미생물법의학: 차세대염기서열분석 방법에 따른 MLVA 결과 비교 및 이를 활용한 DNA 감식
Microbial Forensics: Comparison of MLVA Results According to NGS Methods, and Forensic DNA Analysis Using MLVA
윤형석(국방과학연구소 국방첨단과학기술연구원 Chem-Bio센터); 송동현(국방과학연구소 국방첨단과학기술연구원); 이승호(국방과학연구소 국방첨단과학기술연구원 Chem-Bio센터); 임승현(국방과학연구소); 이대상(국방과학연구소); 구세훈(국방과학연구소); 김정은(국방과학연구소 국방첨단과학기술연구원 Chem-Bio센터); 정주환(국방과학연구소); 김성주(국방과학연구소); 허경행(국방과학연구소 국방첨단과학기술연구원 Chem-Bio센터)
27권 4호, 507~515쪽
초록
Microbial forensics is a scientific discipline for analyzing evidence related to biological crimes by identifying theorigin of microorganisms. Multiple locus variable number tandem repeat analysis(MLVA) is one of the microbiologicalanalysis methods used to specify subtypes within a species based on the number of tandem repeat in the genome, andadvances in next generation sequencing(NGS) technology have enabled in silico anlysis of full-length whole genomesequences. In this paper, we analyzed unknown samples provided by Robert Koch Institute(RKI) through The UnitedNations Secretary-General’s Mechanism(UNSGM)’s external quality assessment exercise(EQAE) project, which weofficially participated in 2023. We confirmed that the 3 unknown samples were B. anthracis through nucleic acidisolation and genetic sequence analysis studies. MLVA results on 32 loci of B. anthracis were analysed by usinggenome sequences obtained from NGS(NextSeq and MinION) and Sanger sequencing. The MLVA typing usingshort-reads based NGS platform(NextSeq) showed a high probability of causing assembly error when a size of thetandem repeats was grater than 200 bp, while long-reads based NGS platform(MinION) showed higher accuracy thanNextSeq, although insertion and deletion was observed. We also showed hybrid assembly can correct most indel errorcaused by MinION. Based on the MLVA results, genetic identification was performed compared to the 2,975 publishedMLVA databases of B. anthracis, and MLVA results of 10 strains were identical with 3 unkonwn samples. As aresult of whole genome alignment of the 10 strains and 3 unknown samples, all samples were identified as B. anthracis strain A4564 which is associated with injectional anthrax isolates in heroin users.
Abstract
Microbial forensics is a scientific discipline for analyzing evidence related to biological crimes by identifying theorigin of microorganisms. Multiple locus variable number tandem repeat analysis(MLVA) is one of the microbiologicalanalysis methods used to specify subtypes within a species based on the number of tandem repeat in the genome, andadvances in next generation sequencing(NGS) technology have enabled in silico anlysis of full-length whole genomesequences. In this paper, we analyzed unknown samples provided by Robert Koch Institute(RKI) through The UnitedNations Secretary-General’s Mechanism(UNSGM)’s external quality assessment exercise(EQAE) project, which weofficially participated in 2023. We confirmed that the 3 unknown samples were B. anthracis through nucleic acidisolation and genetic sequence analysis studies. MLVA results on 32 loci of B. anthracis were analysed by usinggenome sequences obtained from NGS(NextSeq and MinION) and Sanger sequencing. The MLVA typing usingshort-reads based NGS platform(NextSeq) showed a high probability of causing assembly error when a size of thetandem repeats was grater than 200 bp, while long-reads based NGS platform(MinION) showed higher accuracy thanNextSeq, although insertion and deletion was observed. We also showed hybrid assembly can correct most indel errorcaused by MinION. Based on the MLVA results, genetic identification was performed compared to the 2,975 publishedMLVA databases of B. anthracis, and MLVA results of 10 strains were identical with 3 unkonwn samples. As aresult of whole genome alignment of the 10 strains and 3 unknown samples, all samples were identified as B. anthracis strain A4564 which is associated with injectional anthrax isolates in heroin users.
- 발행기관:
- 한국군사과학기술학회
- 분류:
- 기타공학