Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

Summary

Here, we introduce a semiconductor sequencing method for preimplantation genetic testing for aneuploidy (PGT-A) with the advantages of short turnaround time, low cost, and high throughput.

Abstract

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented in human embryos. Preimplantation genetic testing for aneuploidy (PGT-A) is a genetic test that significantly improves reproductive outcomes by detecting chromosomal abnormalities of embryos. Next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A. Here, we present a rapid and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system. Generally, for a PGT-A application, 24 samples can be loaded and sequenced on each chip generating 60−80 million reads at an average read length of 150 base pairs. The method provides a refined protocol for performing template amplification and enrichment of sequencing library, making the PGT-A detection reproducible, high-throughput, cost-efficient, and timesaving. The running time of this semiconductor sequencer is only 2−4 hours, shortening the turnaround time from receiving samples to issuing reports into 5 days. All these advantages make this assay an ideal method to detect chromosomal aneuploidies from embryos and thus, facilitate its wide application in PGT-A.

Introduction

Choosing good-quality viable embryos with normal chromosome copy numbers (euploid) for transfer in assisted reproduction helps to improve pregnancy outcomes. Traditionally, the well-established morphological grading system is widely used for embryo evaluation due to its easy availability and non-invasive nature. However, it has been shown that morphological assessment can only provide limited information on embryo quality¹ and implantation potential². One fundamental reason is its inability in evaluating the chromosomal composition of the embryos.

Chromosomal aneuploidy (abnormal copy number o....

Protocol

Ethical approval was granted by the Joint Chinese University of Hong Kong―New Territories East Cluster Clinical Research Ethics Committee (Reference Number: 2010.432). Research license was approved by the Council on Human Reproductive Technology of Hong Kong (Number R3004).

1. Whole genome amplification

1. Prior to start, check the volume of magnetic beads (Table of Materials) to ensure that there is no less than 135 µL (with a 20% excess) for each sample. .......

Discussion

Different from other sequencing chemistries, the sequencer described here uses semiconductor for the detection of nucleotides. The chip itself is an electronic device that detects hydrogen ions by polymerase-driven base incorporation¹⁷, which enables 2−4 h sequencing time of the Proton program. Besides, the chip is a microwell chip that allows the localization of one target molecule, which is different from the flow cell sequencing chemistry by other sequencer providers. This protocol is a m.......

Materials

Name	Company	Catalog Number	Comments
PCR tubes, 0.2 mL	Axygen	PCR-02D-C
UltraPure 0.5M EDTA, pH 8.0	ThermoFisher	15575020
PBS, pH 7.4, Ca2+ and Mg2+ free	ThermoFisher	10010023
1.0 M NaOH (1.0N) solution	SIGMA-ALDRICH	S2567	For Melt-off solution. Molecular grade
Eppendorf LoBind Tubes, 1.5 mL	Fisher Scientific	13-698-791
Ion Plus Fragment Library Kit	ThermoFisher	4471252
ELGA PURELAB Flex 3 Water Purification System or Equivalent 18 MΩ water system	ThermoFisher	4474524
Ion Plus Fragment Library Kit	ThermoFisher	4471252
PicoPLEX WGA Amplification buffer	Rubicon Genomics	R30050	This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.
PicoPLEX WGA Amplification enzyme	Rubicon Genomics	R30050	This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.
Ion OneTouch Amplification Plate			In kit: Ion OneTouch 2 Supplies (Part No. A26367). Extended kit component in Sheet 5
Ion PI Annealing Buffer
MyOne Beads Capture Solution
Agilent 2100 Bioanalyzer instrument	Agilent	G2939AA
Ion OneTouch Breaking Solution (black cap)			In kit: Ion PI Hi‑Q OT2 Solutions 200 (Part No. A26429). Extended kit component in Sheet 5
Dynabeads MyOne Streptavidin C1	ThermoFisher	65001
PicoPLEX WGA Cell extraction buffer	Rubicon Genomics	R30050	This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-00.
PicoPLEX WGA Cell extraction enzyme	Rubicon Genomics	R30050	This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.
Ion PI Chip Kit v3	ThermoFisher	A26771
Ion Chip Minifuge, 230 V	ThermoFisher	4479673
Ion PI dATP	ThermoFisher	A26772
Ion PI dCTP	ThermoFisher	A26772
Ion PI dGTP	ThermoFisher	A26772
Ion Plus Fragment Library Kit	ThermoFisher	4471252
Ion Plus Fragment Library Kit	ThermoFisher	4471252
ThermoQ–Temperature Dry Bath	TAMAR	HB-T2-A
NEBNext dsDNA Fragmentase	New England Biolabs	M0348L
NEBNext dsDNA Fragmentase	New England Biolabs	M0348L
Ion PI dTTP	ThermoFisher	A26772
Ion OneTouch 2 Instrument	ThermoFisher	INS1005527	ThermoFisher Catalog number: 4474778.
Ion Plus Fragment Library Kit	ThermoFisher	4471252
Ion One Touch ES	ThermoFisher	8441-22	ThermoFisher Catalog number: 4469495. Extended kit component in Sheet 5
Ethanol	SIGMA-ALDRICH	51976	This can be replaced by any brand's molecular grade absolute ethanol.
PicoPLEX WGA Extraction enzyme dilution buffer	Rubicon Genomics	R30050	This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-01.
PicoPLEX WGA Extraction enzyme dilution buffer	Rubicon Genomics	R30050	This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-02.
Qubit 3.0 Fluorometer	ThermoFisher	Q33216	This model has been replaced by Qubit 4 Fluorometer, Catalog number: Q33226.
Qubit ds DNA HS Assay kit	ThermoFisher	M2002-02
Qubit Assay Tubes	ThermoFisher	Q32856
Ion PI Foaming Solution	ThermoFisher	A26772
Index for barcoding of libraries	BaseCare		this is a in-house prepared index. Users can buy commercial product from ThermoFisher Ion Xpress Barcode Adapters Kits (Cat. No. 4474517)
Ion PI Loading Buffer	ThermoFisher	A26772
Solid(TM) Buffer Kit-1X Low TE Buffer	ThermoFisher	4389764
Agencour AMPure XP Kit	Beckman Coulter	A63880
DynaMag-2 magnet (magnetic rack)	ThermoFisher	12321D
Ion PI Master Mix PCR buffer
Sorvall Legend Micro 17 Microcentrifuge	Micro 17	75002430
Ion Plus Fragment Library Kit	ThermoFisher	4471252
Nuclease-free water	ThermoFisher	AM9922	This can be replaced by other brand.
PicoPLEX WGA Nuclease-free water	Rubicon Genomics	R30050	This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.
Ion OneTouch Oil bottle			Ion PI Hi‑Q OT2 Solutions 200 (Part No. A26429). Extended kit component in Sheet 5
Ion Plus Fragment Library Kit	ThermoFisher	4471252	Extended kit component in Sheet 3
double-strand DNA standard			This is a in-house prepared DNA standard for calibration of Qubit before quantification of library.
PicoPLEX WGA Preamplification buffer	Rubicon Genomics	R30050	This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.
PicoPLEX WGA Preamplification enzyme	Rubicon Genomics	R30050	This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.
Library Amplification Primer Mix	ThermoFisher	4471252	Extended kit component in Sheet 3
Ion OneTouch Reaction Filter			Extended kit component in Sheet 5
Recovery Router			Extended kit component in Sheet 5
Recovery Tubes			Extended kit component in Sheet 5
ISP Resuspension Solution
Ion Proton	ThermoFisher	DA8600	This model is imported by Da An Gene Co.,LTD. of Sun Yat-Sen University from ThermoFisher and has been certified by China Food and Drug Administration for clinical application. The catalog number in ThermoFisher is 4476610.
Ion PI Hi‑Q Sequencing Polymerase	ThermoFisher	A26772
Ion PI Sequencing Primer
server for sequencer	Lenovo	T260
Ion PI Sphere Particles
Platinum PCR SuperMix High Fidelity	ThermoFisher	4471252
Nalgene 25mm Syringe Filters	ThermoFisher	724-2045	Pore size: 0.45μm. Specifically for aqueous fluids.
Ion PI Hi‑Q W2 Solution	ThermoFisher	A26772
Ion PI 1X W3 Solution	ThermoFisher	A26772
Ion OneTouch Wash Solution C1
The Ion PGM Hi‑Q View Sequencing Kit	ThermoFisher	A30044	Extended kit component in Sheet 2
Ion Plus Fragment Library Kit	ThermoFisher	4471252	Extended kit component in Sheet 3
Ion PI Hi-Q Sequencing 200 Kit (1 sequencing run per initialization)	ThermoFisher	A26772	Extended kit component in Sheet 4
Ion PI Hi‑Q OT2 200 Kit	ThermoFisher	A26434	Extended kit component in Sheet 5