sureselect automated library prep and capture...

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

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Manual Part NumberG7550-90002

EditionVersion A, October 2012

Printed in USA

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A CAUTION notice denotes a haz-ard. It calls attention to an operat-ing procedure, practice, or the like that, if not correctly performed or adhered to, could result in damage to the product or loss of important data. Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met.

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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.

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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

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www.agilent.com/chem/dnasupport

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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 Begin
This chapter contains information (such as procedural notes, safety information, required reagents and equipment) that you should read and understand before you start an experiment.

2
Using the Agilent NGS Workstation for SureSelect Target Enrichment
This chapter contains an orientation to the Agilent NGS Workstation, an overview of the SureSelect target enrichment protocol, and considerations for designing SureSelect experiments for automated processing using the Agilent NGS Workstation.

3
Sample Preparation
This chapter describes the steps to prepare the DNA samples for target enrichment.

4
Hybridization
This 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
Reference
This 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


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


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


Before You Begin 1 Required Equipment

Required Equipment

SureSelect Automat

Table 4 Required Equipment


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:

[email protected]

Robotic Pipetting Tips (Sterile, Filtered, 250 L) Agilent p/n 19477-022

Eppendorf twin.tec full-skirted 96-well PCR plates Eppendorf p/n 951020401 or 951020619

Thermo Scientific Reservoirs Thermo Scientific p/n 1064156

Nunc DeepWell Plates, sterile, 1.3-mL well volume Thermo Scientific p/n 260251

Axygen 96 Deep Well Plate, 2.2 mL, Square Well (waste reservoirs)

Axygen p/n P-2ML-SQ-C E & K Scientific p/n EK-2440

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 G2964AA or G2965AA





Thermal cycler Life Technologies Veriti Thermal Cycler

Qubit Fluorometer Life Technologies p/n Q32857

Qubit assay tubes Life Technologies p/n Q32856


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)




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.


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


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


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.


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.


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.


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.



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 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.



22

2 If you encounter the W-axis error message shown below, select Retry.



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.


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.


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.


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


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


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.



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.


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.


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


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.

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.


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 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







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 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



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.


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.


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.


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.


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.

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.


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


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.


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





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



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 Column






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 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 Column






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



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 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



48

Prepare the purification reagents

11 Verify that the AMPure XP bead suspension is at room temperature.


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.



Load the Agilent NGS Workstation


Table 17 Initial MiniHub configuration for LibraryPrep_XT_SOLiD_5500_v1.0.rst






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


Empty

Shelf 1 (Bottom) New tip box 70% ethanol reservoir from step 15

Empty Empty tip box



SureSelect Automat


Table 18 Initial BenchCel configuration for LibraryPrep_XT_SOLiD_5500_v1.0.rst


Table 19 Initial Bravo deck configuration for LibraryPrep_XT_SOLiD_5500_v1.0.rst








12 11 Tip boxes 8 Tip boxes Empty Empty

Location Content


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



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.





SureSelect Automat



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

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.



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.



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



Volume for 1 Column






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


* Included with the Herculase II Fusion DNA Polymerase. Do not use the buffer or dNTP mix from any other kit.



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



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 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.


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



56



Table 22 Initial MiniHub configuration for Pre-CapturePCR_XT_SOLiD_5500_v1.0.pro



Shelf 5 (Top)

Empty Empty Empty Empty ABI MicroAmp plate seated in black adapter



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 Automat


Table 23 Initial BenchCel configuration for Pre-CapturePCR_XT_SOLiD_5500_v1.0.pro


Table 24 Initial Bravo deck configuration for Pre-CapturePCR_XT_SOLiD_5500_v1.0.pro









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



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.





SureSelect Automat



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.



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


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.



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.)



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.




3 Sample Preparation Step 6. Purify amplified DNA using AMPure XP beads

62


Table 26 Initial MiniHub configuration for AMPureXP_XT_SOLiD_5500_v1.5.pro


Table 27 Initial BenchCel configuration for AMPureXP_XT_SOLiD_5500_v1.5.pro




Empty Empty Empty



Empty Empty



Empty


Empty Empty











SureSelect Automat


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


4 Amplified DNA libraries in ABI MicroAmp plate seated in black adapter

8 Empty tip box


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





SureSelect Automat



The purification protocol takes approximately 45 minutes. When complete, the amplified DNA samples are in the Eppendorf plate located on Bravo deck position 7.


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.






7 Determine the concentration of the library (ng/µL) by integrating under the peak.

Stopping Point

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.



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.


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.


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.

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.



70


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.


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.


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.



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)


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.





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.


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.



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.


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



Volume for 1 Column






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




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 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



Volume for 1 Column






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


Target size >3.0 Mb



Volume for 1 Column






Nuclease-free water

1.5 µL 25.5 µL 38.3 µL 51.0 µL 63.8 µL 89.3 µL 172.1 µL


0.5 µL 8.5 µL 12.8 µL 17.0 µL 21.3 µL 29.8 µL 57.4 µL


5.0 µL 85.0 µL 127.5 µL 170.0 µL 212.5 µL 297.5 µL 573.8 µL




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



Volume for 1 Column






Nuclease-free water

4.5 µL 9.3 µL 14.1 µL 18.8 µL 23.6 µL 33.2 µL 64.3 µL


0.5 µL 1.0 µL 1.6 µL 2.1 µL 2.6 µL 3.7 µL 7.1 µL


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



Volume for 1 Column






Nuclease-free water

1.5 µL 3.1 µL 4.7 µL 6.3 µL 7.9 µL 11.1 µL 21.4 µL


0.5 µL 1.0 µL 1.6 µL 2.1 µL 2.6 µL 3.7 µL 7.1 µL


5.0 µL 10.3 µL 15.6 µL 20.9 µL 26.3 µL 36.9 µL 71.4 µL




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


Volume for 1 Column






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.



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



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 Automat

Figure 11 Configuration of the master mix source plate for Hybridization_v1.0.pro.


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.



84



Table 37 Initial MiniHub configuration for Hybridization_v1.0.pro


Table 38 Initial BenchCel configuration for Hybridization_v1.0.pro



Shelf 5 (Top) Empty Empty Empty Empty



Shelf 2 Empty tip box Empty Empty Empty

Shelf 1 (Bottom) Empty Empty Empty Empty











SureSelect Automat


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



86

Run VWorks protocol Hybridization_v1.0.pro

16 On the SureSelect setup form, under Select Protocol to Run, select Hybridization_v1.0.pro.





SureSelect Automat



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.



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.


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 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.



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.
WARNINGWarning
Use caution when handling components that contact heated deck positions.

The Agilent NGS Workstation transfers the capture library-hybridization buffer mixture to the wells of the ABI MicroAmp plate, containing the mixture of prepped gDNA samples and blocking agents.



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.


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.


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


Hybridization 4 Step 3. Capture the hybridized DNA

SureSelect Automat



2 Gently wipe down the Labware MiniHub, Bravo decks, and BenchCel with a NucleoClean decontamination wipe.


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






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.)



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






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


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 Automat



Table 44 Initial MiniHub configuration for SureSelect Capture&Wash_v1.5.rst


Table 45 Initial BenchCel configuration for SureSelectCapture&Wash_v1.5.rst



Shelf 5 (Top) Empty Empty Empty Empty


Shelf 3 Empty Eppendorf plate

Empty Wash #1 Eppendorf source plate from step 10

Empty


Empty Empty

Shelf 1 (Bottom) Empty Empty Empty Empty tip box








12 10 Tip boxes 3 Tip boxes Empty Empty



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


4 Empty black ABI adapter

5 Dynabeads streptavidin bead DeepWell source plate

6 Wash #2 DeepWell source plate seated on silver insert



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.





98



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 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.



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.



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.


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


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.



104


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 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



Volume for 1 Column






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


* Included with the Herculase II Fusion DNA Polymerase. Do not use the buffer or dNTP mix from any other kit.



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



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 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.


9 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate any bubbles.



108



Table 51 Initial MiniHub configuration for Post-CaptureBarcoding_XT_SOLiD_5500_v1.5.pro


Table 52 Initial BenchCel configuration for Post-CaptureBarcoding_XT_SOLiD_5500_v1.5.pro



Shelf 5 (Top)

Empty Empty Empty Barcoding primers in ABI MicroAmp plate seated in black adapter



Shelf 2 Empty tip box Empty Empty Empty

Shelf 1 (Bottom)

New tip box Empty Empty Empty tip box











SureSelect Automat


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



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.





SureSelect Automat



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.



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 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



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


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.






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.




Barcoding 5 Step 2. Purify the amplified barcoded libraries using Agencourt AMPure XP beads

SureSelect Automat








Empty Empty Empty



Empty Empty



Empty


Empty Empty











116



Location Content


4 Barcoded library samples in ABI MicroAmp plate seated in black adapter

8 Empty tip box


Barcoding 5 Step 2. Purify the amplified barcoded libraries using Agencourt AMPure XP beads

SureSelect Automat



12 Under Select additional Parameters, select the step Post-Capture PCR Cleanup and the plate type 96 ABI PCR half skirt in black carrier.


NOTE



118





The purification protocol takes approximately 45 minutes. When complete, the purified DNA samples are in the Eppendorf plate located on Bravo deck position 7.


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.






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.




5 Barcoding Step 3. Remove primer-dimers by purification using Agencourt AMPure XP beads

120



9 Load the BenchCel Microplate Handling Workstation according to


Table 60.




Empty Empty Empty



Empty Empty



Empty


Empty Empty











SureSelect Automat



Location Content


4 Barcoded library samples in Eppendorf plate

8 Empty tip box


5 Barcoding Step 3. Remove primer-dimers by purification using Agencourt AMPure XP beads

122



12 Under Select additional Parameters, select the step Primer-Dimer Cleanup and the plate type 96 Eppendorf Twin.tec PCR.


NOTE



SureSelect Automat





The purification protocol takes approximately 45 minutes. When complete, the purified DNA samples are in the Eppendorf plate located on Bravo deck position 7.


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




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



Stopping Point

Barcoding 5 Step 4. Assess barcoded DNA quality

SureSelect Automat

Figure 13 Analysis of barcoded DNA using the High Sensitivity DNA Assay.


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.


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.


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.

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.


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


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


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.


6Reference

Kit Contents 132

Nucleotide Sequences of SureSelect Barcodes 135

This chapter contains reference information, including component kit contents and barcode sequences.


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


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


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


6 Reference Kit Contents

134

Table 67 SureSelectXT Target Enrichment Box #2, S5500 Content


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


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


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


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





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




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





SureSelect Automat

93 GTTACGATGC

94 ATGGGTGTTT

95 GAGTCCGGCA

96 AATCGAAGAG




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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

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