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

Animal forensic genetics plays a critical role in criminal investigations by providing crucial evidence through domestic animal individualization and wildlife species identification. While human forensic genetics benefits from standardized short tandem repeats (STR) genotyping systems, animal forensic applications encounter significant challenges, including the limited availability of validated STR markers, the prevalence of error-prone dinucleotide STRs (di-STRs), and insufficient integration of population data. To address these challenges, we developed pSTRminer, an integrated bioinformatic tool that automates genome-wide STR mining and polymorphism evaluation. By applying pSTRminer to domestic cattle (Bos taurus), we identified 775,444 STRs de novo from the reference genome and genotyped them using whole-genome sequencing data from 60 Chinese and 111 African cattle to evaluate polymorphism across diverse genetic backgrounds. This led to the development of the cattle STR database (CSDB), comprising loci with a genotyping success rate ≥ 40% and polymorphism information content (PIC) ≥ 0.5. Experimental validation of 30 randomly selected tetranucleotide STRs (tetra-STRs) and 33 di-STRs via next-generation sequencing in a local Chinese cattle population (n = 145) confirmed marker reliability. Although tetra-STRs had lower average polymorphism levels, they exhibited significantly lower stutter ratios (p < 0.05), providing a viable path for identifying discriminative markers with fewer artifacts. Systematic screening revealed that certain tetra-STRs could surpass di-STRs in polymorphism. In conclusion, pSTRminer provides a scalable framework for developing standardized STR panels, facilitating the identification of robust and informative markers in forensic applications.