Evaluation of an MPS‐Based Microhaplotype Assay for Non‐Invasive Prenatal Paternity Testing

ABSTRACT Non‐invasive prenatal paternity testing (NIPPT) enables the genotyping of cell‐free DNA (cfDNA) from maternal plasma through deep sequencing. Microhaplotypes (MHs) combine the advantages of short tandem repeats (STRs) and single‐nucleotide polymorphisms (SNPs) and have attracted much attention in NIPPT. In this study, we optimized 45 MHs from our previous study and confirmed the effectiveness of the 45plex MH panel through different kinship inferences using real samples, including duos, trios, full siblings, and second‐to‐fifth‐degree relatives, and excluding unrelated individuals. Furthermore, we tested 11 cfDNA and reference mother–child pairs in the first trimester (7 + 4–12 + 6 weeks) and 11 cfDNA and reference trios in the second trimester (18+ weeks). The R packages Familias and RelMix and the software EuroForMix were used for data interpretation. The results showed that MHs of cfDNA could be effectively detected using our sequencing and genotyping pipelines. We correctly determined paternity in 11 NIPPT cases, with Log 10 LR > 10, which were significantly separated from real unrelated males. Our study indicates that this massively parallel sequencing (MPS)‐based 45plex MH panel provides more robust relationship inference capabilities than standard STR systems, complements NIPPT, and may help solve relevant issues for relative DNA mixtures.