DataSheet12_Simultaneous detection of genomic imbalance in patients receiving preimplantation genetic testing for monogenic diseases (PGT-M).PDF

Background: Preimplantation genetic test for monogenic disorders (PGT-M) has been used to select genetic disease-free embryos for implantation during in vitro fertilization (IVF) treatment. However, embryos tested by PGT-M have risks of harboring chromosomal aneuploidy. Hence, a universal method to detect monogenic diseases and genomic imbalances is required.
Methods: Here, we report a novel PGT-A/M procedure allowing simultaneous detection of monogenic diseases and genomic imbalances in one experiment. Library was prepared in a special way that multiplex polymerase chain reaction (PCR) was integrated into the process of whole genome amplification. The resulting library was used for one-step low-pass whole genome sequencing (WGS) and high-depth target enrichment sequencing (TES).
Results: The TAGs-seq PGT-A/M was first validated with genomic DNA (gDNA) and the multiple displacement amplification (MDA) products of a cell line. Over 90% of sequencing reads covered the whole-genome region with around 0.3–0.4 × depth, while around 5.4%–7.3% of reads covered target genes with >10000 × depth. Then, for clinical validation, 54 embryos from 8 women receiving PGT-M of β-thalassemia were tested by the TAGs-seq PGT-A/M. In each embryo, an average of 20.0 million reads with 0.3 × depth of the whole-genome region was analyzed for genomic imbalance, while an average of 0.9 million reads with 11260.0 × depth of the target gene HBB were analyzed for β-thalassemia. Eventually, 18 embryos were identified with genomic imbalance with 81.1% consistency to karyomapping results. 10 embryos contained β-thalassemia with 100% consistency to conventional PGT-M method.
Conclusion: TAGs-seq PGT-A/M simultaneously detected genomic imbalance and monogenic disease in embryos without dramatic increase of sequencing data output.

背景：植入前单基因遗传病遗传学检测（Preimplantation Genetic Test for Monogenic Disorders, PGT-M）已被广泛应用于体外受精（in vitro fertilization, IVF）治疗周期，用于筛选无遗传疾病的胚胎以进行移植。然而，经PGT-M检测的胚胎仍存在携带染色体非整倍体的风险。因此，亟需一种可同时检测单基因疾病与基因组失衡的通用检测方法。
方法：本研究报道一种新型PGT-A/M流程，可在单次实验中同时实现单基因疾病与基因组失衡的检测。该流程通过特殊方式构建测序文库：将多重聚合酶链式反应（multiplex polymerase chain reaction, PCR）整合至全基因组扩增环节。所得文库可用于一步法低覆盖度全基因组测序（whole genome sequencing, WGS）与高深度目标富集测序（target enrichment sequencing, TES）。
结果：本研究首先以基因组DNA（genomic DNA, gDNA）及某细胞系的多重置换扩增（multiple displacement amplification, MDA）产物对TAGs-seq PGT-A/M进行验证。结果显示，超过90%的测序读段（reads）覆盖全基因组区域，平均测序深度约为0.3~0.4×；另有约5.4%~7.3%的测序读段（reads）覆盖目标基因，测序深度超过10000×。随后开展临床验证：招募8名拟接受β地中海贫血PGT-M治疗的女性，共获取54枚胚胎并采用TAGs-seq PGT-A/M进行检测。针对每枚胚胎，平均通过2000万条测序读段（reads）（全基因组区域测序深度0.3×）分析基因组失衡情况，同时通过平均90万条测序读段（reads）（目标基因HBB测序深度11260.0×）检测β地中海贫血。最终，共鉴定出18枚存在基因组失衡的胚胎，其检测结果与核型定位（karyomapping）结果的一致性达81.1%；另有10枚胚胎携带β地中海贫血，与传统PGT-M方法的检测结果完全一致（一致性100%）。
结论：TAGs-seq PGT-A/M可在不显著增加测序数据产出量的前提下，同时检测胚胎的基因组失衡与单基因疾病。