Typing of Yersinia pestis in Challenging Forensic Samples Through Targeted Next-Generation Sequencing of Multilocus Variable Number Tandem Repeat Regions

Microbial forensics involves analyzing biological evidence to evaluate weaponized microorganisms or their toxins. This study aimed to detect and type <i>Yersinia pestis</i> from four simulated forensic samples-human plasma diluted in phosphate-buffered saline (#24-2), tomato juice (#24-5), grape juice (#24-8), and a surgical mask (#24-10). Notably, samples #24-10 may have contained live bacteria other than <i>Y. pestis</i>. A real-time polymerase chain reaction confirmed the presence of <i>Y. pestis</i> in all samples; however, whole-genome sequencing (WGS) coverage of the <i>Y. pestis</i> chromosome ranged from 0.46% to 97.1%, largely due to host DNA interference and low abundance. To address these limitations and enable strain-level identification, we designed a hybridization-based target enrichment approach focused on multilocus variable number tandem repeat analysis (MLVA). Next-generation sequencing (NGS) using whole-genome amplification revealed that the accuracy of the 25 MLVA profiles of <i>Y. pestis</i> for samples #24-2, #24-5, #24-8, and #24-10 was 4%, 100%, 52%, and 0%, respectively. However, all samples showed 100% accuracy with target-enriched NGS, confirming they all belong to the same strain. These findings demonstrate that a targeted enrichment strategy for MLVA loci can overcome common obstacles in microbial forensics, particularly when working with trace or degraded samples where conventional WGS proves challenging.