Agilent OnePGT Library Preparation for Illumina User Manual

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

+ 44 hidden pages