A novel SNP-based approach for non-invasive prenatal paternity testing using multiplex PCR targeted capture sequencing

Objective: To enhance the safety, simplicity, and efficacy of non-invasive prenatal paternity testing, we developed a method based on multiplex PCR targeted capture sequencing technology utilizing single nucleotide polymorphisms (SNPs) as genetic markers. Method: We screened 627 SNPs from public databases and literature based on specific criteria and population genetic data from 100 unrelated individuals. A total of 15 peripheral blood samples were collected from pregnant women and the suspected father. Paternal alleles were detected and analyzed in the plasma cell-free DNA (cfDNA) of pregnant women, fetal SNP genotypes were obtained, and the combined paternity index (CPI) was calculated for paternity testing. Results: Biological fathers were accurately determined in all cases, with CPI values ranging from 1.05 × 1014 to 2.03 × 1034, consistent with results obtained using polymerase chain reaction-capillary electrophoresis (PCR-CE) with short tandem repeats. Significant differences in CPI between unrelated males and biological fathers allowed for straightforward exclusion. Even cfDNA from maternal plasma as early as five gestational weeks enabled accurate paternity determination. Conclusion: This novel approach demonstrates significant improvements by reducing the number of SNPs, streamlining the research procedure, and lowering costs, yielding substantial advancements in non-invasive prenatal paternity testing.