DataSheet11_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：针对单基因疾病的胚胎植入前遗传学检测（PGT-M）已应用于辅助生殖技术（IVF）治疗过程中对胚胎进行遗传疾病的无害筛选。然而，经PGT-M检测的胚胎存在携带染色体非整倍体的风险。因此，迫切需要一种通用的方法来检测单基因疾病和基因组不平衡。方法：本研究报告了一种新型的PGT-A/M检测程序，该程序可在一次实验中同时检测单基因疾病和基因组不平衡。通过特殊方式制备文库，将多重聚合酶链式反应（PCR）整合到全基因组扩增的过程之中。所得文库被用于一步法低通全基因组测序（WGS）和高深度目标富集测序（TES）。结果：TAGs-seq PGT-A/M首先用细胞系的基因组DNA（gDNA）和多重位移扩增（MDA）产物进行了验证。超过90%的测序读段覆盖了全基因组区域，深度约为0.3–0.4 ×，而约5.4%–7.3%的读段覆盖了目标基因，深度超过10000 ×。随后，为进行临床验证，对8名接受β-地中海贫血PGT-M的妇女的54个胚胎进行了TAGs-seq PGT-A/M检测。在每个胚胎中，平均分析了全基因组区域的2000万条读段，深度为0.3 ×，以检测基因组不平衡，同时分析了目标基因HBB的90万条读段，深度为11260.0 ×，以检测β-地中海贫血。最终，确定了18个存在基因组不平衡的胚胎，与核型映射结果的一致性达到81.1%。10个胚胎含有β-地中海贫血，与传统的PGT-M方法的一致性达到100%。结论：TAGs-seq PGT-A/M可在不显著增加测序数据输出的情况下，同时检测胚胎中的基因组不平衡和单基因疾病。