A relative-independent haplotype derivation method applied for noninvasive prenatal testing for chromosomal rearrangements in a pregnant carrier.

This study aimed to perform noninvasive prenatal testing for structural chromosomal rearrangements (NIPT-SR) for a female pregnant proband carrying a t(4;8) balanced translocation, whose husband exhibited a normal karyotype. NIPT-SR could accurately detect transmission status of structural rearrangements in fetus through Hidden Markov Model (HMM) analysis, which requires the construction of parental haplotypes. To address the challenge of lacking genetic information from other family members of this proband, we developed a novel strategy to infer the fetal inheritance of structural variants by integrating Oxford Nanopore Technologies (ONT) with the NIPT-SR approach. Long-read sequencing was performed on the proband to directly detect the translocation and nearby single nucleotide polymorphisms (SNPs), and to link the structural variants with phased haplotypes. NIPT-SR method was used to infer the fetal inheritance of the constructed haplotypes and to evaluate the potential presence of unbalanced translocation in the fetus. Noninvasive prenatal testing (NIPT) was performed at 12 weeks of gestation, followed by copy number variation sequencing (CNV-seq) and karyotype analysis after birth respectively to confirm the accuracy of NIPT-SR results. Using nanopore sequencing, we identified the precise locations of the breakpoint junctions and successfully established the SNP-based haplotypes that were linked to the breakpoints on chr4 and chr8, without the need for retrieving genetic information of other family members. Haplotype-based analysis of cell-free DNA (cfDNA) indicated that the fetus inherited the normal haplotypes, which was consistent with the NIPT results and confirmed by the postnatal CNV-seq and karyotype analysis. In conclusion, the NIPT-SR method coupled with ONT platform could be used to perform NIPT-SR for those who carries balanced translocation circumventing the need for other family members as reference, providing an important supplement to birth defects prevention.