Short tandem repeats are stretches of DNA in which a short sequence of two to six base pairs is repeated over and over, such as ATATATAT. Because the number of repeats varies widely between individuals, STRs form the backbone of DNA profiling in human forensics. Standardized human STR genotyping systems, built on carefully validated panels of markers, allow laboratories around the world to produce comparable, court-admissible profiles. Animal forensics has never enjoyed that level of coordination. Validated STR markers for most domestic and wild species are scarce, and the markers that do exist are often dinucleotide STRs, repeats of just two base pairs, which are notoriously prone to genotyping artifacts such as stutter, the generation of spurious off-by-one peaks that complicate interpretation. Population data, which allow forensic scientists to calculate the statistical weight of a match, are frequently missing altogether.

The team behind pSTRminer, led by Jiajun Liu, Zhentang Liu, and senior authors Hongyu Sun and Riga Wu of the Faculty of Forensic Medicine at Zhongshan School of Medicine, set out to close these gaps with a single, scalable computational framework. The software automates what has traditionally been a fragmented, largely manual workflow: scanning a reference genome for STR loci, genotyping those loci in large collections of whole-genome sequencing data, and then scoring each locus for the properties that matter in forensic practice, including genotyping success rate and polymorphism information content, a standard measure of how informative a genetic marker is for distinguishing individuals.

To demonstrate the power of the approach, the researchers turned to domestic cattle, Bos taurus, one of the most economically and forensically significant livestock species in the world. Applying pSTRminer to the cattle reference genome, they identified 775,444 STRs de novo, a catalog of repeat loci far exceeding anything previously assembled for the species. They then genotyped this catalog using whole-genome sequencing data from 60 Chinese cattle and 111 African cattle, two populations chosen to represent sharply divergent genetic backgrounds. The logic is straightforward but important: a marker that appears highly variable in only one breed or region may be nearly useless elsewhere, so evaluating polymorphism across diverse lineages is essential before any locus can be recommended for global forensic use.

From this population-scale analysis, the team constructed the cattle STR database, or CSDB, a curated resource containing only those loci that met stringent quality criteria: a genotyping success rate of at least 40 percent and a polymorphism information content of at least 0.5. These thresholds ensure that the database holds markers that both amplify reliably in the laboratory and carry enough variation to discriminate between individuals. The sensitivity of the database to the genotyping success rate threshold was examined in supplementary analyses, giving future users a transparent view of how the marker set changes as criteria are tightened or relaxed.

Computational screening alone, however, is not enough for forensic work. Markers destined for casework must survive contact with real samples. The researchers therefore experimentally validated a panel of loci in a local Chinese cattle population of 145 animals using next-generation sequencing. Thirty tetranucleotide STRs, repeats of four base pairs, and 33 dinucleotide STRs were randomly selected from the database and tested. The validation confirmed that the markers were reliable, and it produced a nuanced picture of the trade-offs between repeat types. Tetranucleotide STRs showed lower average polymorphism than their dinucleotide counterparts, meaning they tend to be somewhat less variable across individuals. But they carried a decisive advantage: significantly lower stutter ratios, a difference the authors report as statistically significant at p less than 0.05. In practical terms, four-base-pair repeats generate fewer genotyping artifacts, producing cleaner, easier-to-interpret profiles.

That finding matters because stutter is one of the most persistent headaches in STR analysis. When a polymerase copies a repeat tract, it occasionally slips, adding or dropping a repeat unit and creating a minor artifact peak one repeat shorter or longer than the true allele. Dinucleotide repeats, with their short two-base motif, are especially vulnerable to this slippage. A marker with high stutter can obscure genuine alleles, particularly in degraded or low-template samples common in forensic contexts. By demonstrating that certain tetranucleotide STRs can actually surpass dinucleotide STRs in polymorphism while producing far fewer artifacts, the study lays out a viable path toward building animal STR panels that are both highly discriminative and technically robust. Systematic screening across the CSDB revealed that such high-performing tetranucleotide loci are not rare exceptions but a discoverable resource waiting to be tapped.

The broader significance of pSTRminer extends well beyond cattle. The software integrates established components of the modern genomics pipeline, drawing on widely used tools for read preprocessing, alignment, and STR genotyping, and wraps them into a reproducible workflow that reduces the manual operations required to move from raw sequencing data to a validated marker panel. The authors provide detailed documentation of the commands needed to reproduce their analyses, and supplementary tables include the formulas used to calculate forensic parameters, the overlap between STRs currently in use and those newly identified in cattle, and recommended analytical thresholds for heterozygote balance at validated loci. In effect, the study offers not just a database but a blueprint that other laboratories can follow to develop standardized STR systems for dogs, cats, horses, yaks, wildlife species, or any organism with a reference genome and population sequencing data.

The need for such tools is well documented in the forensic literature. Individual identification systems based on STR panels have been developed for domestic cats, dogs, and horses, and microsatellite marker sets have been proposed for parentage testing in cattle, yaks, and Chinese Holstein bulls. Yet each of these efforts has relied on comparatively small collections of markers, often selected without genome-wide polymorphism data or population-scale validation. Human forensics, by contrast, has moved decisively toward expanded multiplex systems and sequencing-based genotyping, supported by open population databases built from large-scale sequencing projects such as the 1000 Genomes Project. pSTRminer aims to bring animal forensics closer to that standard, enabling marker discovery at the scale the human field now takes for granted.

The work, supported by the National Natural Science Foundation of China, was approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University, and the authors acknowledge the publicly available whole-genome sequencing data from the NCBI Sequence Read Archive that made the population analyses possible. For forensic scientists, the arrival of pSTRminer and the CSDB marks a shift from ad hoc marker selection to systematic, data-driven panel design. A single hair from a stolen calf, a bloodstain on a suspect’s boot, or a trace of tissue from a poached animal could soon be profiled with the same rigor and statistical confidence that human DNA evidence enjoys. As genome sequencing becomes cheaper and reference genomes accumulate for more species, the framework promises to make animal forensic genetics faster, cleaner, and more defensible, one well-validated repeat at a time.

Subject of Research: Genome-wide identification and population-scale evaluation of polymorphic short tandem repeats for animal forensic genetics

Article Title: pSTRminer: integrated bioinformatic software for genome-wide identification and population-scale evaluation of polymorphic short tandem repeats

Article References: pSTRminer: integrated bioinformatic software for genome-wide identification and population-scale evaluation of polymorphic short tandem repeats. (n.d.). https://doi.org/10.1007/s00414-026-04002-w

Image Credits: AI Generated

DOI: 10.1007/s00414-026-04002-w

Keywords: pSTRminer, short tandem repeats, forensic genetics, cattle, bioinformatics, genotyping, polymorphism, next-generation sequencing, STR database, animal forensics, population genetics, tetranucleotide STRs