The Non-Invasive Prenatal Paternity Test (NIPAT) builds upon the presence of small amounts of cell-free fetal DNA (cffDNA) circulating in maternal blood. Conventional 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 overcome these challenges and increase the accuracy, we developed a new NIPAT test (NEWPAT) based on double nucleotide polymorphisms (DNPs). First, we identified 926 genome-wide DNPs (845 from the autosomes, and 81 from the X chromosome), selected from the Genome Aggregation Database using several filtering criteria. We then performed a target enrichment of these markers (along with 52 Y chromosome SNPs) using the Ion AmpliSeqTM Kit Plus from Thermo Fisher Scientific and sequenced them by NGS using the Ion TorrentTM S5 system. The approach was validated by: (a) sequencing 15 individuals from 4 families at a depth of 5000x, to assess Mendelism and DNP amplification bias (b) re-analysing the same samples at lower depths (500-1000x) to evaluate sensibility; (c) sequencing at high depth (10,000x) 12 pregnant women with decreasing estimated proportions of cffDNA and 12 biological fathers of the fetuses (1,000x) to assess system reliability and estimate combined paternity index (CPI) in real cases. Since DNPs do not represent markers routinely used in most of the genomic analyses, a standard bioinformatic workflow to analyze them from NGS data has not been developed yet. So, we developed an ad hoc pipeline, applying rigorous quality control to ensure the accurate calling of alleles at each position of the DNPs and to check at the same time for contamination. Our approach significantly improved the discrimination between cffDNA alleles and sequencing errors, which is essential for the accurate identification of paternal alleles. The sequencing error rate at most DNPs was close to zero and in any case orders of magnitude lower than commonly used SNPs, emphasizing the improved precision of our sequencing strategy. For all the pregnancies tested, the CPI was well above the threshold for paternity attribution. This innovative method is crucial for the advancement of prenatal diagnostics as it offers a new level of precision and reliability in genetic testing.
NEWPAT: Development of a non-invasive paternity test with high specifity / Pistacchia, Letizia; D’Atanasio, Eugenia; Blandino, Francesca; La Riccia, Pietro; Hajiesmaeil, Mogge; Ravasini, Francesco; Risi, Flavia; Bella, Elisa; Sanzico, Chiara; Meschino, Noemi; Scarabino, Daniela; Spinella, Francesca; Cotroneo, Ettore; Novelletto, Andrea; Trombetta, Beniamino; Cruciani, Fulvio. - (2024). (Intervento presentato al convegno Human Genome Meeting (HUGO 2024) tenutosi a Roma; Italy).
NEWPAT: Development of a non-invasive paternity test with high specifity
Letizia Pistacchia;Eugenia D’Atanasio;Francesca Blandino;Mogge Hajiesmaeil;Francesco Ravasini;Flavia Risi;Elisa Bella;Chiara Sanzico;Noemi Meschino;Beniamino Trombetta;Fulvio Cruciani
2024
Abstract
The Non-Invasive Prenatal Paternity Test (NIPAT) builds upon the presence of small amounts of cell-free fetal DNA (cffDNA) circulating in maternal blood. Conventional 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 overcome these challenges and increase the accuracy, we developed a new NIPAT test (NEWPAT) based on double nucleotide polymorphisms (DNPs). First, we identified 926 genome-wide DNPs (845 from the autosomes, and 81 from the X chromosome), selected from the Genome Aggregation Database using several filtering criteria. We then performed a target enrichment of these markers (along with 52 Y chromosome SNPs) using the Ion AmpliSeqTM Kit Plus from Thermo Fisher Scientific and sequenced them by NGS using the Ion TorrentTM S5 system. The approach was validated by: (a) sequencing 15 individuals from 4 families at a depth of 5000x, to assess Mendelism and DNP amplification bias (b) re-analysing the same samples at lower depths (500-1000x) to evaluate sensibility; (c) sequencing at high depth (10,000x) 12 pregnant women with decreasing estimated proportions of cffDNA and 12 biological fathers of the fetuses (1,000x) to assess system reliability and estimate combined paternity index (CPI) in real cases. Since DNPs do not represent markers routinely used in most of the genomic analyses, a standard bioinformatic workflow to analyze them from NGS data has not been developed yet. So, we developed an ad hoc pipeline, applying rigorous quality control to ensure the accurate calling of alleles at each position of the DNPs and to check at the same time for contamination. Our approach significantly improved the discrimination between cffDNA alleles and sequencing errors, which is essential for the accurate identification of paternal alleles. The sequencing error rate at most DNPs was close to zero and in any case orders of magnitude lower than commonly used SNPs, emphasizing the improved precision of our sequencing strategy. For all the pregnancies tested, the CPI was well above the threshold for paternity attribution. This innovative method is crucial for the advancement of prenatal diagnostics as it offers a new level of precision and reliability in genetic testing.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
