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 Yersinia pestis 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 Y. pestis. A real-time polymerase chain reaction confirmed the presence of Y. pestis in all samples; however, whole-genome sequencing (WGS) coverage of the Y. pestis 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 Y. pestis 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.