Nanopore sequencing with T2T-CHM13 for accurate detection and preventing the transmission of structural rearrangements in highly repetitive heterochromatin regions in human embryos

Abstract

Background: Structural rearrangements in highly repetitive heterochromatin regions can result in miscarriage or foetal malformations; however, detecting and preventing the transmission of these rearrangements has been challenging. Recently, the completion of sequencing of the complete human genome (T2T-CHM13) has made it possible to accurately characterise structural rearrangements in these regions. We developed a method based on T2T-CHM13 and nanopore sequencing to detect and block structural rearrangements in highly repetitive heterochromatin sequences.

Methods: T2T-CHM13-based “Mapping Allele with Resolved Carrier Status” was performed for couples who carry structural rearrangements in heterochromatin regions. Using nanopore sequencing and the T2T-CHM13 reference genome, the precise breakpoints of inversions and translocations close to the centromere were detected and haplotypes were constructed using flanking single-nucleotide polymorphisms (SNPs). Haplotype linkage analysis was then performed by comparing consistent parental SNPs with embryonic SNPs to determine whether the embryos carried hereditary inversions or balanced translocations. Based on copy number variation and haplotype linkage analysis, we transplanted normal embryos, which were further verified by an amniotic fluid test.

Results: To validate this approach, we used nanopore sequencing of families with inversions and reciprocal translocations close to the centromere. Using the T2T-CHM13 reference genome, we accurately detected inversions and translocations in centromeres, constructed haplotypes and prevented the transmission of structural rearrangements in the offspring.

Conclusions: This study represents the first successful application of T2T-CHM13 in human reproduction and provides a feasible protocol for detecting and preventing the transmission of structural rearrangements of heterochromatin in embryos.

Keywords

heterochromatin / nanopore sequencing / structural rearrangements / T2T-CHM13

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2024 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.