Third-generation sequencing: any future opportunities for PGT?
- PDF / 2,004,759 Bytes
- 8 Pages / 595.276 x 790.866 pts Page_size
- 45 Downloads / 158 Views
GENETICS
Third-generation sequencing: any future opportunities for PGT? Sai Liu 1,2 & Hui Wang 1 & Don Leigh 2 & David S. Cram 2 & Li Wang 2 & Yuanqing Yao 1 Received: 24 September 2020 / Accepted: 8 November 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract Purpose To investigate use of the third-generation sequencing (TGS) Oxford Nanopore system as a new approach for preimplantation genetic testing (PGT). Methods Embryos with known structural variations underwent multiple displacement amplification to create fragments of DNA (average ~ 5 kb) suitable for sequencing on a nanopore. Results High-depth sequencing identified the deletion interval for the relatively large HBA1/2–SEA alpha thalassemia deletion. In addition, STRs were able to be identified in the primary sequence data for potential use in conventional PGT-M linkage confirmation. Sequencing of amplified embryo DNA carrying a translocation enabled balanced embryos to be identified and gave the precise identification of translocation breakpoints, offering the opportunity to differentiate carriers from non-carrier embryos. Low-pass sequencing gave reproducible profiles suitable for simple identification of whole-chromosome and segmental aneuploidies. Conclusion TGS on the Oxford Nanopore is a possible alternative and versatile approach to PGT with potential for performing economical workups where the long read sequencing information can be used for assisting in a traditional PGT workup to design an accurate and reliable test. Additionally, application of TGS has the possibility of providing combined PGT-A/SR or in selected stand-alone PGT-M cases involving pathogenic deletions. Both of these applications offer the opportunity for simultaneous aneuploidy detection to select either balanced embryos for transfer or additional carrier identification. The low cost of the instrument offers new laboratories economical entry into onsite PGT. Keywords Third-generation sequencing (TGS) . Preimplantation genetic testing (PGT)
Introduction Sequencing approaches to chromosome analyses in embryo preimplantation assessments have taken over from array CGH [1]. Early arrays used BAC probes that were hybridized with whole-genome amplification (WGA) DNA material from biopsy pieces with the total material costs a combination of the WGA process, the fluorescent labeling, and the array slides and reagents [2]. Major equipment includes hybridization and * Li Wang [email protected] * Yuanqing Yao [email protected] 1
Department of Obstetrics and Gynecology, The First Medical Center of PLA General Hospital, Medical School of Chinese PLA, 28 Fuxing Road, Beijing 100853, People’s Republic of China
2
Reproductive Medicine and Genetic Center, The First Hospital of Kunming Calmette Hospital, Kunming, People’s Republic of China
slide wash systems and moderate- to-high-resolution scanners, depending on the array system chosen. While manageable for many medium to large laboratories, total equipment and maintenance costs were often prohibitive for the
Data Loading...
