ViLR: a novel virtual long read method for breakpoint identification and direct SNP haplotyping in de novo PGT-SR carriers without a proband.

backgroundDespite the gradual application of third-generation long read sequencing (LRS) or reference embryo establishment to preimplantation genetic testing for structural rearrangement (PGT-SR) without familial involvement, there are still limitations to their extensive clinical application yet. This study developed a novel virtual NGS-based long read method (ViLR) and preliminarily evaluated its clinical feasibility of breakpoint characterization and direct SNP haplotyping for de novo chromosomal structural rearrangements (CSR).

methodsA total of 10 families with de novo CSR risk were enrolled in this study for ViLR analysis. In contrast to LRS, ViLR is a virtual long read solution that used the same barcoded labeling and assembly of different long gDNAs differently barcoded. Notably, ViLR could generate an average fragment length of over 30 Kb, with an N50 block size of up to 16 Mb in a single assay, allowing to achieve accurate breakpoint mapping and direct carrier's haplotyping. An approximately 2 Mbp region flanking upstream and downstream of each breakpoint was selected for informative SNP collection. Embryo haplotype determination was based on the established carriers' haplotypes after whole genome amplification and sequencing. To confirm PGT-SR results, we performed prenatal genetic diagnosis.

resultsThis study achieved an average mapping rate of 99.5%, > 90% coverage depth (> 10X), an average number of effective barcode (> 5 kb length) counts of 11,000,000 and an average fragment length of 40 kb, which generated sufficient informative SNPs for breakpoint characterization and haplotype phasing. ViLR analysis of 10 de novo PGT-SR carriers precisely identified breakpoints and haplotypes. Seven families obtained 18 euploid embryos, in which 10 were euploid/normal embryos, 7 were euploid/balanced carrier embryos, and the remaining one unknown was due to homologous recombination of the breakpoint region. Prenatal genetic diagnosis was performed for four women, and the outcomes coincided with the results from embryo PGT-SR. At the time of writing this paper, four healthy babies had been delivered uneventfully.

conclusionHere, we demonstrated the clinical potential of ViLR as a novel solution for breakpoint identification and direct SNP haplotyping in de novo PGT-SR families without proband involvement. CLINICAL TRIAL NUMBER: Not applicable.