Reinvestigation of a paternity case with three CODIS core STR inconsistencies using MPS-based multi‑marker analysis

A routine paternity test using 23 autosomal short tandem repeats (A-STRs) revealed three Mendelian inconsistencies at D21S11, D13S317, and D7S820 between the alleged father and child, yielding a combined paternity index (CPI) below the confirmation threshold (CPI > 10⁴) stipulated in the Chinese technical specifications. Extended capillary electrophoresis typing of 48 A-STRs and 17 Y-STRs eliminated further mismatches and raised the CPI above the standard. Subsequent massively parallel sequencing (MPS) of 54 A-STRs, 27 X-STRs, 48 Y-STRs, 132 identity-informative single nucleotide polymorphisms (iiSNPs), and mitochondrial DNA (mtDNA) provided both length- and sequence-based genotypes. The CPI reached 2.78 × 10²¹ (A-STRs) and 6.28 × 10¹⁷ (iiSNPs), strongly supporting the paternity. All other marker systems were consistent with the alleged relationships. The three observed incompatibilities were attributed to single-step paternal mutations. This case highlights the risk of misinterpreting multi-locus STR mismatches and demonstrates the value of MPS-based genotyping in resolving complex paternity evaluations.