Background: Massively parallel sequencing (MPS) is a promising supplementary method for forensic casework in short tandem repeats (STRs) genotyping, owing to several advantageous features in comparison to traditional capillary electrophoresis (CE). However, the application of MPS in casework requires accessible datasets from the worldwide population to enrich the allele frequencies of sequence‐based STR genotypes.Methods: In this study, we report the characterization of sequence‐based allele frequencies of 58 STRs from a Tibetan population comprising 120 unrelated individuals using the ForenSeq™ DNA Signature Prep Kit. A concordance study evaluating MPS and CE allele data was performed to ensure that MPS is compatible with current CE‐based forensic databases. The diversity of observed alleles, allele frequencies, and forensic parameters per locus by length (LB), sequence without flanking region (RSB), and sequence with flanking region (FSB) were analyzed and compared.Results: The concordance study demonstrated a concordance rate exceeding 99%. The combined random match probability (RMP) for the 26 A‐STRs was 2.04 × 10^–29, 1.93 × 10^–31, and 9.56 × 10^–33 for LB, RSB, and FSB, respectively. Similar trends were observed in other forensic parameters resulting from the increase in the number of unique alleles available. A total of 111 and 113 unique haplotypes in the Y‐STR loci were observed when using length‐based and sequence‐based alleles, respectively. In addition, we identified 35 novel alleles at 25 loci and 25 polymorphisms in the flanking regions at 17 STRs.Conclusions: Our data suggest that MPS‐ and CE‐derived alleles are compatible. MPS‐based analysis of the STR data substantially increased the allele diversity and improved the forensic parameters, which clearly demonstrated the advantages of MPS in comparison to CE. With more pooled data and larger‐scale validation, MPS could play a valuable role in forensic genetics and might be an additional tool for routine casework.

Short tandem repeats have been essential and fundamental genetic markers used in forensic individual discrimination and paternity testing since their discovery, especially those used in the Combined DNA Index System. Nevertheless, in cases of complex kinship identification, such as full-sibling, half-sibling, and uncle–niece relationships, the combined application of additional short tandem repeat loci is necessary to reach reliable identification conclusions. In this study, we evaluated the efficiency of an updated novel short tandem repeat genotyping system for kinship identification in the Eastern Chinese Han population. This 23-plex short tandem repeat system demonstrated strong discrimination power among individuals in the target population, with a combined power of discrimination and cumulative probability of exclusion of 1–2.107 1 × 10−27 and 0.999 999 999 800, respectively. When 74 short tandem repeats were used and the threshold log10(likelihood ratio) was set to 4, the system efficiency reached 0.999 9 and 0.707 3 for simulated full-sibling and half-sibling pairs, respectively. Furthermore, in two real secondary kinship identification cases, incorporation of the novel 23-plex short tandem repeat system increased the probabilities of the prior kinship hypotheses from 154.259 5 and 1 031.699 5 to 56 597.118 4 and 134 829.791 5, respectively, yielding reliable identification conclusions. Hence, it is evident that the novel 23-plex short tandem repeat system has notable potential as a tool for forensic kinship identification in the Eastern Chinese Han population and could serve as a complementary set of short tandem repeat loci for the identification of distant kinship.