Objectives: Animal forensic genetics is hindered by a lack of validated short tandem repeat (STR) markers and some species rely on error-prone dinucleotide STRs (di-STRs) with high stutter artifacts. This study aimed to develop a scalable bioinformatic workflow for genome-wide STR mining, population-scale genotyping and forensic parameter evaluation, and to validate its efficacy in cattle (Bos taurus)—a representative species commonly genotyped by di-STRs.

Material and methods: This pipeline integrated Tandem Repeat Finder for de novo STR mining and HipSTR for high-accuracy genotyping. Custom scripts were developed to streamline workflow configuration and calculation of core forensic parameters (e.g., polymorphism information content [PIC], genotyping success rate). We mined STRs from the cattle reference genome and genotyped these loci using whole-genome sequencing data from 60 Chinese and 111 African cattle populations. STRs with genotyping success rate ≥40% and PIC ≥0.5 were selected to build a cattle STR database (CSDB) for practical forensic and population genetic research. Experimental validation was conducted via next-generation sequencing on 30 randomly selected tetranucleotide STRs (tetra-STRs) and 33 in-use di-STRs in the Chinese cattle population, with stutter ratios and polymorphism systematically compared between STR types.

Results and conclusions: The CSDB for the studied Chinese and African cattle population each contained 69,376 and 89,699 STRs, respectively. Of the 33 commonly used cattle di-STRs studied here, 31 loci were successfully identified by our pipeline, while two were excluded due to flanking sequence complexity. Experimental validation showed that di-STRs had a higher average PIC than tetra-STRs, yet tetra-STRs exhibited significantly lower N-1 stutter ratios (1.77%–1.85% vs. 26.88%–33.74% for di-STRs, *p*<0.05). Most loci achieved >99% experimental genotyping success, far exceeding in silico predictions based on WGS data. This pipeline provides a reproducible, species-independent framework for developing STR panels, enabling screening of stutter-less tetra/penta-STRs and addressing gaps in animal forensic genetics. This workflow could facilitate efficient STR marker discovery for forensic, population genetic and ecological studies in non-model species.

Authors

• Jiajun Liu (Faculty of Forensic Medicine, Zhongshan School of Medicine, Sun Yat-sen University, China)
• Zhentang Liu (Faculty of Forensic Medicine, Zhongshan School of Medicine, Sun Yat-sen University, China)
• Hongyu Sun (Faculty of Forensic Medicine, Zhongshan School of Medicine, Sun Yat-sen University, China)
• Riga Wu (Faculty of Forensic Medicine, Zhongshan School of Medicine, Sun Yat-sen University, China)