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Agilent HaloPlex Target Enrichment System Handler Protocol PDF

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Summary of Content for Agilent HaloPlex Target Enrichment System Handler Protocol PDF

HaloPlex Target Enrichment System

Automation Protocol

For Illumina Sequencing

Protocol Version G0, July 2018

For Research Use Only. Not for use in diagnostic procedures.

Agilent Technologies

Notices Agilent Technologies, Inc. 2015, 2018

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Manual Part Number G9900-90020

Edition Version G0, July 2018

Printed in USA

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2 HaloPlex Target Enrichment System Automation Protocol-ILM

In this Guide...

HaloPlex Target Enrichment System

This guide describes an optimized automation protocol for using the HaloPlex target enrichment system to prepare sequencing library samples for Illumina paired- end multiplexed sequencing platforms. Sample processing steps are automated using the Agilent NGS Bravo Option A.

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 Bravo for HaloPlex Target Enrichment

This chapter contains an orientation to the Agilent NGS Bravo Option A, an overview of the HaloPlex target enrichment protocol, and considerations for designing HaloPlex experiments for automated processing using the Agilent NGS Bravo Option A.

3

Sample Preparation

This chapter describes the steps of the automated HaloPlex workflow to prepare target- enriched sequencing libraries for the Illumina platform.

4

Appendix: Provisional Adaptor-Dimer Removal Protocol

This chapter describes a protocol used to remove adaptor- dimer (125 bp) molecules that may be observed for some designs.

5

Reference

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

Automation Protocol-ILM 3

Whats New in Version G0

4

Support for Tier 1 Plus probe designs (see Table 2 on page 10, Table 19 on page 49, and Table 34 on page 82)

Removal of support information for discontinued kits p/n G9903B, G9908B, and G9913B (see Table 2 on page 10 and Table 35 on page 82)

Updates to sequencing support details (see page 73 through page 76)

Update to acceptable electrophoresis result description for restriction digest G (see Note on page 40)

Support for the Agilent 4200 TapeStation system (see Table 4 on page 12 and see page 42 and page 71)

Updates to Agilent 2100 Bioanalyzer system ordering information (see Table 4 on page 12)

Updates to supplier name for materials purchased from Thermo Fisher Scientific (see Table 1 on page 9, Table 3 on page 11, and Table 4 on page 12)

Support for VWorks software version 13.1.0.1366 (see Table 3 on page 11)

Updates to information for Agilent NGS Workstation component user guides (see Table 6 on page 16)

Removal of reference information for obsolete kits containing 8- bp indexing primers 196, provided in a clear plate (typically received before December 2014). If you need assistance with kits containing these obsolete indexing primer components, please contact Technical Support.

Updates to Technical Support contact information (see page 2)

HaloPlex Target Enrichment System Automation Protocol-ILM

Content

1 Before You Begin

Procedural Notes 8 Safety Notes 8 Required Reagents 9 Required Equipment 11 Optional Validation Reagents and Equipment 12 Using the Agilent NGS Workstation Option B for HaloPlex Automation 13

2 Using the Agilent NGS Bravo for HaloPlex Target Enrichment

About the NGS Bravo Option A 16

About the Bravo Platform 16 VWorks Automation Control Software 20

Overview of the HaloPlex Target Enrichment Procedure 26

Experimental Setup Considerations for Automated Runs 28

Considerations for Placement of gDNA Samples in 96-well Plates for Automated Processing 29

Considerations for Equipment Setup 29 Run Time Considerations 29

3 Sample Preparation

Step 1. Digest genomic DNA with restriction enzymes 32 Step 2. Hybridize digested DNA to HaloPlex or ClearSeq probe for target

enrichment and sample indexing 44 Step 3. Capture and amplify the target DNA 50 Step 4. Purify the amplified target DNA 65 Step 5. Validate enrichment and quantify enriched target DNA 69 Step 6. Pool samples with different indexes for multiplexed sequencing 73

4 Appendix: Provisional Adaptor-Dimer Removal Protocol

Purify the enriched library pool using AMPure XP beads 78

HaloPlex Target Enrichment System Automation Protocol-ILM 5

5 Reference

Kit Contents 82

Nucleotide Sequences of HaloPlex Indexes 85

Qualitative analysis of enrichment by gel electrophoresis 86

6 HaloPlex Target Enrichment System Automation Protocol-ILM

HaloPlex Target Enrichment System Protocol

1 Before You Begin

Procedural Notes 8

Safety Notes 8

Required Reagents 9

Required Equipment 11

Optional Validation Reagents and Equipment 12

Using the Agilent NGS Workstation Option B for HaloPlex Automation 13

Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an experiment.

7Agilent Technologies

1 Before You Begin Procedural Notes

Procedural Notes

8

The 96 reaction kit contains enough reagents to prepare master mixes for four runs of 3 columns of samples (24 samples) per run. When processing samples using runs with fewer than 24 samples, some reagents may be depleted before 96 samples are run.

The HaloPlex protocol is optimized for digestion of 200 ng of genomic DNA (split among 8 different restriction digestion reactions) plus 25 ng excess DNA, for a total of 225 ng genomic DNA. Using lower amounts of DNA in the enrichment protocol can adversely affect your results. Use a fluorometry- based DNA quantitation method, such as PicoGreen stain or Qubit fluorometry to quantify the DNA starting material.

Always keep pre- amplification and post- amplification DNA samples in separate work areas. Perform the enrichment procedure in the pre- amplification area. Open and store the amplified, enriched DNA samples only in the post- amplification area.

Possible stopping points, where DNA samples may be stored between steps, are marked in the protocol. Store the samples at 20C, but do not subject the samples to multiple freeze/thaw cycles.

Ensure that master mixes are thoroughly mixed, by pipetting up- and- down or by gentle vortexing, before distributing to the samples.

In general, follow Biosafety Level 1 (BL1) safety rules.

Safety Notes

Wear appropriate personal protective equipment (PPE) when working in the laboratory.

CAUTION

HaloPlex Target Enrichment System Automation Protocol-ILM

Before You Begin 1 Required Reagents

Required Reagents

HaloPlex Target Enr

Table 1 Required Reagents for HaloPlex Target Enrichment

Description Vendor and part number

HaloPlex Target Enrichment System Kit or ClearSeq Target Enrichment System Kit

Select the appropriate kit for your probe design from Table 2

Herculase II Fusion Enzyme with dNTPs (100 mM; 25 mM for each nucleotide), 200 reactions

Agilent p/n 600677

Nuclease-free Water (not DEPC-treated) Thermo Fisher Scientific p/n AM9930

Agencourt AMPure XP Kit 5 mL 60 mL 450 mL

Beckman Coulter Genomics p/n A63880 p/n A63881 p/n A63882

10 M NaOH, molecular biology grade Sigma, p/n 72068

2 M acetic acid Sigma, p/n A8976

10 mM Tris-HCl, pH 8.0 or 10 mM Tris-acetate, pH 8.0 General laboratory supplier

100% Ethanol, molecular biology grade Sigma-Aldrich p/n E7023

Quant-iT dsDNA BR Assay Kit, for use with the Qubit fluorometer

100 assays, 2-1000 ng 500 assays, 2-1000 ng

Thermo Fisher Scientific p/n Q32850 Thermo Fisher Scientific p/n Q32853

ichment System Automation Protocol-ILM 9

1 Before You Begin Required Reagents

10

Before ordering a HaloPlex Target Enrichment System Reagent Kit, use Agilents SureDesign tool at www.agilent.com/genomics/suredesign to design a custom HaloPlex probe or to select a pre- designed ClearSeq disease research probe. Reagent kit ordering information is supplied as part of the SureDesign process and is summarized in Table 2 below.

Table 2 HaloPlex and ClearSeq Target Enrichment System Kits for Illumina Sequencing

Probe Design Part Number-96 Reactions

HaloPlex Custom Panel Tier 1*, ILMFST

* Tier 1 designs are 1-500 kb with <15,000 probes and require a 3-hour hybridization protocol. Tier 1 Plus designs are also 1-500 kb, but contain 15,000 to 20,000 probes and require a 16-hour hybridization protocol.

G9901B

HaloPlex Custom Panel Tier 1 Plus*, ILM G9961B

HaloPlex Custom Panel Tier 2, ILM

Tier 2 designs are 0.5-2.5 Mb OR 1-500 kb with >20,000 probes.

G9911B

HaloPlex Custom Panel Tier 3, ILM

Tier 3 designs are 2.6 Mb-5 Mb.

G9921B

ClearSeq ICCG, ILM G9953B**

** Select the appropriate made-to-order probe option in SureDesign.

ClearSeq Connective Disorder, ILM G9953B**

ClearSeq Arrhythmia, ILM G9953B**

ClearSeq Noonan Syndrome, ILM G9953B**

ClearSeq Chromosome X, ILM G9953B**

NOTE Kits contain enough reagents for 96 reactions total, including one or more control reactions using Enrichment Control DNA (ECD) samples. Each run of up to 96 samples should include one ECD control enrichment reaction.

HaloPlex Target Enrichment System Automation Protocol-ILM

Before You Begin 1 Required Equipment

Required Equipment Table 3 Required Equipment for HaloPlex Target Enrichment Automated Protocols

Description Vendor and part number

Agilent NGS Bravo Option A* with VWorks software version 13.1.0.1366 or 11.3.0.1195

* Protocols are also compatible with Agilent NGS Workstation Option B. See page 13 for more information.

Contact Agilent Automation Solutions for ordering information: Customerservice.automation@agilent.com

PlateLoc Thermal Microplate Sealer with Small Hotplate Agilent p/n G5402#226

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

Thermal Cycler Agilent SureCycler 8800, p/n G8800A, 96 well plate module, p/n G8810A, and 384 well plate module, p/n G8820A, or equivalent thermal cycler and accessories

Thermal cycler must have a maximum reaction volume specification of at least 100 L and be compatible with 0.2 mL tubes.

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

Eppendorf twin.tec full-skirted 384-well PCR plates* Eppendorf p/n 951020702

Eppendorf twin.tec half-skirted 96-well PCR plates

Compatible with Agilent SureCycler 8800.

Eppendorf p/n 951020303

Thermo Scientific Reservoirs Thermo Fisher Scientific p/n 1064156

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

Axygen 96 Deep Well Plate, 2 mL, Square Well (waste reservoirs; working volume 2.2 mL)

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

Magnetic separator **

1.5 mL tube-compatible separator or Conical vial-compatible separator

** Select the appropriate device based on run size. See page 55 to determine magnetic bead volume to be used for your run size.

Thermo Fisher Scientific DynaMag-2 magnet, p/n 12321D or equivalent DynaMag-15 magnet, p/n 12301D or equivalent

Benchtop microcentrifuge VWR p/n 93000-196, or equivalent

Benchtop plate centrifuge Labnet International MPS1000 Mini Plate Spinner p/n C1000, or equivalent

P10, P20, P200 and P1000 pipettes Pipetman P10, P20, P200, P1000 or equivalent

Nucleic acid surface decontamination wipes Thermo Fisher Scientific p/n 7008, or equivalent

Qubit Fluorometer Thermo Fisher Scientific p/n Q33226

Qubit assay tubes Thermo Fisher Scientific p/n Q32856

Vortex mixer General laboratory supplier

HaloPlex Target Enrichment System Automation Protocol-ILM 11

1 Before You Begin Optional Validation Reagents and Equipment

Optional Validation Reagents and Equipment

12

Table 4 Reagents and Equipment for Optional Validation Methods

Description Vendor and part number

Agilent 4200 TapeStation*

96-well sample plates

96-well plate foil seals

8-well tube strips

8-well tube strip caps

High Sensitivity D1000 ScreenTape

High Sensitivity D1000 Reagents

* DNA samples may also be analyzed using the Agilent 2200 TapeStation, p/n G2964AA or G2965AA. ScreenTape devices and associated reagents listed in this table are compatible with both platforms.

Agilent p/n G2991AA

Agilent p/n 5042-8502

Agilent p/n 5067-5154

Agilent p/n 401428

Agilent p/n 401425

Agilent p/n 5067-5584

Agilent p/n 5067-5585

Agilent 2100 Bioanalyzer Instrument

Agilent 2100 Expert SW Laptop Bundle (optional)

High Sensitivity DNA Kit

Agilent p/n G2939BA

Agilent p/n G2953CA

Agilent p/n 5067-4626

Gel Electrophoresis Platform and Consumables

XCell SureLock Mini-cell

Novex 6% Polyacrylamide, TBE Pre-cast Gels

Novex TBE Running Buffer, 5X

Novex High-density TBE Sample Buffer, 5X

GelRed Nucleic Acid Stain, 3X in water

DNA molecular weight markers

UV-transilluminator

Thermo Fisher Scientific p/n EI0001

Thermo Fisher Scientific p/n EC62655BOX

Thermo Fisher Scientific p/n LC6675

Thermo Fisher Scientific p/n LC6678

Biotium p/n 41001

General laboratory supplier

General laboratory supplier

HaloPlex Target Enrichment System Automation Protocol-ILM

Before You Begin 1 Using the Agilent NGS Workstation Option B for HaloPlex Automation

Using the Agilent NGS Workstation Option B for HaloPlex Automation

HaloPlex Target Enr

HaloPlex target enrichment protocols are compatible with the Agilent NGS Workstation Option B. Depending on the configuration of the system purchased, however, additional adapters may be required. Before initiating experiments, see Table 5 below, and verify that the listed adapters are available for your workstation.

Table 5 Adapter checklist for HaloPlex automation using Agilent NGS Workstation Option B

Adapter Description Quantity Required for HaloPlex Automation

Agilent part number (single adapter)

384-well plate insert 2 G5498B#60

96-well PCR plate insert (red)*

* If your NGS Workstation Option B system is already equipped with one red insert, purchase one additional insert using the ordering information shown.

2 G5498B#13

ichment System Automation Protocol-ILM 13

1 Before You Begin Using the Agilent NGS Workstation Option B for HaloPlex Automation

14

HaloPlex Target Enrichment System Automation Protocol-ILM

HaloPlex Target Enrichment System Protocol

2 Using the Agilent NGS Bravo for HaloPlex Target Enrichment

About the NGS Bravo Option A 16

Overview of the HaloPlex Target Enrichment Procedure 26

Experimental Setup Considerations for Automated Runs 28

This chapter contains an orientation to the Agilent NGS Bravo (Option A), an overview of the HaloPlex target enrichment protocol, and considerations for designing HaloPlex experiments for automated processing using the Agilent NGS Bravo.

15Agilent Technologies

2 Using the Agilent NGS Bravo for HaloPlex Target Enrichment About the NGS Bravo Option A

About the NGS Bravo Option A

CAUTION Before you begin, make sure that you have read and understand operating, maintenance and safety instructions for using the Bravo platform and additional devices and software. Refer to the user guides listed in Table 6.

16

Review the user guides listed in Table 6 (available at Agilent.com) to become familiar with the general features and operation of the automation components. Instructions for using the Bravo platform and other automation components in the HaloPlex Target Enrichment workflow are detailed in this user guide.

Table 6 Agilent NGS Bravo User Guide reference information

Device User Guide part number

Bravo Platform G5562-90000

VWorks Software G5415-90063

PlateLoc Thermal Microplate Sealer G5402-90001

About the Bravo Platform

The Bravo platform is a versatile liquid handler with a nine plate- location platform deck, suitable for handling 96- well, 384- well, and 1536- well plates. The Bravo platform is controlled by the VWorks Automation Control software. Fitted with a choice of seven interchangeable fixed- tip or disposable- tip pipette heads, it accurately dispenses fluids from 0.1 l to 250 l.

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.

HaloPlex Target Enrichment System Automation Protocol-ILM

Using the Agilent NGS Bravo for HaloPlex Target Enrichment 2 About the Bravo Platform

HaloPlex Target Enr

Figure 1 Bravo platform deck

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 low- temperature (4C) or high- temperature (54C) incubation steps may be expedited by pre- setting the temperature of the affected block before starting the run.

Bravo deck heat block temperatures may be changed using the Inheco Multi TEC Control device touchscreen as described in the steps below. See Table 7 for designations of the heat block- containing Bravo deck positions on the Multi TEC control device.

ichment System Automation Protocol-ILM 17

2 Using the Agilent NGS Bravo for HaloPlex Target Enrichment About the Bravo Platform

18

1 Using the arrow buttons, select the appropriate block (CPAC 2 block 1 or CPAC 2 block 2).

2 To set the temperature of the selected block, press the SET button.

Table 7 Inheco Multi TEC Control touchscreen designations

Bravo Deck Position Designation on Inheco Multi TEC Control Screen

4 CPAC 2 1

6 CPAC 2 2

HaloPlex Target Enrichment System Automation Protocol-ILM

Using the Agilent NGS Bravo for HaloPlex Target Enrichment 2 About the Bravo Platform

HaloPlex Target Enr

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.

ichment System Automation Protocol-ILM 19

2 Using the Agilent NGS Bravo for HaloPlex Target Enrichment VWorks Automation Control Software

VWorks Automation Control Software

20

VWorks software, included with your Agilent NGS Bravo, allows you to control the robot and integrated devices using a PC. The Agilent NGS Bravo Option A is preloaded with VWorks software containing all of the necessary HaloPlex 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 HaloPlex 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 13.1.0.1366 or 11.3.0.1195.

If you have questions about VWorks version compatibility, please contact service.automation@agilent.com.

Logging in to the VWorks software

1 Double- click the VWorks icon or the HaloPlex.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.)

HaloPlex Target Enrichment System Automation Protocol-ILM

Using the Agilent NGS Bravo for HaloPlex Target Enrichment 2 VWorks Automation Control Software

HaloPlex Target Enr

Using the HaloPlex.VWForm to setup and start a run

Use the VWorks form HaloPlex.VWForm, shown below, to set up and start each HaloPlex automation protocol.

1 Open the form using the HaloPlex.VWForm shortcut on your desktop.

2 Use the drop- down menus on the form to select the appropriate HaloPlex workflow step and number of columns of samples for the run.

3 Once all run parameters have been specified on the form, click Update layout and information.

The displayed protocol will not run unless the Update layout and information button has been clicked.

NOTE

4 The Bravo Deck Setup region of the form will then display the required placement of reaction components and labware on the NGS Bravo deck for the specified run parameters.

ichment System Automation Protocol-ILM 21

2 Using the Agilent NGS Bravo for HaloPlex Target Enrichment VWorks Automation Control Software

22

5 Verify that the Current Tip State indicator on the form (shown below) matches the configuration of unused tips in the tip box at Bravo Deck position 2.

For a fresh tip box, containing 12 columns of tips, all positions of the Current Tip State unused tip indicator (top portion, Box 2) should be selected, as shown below. Clicking Reset selects all columns for position 2.

Also verify that the used tip indicator (bottom portion, Box 8) matches the configuration of used tips in the tip box at Bravo Deck position 8.

For an empty tip box, all positions of the Current Tip State used tip indicator (bottom portion, Box 8) should be cleared, as shown above. Clicking Reset clears all columns for position 8.

It is important that the Current Tip State indicator matches the configuration of tips present at Bravo Deck positions 2 and 8 when initiating the run. Tips that are inappropriately loaded onto the Bravo platform pipette head, or tips missing from the pipette head, will interfere with automated processing steps.

You can use partial tip boxes for HaloPlex automation protocols, as long as positions of available tips are accurately indicated during run setup.

NOTE

6 After verifying that the NGS Bravo has been set up correctly, click Start in the Controls section of the form to begin the run. Do not use the Start button on the VWorks Control Toolbar; runs must be initiated using the start button on the HaloPlex.VW Form, shown below.

HaloPlex Target Enrichment System Automation Protocol-ILM

Using the Agilent NGS Bravo for HaloPlex Target Enrichment 2 VWorks Automation Control Software

HaloPlex Target Enr

Error messages encountered at start of run

After starting the run, you may see the error messages displayed below. When encountered, make the indicated selections and proceed with the run. Encountering either or both of these error messages is not indicative of a problem with the NGS Bravo or your run setup.

1 If you encounter the G- axis error message shown below, select Ignore and Continue, leaving device in current state.

ichment System Automation Protocol-ILM 23

2 Using the Agilent NGS Bravo for HaloPlex Target Enrichment VWorks Automation Control Software

24

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

HaloPlex Target Enrichment System Automation Protocol-ILM

Using the Agilent NGS Bravo for HaloPlex Target Enrichment 2 VWorks Automation Control Software

HaloPlex Target Enr

Verifying the Simulation setting

VWorks software may be run in simulation mode, during which commands entered on screen are not completed by the NGS Bravo. If NGS Bravo devices do not respond when you start a run, verify the simulation mode status in VWorks using the following steps.

1 Verify that Simulation is off is displayed on the status indicator (accessible by clicking View > Control Toolbar).

2 If the indicator displays Simulation is on, click the status indicator button to turn off the simulation mode.

If you cannot see the toolbar above the HaloPlex. VWorks form, click Screen in the Controls section of the formto 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

ichment System Automation Protocol-ILM 25

2 Using the Agilent NGS Bravo for HaloPlex Target Enrichment Overview of the HaloPlex Target Enrichment Procedure

Overview of the HaloPlex Target Enrichment Procedure

26

Figure 2 summarizes the HaloPlex target enrichment workflow. For each sample to be sequenced, individual HaloPlex- enriched, indexed libraries are prepared. Depending on the specific sequencing platform used, up to 96 samples can be pooled and sequenced in a single lane.

Table 8 summarizes how the VWorks automation protocols are integrated into the HaloPlex workflow. See the Sample Preparation chapter for complete instructions for use of the VWorks protocols for sample processing.

Table 8 Overview of VWorks protocols used during the workflow

Workflow Step VWorks Protocol used for Automation

Digest genomic DNA Digestion.pro

Hybridize to HaloPlex or ClearSeq probe and index samples

Hybridization.pro

Capture and amplify enriched DNA Capture_v1.1.pro

Purify amplified libraries Purification_v1.1.pro

HaloPlex Target Enrichment System Automation Protocol-ILM

Using the Agilent NGS Bravo for HaloPlex Target Enrichment 2 Overview of the HaloPlex Target Enrichment Procedure

HaloPlex Target Enr

Figure 2 Overall HaloPlex target-enriched sequencing sample preparation workflow.

ichment System Automation Protocol-ILM 27

2 Using the Agilent NGS Bravo for HaloPlex Target Enrichment Experimental Setup Considerations for Automated Runs

Experimental Setup Considerations for Automated Runs

28

HaloPlex Automated Target Enrichment System runs may include 1, 2, 3, 4, 6, or 12 columns (equivalent to 8, 16, 24, 32, 48, or 96 wells) of gDNA samples to be enriched for sequencing on the Illumina platform. Plan your experiments using complete columns of samples.

The number of columns or samples that may be processed using the supplied reagents (see page 82) 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 9 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

HaloPlex Target Enrichment System Automation Protocol-ILM

Using the Agilent NGS Bravo for HaloPlex Target Enrichment 2 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

HaloPlex Target Enr

The Agilent NGS Bravo processes samples column- wise beginning at column 1. gDNA samples should be loaded into 96- well plates column- wise, in well order A1 to H1, then A2 to H2, ending with A12 to H12. When processing partial runs with <12 sample columns, do not leave empty columns between sample columns; always load the plate using the left- most column that is available.

For sample indexing during hybridization to the HaloPlex or ClearSeq probe (see Figure 2), you will need to prepare a separate plate containing the HaloPlex Indexing Primers. Assign the wells to be indexed with their respective indexing primers during experimental design. See the Reference chapter for nucleotide sequences of the 96 indexes used in the HaloPlex Target Enrichment System.

Considerations for Equipment Setup

Some workflow steps require the rapid transfer of sample plates between the Bravo deck and a thermal cycler. Locate your thermal cycler in close proximity to the Agilent NGS Bravo to allow rapid and efficient plate transfer.

Some workflow steps require that the sample plate be sealed, then centrifuged to collect any dispersed liquid, before being transfered between instruments. To maximize efficiency, locate the PlateLoc thermal microplate sealer and the centrifuge in close proximity to the NGS Bravo and thermal cycler.

Run Time Considerations

Before you begin, refer to the Certificate of Analysis provided with Box 1 of your kit to determine the hybridization duration appropriate for your design. After reviewing the duration of this and other steps in the protocol, plan the start time for your experiment accordingly.

ichment System Automation Protocol-ILM 29

2 Using the Agilent NGS Bravo for HaloPlex Target Enrichment Run Time Considerations

30

Designs containing <15,000 probes use a 3- hour hybridization time. For these designs DNA digestion through PCR protocols (see Figure 2) are typically run on the same day with the DNA digestion protocol initiated early in the day.

Designs containing 15,000 probes use a 16- hour hybridization time, which is typically completed overnight. Calculate the appropriate start time for the DNA digestion protocol, based on your run size and the run time estimates provided in the HaloPlex form in the VWorks software (HaloPlex.VWForm), to allow overnight hybridization.

HaloPlex Target Enrichment System Automation Protocol-ILM

HaloPlex Target Enrichment System Protocol

3 Sample Preparation

Step 1. Digest genomic DNA with restriction enzymes 32

Step 2. Hybridize digested DNA to HaloPlex or ClearSeq probe for target enrichment and sample indexing 44

Step 3. Capture and amplify the target DNA 50

Step 4. Purify the amplified target DNA 65

Step 5. Validate enrichment and quantify enriched target DNA 69

Step 6. Pool samples with different indexes for multiplexed sequencing 73

This section contains instructions for gDNA library target enrichment for sequence analysis using the Illumina platform. For each sample to be sequenced, an individual target- enriched, indexed library is prepared.

The target region can vary from 1 kb to 5 Mb. Custom HaloPlex probes must be designed before purchasing the kit using Agilents SureDesign tool at www.agilent.com/genomics/suredesign.

The HaloPlex Target Enrichment System amplifies thousands of targets in the same reaction, incorporating standard Illumina paired- end sequencing motifs in the process. During hybridization, each sample can be uniquely indexed, allowing for pooling of up to 96 samples per sequencing lane.

31Agilent Technologies

3 Sample Preparation Step 1. Digest genomic DNA with restriction enzymes

Step 1. Digest genomic DNA with restriction enzymes

32

In this step, gDNA samples are digested by 16 different restriction enzymes to create a library of gDNA restriction fragments. The gDNA is digested in eight different restriction reactions, each containing two restriction enzymes. The 16 restriction enzymes are provided in two 8- well strip tubes that are distinguished by red and green color markers. Enzymes are combined from corresponding wells of the red- and green- marked strip tubes to make eight different RE Master Mixes, which are then combined with each DNA sample in the run.

NOTE Successful enrichment using the protocol in this guide requires high-quality DNA samples. Before you begin, verify that the genomic DNA samples have an OD 260/280 ratio ranging from 1.8 to 2.0. Verify the size distribution of DNA in each DNA preparation by gel electrophoresis. Any smearing below 2.5 kb indicates sample degradation.

For HaloPlex target enrichment of FFPE-derived DNA samples, see Agilent publication no. G9900-90050, available at http://www.genomics.agilent.com. This publication provides a PCR-based protocol for assessment of DNA integrity and provides HaloPlex protocol modifications for improved performance from lower-quality DNA samples.

Prepare the NGS Bravo

1 Gently wipe down the Bravo deck with a surface decontamination wipe.

2 Place red aluminum inserts on Bravo deck positions 4 and 9.

3 Turn on the chiller, set to 0C, at position 9 of the Bravo deck. Be sure that the chiller reservoir contains at least 300 mL of 25% ethanol.

4 Place a 384- well adapter insert on Bravo deck position 6.

5 Pre- set the temperature of Bravo deck positions 4 and 6 to 4C using the Inheco Multi TEC control touchscreen, as described in Setting the Temperature of Bravo Deck Heat Blocks.

NOTE To expedite thermal cycler warm-up for the restriction digest incubation on page 38, you can enter and initiate the digestion program on the thermal cycler now, and then pause the program until you are instructed to transfer the reaction plate. Be sure to pause the thermal cycler before the initiation of the 37C incubation segment. Release the pause immediately after transferring the plate to the thermal cycler in step 14 on page 38. Be sure that the 384-well block is in the thermal cycler before initiating the program for warm-up.

HaloPlex Target Enrichment System Automation Protocol-ILM

Sample Preparation 3 Step 1. Digest genomic DNA with restriction enzymes

HaloPlex Target Enr

Prepare the DNA Sample Source Plate

In the protocol below, 200 ng genomic DNA is split among eight different restriction digests, with an additional 25 ng excess DNA included to allow for pipetting losses. Using <225 ng DNA in the enrichment protocol can result in low yield and can potentiate rare allele dropouts. Use a fluorometry-based DNA quantitation method, such as Qubit fluorometry or PicoGreen staining, to accurately quantify the DNA starting material.

NOTE

1 Use the Qubit dsDNA BR Assay or PicoGreen staining kit to determine the concentration of your gDNA samples.

Follow the manufacturers instructions for the kits and instruments.

2 Prepare the DNA sample plate for the run, containing up to 95 gDNA samples and the Enrichment Control DNA sample, using a full- skirted 96- well Eppendorf twin.tec plate.

HaloPlex Automated Target Enrichment System runs may include 1, 2, 3, 4, 6, or 12 columns of the plate. Use full columns of DNA samples for each run.

NOTE

a In well A1 of a 96- well twin.tec plate, dispense 45 L of the supplied Enrichment Control DNA (ECD). Store on ice.

b In separate wells of the same 96- well twin.tec plate, dilute 225 ng of each gDNA sample in 45 L nuclease- free water, for a final DNA concentration of 5 ng/L. Continue to store on ice.

For automated processing, fill plate wells column- wise in well order A1 to H1, then A2 to H2, ending with A12 to H12.

Prepare the RE Master Mix Source Plate

1 Prepare the appropriate amount of RE Buffer +BSA mixture, according to the table below.

Table 10 Preparation of RE Buffer + BSA mixture for Digestion.pro protocol

Reagent Volume for 1 Library

Volume for 1 Column

Volume for 2 Columns

Volume for 3 Columns

Volume for 4 Columns

Volume for 6 Columns

Volume for 12 Columns

RE Buffer 34 L 408 L 680 L 952 L 1224 L 1768 L 3536 L

BSA Solution 0.85 L 10.2 L 17 L 23.8 L 30.6 L 44.2 L 88.4 L

Total Volume 34.85 L 418.2 L 697 L 975.8 L 1254.6 L 1812.2 L 3624.4 L

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3 Sample Preparation Step 1. Digest genomic DNA with restriction enzymes

34

2 Obtain the two provided green- and red- marked Enzyme Strips from Box 1. For each strip, label the color- marked tube with A, then continue labeling the remaining tubes with B through H, in order. Keep the strips on ice.

CAUTION It is important to use the restriction enzyme tube strips in the proper orientation when preparing the RE Master Mixes as described below. The red or green color marker on the tube strip and cap strip are used to mark well A of each enzyme strip.

3 In eight individual tubes, prepare the eight Restriction Enzyme Master Mixes A, B, C, D, E, F, G, and H according to the table below. To prepare Master Mix A, combine RE Buffer + BSA from step 1 with the indicated volumes of enzyme solution from well A of the Green Enzyme Strip and from well A of the Red Enzyme Strip. Prepare Master Mixes BH by repeating this process using enzyme solutions from the corresponding wells B- H of each provided Enzyme Strip.

Table 11 Preparation of RE Master Mixes AH for Digestion.pro protocol

Reagent Volume for 1 Library

Volume for 1 Column

Volume for 2 Columns

Volume for 3 Columns

Volume for 4 Columns

Volume for 6 Columns

Volume for 12 Columns

RE Buffer + BSA 4.0 51.0 L 85.0 L 119.0 L 153.0 L 221.0 L 442.0 L

Green Enzyme Strip enzyme AH

0.5 6.4 L 10.6 L 14.9 L 19.1 L 27.6 L 55.25 L

Red Enzyme Strip enzyme AH

0.5 6.4 L 10.6 L 14.9 L 19.1 L 27.6 L 55.25 L

Total Volume for each Master Mix A, B, C, D, E, F, G, or H

5 L 63.8 L 106.2 L 148.8 L 191.2 L 276.2 L 552.5 L

For 1-4 column runs, RE master mixes A-H may be prepared in a 8 x 0.2-mL well strip tube,

NOTE using a multichannel pipette to transfer volumes from Enzyme Strips 1 and 2 to the RE master mix strip. For 6- or 12-column runs, prepare the master mixes in 1.5-mL tubes.

4 Mix by gentle vortexing and then spin briefly. Keep on ice.

HaloPlex Target Enrichment System Automation Protocol-ILM

Sample Preparation 3 Step 1. Digest genomic DNA with restriction enzymes

HaloPlex Target Enr

Prepare the RE master mix source plate

1 Aliquot the Restriction Enzyme Master Mixes to a full- skirted 96- well Eppendorf twin.tec plate as shown in Figure 3. Add the volumes indicated in Table 12 of each master mix AH to each well of the indicated column of the twin.tec plate. Keep the master mixes on ice during the aliquoting steps.

Table 12 Preparation of the RE Master Mix Source Plate for Digestion.pro

Master Mix Solution

Position on Source Plate

Volume of Master Mix added per Well of Source Plate

1-Column Runs

2-Column Runs

3-Column Runs

4-Column Runs

6-Column Runs

12-Column Runs

RE Master Mix A Column 1 (A1-H1) 7.3 L 12.7 L 18.0 L 23.3 L 33.9 L 68.4 L

RE Master Mix B Column 2 (A2-H2) 7.3 L 12.7 L 18.0 L 23.3 L 33.9 L 68.4 L

RE Master Mix C Column 3 (A3-H3) 7.3 L 12.7 L 18.0 L 23.3 L 33.9 L 68.4 L

RE Master Mix D Column 4 (A4-H4) 7.3 L 12.7 L 18.0 L 23.3 L 33.9 L 68.4 L

RE Master Mix E Column 5 (A5-H5) 7.3 L 12.7 L 18.0 L 23.3 L 33.9 L 68.4 L

RE Master Mix F Column 6 (A6-H6) 7.3 L 12.7 L 18.0 L 23.3 L 33.9 L 68.4 L

RE Master Mix G Column 7 (A7-H7) 7.3 L 12.7 L 18.0 L 23.3 L 33.9 L 68.4 L

RE Master Mix H Column 8 (A8-H8) 7.3 L 12.7 L 18.0 L 23.3 L 33.9 L 68.4 L

Figure 3 Preparation of the RE Master Mix source plate for automation protocol Digestion.pro.

ichment System Automation Protocol-ILM 35

3 Sample Preparation Step 1. Digest genomic DNA with restriction enzymes

36

Load the NGS Bravo and Run the Digestion.pro VWorks Protocol

1 Open the HaloPlex setup form using the HaloPlex.VWForm shortcut on your desktop.

2 Log in to the VWorks software.

3 On the setup form, under Step, select 01 Digestion.pro.

4 Select the number of columns of samples to be processed. Runs must include 1, 2, 3, 4, 6, or 12 columns.

5 Click Update layout and information.

6 Load the Bravo deck according to Table 13.

Table 13 Initial Bravo deck configuration for Digestion.pro

7 Verify that the NGS Bravo has been set up as displayed in the Bravo Deck Setup and Information regions of the form.

Location Content

1 (empty)

2 New tip box

3 For 12-column runs only: Empty 384-well Eppendorf twin.tec plate (no 384-well insert required)

For 1- to 6-column runs: empty

4 gDNA samples in full-skirted 96-well Eppendorf twin.tec plate seated on red insert

5 (empty)

6 Empty 384-well Eppendorf twin.tec plate seated on 384-well insert

7 (empty)

8 Empty tip box

9 RE Master Mix source plate (full-skirted 96-well Eppendorf twin.tec plate) seated on red insert

HaloPlex Target Enrichment System Automation Protocol-ILM

Sample Preparation 3 Step 1. Digest genomic DNA with restriction enzymes

HaloPlex Target Enr

8 Verify that the Current Tip State indicator on the form matches the configuration of unused and used tips in the tip boxes at Bravo Deck positions 2 and 8, respectively. See page 22 for more information on using this segment of the form during the run.

9 When verification is complete, click Start to start the run.

NOTE If Bravo 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 25 for more information.

10 When prompted by VWorks as shown below, replace the tip box at position 2 with a new tip box and replace the used tip box at position 8 with an empty tip box. After both tip boxes are in place, click Reset under Current Tip State on the form. Verify that the tip state was updated and then click Continue on the prompt shown below.

Depending on the run size, you may be prompted to change tip boxes multiple times during the run.

The NGS Bravo combines each gDNA sample with each RE Master Mix in wells of a 384- well reaction plate. For 1- to 6- column runs, a single 384- well restriction digest plate is prepared; for 12 column runs, two 384- well restriction digest plates are prepared.

ichment System Automation Protocol-ILM 37

3 Sample Preparation Step 1. Digest genomic DNA with restriction enzymes

38

11 When the NGS Bravo has finished preparing each 384- well restriction digest plate for the run, you will be prompted by VWorks as shown below.

The final Bravo deck position of the prepared restriction digest plate varies for different run sizes.

12 Remove the 384- well plate from the Bravo deck position indicated in the prompt.

13 Seal the sample plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165C and 3.0 sec. Spin the plate briefly to release any bubbles trapped in the liquid.

14 Transfer the sealed plate to a thermal cycler and run the digestion program shown in Table 14, using a heated lid. After transferring the plate, click Continue on the prompt.

Table 14 Thermal cycler program for restriction digestion

Preparation of each restriction digest reaction plate takes approximately 30- 45 minutes.

Step Temperature Time

Step 1 37C 30 minutes

Step 2 8C Hold

HaloPlex Target Enrichment System Automation Protocol-ILM

Sample Preparation 3 Step 1. Digest genomic DNA with restriction enzymes

HaloPlex Target Enr

For 12 column- runs, the two 384- well plates are prepared sequentially, for a total run time of approximately 90 minutes. Run the thermal cycler digestion program for each plate as soon as prompted. During the 30- minute incubation of plate 1 in the thermal cycler, the NGS Bravo begins preparation of the digestion reactions in plate 2. Once the thermal cycler program is complete for plate 1, store the digested DNA in plate 1 on ice until the Digestion.pro protocol and thermal cycler program for plate 2 is finished.

15 Validate the restriction digestion reaction by electrophoretic analysis of the Enrichment Control DNA (ECD) reactions.

a Transfer 4 L of each ECD digestion reaction from the wells of the 384- well reaction plate indicated in Table 15 to fresh 0.2- mL PCR tubes. Note that for 12- column runs, four of the eight ECD digests are found on the first 384- well plate, and the remaining four digests are on the second 384- well plate.

Table 15 Position of ECD digestion reactions for obtaining validation samples

b Incubate the removed 4- L samples at 80C for 5 minutes to inactivate the restriction enzymes.

c Analyze the prepared samples by electrophoresis using the Agilent 2100 Bioanalyzer (see page 41), or the Agilent 4200 TapeStation (see page 42), or by gel electrophoresis (see page 43).

Restriction Enzyme Master Mix to be Validated

Position of ECD Digestion Reaction in 384-Well Plates

1-6 Column Runs 12-Column Runs (two 384-well plates produced)

RE Master Mix A A1 A1 (plate 1)

RE Master Mix B A2 A2 (plate 1)

RE Master Mix C B1 B1 (plate 1)

RE Master Mix D B2 B2 (plate 1)

RE Master Mix E A13 A1 (plate 2)

RE Master Mix F A14 A2 (plate 2)

RE Master Mix G B13 B1 (plate 2)

RE Master Mix H B14 B2 (plate 2)

ichment System Automation Protocol-ILM 39

3 Sample Preparation Step 1. Digest genomic DNA with restriction enzymes

40

The ECD sample contains genomic DNA mixed with an 800- bp PCR product that contains restriction sites for all the enzymes used in the digestion protocol. When analyzing validation results, the undigested control should have gDNA bands at >2.5 kbp and a PCR product band at 800 bp. Each of the eight digested ECD samples should have a smear of gDNA restriction fragments between 100 and 2500 bp, overlaid with three predominant bands at approximately 125, 225 and 450 bp. These three bands correspond to the 800- bp PCR product- derived restriction fragments, and precise sizes will differ after digestion in each of the eight RE master mixes.

In addition to the three predominant bands at approximately 125, 225 and 450 bp, you may detect additional, minor bands in the digested ECD sample lanes.

Successful digestion is indicated by the appearance of the three predominant bands. The presence of additional minor bands, with relative abundance similar to the additional bands visible in Figure 4, Figure 5 and Figure 6, does not impact enrichment results.

It is acceptable for band intensities in digestion reactions B and G to be slightly reduced, compared to the other digestion reactions.

NOTE

HaloPlex Target Enrichment System Automation Protocol-ILM

Sample Preparation 3 Step 1. Digest genomic DNA with restriction enzymes

HaloPlex Target Enr

Option 1: Validation by Agilent 2100 Bioanalyzer analysis

Use a High Sensitivity DNA Kit (p/n 5067- 4626) and the 2100 Bioanalyzer system with 2100 Expert Software (version B.02.07 or higher required to run the High Sensitivity Kit). See the High Sensitivity DNA Kit Guide at www.genomics.agilent.com.

Prepare an undigested DNA gel control by combining 0.5 L of the Enrichment Control DNA stock solution and 3.5 L of nuclease- free water.

Prepare the chip, samples and ladder as instructed in the reagent kit guide, using 1 L of each ECD sample and undigested DNA control for the analysis.

When loading samples on the chip, load the DNA ladder in the ladder sample well marked on the chip. Load the eight ECD digest samples (A to H) in sample wells 1 to 8, and load the undigested ECD sample in sample well 9. Do not run the undigested ECD control in sample well 1.

Place the prepared chip into the 2100 Bioanalyzer instrument and start the run within five minutes after preparation.

See Figure 4 for sample Bioanalyzer electrophoresis results.

Figure 4 Validation of restriction digestion by 2100 Bioanalyzer system analysis. Lane 1: 50-bp DNA ladder, Lanes 2-9: ECD digestion reactions AH, Lane 10: Undi- gested Enrichment Control DNA.

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3 Sample Preparation Step 1. Digest genomic DNA with restriction enzymes

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Option 2: Validation by Agilent 4200 TapeStation analysis

Use a High Sensitivity D1000 ScreenTape and reagent kit. For more information to do this step, see the Agilent High Sensitivity D1000 Assay Quick Guide for 4200 TapeStation System.

Prepare an undigested DNA gel control by combining 1 L of the Enrichment Control DNA stock solution and 1 L of nuclease- free water.

Prepare the TapeStation samples as instructed in assay Quick Guide. Use 2 L of each ECD sample diluted with 2 L of High Sensitivity D1000 sample buffer in separate wells of a tube strip for the analysis.

Load the sample tube strip, the High Sensitivity D1000 ScreenTape, and loading tips into the 4200 TapeStation and start the run.

See Figure 5 for sample TapeStation electrophoresis results.

Figure 5 Validation of restriction digestion by TapeStation analysis. Lane 1: High-Sensitivity Ladder, Lane 2: Undigested Enrichment Control DNA, Lanes 310: ECD digestion reactions AH.

HaloPlex Target Enrichment System Automation Protocol-ILM

Sample Preparation 3 Step 1. Digest genomic DNA with restriction enzymes

HaloPlex Target Enr

Option 3: Validation by gel electrophoresis

Use a Novex 6% polyacrylamide TBE pre- cast gel and 1X Novex TBE Running Buffer. For more information to do this step, consult the manufacturers recommendations.

Prepare an undigested DNA gel control by combining 2 L of the Enrichment Control DNA stock solution and 2 L of nuclease- free water.

Add 1 L of Novex Hi- Density TBE Sample Buffer (5X) to each 4- L ECD sample.

Load 5 L of each sample on the gel. In one or more adjacent lanes, load 200 ng of a 50- bp DNA ladder.

Run the gel at 210 V for approximately 15 minutes.

Stain the gel in 3X GelRed Nucleic Acid Stain for 10 minutes, and visualize bands under UV radiation.

See Figure 6 for sample gel results.

Figure 6 Validation of restriction digestion by gel electrophoresis. Lanes 18: ECD di- gestion reactions AH, Lane 9: Undigested Enrichment Control DNA, Lane 10: 25-bp DNA ladder.

Stopping Point

If you do not continue to the next step, samples may be stored at 20C for long term storage. There are no more long- term stopping points until after the PCR amplification step on page 64.

ichment System Automation Protocol-ILM 43

3 Sample Preparation Step 2. Hybridize digested DNA to HaloPlex or ClearSeq probe for target enrichment and sample indexing

Step 2. Hybridize digested DNA to HaloPlex or ClearSeq probe for target enrichment and sample indexing

44

In this step, the collection of gDNA restriction fragments is hybridized to the HaloPlex or ClearSeq probe capture library. HaloPlex and ClearSeq probes are designed to hybridize selectively to fragments originating from target regions of the genome and to direct circularization of the targeted DNA fragments. During the hybridization process, Illumina sequencing motifs including index sequences are incorporated into the targeted fragments.

The duration of the hybridization reaction is determined by the probe density of your design. Refer to the Certificate of Analysis provided with Box 1 of your kit to determine the hybridization conditions appropriate for your design.

For sample indexing primer assignments, see the Reference chapter for nucleotide sequences of the 96 indexes used in the HaloPlex Target Enrichment System.

Prepare the NGS Bravo

1 Gently wipe down the Bravo deck with a surface decontamination wipe.

2 Place a red insert on Bravo deck position 1.

3 Place a silver Nunc plate insert on Bravo deck position 9.

4 Turn on the chiller, set to 0C, at position 9 of the Bravo deck. Be sure that the chiller reservoir contains at least 300 mL of 25% ethanol.

5 For all run sizes, place a 384- well adapter insert on Bravo deck position 4. Pre- set the temperature of Bravo deck position 4 to 4C using the Inheco Multi TEC control touchscreen, as described in Setting the Temperature of Bravo Deck Heat Blocks.

For 12- column runs only, place a second 384- well adapter insert on Bravo deck position 6 and pre- set the temperature of Bravo deck position 6 to 4C.

HaloPlex Target Enrichment System Automation Protocol-ILM

Sample Preparation 3 Step 2. Hybridize digested DNA to HaloPlex or ClearSeq probe for target enrichment and sample indexing

HaloPlex Target Enr

To expedite thermal cycler warm-up for the hybridization reaction on page 49, you can enter and initiate the hybridization program on the thermal cycler now, and then pause the program until you are instructed to transfer the reaction plate. Be sure to pause the thermal cycler before the initiation of the 95C incubation segment. Release the pause immediately after transferring the plate to the thermal cycler in step 11 on page 49. Be sure that the 96-well block is in the thermal cycler before initiating the program for warm-up.

NOTE

Prepare the Master Mix Source Plate for Hybridization.pro

1 Prepare the appropriate amount of Hybridization Master Mix, according to the table below.

Mix well by gentle vortexing, then spin the tube briefly.

Table 16 Preparation of Hybridization Master Mix for Hybridization.pro

Reagent Volume for 1 Library

Volume for 1 Column

Volume for 2 Columns

Volume for 3 Columns

Volume for 4 Columns

Volume for 6 Columns

Volume for 12 Columns

HaloPlex or ClearSeq Probe

20 L 255 L 425 L 595 L 765 L 1105 L 2210 L

Hybridization Solution

50 L 637.5 L 1062.5 L 1487.5 L 1912.5 L 2762.5 L 5525 L

Total Volume 70 L 892.5 L 1487.5 L 2082.5 L 2677.5 L 3867.5 L 7735 L

ichment System Automation Protocol-ILM 45

3 Sample Preparation Step 2. Hybridize digested DNA to HaloPlex or ClearSeq probe for target enrichment and sample indexing

46

2 In a Nunc DeepWell plate, prepare the Hybridization Master Mix source plate. Add the volumes indicated in Table 17 of the Hybridization Master Mix to all wells of the indicated column of the Nunc DeepWell plate.

Table 17 Preparation of the Master Mix Source Plate for Hybridization.pro

Master Mix Solution

Position on Source Plate

Volume of Master Mix added per Well of Nunc DeepWell Source Plate

1-Column Runs

2-Column Runs

3-Column Runs

4-Column Runs

6-Column Runs

12-Column Runs

Hybridization Master Mix

Column 1

(A1-H1)

102.8 L 177.2 L 251.6 L 325.9 L 474.7 L 958.1 L

Prepare the Hybridization Reaction Plate with Indexing Primers

1 In a half- skirted 96- well Eppendorf twin.tec plate, aliquot 10 L of the appropriate HaloPlex Indexing Primer to each intended sample indexing well position. Keep the plate on ice.

Be sure to add only one specific Indexing Primer to each well, using different indexes for each sample to be multiplexed. Record the identity of the index assigned to each well for later sequence analysis.

2 If the run includes an ECD control sample that was analyzed as described on page 39, add 32 L of nuclease- free water to well A1 of the hybridization reaction plate. (Well A1 should also contain 10 L of indexing primer from step 1 above.) The 32 L of water added- back here compensates for the combined volume removed from the eight ECD digest wells during validation.

Load the Agilent NGS Bravo and Run the Hybridization.pro VWorks Protocol

1 On the VWorks HaloPlex form, under Step, select 02 Hybridization.pro.

2 Select the number of columns of samples to be processed. Runs must include 1, 2, 3, 4, 6, or 12 columns.

3 Click Update layout and information.

HaloPlex Target Enrichment System Automation Protocol-ILM

Sample Preparation 3 Step 2. Hybridize digested DNA to HaloPlex or ClearSeq probe for target enrichment and sample indexing

HaloPlex Target Enr

4 Load the Bravo deck according to Table 18.

Table 18 Initial Bravo deck configuration for Hybridization.pro

5 Verify that the NGS Bravo has been set up as displayed in the Bravo Deck Setup and Information regions of the form.

6 Verify that the Current Tip State indicator on the form matches the configuration of unused and used tips in the tip boxes at Bravo Deck positions 2 and 8, respectively. See page 22 for more information on using this segment of the form during the run.

7 When verification is complete, click Start to start the run.

The NGS Bravo combines all eight digestion reactions for each gDNA sample with Hybridization Master Mix and the appropriate Indexing Primer in wells of a 96- well plate.

Location Content

1 HaloPlex Indexing Primer source plate (half-skirted 96-well Eppendorf twin.tec plate) seated on red insert

2 New tip box

3 (empty)

4 Digested DNA in 384-well plate, seated on 384-well insert

5 Empty full-skirted 96-well Eppendorf twin.tec plate

6 For 12-column runs only: Digested DNA in 384-well plate (digest plate 2), seated on 384-well insert

For 1- to 6-column runs: empty

7 (empty)

8 Empty tip box

9 Hybridization Master Mix source plate (Nunc DeepWell plate) seated on silver insert

ichment System Automation Protocol-ILM 47

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48

8 When the NGS Bravo has finished preparing the hybridization plate for the run, you will be prompted by VWorks as shown below.

9 Seal the sample plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165C and 3.0 sec.

10 Spin the plate briefly.

HaloPlex Target Enrichment System Automation Protocol-ILM

Sample Preparation 3 Step 2. Hybridize digested DNA to HaloPlex or ClearSeq probe for target enrichment and sample indexing

HaloPlex Target Enr

11 Transfer the sealed plate to a thermal cycler and run the appropriate program in Table 19, using the hybridization duration listed on the Certificate of Analysis. After transferring the plate, click Continue on the VWorks prompt to finish the protocol.

Use a heated lid. Do not include a low- temperature hold step in the thermal cycler program. Incubation at 54C for more than the indicated time is not recommended.

Table 19 Thermal cycler program* for probe hybridization

* Thermal cyclers that use calculated temperature methods cannot be set to 160 L reaction volumes. In that case, enter the maximum possible volume.

Step Temperature Time (Duration of Step)

Designs with <15,000 probes (see Certificate of Analysis)

Designs with 15,000 probes (see Certificate of Analysis)

Step 1 95C 10 minutes 10 minutes

Step 2 54C 3 hours 16 hours

CAUTION Make sure that the thermal cycler has a maximum reaction volume specification of at least 100 L.

The 160-L hybridization reaction conditions have been optimized with the SureCycler thermal cycler (with volume specification of 10-100 L for PCR reactions). The performance of other thermal cyclers for this application should be verified before use.

ichment System Automation Protocol-ILM 49

3 Sample Preparation Step 3. Capture and amplify the target DNA

Step 3. Capture and amplify the target DNA

50

In this step, the circularized target DNA- probe hybrids, containing biotin, are captured on streptavidin beads. After capture, DNA ligase is added to seal nicks, then target DNA is eluted and PCR- amplified.

Assemble reagents for the run

1 Remove reagents to be used in upcoming protocol steps from cold storage and allow the solutions to reach room temperature:

From 20C storage, remove the Capture Solution, Wash Solution, Ligation Solution and SSC Buffer.

From +4C storage, remove the HaloPlex Magnetic Beads.

2 Prepare 30 L per sample, plus excess, of fresh 50 mM NaOH for use in the DNA elution step on page 52.

Prepare the 50 mM NaOH solution from a 10M NaOH stock solution.

Do not use stock NaOH solutions that were stored at concentrations below 10 M to prepare the 50 mM NaOH solution.

Keep the 50 mM NaOH solution container sealed when not in use, especially when processing large numbers of samples per run.

Table 20 Amount of 50mM NaOH required per run size

CAUTION Using high-quality NaOH is critical for optimal DNA elution and recovery.

Volume for 1 Library

Volume for 1 Column

Volume for 2 Columns

Volume for 3 Columns

Volume for 4 Columns

Volume for 6 Columns

Volume for 12 Columns

30 L 270 L 510 L 750 L 990 L 1470 L 2940 L

3 Obtain or prepare 0.5 L per sample, plus excess, of 2 M acetic acid, for use in the PCR master mix on page 53.

CAUTION It is critical to use high-quality acetic acid at 2 M concentration in this step to ensure neutralization of the NaOH used for elution.

See Table 1 on page 9 for 2 M acetic acid supplier information, or prepare 2 M acetic acid from high-quality glacial acetic acid.

HaloPlex Target Enrichment System Automation Protocol-ILM

Sample Preparation 3 Step 3. Capture and amplify the target DNA

HaloPlex Target Enr

Prepare the NGS Bravo

1 Gently wipe down the Bravo deck with a surface decontamination wipe.

2 Place a red insert on Bravo deck position 4.

3 Pre- set the temperature of Bravo deck position 4 to 54C using the Inheco Multi TEC control touchscreen, as described in Setting the Temperature of Bravo Deck Heat Blocks.

4 Place a second red insert on Bravo deck position 6.

5 Pre- set the temperature of Bravo deck position 6 to 4C using the Inheco Multi TEC control touchscreen, as described in Setting the Temperature of Bravo Deck Heat Blocks.

6 Place the silver Nunc plate insert on Bravo deck position 9.

7 Turn on the chiller, set to 0C, at position 9 of the Bravo deck. Be sure that the chiller reservoir contains at least 300 mL of 25% ethanol.

Prepare the HaloPlex Magnetic Beads Source Plate

1 Vigorously resuspend the HaloPlex Magnetic Beads on a vortex mixer. The beads settle during storage.

2 Wash the magnetic beads.

a Transfer 40 L per sample of the HaloPlex Magnetic Beads suspension to a 1.5- mL tube or conical vial, using volumes provided in Table 21.

Table 21 Volume of HaloPlex Magnetic Bead suspension for capture

Reagent Volume for 1 Library

Volume for 1 Column

Volume for 2 Columns

Volume for 3 Columns

Volume for 4 Columns

Volume for 6 Columns

Volume for 12 Columns

HaloPlex Magnetic Beads

0.04 mL 0.36 mL 0.68 mL 1.0 mL 1.32 mL 1.96 mL 3.92 mL

ichment System Automation Protocol-ILM 51

3 Sample Preparation Step 3. Capture and amplify the target DNA

52

b Put the vial into a compatible magnetic device for 5 minutes.

c After verifying that the solution has cleared, carefully remove and discard the supernatant using a pipette.

d Add an equivalent volume of Capture Solution (see Table 22) to the beads and resuspend by pipetting up and down.

Table 22 Volume of Capture Solution used for bead resuspension

Reagent Volume for 1 Library

Volume for 1 Column

Volume for 2 Columns

Volume for 3 Columns

Volume for 4 Columns

Volume for 6 Columns

Volume for 12 Columns

Capture Solution 0.04 mL 0.36 mL 0.68 mL 1.0 mL 1.32 mL 1.96 mL 3.92 mL

3 Prepare a Nunc DeepWell source plate for the washed HaloPlex streptavidin bead suspension. Add 40 L of the homogeneous bead suspension to all wells of the Nunc DeepWell plate that correspond to sample- containing wells on the hybridization plate.

4 Place the streptavidin bead source plate at position 5 of the Bravo deck.

Prepare wash and elution solution source plates

Prepare a separate source plate for each of the solutions listed in Table 23. Use full- skirted 96- well Eppendorf twin.tec plates to prepare all three source plates. For all sample- containing wells of the hybridization plate, add the specified volume of solution to all corresponding wells of the solution source plate.

Seal the 50 mM NaOH source plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165C and 1.0 sec. Leave sealed until you are prompted to add the plate to the Bravo deck in step 15 on page 62.

Table 23 Preparation of solution source plates for Capture_v1.1.pro protocol

Solution Volume to dispense per well of source plate

Wash Solution 110 L

SSC Buffer 110 L

50 mM NaOH 30 L

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Prepare the Master Mixes for Capture_v1.1.pro protocol

1 Prepare the appropriate amount of PCR Master Mix, according to the table below.

Mix well by gentle vortexing, then spin the tube briefly.

Table 24 Preparation of PCR Master Mix for Capture_v1.1.pro

Reagent Volume for 1 Library

Volume for 1 Column

Volume for 2 Columns

Volume for 3 Columns

Volume for 4 Columns

Volume for 6 Columns

Volume for 12 Columns

Nuclease-free water 16.1 L 205.3 L 342.1 L 479 L 615.8 L 889.5 L 1779 L

5X Herculase II Reaction Buffer

10 L 127.5 L 212.5 L 297.5 L 382.5 L 552.5 L 1105 L

dNTPs (100 mM)* 0.4 L 5.1 L 8.5 L 11.9 L 15.3 L 22.1 L 44.2 L

Primer 1 1 L 12.75 L 21.3 L 29.8 L 38.3 L 55.3 L 110.5 L

Primer 2 1 L 12.75 L 21.3 L 29.8 L 38.3 L 55.3 L 110.5 L

2 M Acetic acid 0.5 L 6.4 L 10.6 L 14.9 L 19.1 L 27.6 L 55.3 L

Herculase II Fusion DNA Polymerase

1 L 12.75 L 21.3 L 29.8 L 38.3 L 55.3 L 110.5 L

Total Volume 30 L 382.5 L 637.6 L 892.7 L 1147.6 L 1657.6 L 3315 L

* Be sure to use dNTPs at 100 mM concentration (25 mM for each nucleotide), like those provided with the Herculase II Fusion Enzyme with dNTPs (Agilent p/n 600677 or 600679).

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2 Prepare the appropriate amount of Ligation Master Mix, according to the table below.

Mix well by gentle vortexing, then spin the tube briefly. Store the master mix on ice until it is used on page 58. The Ligation Master Mix is added to the Master Mix Source Plate just before it is used in the Capture_v1.1.pro protocol. Do not add this master mix to the source plate before starting the run.

Table 25 Preparation of Ligation Master Mix for Capture_v1.1.pro

Reagent Volume for 1 Library

Volume for 1 Column

Volume for 2 Columns

Volume for 3 Columns

Volume for 4 Columns

Volume for 6 Columns

Volume for 12 Columns

Ligation Solution 47.5 L 605.6 L 1009 L 1413 L 1817 L 2624 L 5249 L

DNA Ligase 2.5 L 31.9 L 53.1 L 74.4 L 95.6 L 138.1 L 276.3 L

Total Volume 50 L 637.5 L 1062.1 L 1487.4 L 1912.6 L 2762.1 L 5525.3 L

Prepare the Master Mix Source Plate for Capture_v1.1.pro

Using the same Nunc DeepWell plate that was used for the Hybridization.pro run, prepare the Master Mix source plate for Capture_v1.1.pro. Add the volume indicated in Table 26 of PCR Master Mix to all wells of column 3 of the Nunc DeepWell plate.

Table 26 Preparation of the Master Mix Source Plate for Capture_v1.1.pro

Master Mix Solution

Position on Source Plate

Volume of Master Mix added per Well of Nunc Deep Well Source Plate

1-Column Runs

2-Column Runs

3-Column Runs

4-Column Runs

6-Column Runs

12-Column Runs

PCR Master Mix Column 3

(A3-H3)

44.1 L 75.9 L 107.8 L 139.7 L 203.4 L 410.6 L

NOTE Column 2 of the Master Mix source plate must remain empty at this step. You will be prompted to add Ligation Master Mix to Column 2 at the appropriate time during the Capture_v1.1.pro protocol. Column 1 was used during the Hybridization.pro protocol.

If you are using a new DeepWell plate for the Capture_v1.1.pro Master Mix source plate, be sure to leave columns 1 and 2 empty at this time, adding the PCR Master Mix to column 3 of the new plate.

HaloPlex Target Enrichment System Automation Protocol-ILM

Sample Preparation 3 Step 3. Capture and amplify the target DNA

HaloPlex Target Enr

Load the Agilent NGS Bravo and Run the Capture_v1.1.pro VWorks Protocol

1 On the VWorks HaloPlex form, under Step, select 03 Capture_v1.1.pro.

2 Select the number of columns of samples to be processed. Runs must include 1, 2, 3, 4, 6, or 12 columns.

3 Click Update layout and information.

4 Load the Bravo deck according to Table 27.

Table 27 Initial Bravo deck configuration for Capture_v1.1.pro

5 Verify that the NGS Bravo has been set up as displayed in the Bravo Deck Setup and Information regions of the form.

6 Verify that the Current Tip State indicator on the form matches the configuration of unused and used tips in the tip boxes at Bravo Deck positions 2 and 8, respectively. See page 22 for more information on using this segment of the form during the run.

7 When verification is complete, click Start to start the run.

The NGS Bravo completes the liquid- handling steps for capture of the target DNA- HaloPlex probe hybrids on the streptavidin beads.

Location Content

1 Empty Axygen 96 Deep Well Plate (square wells) for waste

2 New tip box

3 Wash Solution source plate (full-skirted 96-well Eppendorf twin.tec plate)

4 Hybridized sample plate seated on red insert

5 HaloPlex magnetic streptavidin bead source plate (Nunc DeepWell plate)

6 Empty half-skirted 96-well Eppendorf twin.tec plate seated on red insert

7 (empty)

8 Empty tip box

9 Master Mix source plate (Nunc DeepWell plate) seated on silver insert

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NOTE To expedite thermal cycler warm-up for the subsequent wash program on page 57, you can enter and initiate the wash program on the thermal cycler now, and then pause the program until you are instructed to transfer the reaction plate. Be sure to pause the thermal cycler before the initiation of the 46C incubation segment. Release the pause immediately after transferring the plate to the thermal cycler in step 9.

8 When prompted by VWorks as shown below, remove and discard the hybridization plate from position 4 of the Bravo deck. Place a fresh half- skirted 96- well Eppendorf twin.tec plate at position 4 for use in the wash segment of the protocol.

HaloPlex Target Enrichment System Automation Protocol-ILM

Sample Preparation 3 Step 3. Capture and amplify the target DNA

HaloPlex Target Enr

9 When the NGS Bravo has finished preparing the capture wash plate, you will be prompted by VWorks as shown below.

a Get the sample plate from position 4 and seal the plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165C and 3.0 sec.

Transfer the sealed plate to a thermal cycler and run the wash program shown in Table 28, using a heated lid.

Do not include a low- temperature hold step in the thermal cycler program following the 10- minute incubation.

Table 28 Thermal cycler program for Capture_v1.1.pro wash step

b Remove and discard the Wash Solution plate from position 3 of the Bravo deck. Place the SSC Buffer source plate at position 3 for use in the ligation segment of the protocol.

c Remove and discard the bead source plate from position 5 of the Bravo deck. Place a fresh Nunc DeepWell plate at position 5.

d After completing all steps, click Continue on the VWorks prompt to continue the automation protocol. Do not wait for the conclusion of the thermal cycler wash program to continue the protocol.

Step Temperature Time

Step 1 46C 10 minutes

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10 During the 10- minute incubation of the sample plate on the thermal cycler, you will be prompted to add the Ligation Master Mix to the Master Mix source plate as shown below.

Add the volume of Ligation Master Mix indicated in Table 29 to all wells of column 2 of the Nunc DeepWell Master Mix source plate on Bravo deck position 9.

of Ligation Master Mix to the Master Mix Source Plate for Capture_v1.1.pro

Table 29 Addition

The Master Mix source plate at postition 9 should already contain the PCR Master Mix in Column 3 and the depleted Hybridization Master Mix from the Hybridization.pro protocol in Column 1. Be sure to add the Ligation Master Mix to Column 2 of the source plate at this step.

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

Ligation Master Mix

Column 2

(A2-H2)

73.4 L 126.6 L 179.7 L 232.8 L 339.1 L 684.4 L

NOTE

HaloPlex Target Enrichment System Automation Protocol-ILM

Sample Preparation 3 Step 3. Capture and amplify the target DNA

HaloPlex Target Enr

11 Once the wash program in Table 28 has finished and you are prompted by VWorks, transfer the plate from the thermal cycler to Bravo deck position 4. Carefully unseal the plate, then click Continue on the VWorks prompt to resume the Capture_v1.1.pro protocol.

The NGS Bravo completes the liquid- handling steps for ligation of the captured target DNA.

To expedite thermal cycler warm-up for the subsequent ligation program on page 61, you

NOTE can enter and initiate the wash program on the thermal cycler now, and then pause the program until you are instructed to transfer the reaction plate. Be sure to pause the thermal cycler before the initiation of the 55C incubation segment. Release the pause immediately after transferring the plate to the thermal cycler in step 13.

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12 When prompted by VWorks as shown below, remove and discard the plate from position 4. Click Continue on the prompt to resume the protocol.

HaloPlex Target Enrichment System Automation Protocol-ILM

Sample Preparation 3 Step 3. Capture and amplify the target DNA

HaloPlex Target Enr

13 When the NGS Bravo has finished preparing the ligation plate, you will be prompted by VWorks as shown below.

a Get the sample plate from position 6 and seal the plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165C and 3.0 sec.

b Transfer the sealed plate to a thermal cycler and run the ligation program shown in Table 30, using a heated lid.

Table 30 Thermal cycler program for Capture_v1.1.pro ligation step

c Remove and discard the Nunc DeepWell plate from position 5 of the Bravo deck. Place a fresh Nunc DeepWell plate at position 5 for use in the ligation purification steps of the protocol.

d Place a fresh half- skirted 96- well Eppendorf twin.tec plate at position 6 for use in the following PCR protocol steps.

e After completing all steps, click Continue on the VWorks prompt to continue the automation protocol. Do not wait for the conclusion of the thermal cycler ligation program to continue the protocol.

Step Temperature Time

Step 1 55C 10 minutes

Step 2 4C Hold

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14 Once the ligation program in Table 30 is finished and you are prompted by VWorks, transfer the plate from the thermal cycler to Bravo deck position 4. Carefully unseal the plate, then click Continue on the VWorks prompt to resume the Capture_v1.1.pro protocol.

15 When prompted by VWorks as shown below, remove and discard the SSC Buffer plate from position 3 of the Bravo deck. Place the 50 mM NaOH source plate at position 3 for use in the elution steps of the protocol. After carefully unsealing the source plate, click Continue on the prompt to resume the protocol.

The NGS Bravo completes the liquid- handling steps for elution of the captured target DNA followed by preparation of PCR reactions for amplification.

HaloPlex Target Enrichment System Automation Protocol-ILM

Sample Preparation 3 Step 3. Capture and amplify the target DNA

HaloPlex Target Enr

NOTE To expedite thermal cycler warm-up for the subsequent PCR program on page 64, you can enter and initiate the PCR program on the thermal cycler now, and then pause the program until you are instructed to transfer the reaction plate. Be sure to pause the thermal cycler before the initiation of the 98C denaturation segment. Release the pause immediately after transferring the plate to the thermal cycler in step 16.

16 When the NGS Bravo has finished preparing the PCR amplification reactions, you will be prompted by VWorks as shown below.

a Get the sample plate from position 6 and seal the plate using the PlateLoc Thermal Microplate Sealer, with sealing settings of 165C and 3.0 sec.

b Transfer the sealed plate to a thermal cycler and run the PCR program in Table 31, using a heated lid.

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The optimal amplification cycle number varies for each HaloPlex or ClearSeq Probe design. Consult the Certificate of Analysis (provided with HaloPlex Target Enrichment System Box 1) for the PCR cycling recommendation for your probe.

Table 31 HaloPlex post-capture DNA amplification PCR program

c After initiating the PCR program in the thermal cycler, click Continue on the VWorks prompt to finish the automation protocol.

d If you are continuing to the next step of PCR product purification, remove the Agencourt AMPure XP Beads from +4C storage for use on page 65. Let the beads come to room temperature for the remainder of the amplification program.

Segment Number of Cycles Temperature Time

1 1 98C 2 minutes

2 Obtain cycle number from Certificate of Analysis

98C 30 seconds

60C 30 seconds

72C 1 minute

3 1 72C 10 minutes

4 1 8C Hold

Stopping Point

If you do not continue to the next step, PCR products may be stored at 20C for up to 72 hours or at 8C overnight. For best results, however, purify PCR products as soon as possible.

HaloPlex Target Enrichment System Automation Protocol-ILM

Sample Preparation 3 Step 4. Purify the amplified target DNA

Step 4. Purify the amplified target DNA

HaloPlex Target Enr

In this step, the NGS Bravo does the liquid handling steps to purify the amplified target DNA sample using AMPure XP beads.

Prepare the NGS Bravo and reagents

1 Gently wipe down the Bravo deck with a surface decontamination wipe.

2 Let the AMPure XP beads come to room temperature for at least 30 minutes.

Do not freeze the AMPure XP beads at any time.

3 Mix the room- temperature bead suspension well so that the reagent appears homogeneous and consistent in color.

4 Prepare a Nunc DeepWell source plate containing AMPure XP beads. For each well to be processed, add 100 L of homogeneous AMPure XP beads per well to the Nunc DeepWell plate.

5 Place a red insert on Bravo deck position 6.

6 Pre- set the temperature of Bravo deck position 6 to 4C using the Inheco Multi TEC control touchscreen, as described in Setting the Temperature of Bravo Deck Heat Blocks.

7 Prepare a Thermo Scientific reservoir containing 15 mL of nuclease- free water.

8 Prepare a Thermo Scientific reservoir containing 15 mL of the final sample elution buffer [nuclease- free 10 mM Tris- acetate or Tris- HCl buffer (pH 8.0)].

9 Prepare a separate Thermo Scientific reservoir containing 45 mL of freshly- prepared 70% ethanol.

Load the Agilent NGS Bravo and Run the Purification_v1.1.pro VWorks Protocol

1 On the VWorks HaloPlex form, under Step, select 04 Purification_v1.1.pro.

2 Select the number of columns of samples to be processed. Runs must include 1, 2, 3, 4, 6, or 12 columns.

3 Click Update layout and information.

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4 Load the Bravo deck according to Table 32.

Table 32 Initial Bravo deck configuration for Purification_v1.1.pro

5 Verify that the NGS Bravo has been set up as displayed in the Bravo Deck Setup region of the form.

6 Verify that the Current Tip State indicator on the form matches the configuration of unused and used tips in the tip boxes at Bravo Deck positions 2 and 8, respectively. See page 22 for more information on using this segment of the form during the run.

7 When verification is complete, click Start to start the run.

Location Content

1 Empty Axygen 96 Deep Well Plate (square wells) for waste

2 New tip box

3 Empty full-skirted 96-well Eppendorf twin.tec plate

4 (empty)

5 AMPure XP beads in Nunc DeepWell source plate

6 Amplified DNA samples in half-skirted 96-well Eppendorf twin.tec plate seated on red insert

7 (empty)

8 Empty tip box

9 Nuclease-free water in Thermo Scientific reservoir

HaloPlex Target Enrichment System Automation Protocol-ILM

Sample Preparation 3 Step 4. Purify the amplified target DNA

HaloPlex Target Enr

8 When prompted by VWorks as shown below, remove the water reservoir from position 9 of the Bravo deck and replace it with the 70% ethanol reservoir.

When finished, click Continue on the VWorks prompt.

9 When prompted by VWorks as shown below, remove the 70% ethanol reservoir from position 9 of the Bravo deck and replace it with the final sample elution buffer reservoir.

When finished, click Continue on the VWorks prompt.

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The NGS Bravo completes the liquid- handling steps for elution of the captured target DNA.

10 When the NGS Bravo has finished preparing the final eluted sample plate, you will be prompted by VWorks as shown below. Click Continue on the VWorks prompt to finish the protocol.

Stopping Point

If you do not continue to the next step, samples may be stored at 20C for long- term storage (up to one year). Avoid subjecting the stored DNA samples to multiple freeze- thaw cycles.

HaloPlex Target Enrichment System Automation Protocol-ILM

Sample Preparation 3 Step 5. Validate enrichment and quantify enriched target DNA

Step 5. Validate enrichment and quantify enriched target DNA

HaloPlex Target Enr

Prior to sample pooling and sequencing sample preparation, validate enrichment and quantify the enriched target DNA in each library sample by microfluidic analysis using the Agilent 2100 Bioanalyzer (see page 70) 4200 TapeStation (see page 71).

Enriched library samples may also be qualitatively analyzed using gel electrophoresis. Sample gel electrophoresis results are provided in the Reference section on page 86.

Expected Results

Each amplicon in the prepared library contains one target insert surrounded by sequence motifs required for multiplexed sequencing using the Illumina platform. Amplicons include 50 to 500 bp of target DNA insert and 125 bp of sequencing motifs, as shown in Figure 7.

Figure 7 Content of target-enriched amplicons. Each amplicon contains one target in- sert (blue) surrounded by the Illumina paired-end sequencing elements (black), the sample index (red) and the library bridge PCR primers (yellow).

The amplicons should range from 175 to 625 bp in length, with the majority of products sized 225 to 525 bp. Amplicons in the 175 to 625 bp size range should be included for quantitation of the enriched target DNA in each sample. Any spurious DNA products outside of this size range in any sample should be excluded from the target DNA quantitation results.

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Option 1: Analysis using the 2100 Bioanalyzer System

Use a Bioanalyzer High Sensitivity DNA Assay kit and the 2100 Bioanalyzer instrument with 2100 Expert Software (version B.02.07 or higher required to run the High Sensitivity Kit). See the High Sensitivity DNA Kit Guide at www.genomics.agilent.com for more information on doing this step.

1 Prepare the chip, samples and ladder as instructed in the reagent kit guide, using 1 L of enriched library sample for the analysis.

2 Load the prepared chip into the instrument and start the run within five minutes after preparation.

3 Analyze the electropherogram for each sample according to the analysis guidelines on page 72.

See Figure 8 for a sample electropherogram.

If the concentration determined by Bioanalyzer analysis is > 10 ng/L, repeat the analysis using a 1:10 dilution of the sample. Dilute 1 L of the sample in 9 L of 10 mM Tris, 1 mM EDTA and then mix well by vortexing at 2000 rpm on the IKA vortex supplied with the Bioanalyzer before analyzing the diluted sample.

NOTE

Figure 8 Validation of HaloPlex enrichment by Agilent 2100 Bioanalyzer system analy- sis.

HaloPlex Target Enrichment System Automation Protocol-ILM

Sample Preparation 3 Step 5. Validate enrichment and quantify enriched target DNA

HaloPlex Target Enr

Option 2: Analysis using the Agilent 4200 TapeStation

Use a High Sensitivity D1000 ScreenTape and reagent kit. For more information to do this step, see the Agilent High Sensitivity D1000 Assay Quick Guide for 4200 TapeStation System.

1 Prepare the TapeStation samples as instructed in tthe reagent Quick Guide. Use 2 L of each enriched library sample diluted with 2 L of High Sensitivity D1000 sample buffer in separate wells of a tube strip for the analysis.

2 Load the sample tube strip, the High Sensitivity D1000 ScreenTape, and loading tips into the 4200 TapeStation and start the run.

3 Analyze the electropherogram for each sample according to the analysis guidelines on page 72.

See Figure 9 for a sample electropherogram.

Figure 9 Validation of HaloPlex enrichment by TapeStation analysis.

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Analysis of Electropherogram Results

Check that the electropherogram shows a peak fragment size between approximately 225 to 525 bp.

Determine the concentration of enriched target DNA in the sample by integration under the peak between 175 and 625 bp. Peaks at <150 bp may be observed, but should be excluded from quantitation.

Some designs may generate a peak at about 125 bp. This peak is associated with an adaptor- dimer product which will cluster and generate sequence that does not map to the genome. If the molar fraction of the 125 bp peak is greater than 10%, do another round of AMPure purification after pooling samples. First, pool equimolar amounts of libraries to be multiplexed, using concentrations determined for the 175625 peak of each sample. Using 40 L of the pooled libraries, purify the DNA using AMPure XP beads according to the protocol starting on page 77.

HaloPlex Target Enrichment System Automation Protocol-ILM

Sample Preparation 3 Step 6. Pool samples with different indexes for multiplexed sequencing

Step 6. Pool samples with different indexes for multiplexed sequencing

HaloPlex Target Enr

Use the following guidelines to design your sample pooling strategy:

Use the Bioanalyzer- or TapeStation- measured concentration of 175- 625 bp products in each sample to pool equimolar amounts of differentially indexed samples in order to optimize the use of sequencing capacity.

The final HaloPlex enrichment pool is ready for direct sequencing using standard Illumina paired- end primers and chemistry on the Illumina HiSeq or MiSeq platform. See additional guidelines for the MiSeq platform (below) and HiSeq platform (page 76).

Use 100 + 100 bp or 150 + 150 bp paired- end sequencing, depending on the selection made during probe design. Since the read length affects maximum achievable coverage, check the design report to verify read length selected in probe design.

Sequencing runs must be set up to perform an 8- nt index read. For complete index sequence information, see the Reference chapter starting on page 81.

Before aligning reads to the reference genome, trim the reads from Illumina adaptor sequences.

MiSeq platform sequencing run setup guidelines

Setting up a custom Sample Sheet:

1 In the IEM software, create a Sample Sheet for the MiSeq platform using the following Workflow selections.

Under Category, select Other.

Under Application, select FASTQ Only.

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2 On the Workflow Parameters screen, enter the run information, making sure to specify the key parameters highlighted below:

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Sample Preparation 3 Step 6. Pool samples with different indexes for multiplexed sequencing

HaloPlex Target Enr

3 Using the Sample Plate Wizard, set up a New Plate, entering the required information for each sample to be sequenced. In the Index 1(17) column of the table, assign each sample to any of the Illumina 17 indexes. The index will be corrected to a HaloPlex index at a later stage.

4 Finish the sample plate setup tasks and save the sample plate file.

5 Using the Sample Sheet Wizard, select the samples to include in the run and save the Sample Sheet file.

Editing the Sample Sheet to Include HaloPlex indexes:

1 Open the Sample Sheet file in a text editor. For each sample, select the text for the 6- nucleotide index (highlighted below), and replace with the appropriate 8- nucleotide HaloPlex index sequence.

2 Save the edited Sample Sheet in an appropriate file location for use in the MiSeq platform run.

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HiSeq platform sequencing run setup guidelines

Set up sequencing runs to perform an 8- nt index read using the Cycles settings shown in Table 33. Cycle number settings can be specified on the Run Configuration screen of the instrument control software interface after choosing Custom from the index type selection buttons.

Sequence analysis resources

Agilents SureCall data analysis software is available to simplify the sequencing data analysis workflow after HaloPlex target enrichment. To learn more about this resource and download the SureCall software free of charge, visit www.agilent.com/genomics/surecall.

Table 33 HiSeq platform Run Configuration screen Cycle Number settings

Run Segment Cycle Number

Read 1 100

Index 1 (i7) 8

Index 2 (i5) 0

Read 2 100

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HaloPlex Target Enrichment System Protocol

4 Appendix: Provisional Adaptor-Dimer Removal Protocol

Purify the enriched library pool using AMPure XP beads 78

This section contains a protocol for purification of the target- enriched library pool to remove adaptor- dimer molecules of approximately 125 bp size. Only do this protocol if electrophoretic analysis of the target- enriched library samples shows a peak at approximately 125 bp which represents a molar fraction of >10% of DNA in the sample (see page 70 to page 72.)

77Agilent Technologies

4 Appendix: Provisional Adaptor-Dimer Removal Protocol Purify the enriched library pool using AMPure XP beads

Purify the enriched library pool using AMPure XP beads

78

In this step, a 40- L pool of target- enriched DNA libraries is purified using AMPure XP beads using manual sample processing.

NOTE This protocol requires a 0.2 mL tube-compatible magnetic separation device, such as the Agencourt SPRIPlate Super Magnet Plate, Agencourt p/n A32782, or equivalent.

1 Let the AMPure XP beads come to room temperature for at least 30 minutes.

2 Prepare 400 L of 70% ethanol per sample, plus excess, for use in step 10.

3 Transfer 40 L of each target- enriched library pool to a fresh 0.2- mL tube.

4 Mix the AMPure XP bead suspension well, until the suspension appears homogeneous and consistent in color.

5 For each sample to be purified, prepare a bead mix by combining 40 L of nuclease- free water and 100 L of the homogeneous AMPure XP bead suspension. Mix well, until the bead mix suspension appears homogeneous.

6 Add 140 L of the homogeneous bead suspension prepared in step 5 to each 40- L DNA sample. Vortex thoroughly.

Using this bead- to- sample volume ratio is imperative to ensure optimal purification results.

7 Incubate samples for 5 minutes at room temperature with continuous shaking.

Make sure the samples are properly mixing in the wells during the 5- minute incubation.

8 Spin briefly to collect the liquid, then place the tubes in the magnetic separation device. Wait for the solution to clear (approximately 5 minutes).

9 Keep the tubes in the magnetic device. Carefully remove and discard the cleared solution from each tube using a 200- L pipette set to 180 L. Do not touch the beads while removing the solution.

10 Continue to keep the tubes in the magnetic device while you add 200 L of 70% ethanol into the tubes.

Use fresh 70% ethanol for optimal results.

HaloPlex Target Enrichment System Automation Protocol-ILM

Appendix: Provisional Adaptor-Dimer Removal Protocol 4 Purify the enriched library pool using AMPure XP beads

HaloPlex Target Enr

11 Wait for 30 seconds to allow any disturbed beads to settle, then remove the ethanol using a 200- L pipette set to 200 L.

12 Repeat step 10 and step 11 once for a total of two washes.

13 Remove any residual ethanol with a 20- L volume pipette.

14 Air- dry the tubes with open lids at room temperature until the residual ethanol completely evaporates.

Make sure all ethanol has evaporated before continuing.

15 Remove tubes from the magnetic device and add 40 L of 10 mM Tris- acetate or Tris- HCl buffer (pH 8.0) to each sample.

Use room-temperature Tris-acetate or Tris-HCl buffer for elution at this step.NOTE

16 Mix thoroughly by pipetting up and down 15 times using a 100- L pipette set to 30 L.

17 Incubate for 2 minutes at room temperature to allow elution of DNA.

18 Put the tube in the magnetic device and leave for 2 minutes or until the solution is clear.

19 Remove the cleared supernatant (approximately 40 L) to a fresh tube. You can discard the beads at this time.

Stopping Point

If you do not continue to the next step, samples may be stored at 20C for long- term storage (up to one year). Avoid subjecting the stored DNA samples to multiple freeze- thaw cycles.

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HaloPlex Target Enrichment System Automation Protocol-ILM

HaloPlex Target Enrichment System Protocol

5 Reference

Kit Contents 82

Nucleotide Sequences of HaloPlex Indexes 85

Qualitative analysis of enrichment by gel electrophoresis 86

This chapter contains reference information, including component kit contents, index sequences, and optional gel validation instructions.

81Agilent Technologies

5 Reference Kit Contents

Kit Contents

82

Component kits supplied with the HaloPlex Target Enrichment System are listed in Table 34 for custom probe designs and in Table 35 for ClearSeq Disease Research Panels. Reagents included in the HaloPlex Target Enrichment System- ILM Box 1 are listed in Table 36 on page 83.

Table 34 HaloPlex Target Enrichment System Kit Contents-Custom Designs

Design Type HaloPlex Target Enrichment System-ILM, Box 1

HaloPlex Magnetic Beads Box 2

Store at 20C Store at +4C

HaloPlex 1-500 kb with <15,000 Probes, ILM, 96 Reactions

5190-8050 OR 5190-8051*

* Part number 5190-8050, 5191-4068, 5190-8052, or 5190-8054 is provided for the first order of a spe- cific HaloPlex Probe design. Re-order kits, containing previously-purchased HaloPlex Probe designs, include Box 1 part number 5190-8051, 5191-4069, 5190-8053, or 5190-8055.

5190-5386

HaloPlex 1-500 kb with 15,000-20,000 Probes, ILM, 96 Reactions

5191-4068 OR 5191-4069 5190-5386

HaloPlex 0.5-2.5 Mb OR <0.5 Mb with >20,000 probes, ILM, 96 Reactions

5190-8052 OR 5190-8053 5190-5386

HaloPlex 2.6 Mb-5 Mb, ILM, 96 Reactions 5190-8054 OR 5190-8055 5190-5386

Table 35 HaloPlex Target Enrichment System Kit Contents-ClearSeq Disease Research Panels

Design Type HaloPlex Target Enrichment System-ILM, Box 1

HaloPlex Magnetic Beads Box 2

Store at 20C Store at +4C

ClearSeq ICCG, ILM, 96 Reactions 5190-9164 5190-9224

ClearSeq Connective Disorder, ILM, 96 Reactions 5190-9168 5190-9224

ClearSeq Arrhythmia, ILM, 96 Reactions 5190-9162 5190-9224

ClearSeq Noonan Syndrome, ILM, 96 Reactions 5190-9170 5190-9224

ClearSeq Chromosome-X, ILM, 96 Reactions 5190-9166 5190-9224

HaloPlex Target Enrichment System Automation Protocol-ILM

Reference 5 Kit Contents

HaloPlex Target Enr

The contents of the HaloPlex Target Enrichment System Box 1 included with each kit are detailed in the table below:

Table 36 HaloPlex Target Enrichment System Box 1 Contents

Included Reagents Format

Hybridization Solution bottle

Ligation Solution bottle

Wash Solution bottle

Capture Solution bottle

SSC Buffer bottle

RE Buffer bottle

BSA Solution tube with clear cap

DNA Ligase tube with red cap

Enrichment Control DNA tube with orange cap

Primer 1 tube with yellow cap

Primer 2 tube with blue cap

HaloPlex Indexing Primers 96-well plate with Indexing Primer A01 to H12 (blue plate)*

* See Table 37 for a plate map.

Enzyme Strip 1 8-well strip tube with green label

Enzyme Strip 2 8-well strip tube with red label

HaloPlex or ClearSeq Probe tube with pink cap

ichment System Automation Protocol-ILM 83

5 Reference Kit Contents

Table 37 Plate map for HaloPlex Indexing Primers A01 through H12 provided in blue plate

1 2 3 4 5 6 7 8 9 10 11 12

A A01 A02 A03 A04 A05 A06 A07 A08 A09 A10 A11 A12

B B01 B02 B03 B04 B05 B06 B07 B08 B09 B10 B11 B12

C C01 C02 C03 C04 C05 C06 C07 C08 C09 C10 C11 C12

D D01 D02 D03 D04 D05 D06 D07 D08 D09 D10 D11 D12

E E01 E02 E03 E04 E05 E06 E07 E08 E09 E10 E11 E12

F F01 F02 F03 F04 F05 F06 F07 F08 F09 F10 F11 F12

G G01 G02 G03 G04 G05 G06 G07 G08 G09 G10 G11 G12

H H01 H02 H03 H04 H05 H06 H07 H08 H09 H10 H11 H12

84 HaloPlex Target Enrichment System Automation Protocol-ILM

Reference 5 Nucleotide Sequences of HaloPlex Indexes

Nucleotide Sequences of HaloPlex Indexes

he nucleotide sequence of the 8- nucleotide index portion of each HaloPlex Indexing Primer is provided in the table below.

HaloPlex Target Enr

Table 38 HaloPlex Indexes, for indexing primers provided in blue 96-well plate

Index Sequence Index Sequence Index Sequence Index Sequence

A01 ATGCCTAA A04 AACTCACC A07 ACGTATCA A10 AATGTTGC

B01 GAATCTGA B04 GCTAACGA B07 GTCTGTCA B10 TGAAGAGA

C01 AACGTGAT C04 CAGATCTG C07 CTAAGGTC C10 AGATCGCA

D01 CACTTCGA D04 ATCCTGTA D07 CGACACAC D10 AAGAGATC

E01 GCCAAGAC E04 CTGTAGCC E07 CCGTGAGA E10 CAACCACA

F01 GACTAGTA F04 GCTCGGTA F07 GTGTTCTA F10 TGGAACAA

G01 ATTGGCTC G04 ACACGACC G07 CAATGGAA G10 CCTCTATC

H01 GATGAATC H04 AGTCACTA H07 AGCACCTC H10 ACAGATTC

A02 AGCAGGAA A05 AACGCTTA A08 CAGCGTTA A11 CCAGTTCA

B02 GAGCTGAA B05 GGAGAACA B08 TAGGATGA B11 TGGCTTCA

C02 AAACATCG C05 CATCAAGT C08 AGTGGTCA C11 CGACTGGA

D02 GAGTTAGC D05 AAGGTACA D08 ACAGCAGA D11 CAAGACTA

E02 CGAACTTA E05 CGCTGATC E08 CATACCAA E11 CCTCCTGA

F02 GATAGACA F05 GGTGCGAA F08 TATCAGCA F11 TGGTGGTA

G02 AAGGACAC G05 CCTAATCC G08 ATAGCGAC G11 AACAACCA

H02 GACAGTGC H05 CTGAGCCA H08 ACGCTCGA H11 AATCCGTC

A03 ATCATTCC A06 AGCCATGC A09 CTCAATGA A12 CAAGGAGC

B03 GCCACATA B06 GTACGCAA B09 TCCGTCTA B12 TTCACGCA

C03 ACCACTGT C06 AGTACAAG C09 AGGCTAAC C12 CACCTTAC

D03 CTGGCATA D06 ACATTGGC D09 CCATCCTC D12 AAGACGGA

E03 ACCTCCAA E06 ATTGAGGA E09 AGATGTAC E12 ACACAGAA

F03 GCGAGTAA F06 GTCGTAGA F09 TCTTCACA F12 GAACAGGC

G03 ACTATGCA G06 AGAGTCAA G09 CCGAAGTA G12 AACCGAGA

H03 CGGATTGC H06 CCGACAAC H09 CGCATACA H12 ACAAGCTA

ichment System Automation Protocol-ILM 85

5 Reference Qualitative analysis of enrichment by gel electrophoresis

Qualitative analysis of enrichment by gel electrophoresis

86

Enrichment products may be qualitatively analyzed by gel electrophoresis. Analyze 5 L of each enriched library sample (enriched ECD sample or experimental enriched libraries) by electrophoresis on a Novex 6% polyacrylamide TBE pre- cast gel. See page 43 for additional gel analysis protocol recommendations.

Successful enrichment is indicated by the presence of a smear of amplicons from approximately 225 to 525 bp in each enrichment library lane. For some probe designs, low molecular weight (<150 bp) bands may also be visible, but should not be included in enriched sample quantitation. See Figure 10 for a sample gel analysis image.

Figure 10 Validation of HaloPlex enrichment process by gel electrophoresis. Lane 1: 25-bp DNA ladder, Lanes 2-7: enriched library samples.

HaloPlex Target Enrichment System Automation Protocol-ILM

www.agilent.com

In This Book

This guide contains information to run the HaloPlex Target Enrichment System automation protocol.

Agilent Technologies, Inc. 2015, 2018

Version G0, July 2018

*G9900-90

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