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

Abstract

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.

Obstetric Markers as a Diagnostic Forensic Tool

“…A recent study done by Tam et al developed a systematic SNPs selection procedure which reduced the number of target-SNPs for sequencing analysis to an average of 148 effective SNPs to calculate the probability of paternity. But the possible drawback is that in order to perform the test, a large number of loci is required [22]. But this is the mainstay for noninvasive paternity testing as of now.…”

Improved library preparation protocols for amplicon sequencing-based noninvasive fetal genotyping for RHD-positive D antigen-negative alleles

“…The limitation of our amplicon-sequencing-based fetal RHD genotyping is that the method by itself cannot determine FF, which is common to all amplicon-based NIPT methods. To complement this limitation, we plan to adopt an amplicon-sequencing method for multiple SNPs [ 16 ] when FF of a cfDNA sample needs to be precisely determined.…”

Set of 15 SNP-SNP Markers for Detection of Unbalanced Degraded DNA Mixtures and Noninvasive Prenatal Paternity Testing

“…In addition, stutter products interfere the analysis of cffDNA. SNP, as a single base variation and no stutter products present during the PCR amplification, is considered more appropriate for NIPPT ( Chang et al, 2019 ; Tam et al, 2020 ). However, the biallelic nature of SNP results in limited polymorphism at a single locus.…”