Non-invasive prenatal paternity testing (NIPAT) relies on detecting cell-free fetal DNA (cffDNA) in maternal plasma. Traditional approaches based on single nucleotide polymorphisms (SNPs) face challenges due to low fetal DNA fractions and sequencing errors. To improve accuracy, we developed NEWPAT, a novel method using Double Nucleotide Polymorphisms (DNPs), genetic markers where two consecutive nucleotide changes occur on the same haplotype. DNPs are strictly biallelic, existing in only two haplotypic forms, which reduces the possibility of sequencing errors being misinterpreted as true variants. The probability of both bases being miscalled simultaneously is extremely low, making DNPs highly reliable for paternal allele detection. We selected 845 genome-wide DNPs on autosomes and 80 DNPs on the X chromosome from the Genome Aggregation Database. To enhance analysis and provide internal controls, we included 54 Y-linked SNPs. Target enrichment was performed using the Ion AmpliSeqTM Kit Plus, and sequencing was conducted on the Ion TorrentTM S5 platform. Validation encompassed 42 individuals analyzed across four batches: 1) 15 individuals from 4 families sequenced at 5,000× to assess Mendelism and DNP amplification bias; 2) two artificial mixtures mimicking cffDNA; 3) 12 pregnant women sequenced at 10,000× and fathers at 1,000× to evaluate system reliability and paternity index estimation; and 4) 3 unrelated males as external controls. NEWPAT reliably detected cffDNA even at very low concentration (<1%). In all pregnancies, CPI exceeded 1035, far surpassing the standard threshold for paternity attribution. The ratio of X- and Y-linked reads to autosomal coverage was used to determine fetal sex and validate data consistency. Any deviation from expected values flagged potential technical artifacts or external contamination. These results confirm the accuracy of DNP-based detection, establishing NEWPAT as a key advancement in prenatal paternity testing by improving fetal DNA detection, reducing errors, and enhancing sequencing efficiency for forensic use.
NEWPAT: A Non-Invasive Prenatal Paternity Test Using Double Nucleotide Polymorphisms (DNPs) / Pistacchia, Letizia; D’Atanasio, Eugenia; Blandino, Francesca; La Riccia, Pietro; Hajiesmaeil, Mogge; Ravasini, Francesco; Risi, Flavia; Meschino, Noemi; Scarabino, Daniela; Spinella, Francesca; Cotroneo, Ettore; Sanzico, Chiara; Bella, Elisa; Novelletto, Andrea; Trombetta, Beniamino; Cruciani, Fulvio. - (2025). (Intervento presentato al convegno 13th International Congress on Haploid Markers (HM13) tenutosi a Belgrade).
NEWPAT: A Non-Invasive Prenatal Paternity Test Using Double Nucleotide Polymorphisms (DNPs)
Letizia Pistacchia;Eugenia D’Atanasio;Francesca Blandino;Mogge Hajiesmaeil;Francesco Ravasini;Flavia Risi;Noemi Meschino;Chiara Sanzico;Elisa Bella;Beniamino Trombetta;Fulvio Cruciani
2025
Abstract
Non-invasive prenatal paternity testing (NIPAT) relies on detecting cell-free fetal DNA (cffDNA) in maternal plasma. Traditional approaches based on single nucleotide polymorphisms (SNPs) face challenges due to low fetal DNA fractions and sequencing errors. To improve accuracy, we developed NEWPAT, a novel method using Double Nucleotide Polymorphisms (DNPs), genetic markers where two consecutive nucleotide changes occur on the same haplotype. DNPs are strictly biallelic, existing in only two haplotypic forms, which reduces the possibility of sequencing errors being misinterpreted as true variants. The probability of both bases being miscalled simultaneously is extremely low, making DNPs highly reliable for paternal allele detection. We selected 845 genome-wide DNPs on autosomes and 80 DNPs on the X chromosome from the Genome Aggregation Database. To enhance analysis and provide internal controls, we included 54 Y-linked SNPs. Target enrichment was performed using the Ion AmpliSeqTM Kit Plus, and sequencing was conducted on the Ion TorrentTM S5 platform. Validation encompassed 42 individuals analyzed across four batches: 1) 15 individuals from 4 families sequenced at 5,000× to assess Mendelism and DNP amplification bias; 2) two artificial mixtures mimicking cffDNA; 3) 12 pregnant women sequenced at 10,000× and fathers at 1,000× to evaluate system reliability and paternity index estimation; and 4) 3 unrelated males as external controls. NEWPAT reliably detected cffDNA even at very low concentration (<1%). In all pregnancies, CPI exceeded 1035, far surpassing the standard threshold for paternity attribution. The ratio of X- and Y-linked reads to autosomal coverage was used to determine fetal sex and validate data consistency. Any deviation from expected values flagged potential technical artifacts or external contamination. These results confirm the accuracy of DNP-based detection, establishing NEWPAT as a key advancement in prenatal paternity testing by improving fetal DNA detection, reducing errors, and enhancing sequencing efficiency for forensic use.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
