Agilent OnePGT Library Preparation for Illumina User Manual

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OnePGT Library Preparation for Illumina Sequencing

Protocol

Version C1, December 2020

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

Agilent Technologies

Notices

No part of this manual may be reproduced in any form or by any means (including electronic storage and retrieval or translation into a foreign language) without prior agreement and written consent from Agilent Technologies, Inc. as governed by United States and international copyright laws.

Manual Part Number

G9425-90000

Edition

Version C1, December 2020

Agilent Technologies, Inc. 5301 Stevens Creek Blvd Santa Clara, CA 95051 USA

Technical Support

For support with setup and use of Agilent OnePGT Solution, contact us using the following e-mail address:

onepgt@agilent.com

Safety Notices

CAUTION

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

WARNING

A WARNING notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly performed or adhered to, could result in personal injury or death. Do not proceed beyond a WARNING notice until the indicated conditions are fully understood and met.

2 OnePGT Library Preparation for Illumina Sequencing

In this Guide...

This guide describes the optimized workflow for generation of OnePGT libraries compatible with Illumina NextSeq 500/550 and HiSeq 2500 sequencing platforms. NGS data obtained after completing the workflow and subsequent sequencing needs to be analyzed with Agilent’s Alissa OnePGT software for reporting of preimplantation genetic testing data.

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 the procedure.

2 Whole Genome Amplification of Biopsy Samples using REPLI-g

Single Cell Kit

This chapter describes the steps to prepare amplified DNA from a biopsy sample using the REPLI- g Single Cell Kit according to a modified protocol with a two- hour amplification step.

3 Library Preparation

This chapter describes the steps to prepare OnePGT libraries for DNA sequencing.

4 Reference

This chapter contains reference information, including component kit contents, a troubleshooting guide, and abbreviated quick reference protocols for experienced users.

OnePGT Library Preparation for Illumina Sequencing 3

What’s New in Version C1

• Updates to thermal cycler recommendations and usage

• Update to Qubit instrument ordering information in

• Addition of unamplified reference gDNA concentration

• Update to order of operations in step 13 on page 31

• Updates to support for downstream NGS demultiplexing

What’s New in Version C0

• Updates to Reverse PCR Primer plate orientation

• Updates to p/n and content details for the REPLI- g

• Support for 4150 TapeStation (see Table 2 on page 12)

• Update to page 20 to indicate optional sample storage

• Update to headings on page 30 and page 38

• Minor updates to 2100 Bioanalyzer, 4200 TapeStation and

• Updates to instructions for dilution of Custom Read 1

instructions (see Table 2 on page 12, procedural note 13 on page 10, and step 3 on page 18)

Table 2 on page 12

(

29.4 ng/µl) to page 23

methods (see step 1 on page 47 and Troubleshooting on

page 58)

information (see Caution on page 37 and figure on

page 51)

Single Cell Kit supplied with Agilent OnePGT Solution (see Table 27 on page 49 and Table 30 on page 50)

after DNA amplification as a Stopping Point

4150 TapeStation reference document links (see page 40)

Sequencing Primer during sequencing run setup (see

page 44 and page 45)

OnePGT Library Preparation for Illumina Sequencing 4

Content

1 Before You Begin 7

Product Description 8

Safety Notes 9

Procedural Notes 9

Disposal 10

Required Reagents 11

Required Equipment 12

2 Whole Genome Amplification of Biopsy Samples using REPLI-g Single Cell

Kit 15

Material Preparation 16

Whole Genome Amplification Protocol 17

OnePGT Library Preparation for Illumina Sequencing 5

3 Library Preparation 21

Overview of the Workflow 22

Protocol 23

Step 1. Prepare DNA samples 23 Step 2. Fragment the DNA 24 Step 3. Add adapters to fragmented DNA 26 Step 4. Ligate the adapters 28 Step 5. Purify the DNA using SPRI technology 30 Step 6. Size-select the DNA fragments 33 Step 7. Suppression PCR-amplify the size-selected DNA 35 Step 8. Purify the DNA using SPRI technology 38 Step 9. Quantify and qualify the OnePGT libraries 40 Step 10. Pool libraries for multiplexed sequencing 42 Step 11. Set up the sequencing run 44 Step 12. Process sequencing data and upload to Agilent Alissa OnePGT 47

Contents

4 Reference 48

Kit Contents and Supported Configurations 49

Reference Information for OnePGT Indexes 51

Guidelines for Optimal Index Multiplexing 53

Troubleshooting Guide 54

Quick Reference Protocols 59

OnePGT Library Preparation for Illumina Sequencing 6

OnePGT Library Preparation for Illumina Sequencing Protocol

1 Before You Begin

Product Description 8 Safety Notes 9 Procedural Notes 9 Disposal 10 Required Reagents 11 Required Equipment 12

Make sure you read and understand the information in this section and have the necessary equipment and reagents listed available before you begin the procedure.

Agilent Technologies

Product Description

Agilent OnePGT Solution is a genome- wide, next- generation sequencing (NGS)- based system designed to integrate pre- implantation genetic testing (PGT) for monogenic disorders (PGT- M), translocations (PGT- SR), and aneuploidy screening (PGT- A) in a single workflow. Agilent OnePGT Solution includes the REPLI- g Single Cell Kit for whole genome amplification, the Agilent OnePGT Library Prep Kit for the generation of NGS- ready libraries, and the Agilent Alissa OnePGT software for data analysis and reporting.

Agilent OnePGT Solution is intended for PGT analysis of DNA derived from a blastomere (i.e. a single cell of a human cleavage- stage embryo) or a trophectoderm biopsy (i.e. 3- 10 cells of the trophectoderm of a human blastocyst- stage embryo). The protocols are not compatible with DNA derived from polar bodies or other sources.

This publication includes directions for using the REPLI- g Single Cell Kit for whole genome amplification of biopsy samples and for using the Agilent OnePGT Library Prep Kit for sequencing library preparation. The Library Preparation protocol is used to prepare sequencing libraries both from whole genome amplified biopsy samples and from unamplified reference family genomic DNA samples (required only for PGT- M applications).

Before You Begin 1

Product Description

Use of the Agilent Alissa OnePGT software for data analysis and reporting is described in separate documentation, available through the Agilent Alissa OnePGT software application. Contact onepgt@agilent.com for assistance with setting up your Agilent Alissa environment.

NOTE

OnePGT Library Preparation for Illumina Sequencing 8

If you are using this product for embryo screening please make sure you adhere to your country specific laws and regulations for human assisted reproductive technologies. Your country might have banned sex selection for non-medical purposes, as well as the commercial use of gametes, zygotes, and embryos. Agilent shall have no liability for any direct, indirect, consequential, or incidental damages arising out of the use, the results of use, or the inability to use this product.

Safety Notes

1 Specimens should be handled as if infectious using safe laboratory

procedures such as those outlined in Biosafety in Microbiological and Biomedical Laboratories and in the CLSI Document M29- A. Thoroughly clean and disinfect all work surfaces with a freshly prepared solution of 70% ethanol in deionized or distilled water.

2 Wear appropriate personal protective equipment (PPE) – including

disposable gloves, laboratory coat, and eye protection – when working in the laboratory or when handling specimens and reagents.

3 Material Safety Data Sheets (MSDS) are available from the Agilent

website at:

www.chem.agilent.com/en- US/Search/Library/Pages/MsdsSearch.aspx.

Procedural Notes

Use Good Laboratory Practice (GLP) principles at all times, including the procedures outlined below.

1 Do not pool reagents from different lots or from different bottles of the

same lot.

2 Do not use assay materials after their expiration dates.

3 All volumes stated in the instructions are intended to be used as

specified within the tolerance ranges for standard micropipettors. Make sure that all pipettors are calibrated and operating within manufacturer’s specifications.

4 Workflow in the laboratory must proceed in a uni- directional manner,

beginning in the whole genome amplification or gDNA sample preparation area and moving to the library preparation area.

5 Supplies and equipment for DNA isolation must be dedicated to that

activity and not used for other activities or moved between areas.

6 Powder- free gloves must be worn in each area and must be changed

before leaving that area.

7 Equipment and supplies used for reagent preparation must not be used

for specimen preparation activities or for pipetting or processing amplified DNA or other sources of target DNA.

Before You Begin 1

Safety Notes

OnePGT Library Preparation for Illumina Sequencing 9

Before You Begin 1

Disposal

8 Use best- practices to prevent PCR product contamination of samples

throughout the workflow:

a Assign separate pre- PCR and post- PCR work areas and use

dedicated equipment, supplies, and reagents in each area. In particular, never use materials designated to post- PCR work areas for pre- PCR segments of the workflow.

b Maintain clean work areas. Clean pre- PCR surfaces that pose the

highest risk of contamination daily using a 10% bleach solution.

c Always use dedicated pre- PCR pipettors with nuclease- free

aerosol- resistant tips to pipette dedicated pre- PCR solutions.

d Use good laboratory hygiene, including changing gloves after contact

with any potentially- contaminated surfaces.

9 Follow your institution’s procedures or common practices for tracking

samples throughout the assay.

10 Possible stopping points, where DNA samples may be stored at 4°C or

–20°C, are marked in the protocol.

11 Avoid repeated freeze- thaw cycles of solutions containing gDNA or

enzymes.

12 When preparing frozen reagent stock solutions not containing gDNA or

enzymes for use:

a Thaw the aliquot as quickly as possible without heating above room

temperature (15°C to 30°C).

b Mix briefly on a vortex mixer, then spin in a microcentrifuge for 5 to

10 seconds to drive the contents off the walls and lid.

c Store on ice or in a cold rack until use.

13 For incubation or amplification steps performed using a thermal cycler

with heated lid ON, use a lid temperature of 105°C.

Disposal

Dispose of unused reagents, waste, and specimens in accordance with country, federal, state and local regulations.

OnePGT Library Preparation for Illumina Sequencing 10

Before You Begin 1

Required Reagents

Table 1 Reagents Required for OnePGT Library Preparation

Description Vendor and part number

Agilent OnePGT Solution (see page 49 for list of materials provided) Agilent p/n G9426AA

Nuclease-free water Thermo Fisher Scientific p/n AM9930, or

equivalent

Agencourt AMPure XP magnetic particle solution 5 ml 60 ml 450 ml

Ethanol, 96%–100% general laboratory supplier

Qubit dsDNA BR Assay Kit, or equivalent Thermo Fisher Scientific p/n Q32850

Qubit dsDNA HS Assay Kit, or equivalent Thermo Fisher Scientific p/n Q32851

Control gDNA (whole genome amplification control) Agilent OneSeq Reference DNA, Male,

†

Beckman Coulter Genomics

p/n A63880 p/n A63881 p/n A63882

p/n 5190-8848, or equivalent

* This Protocol also supports use of Agilent p/n G9427AA (Agilent OnePGT Solution without REPLI-g) plus two REPLI-g Single

Cell Kits p/n 5191-4065 (48 reactions/kit).

† Alternatively, SPRIselect Reagent (Beckman Coulter Genomics p/n B23317) may be used for DNA purification steps with the

minor protocol modification detailed in the footnote to Table 17 on page 30.

OnePGT Library Preparation for Illumina Sequencing 11

Required Equipment

Table 2 Equipment Required for OnePGT Library Preparation

Description Vendor and part number

Thermal Cycler with 96-well, 0.2 ml block Various suppliers

Before You Begin 1

Required Equipment

Plasticware compatible with the selected thermal cycler:

Polypropylene 96-well PCR plates or 8-well strip tubes 8-well strip tube caps

Magnetic separator DynaMag-96 Side magnet, Thermo Fisher

1.5-mL, PCR clean tubes Eppendorf p/n 022431021 or equivalent

PippinHT size selection device and consumables

PippinHT instrument

1.5% Agarose 300-1500 bp 15C cassette, with electrophoresis buffer and Marker 15 C

Qubit Fluorometric Quantitation System, or equivalent Thermo Fisher Scientific p/n Q33238 and Q32856

DNA Analysis Platform and Consumables

Agilent 2100 Bioanalyzer Instrument

Agilent 2100 Expert SW Laptop Bundle (optional)

DNA 1000 Kit

Agilent TapeStation Instrument and Consumables

96-well sample plates

96-well plate foil seals

8-well tube strips

8-well tube strip caps

D1000 ScreenTape

D1000 Reagents

Consult the thermal cycler manufacturer’s

recommendations

Scientific p/n 12331D, or equivalent

Sage Science

p/n HTP0001

p/n HTC1510

Agilent p/n G2939BA

Agilent p/n G2953CA

Agilent p/n 5067-1504

Agilent 4200 TapeStation p/n G2991AA OR

Agilent 4150 TapeStation p/n G2992AA

Agilent p/n 5042-8502

Agilent p/n 5067-5154

Agilent p/n 401428

Agilent p/n 401425

Agilent p/n 5067-5582

Agilent p/n 5067-5583

Centrifuge Eppendorf Centrifuge model 5804 or equivalent

OnePGT Library Preparation for Illumina Sequencing 12

Before You Begin 1

Required Equipment

Table 2 Equipment Required for OnePGT Library Preparation

Description Vendor and part number

Plate or strip tube centrifuge Labnet International MPS1000 Mini Plate Spinner,

p/n C1000 (requires adapter, p/n C1000-ADAPT, for use with strip tubes) or equivalent

Multichannel pipette general laboratory supplier

P10, P20, P200 and P1000 pipettes general laboratory supplier

Sterile, nuclease-free aerosol barrier pipette tips general laboratory supplier

Vortex mixer general laboratory supplier

Ice bucket general laboratory supplier

Powder-free gloves general laboratory supplier

Freezer, set to –20°C (acceptable range –25°C to –15°C) general laboratory supplier

Freezer, set to –80°C (acceptable range –84°C to –67°C) general laboratory supplier

Refrigerator, set to +4°C (acceptable range +2°C to +8°C) general laboratory supplier

OnePGT Library Preparation for Illumina Sequencing 13

Before You Begin 1

Required Equipment

OnePGT Library Preparation for Illumina Sequencing 14

OnePGT Library Preparation for Illumina Sequencing Protocol

2 Whole Genome Amplification of Biopsy Samples using REPLI-g Single Cell Kit

Material Preparation 16 Whole Genome Amplification Protocol 17

This section contains instructions for amplification of DNA from human embryo biopsy samples using the REPLI- g Single Cell Kit (Agilent p/n 5191- 4065) and using a modified two- hour DNA amplification protocol. The protocol is intended for use with blastomere (i.e. a single cell of a human cleavage- stage embryo) or trophectoderm (i.e. 3–10 cells of the trophectoderm of a human blastocyst- stage embryo) biopsy samples.

CAUTION

Do not use other whole genome amplification (WGA) methods to prepare DNA samples for use in the OnePGT Library Preparation protocol. Use only REPLI-g Single Cell Kits purchased from Agilent and prepare samples according to the two-hour protocol provided in this chapter. Use of REPLI-g Single Cell Kits purchased directly from Qiagen, and use of WGA protocols provided by Qiagen, are not supported and may cause loss of samples or data quality.

Agilent Technologies

Whole Genome Amplification of Biopsy Samples using REPLI-g Single Cell Kit 2

Material Preparation

This protocol uses the reagents from the REPLI- g Single Cell Kit listed in

Table 3. See Table 1 on page 11 for kit ordering information.

Before starting each protocol step, prepare the reagents as described below.

Table 3 Reagents for whole genome amplification

Material Preparation

REPLI-g Single Cell Kit Component

Buffer DLB Provided lyophilized. For first use, resuspend as directed in

O sc Thaw at room temperature. page 16, page 19

DTT, 1 M Thaw at room temperature, then vortex and centrifuge briefly. page 18

PBS sc Thaw at room temperature, then vortex and centrifuge briefly. page 17

Stop Solution Thaw at room temperature, then vortex and centrifuge briefly. page 19

REPLI-g sc Reaction Buffer Thaw at room temperature, just prior to use. Once thawed,

REPLI-g sc DNA Polymerase Thaw on ice, just prior to use. Once thawed, mix well by

Preparation Steps Where Used in Protocol

page 16 (lyophilized), “Reconstitution of Buffer DLB” below, then store any unused

material at –20°C. For subsequent use, thaw at room temperature then vortex to mix before use.

vortex and centrifuge briefly. If a precipitate is present, vortex the tube for an additional 10 seconds to dissolve the material.

inverting the tube and centrifuge briefly.

page 18 (reconstituted)

page 19

Reconstitution of Buffer DLB

During first use of each Buffer DLB vial, reconstitute the lyophilized material by adding 500 µl of H2O sc to the tube. Mix thoroughly to dissolve and then centrifuge briefly.

NOTE

The reconstituted Buffer DLB, which is pH labile, may be stored for 6 months at –20°C.

OnePGT Library Preparation for Illumina Sequencing 16

Whole Genome Amplification of Biopsy Samples using REPLI-g Single Cell Kit 2

Whole Genome Amplification Protocol

1 Prepare each biopsy sample to be processed as a cell suspension in PBS

solution with a maximum volume of 4 µl, in a microcentrifuge tube compatible with your thermal cycler.

If using <4 µl of cell material, add a sufficient volume of kit- supplied PBS sc to bring the volume to 4 µl.

Keep the samples on ice until they are used in step 5.

Whole Genome Amplification Protocol

CAUTION

Due to the small number of cells in the sample, it is important to use the liquid handling methods below to prevent sample loss:

• When adding solutions to tubes containing the cell suspension, pipette the solutions onto the side of the tube. Do not insert the pipette tip into the cell suspension liquid, since cells may adhere to the tip and be removed from the sample.

• Mixtures containing the cell suspension must be mixed as specified in the protocol. Do not mix liquids into the cell suspension by vortexing or by pipetting up and down during any of the protocol steps below. (After amplification, solutions containing the amplified DNA may be mixed using these methods as specified in the library preparation protocol starting on page 23.)

2 Prepare positive and negative control samples in microcentrifuge tubes

compatible with your thermal cycler.

a Positive control: 4 µl of well- characterized control gDNA (see

Table 1 on page 11 for a recommended source) diluted to 15 pg/µl in

PBS sc

b Collection buffer negative control: 4 µl of the embryo biopsy

collection buffer

c NTC negative control: 4 µl of PBS sc

OnePGT Library Preparation for Illumina Sequencing 17

Whole Genome Amplification of Biopsy Samples using REPLI-g Single Cell Kit 2

Whole Genome Amplification Protocol

3 Preprogram a thermal cycler, with the heated lid ON, using the program

in Table 4. Start the program, then immediately pause the program to allow the heated lid to reach temperature while you prepare Buffer D2. Follow the manufacturer’s instructions for pausing the PCR program.

Table 4 Thermal cycler program for cell lysis and DNA denaturation

Step Temperature Time

Step 1 65°C 10 minutes

Step 2 4°C Hold

4 Prepare the appropriate volume of Buffer D2 (denaturation buffer) in a

1.5- ml tube, as described in Table 5. Mix by vortexing, then spin the tube briefly to collect the liquid.

Table 5 Preparation of Buffer D2

NOTE

Reagent Volume for 12 samples

DTT, 1 M 3 µl

Reconstituted Buffer DLB (prepared on page 16) 33 µl

Total 36 µl

* If processing fewer than 12 samples, store the remaining Buffer D2 at –20°C for up to three months.

5 Add 3 µl Buffer D2 (prepared in Table 5) to each 4- µl cell sample and

each control sample. Pipette the Buffer D2 onto the wall of the tube above the liquid surface, then mix by flicking the tubes carefully. Spin the tubes briefly to collect the liquid.

Before continuing to the next step, verify that the cell material in the tube is suspended in liquid and is not adhering to the tube wall above the liquid surface.

6 Place the samples in the thermal cycler. Close the lid, then resume the

cell lysis/DNA denaturation program in Table 4.

OnePGT Library Preparation for Illumina Sequencing 18

Whole Genome Amplification of Biopsy Samples using REPLI-g Single Cell Kit 2

Whole Genome Amplification Protocol

7 Once the thermal cycler reaches the 4°C Hold step, remove the samples

and add 3 µl of the kit-

provided Stop Solution to each tube. Pipette the Stop Solution onto the wall of the tube above the liquid surface, then mix by flicking the tube carefully. Spin the tubes briefly to collect the liquid. Keep the samples on ice.

8 Thaw the REPLI-

g sc DNA Polymerase on ice, mix well by inverting the tube, and keep on ice until use in step 10. Thaw the REPLI- g sc Reaction Buffer at room temperature, mix by vortexing, and keep at room temperature until use in step 10. Spin the reagent tubes briefly to collect the liquid before use.

9 Preprogram the thermal cycler, with the heated lid ON, using the

program in Table 6. Start the program, then immediately pause the program to allow the heated lid to reach temperature while you set up the reactions.

Table 6 Thermal cycler program for DNA amplification

Step Temperature Time

Step 1 30°C 2 hours

Step 2 65°C 3 minutes

Step 3 4°C Hold

10 Prepare the appropriate volume of amplification master mix in a 1.5- ml

tube, as described in Table 7. First combine the kit- supplied H2O sc and the REPLI- g sc Reaction Buffer, then mix by vortexing, and spin the tube briefly. Just before use of the master mix in step 11, add the REPLI- g sc DNA Polymerase and mix well by pipetting up and down. Keep the master mix on ice and proceed immediately to step 11.

Table 7 Preparation of amplification master mix

Reagent Volume for 1 sample Volume for 12 samples

(includes excess)

O sc 9 µl 117 µl

REPLI-g sc Reaction Buffer 29 µl 377 µl

REPLI-g sc DNA Polymerase 2 µl 26 µl

Total 40 µl 520 µl

OnePGT Library Preparation for Illumina Sequencing 19

Whole Genome Amplification of Biopsy Samples using REPLI-g Single Cell Kit 2

Whole Genome Amplification Protocol

11 To each 10-

master mix prepared in Table 7. Pipette the master mix onto the wall of the tube above the liquid surface, then mix by flicking the tube carefully. Briefly spin the tubes to collect the liquid.

12 Place the samples in the thermal cycler. Close the lid, then resume the

DNA amplification program in Table 6.

NOTE

Stopping Point If the amplified DNA samples will not be used immediately, store the

The DNA polymerase is inactivated during incubation at 65°C in Step 2 of this program.

13 Once the thermal cycler reaches the 4°C Hold step, proceed to the DNA

library preparation protocol on page 23.

samples at 4°C for up to 3 days or at –20°C for up to 1 year.

µl denatured DNA sample, add 40 µl of the amplification

OnePGT Library Preparation for Illumina Sequencing 20

OnePGT Library Preparation for Illumina Sequencing Protocol

3 Library Preparation

Overview of the Workflow 22 Protocol 23

OnePGT 47

This section contains instructions for the preparation of OnePGT libraries compatible with sequencing on Illumina NGS systems.

Agilent Technologies

Overview of the Workflow

The OnePGT library preparation workflow is summarized in Figure 1. The estimated hands- on time (HOT) and estimated total turnaround time (TAT) are shown for each step, when processing 24 samples and using a TapeStation 4200 instrument for QC size profiling steps.

Library Preparation 3

Overview of the Workflow

Figure 1 OnePGT library preparation workflow and time requirements.

OnePGT Library Preparation for Illumina Sequencing 22

Protocol

Library Preparation 3

Protocol

This protocol describes the steps to prepare sequencing libraries from whole genome amplified DNA samples, derived from human blastomere or trophectoderm biopsies. The protocol is also used to prepare sequencing libraries from unamplified family reference gDNA prepared from blood samples with initial concentration applications). The protocol is not compatible with DNA derived from polar bodies or other sources.

Biopsy- derived DNA samples must be amplified using the REPLI- g Single Cell Kit supplied by Agilent, using the protocol provided in Chapter 2, “Whole Genome Amplification of Biopsy Samples using REPLI- g Single Cell Kit”. The concentration of the amplified product should be 200 ng/µl by fluorometric quantitation. Do not use other whole genome amplification

(WGA) methods to prepare DNA samples for use in this protocol.

29.4 ng/µl (required only for PGT- M

Step 1. Prepare DNA samples

The protocol requires 500 ng DNA per sample. Make sure the gDNA samples are of high quality, with OD

1 Mix the DNA sample by vortexing. Spin the sample tube briefly to

collect the liquid and keep on ice.

2 Use the Qubit dsDNA BR Assay to determine the initial concentration

of each DNA sample. Follow the manufacturer’s instructions.

3 Prepare 20 µl of 29.4 ng/µl DNA for each sample by diluting the DNA

samples with nuclease- free H2O. Place the diluted samples in wells of a PCR plate or strip tube and keep on ice.

260

/OD

ratio 1.8.

280

NOTE

OnePGT Library Preparation for Illumina Sequencing 23

Do not use the Nuclease-Free Water provided in Agilent OnePGT Library Prep Kit Box 2 for this step. The kit-supplied water is intended for use only during the ligation step on page 29.

Library Preparation 3

Step 2. Fragment the DNA

In this step, the DNA is enzymatically fragmented. This step uses the components listed in Table 8.

Table 8 Reagents for DNA fragmentation

Kit Component Storage Location

Restriction Enzyme 1 Agilent OnePGT Library Prep Kit Box 1, –80°C

Restriction Enzyme 2 Agilent OnePGT Library Prep Kit Box 2, –20°C

Restriction Enzyme Buffer Agilent OnePGT Library Prep Kit Box 2, –20°C

1 Thaw the vial of Restriction Enzyme Buffer completely before use. Mix

by vortexing. Spin the vial briefly to collect the liquid and keep on ice.

2 Preprogram a thermal cycler, with the heated lid ON, using the program

in Table 9. Start the program, then immediately pause the program to allow the heated lid to reach temperature while you set up the reactions. Follow the manufacturer’s instructions for pausing the PCR program.

Table 9 Thermal cycler program for DNA fragmentation

Step Temperature Time

Step 1 37°C 15 minutes

Step 2 65°C 20 minutes

Step 3 4°C 1 minute

Step 4 4°C Hold

3 Place vials of Restriction Enzyme 1 and Restriction Enzyme 2 on ice

and verify that the contents are thawed. Spin the tubes briefly to collect the liquid and keep on ice.

NOTE

OnePGT Library Preparation for Illumina Sequencing 24

Return Restriction Enzyme 1 and Restriction Enzyme 2 vials to the storage locations listed in

Table 8 as quickly as possible after use in step 4.

Library Preparation 3

Step 2. Fragment the DNA

4 Prepare the appropriate volume of restriction digest mix in a 1.5-

ml tube, as described in Table 10. Mix by pipetting up and down at least 10 times. Do not vortex. Spin the tube briefly to collect the liquid and keep on ice.

Table 10 Preparation of restriction digest mix

Reagent Volume for 1 sample Volume for 12 samples (includes excess)

Restriction Enzyme Buffer 2 µl 28 µl

Restriction Enzyme 1 0.5 µl 7 µl

Restriction Enzyme 2 0.5 µl 7 µl

Total 3 µl 42 µl

5 Set up the digestion reactions using a PCR plate or strip tube.

a To each sample well, add 3 µl of the restriction digest mix prepared

in Table 10.

b Using a multichannel pipette, add 17 µl of each 29.4 ng/µl DNA

sample to its assigned sample well, for a total reaction volume of 20 µl.

c Mix by pipetting up and down at least 10 times. Do not vortex.

6 Cap the wells, then place the plate or strip tube in the thermal cycler.

Close the lid, then resume the thermal cycling program in Table 9.

OnePGT Library Preparation for Illumina Sequencing 25

Library Preparation 3

Step 3. Add adapters to fragmented DNA

In this step, adapters are added to the DNA fragments. The mixture is heated to 65°C in order to increase adapter- fragment interactions and decrease fragment- fragment interactions. This step uses the components listed in Table 11.

Table 11 Reagents for adapter addition

Kit Component Storage Location

Adapter 1 Agilent OnePGT Library Prep Kit Box 2, –20°C

Adapter 2 Agilent OnePGT Library Prep Kit Box 2, –20°C

1 Thaw vials of Adapter 1 and Adapter 2 on ice.

2 Once the thermal cycler reaches the 4°C Hold step (Step 4 in Table 9),

remove the fragmented DNA samples, briefly spin the samples to collect the liquid, then place the samples on ice.

3 Preprogram the thermal cycler (with the heated lid ON) with the

program in Table 12. Start the program, then immediately pause the program to allow the heated lid to reach temperature while you set up the reactions.

Table 12 Thermal cycler program for adapter addition

Step Temperature Time

Step 1 65°C 10 minutes

Step 2 4°C 1 minute

Step 3 4°C Hold

OnePGT Library Preparation for Illumina Sequencing 26

Library Preparation 3

Step 3. Add adapters to fragmented DNA

4 Combine appropriate volumes of Adapter 1 and Adapter 2, in a 1.5- ml

tube, as described in Table 13. Mix by vortexing for 2 seconds, then spin the tube briefly and keep on ice.

Table 13 Preparation of adapter mixture

Reagent Volume for 1 sample Volume for 12 samples (includes excess)

Adapter 1 2.5 µl 32.5 µl

Adapter 2 2.5 µl 32.5 µl

Total 5 µl 65 µl

5 Add 5 µl of the adapter mixture to each DNA sample well. Mix by

pipetting up and down at least 10 times.

6 Cap the wells, then place the plate or strip tube in the thermal cycler.

Close the lid, then resume the thermal cycling program in Table 12.

OnePGT Library Preparation for Illumina Sequencing 27

Library Preparation 3

Step 4. Ligate the adapters

In this step, the adapters are ligated to the DNA fragments. This step uses the components listed in Table 14.

Table 14 Reagents for adapter ligation

Kit Component Storage Location

DNA Ligase Agilent OnePGT Library Prep Kit Box 2, –20°C

Ligase Buffer Agilent OnePGT Library Prep Kit Box 2, –20°C

Nuclease-Free Water Agilent OnePGT Library Prep Kit Box 2, –20°C

1 Once the thermal cycler reaches the 4°C Hold step (Step 3 in Table 12),

remove the samples (DNA + adapters), briefly spin the samples to collect the liquid, then place the samples on ice.

2 Thaw vial of Ligase Buffer on ice and place vial DNA Ligase on ice.

Thaw vial of Nuclease- Free Water at room temperature and then keep on ice.

3 Preprogram the thermal cycler (with the heated lid ON) with the

program in Table 15. Start the program, then immediately pause the program to allow the heated lid to reach temperature while you set up the reactions.

Table 15 Thermal cycler program for adapter ligation

Step Temperature Time

Step 1 22°C 15 minutes

Step 2 65°C 10 minutes

Step 3 4°C 1 minute

Step 4 4°C Hold

OnePGT Library Preparation for Illumina Sequencing 28

Library Preparation 3

Step 4. Ligate the adapters

4 Prepare the appropriate volume of ligation reagent mix in a 1.5-

ml tube, as described in Table 16. Mix well by pipetting up and down at least 10 times. Do not vortex. Spin the tube briefly to collect the liquid and keep on ice.

Table 16 Preparation of ligation reagent mix

Reagent Volume for 1 sample Volume for 12 samples (includes excess)

Nuclease-Free Water 1.5 µl 21 µl

Ligase Buffer 3.0 µl 42 µl

DNA Ligase 0.5 µl 7 µl

Total 5 µl 70 µl

5 Add 5 µl of the ligation reagent mix to each DNA sample well. Mix by

pipetting up and down at least 10 times. Do not vortex.

6 Cap the wells, then place the plate or strip tube in the thermal cycler.

Close the lid, then resume the thermal cycling program in Table 15.

7 Once the thermal cycler reaches the 4°C Hold step (Step 4 in Table 15),

remove the samples, briefly spin the samples to collect the liquid, and place on ice.

OnePGT Library Preparation for Illumina Sequencing 29

Library Preparation 3

Step 5. Purify the DNA using SPRI technology

In this step, the adapter tagged DNA is purified, using a solid- phase reversible immobilization (SPRI) system, to remove excess unligated adapters and adapter- dimers. This step uses the components listed in

Table 17.

Table 17 Reagents for SPRI purification

Kit Component Storage Location

TE Agilent OnePGT Library Prep Kit Box 2, –20°C

AMPure XP beads

Ethanol, 96%–100% User-determined (component not provided)

Nuclease-free H

* Beckman Coulter’s SPRIselect Reagent can be used instead of AMPure XP beads at this step. If us-

ing SPRIselect Reagent, replace the 70% ethanol with 85% ethanol in all protocol steps below.

† Do not use the Nuclease-Free Water provided in Agilent OnePGT Library Prep Kit Box 2, which is

supplied for use only in the ligation reactions (see page 29).

O User-determined (component not provided)

Consult manufacturer’s storage conditions; keep at room temperature for at least 30 minutes before use

†

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

30 minutes before use. Do not freeze the beads at any time.

2 Prepare 400 µl of fresh 70% ethanol per sample for use in step 9 and

step 11.

NOTE

When the complete library preparation protocol is performed on the same day, you can prepare 0.8 ml of fresh 70% ethanol per sample for use in both SPRI purification procedures in the workflow (steps below and on page 38).

When both SPRI purification procedures are performed on the same day, also retain the AMPure XP bead suspension at room temperature through use on page 38.

3 Add 20 µl of nuclease- free H2O to each sample well containing

adapter- tagged DNA.

4 Mix the room- temperature AMPure XP bead suspension well so that the

reagent appears homogeneous and consistent in color.

OnePGT Library Preparation for Illumina Sequencing 30

Library Preparation 3

Step 5. Purify the DNA using SPRI technology

5 Add 50 µl of the homogeneous bead suspension to each sample well

containing the 50least 10 times.

Check that the beads are in a homogeneous suspension in the sample wells. Each well should have a uniform color with no layers of beads or clear liquid present.

6 Incubate samples for 5 minutes at room temperature.

7 Put the plate or strip tube on the magnetic stand at room temperature,

and incubate the samples on the stand for 2 minutes.

µl DNA samples. Mix by pipetting up and down at

NOTE

The plate or strip tube remains on magnetic stand until step 13.

8 While keeping the samples on the magnetic stand, carefully remove and

discard 90 µl of cleared solution from each well. Do not disturb the beads while removing the solution.

9 Continue to keep the samples on the magnetic stand while you dispense

180 µl of fresh 70% ethanol in each sample well.

10 Wait for up to 30 seconds to allow any disturbed beads to settle, then

remove the ethanol.

11 Repeat step 9 and step 10 once for a total of two washes. Make sure to

remove all of the ethanol at each wash step.

Retention of ethanol in the sample wells can cause incomplete elution of DNA from the beads and sample loss. At the end of the final wash step, use a P10 pipette to remove any traces of ethanol from the wells without disturbing the beads.

12 Allow the beads to air- dry for 8 minutes at room temperature, keeping

the plate or strip tube on the magnetic stand.

Do not exceed 8 minutes. Over-drying the beads can reduce sample recovery.

13 Add 25 µl of TE to each sample well, then remove the samples from the

magnetic stand. Mix by pipetting up and down at least 10 times.

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

OnePGT Library Preparation for Illumina Sequencing 31

Library Preparation 3

Step 5. Purify the DNA using SPRI technology

15 Put the plate or strip tube in the magnetic stand at room temperature

and leave for 1 minute or until the solution in each well is clear.

16 Remove 20 µl of each cleared supernatant to wells of a fresh plate or

strip tube and keep on ice. You can discard the beads at this time.

CAUTION

Stopping Point If you will not continue to the next protocol step within two hours, cap

Take care to avoid transferring any of the beads to the final DNA sample wells.

the plate or strip tube wells, and store the samples at 4°C or –20°C for up to one week.

OnePGT Library Preparation for Illumina Sequencing 32

Library Preparation 3

Step 6. Size-select the DNA fragments

In this step, the adapter- tagged DNA fragments are size- selected by agarose gel electrophoresis using the PippinHT system (see supplier information on page 12).

Before you begin, make sure you understand the PippinHT system instructions for use provided by the manufacturer.

1 Transfer the PippinHT system reagents from cold storage to room

temperature at least 30 minutes before use.

2 Set up the PippinHT system, using a 1.5% Agarose 300- 1500 bp 15C

cassette, according to the manufacturer’s instructions.

Make sure to calibrate the PippinHT instrument as directed in the Optical Calibration section of the PippinHT Operations Manual.

3 Prepare the cassette as described in the Preparing a Cassette section of

the PippinHT Operations Manual.

4 Perform a continuity test as described in the Continuity Test section of

the PippinHT Operations Manual.

5 Program the run using the PippinHT user interface making the

selections provided below. Refer to the Programming a Protocol section of the PippinHT Operations Manual for more information.

a Select cassette definition 1.5% Agarose 300-1500 bp 15C

b For each set of lane pairs, select Range from the programming mode

options (Tight/Range/Time) and enter size range of 335- 575 bp

c Select Use Internal Standards

6 Add 5 µl of Internal Marker 15C to each 20- µl purified DNA sample in

the PCR plates or strip tubes. Mix thoroughly by pipetting up and down at least 15 times or vortexing then spin the samples briefly to collect the liquid.

Refer to the Sample Preparation section of the PippinHT Operations Manual for more information.

NOTE

OnePGT Library Preparation for Illumina Sequencing 33

Insufficient mixing can result in incorrect marker detection in the run and sample loss.

Library Preparation 3

Step 6. Size-select the DNA fragments

7 For each sample to be run, remove 30 µl of electrophoresis buffer from

a loading well of the cassette. Refer to the Loading Samples section of the PippinHT Operations Manual for more information.

NOTE

CAUTION

Take care to avoid damaging the agarose surrounding the loading well during sample loading steps. Consult the manufacturer’s instructions for more information.

Loading using a multi-channel pipette is preferred for greatest efficiency. If loading using a single-channel pipette, process samples in sets of up to four at a time and complete multiple rounds of step 7 and step 8 until all samples have been loaded.

8 Load each 25- µl DNA sample into a separate loading well. Make sure to

use the wells from which the buffer was removed in step 7.

9 Press Start to start the electrophoresis run.

The run must be started within 10 minutes of loading the samples.

10 Once the run is finished, transfer the 30- µl size- selected DNA samples

from the elution wells of the cassette to a fresh PCR plate or strip tube. Keep the samples on ice.

11 Remove 2 µl of each size- selected DNA sample and measure the DNA

concentration using the Qubit dsDNA HS assay, according to the manufacturer’s instructions (see supplier information on page 12).

The minimum acceptable concentration is 0.2 ng/µl. A sample with concentration <0.2 ng/µl should be discarded, and the preparation steps for the sample repeated.

Stopping Point If you will not continue to the next protocol step within two hours, cap

the plate or strip tube wells, and store the samples at 4°C for up to 3 days.

OnePGT Library Preparation for Illumina Sequencing 34

Library Preparation 3

Step 7. Suppression PCR-amplify the size-selected DNA

In this step, the size- selected DNA libraries are amplified in PCR reactions using primers designed for the following purposes:

• Elongation of adapters to full- length adapter sequences required for

sequencing on the Illumina platform

• Indexing using sample- specific reverse primers

• Selective amplification of only fragments ligated to both Adapter 1 and

Adapter 2

This step uses the components listed in Table 18.

Table 18 Reagents for adapter PCR amplification and indexing

Kit Component Storage Location

PCR Mix Agilent OnePGT Library

Prep Kit Box 2, –20°C

Forward PCR Primer Agilent OnePGT Library

Prep Kit Box 2, –20°C

CAUTION

Reverse PCR Primers-index 1-96 (96 primers containing sample

indexing sequences, supplied in 96-well plate)

Reverse NTC PCR Primer Agilent OnePGT Library

Nuclease-free H

O User-determined

To avoid cross-contaminating libraries, set up PCR reactions in a dedicated clean area or PCR hood with UV sterilization and positive air flow.

Agilent OnePGT Library Prep Kit Box 2, –20°C

Prep Kit Box 2, –20°C

(component not provided)

No- template controls: Each time you run suppression PCR, include a no- template control (NTC), using the same source of nuclease- free H2O that was used to dilute DNA samples on page 23 (step 3). The reverse PCR primer for NTC reactions is the kit- supplied Reverse NTC PCR

Primer, which is supplied in an amount sufficient for 8 PCR reactions.

OnePGT Library Preparation for Illumina Sequencing 35

Library Preparation 3

Step 7. Suppression PCR-amplify the size-selected DNA

1 Determine the appropriate index assignments for each sample, using the

considerations below, and record the index assignments.

• Use a different index (included in reverse primers used at this step)

for each sample to be sequenced in the same lane.

• Refer to “Guidelines for Optimal Index Multiplexing” on page 53 for

specific index multiplexing recommendations for OnePGT sequencing data processing.

2 Using the starting DNA concentration determined by fluorometry in

step 11 on page 34, dilute each DNA sample to 0.625 ng/µl using

nuclease- free H2O. Prepare at least 25 µl of diluted DNA for each sample. Keep the samples on ice.

Samples with starting concentration between 0.2 ng/µl and 0.625 ng/µl can be added to the PCR reaction (step 8, below) without dilution.

3 Thaw the vial of PCR Mix on ice. Once thawed, mix the reagent well by

pipetting up and down at least 10 times.

NOTE

The vial of PCR mix may initially contain a precipitate, which should be dissolved by thawing and mixing the vial contents.

4 Preprogram the thermal cycler (with the heated lid ON) with the

program in Table 19. Start the program, then immediately pause the program to allow the heated lid to reach temperature while you set up the reactions.

Table 19 Thermal cycler program for suppression PCR

Segment Number Number of Cycles Temperature Time

1 1 98°C 45 seconds

98°C 15 seconds

3 1 72°C 1 minute

4 1 4°C 1 minute

5 1 4°C Hold

64°C 30 seconds

72°C 30 seconds

OnePGT Library Preparation for Illumina Sequencing 36

Library Preparation 3

Step 7. Suppression PCR-amplify the size-selected DNA

5 Prepare the appropriate volume of PCR reagent mix, as described in

Table 20, on ice. Mix by pipetting up and down at least 10 times.

Table 20 Preparation of PCR reagent mix

CAUTION

Reagent Volume for 1 reaction Volume for 13 reactions (12 library amplifications

+ 1 NTC; includes excess

PCR Mix 25 µl 375 µl

Forward PCR Primer 2.5 µl 37.5 µl

Total 27.5 µl 412.5 µl

* When preparing reagent mixtures for <24 samples, include excess reagent volumes equivalent to

2 additional reactions. For 25–48 samples include excess of 4 additional reactions, for 49–72 samples include excess of 6 additional reactions, and for 73–96 samples include excess of 8 additional reactions.

)

6 Dispense 27.5 µl of the PCR reagent mix prepared in Table 20 into each

sample well of a fresh PCR plate or strip tube.

7 To each well add 2.5 µl of the appropriate Reverse PCR Primer (using

the specific indexed primer assigned to the sample in step 1 on page 36 or using Reverse NTC PCR Primer for the no- template control). For a map of plate positions for the indexed Reverse PCR primers, see

Table 31 on page 51.

Check the orientation of the Reverse PCR Primer plate before removing the indexed primer assigned to the sample. Orient the plate with the notched corner (proximal to well H01) at the bottom, left position. See page 51 for a plate map and orientation image.

8 Add 20 µl of each DNA sample (0.2–0.625 ng/µl) to the appropriate

well. For the no- template control well, add 20 µl of the nuclease- free H2O that was used to dilute the DNA samples. Mix by pipetting up and down at least 10 times.

9 Cap the wells, then place the plate or strip tube in the thermal cycler.

Close the lid, then resume the thermal cycling program in Table 19.

10 Once the thermal cycler reaches the 4°C Hold step (Segment 5 in

Table 19), remove the samples, and briefly spin the samples to collect

the liquid.

OnePGT Library Preparation for Illumina Sequencing 37

Library Preparation 3

Step 8. Purify the DNA using SPRI technology

In this step, the amplified DNA is SPRI- purified to remove excess primers. Before you begin, verify that the AMPure XP beads have been kept at room temperature for at least 30 minutes and that fresh 70% ethanol has been prepared.

This step uses the components listed in Table 17.

Table 21 Reagents for SPRI purification

Kit Component Storage Location

AMPure XP beads

70% ethanol Prepared on page 30; if samples were stored prior to

Transferred to room temperature on page 29

suppression PCR, prepare fresh 70% ethanol

†

NOTE

Nuclease-free H

* Beckman Coulter’s SPRIselect Reagent can be used instead of AMPure XP beads at this step. If us-

ing SPRIselect Reagent, replace the 70% ethanol with 85% ethanol in all protocol steps below.

† Ensure that the AMPure XP beads have been held at room temperature for at least 30 minutes be-

fore use if they were returned to cold storage after use on page 30.

O User-determined (component not provided)

1 Mix the room- temperature AMPure XP bead suspension well so that the

reagent appears homogeneous and consistent in color.

2 Add 50 µl of the homogeneous bead suspension to each sample well

containing the 50- µl amplified DNA samples. Mix by pipetting up and down at least 10 times.

Check that the beads are in a homogeneous suspension in the sample wells. Each well should have a uniform color with no layers of beads or clear liquid present.

3 Incubate samples for 5 minutes at room temperature.

4 Put the plate or strip tube on the magnetic stand at room temperature,

and incubate the samples on the stand for 2 minutes.

The plate or strip tube remains on magnetic stand until step 10.

OnePGT Library Preparation for Illumina Sequencing 38

Library Preparation 3

Step 8. Purify the DNA using SPRI technology

5 While keeping the samples on the magnetic stand, carefully remove and

discard 90 µl of cleared solution from each well. Do not disturb the beads while removing the solution.

6 Continue to keep the samples on the magnetic stand while you dispense

180 µl of fresh 70% ethanol in each sample well.

7 Wait for up to 30 seconds to allow any disturbed beads to settle, then

remove the ethanol.

8 Repeat step 6 and step 7 once for a total of two washes. Make sure to

remove all of the ethanol at each wash step.

NOTE

CAUTION

9 Allow the beads to air- dry for 8 minutes at room temperature, keeping

the plate or strip tube on the magnetic stand.

Do not exceed 8 minutes. Over-drying the beads can reduce sample recovery.

10 Remove the samples from the magnetic stand, then add 50 µl of

nuclease- free H2O to each sample well. Mix by pipetting up and down at least 10 times.

11 Incubate for 2 minutes at room temperature.

12 Put the plate or tubes in the magnetic stand at room temperature and

leave for 1 minute or until the solution in each well is clear.

13 Remove 45 µl of each cleared supernatant to wells of a fresh plate or

strip tube and keep on ice. You can discard the beads at this time.

Take care to avoid transferring any of the beads to the final DNA sample wells.

OnePGT Library Preparation for Illumina Sequencing 39

Library Preparation 3

Step 9. Quantify and qualify the OnePGT libraries

In this step, the purified DNA library is quantified by fluorometry and library quality is verified using Agilent’s 2100 Bioanalyzer or 4200 TapeStation.

1 Remove 2 µl of each purified library DNA sample and measure the DNA

concentration using the Qubit dsDNA HS assay, according to the manufacturer’s instructions (see supplier information on page 12).

NOTE

DNA concentration must be determined fluorometrically, using the Qubit dsDNA HS assay. Do not use the concentration reported by the TapeStation system or Bioanalyzer system when pooling samples for sequencing.

2 Remove 1 µl of each purified DNA sample and analyze the fragment size

profile of the library using one of the methods below.

• Option 1: Agilent’s 4200 TapeStation or 4150 TapeStation and

D1000 ScreenTape. See the Agilent D1000 Assay Quick Guide for

more information.

• Option 2: Agilent’s 2100 Bioanalyzer and DNA 1000 Assay. See the

Agilent DNA 1000 Kit Guide for more information.

3 Analyze the library DNA size profile results using the guidelines below.

• Assess and record the Average Size [bp] of DNA fragments in each

library.

• Analyze the distribution of fragments in the library using the

electropherogram. A high- quality sample electropherogram is shown in Figure 2 on page 41.

High- quality libraries should produce an electropherogram showing a major peak with an average size of 500 bp ±40 bp and with a peak width of 285 bp ±50 bp. It is also acceptable for the peak to be bifurcated.

A peak in the electropherogram at approximately 58 bp or 84 bp indicates incomplete removal of primers or primer- dimers from the sample. When present, the residual primers or primer- dimers should be removed by subjecting the sample to an additional round of SPRI bead purification, using the protocol on page 38.

OnePGT Library Preparation for Illumina Sequencing 40

Library Preparation 3

Step 9. Quantify and qualify the OnePGT libraries

Figure 2 Analysis of the DNA size profile for a representative OnePGT library using the

4200 TapeStation.

OnePGT Library Preparation for Illumina Sequencing 41

Library Preparation 3

Step 10. Pool libraries for multiplexed sequencing

In this step, the molar DNA concentration is calculated for each indexed library, and libraries are pooled for multiplexed sequencing, using equimolar amounts of each index in the pool (see Table 26 on page 45 for pool size guidelines).

1 Determine the molarity of DNA fragments (nM) in each indexed library

according to the formula below.

Molar concentration (nM)

where [DNA] is the DNA concentration in ng/µl determined by fluorometry on page 40 (step 1) and

average size (bp) is the average size determined by Bioanalyzer or TapeStation analysis on page 40 (step 3)

The minimum acceptable concentration is 2 nM. A library with concentration <2 nM should be discarded, and the library preparation for the sample repeated.

2 Determine the appropriate pooling strategy using the following

considerations.

a The number of indexed libraries that may be multiplexed in a single

sequencing lane is determined by the output specifications of the platform used, together with the amount of sequencing data required for your research design. Calculate the number of indexes that can be combined per lane, according to the capacity of your platform and the amount of sequencing data required per sample (see Table 26 on page 45 for guidelines).

b Combine the libraries such that each index- tagged sample is present

in equimolar amounts in the pool using one of the following methods:

Method 1: Dilute each sample to be pooled to the same final concentration (typically 2 nM–15 nM, or the concentration of the most dilute sample), then combine equal volumes of all samples to create the final pool. Select the diluent according to the requirements of the sequencing facility.

DNA1 000 000

----------------------------------------------------------=

averagesize bp650

OnePGT Library Preparation for Illumina Sequencing 42

Library Preparation 3

Step 10. Pool libraries for multiplexed sequencing

Method 2: Starting with samples at different concentrations, add the appropriate volume of each sample to achieve equimolar concentration in the pool, then adjust the pool to the desired final volume. Select the diluent according to the requirements of the sequencing facility. The formula below is provided for determination of the amount of each indexed sample to add to the pool.

Volume of Index

Vf Cf

---------------------------------=

# Ci

where V(f) is the final desired volume of the pool,

C(f) is the desired final concentration of all the DNA in the pool

(typically 2 nM–15 nM or the concentration of the most dilute sample)

# is the number of indexes, and

C(i) is the initial concentration of each indexed sample

Table 22 shows an example of amounts of 5 libraries needed for a

final volume V(f) of 100 µl at final concentration C(f) of 10 nM. In this example, the total volume of combined libraries is 69.3 µl, thus

30.7 µl of diluent should be added for a final volume of 100 µl. Select the diluent according to the requirements of the sequencing facility.

Table 22 Example of indexed library volume calculation for total volume of 100 µl

Sample C(i) V(f) C(f) Volume to add to pool (µl)

Sample 1 14.0 nM 100 µl 10.0 nM 14.3

Sample 2 18.8 nM 100 µl 10.0 nM 10.4

Sample 3 17.7 nM 100 µl 10.0 nM 11.3

Sample 4 15.0 nM 100 µl 10.0 nM 13.3

Sample 5 10.0 nM 100 µl 10.0 nM 20

Diluent — — — 30.7

OnePGT Library Preparation for Illumina Sequencing 43

Library Preparation 3

Step 11. Set up the sequencing run

Supported sequencing platforms include Illumina’s NextSeq 500/550 and HiSeq 2500 instruments. Refer to Illumina’s protocols to set up a single- indexed, paired- end sequencing run, using the additional guidelines outlined below.

1 The sample- level index (i7) requires an 8- bp index read. See Table 32

on page 52 for index sequences.

2 The OnePGT kit Custom Read 1 Sequencing Primer is provided at

100 µM and must be diluted with Illumina Read 1 Primer to the final concentration shown in Table 23 for each platform. Combine the Read 1 primers using the platform- specific instructions below.

Table 23 Custom Read 1 Sequencing Primer concentration requirements

Platform Run type Custom Read 1 Sequencing Primer

final concentration

NextSeq 500/550 High and Mid Output 0.3 µM

HiSeq 2500 Rapid Run 0.5 µM

HiSeq 2500 Platform

a Transfer the entire volume of Illumina’s Read 1 TruSeq Primer HP10

from position 18 to an empty Eppendorf tube.

b In a fresh Eppendorf tube, combine the amounts of OnePGT kit

Custom Read 1 Sequencing Primer and TruSeq Primer HP10 shown

in Table 24. Mix well by vortexing.

c Transfer the entire volume of the Read 1 Primer mixture (1.75 ml)

prepared in step b back into position 18.

Table 24 HiSeq 2500 Custom Read 1 sequencing primer dilution

Run Type Volume of Agilent OnePGT System Primer Volume of Illumina TruSeq Primer Total Volume

Rapid Mode 8.8 µl Custom Read Primer 1 Sequencing Primer 1741.2 µl HP10 (PE Rack-position 18) 1.75 ml

OnePGT Library Preparation for Illumina Sequencing 44

Library Preparation 3

Step 11. Set up the sequencing run

NextSeq 500/550 Platform

a Transfer the entire volume of Illumina’s Read 1 Primer BP10 from

well 20 to an empty Eppendorf tube.

b In a fresh Eppendorf tube, combine the amounts of OnePGT kit

Custom Read 1 Sequencing Primer and Primer BP10 shown in Table 25. Mix well by vortexing.

c Transfer the entire volume of the Read 1 Primer mixture prepared in

step b back into well 20.

Table 25 NextSeq 500/550 Custom Read 1 sequencing primer dilution

Run Type Volume of Agilent OnePGT System Primer Volume of Illumina Primer Total Volume

High Output 3.9 µl Custom Read Primer 1 Sequencing Primer 1296.1 µl BP10 (well 20) 1.3 ml

Mid Output 2.7 µl Custom Read Primer 1 Sequencing Primer 897.3 µl BP10 (well 20) 0.9 ml

3 Set up the sequencing run using the parameters in Table 26. Follow

Illumina’s recommendation for a PhiX control in a low- concentration spike- in for improved sequencing quality control.

Table 26 Sequencing run setup guidelines

Application Platform Run type Read length Number of

Samples

PGT-M (with PGT-A and/or PGT-SR)

PGT-SR and PGT-A NextSeq

PGT-SR and PGT-A HiSeq 2500 Rapid Run Mode, SBS Kit v2

* Seeding concentrations are provided as guidelines and may require optimization.

CAUTION

NextSeq 500/550

HiSeq 2500 Rapid Run Mode, SBS Kit v2

500/550

During run setup, leave the Custom Primer for Read 1 checkbox cleared (default state), since the OnePGT Custom Read 1 Sequencing Primer is spiked into Illumina’s Read 1 Primer at the default Read 1 Primer location.

High Output (400M PE-reads)

(300M PE-reads per flow cell)

Mid Output (130M PE-reads)

(300M PE-reads per flow cell)

2 × 150 bp 24 1.4 pM 1%

2 × 150 bp 18 10 pM 1%

2 × 75 bp 96 1.4 pM 1%

2 × 75 bp 96 10 pM 1%

Seeding concentration

%Phi X

OnePGT Library Preparation for Illumina Sequencing 45

Library Preparation 3

Step 11. Set up the sequencing run

A successful sequencing run is characterized by the following attributes:

• 75% bases higher than Q30, averaged across the entire run

• 16 x 106 paired- end reads per library for PGT- M analysis or

1.3 x 10

paired- end reads per library for PGT- A/PGT- SR analysis

OnePGT Library Preparation for Illumina Sequencing 46

Library Preparation 3

Step 12. Process sequencing data and upload to Agilent Alissa OnePGT

The sequencing data must be demultiplexed and then uploaded to the Agilent Alissa OnePGT software for PGT- M, PGT- SR, and PGT- A analysis as outlined below. For more information on the Agilent Alissa OnePGT software setup and analysis steps, refer to the Agilent Alissa OnePGT software documentation.

1 Demultiplex the sequencing data using Illumina’s bcl2fastq software or

similar tool, as appropriate for your NGS pipeline, to generate .fastq formatted files for uploading to Alissa OnePGT. Using the standard Alissa OnePGT upload process, all data for one sample should be captured in one .fastq file per lane for the forward read and one .fastq file per lane for the reverse read. If your pipeline is not compatible with .fastq file merging, see Troubleshooting on page 58 for more information.

2 The .fastq files are automatically uploaded to the Agilent Alissa OnePGT

platform when the OnePGT Upload Module is installed on your local system, and the .fastq files are placed in the correct directory. Refer to Agilent Alissa OnePGT software documentation topic Uploading NGS data files for more information.

3 Once uploaded to the Agilent Alissa OnePGT platform, the sequencing

data needs to be associated with an Embryo ID or family member (Mother ID, Father ID or Family member ID) to enable analysis. Refer to Agilent Alissa OnePGT software documentation topic Associating NGS data to Family Members and Embryos for more information.

OnePGT Library Preparation for Illumina Sequencing 47

OnePGT Library Preparation for Illumina Sequencing Protocol

4 Reference

Kit Contents and Supported Configurations 49 Reference Information for OnePGT Indexes 51 Guidelines for Optimal Index Multiplexing 53 Troubleshooting Guide 54 Quick Reference Protocols 59

This section contains reference information including kit contents, index assignment and pooling information, troubleshooting information, and abbreviated quick reference protocols for experienced users.

Agilent Technologies

Kit Contents and Supported Configurations

Agilent OnePGT Solution includes the reagent component kits listed in

Table 27.

Table 27 OnePGT Solution Component Kits

Reference 4

Component Kit Name Storage

Condition

Agilent OnePGT Library Prep Kit Box 1 –80°C 5191-4001 1kit

Agilent OnePGT Library Prep Kit Box 2 –20°C 5191-4002 1kit

REPLI-g Single Cell Kit (48 reactions) –20°C 5191-4065 2kits

Component Kit p/n

Quantity Provided

The library preparation reagents are sufficient for 96 libraries, with enough reagents for 8 runs containing 12 samples per run. When processing samples using runs with fewer than 12 samples, some reagents may be depleted before 96 samples are run. Contents of each of the component kits listed in Table 27 are detailed in Table 28 and Table 29.

Table 28 Agilent OnePGT Library Prep Kit Box 1 Content (stored at –80°C)

Kit Component Configuration

Restriction Enzyme 1 1 vial

OnePGT Library Preparation for Illumina Sequencing 49

Reference 4

Kit Contents and Supported Configurations

Table 29

Kit component Configuration

TE 4 vials

Restriction Enzyme 2 1 vial

Restriction Enzyme Buffer 1 vial

Adapter 1 1 vial

Adapter 2 1 vial

DNA Ligase 1 vial

Ligase Buffer 1 vial

PCR Mix 4 vials

Forward PCR Primer 1 vial

Reverse PCR Primers-index 1-96 Plate containing one indexed Reverse PCR Primer per well;

Reverse NTC PCR Primer 1 vial

Nuclease-Free Water 1 vial

Custom Read 1 Sequencing Primer 1 vial

Agilent OnePGT Library Prep Kit Box 2 Content (stored at –20°C)

see Table 31 for plate map

Each of the two provided REPLI- g Single Cell Kits (p/n 5191- 4065) includes reagents for whole genome amplification from 48 samples. Kits contain the components listed in Table 30.

Table 30 REPLI-g Single Cell Kit Content (p/n 5191-4065 stored at –20°C)

Kit Component Configuration

Buffer DLB 2 vials; provided lyophilized

O sc 2 vials

DTT, 1 M 1 vial

PBS sc 2 vials

Stop Solution 1 vial

REPLI-g sc Reaction Buffer 3 vials

REPLI-g sc DNA Polymerase 2 vials

OnePGT Library Preparation for Illumina Sequencing 50

Reference 4

Reference Information for OnePGT Indexes

When assigning the indexes to samples in the Agilent Alissa OnePGT software for analysis, enter index designations using the well position format of A01 through H12 shown in Table 31 below. Orient the plate as shown in Figure 3 when removing the indexed primers for use.

The nucleotide sequence of the index portion of each Reverse PCR Primer is shown in Table 32 on page 52.

Table 31 Plate map for Reverse PCR Primer indexes

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

Figure 3 Orientation of Reverse PCR Primer plate

OnePGT Library Preparation for Illumina Sequencing 51

Reference 4

Reference Information for OnePGT Indexes

Table 32 Nucleotide sequences of OnePGT Indexes in Reverse PCR Primers

Index Ref # Sequence Index Ref # Sequence Index Ref # Sequence Index Ref # Sequence

A01 1 ATGCCTAA

B01 2 GAATCTGA

C01 3 AACGTGAT

D01 4 CACTTCGA

E01 5 GCCAAGAC

F01 6 GACTAGTA

G01 7 ATTGGCTC

H01 8 GATGAATC

A02 9 AGCAGGAA

B02 10 GAGCTGAA

C02 11 AAACATCG

D02 12 GAGTTAGC

E02 13 CGAACTTA

F02 14 GATAGACA

G02 15 AAGGACAC

H02 16 GACAGTGC

A03 17 ATCATTCC

B03 18 GCCACATA

C03 19 ACCACTGT

D03 20 CTGGCATA

E03 21 ACCTCCAA

F03 22 GCGAGTAA

G03 23 ACTATGCA

H03 24 CGGATTGC

A04 25 AACTCACC A07 49 ACGTATCA A10 73 AATGTTGC

B04 26 GCTAACGA B07 50 GTCTGTCA B10 74 TGAAGAGA

C04 27 CAGATCTG C07 51 CTAAGGTC C10 75 AGATCGCA

D04 28 ATCCTGTA D07 52 CGACACAC D10 76 AAGAGATC

E04 29 CTGTAGCC E07 53 CCGTGAGA E10 77 CAACCACA

F04 30 GCTCGGTA F07 54 GTGTTCTA F10 78 TGGAACAA

G04 31 ACACGACC G07 55 CAATGGAA G10 79 CCTCTATC

H04 32 AGTCACTA H07 56 AGCACCTC H10 80 ACAGATTC

A05 33 AACGCTTA A08 57 CAGCGTTA A11 81 CCAGTTCA

B05 34 GGAGAACA B08 58 TAGGATGA B11 82 TGGCTTCA

C05 35 CATCAAGT C08 59 AGTGGTCA C11 83 CGACTGGA

D05 36 AAGGTACA D08 60 ACAGCAGA D11 84 CAAGACTA

E05 37 CGCTGATC E08 61 CATACCAA E11 85 CCTCCTGA

F05 38 GGTGCGAA F08 62 TATCAGCA F11 86 TGGTGGTA

G05 39 CCTAATCC G08 63 ATAGCGAC G11 87 AACAACCA

H05 40 CTGAGCCA H08 64 ACGCTCGA H11 88 AATCCGTC

A06 41 AGCCATGC A09 65 CTCAATGA A12 89 CAAGGAGC

B06 42 GTACGCAA B09 66 TCCGTCTA B12 90 TTCACGCA

C06 43 AGTACAAG C09 67 AGGCTAAC C12 91 CACCTTAC

D06 44 ACATTGGC D09 68 CCATCCTC D12 92 AAGACGGA

E06 45 ATTGAGGA E09 69 AGATGTAC E12 93 ACACAGAA

F06 46 GTCGTAGA F09 70 TCTTCACA F12 94 GAACAGGC

G06 47 AGAGTCAA G09 71 CCGAAGTA G12 95 AACCGAGA

H06 48 CCGACAAC H09 72 CGCATACA H12 96 ACAAGCTA

OnePGT Library Preparation for Illumina Sequencing 52

Guidelines for Optimal Index Multiplexing

The provided 96- well plate contains 96 reverse primers, each with a unique index, for suppression PCR (see page 35). Use the considerations below when determining which reverse primer to include in the suppression PCR reaction for each sample.

• Each reverse primer in the 96- well plate should only be used once.

• Design the sample indexing and pooling strategies so that indexes are

combined in the ordered combinations specified below. Pool indexes column- wise (from A to H), and then from left to right (from 1 to 12).

Table 33 shows example index pooling configurations for pools

containing 12 libraries. A similar strategy, using indexes pooled in order by column, should be applied to pools of different sizes.

Table 33 Recommended index pooling order for 12-library pools

Reference 4

Libraries in pool

12 8 A01 to H01 and A02 to D02 (full column 1 + first half column 2)

Number of pools from single primer plate

Indexes to combine in each pool

E02 to H02 and A03 to H03 (second half column 2 + full column 3)

A04 to H04 and A05 to D05 (full column 4 + first half column 5)

E05 to H05 and A06 to H06 (second half column 5 + full column 6)

A07 to H07 and A08 to D08 (full column 7 + first half column 8)

E08 to H08 and A09 to H09 (second half column 8 + full column 9)

A10 to H10 and A11 to D11 (full column 10 + first half column 11)

E08 to H08 and A09 to H09 (second half column 11 + full column 12)

OnePGT Library Preparation for Illumina Sequencing 53

Troubleshooting Guide

If sample yield after whole genome amplification is low (<200 ng/µl)

✔ Ensure that at least 1 embryo cell is collected during the biopsy.

✔ Use only REPLI- g Single Cell Kits purchased from Agilent and prepare

samples using the two- hour incubation step shown in Table 6 on page 19. Do not use WGA protocols provided by Qiagen.

✔ Ensure reconstituted buffer DLB has not been stored longer than the

recommended 6 months at –20°C, or prepare fresh buffer DLB.

✔ Ensure WGA reagents are stored properly, including thawing the

amplification REPLI- g sc DNA polymerase on ice and keeping samples and reagents on ice during use.

✔ Ensure all vials containing biopsies are spun down so that all material

is at the bottom of the vial.

✔ Dispense all WGA reagents onto the side of the vial and spin down.

Never make direct contact with the sample by the pipette tip.

✔ Ensure precise adherence to all sample mixing instructions. Mix

samples containing single cells (i.e. during WGA steps) by flicking the tube, not by pipetting up and down.

Reference 4

Troubleshooting Guide

If lane failure occurs during the PippinHT continuity test

✔ Buffer levels inside the cassette may be too low. Refer to the Continuity

test failure and troubleshooting section of the PippinHT manual. If the

issue persists, do not use the affected lane.

✔ Ensure that the cassettes have been stored at the temperature specified

by the supplier.

✔ Contact the PippinHT manufacturer, Sage Science, for additional lane

failure troubleshooting.

If lane failure occurs during the PippinHT run (indicated by Elution Timer box turning red) with no marker peaks visible

✔ Ensure that 5 µl of marker mix 15C is added to each sample before

loading into the PippinHT cassette. Never use the Loading Solution for External Marker Size Selection.

✔ Contact the PippinHT manufacturer, Sage Science, for additional lane

failure troubleshooting.

OnePGT Library Preparation for Illumina Sequencing 54

Reference 4

Troubleshooting Guide

If lane failure occurs during the PippinHT run (indicated by Elution Timer box turning red) with one or two rounded or merged marker peaks visible instead of two individual sharp marker peaks

✔ Ensure that the time between sample loading into the PippinHT cassette

and start of the run is <10 minutes. Pre- program the PippinHT run before loading the samples and use a multichannel pipette for sample loading.

✔ Ensure that the marker mix 15C solution is brought to room

temperature for at least 15 minutes before use. Ensure that the sample and marker are mixed thoroughly either by vortexing or by pipetting at least 15 times.

✔ Contact the PippinHT manufacturer, Sage Science, for additional lane

failure troubleshooting.

If the barcode of the cassette is not correctly identified

✔ The PippinHT system automatically registers the barcode of the

cassette. When the barcode is damaged, however, no unique cassette barcode number is registered. Failure to register the barcode does not affect sample separation and no action is required.

If sample yield is low after PippinHT size selection

✔ Ensure that the protocol is executed with 500 ng of input DNA as

measured by Qubit. Do not use an absorbance- based method to determine input DNA concentration.

✔ Ensure that the plasticware specified in Table 2 on page 12 is used in

all protocol steps.

✔ Ensure that all reagents, including both kit- supplied and user- supplied

materials, are used prior to the expiry date and are stored in adherence with the manufacturer- specified requirements.

✔ Ensure that the amplified DNA samples and AMPure XP beads are

mixed to homogeneity during SPRI purification (see step 2 on page 38).

✔ Ensure that all ethanol is removed after each wash step during SPRI

purification (see “Step 5. Purify the DNA using SPRI technology” on page 30). Any residual ethanol may reduce sample recovery.

✔ Ensure that freshly- prepared 70% ethanol is used during the SPRI

purification step.

OnePGT Library Preparation for Illumina Sequencing 55

Reference 4

Troubleshooting Guide

✔ Ensure that the AMPure XP beads are not over- dried prior to sample

elution during the SPRI purification step.

✔ Ensure that the sample volume is 20 µl after SPRI purification. Adjust

the volume with TE buffer if <20 µl is recovered from purification. Never use electrophoresis buffer or another high- salt buffer for sample elution or volume adjustments.

If concentration of the final library is low (< 2 nM)

✔ Ensure that the correct volume of indexed Reverse PCR Primer is added

to each reaction by briefly spinning down the Reverse PCR Primer plate to remove any air bubbles before removing an aliquot for use.

✔ Ensure that the PCR components are stored at the correct temperature.

✔ Ensure that all ethanol is removed after each wash step during SPRI

purification (see “Step 8. Purify the DNA using SPRI technology” on page 38). Any residual ethanol may reduce sample recovery.

If the size profile of the final library is different than expected

✔ A size profile similar to Figure 2 on page 41 should be observed in the

final library after PCR. Slight shifts in peak position (either leftward or rightward) within the error range of the instrument are also acceptable.

✔ A size profile similar to the electropherogram below may indicate low

quality starting material, resulting in a sequencing library containing primarily mitochondrial DNA. It is unlikely that samples with this type of profile will provide valid PGT conclusions.

OnePGT Library Preparation for Illumina Sequencing 56

Reference 4

Troubleshooting Guide

If an additional low molecular weight peak is present in the final library electropherogram

✔ The presence of a low molecular weight peak indicates the presence of

adaptor- dimers in the library. A low abundance (<10% of total library DNA fragments) of adaptor- dimers is acceptable. If excessive adaptor- dimers are observed, adjust the sample volume to 50 µl with nuclease- free water, then repeat the SPRI purification procedure detailed on page 38.

If cluster density is too low in sequencing results

✔ Ensure that Illumina’s denaturation protocol is performed exactly as

directed. In particular, verify that the NaOH reagent was freshly prepared at the correct concentration and was stored as directed.

✔ The library concentration may be too low. For each final library

remeasure the concentration, using Qubit dsDNA HS Assay, and the average DNA fragment size (see page 40). Recalculate the molar DNA concentration for each indexed library, and then pool using equimolar amounts of each index in the pool as directed on page 42.

✔ Consider using a higher seeding concentration. Seeding concentrations

provided in Table 26 on page 45 are guidelines and may require optimization.

If cluster density is too high in sequencing results

✔ The library concentration may be too high. For each final library

✔ Consider using a lower seeding concentration. Seeding concentrations

provided in Table 26 on page 45 are guidelines and may require optimization.

OnePGT Library Preparation for Illumina Sequencing 57

Reference 4

Troubleshooting Guide

If your NGS demultiplexing pipeline produces unmerged FASTQ files

✔ Alissa OnePGT allows the upload of either unmerged or merged FASTQ

files. Use the appropriate supported FASTQ file naming convention shown below, which allows the software to automatically assign the lane when required. Refer to Agilent Alissa OnePGT software documentation topic Uploading NGS data files for more information.

• Example 1–Unmerged Files

Read group R1:

<Sample ID>_<***>_<Lane1>_<R1>_001.fastq

<Sample ID>_<***>_<Lane2>_<R1>_001.fastq

Read group R2:

<Sample ID>_<***>_<Lane1>_<R2>_001.fastq

<Sample ID>_<***>_<Lane2>_<R2>_001.fastq

• Example 2–Merged Files

Forward read:

<Sample ID>_<***>_<R1>_001.fastq

Reverse read:

<Sample ID>_<***>_<R2>_001.fastq

If demultiplexing in BaseSpace trims the OnePGT adapters

✔ Downstream analysis in Alissa OnePGT is compatible with sequences

demultiplexed in BaseSpace either with or without adapter trimming. Adapter trimming during demultiplexing in BaseSpace is optional but is switched on by default. If you wish to disable adapter trimming during demultiplexing in BaseSpace, go to the Settings section of the index sheet and clear (leave blank) both AdapterSequenceRead1 and AdapterSequenceRead2 fields.

OnePGT Library Preparation for Illumina Sequencing 58

Quick Reference Protocols

The Quick Reference Protocols listed below, showing abbreviated summaries of the protocol steps, are provided on the following pages for experienced users.

• Whole Genome Amplification Protocol (page 60). Use the complete

protocol on page 16 to page 20 until you are familiar with all protocol details.

• Library Preparation Protocol (page 61 to page 63). Use the complete

protocol on page 21 to page 46 until you are familiar with all protocol details such as reagent mixing instructions and instrument settings.

Reference 4

Quick Reference Protocols

NOTE

Most Library Preparation protocol steps use customer-supplied nuclease-free H20. The vial of Nuclease-Free Water supplied with Agilent OnePGT Library Prep Kit Box 2 is intended for use only during the ligation step, as specified in the full protocol.

OnePGT Library Preparation for Illumina Sequencing 59

Quick Reference Protocols

Quick Reference Protocol: Whole-Genome Amplification

Reference 4

Step 1: Prepare Buffer DLB

Reconstitute lyophilized Buffer DLB by adding

500 μl of kit-supplied H

O sc.

Step 2. Prepare controls

• Positive control: 4 μl of gDNA diluted to

15 pg/μl in PBS sc

• Collection buffer negative control: 4 μl of the embryo biopsy collection buffer

• NTC negative control: 4 μl of PBS sc

Step 3. Prepare Buffer D2

For 12 samples:

• 3 μl of 1M DTT

• 33 μl of reconstituted Buffer DLB

Step 4. Add Buffer D2 to samples

Add 3 μl of Buffer D2 to each 4-μl sample.

Step 5. Lyse cells and denature DNA

1. Place in thermal cycler and run program below with heated lid:

• 65°C for 10 min

• Hold at 4°C

2. Add 3 μl of Stop Solution to each sample.

Step 6. PCR-amplify the sample DNA

1. Prepare PCR reagent mix (per rxn):

• 9 μl of H

O sc

• 29 μl of REPLI-g sc Reaction Buffer

• 2 μl of REPLI-g sc DNA Polymerase

2. Add 40 μl of the PCR reagent mix to each sample.

3. Place in thermal cycler and run program below with heated lid:

• 30°C for 2 h

• 65°C for 3 min

• Hold at 4°C

4. Store amplified DNA at -20°C until ready to proceed with OnePGT workflow.

OnePGT Library Preparation for Illumina Sequencing 60

Quick Reference Protocols

Quick Reference Protocol: Library Preparation

Reference 4

Step 1: Prepare DNA samples

1. Measure the concentration with Qubit dsDNA BR assay.

2. Dilute the samples to 29.4 ng/μl with nuclease-free H

Step 2. Digest the DNA

1. Prepare digestion mix (per rxn):

• 2 μl of Restriction Enzyme Buffer

• 0.5 μl of Restriction Enzyme 1

• 0.5 μl of Restriction Enzyme 2

2. Mix 17 μl of diluted DNA sample with 3 μl of digestion mix.

3. Run thermal cycler with heated lid:

• 37°C for 15 min

• 65°C for 20 min

• 4°C for 1 min

• Hold at 4°C

Step 3. Add adapters

1. Prepare adapter mix (per rxn):

• 2.5 μl of Adapter 1

• 2.5 μl of Adapter 2

2. Add 5 μl of adapter mix to sample.

3. Run thermal cycler with heated lid:

• 65°C for 10 min

• 4°C for 1 min

• Hold at 4°C

Step 4. Ligate the adapters

1. Prepare ligation mix (per rxn):

• 1.5 μl of Nuclease-Free Water (from kit)

• 3.0 μl of Ligase Buffer

• 0.5 μl of DNA Ligase

2. Add 5 μl of ligation mix to sample.

3. Run thermal cycler with heated lid:

• 22°C for 15 min

• 65°C for 10 min

• 4°C for 1 min

• Hold at 4°C

Step 5. SPRI-purify the DNA

1. Prepare 70% ethanol.

2. Add 20 μl of nuclease-free H

O to sample.

3. Add 50 μl of premixed AMPure XP beads to sample.

4. Incubate:

• 5 min off-magnet at room temperature (RT)

• 2 min on-magnet at RT

5. Remove 90 μl of supernatant.

6. Wash 2 times on-magnet with 180 μl of 70% ethanol.

7. Remove all residual ethanol.

8. Air dry on-magnet for 8 min at RT.

9. Resuspend in 25 μl of TE.

10. Incubate:

• 2 minutes off-magnet at RT

• 1 minute on-magnet at RT

11. Transfer 20 μl of sample to a fresh PCR plate or strip tube well.

SAFE STOPPING POINT: UP TO 7 DAYS AT +4˚C OR -20˚C

OnePGT Library Preparation for Illumina Sequencing 61

Quick Reference Protocols

Quick Reference Protocol: Library Preparation

Reference 4

Step 6. Size-select the DNA fragments using PippinHT

1. Set up PippinHT instrument per supplier’s instructions:

• Calibrate the instrument

• Prepare the cassette

• Perform a continuity test

2. Program the PippinHT instrument:

• Select cassette type:

1.5% Agarose 300-1500 bp 15 C

• Select Use Internal Standards

• Select Range and enter range 335-575 bp

3. Add 5 μl of internal marker to sample. Mix thoroughly.

4. Remove 30 μl of buffer from the loading well.

5. Load 25 μl of sample into the loading well.

6. Press Start to begin the electrophoresis run.

7. At end of run, transfer 30 μl of size-selected sample to a fresh PCR plate or strip tube well.

SAFE STOPPING POINT: UP TO 3 DAYS AT +4˚C

Step 7. Suppression PCR-amplify the size-selected DNA

1. Dilute size-selected DNA if required:

• For samples >0.625 ng/μl, dilute using nuclease-free H

O to 0.625 ng/μl

• For samples 0.2–0.625 ng/μl, use undiluted

2. Prepare PCR reagent mix (per rxn):

• 25 μl of PCR mix

• 2.5 μl of Forward PCR primer

3. Put 27.5 μl of PCR reagent mix in a fresh PCR plate or strip tube well.

4. For each sample, add 2.5 μl of unique reverse primer (index A01-H12) from the Reverse PCR primer plate.

5. Add 20 μl of size-selected DNA.

6. Run thermal cycler with heated lid:

• 98°C for 45 sec

• 7 cycles of:

98°C for 15 sec 64°C for 30 sec 72°C for 30 sec

• 72°C for 1 min

• 4°C for 1 min

• Hold at 4°C

OnePGT Library Preparation for Illumina Sequencing 62

Quick Reference Protocols

Quick Reference Protocol: Library Preparation

Reference 4

Step 8. SPRI-purify the DNA

1. Add 50 μl of premixed AMPure XP beads to sample.

2. Incubate:

• 5 min off-magnet at RT

• 2 min on-magnet at RT

3. Remove 90 μl of supernatant.

4. Wash 2 times on-magnet with 180 μl of 70% ethanol.

5. Remove all residual ethanol.

6. Air dry on-magnet for 8 min at RT.

7. Resuspend in 50 μl of nuclease-free H

8. Incubate:

• 2 minutes off-magnet at RT

• 1 minute on-magnet at RT

11. Transfer 45 μl of sample to a fresh PCR plate or strip tube well.

Step 9. Quantify and qualify the library DNA

1. Analyze 2 μl of library DNA with Qubit dsDNA HS assay.

2. Analyze 1 μl of library DNA with Agilent’s 4200 TapeStation or 2100 Bioanalyzer instrument.

Step 10. Pool libraries for multiplexed sequencing

1. Calculate the molarity of each sample.

2. Pool equimolar amounts of each library (see

table below).

Step 11. Set up sequencing

1. Choose the read length according to your desired application:

• PGT-M (with PGT-A/SR) 2 × 150 bp

• PGT-SR or PGT-A 2 × 75 bp

2. Spike in the Custom Read 1 Sequencing Primer (provided at 100 μM) into Illumina’s Read 1 primer and set up sequencing reactions for the run as shown in table below.

Sequencing Parameter Guidelines

Application Platform & Run Type Number of

Samples Pooled

PGT-M (with PGT-A/SR) NextSeq 500/550 High

Output

PGT-M (with PGT-A/SR) HiSeq 2500 Rapid Run 18 10 pM 1% 0.5 µM

PGT-SR or PGT-A only NextSeq 500/550 Mid

Output

PGT-SR or PGT-A only HiSeq 2500 Rapid Run 96 10 pM 1% 0.5 µM

OnePGT Library Preparation for Illumina Sequencing 63

24 1.4 pM 1% 0.3 µM

96 1.4 pM 1% 0.3 µM

Seeding Conc.

%Phi X Final Concentration

of Custom Read 1 Sequencing Primer

www.agilent.com

In This Book

This guide contains information to run the OnePGT Library Preparation for Illumina Sequencing protocol.

Agilent Technologies, Inc. 2018-2020

Version C1, December 2020

*G9425-90000*

p/n G9425-90000

Agilent Technologies

Agilent OnePGT Library Preparation for Illumina User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

Product Description

Safety Notes

Procedural Notes

Disposal

Required Reagents

Required Equipment

Material Preparation

Whole Genome Amplification Protocol

Overview of the Workflow

Protocol

Step 1. Prepare DNA samples

Step 2. Fragment the DNA

Step 3. Add adapters to fragmented DNA

Step 4. Ligate the adapters

Step 5. Purify the DNA using SPRI technology

Step 6. Size-select the DNA fragments

Step 7. Suppression PCR-amplify the size-selected DNA

Step 8. Purify the DNA using SPRI technology

Step 9. Quantify and qualify the OnePGT libraries

Step 10. Pool libraries for multiplexed sequencing

Step 11. Set up the sequencing run

Step 12. Process sequencing data and upload to Agilent Alissa OnePGT

Kit Contents and Supported Configurations

Reference Information for OnePGT Indexes

Guidelines for Optimal Index Multiplexing

Troubleshooting Guide

Quick Reference Protocols