Noninvasive prenatal paternity testing by means of SNP-based targeted sequencing

Objective To develop a method for noninvasive prenatal paternity testing based on targeted sequencing of single nucleotide polymorphisms (SNPs). Method SNPs were selected based on population genetics data. Target-SNPs in cell-free DNA extracted from maternal blood (maternal cfDNA) were analyzed by targeted sequencing wherein target enrichment was based on multiplex amplification using QIAseq Targeted DNA Panels with Unique Molecular Identifiers. Fetal SNP genotypes were called using a novel bioinformatics algorithm, and the combined paternity indices (CPIs) and resultant paternity probabilities were calculated. Results Fetal SNP genotypes obtained from targeted sequencing of maternal cfDNA were 100% concordant with those from amniotic fluid-derived fetal genomic DNA. From an initial panel of 356 target-SNPs, an average of 148 were included in paternity calculations in 15 family trio cases, generating paternity probabilities of greater than 99.9999%. All paternity results were confirmed by short-tandem-repeat analysis. The high specificity of the methodology was validated by successful paternity discrimination between biological fathers and their siblings and by large separations between the CPIs calculated for the biological fathers and those for 60 unrelated men. Conclusion The novel method is highly effective, with substantial improvements over similar approaches in terms of reduced number of target-SNPs, increased accuracy, and reduced costs.