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

The Non-Invasive Prenatal Paternity Test (NIPAT) builds upon the discovery of small amounts of cell-free fetal DNA (cffDNA) circulating in maternal blood. The current non-invasive tests rely on single nucleotide polymorphisms (SNPs), with limitations due to low fetal DNA fractions and high error rate of targeted next-generation sequencing (NGS). To address this, our project aims to exploit a new class of genetic markers, biallelic double nucleotide polymorphisms (DNPs). The probability of being sequenced incorrectly is the product of the individual base sequencing error probabilities, reducing the NGS error rate by two orders of magnitude. First, a bioinformatic approach has been used to identify 978 genome-wide DNPs from the large amount of whole genome sequences in the Genome Aggregation Database. These markers were analyzed in 15 individuals from 4 families, amplifying their DNA using Ion AmpliSeq™ Kit Plus from Thermo Fisher Scientific. Different depths of coverage were explored for the same samples, reaching a maximum of 5000x, while 6 full-risk samples with varying fetal fractions were tested to validate the minimum detectable and informative quantity. The NGS amplification quality of the selected DNPs in all the samples has been evaluated in different steps, by performing a depth analysis and by calling all the alleles at each position in order to estimate the background noise and distinguish the false calls due to sequencing errors from the true cffDNA alleles.