Microbial Forensics: Comparison of MLVA Results According to NGS Methods, and Forensic DNA Analysis Using MLVA

Microbial forensics is a scientific discipline for analyzing evidence related to biological crimes by identifying the origin of microorganisms. Multiple locus variable number tandem repeat analysis(MLVA) is one of the microbiological analysis methods used to specify subtypes within a species based on the number of tandem repeat in the genome, and advances in next generation sequencing(NGS) technology have enabled <i>in silico</i> anlysis of full-length whole genome sequences. In this paper, we analyzed unknown samples provided by Robert Koch Institute(RKI) through The United Nations Secretary-General’s Mechanism(UNSGM)’s external quality assessment exercise(EQAE) project, which we officially participated in 2023. We confirmed that the 3 unknown samples were <i>B. anthracis</i> through nucleic acid isolation and genetic sequence analysis studies. MLVA results on 32 loci of <i>B. anthracis</i> were analysed by using genome sequences obtained from NGS(NextSeq and MinION) and Sanger sequencing. The MLVA typing using short-reads based NGS platform(NextSeq) showed a high probability of causing assembly error when a size of the tandem repeats was grater than 200 bp, while long-reads based NGS platform(MinION) showed higher accuracy than NextSeq, although insertion and deletion was observed. We also showed hybrid assembly can correct most indel error caused by MinION. Based on the MLVA results, genetic identification was performed compared to the 2,975 published MLVA databases of <i>B. anthracis</i>, and MLVA results of 10 strains were identical with 3 unkonwn samples. As a result of whole genome alignment of the 10 strains and 3 unknown samples, all samples were identified as <i>B. anthracis</i> strain A4564 which is associated with injectional anthrax isolates in heroin users.