Sai Liu1,2, Hui Wang1, Don Leigh2, David S Cram2, Li Wang3, Yuanqing Yao4. 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. 3. Reproductive Medicine and Genetic Center, The First Hospital of Kunming Calmette Hospital, Kunming, People's Republic of China. pkulwang@126.com. 4. 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. yqyao_ghpla@163.com.
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.
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.
Authors: Jason D Merker; Aaron M Wenger; Tam Sneddon; Megan Grove; Zachary Zappala; Laure Fresard; Daryl Waggott; Sowmi Utiramerur; Yanli Hou; Kevin S Smith; Stephen B Montgomery; Matthew Wheeler; Jillian G Buchan; Christine C Lambert; Kevin S Eng; Luke Hickey; Jonas Korlach; James Ford; Euan A Ashley Journal: Genet Med Date: 2017-06-22 Impact factor: 8.822