DataSheet10_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：单基因疾病的植入前遗传学检测（PGT-M）已被应用于选择在体外受精（IVF）治疗中植入的无遗传疾病胚胎。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：在本研究中，我们报道了一种新型的PGT-A/M检测程序，该程序允许在一次实验中同时检测单基因疾病和基因组不平衡。图书馆以特殊方式制备，将多重聚合酶链反应（PCR）整合到全基因组扩增的过程中。得到的图书馆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首先通过细胞系的基因组DNA（gDNA）和多重置换扩增（MDA）产物进行验证。超过90%的测序读段覆盖了整个基因组区域，深度约为0.3–0.4倍，而大约5.4%–7.3%的读段覆盖了目标基因，深度超过10000倍。随后，为进行临床验证，对接受PGT-M治疗的8名妇女的54个β-地中海贫血胚胎进行了TAGs-seq PGT-A/M检测。在每一个胚胎中，平均分析了约2000万个读段，覆盖全基因组区域的深度为0.3倍，同时分析了平均90万个读段，覆盖目标基因HBB的深度为11260.0倍。最终，识别出18个存在基因组不平衡的胚胎，与核型图谱结果的一致性达到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能够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。