Third-Generation Single-Molecule Sequencing for Preimplantation Genetic Testing of Aneuploidy and Segmental Imbalances

Vivienne J Tan; Timing Liu; Zainul Arifin; Beatrice Pak; Arnold S C Tan; Simin Wong; Chiea-Chuen Khor; Henry Yang; Caroline G Lee; Zhongwei Huang; Mahesh A Choolani; Samuel S Chong

doi:10.1093/clinchem/hvad062

Third-Generation Single-Molecule Sequencing for Preimplantation Genetic Testing of Aneuploidy and Segmental Imbalances

Clin Chem. 2023 Aug 2;69(8):881-889. doi: 10.1093/clinchem/hvad062.

Authors

Vivienne J Tan¹, Timing Liu¹, Zainul Arifin¹, Beatrice Pak¹, Arnold S C Tan¹, Simin Wong², Chiea-Chuen Khor³, Henry Yang⁴, Caroline G Lee⁵, Zhongwei Huang⁶, Mahesh A Choolani⁶, Samuel S Chong^{1

2

6

7}

Affiliations

¹ Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
² Preimplantation Genetic Diagnosis Centre, Department of Obstetrics and Gynaecology, National University Hospital, Singapore.
³ Division of Human Genetics, Genome Institute of Singapore, Singapore.
⁴ Cancer Science Institute of Singapore, Centre for Translational Medicine, National University of Singapore, Singapore.
⁵ Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
⁶ Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
⁷ Department of Laboratory Medicine, National University Hospital, Singapore.

PMID: 37477572
DOI: 10.1093/clinchem/hvad062

Abstract

Background: Current strategies for preimplantation genetic testing for aneuploidy or structural rearrangements (PGT-A/SR) rely mainly on next-generation sequencing (NGS) and microarray platforms, which are robust but require expensive instrumentation. We explored the suitability of third-generation single-molecule sequencing as a PGT-A/SR screening platform for both aneuploidy and segmental imbalance.

Methods: Single-cell and multicell replicates from aneuploid or segmentally unbalanced cell lines (n = 208) were SurePlex-amplified, randomized, and subjected to (a) Nanopore-based single-molecule sequencing (Oxford Nanopore Technologies) and (b) NGS using a leading commercial PGT-A solution (Illumina VeriSeq PGS). Archival SurePlex-amplified trophectoderm biopsy samples (n = 96) previously analyzed using the commercial kit were blinded and reanalyzed using Nanopore.

Results: Nanopore-based PGT-A identified the specific aberration in 95.45% (84/88) and 97.78% (88/90) of single-/multicells with an aneuploidy or segmental imbalance (10-30.5 Mb), respectively. Comparison against the commercial kit's results revealed concordances of 98.86% (87/88) and 98.89% (89/90) for the aneuploid and segmentally unbalanced (10-30.5 Mb aberration) samples, respectively. Detection sensitivity for smaller segmental imbalances (5-5.8 Mb aberration, n = 30) decreased markedly on both platforms. Nanopore-based PGT-A reanalysis of trophectoderm biopsy samples was 97.92% (94/96) concordant with the commercial kit results.

Conclusion: Up to 24 SurePlex-amplified single-cell, multicell, or trophectoderm samples could be sequenced in a single MinION flow-cell for subsequent preimplantation genetic testing for aneuploidy or structural rearrangements (PGT-A/SR) analysis, with results obtainable in ≤3 days and at per-sample costs that are competitive with commercial offerings. Nanopore's third-generation single-molecule sequencing represents a viable alternative to current commercial NGS-based PGT-A solutions for aneuploidy and segmental imbalance (≥10 Mb) screening of single-/multicell or trophectoderm biopsy samples.

American Association for Clinical Chemistry 2023.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Aneuploidy
Female
Gene Rearrangement
Genetic Testing / methods
High-Throughput Nucleotide Sequencing / methods
Humans
Pregnancy
Preimplantation Diagnosis* / methods