NEWPAT: Development of a non-invasive paternity test with high specificity

The non-invasive prenatal paternity test (NIPAT) leverages small amounts of cell-free fetal DNA (cffDNA) present in maternal blood. Conventional non-invasive tests rely on single nucleotide polymorphisms (SNPs), but face challenges such as low fetal DNA fractions and high error rates stemming from targeted next-generation sequencing (NGS). To enhance accuracy, we introduced a pioneering NIPAT approach (NEWPAT) employing double nucleotide polymorphisms (DNPs). We selected 978 genome-wide DNPs from the Genome Aggregation Database, amplified these markers using the Ion AmpliSeq™ Kit Plus from Thermo Fisher Scientific, and sequenced them with the Ion Torrent™ S5 platform. System validation encompassed: 1) 5000x sequencing across 4 families, to assess Mendelism and DNP amplification bias; 2) re-sequencing at 500-1000x, to evaluate sensitivity; and 3) ultra-high depth (10000x) sequencing of 12 pregnant women and the putative fathers, to evaluate system reliability and estimate combined paternity index (CPI) in real-word scenarios. Since DNPs are not routinely used as markers in most genomic analyses, a standardized bioinformatic workflow for their analysis from NGS data has yet to be established. We developed an ad hoc pipeline and applied rigorous quality control measures to ensure precise allele calling at each DNP position while safeguarding against contamination. Our methodology significantly enhanced the discrimination between cffDNA alleles and sequencing artefacts, crucial for paternal allele detection. Sequencing error rates for most DNPs were negligible and significantly lower compared to SNPs, highlighting the precision of our approach. For all the pregnancies tested, we obtained CPIs > 10²⁵, far exceeding the thresholds for paternity attribution. This innovative methodology represents a pivotal advancement in prenatal diagnostics, offering a new level of accuracy and reliability in genetic testing.