Agilent HaloPlex Sequencer Protocol Manual PDF

HaloPlex Target Enrichment System

For Illumina Sequencing

Protocol Version G0, July 2018

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

Agilent Technologies

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2 HaloPlex Target Enrichment System-ILM

In this Guide...

HaloPlex Target Enrichment System

This guide describes an optimized protocol for using the HaloPlex target enrichment system to prepare sequencing library samples for Illumina paired- end multiplexed sequencing platforms.

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

Sample Preparation

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

3

Reference

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

-ILM 3

Whats New in Version G0

4

Support for new Tier 1 Plus probe designs (see Table 2 on page 10, Table 7 on page 28, and Table 12 on page 48)

Removal of support information for discontinued kits p/n G9903A, G9903B, G9908A, G9908B, G9913A, G9913B (Table 2 on page 10, Table 13 on page 49 and Table 14 on page 50)

Updates to sequencing support details (see page 42 through page 45)

Update to acceptable electrophoresis result description for restriction digest G (see Note on page 22)

Support for the Agilent 4200 TapeStation system (see Table 4 on page 12 and see page 24 and page 40)

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)

Removal of reference information for obsolete kits containing 8- bp indexing primers 196, provided in clear- capped tubes or 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)

Whats New in Version F1

Support for ClearSeq Disease Research Panels (see

Table 2 on page 10 and Table 13 on page 49)

Updates to format of hybridization instructions (see Table 7 on page 28)

Update to name of indexing primers (see Table 14 on page 50)

HaloPlex Target Enrichment System-ILM

Content

1 Before You Begin

Procedural Notes 8 Safety Notes 8 Required Reagents 9 Required Equipment 11 Optional Validation Reagents and Equipment 12

2 Sample Preparation

Run Size Considerations 15 Run Time Considerations 15 Step 1. Digest genomic DNA with restriction enzymes 16 Step 2. Hybridize digested DNA to HaloPlex or ClearSeq probe for target

enrichment and sample indexing 26 Step 3. Capture the target DNA 29 Step 4. Ligate the captured, circularized fragments 32 Step 5. Prepare the PCR Master Mix 33 Step 6. Elute captured DNA with NaOH 34 Step 7. PCR amplify the captured target libraries 35 Step 8. Purify the amplified target libraries 36 Step 9. Validate enrichment and quantify enriched target DNA 38 Step 10. Pool samples with different indexes for multiplexed sequencing 42

3 Reference

Kit Contents 48 Nucleotide Sequences of HaloPlex Indexes 52 Qualitative analysis of enrichment by gel electrophoresis 53

HaloPlex Target Enrichment System-ILM 5

6 HaloPlex Target Enrichment System-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

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 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-ILM

Before You Begin 1 Required Reagents

Required Reagents

HaloPlex Target Enr

Table 1 Required Reagents

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*

* Also available separately as Herculase II Fusion DNA Polymerase, 40 reactions (Agilent p/n 600675) and 100 mM dNTP Mix (Agilent p/n 200415, sufficient for 1000 enrichment 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

Beckman Coulter Genomics p/n A63880 p/n A63881

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-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

Part Number

Probe Design 96 Reactions 48 Reactions

HaloPlex Custom Panel Tier 1*, ILM

* 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 G9901C

HaloPlex Custom Panel Tier 1 Plus*, ILM G9961B G9961C

HaloPlex Custom Panel Tier 2, ILM

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

G9911B G9911C

HaloPlex Custom Panel Tier 3, ILM

Tier 3 designs are 2.6 Mb-5 Mb.

G9921B G9921C

ClearSeq ICCG, ILM G9953B**

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

G9953C**

ClearSeq Connective Disorder, ILM G9953B** G9953C**

ClearSeq Arrhythmia, ILM G9953B** G9953C**

ClearSeq Noonan Syndrome, ILM G9953B** G9953C**

ClearSeq Chromosome X, ILM G9953B** G9953C**

NOTE Kits contain enough reagents for 96 or 48 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-ILM

Before You Begin 1 Required Equipment

Required Equipment

HaloPlex Target Enr

Table 3 Required Equipment for HaloPlex Target Enrichment

Description Vendor and part number

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

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

Thermal cycler-compatible 96-well plates Agilent p/n 401333 (for SureCycler 8800) or see manufacturers recommendations

8-well PCR strip tubes with caps Nippon Genetics, p/n FG-088WF, or equivalent

96-well plate and strip tube-compatible magnetic separator

Agencourt SPRIPlate Super Magnet Plate p/n A32782, or equivalent

1.5 mL tube-compatible magnetic separator DynaMag-2 magnet, Thermo Fisher Scientific p/n 12321D, 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

Multichannel pipettes (10-L and 100-L volume) Pipetman or equivalent

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

Adhesive seals for 96-well PCR plates Agilent p/n 410186, or equivalent

Qubit Fluorometer Thermo Fisher Scientific p/n Q33226

Qubit assay tubes Thermo Fisher Scientific p/n Q32856

Ice bucket General laboratory supplier

Vortex mixer General laboratory supplier

ichment System-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-ILM

HaloPlex Target Enrichment System Protocol

2 Sample Preparation

Run Size Considerations 15

Run Time Considerations 15

Step 1. Digest genomic DNA with restriction enzymes 16

Step 2. Hybridize digested DNA to HaloPlex or ClearSeq probe for target enrichment and sample indexing 26

Step 3. Capture the target DNA 29

Step 4. Ligate the captured, circularized fragments 32

Step 5. Prepare the PCR Master Mix 33

Step 6. Elute captured DNA with NaOH 34

Step 7. PCR amplify the captured target libraries 35

Step 8. Purify the amplified target libraries 36

Step 9. Validate enrichment and quantify enriched target DNA 38

Step 10. Pool samples with different indexes for multiplexed sequencing 42

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.

See Figure 1 for a summary of the overall HaloPlex target enrichment workflow.

13Agilent Technologies

2 Sample Preparation

14

Figure 1 Overall HaloPlex target-enriched sequencing sample preparation workflow.

HaloPlex Target Enrichment System-ILM

Sample Preparation 2 Run Size Considerations

Run Size Considerations

HaloPlex Target Enr

Kits contain enough reagents for 48 or 96 reactions total, including control reactions using the provided Enrichment Control DNA (ECD). Each run that uses independently- prepared reagent master mixes should include one ECD control enrichment reaction.

The following protocol includes volumes appropriate for 12- sample runs. When planning a run size different from 12 samples, you will need to adjust volumes of components accordingly. Calculate the amount of each solution needed for the number of reactions in your run, plus 2 reactions excess for the restriction digestion steps and 1 reaction excess for the remaining steps. For example, for a 24 reaction run, calculate amounts of each solution by multiplying the single reaction value by 26 for restriction digestion steps and by 25 for hybridization and later steps.

A 96- reaction kit contains enough reagents to prepare master mixes for eight runs of 12 samples each for a total of 96 samples. When processing samples using runs with fewer than 12 samples, some reagents may be depleted before 96 samples are run.

A 48- reaction kit contains enough reagents to prepare master mixes for four runs of 12 samples each, for a total of 48 samples. When processing samples using runs with fewer than 12 samples, some reagents may be depleted before 48 samples are run.

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.

Designs containing <15,000 probes use a 3- hour hybridization time, and DNA digestion through PCR steps (see Figure 1) are typically run in the same day. Designs containing 15,000 probes use a 16- hour hybridization time, which is typically completed overnight, with the DNA digestion step started in the afternoon.

ichment System-ILM 15

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

Step 1. Digest genomic DNA with restriction enzymes

16

In this step, gDNA samples are digested by 16 different restriction enzymes to create a library of gDNA restriction fragments.

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.

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.

Use a fluorometry-based DNA quantitation method, such as Qubit fluorometry or PicoGreen staining, to accurately quantify the DNA starting material.

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.

NOTE

2 Prepare the DNA samples for the run. For 12- reaction runs, prepare 11 gDNA samples and one Enrichment Control DNA sample.

a In separate 0.2- mL PCR tubes, dilute 225 ng of each gDNA sample in 45 L nuclease- free water, for a final DNA concentration of 5 ng/L. Store on ice.

b In a separate 0.2- mL PCR tube, dispense 45 L of the supplied Enrichment Control DNA (ECD). Store on ice.

Although specific instructions are provided for the typical 12-sample run size, runs may include up to 96 samples. Include one ECD control sample per run of 2-96 samples. See page 15 for additional run size considerations.

NOTE

HaloPlex Target Enrichment System-ILM

Sample Preparation 2 Step 1. Digest genomic DNA with restriction enzymes

HaloPlex Target Enr

3 Prepare the Restriction Enzyme Master Mix strip.

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, along with restriction buffer and BSA to make eight different RE Master Mixes. Figure 2 illustrates how to prepare the 8- well Restriction Enzyme Master Mix strip for a 12- sample run using the steps detailed on page 18.

Figure 2 Preparation of the Restriction Enzyme Master Mix Strip for 12-sample run.

ichment System-ILM 17

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

18

a Combine the amounts of RE Buffer and BSA Solution indicated in the table below in a 1.5- mL tube. Mix by vortexing briefly.

b To begin preparation of the Restriction Enzyme Master Mix Strip, dispense the appropriate volume of the RE Buffer/BSA mixture to each well of an 8- well strip tube.

Reagent Volume for 1 Reaction Volume for 12 Reactions (includes excess)

RE Buffer 34.0 L 476 L

BSA Solution 0.85 L 11.9 L

Total Volume 34.85 L 487.9 L

Reagent Volume for 1 Reaction Volume for 12 Reactions (includes excess)

RE Buffer/BSA mixture

4 L 56 L

CAUTION It is important to use the restriction enzyme tube strip 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 is positioned adjacent to well A of each enzyme strip.

c Using a multichannel pipette, add the appropriate volume of each enzyme from the Green Enzyme Strip, with green marker aligned with tube A, to corresponding tubes A to H of the Restriction Enzyme Master Mix Strip.

Reagent Volume for 1 Reaction Volume for 12 Reactions (includes excess)

RE Enzymes from Green Enzyme Strip

0.5 L 7 L

HaloPlex Target Enrichment System-ILM

Sample Preparation 2 Step 1. Digest genomic DNA with restriction enzymes

HaloPlex Target Enr

d Using a multichannel pipette, add the appropriate volume of each enzyme from the Red Enzyme Strip, with red marker aligned with tube A, to each corresponding tube A to H of the same Restriction Enzyme Master Mix Strip.

e Mix by gentle vortexing and then spin briefly.

f Keep the Restriction Enzyme Master Mix Strip on ice until it is used in step 4.

4 Aliquot the Restriction Enzyme Master Mixes to the rows of a 96- well plate to be used as the restriction digest reaction plate.

a Align the Restriction Enzyme Master Mix Strip, prepared in step 3, along the vertical side of a 96- well PCR plate as shown below.

b Using a multichannel pipette, carefully distribute 5 L of each RE master mix row- wise into each well of the plate.

For runs with >12 samples, continue distributing 5 L from the same RE Master Mix strip row- wise into each well of the additional plates.

Visually inspect pipette tips for equal volumes before dispensing to the plate(s).

Reagent Volume for 1 Reaction Volume for 12 Reactions (includes excess)

RE Enzymes from Red Enzyme Strip

0.5 L 7 L

Each row of the 96- well plate now contains 5 L per well of the same restriction enzyme combination.

ichment System-ILM 19

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

20

5 Aliquot DNA samples into the 96- well Restriction Digest Reaction Plate(s).

a Align the DNA samples (11 gDNA samples and the ECD sample), prepared in step 2, along the horizontal side of the digestion reaction plate(s) as shown below.

b Carefully distribute 5 L of DNA samples column- wise into each well of the digestion reaction plate.

If using a multichannel pipette, visually inspect pipette tips for equal volumes before dispensing.

c Seal the plate thoroughly with adhesive plastic film.

6 Carefully vortex the plate to mix the digestion reactions.

7 Briefly spin the plate in a plate centrifuge.

Wells of the prepared 96- well plate now contain complete 10- L restriction digestion reactions. In this format, each column corresponds to one DNA sample digested in eight different restriction reactions.

HaloPlex Target Enrichment System-ILM

Sample Preparation 2 Step 1. Digest genomic DNA with restriction enzymes

HaloPlex Target Enr

8 Place the Restriction Digest Reaction Plate in a thermal cycler and run the program in Table 5, using a heated lid.

Table 5 Thermal cycler program for restriction digestion

Step Temperature Time

Step 1 37C 30 minutes

Step 2 8C Hold

NOTE Do not pool the eight restriction digests for a single DNA sample at this time. Restriction enzymes are still active and will catalyze inappropriate cleavage events if DNA samples are pooled before enzyme inactivation. DNA samples are pooled during the hybridization step on page 26, upon which restriction enzymes are inactivated by the reaction conditions.

ichment System-ILM 21

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

22

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

Keep the Restriction Digest Reaction Plate on ice during validation.

a Transfer 4 L of each ECD digestion reaction from wells of the digestion reaction plate to fresh 0.2- mL PCR tubes.

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 23), the Agilent 4200 TapeStation (see page 24). or by gel electrophoresis (see page 25).

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 3, Figure 4, and Figure 5 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-ILM

Sample Preparation 2 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 3 for sample Bioanalyzer electrophoresis results.

Figure 3 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.

ichment System-ILM 23

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

24

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 solution and 1 L of nuclease- free water.

Prepare the TapeStation samples as instructed in the 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.

CAUTION Make sure that you thoroughly mix the combined DNA and High Sensitivity D1000 sample buffer on a vortex mixer for 5 seconds for accurate results.

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

See Figure 4 for sample TapeStation electrophoresis results.

.

Figure 4 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-ILM

Sample Preparation 2 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 5 for sample gel results.

Figure 5 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 35.

ichment System-ILM 25

2 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

26

In this step, the collection of gDNA restriction fragments is hybridized to the HaloPlex or ClearSeq probe capture library. 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.

HaloPlex or 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.

1 Prepare a Hybridization Master Mix by combining the reagents in Table 6. Mix well by gentle vortexing, then spin the tube briefly.

Table 6 Hybridization Master Mix

2 Distribute 70 L of the Hybridization Master Mix to each of 12 0.2- mL tubes.

3 Add 10 L of the appropriate Indexing Primer to each tube containing Hybridization Master Mix.

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

4 Transfer digested DNA samples from the 96- well Restriction Digest Reaction Plate(s) directly into the hybridization reaction tubes prepared in step 3.

Reagent Volume for 1 Reaction Volume for 12 Reactions (includes excess)

Hybridization Solution 50 L 650 L

HaloPlex or ClearSeq Probe

20 L 260 L

Total Volume 70 L 910 L

HaloPlex Target Enrichment System-ILM

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

HaloPlex Target Enr

Transfer all eight digestion reactions that correspond to one DNA sample into the appropriate hybridization reaction tube. After addition of each individual digest reaction to the hybridization solution, mix by pipetting before adding the next digest reaction to ensure complete inactivation of the enzymes.

CAUTION Do not pool the digestion samples before adding to the hybridization reaction mixture as restriction enzymes are still active and may catalyze inappropriate cleavage events.

For the ECD sample, add 32 L of nuclease- free water, in addition to the digested DNA samples, to compensate for the volume removed for digest validation.

After pooling, each hybridization reaction contains the following components:

70 L Hybridization Master Mix

10 L Indexing Primer

approximately 80 L pooled digested DNA samples

Due to partial evaporation of samples, you may recover less than 10 L of each restriction digest. Minor reductions to the digested DNA pool volume will not impact hybridization performance; you do not need to compensate for any sample evaporation volume losses in the final pool.

NOTE

5 Vortex the mixtures briefly and then spin tubes briefly.

ichment System-ILM 27

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

28

6 Place the hybridization reaction tubes in a thermal cycler. Run the appropriate program in Table 7, using the hybridization duration listed on the Certificate of Analysis.

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 7 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.

HaloPlex Target Enrichment System-ILM

Sample Preparation 2 Step 3. Capture the target DNA

Step 3. Capture the target DNA

HaloPlex Target Enr

In this step, the circularized target DNA- probe hybrids, containing biotin, are captured on streptavidin beads.

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 Obtain or prepare 0.5 L per sample, plus excess, of 2 M acetic acid, for use on page 33.

Use a high-quality 2 M acetic acid solution. 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.

NOTE

3 Prepare 25 L per sample, plus excess, of fresh 50 mM NaOH for use in the DNA elution step on page 34.

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.

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

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

ichment System-ILM 29

2 Sample Preparation Step 3. Capture the target DNA

30

5 Prepare 40 L (1 Volume) of HaloPlex Magnetic Beads per hybridization sample, plus excess, for the capture reaction:

a Transfer the appropriate volume of bead suspension to a 1.5- mL tube.

b Put the tube into a 1.5 mL tube- compatible magnetic rack 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 to the beads and resuspend by pipetting up and down.

6 Remove the hybridization reactions from the thermal cycler and immediately add 40 L of the prepared bead suspension to each 160- L hybridization reaction.

Reagent Volume for 1 Reaction Volume for 12 Reactions (includes excess)

HaloPlex Magnetic Bead suspension

40 L 520 L

Reagent Volume for 1 Reaction Volume for 12 Reactions (includes excess)

Capture Solution 40 L 520 L

When adding beads to the hybridization reactions, visually inspect the bead preparation to ensure a homogenous suspension with no aggregated bead mass at the bottom of the tube. If aggregation is present, thoroughly resuspend the beads by vortexing and pipetting up and down before use.

NOTE

7 After adding the magnetic beads, mix the capture reactions thoroughly by pipetting up and down 15 times using a 100- L pipette set to 80 L.

8 Incubate the capture reactions at room temperature for 15 minutes.

9 Briefly spin the tubes in a desktop centrifuge and then transfer the tubes to the Agencourt SPRIPlate Super Magnet magnetic plate.

Use the Agencourt SPRIPlate Super Magnet magnetic plate for the remainder of magnetic bead collection steps for samples in PCR tubes or strip tubes.

NOTE

HaloPlex Target Enrichment System-ILM

Sample Preparation 2 Step 3. Capture the target DNA

HaloPlex Target Enr

10 Wait for the solution to clear (about 30 seconds), then remove and discard the supernatant using a pipette set to 200 L.

11 Wash the bead- bound samples:

a Remove the capture reaction tubes from the magnetic plate and add 100 L of Wash Solution to each tube.

b Resuspend the beads thoroughly by pipetting up and down 10 times using a 100- L multichannel pipette set to 80 L.

c Incubate the tubes in a thermal cycler at 46C for 10 minutes, using a heated lid.

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

d Briefly spin the tubes in a desktop centrifuge at room temperature and then transfer the tubes to the magnetic plate.

e Wait for the solution to clear (about 30 seconds), then carefully remove and discard the supernatant using a pipette set to 120 L. If necessary, carefully remove any residual liquid with a 20- L volume pipette.

ichment System-ILM 31

2 Sample Preparation Step 4. Ligate the captured, circularized fragments

Step 4. Ligate the captured, circularized fragments

32

In this step, DNA ligase is added to the capture reaction to close nicks in the circularized probe- target DNA hybrids.

1 Prepare a DNA ligation master mix by combining the reagents in the following table.

Mix the components thoroughly by gentle vortexing then spin the tube briefly.

Table 8 Preparation of DNA ligation master mix

2 Add 50 L of the DNA ligation master mix to the beads in each DNA capture reaction tube.

3 Resuspend the beads thoroughly by pipetting up and down 15 times using a 100- L multichannel pipette set to 40 L.

4 Incubate the tubes in a thermal cycler at 55C for 10 minutes, using a heated lid.

The thermal cycler may be programmed to include a 4C hold step following the 10- minute incubation. During the 10- minute incubation, prepare the PCR master mix as specified in the following step.

Reagent Volume for 1 Reaction Volume for 12 Reactions (includes excess)

Ligation Solution 47.5 L 617.5 L

DNA Ligase 2.5 L 32.5 L

Total Volume 50 L 650 L

HaloPlex Target Enrichment System-ILM

Sample Preparation 2 Step 5. Prepare the PCR Master Mix

Step 5. Prepare the PCR Master Mix

HaloPlex Target Enr

In this step, you prepare a PCR master mix for the captured target DNA amplification step on page 35.

CAUTION It is critical to include Acetic acid at 2 M concentration in this step to ensure neutralization of the NaOH used for elution on page 34.

1 Prepare the PCR master mix by combining the reagents in the following table.

2 Mix the master mix components by gentle vortexing, then distribute 30- L aliquots to fresh 0.2- mL reaction tubes.

3 Store the tubes on ice until they are used in Step 7. PCR amplify the captured target libraries on page 35.

Table 9 Preparation of PCR master mix

Reagent Volume for 1 reaction Volume for 12 reactions (includes excess)

Nuclease-free water 16.1 L 209.3 L

5X Herculase II Reaction Buffer

10 L 130 L

dNTPs (100 mM, 25 mM for each dNTP)

0.4 L 5.2 L

Primer 1 (25 M) 1 L 13 L

Primer 2 (25 M) 1 L 13 L

2 M Acetic acid 0.5 L 6.5 L

Herculase II Fusion DNA Polymerase

1 L 13 L

Total 30 L 390 L

ichment System-ILM 33

2 Sample Preparation Step 6. Elute captured DNA with NaOH

Step 6. Elute captured DNA with NaOH

34

When the 10- minute ligation reaction period is complete, proceed with the following steps to elute the captured DNA libaries.

CAUTION Using a high-quality NaOH solution for this step is critical for optimal DNA elution and recovery.

Be sure to use freshly-prepared 50 mM NaOH, prepared from 10 M NaOH according to the instructions on page 29.

1 Briefly spin the ligation reaction tubes in a desktop centrifuge and then transfer the tubes to the magnetic plate.

2 Wait for the solution to clear (about 30 seconds), then carefully remove and discard the supernatant using a pipette set to 50 L.

3 Remove the tubes from the magnetic plate and add 100 L of the SSC Buffer provided with the kit to each tube.

4 Resuspend the beads thoroughly by pipetting up and down 10 times using a 100- L multichannel pipette set to 80 L.

5 Briefly spin the tubes and then return the tubes to the magnetic plate.

6 Wait for the solution to clear (about 30 seconds), then carefully remove and discard the SSC Buffer using a multichannel pipette set to 120 L.

If necessary, carefully remove any residual liquid with a 20- L volume pipette.

7 Add 25 L of 50 mM NaOH, which was freshly- prepared on page 29, to each tube.

8 Resuspend the beads thoroughly by pipetting up and down 10 times using a 100- L multichannel pipette set to 15 L.

9 Incubate samples for 1 minute at room temperature to allow elution of the captured DNA.

10 Briefly spin the tubes and then transfer the tubes to the magnetic plate.

Proceed immediately to PCR amplification in the following section.

HaloPlex Target Enrichment System-ILM

Sample Preparation 2 Step 7. PCR amplify the captured target libraries

Step 7. PCR amplify the captured target libraries

HaloPlex Target Enr

1 Prepare amplification reactions by transferring 20 L of cleared supernatant from each tube on the magnetic plate to a PCR Master Mix tube held on ice (from page 33).

2 Mix by gentle vortexing and then spin briefly to collect the liquid.

3 Place the amplification reaction tubes in a thermal cycler and run the program in Table 10, using a heated lid.

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

. Table 10 Post-capture DNA amplification PCR 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.

ichment System-ILM 35

2 Sample Preparation Step 8. Purify the amplified target libraries

Step 8. Purify the amplified target libraries

36

In this step, the amplified target DNA is purified using AMPure XP beads.

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 PCR reaction sample to a fresh 0.2- mL tube. Store the remaining volume of each sample at 20C for troubleshooting.

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 homogenous 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 amplified library 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 plate. Wait for the solution to clear (approximately 5 minutes).

9 Keep the tubes in the magnetic plate. 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 plate while you add 200 L of 70% ethanol into the tubes.

Use fresh 70% ethanol for optimal results.

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.

HaloPlex Target Enrichment System-ILM

Sample Preparation 2 Step 8. Purify the amplified target libraries

HaloPlex Target Enr

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 plate 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 plate 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.

ichment System-ILM 37

2 Sample Preparation Step 9. Validate enrichment and quantify enriched target DNA

Step 9. Validate enrichment and quantify enriched target DNA

38

Prior to sample pooling and sequencing sample preparation, validate enrichment and quantify the enriched target DNA in each library sample using the Agilent 2100 Bioanalyzer (see page 39) or 4200 TapeStation (see page 40).

Enriched library samples may also be qualitatively analyzed using gel electrophoresis. Sample gel electrophoresis results are provided in the Reference section on page 53.

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 6.

Figure 6 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.

HaloPlex Target Enrichment System-ILM

Sample Preparation 2 Step 9. Validate enrichment and quantify enriched target DNA

HaloPlex Target Enr

Option 1: Analysis using the Agilent 2100 Bioanalyzer

Use a Bioanalyzer High Sensitivity DNA Assay kit and the Agilent 2100 Bioanalyzer 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 using the analysis guidelines on page 41.

See Figure 7 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 7 Validation of enrichment by Agilent 2100 Bioanalyzer analysis.

ichment System-ILM 39

2 Sample Preparation Step 9. Validate enrichment and quantify enriched target DNA

40

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 the 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.

CAUTION Make sure that you thoroughly mix the combined DNA sample and High Sensitivity D1000 sample buffer on a vortex mixer for 5 seconds for accurate results.

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 using the analysis guidelines on page 41.

See Figure 8 for a sample electropherogram.

Figure 8 Validation of enrichment by TapeStation analysis.

HaloPlex Target Enrichment System-ILM

Sample Preparation 2 Step 9. Validate enrichment and quantify enriched target DNA

HaloPlex Target Enr

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 on page 36.

ichment System-ILM 41

2 Sample Preparation Step 10. Pool samples with different indexes for multiplexed sequencing

Step 10. Pool samples with different indexes for multiplexed sequencing

42

Use the following guidelines to design your sample pooling and sequencing 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 45).

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 47.

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

MiSeq platform sequencing run setup guidelines

Use the Illumina Experiment Manager (IEM) software to generate a custom Sample Sheet according to the guidelines below. Once a Sample Sheet has been generated, index sequences need to be manually changed to the HaloPlex indexes used for each sample. See the Reference chapter, starting on page 47, for nucleotide sequences of the HaloPlex system indexes.

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.

HaloPlex Target Enrichment System-ILM

Sample Preparation 2 Step 10. Pool samples with different indexes for multiplexed sequencing

HaloPlex Target Enr

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

ichment System-ILM 43

2 Sample Preparation Step 10. Pool samples with different indexes for multiplexed sequencing

44

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.

HaloPlex Target Enrichment System-ILM

Sample Preparation 2 Step 10. Pool samples with different indexes for multiplexed sequencing

HaloPlex Target Enr

HiSeq platform sequencing run setup guidelines

Set up sequencing runs to perform an 8- nt index read using the Cycles settings shown in Table 11. 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 11 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

ichment System-ILM 45

2 Sample Preparation Step 10. Pool samples with different indexes for multiplexed sequencing

46

HaloPlex Target Enrichment System-ILM

HaloPlex Target Enrichment System Protocol

3 Reference

Kit Contents 48

Nucleotide Sequences of HaloPlex Indexes 52

Qualitative analysis of enrichment by gel electrophoresis 53

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

47Agilent Technologies

3 Reference Kit Contents

Kit Contents

HaloPlex Target Enrichment System Kits for custom designs include the component kits listed in Table 12 below. Kits for ClearSeq Disease Research Panels include the component kits listed in Table 13 below.

Table 12 HaloPlex Target Enrichment System Kit Contents-Custom Designs

Design Type Reaction Number

HaloPlex Target Enrichment System-ILM, Box 1*

* See Table 14 for list of included reagents.

HaloPlex Magnetic Beads Box 2

Store at 20C Store at +4C

HaloPlex 1-500 kb with <15,000 Probes, ILM 48 Reactions 5190-8044 OR 5190-8045

Part number 5190-8044, 5190-8050, 5191-4066, 5191-4068, 5190-8046, 5190-8052, 5190-8048 or 5190-8054 is provided for the first order of a specific HaloPlex Custom Probe design. Re-order kits, containing previously-purchased Custom Probe designs, include Box 1 part number 5190-8045, 5190-8051, 5191-4067, 5191-4069, 5190-8047, 5190-8053, 5190-8049 or 5190-8055.

5190-5976

96 Reactions 5190-8050 OR 5190-8051 5190-5386

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

48 Reactions 5191-4066 OR 5191-4067 5190-5976

96 Reactions 5191-4068 OR 5191-4069 5190-5386

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

48 Reactions 5190-8046 OR 5190-8047 5190-5976

96 Reactions 5190-8052 OR 5190-8053 5190-5386

HaloPlex 2.6 Mb-5 Mb, ILM 48 Reactions 5190-8048 OR 5190-8049 5190-5976

96 Reactions 5190-8054 OR 5190-8055 5190-5386

48 HaloPlex Target Enrichment System-ILM

Reference 3 Kit Contents

Table 13 HaloPlex Target Enrichment System Kit Contents-ClearSeq Disease Research Panels

Design Type Reaction Number

HaloPlex Target Enrichment System-ILM, Box 1*

* See Table 14 for list of included reagents.

HaloPlex Magnetic Beads Box 2

Store at 20C Store at +4C

ClearSeq ICCG, ILM 48 Reactions 5190-9146 5190-9223

96 Reactions 5190-9164 5190-9224

ClearSeq Connective Disorder, ILM 48 Reactions 5190-9150 5190-9223

96 Reactions 5190-9168 5190-9224

ClearSeq Arrhythmia, ILM 48 Reactions 5190-9144 5190-9223

96 Reactions 5190-9162 5190-9224

ClearSeq Noonan Syndrome, ILM 48 Reactions 5190-9152 5190-9223

96 Reactions 5190-9170 5190-9224

ClearSeq Chromosome-X, ILM 48 Reactions 5190-9148 5190-9223

96 Reactions 5190-9166 5190-9224

HaloPlex Target Enrichment System-ILM 49

3 Reference Kit Contents

50

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

Table 14 HaloPlex Target Enrichment System Box 1 Contents

Included Reagents 48 Reaction Kit 96 Reaction Kit

Hybridization Solution bottle bottle

Ligation Solution bottle bottle

Wash Solution bottle bottle

Capture Solution bottle bottle

SSC Buffer bottle bottle

RE Buffer bottle bottle

BSA Solution tube with clear cap tube with clear cap

DNA Ligase tube with red cap tube with red cap

Enrichment Control DNA tube with orange cap tube with orange cap

Primer 1 tube with yellow cap tube with yellow cap

Primer 2 tube with blue cap tube with blue cap

HaloPlex Indexing Primers 96-well plate with Indexing Primer A01 to H06 (blue plate)*

* See Table 15 for a plate map.

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

See Table 16 for a plate map.

Enzyme Strip 1 8-well strip tube with green label

8-well strip tube with green label

Enzyme Strip 2 8-well strip tube with red label

8-well strip tube with red label

HaloPlex or ClearSeq Probe tube with pink cap tube with pink cap

HaloPlex Target Enrichment System-ILM

Reference 3 Kit Contents

Table 15 Plate map for HaloPlex Indexing Primers A01 through H06 provided with 48-reaction kits; wells in columns 7 through 12 are empty

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

A A01 A02 A03 A04 A05 A06

B B01 B02 B03 B04 B05 B06

C C01 C02 C03 C04 C05 C06

D D01 D02 D03 D04 D05 D06

E E01 E02 E03 E04 E05 E06

F F01 F02 F03 F04 F05 F06

G G01 G02 G03 G04 G05 G06

H H01 H02 H03 H04 H05 H06

Table 16 Plate map for HaloPlex Indexing Primers A01 through H12 provided with 96-reaction kits

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

HaloPlex Target Enrichment System-ILM 51

3 Reference Nucleotide Sequences of HaloPlex Indexes

Nucleotide Sequences of HaloPlex Indexes

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

52

Table 17 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

HaloPlex Target Enrichment System-ILM

Reference 3 Qualitative analysis of enrichment by gel electrophoresis

Qualitative analysis of enrichment by gel electrophoresis

HaloPlex Target Enr

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 22 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 9 for a sample gel analysis image.

Figure 9 Validation of HaloPlex enrichment process by gel electrophoresis. Lane 1: 25-bp DNA ladder, Lanes 2-7: enriched library samples.

ichment System-ILM 53

www.agilent.com

In This Book

This guide contains information to run the HaloPlex Target Enrichment System protocol for the Illumina sequencing platform.

Agilent Technologies, Inc. 2015, 2018

Version G0, July 2018

*G9900-90001* G9900-90001

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