Noninvasive prenatal paternity testing by target sequencing microhaps

Microhaplotypes (i.e.,microhaps or MHs) are emerging multi-allelic markers with at least two single nucleotide polymorphisms (SNPs) within ∼ 200 bp that have alleles of the same length and do not generate stutter products. Based on massively parallel sequencing (MPS) technology, microhaps have proven applicability in forensics for different application purposes. Here we evaluate the feasibility of non-invasive prenatal paternity testing (NIPPT) with a panel of polymorphic microhap markers, using cell-free DNA (cfDNA) in the maternal circulation. A custom MPS-based assay targeting 60 microhaps was developed in our previous study. Herein, we applied the developed assay to cfDNA samples in 15 NIPPT cases in the first trimester of pregnancy (6∼13 weeks). The R package relMix was employed for data interpretation, with a regression dropout estimating model. As a result, the targeted sequencing wherein target enrichment is by hybridization capture can be effectively employed for microhap sequencing with cfDNA samples. With the combined use of relMix, the paternity of the biological fathers in 15 cases was correctly determined, with the combined paternity index (CPI) value > 10¹². Moreover, the specificity of this approach was validated by the successful paternity exclusion of 3 close relatives (father, full sibling and uncle) of the biological father in one case, and further by the significant separation in CPI distribution between the biological father and 112 unrelated males in each cases. Our results indicate that this MPS-based microhap sequencing strategy could be utilized in NIPPT. This method may contribute to developments in NIPPT and to the resolution of issues related to DNA mixtures of close relatives for specific purposes.