. Amplify adaptor-ligated libraries
SureSelect Automat
9 Load the BenchCel Microplate Handling Workstation according to Table 23.
Table 23 Initial BenchCel configuration for Pre-CapturePCR_XT_SOLiD_5500_v1.0.pro
10 Load the Bravo deck according to Table 24.
Table 24 Initial Bravo deck configuration for Pre-CapturePCR_XT_SOLiD_5500_v1.0.pro
No. of Columns Processed
Rack 1 Rack 2 Rack 3 Rack 4
1 1 Tip box Empty Empty Empty
2 1 Tip box Empty Empty Empty
3 1 Tip box Empty Empty Empty
4 1 Tip box Empty Empty Empty
6 1 Tip box Empty Empty Empty
12 1 Tip box Empty Empty Empty
Location Content
7 Eppendorf plate containing adaptor-ligated DNA samples, oriented with well A1 in the upper-left
9 Master mix plate containing PCR Master Mix in Column 4 seated on silver insert
ed Library Prep and Capture System 57
3 Sample Preparation Step 5. Amplify adaptor-ligated libraries
58
Run VWorks protocol Pre-CapturePCR_XT_SOLiD_5500_v1.0.pro
11 On the SureSelect setup form, under Select Protocol to Run, select Pre-CapturePCR_XT_SOLiD_5500_v1.0.pro.
12 Select the number of columns of samples to be processed. Runs must include 1, 2, 3, 4, 6, or 12 columns.
13 Click Display Initial Workstation Setup.
SureSelect Automated Library Prep and Capture System
Sample Preparation 3 Step 5. Amplify adaptor-ligated libraries
SureSelect Automat
14 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form.
15 When verification is complete, click Run Selected Protocol.
Running the Pre-CapturePCR_XT_SOLiD_5500_v1.0.pro protocol takes approximately 15 minutes. Once complete, the PCR-ready samples, containing prepped DNA and PCR master mix, are located in the ABI MicroAmp plate at position 5 of the Bravo deck. The Eppendorf plate containing the remaining prepped DNA samples, which may be stored for future use at 4°C overnight, or at –20°C for long-term storage, is located at position 7 of the Bravo deck.
16 When you see the following prompt, remove the PCR plate from position 5 of the Bravo deck and seal the plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165°C and 3.0 seconds.
ed Library Prep and Capture System 59
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60
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 25.
Table 25 Pre-Capture PCR cycling program
Segment Number of Cycles
Temperature Time
1 1 98°C 2 minutes
2 4–6 98°C 30 seconds
54°C 10 seconds
72°C 60 seconds
3 1 72°C 10 minutes
4 1 4°C Hold
Different library preparations can produce slightly different results, based on varying DNA quality. In most cases, 5 cycles will produce an adequate yield for subsequent capture without introducing bias or non-specific products. If yield is too low or non-specific high molecular weight products are observed, adjust the number of cycles accordingly with the remaining library template.
NOTE
SureSelect Automated Library Prep and Capture System
Sample Preparation 3 Step 6. Purify amplified DNA using AMPure XP beads
Step 6. Purify amplified DNA using AMPure XP beads
SureSelect Automat
In this step, the Agilent NGS Workstation transfers AMPure XP beads and amplified adaptor-ligated DNA to a Nunc DeepWell plate and then collects and washes the bead-bound DNA.
Prepare the workstation and reagents
1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes.
2 Verify that the AMPure XP bead suspension is at room temperature. (If necessary, allow the bead solution to come to room temperature for at least 30 minutes.)
3 Pre-set the temperature of Bravo deck position 4 to 4°C using the Inheco Multi TEC control touchscreen, as described in Setting the Temperature of Bravo Deck Heat Blocks. Bravo deck position 4 corresponds to CPAC 2, position 1 on the Multi TEC control touchscreen.
4 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color. Do not freeze.
5 Prepare a Nunc DeepWell source plate for the beads by adding 95 µL of homogenous AMPure XP beads per well, for each well to be processed.
6 Prepare a Thermo Scientific reservoir containing 15 mL of nuclease-free water.
7 Prepare a separate Thermo Scientific reservoir containing 45 mL of freshly-prepared 70% ethanol.
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62
8 Load the Labware MiniHub according to Table 26, using the plate orientations shown in Figure 3.
Table 26 Initial MiniHub configuration for AMPureXP_XT_SOLiD_5500_v1.5.pro
9 Load the BenchCel Microplate Handling Workstation according to Table 27.
Table 27 Initial BenchCel configuration for AMPureXP_XT_SOLiD_5500_v1.5.pro
Vertical Shelf Position
Cassette 1 Cassette 2 Cassette 3 Cassette 4
Shelf 5 (Top) Empty Nunc DeepWell plate
Empty Empty Empty
Shelf 4 Empty Empty Empty Empty
Shelf 3 Empty Empty Eppendorf Plate
Empty Empty
Shelf 2 Empty Nuclease-free water reservoir from step 6
AMPure XP beads in Nunc DeepWell plate from step 5
Empty
Shelf 1 (Bottom) Empty 70% ethanol reservoir from step 7
Empty Empty
No. of Columns Processed
Rack 1 Rack 2 Rack 3 Rack 4
1 1 Tip box Empty Empty Empty
2 1 Tip box Empty Empty Empty
3 2 Tip boxes Empty Empty Empty
4 2 Tip boxes Empty Empty Empty
6 3 Tip boxes Empty Empty Empty
12 6 Tip boxes Empty Empty Empty
SureSelect Automated Library Prep and Capture System
Sample Preparation 3 Step 6. Purify amplified DNA using AMPure XP beads
SureSelect Automat
10 Load the Bravo deck according to Table 28.
Table 28 Initial Bravo deck configuration for AMPureXP_XT_SOLiD_5500_v1.5.pro
Run VWorks protocol AMPureXP_XT_SOLiD_5500_v1.5.pro
11 On the SureSelect setup form, under Select Protocol to Run, select AMPureXP_XT_SOLiD_5500_v1.5.pro.
Location Content
1 Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)
4 Amplified DNA libraries in ABI MicroAmp plate seated in black adapter
8 Empty tip box
ed Library Prep and Capture System 63
3 Sample Preparation Step 6. Purify amplified DNA using AMPure XP beads
64
12 Under Select additional Parameters, select the step Pre-Capture PCR Cleanup and the plate type 96 ABI PCR half skirt in black carrier.
AMPureXP bead purification protocols are used during multiple steps of the SureSelect automation workflow. Be sure to select the correct protocol name, workflow step, and plate type when initiating the automation protocol.
NOTE
13 Select the number of columns of samples to be processed. Runs must include 1, 2, 3, 4, 6, or 12 columns.
14 Click Display Initial Workstation Setup.
SureSelect Automated Library Prep and Capture System
Sample Preparation 3 Step 6. Purify amplified DNA using AMPure XP beads
SureSelect Automat
15 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form.
16 When verification is complete, click Run Selected Protocol.
The purification protocol takes approximately 45 minutes. When complete, the amplified DNA samples are in the Eppendorf plate located on Bravo deck position 7.
ed Library Prep and Capture System 65
3 Sample Preparation Step 7. Assess prepared library DNA quantity and quality
Step 7. Assess prepared library DNA quantity and quality
66
The hybridization protocol in the following section requires 500 ng of each amplified DNA library. Measure the concentration of each library using one of the methods detailed below. Once DNA concentration for each sample is determined, calculate the volume of the library to be used for hybridization using the following formula:
Volume (µL) = 500 ng/concentration (ng/µL)
Option 1: Analysis using the 2100 Bioanalyzer System and DNA 1000 Assay
Use a Bioanalyzer DNA 1000 chip and reagent kit. For more information to do this step, see the Agilent DNA 1000 Kit Guide.
1 Set up the 2100 Bioanalyzer system 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 instrument and start the run within five minutes after preparation.
6 Verify that the electropherogram shows an average DNA fragment size of approximately 200 bp. A sample electropherogram is shown in Figure 8.
7 Determine the concentration of the library (ng/µL) by integrating under the peak.
Stopping Point
If you do not continue to the next step, seal the plate and store at 4°C overnight or at –20°C for prolonged storage.SureSelect Automated Library Prep and Capture System
Sample Preparation 3 Step 7. Assess prepared library DNA quantity and quality
SureSelect Automat
Figure 8 Analysis of amplified prepped library DNA using a DNA 1000 assay.
ed Library Prep and Capture System 67
3 Sample Preparation Step 7. Assess prepared library DNA quantity and quality
68
Option 2: Analysis using the 2200 TapeStation and D1K ScreenTape
Use a D1K ScreenTape (p/n 5067-5361) and associated reagent kit (p/n 5067-5362) to analyze the amplified libraries. For more information to do this step, see the Agilent 2200 TapeStation User Manual.
1 Seal the DNA sample plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165°C and 1.0 sec.
2 Vortex the plate to mix samples in each well, then centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal.
3 Prepare the TapeStation samples as instructed in the Agilent 2200 TapeStation User Manual. Use 1 µL of each amplified library DNA sample diluted with 3 µL of D1K sample buffer for the analysis.
CAUTION Make sure that you thoroughly mix the combined DNA and D1K sample buffer on a vortex mixer for 5 seconds for accurate quantitation.
4 Load the sample plate or tube strips from step 3, the D1K ScreenTape, and loading tips into the 2200 TapeStation as instructed in the Agilent 2200 TapeStation User Manual. Start the run.
5 Verify that the electropherogram shows an average DNA fragment size of approximately 200 bp. A sample electropherogram is shown in Figure 9.
Stopping Point
If you do not continue to the next step, seal the library DNA sample plate and store at 4°C overnight or at –20°C for prolonged storage.SureSelect Automated Library Prep and Capture System
Sample Preparation 3 Step 7. Assess prepared library DNA quantity and quality
SureSelect Automat
Figure 9 Analysis of amplified library DNA using the 2200 TapeStation with a D1K ScreenTape.
ed Library Prep and Capture System 69
3 Sample Preparation Step 7. Assess prepared library DNA quantity and quality
70
SureSelect Automated Library Prep and Capture SystemSureSelect Automated Library Prep and Capture System Protocol
4Hybridization
Step 1. Aliquot prepped DNA samples for hybridization 72
Step 2. Hybridize the gDNA library and SureSelect Capture Library 76
Step 3. Capture the hybridized DNA 92
This chapter describes the steps to hybridize the prepped gDNA library with the SureSelect capture library and then capture the hybridized target DNA. Each DNA library sample must be hybridized and captured individually prior to addition of the barcode tag by PCR.
CAUTION The ratio of SureSelect capture library to prepped library is critical for successful capture.
CAUTION You must avoid evaporation from the small volumes of the hybridization reaction during the 24 hour or greater incubation.
If you want to use a duration of hybridization >24 hours, first test the conditions. Incubate 35 µL of SureSelect Hybridization Buffer (without DNA) at 65°C for 24 hours (or longer, if applicable) as a test. Include buffer in each well that you might use, including those in the center and those on the edges. Check that you do not get extensive evaporation. Evaporation should not exceed 3 to 4 µL.
71Agilent Technologies
4 Hybridization Step 1. Aliquot prepped DNA samples for hybridization
Step 1. Aliquot prepped DNA samples for hybridization
72
For each sample library prepared, do one hybridization and capture. Do not pool samples at this stage.
Each hybridization reaction will contain 500 ng of the prepped gDNA sample. Before starting the hybridization step, you must create a table containing instructions for the Agilent NGS Workstation indicating the volume of each sample required for a 500-ng aliquot.
1 Create a .csv (comma separated value) file with the headers shown in Figure 10. The header text must not contain spaces. The table may be created using a spreadsheet application, such as Microsoft Excel software, and then saved in .csv format. The file must include rows for all 96 wells of the plate.
2 Enter the information requested in the header for each DNA sample.
• In the SourceBC field, enter the sample plate description or barcode. The SourceBC field contents must be identical for all rows.
• In the SourceWell and DestinationWell fields, enter each well position for the plate. SourceWell and DestinationWell field contents must be identical for a given sample.
• In the Volume field, enter the volume (in µL) equivalent to 500 ng DNA for each sample. These values are determined from the concentration values obtained from Bioanalyzer or TapeStation system results in the previous section. For all empty wells on the plate, enter the value 0, as shown in Figure 10; do not delete rows for empty wells.
Figure 10 Sample spreadsheet for 500-ng sample aliquot for 1-column run.
SureSelect Automated Library Prep and Capture System
Hybridization 4 Step 1. Aliquot prepped DNA samples for hybridization
SureSelect Automat
NOTE You can find a sample spreadsheet in the directory C: > VWorks Workspace > NGS Option B > SureSelectXT_SOLiD5500 > Aliquot Library Input Files > 500ng_transfer_full_plate_template_xlsx.
The 500ng_transfer_full_plate_template.xlsx file may be copied and used as a template for creating the .csv files for each 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.
3 Load the .csv file onto the PC containing the VWorks software into a suitable folder, such as C: > VWorks Workspace > NGS Option B > SureSelectXT_SOLiD5500 > Aliquot Library Input Files.
4 Turn on the chiller, set to 0°C, at position 9 of the Bravo deck. Be sure that the chiller reservoir contains at least 300 mL of 25% ethanol.
5 Load the Bravo deck according to Table 29.
Table 29 Initial Bravo deck configuration for Aliquot_Libraries_v1.0.pro
Location Content
5 Empty ABI MicroAmp plate seated in black adapter (oriented with well A1 in the upper-left)
6 Empty tip box
8 New tip box
9 Prepped library DNA in Eppendorf plate (oriented with well A1 in the upper-left)
ed Library Prep and Capture System 73
4 Hybridization Step 1. Aliquot prepped DNA samples for hybridization
74
6 On the SureSelect setup form, under Select Protocol to Run, select Aliquot_Libraries_v1.0.pro.
7 Click Display Initial Workstation Setup.
8 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form.
9 When verification is complete, click Run Selected Protocol.
SureSelect Automated Library Prep and Capture System
Hybridization 4 Step 1. Aliquot prepped DNA samples for hybridization
SureSelect Automat
10 When prompted by the dialog below, browse to the .csv file created for the source plate of the current run, and then click OK to start the run.
The 500 ng aliquoting protocol takes approximately 1 hour for 96 samples. When complete, the 500-ng samples are in the ABI MicroAmp plate located on Bravo deck position 5.
11 Remove the 500-ng sample plate from the Bravo deck and use a vacuum concentrator to dry the sample at 45°C.
12 Reconstitute each dried sample with 3.4 µL of nuclease-free water to bring the final concentration to 147 ng/µL. Pipette up and down along the sides of each well for optimal recovery.
13 Seal the plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165°C and 1.0 sec.
14 Vortex the plate for 30 seconds to ensure complete reconstitution, then centrifuge the plate for 1 minute to drive the well contents off the walls and plate seal.
ed Library Prep and Capture System 75
4 Hybridization Step 2. Hybridize the gDNA library and SureSelect Capture Library
Step 2. Hybridize the gDNA library and SureSelect Capture Library
76
In this step, the Agilent NGS Workstation completes the liquid handling steps in preparation for hybridization of the prepared DNA samples to one or more SureSelect capture libraries. Afterward, you transfer the sample plate to a thermal cycler, held at 65°C, to allow hybridization of the DNA sample to the SureSelect capture library.
Prepare the workstation
1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes.
2 Gently wipe down the Labware MiniHub, Bravo decks, and BenchCel with a NucleoClean decontamination wipe.
3 Pre-set the temperature of Bravo deck position 4 to 85°C using the Inheco Multi TEC control touchscreen (see Setting the Temperature of Bravo Deck Heat Blocks). Bravo deck position 4 corresponds to CPAC 2, position 1 on the Multi TEC control touchscreen.
4 Turn on the ThermoCube and set to 25°C for position 9 of the Bravo deck.
5 Place the silver Nunc DeepWell plate insert on position 9 of the Bravo deck. This insert is required to facilitate heat transfer to DeepWell source plate wells during the Hybridization protocol.
SureSelect Automated Library Prep and Capture System
Hybridization 4 Step 2. Hybridize the gDNA library and SureSelect Capture Library
SureSelect Automat
Prepare the SureSelect Block master mix
6 Prepare the appropriate volume of SureSelect Block master mix, on ice, as indicated in Table 30.
Table 30 Preparation of SureSelect Block Master Mix
SureSelectXT Reagent
Volume for 1 Library
Volume for 1 Column
Volume for 2 Columns
Volume for 3 Columns
Volume for 4 Columns
Volume for 6 Columns
Volume for 12 Columns
Nuclease-free water
6.0 µL 76.5 µL 127.5 µL 178.5 µL 229.5 µL 331.5 µL 663.0 µL
SureSelect Indexing Block #1 (green cap)
2.5 µL 31.9 µL 53.1 µL 74.4 µL 95.6 µL 138.1 µL 276.3 µL
SureSelect Block #2 (blue cap)
2.5 µL 31.9 µL 53.1 µL 74.4 µL 95.6 µL 138.1 µL 276.3 µL
SureSelect LT Indexing Block #3 (brown cap)
0.6 µL 7.6 12.8 µL 17.8 µL 23.0 µL 33.2 µL 66.3 µL
Total Volume 11.6 µL 147.9 µL 246.5 µL 345.1 µL 443.7 µL 640.9 µL 1281.9 µL
ed Library Prep and Capture System 77
4 Hybridization Step 2. Hybridize the gDNA library and SureSelect Capture Library
78
Prepare one or more SureSelect Capture Library master mixes
7 Prepare the appropriate volume of SureSelect capture library master mix for each of the capture libraries that will be used for hybridization as indicated in Table 31 to Table 34. Mix the components by pipetting. Keep the master mixes on ice during preparation and aliquoting.
Each row of the prepped gDNA sample plate may be hybridized to a different SureSelect Capture Library. However, capture libraries of different sizes require different post-capture amplification cycles. Plan experiments such that similar-sized libraries are hybridized on the same plate.
For runs that use a single capture library for all rows of the plate, prepare the master mix as described in Step a (Table 31 or Table 32) below.
For runs that use different capture libraries for individual rows, prepare each master mix as described in Step b (Table 33 or Table 34) below.
NOTE
SureSelect Automated Library Prep and Capture System
Hybridization 4 Step 2. Hybridize the gDNA library and SureSelect Capture Library
SureSelect Automat
a For runs that use a single capture library for all rows, prepare the SureSelect Capture Library Master Mix as listed in Table 31 or Table 32, based on the Mb target size of your design.
Table 31 Preparation of Capture Library Master Mix for target sizes <3.0 Mb, 8 rows of wells
Table 32 Preparation of Capture Library Master Mix for target sizes >3.0 Mb, 8 rows of wells
Target size <3.0 Mb
SureSelectXT Reagent
Volume for 1 Library
Volume for 1 Column
Volume for 2 Columns
Volume for 3 Columns
Volume for 4 Columns
Volume for 6 Columns
Volume for 12 Columns
Nuclease-free water
4.5 µL 76.5 µL 114.8 µL 153.0 µL 191.3 µL 267.8 µL 516.4 µL
RNase Block (purple cap)
0.5 µL 8.5 µL 12.8 µL 17.0 µL 21.3 µL 29.8 µL 57.4 µL
SureSelect Capture Library
2.0 µL 34.0 µL 51.0 µL 68.0 µL 85.0 µL 119.0 µL 229.5 µL
Total Volume 7.0 µL 119.0 µL 178.6 µL 238.0 µL 297.6 µL 416.6 µL 803.3 µL
Target size >3.0 Mb
SureSelectXT Reagent
Volume for 1 Library
Volume for 1 Column
Volume for 2 Columns
Volume for 3 Columns
Volume for 4 Columns
Volume for 6 Columns
Volume for 12 Columns
Nuclease-free water
1.5 µL 25.5 µL 38.3 µL 51.0 µL 63.8 µL 89.3 µL 172.1 µL
RNase Block (purple cap)
0.5 µL 8.5 µL 12.8 µL 17.0 µL 21.3 µL 29.8 µL 57.4 µL
SureSelect Capture Library
5.0 µL 85.0 µL 127.5 µL 170.0 µL 212.5 µL 297.5 µL 573.8 µL
Total Volume 7.0 µL 119.0 µL 178.6 µL 238.0 µL 297.6 µL 416.6 µL 803.3 µL
ed Library Prep and Capture System 79
4 Hybridization Step 2. Hybridize the gDNA library and SureSelect Capture Library
80
b For runs that use different capture libraries in individual rows, prepare a SureSelect Capture Library Master Mix for each capture library as listed in Table 33 or Table 34, based on the Mb target size of your design. 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 target sizes <3.0 Mb, single row of wells
Table 34 Preparation of Capture Library Master Mix for target sizes >3.0 Mb, single row of wells
Target size <3.0 Mb
SureSelectXT Reagent
Volume for 1 Library
Volume for 1 Column
Volume for 2 Columns
Volume for 3 Columns
Volume for 4 Columns
Volume for 6 Columns
Volume for 12 Columns
Nuclease-free water
4.5 µL 9.3 µL 14.1 µL 18.8 µL 23.6 µL 33.2 µL 64.3 µL
RNase Block (purple cap)
0.5 µL 1.0 µL 1.6 µL 2.1 µL 2.6 µL 3.7 µL 7.1 µL
SureSelect Capture Library
2.0 µL 4.1 µL 6.3 µL 8.4 µL 10.5 µL 14.8 µL 28.6 µL
Total Volume 7.0 µL 14.4 µL 22.0 µL 29.3 µL 36.7 µL 51.7 µL 100 µL
Target size >3.0 Mb
SureSelectXT Reagent
Volume for 1 Library
Volume for 1 Column
Volume for 2 Columns
Volume for 3 Columns
Volume for 4 Columns
Volume for 6 Columns
Volume for 12 Columns
Nuclease-free water
1.5 µL 3.1 µL 4.7 µL 6.3 µL 7.9 µL 11.1 µL 21.4 µL
RNase Block (purple cap)
0.5 µL 1.0 µL 1.6 µL 2.1 µL 2.6 µL 3.7 µL 7.1 µL
SureSelect Capture Library
5.0 µL 10.3 µL 15.6 µL 20.9 µL 26.3 µL 36.9 µL 71.4 µL
Total Volume 7.0 µL 14.4 µL 21.9 µL 29.3 µL 36.8 µL 51.7 µL 99.9 µL
SureSelect Automated Library Prep and Capture System
Hybridization 4 Step 2. Hybridize the gDNA library and SureSelect Capture Library
SureSelect Automat
Prepare the Hybridization Buffer master mix
8 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
SureSelectXT Reagent
Volume for 1 Column
Volume for 2 Columns
Volume for 3 Columns
Volume for 4 Columns
Volume for 6 Columns
Volume for 12 Columns
SureSelect Hyb # 1 212.5 µL 212.5 µL 212.5 µL 425 µL 425 µL 850 µL
SureSelect Hyb # 2 (red cap)
8.5 µL 8.5 µL 8.5 µL 17 µL 17 µL 34 µL
SureSelect Hyb # 3 (yellow cap)
85 µL 85 µL 85 µL 170 µL 170 µL 340 µL
SureSelect Hyb # 4 110.5 µL 110.5 µL 110.5 µL 221 µL 221 µL 442 µL
Total Volume 416.5 µL 416.5 µL 416.5 µL 833 µL 833 µL 1666 µL
9 If precipitate forms, warm the hybridization buffer at 65°C for 5 minutes.
ed Library Prep and Capture System 81
4 Hybridization Step 2. Hybridize the gDNA library and SureSelect Capture Library
82
Prepare the master mix source plate
10 In a Nunc DeepWell plate, prepare the master mix source plate containing the master mixes prepared in steps 4 to 6 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 11.
Table 36 Preparation of the Master Mix Source Plate for Hybridization_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
(A1-H1)
17 µL 29.4 µL 41.7µL 54 µL 78.7 µL 158.8 µL
Capture Library Master Mix
Column 2
(A2-H2)
14 µL 21.4 µL 28.9 µL 36.3 µL 51.2 µL 99.5 µL
Hybridization Buffer Master Mix
Column 3
(A3-H3)
45.9 µL 45.9 µL 45.9 µL 98 µL 98 µL 202.1 µL
SureSelect Automated Library Prep and Capture System
Hybridization 4 Step 2. Hybridize the gDNA library and SureSelect Capture Library
SureSelect Automat
Figure 11 Configuration of the master mix source plate for Hybridization_v1.0.pro.
11 Seal the master mix source plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165°C and 1.0 sec.
12 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.
ed Library Prep and Capture System 83
4 Hybridization Step 2. Hybridize the gDNA library and SureSelect Capture Library
84
Load the Agilent NGS Workstation
13 Load the Labware MiniHub according to Table 37, using the plate orientations shown in Figure 3.
Table 37 Initial MiniHub configuration for Hybridization_v1.0.pro
14 Load the BenchCel Microplate Handling Workstation according to Table 38.
Table 38 Initial BenchCel configuration for Hybridization_v1.0.pro
Vertical Shelf Position
Cassette 1 Cassette 2 Cassette 3 Cassette 4
Shelf 5 (Top) Empty Empty Empty Empty
Shelf 4 Empty Empty Empty Empty
Shelf 3 Empty Empty Empty Empty
Shelf 2 Empty tip box Empty Empty Empty
Shelf 1 (Bottom) Empty Empty Empty Empty
No. of Columns Processed
Rack 1 Rack 2 Rack 3 Rack 4
1 2 Tip boxes Empty Empty Empty
2 2 Tip boxes Empty Empty Empty
3 2 Tip boxes Empty Empty Empty
4 3 Tip boxes Empty Empty Empty
6 3 Tip boxes Empty Empty Empty
12 5 Tip boxes Empty Empty Empty
SureSelect Automated Library Prep and Capture System
Hybridization 4 Step 2. Hybridize the gDNA library and SureSelect Capture Library
SureSelect Automat
15 Load the Bravo deck according to Table 39.
Table 39 Initial Bravo deck configuration for Hybridization_v1.0.pro
Location Content
5 500-ng aliquots of prepped gDNA (reconstituted at 147 ng/µL), in ABI MicroAmp plate seated in black adapter
6 Empty Eppendorf plate
8 Empty tip box
9 Hybridization Master Mix source plate seated on silver insert
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4 Hybridization Step 2. Hybridize the gDNA library and SureSelect Capture Library
86
Run VWorks protocol Hybridization_v1.0.pro
16 On the SureSelect setup form, under Select Protocol to Run, select Hybridization_v1.0.pro.
17 Select the number of columns of samples to be processed. Runs must include 1, 2, 3, 4, 6, or 12 columns.
18 Click Display Initial Workstation Setup.
SureSelect Automated Library Prep and Capture System
Hybridization 4 Step 2. Hybridize the gDNA library and SureSelect Capture Library
SureSelect Automat
19 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form.
20 When verification is complete, click Run Selected Protocol.
The Agilent NGS Workstation transfers SureSelect Block Master Mix to the prepped gDNA-containing wells of the ABI MicroAmp plate. When this process is complete, you will be prompted to transfer the plate to the thermal cycler for sample denaturation prior to hybridization.
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4 Hybridization Step 2. Hybridize the gDNA library and SureSelect Capture Library
88
21 When prompted by VWorks as shown below, remove the ABI MicroAmp plate from position 5 of the Bravo deck, leaving the black adapter in place.
22 Seal the sample plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165°C and 3.0 sec.
23 Transfer the sealed plate to a thermal cycler and run the following program shown in Table 40. After transferring the plate, click Continue on the VWorks screen.
Table 40 Thermal cycler program used for sample denaturation prior to hybridization
While the sample plate incubates on the thermal cycler, the Agilent NGS Workstation combines aliquots of the SureSelect Capture Library master mix and Hybridization Buffer master mix.
Step Temperature Time
Step 1 95°C 5 minutes
Step 2 65°C Hold
SureSelect Automated Library Prep and Capture System
Hybridization 4 Step 2. Hybridize the gDNA library and SureSelect Capture Library
SureSelect Automat
CAUTION You must complete step 24 to step 28 quickly, and immediately after being prompted by the VWorks software. It is important that sample temperature remains approximately 65°C during transfers between the Agilent NGS Workstation and thermal cycler.
24 When the workstation has finished aliquoting the Capture Library and Hybridization Buffer master mixes, you will be prompted by VWorks as shown below. When the thermal cycler reaches the 65°C hold step, click Continue. Leave the sample plate in the thermal cycler until you are notified to move it.
ed Library Prep and Capture System 89
4 Hybridization Step 2. Hybridize the gDNA library and SureSelect Capture Library
90
25 When prompted by VWorks as shown below, quickly remove the sample plate from the thermal cycler, unseal the plate carefully to avoid splashing, and transfer the plate to position 4 of the Bravo deck, seated in the black ABI adapter. Click Continue.
Bravo deck position 4 will be hot.
WARNINGWarningUse caution when handling components that contact heated deck positions.
The Agilent NGS Workstation transfers the capture library-hybridization buffer mixture to the wells of the ABI MicroAmp plate, containing the mixture of prepped gDNA samples and blocking agents.
SureSelect Automated Library Prep and Capture System
Hybridization 4 Step 2. Hybridize the gDNA library and SureSelect Capture Library
SureSelect Automat
26 When prompted by VWorks as shown below, quickly remove the ABI MicroAmp sample plate from Bravo deck position 4, leaving the black ABI adapter in place.
27 Seal the sample plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165°C and 3.0 sec.
28 Quickly transfer the plate back to the thermal cycler, held at 65°C. After transferring the plate, click Continue on the VWorks screen.
29 To finish the VWorks protocol, click Continue in the Unused Tips and Empty Tip box dialogs, and then click Yes in the Protocol Complete dialog.
CAUTION The temperature of the plate in the thermal cycler should be held at 65°C using a heated lid at 105°C. The lid of the thermal cycler is hot and can cause burns. Use caution when working near the lid.
30 Incubate the hybridization mixture in the thermal cycler for 24 hours at 65°C with a heated lid at 105°C.
Samples may be hybridized for up to 72 hours, but you must verify that the extended hybridization does not cause extensive evaporation in the sample wells.
ed Library Prep and Capture System 91
4 Hybridization Step 3. Capture the hybridized DNA
Step 3. Capture the hybridized DNA
92
In this step, the gDNA-capture library hybrids are captured using streptavidin-coated magnetic beads. This step is run immediately after the 24-hour hybridization period.
This step is automated by the NGS workstation using the SureSelectCapture&Wash_v1.5.rst runset, with a total duration of approximately 3.5 hours. A workstation operator must be present to complete two actions during the runset, at the time points in the table below. The times provided are approximate; each action is completed in response to a VWorks prompt at the appropriate time in the runset.
Table 41
Operator action Approximate time after run start
Transfer hybridization reactions from thermal cycler to NGS workstation
<5 minutes
Replace PCR plate with red aluminum insert
5-10 minutes
SureSelect Automated Library Prep and Capture System
Hybridization 4 Step 3. Capture the hybridized DNA
SureSelect Automat
Prepare the workstation
1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes.
2 Gently wipe down the Labware MiniHub, Bravo decks, and BenchCel with a NucleoClean decontamination wipe.
3 Pre-set the temperature of Bravo deck position 4 to 85°C using the Inheco Multi TEC control touchscreen, as described in Setting the Temperature of Bravo Deck Heat Blocks. Bravo deck position 4 corresponds to CPAC 2, position 1 on the Multi TEC control touchscreen.
Prepare the Dynabeads streptavidin beads
4 Vigorously resuspend the Dynabeads MyOne Streptavidin T1 magnetic beads on a vortex mixer. The beads settle during storage.
5 Wash the magnetic beads.
a In a conical vial, combine the components listed in Table 42. The volumes below include the required overage.
Table 42 Components required for magnetic bead washing procedure
Reagent Volume for 1 Library
Volume for 1 Column
Volume for 2 Columns
Volume for 3 Columns
Volume for 4 Columns
Volume for 6 Columns
Volume for 12 Columns
Dynabeads MyOne Streptavidin T1 bead suspension
50 µL 425 µL 825 µL 1225 µL 1.65 mL 2.5 mL 5.0 mL
SureSelect Binding Buffer
0.2 mL 1.7 mL 3.3 mL 4.9 mL 6.6 mL 10 mL 20 mL
Total Volume 0.25 mL 2.125 mL 4.125 mL 6.125 mL 8.25 mL 12.5 mL 25 mL
b Mix the beads on a vortex mixer for 5 seconds.
c Put the vial into a magnetic device, such as the Dynal magnetic separator (Invitrogen).
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.)
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4 Hybridization Step 3. Capture the hybridized DNA
94
6 Resuspend the beads in SureSelect Binding buffer, according to Table 43 below.
Table 43 Preparation of magnetic beads for SureSelect Capture&Wash_v1.5.rst
Reagent Volume for 1 Library
Volume for 1 Column
Volume for 2 Columns
Volume for 3 Columns
Volume for 4 Columns
Volume for 6 Columns
Volume for 12 Columns
SureSelect Binding Buffer
0.2 mL 1.7 mL 3.3 mL 4.9 mL 6.6 mL 10 mL 20 mL
7 Prepare a Nunc DeepWell source plate for the washed streptavidin bead suspension. For each well to be processed, add 200 µL of the homogenous bead suspension to the Nunc DeepWell plate.
8 Place the streptavidin bead source plate at position 5 of the Bravo deck.
Prepare capture and wash solution source plates
9 Prepare a Thermo Scientific reservoir containing 15 mL of nuclease-free water.
10 Prepare an Eppendorf source plate labeled Wash #1. For each well to be processed, add 160 µL of SureSelect Wash Buffer #1.
11 Prepare a Nunc DeepWell source plate labeled Wash #2. For each well to be processed, add 1150 µL of SureSelect Wash Buffer #2.
12 Place the silver Nunc DeepWell plate insert on position 6 of the Bravo deck. This insert is required to facilitate heat transfer to DeepWell source plate wells during the Capture&Wash runset.
13 Place the Wash #2 source plate on the silver insert at position 6 of the Bravo deck. Make sure the plate is seated properly on the silver DeepWell insert.
SureSelect Automated Library Prep and Capture System
Hybridization 4 Step 3. Capture the hybridized DNA
SureSelect Automat
Load the Agilent NGS Workstation
14 Load the Labware MiniHub according to Table 44, using the plate orientations shown in Figure 3.
Table 44 Initial MiniHub configuration for SureSelect Capture&Wash_v1.5.rst
15 Load the BenchCel Microplate Handling Workstation according to Table 45.
Table 45 Initial BenchCel configuration for SureSelectCapture&Wash_v1.5.rst
Vertical Shelf Position
Cassette 1 Cassette 2 Cassette 3 Cassette 4
Shelf 5 (Top) Empty Empty Empty Empty
Shelf 4 Empty Empty Empty Empty
Shelf 3 Empty Eppendorf plate
Empty Wash #1 Eppendorf source plate from step 10
Empty
Shelf 2 Empty Nuclease-free water reservoir from step 9
Empty Empty
Shelf 1 (Bottom) Empty Empty Empty Empty tip box
No. of Columns Processed
Rack 1 Rack 2 Rack 3 Rack 4
1 2 Tip boxes Empty Empty Empty
2 3 Tip boxes Empty Empty Empty
3 4 Tip boxes Empty Empty Empty
4 5 Tip boxes Empty Empty Empty
6 7 Tip boxes Empty Empty Empty
12 10 Tip boxes 3 Tip boxes Empty Empty
ed Library Prep and Capture System 95
4 Hybridization Step 3. Capture the hybridized DNA
96
16 Load the Bravo deck according to Table 46 (positions 5 and 6 should already be loaded).
Table 46 Initial Bravo deck configuration for SureSelectCapture&Wash_v1.5.rst
Location Content
1 Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)
4 Empty black ABI adapter
5 Dynabeads streptavidin bead DeepWell source plate
6 Wash #2 DeepWell source plate seated on silver insert
SureSelect Automated Library Prep and Capture System
Hybridization 4 Step 3. Capture the hybridized DNA
SureSelect Automat
Run VWorks runset SureSelectCapture&Wash_v1.5.rst
17 On the SureSelect setup form, under Select Protocol to Run, select SureSelectCapture&Wash_v1.5.rst.
18 Select the number of columns of samples to be processed. Runs must include 1, 2, 3, 4, 6, or 12 columns.
19 Click Display Initial Workstation Setup.
ed Library Prep and Capture System 97
4 Hybridization Step 3. Capture the hybridized DNA
98
20 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form.
21 When verification is complete, click Run Selected Protocol.
22 When ready to begin the run, click OK in the following window. If the temperature of Bravo deck position 4 was not pre-set to 85°C, the runset will pause while position 4 reaches temperature.
SureSelect Automated Library Prep and Capture System
Hybridization 4 Step 3. Capture the hybridized DNA
SureSelect Automat
CAUTION It is important to complete step 23 quickly and carefully. Transfer the sample plate to the Bravo platform quickly to retain the 65°C sample temperature. Unseal the plate without tilting or jerking the plate to avoid sample splashing. Make sure that the Agilent NGS Workstation is completely prepared, with deck platforms at temperature and all components in place, before you transfer the sample plate to the Bravo deck.
23 When prompted by VWorks as shown below, quickly remove the ABI MicroAmp 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 black ABI adapter. Click Continue to resume the runset.
WARNINGWarning
Bravo deck position 4 will be hot.
Use caution when handling components that contact heated deck positions.
ed Library Prep and Capture System 99
4 Hybridization Step 3. Capture the hybridized DNA
100
24 When prompted by VWorks as shown below, install the red aluminum insert at position 4. When finished, click Continue to resume the runset.
The remainder of the SureSelectCapture&Wash_v1.5.rst runset takes approximately 3 hours. Once the runset is complete, the captured, bead-bound DNA samples are located in the Eppendorf plate at position 9 of the Bravo deck.
When the runset is complete, seal the plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165°C and 1.0 sec and store the plate on ice while setting up the next automation protocol.
NOTE Captured DNA is retained on the streptavidin beads during the post-capture amplification step.
SureSelect Automated Library Prep and Capture System
SureSelect Automated Library Prep and Capture System Protocol
5Barcoding
Step 1. Amplify the captured libraries to add barcode tags 102
Step 2. Purify the amplified barcoded libraries using Agencourt AMPure XP beads 114
Step 3. Remove primer-dimers by purification using Agencourt AMPure XP beads 119
Step 4. Assess barcoded DNA quality 124
Step 5. Assess the quantity of each barcode-tagged library by QPCR 128
Step 6. Pool samples for multiplexed sequencing 129
This chapter describes the steps to add barcode tags by amplification, purify, assess quality and quantity of the libraries, and pool barcoded samples for multiplexed sequencing.
The size of your SureSelect Capture Library determines the amplification cycle number used for barcoding. Plan your experiments for amplification of samples prepared using SureSelect Capture Libraries of similar sizes on the same plate. See Table 55 on page 113 for cycle number recommendations.
101Agilent Technologies
5 Barcoding Step 1. Amplify the captured libraries to add barcode tags
Step 1. Amplify the captured libraries to add barcode tags
102
In this step, the Agilent NGS Workstation completes the liquid handling steps for PCR-based addition of barcoding tags to the SureSelect-enriched DNA samples. After the PCR plate is prepared by the Agilent NGS Workstation, you transfer the plate to a thermal cycler for amplification.
Assign barcodes to DNA samples
Select the appropriate barcoding primer(s) for each sample. Nucleotide sequences of the 96 barcodes provided with the SureSelectXT system are provided in Table 70 on page 135.
1 Use a different barcoding primer for each sample to be sequenced in the same lane.
2 The number of samples that may be combined per lane depends on the sequencing platform performance and the Capture Library size. See Table 47 for sequence data requirement guidelines. Calculate the number of barcodes that can be combined per lane based on these guidelines.
Table 47 Sequencing data requirement guidelines
Capture Library Size Recommended Amount of Sequencing Data per Sample
1 kb up to 0.5 Mb 0.1 to 50 Mb*
* For custom libraries, Agilent recommends analyzing 100X amount of sequencing data com-pared to the Capture Library size for each sample. Pool samples according to your expected sequencing output.
0.5 Mb up to 2.9 Mb 50 to 290 Mb*
3 Mb up to 5.9 Mb 300 to 590 Mb*
6 Mb up to 11.9 Mb 600 to 1190 Mb*
12 Mb up to 24 Mb 1.2 to 2.4 Gb*
Human All Exon v4 4 Gb
Human All Exon v4 + UTRs 6 Gb
Human All Exon 50 Mb 5 Gb
Human DNA Kinome 320 Mb
Mouse All Exon 5 Gb
SureSelect Automated Library Prep and Capture System
Barcoding 5 Step 1. Amplify the captured libraries to add barcode tags
SureSelect Automat
3 Include full sets of 4 barcodes in each lane. A full set of barcodes refers to BC1 through BC4, BC5 through BC8, BC9 through BC12, etc.
If the number of libraries to be combined in a sequencing lane is not a multiple of 4 (based on guidelines in Table 47), then use multiple barcoding primers, in equal ratios, to amplify a single library. For example, for a sequencing sample designed to contain 2 libraries, amplify each library using 2 barcoding primers. For 3 libraries, amplify each library using 4 barcoding primers, for a total of 12 barcodes in the sequencing sample.
ed Library Prep and Capture System 103
5 Barcoding Step 1. Amplify the captured libraries to add barcode tags
104
Prepare the workstation
1 Turn on the ThermoCube, set to 0°C, at position 9 of the Bravo deck. Be sure that the chiller reservoir contains at least 300 mL of 25% ethanol.
2 Clear the Labware MiniHub and BenchCel of plates and tip boxes.
3 Gently wipe down the Labware MiniHub, Bravo decks, and BenchCel with a Nucleoclean decontamination wipe.
Prepare barcoding primers and PCR master mix
CAUTION Do not use amplification enzymes other than Herculase II Fusion DNA Polymerase. Other enzymes have not been validated.
CAUTION 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 Dilute each barcoding primer to be used in the run according to Table 48. The volumes below include the required excess.
Table 48 Preparation of barcoding primer dilutions
5 In an ABI MicroAmp plate, aliquot 9 µL of the appropriate barcoding primer dilution to the intended sample barcoding well position. Keep the plate on ice.
SureSelectXT Reagent Volume to Barcode 1 Sample Volume to Barcode 12 Samples
Nuclease-free water 7.0 µL 87.5 µL
Barcode PCR primer (BC1-BC16)
2.0 µL 25 µL
Total Volume 9.0 µL 112.5 µL
SureSelect Automated Library Prep and Capture System
Barcoding 5 Step 1. Amplify the captured libraries to add barcode tags
SureSelect Automat
6 Prepare the appropriate volume of PCR master mix, according to Table 49. Mix well using a vortex mixer and keep on ice.
Table 49 Preparation of PCR Master Mix for Post-CaptureBarcoding_XT_SOLiD_5500_v1.5.pro
SureSelectXT Reagent
Volume for 1 Library
Volume for 1 Column
Volume for 2 Columns
Volume for 3 Columns
Volume for 4 Columns
Volume for 6 Columns
Volume for 12 Columns
Nuclease-free water
15.5 µL 197.6 µL 329.4 µL 461.1 µL 592.9 µL 856.4 µL 1713 µL
Herculase II 5X Reaction Buffer*
10.0 µL 127.5 µL 212.5 µL 297.5 µL 382.5 µL 552.5 µL 1105 µL
dNTP mix* 0.5 µL 6.4 µL 10.6 µL 14.9 µL 19.1 µL 27.6 µL 55.3 µL
Herculase II polymerase
1.0 µL 12.8 µL 21.3 µL 29.8 µL 38.3 µL 55.3 µL 110.5 µL
Total Volume 27.0 µL 344.3 µL 573.8 µL 803.3 µL 1032.8 µL 1491.8 µL 2983.8 µL
* Included with the Herculase II Fusion DNA Polymerase. Do not use the buffer or dNTP mix from any other kit.
ed Library Prep and Capture System 105
5 Barcoding Step 1. Amplify the captured libraries to add barcode tags
106
7 Using the same Nunc DeepWell master mix source plate that was used for the Hybridization_v1.0.pro protocol, add the volume of PCR master mix indicated in Table 50 to all wells of column 4 of the master mix source plate. The final configuration of the master mix source plate is shown in Figure 12.
Table 50 Preparation of the Master Mix Source Plate for Post-CaptureBarcoding_XT_SOLiD_5500_v1.5.pro
If you are using a new DeepWell plate for the post-capture PCR source plate (for example, when amplifying the second half of the captured DNA sample), leave columns 1 to 3 empty and add the PCR Master Mix to column 4 of the new plate.
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
(A4-H4)
39.7 µL 68.3 µL 97.0 µL 125.7 µL 183.1 µL 369.6 µL
NOTE
SureSelect Automated Library Prep and Capture System
Barcoding 5 Step 1. Amplify the captured libraries to add barcode tags
SureSelect Automat
Figure 12 Configuration of the master mix source plate for Post-CaptureBarcoding_XT_SOLiD_5500_v1.5.pro. Columns 1-3 were used to dispense master mixes for the Hybridization_v1.0.pro protocol.
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.
ed Library Prep and Capture System 107
5 Barcoding Step 1. Amplify the captured libraries to add barcode tags
108
Load the Agilent NGS Workstation
10 Load the Labware MiniHub according to Table 51, using the plate orientations shown in Figure 3.
Table 51 Initial MiniHub configuration for Post-CaptureBarcoding_XT_SOLiD_5500_v1.5.pro
11 Load the BenchCel Microplate Handling Workstation according to Table 52.
Table 52 Initial BenchCel configuration for Post-CaptureBarcoding_XT_SOLiD_5500_v1.5.pro
Vertical Shelf Position
Cassette 1 Cassette 2 Cassette 3 Cassette 4
Shelf 5 (Top)
Empty Empty Empty Barcoding primers in ABI MicroAmp plate seated in black adapter
Shelf 4 Empty Empty Empty Empty
Shelf 3 Empty Empty Empty Empty
Shelf 2 Empty tip box Empty Empty Empty
Shelf 1 (Bottom)
New tip box Empty Empty Empty tip box
No. of Columns Processed
Rack 1 Rack 2 Rack 3 Rack 4
1 1 Tip box Empty Empty Empty
2 1 Tip box Empty Empty Empty
3 1 Tip box Empty Empty Empty
4 1 Tip box Empty Empty Empty
6 1 Tip box Empty Empty Empty
12 1 Tip box Empty Empty Empty
SureSelect Automated Library Prep and Capture System
Barcoding 5 Step 1. Amplify the captured libraries to add barcode tags
SureSelect Automat
12 Load the Bravo deck according to Table 53.
Table 53 Initial Bravo deck configuration for Post-CaptureBarcoding_XT_SOLiD_5500_v1.5.pro
Location Content
4 Captured DNA bead suspensions in Eppendorf twin.tec plate
9 Master mix plate, containing PCR Master Mix in Column 4 of Nunc DeepWell plate, seated on silver insert
ed Library Prep and Capture System 109
5 Barcoding Step 1. Amplify the captured libraries to add barcode tags
110
Run VWorks protocol Post-CaptureBarcoding_XT_SOLiD_5500_v1.5.pro
13 On the SureSelect setup form, under Select Protocol to Run, select Post-CaptureBarcoding_XT_SOLiD_5500_v1.5.pro.
14 Select the number of columns of samples to be processed. Runs must include 1, 2, 3, 4, 6, or 12 columns.
15 Click Display Initial Workstation Setup.
SureSelect Automated Library Prep and Capture System
Barcoding 5 Step 1. Amplify the captured libraries to add barcode tags
SureSelect Automat
16 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form.
17 When verification is complete, click Run Selected Protocol.
Running the Post-CaptureBarcoding_XT_SOLiD_5500_v1.5.pro protocol takes approximately 15 minutes. Once complete, the PCR-ready samples, containing captured DNA and PCR master mix, are located in the ABI MicroAmp plate at position 5 of the Bravo deck. The Eppendorf plate containing the remaining bead-bound captured DNA samples, which may be stored for future use at 4°C overnight, or at –20°C for long-term storage, is located at position 4 of the Bravo deck.
ed Library Prep and Capture System 111
5 Barcoding Step 1. Amplify the captured libraries to add barcode tags
112
18 When you see the following prompt, remove the PCR plate from position 5 of the Bravo deck and seal the plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165°C and 3.0 seconds.
19 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate air bubbles.
SureSelect Automated Library Prep and Capture System
Barcoding 5 Step 1. Amplify the captured libraries to add barcode tags
SureSelect Automat
20 Transfer the PCR plate to a thermal cycler and run the PCR amplification program shown in Table 54 using the cycle number specified in Table 55.
Table 54 Post-Capture PCR cycling program
Table 55 Recommended cycle number based on SureSelect Capture Library size
Segment Number of Cycles
Temperature Time
1 1 98°C 2 minutes
2 8-12
see Table 55
98°C 30 seconds
54°C 10 seconds
72°C 60 seconds
3 1 72°C 10 minutes
4 1 4°C Hold
Size of SureSelect Capture Library
Cycles
0.2 to 0.49 Mb 12 cycles
0.5 to 1.49 Mb 10 cycles
1.5 to 2.99 Mb 9 cycles
3 to 50 Mb 8 cycles
Amplify the captured DNA using a minimal number of PCR cycles. If yield is too low or non-specific high molecular weight products are observed, adjust the number of cycles accordingly with the remaining captured DNA template.
Nee
NOTE
ed Library Prep and Capture System 113
5 Barcoding Step 2. Purify the amplified barcoded libraries using Agencourt AMPure XP beads
Step 2. Purify the amplified barcoded libraries using Agencourt AMPure XP beads
114
In this step, the Agilent NGS Workstation transfers AMPure XP beads and barcoded DNA samples to a Nunc DeepWell plate and then collects and washes the bead-bound DNA.
Prepare the workstation and reagents
1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes.
2 Gently wipe down the Labware MiniHub, Bravo decks, and BenchCel with a Nucleoclean decontamination wipe.
3 Let the AMPure XP beads come to room temperature for at least 30 minutes.
4 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color. Do not freeze.
5 Prepare a Nunc DeepWell source plate containing AMPure XP beads. For each well to be processed, add 65 µL of homogenous AMPure XP beads per well to the Nunc DeepWell plate.
6 Prepare a Thermo Scientific reservoir containing 15 mL of nuclease-free water.
7 Prepare a separate Thermo Scientific reservoir containing 45 mL of freshly-prepared 70% ethanol.
SureSelect Automated Library Prep and Capture System
Barcoding 5 Step 2. Purify the amplified barcoded libraries using Agencourt AMPure XP beads
SureSelect Automat
8 Load the Labware MiniHub according to Table 56, using the plate orientations shown in Figure 3.
Table 56 Initial MiniHub configuration for AMPureXP_XT_SOLiD_5500_v1.5.pro
9 Load the BenchCel Microplate Handling Workstation according to Table 57.
Table 57 Initial BenchCel configuration for AMPureXP_XT_SOLiD_5500_v1.5.pro
Vertical Shelf Position
Cassette 1 Cassette 2 Cassette 3 Cassette 4
Shelf 5 (Top) Empty Nunc DeepWell plate
Empty Empty Empty
Shelf 4 Empty Empty Empty Empty
Shelf 3 Empty Empty Eppendorf Plate
Empty Empty
Shelf 2 Empty Nuclease-free water reservoir from step 6
AMPure XP beads in Nunc DeepWell plate from step 5
Empty
Shelf 1 (Bottom) Empty 70% ethanol reservoir from step 7
Empty Empty
No. of Columns Processed
Rack 1 Rack 2 Rack 3 Rack 4
1 1 Tip box Empty Empty Empty
2 1 Tip box Empty Empty Empty
3 2 Tip boxes Empty Empty Empty
4 2 Tip boxes Empty Empty Empty
6 3 Tip boxes Empty Empty Empty
12 6 Tip boxes Empty Empty Empty
ed Library Prep and Capture System 115
5 Barcoding Step 2. Purify the amplified barcoded libraries using Agencourt AMPure XP beads
116
10 Load the Bravo deck according to Table 58.
Table 58 Initial Bravo deck configuration for AMPureXP_XT_SOLiD_5500_v1.5.pro
Location Content
1 Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)
4 Barcoded library samples in ABI MicroAmp plate seated in black adapter
8 Empty tip box
SureSelect Automated Library Prep and Capture System
Barcoding 5 Step 2. Purify the amplified barcoded libraries using Agencourt AMPure XP beads
SureSelect Automat
Run VWorks protocol AMPureXP_XT_SOLiD_5500_v1.5.pro
11 On the SureSelect setup form, under Select Protocol to Run, select AMPureXP_XT_SOLiD_5500_v1.5.pro.
12 Under Select additional Parameters, select the step Post-Capture PCR Cleanup and the plate type 96 ABI PCR half skirt in black carrier.
AMPureXP bead purification protocols are used during multiple steps of the SureSelect automation workflow. Be sure to select the correct protocol name, workflow step, and plate type when initiating the automation protocol.
NOTE
ed Library Prep and Capture System 117
5 Barcoding Step 2. Purify the amplified barcoded libraries using Agencourt AMPure XP beads
118
13 Select the number of columns of samples to be processed. Runs must include 1, 2, 3, 4, 6, or 12 columns.
14 Click Display Initial Workstation Setup.
15 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form.
16 When verification is complete, click Run Selected Protocol.
The purification protocol takes approximately 45 minutes. When complete, the purified DNA samples are in the Eppendorf plate located on Bravo deck position 7.
SureSelect Automated Library Prep and Capture System
Barcoding 5 Step 3. Remove primer-dimers by purification using Agencourt AMPure XP beads
Step 3. Remove primer-dimers by purification using Agencourt AMPure XP beads
SureSelect Automat
In this step, the Agilent NGS Workstation does a primer-dimer purification step using AMPure XP beads.
Prepare the workstation and reagents
1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes.
2 Gently wipe down the Labware MiniHub, Bravo decks, and BenchCel with a Nucleoclean decontamination wipe.
3 Let the AMPure XP beads come to room temperature for at least 30 minutes.
4 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color. Do not freeze.
5 Prepare a Nunc DeepWell source plate containing AMPure XP beads. For each well to be processed, add 65 µL of homogenous AMPure XP beads per well to the Nunc DeepWell plate.
6 Prepare a Thermo Scientific reservoir containing 15 mL of nuclease-free water.
7 Prepare a separate Thermo Scientific reservoir containing 45 mL of freshly-prepared 70% ethanol.
ed Library Prep and Capture System 119
5 Barcoding Step 3. Remove primer-dimers by purification using Agencourt AMPure XP beads
120
8 Load the Labware MiniHub according to Table 59, using the plate orientations shown in Figure 3.
Table 59 Initial MiniHub configuration for AMPureXP_XT_SOLiD_5500_v1.5.pro
9 Load the BenchCel Microplate Handling Workstation according to
Table 60 Initial BenchCel configuration for AMPureXP_XT_SOLiD_5500_v1.5.pro
Table 60.
Vertical Shelf Position
Cassette 1 Cassette 2 Cassette 3 Cassette 4
Shelf 5 (Top) Empty Nunc DeepWell plate
Empty Empty Empty
Shelf 4 Empty Empty Empty Empty
Shelf 3 Empty Empty Eppendorf Plate
Empty Empty
Shelf 2 Empty Nuclease-free water reservoir from step 6
AMPure XP beads in Nunc DeepWell plate from step 5
Empty
Shelf 1 (Bottom) Empty 70% ethanol reservoir from step 7
Empty Empty
No. of Columns Processed
Rack 1 Rack 2 Rack 3 Rack 4
1 1 Tip box Empty Empty Empty
2 1 Tip box Empty Empty Empty
3 2 Tip boxes Empty Empty Empty
4 2 Tip boxes Empty Empty Empty
6 3 Tip boxes Empty Empty Empty
12 6 Tip boxes Empty Empty Empty
SureSelect Automated Library Prep and Capture System
Barcoding 5 Step 3. Remove primer-dimers by purification using Agencourt AMPure XP beads
SureSelect Automat
10 Load the Bravo deck according to Table 61.
Table 61 Initial Bravo deck configuration for AMPureXP_XT_SOLiD_5500_v1.5.pro
Location Content
1 Empty waste reservoir (Axygen 96 Deep Well Plate, square wells)
4 Barcoded library samples in Eppendorf plate
8 Empty tip box
ed Library Prep and Capture System 121
5 Barcoding Step 3. Remove primer-dimers by purification using Agencourt AMPure XP beads
122
Run VWorks protocol AMPureXP_XT_SOLiD_5500_v1.5.pro
11 On the SureSelect setup form, under Select Protocol to Run, select AMPureXP_XT_SOLiD_5500_v1.5.pro.
12 Under Select additional Parameters, select the step Primer-Dimer Cleanup and the plate type 96 Eppendorf Twin.tec PCR.
AMPureXP bead purification protocols are used during multiple steps of the SureSelect automation workflow. Be sure to select the correct protocol name, workflow step, and plate type when initiating the automation protocol.
NOTE
SureSelect Automated Library Prep and Capture System
Barcoding 5 Step 3. Remove primer-dimers by purification using Agencourt AMPure XP beads
SureSelect Automat
13 Select the number of columns of samples to be processed. Runs must include 1, 2, 3, 4, 6, or 12 columns.
14 Click Display Initial Workstation Setup.
15 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form.
16 When verification is complete, click Run Selected Protocol.
The purification protocol takes approximately 45 minutes. When complete, the purified DNA samples are in the Eppendorf plate located on Bravo deck position 7.
ed Library Prep and Capture System 123
5 Barcoding Step 4. Assess barcoded DNA quality
Step 4. Assess barcoded DNA quality
124
Option 1: Analysis using the 2100 Bioanalyzer system and High Sensitivity DNA Assay
1 Set up the 2100 Bioanalyzer system as instructed in the High Sensitivity DNA Assay kit guide.
Version B.02.07 or higher of the Agilent 2100 Expert Software is required for High Sensitivity DNA Assay Kit runs.
NOTE
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.
For some samples, Bioanalyzer system 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 system before analyzing the diluted samples.
NOTE
5 Load the prepared chip into the 2100 Bioanalyzer instrument and start the run within five minutes after preparation.
6 Verify that the electropherogram shows an average DNA fragment size of approximately 260 bp. A sample electropherogram is shown in Figure 13.
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.SureSelect Automated Library Prep and Capture System
Barcoding 5 Step 4. Assess barcoded DNA quality
SureSelect Automat
Figure 13 Analysis of barcoded DNA using the High Sensitivity DNA Assay.
ed Library Prep and Capture System 125
5 Barcoding Step 4. Assess barcoded DNA quality
126
Option 2: Analysis using the 2200 TapeStation and High Sensitivity D1K ScreenTape
Use a High Sensitivity D1K ScreenTape (p/n 5067-5363) and reagent kit (p/n 5067-5364) to analyze the barcoded DNA. For more information to do this step, see the Agilent 2200 TapeStation User Manual.
1 Seal the DNA sample plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165°C and 1.0 sec.
2 Vortex the plate to mix samples in each well, then centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal.
3 Prepare the TapeStation samples as instructed in the Agilent 2200 TapeStation User Manual. Use 2 µL of each barcoded DNA sample diluted with 2 µL of High Sensitivity D1K sample buffer for the analysis.
CAUTION Make sure that you thoroughly mix the combined DNA and High Sensitivity D1K 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 D1K ScreenTape, and loading tips into the 2200 TapeStation as instructed in the Agilent 2200 TapeStation User Manual. Start the run.
5 Verify that the electropherogram shows an average DNA fragment size of approximately 260 bp. A sample electropherogram is shown in Figure 14.
Stopping Point
If you do not continue to the next step, seal the barcoded DNA sample plate and store at 4°C overnight or at –20°C for prolonged storage.SureSelect Automated Library Prep and Capture System
Barcoding 5 Step 4. Assess barcoded DNA quality
SureSelect Automat
Figure 14 Analysis of barcoded DNA using the 2200 TapeStation with a High Sensitivity D1K ScreenTape.
ed Library Prep and Capture System 127
5 Barcoding Step 5. Assess the quantity of each barcode-tagged library by QPCR
Step 5. Assess the quantity of each barcode-tagged library by QPCR
128
Refer to the protocol that is included with the Agilent QPCR NGS Library Quantification Kit (p/n G4881A) for more details to do this step.
1 Use the Agilent QPCR NGS Library Quantification Kit (for SOLiD) to determine the concentration of each barcode-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 barcode-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 SOLiD 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 barcode-tagged library, in nM.
The concentration will be used to accurately pool samples for multiplexed sequencing.
In most cases, the cycle numbers in Table 55 will produce an adequate yield for sequencing without introducing bias or non-specific products. If yield is too low or non-specific products are observed, adjust the number of cycles accordingly with the remaining captured DNA template.
NOTE
SureSelect Automated Library Prep and Capture System
Barcoding 5 Step 6. Pool samples for multiplexed sequencing
Step 6. Pool samples for multiplexed sequencing
SureSelect Automat
1 Combine the libraries such that each barcode-tagged sample is present in equimolar amounts in the pool. For each library, use the formula below to determine the amount of barcoded sample to use.
Volume of Index V f C f # C i
---------------------------------= where
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, for example, 500 pM for the standard SOLiD protocol
# is the number of samples to be combined, and
C(i) is the initial concentration of each barcoded-sample.
See Table 62 for the approximate volume of sample to use.
Table 62 Approximate volume of sample to use
Table 63 shows an example of the amount of 4 barcoded samples (of different concentrations) and Low TE needed for a final volume of 40 µL at 500 pM.
SOLiD Sequencing Capacity
Approximate Sample Volume Final Concentration
1 lane 50 µL 500 pM
Table 63 Example of barcoded sample volume calculation for a total volume of 40 µL
Component V(f) C(i) C(f) # Volume to use (µL)
Sample 1 40 µL 921 pM 500 pM 4 5.4
Sample 2 40 µL 1050 pM 500 pM 4 4.8
Sample 3 40 µL 1352 pM 500 pM 4 3.7
Sample 4 40 µL 684 pM 500 pM 4 7.3
Low TE 18.8
ed Library Prep and Capture System 129
5 Barcoding Step 6. Pool samples for multiplexed sequencing
130
2 Adjust the final volume of the pooled library to the desired final concentration.
• If the final volume of the combined barcode-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 barcode-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.
4 Proceed to emulsion PCR. Refer to the appropriate SOLiD 5500 protocol.
Stopping Point
If you do not do an emulsion PCR at this time, store the DNA at 4°C overnight or at –20°C for prolonged storage.SureSelect Automated Library Prep and Capture System
SureSelect Automated Library Prep and Capture System Protocol
6Reference
Kit Contents 132
Nucleotide Sequences of SureSelect Barcodes 135
This chapter contains reference information, including component kit contents and barcode sequences.
131Agilent Technologies
6 Reference Kit Contents
Kit ContentsSureSelect capture libraries and reagents must be used within one year of receipt. NOTE
132
Each SureSelectXT Reagent Kit-5500 contains the following component kits:
Table 64 SureSelectXT Automation Reagent Kit Contents
Product Storage Condition Agilent p/n
SureSelectXT Library Prep Kit, S5500 -20°C 5500-0113
SureSelectXT Target Enrichment Box #1, S5500 Room Temperature 5190-5932
SureSelectXT Target Enrichment Box #2, S5500 -20°C 5190-5930
SureSelect S5500 Indexing Construction Kit -20°C 5190-5456
SureSelect Automated Library Prep and Capture System
Reference 6 Kit Contents
SureSelect Automat
The contents of each of the component kits listed in Table 64 are described in the tables below.Table 65 SureSelectXT Library Prep Kit-S5500 Content
Kit Component Format
10X End Repair Buffer tube with clear cap
10X Klenow Polymerase Buffer tube with blue cap
5X T4 DNA Ligase Buffer tube with green cap
T4 DNA Ligase tube with red cap
Exo(-) Klenow tube with red cap
T4 DNA Polymerase tube with purple cap
Klenow DNA Polymerase tube with yellow cap
T4 Polynucleotide Kinase tube with orange cap
dATP tube with green cap
dNTP Mix tube with green cap
Table 66 SureSelectXT Target Enrichment Box #1, S5500 Content
Kit Component Format
SureSelect Hyb # 1 bottle
SureSelect Hyb # 2 tube with red cap
SureSelect Hyb # 4 tube with black cap
SureSelect Binding Buffer bottle
SureSelect Wash Buffer 1 bottle
SureSelect Wash Buffer 2 bottle
ed Library Prep and Capture System 133
6 Reference Kit Contents
134
Table 67 SureSelectXT Target Enrichment Box #2, S5500 Content
Kit Component Format
SureSelect Hyb # 3 tube with yellow cap
SureSelect Indexing Block #1 tube with green cap
SureSelect Block #2 tube with blue cap
SureSelect LT Indexing Block #3 tube with brown cap
SureSelect RNase Block tube with purple cap
Table 68 SureSelect S5500 Indexing Construction Kit Content
Kit Component Format
SureSelect LTI5500 P1 tube with purple cap
SureSelect LTI5500 IA tube with blue cap
SureSelect LTI Precapture Primer tube with green cap
SureSelect LTI5500 BC1-BC96 plate*
* See Table 69 for a plate map.
96-well plate containing one barcoding primer per well
Table 69 SureSelect LTI5500 BC1-BC96 plate map
1 2 3 4 5 6 7 8 9 10 11 12A BC1 BC9 BC17 BC25 BC33 BC41 BC49 BC57 BC65 BC73 BC81 BC89B BC2 BC10 BC18 BC26 BC34 BC42 BC50 BC58 BC66 BC74 BC82 BC90C BC3 BC11 BC19 BC27 BC35 BC43 BC51 BC59 BC67 BC75 BC83 BC91D BC4 BC12 BC20 BC28 BC36 BC44 BC52 BC60 BC68 BC76 BC84 BC92E BC5 BC13 BC21 BC29 BC37 BC45 BC53 BC61 BC69 BC77 BC85 BC93F BC6 BC14 BC22 BC30 BC38 BC46 BC54 BC62 BC70 BC78 BC86 BC94G BC7 BC15 BC23 BC31 BC39 BC47 BC55 BC63 BC71 BC79 BC87 BC95H BC8 BC16 BC24 BC32 BC40 BC48 BC56 BC64 BC72 BC80 BC88 BC96
SureSelect Automated Library Prep and Capture System
Reference 6 Nucleotide Sequences of SureSelect Barcodes
Nucleotide Sequences of SureSelect Barcodes
SureSelect Automat
The nucleotide sequence of each SureSelectXT Barcode is provided in the table below. Barcode sets are delineated in the table.
Table 70 SureSelectXT Barcodes 1-96
Barcode Number Sequence
1 GTGTAAGAGG
2 AGGGAGTGGT
3 ATAGGTTATA
4 GGATGCGGTC
5 GTGGTGTAAG
6 GCGAGGGACA
7 GGGTTATGCC
8 GAGCGAGGAT
9 AGGTTGCGAC
10 GCGGTAAGCT
11 GTGCGACACG
12 AAGAGGAAAA
13 GCGGTAAGGC
14 GTGCGGCAGA
15 GAGTTGAATG
16 GGGAGACGTT
17 GGCTCACCGC
18 AGGCGGATGA
19 ATGGTAACTG
20 GTCAAGCTTT
ed Library Prep and Capture System 135
6 Reference Nucleotide Sequences of SureSelect Barcodes
136
21 GTGCGGTTCC
22 GAGAAGATGA
23 GCGGTGCTTG
24 GGGTCGGTAT
25 AACATGATGA
26 CGGGAGCCCG
27 CAGCAAACTT
28 AGCTTACTAC
29 GAATCTAGGG
30 GTAGCGAAGA
31 GCTGGTGCGT
32 GGTTGGGTGC
33 CGTTGGATAC
34 TCGTTAAAGG
35 AAGCGTAGGA
36 GTTCTCACAT
37 CTGTTATACC
38 GTCGTCTTAG
39 TATCGTGAGT
40 AAAAGGGTTA
41 TGTGGGATTG
42 GAATGTACTA
43 CGCTAGGGTT
44 AAGGATGATC
Table 70 SureSelectXT Barcodes 1-96
Barcode Number Sequence
SureSelect Automated Library Prep and Capture System
Reference 6 Nucleotide Sequences of SureSelect Barcodes
SureSelect Automat
45 GTACTTGGCT
46 GGTCGTCGAA
47 GAGGGATGGC
48 GCCGTAAGTG
49 ATGTCATAAG
50 GAAGGCTTGC
51 AAGCAGGAGT
52 GTAATTGTAA
53 GTCATCAAGT
54 AAAAGGCGGA
55 AGCTTAAGCG
56 GCATGTCACC
57 CTAGTAAGAA
58 TAAAGTGGCG
59 AAGTAATGTC
60 GTGCCTCGGT
61 AAGATTATCG
62 AGGTGAGGGT
63 GCGGGTTCGA
64 GTGCTACACC
65 GGGATCAAGC
66 GATGTAATGT
67 GTCCTTAGGG
68 GCATTGACGA
Table 70 SureSelectXT Barcodes 1-96
Barcode Number Sequence
ed Library Prep and Capture System 137
6 Reference Nucleotide Sequences of SureSelect Barcodes
138
69 GATATGCTTT
70 GCCCTACAGA
71 ACAGGGAACG
72 AAGTGAATAC
73 GCAATGACGT
74 AGGACGCTGA
75 GTATCTGGGC
76 AAGTTTTAGG
77 ATCTGGTCTT
78 GGCAATCATC
79 AGTAGAATTA
80 GTTTACGGTG
81 GAACGTCATT
82 GTGAAGGGAG
83 GGATGGCGTA
84 GCGGATGAAC
85 GGAAAGCGTT
86 AGTACCAGGA
87 ATAGCAAAGC
88 GTTGATCATG
89 AGGCTGTCTA
90 GTGACCTACT
91 GCGTATTGGG
92 AAGGGATTAC
Table 70 SureSelectXT Barcodes 1-96
Barcode Number Sequence
SureSelect Automated Library Prep and Capture System
Reference 6 Nucleotide Sequences of SureSelect Barcodes
SureSelect Automat
93 GTTACGATGC
94 ATGGGTGTTT
95 GAGTCCGGCA
96 AATCGAAGAG
Table 70 SureSelectXT Barcodes 1-96
Barcode Number Sequence
ed Library Prep and Capture System 139
www.agilent.com
In This Book
This guide contains information to run the SureSelect Automated Library Prep and Capture System protocol using automation protocols provided with the Agilent NGS Workstation.
Agilent Technologies, Inc. 2012
Version A, October 2012
*G7550-90002*G7550-90002
Agilent Technologies