Objectives To assess the efficacy of Y-chromosome mini-STR-based next-generation sequencing (NGS) for non-invasive prenatal paternity testing (NIPPT). Methods DNA was extracted from the plasma of 24 pregnant women, and cell-free fetal DNA (cffDNA) haplotyping was performed at 12 Y-chromosome mini-STR loci using the Illumina NextSeq 500 system. The cffDNA haplotype was validated by the paternal haplotype. Subsequentlly, the paternity testing parameters were attributed to each case quantitatively. Results The biological relationship between the alleged fathers and infants in all 24 family cases were confirmed by capillary electrophoresis (CE). The Y-chromosome mini-STR haplotypes of all 14 male cffDNA were obtained by NGS without any missing loci. The alleles of cffDNA and paternal genomic DNA were matched in 13 cases, and a mismatched allele was detected at the DYS393 locus in one case and considered as mutation. No allele was detected in the 10 female cffDNA. The combined paternity index (CPI) and probability of paternity calculation was based on 6 loci Y-haplotype distributions of a local population. The probability of paternity was 98.2699–99.8828% for the cases without mutation, and 14.8719% for the case harboring mutation. Conclusions Our proof-of-concept study demonstrated that Y-chromosome mini-STR can be used for NGS-based NIPPT with high accuracy in real cases, and is a promising tool for familial searching, paternity exclusion and sex selection in forensic and medical applications.

Objectives To assess the efficacy of a mini-STR-based next-generation sequencing (NGS) method for non-invasive prenatal paternity testing (NIPPT). Methods Plasma DNA from 28 pregnant women was extracted and cell-free foetal DNA (cffDNA) genotyping was performed at 23 mini-STR loci using the Illumina NextSeq 500 system. For each mini-STR locus, the cffDNA genotype was validated by determining infant DNA genotype. The mini-STR loci with high concordance rates were selected for the comparison of STR genotyping results between cffDNA and biological father DNA or random male DNA for each family. Results The biological relationship was identified between alleged fathers and infants in all 28 families using the capillary electrophoresis (CE) method. Moreover, the concordance rates of STR genotypes D5S818, D19S253, and D21S1270 were less than 50% in 23 autosomal STR loci. The STR genotype matching probability was calculated using 20 STR loci with more than 60% concordance rate. There was a significant difference in the STR genotype matching probability between cffDNA and the DNA from the biological father (75–100%) or from random males (25–70%) (p<0.0001). Conclusions Our study demonstrated that mini-STR can be used for NGS-based NIPPT. Furthermore, this method can be used for crime control purposes using the STR data available from the national forensic DNA databases.