Sekizuka Tsuyoshi, Yamashita Akifumi, Murase Yoshiro, Iwamoto Tomotada, Mitarai Satoshi, Kato Seiya, Kuroda Makoto
Pathogen Genomics Center, National Institute of Infectious Diseases, Shinjyuku-ku, Tokyo, Japan.
Molecular Epidemiology Division, The Research Institute of Tuberculosis/Japan Anti-Tuberculosis Association, Kiyose, Tokyo, Japan.
PLoS One. 2015 Nov 13;10(11):e0142951. doi: 10.1371/journal.pone.0142951. eCollection 2015.
Whole-genome sequencing (WGS) with next-generation DNA sequencing (NGS) is an increasingly accessible and affordable method for genotyping hundreds of Mycobacterium tuberculosis (Mtb) isolates, leading to more effective epidemiological studies involving single nucleotide variations (SNVs) in core genomic sequences based on molecular evolution. We developed an all-in-one web-based tool for genotyping Mtb, referred to as the Total Genotyping Solution for TB (TGS-TB), to facilitate multiple genotyping platforms using NGS for spoligotyping and the detection of phylogenies with core genomic SNVs, IS6110 insertion sites, and 43 customized loci for variable number tandem repeat (VNTR) through a user-friendly, simple click interface. This methodology is implemented with a KvarQ script to predict MTBC lineages/sublineages and potential antimicrobial resistance. Seven Mtb isolates (JP01 to JP07) in this study showing the same VNTR profile were accurately discriminated through median-joining network analysis using SNVs unique to those isolates. An additional IS6110 insertion was detected in one of those isolates as supportive genetic information in addition to core genomic SNVs. The results of in silico analyses using TGS-TB are consistent with those obtained using conventional molecular genotyping methods, suggesting that NGS short reads could provide multiple genotypes to discriminate multiple strains of Mtb, although longer NGS reads (≥ 300-mer) will be required for full genotyping on the TGS-TB web site. Most available short reads (~100-mer) can be utilized to discriminate the isolates based on the core genome phylogeny. TGS-TB provides a more accurate and discriminative strain typing for clinical and epidemiological investigations; NGS strain typing offers a total genotyping solution for Mtb outbreak and surveillance. TGS-TB web site: https://gph.niid.go.jp/tgs-tb/.
采用下一代DNA测序（NGS）的全基因组测序（WGS）是一种越来越容易获得且成本较低的方法，可对数百株结核分枝杆菌（Mtb）进行基因分型，从而开展更有效的流行病学研究，这些研究基于分子进化分析核心基因组序列中的单核苷酸变异（SNV）。我们开发了一种基于网络的一体化Mtb基因分型工具，称为结核病全基因分型解决方案（TGS-TB），通过用户友好的简单点击界面，促进使用NGS的多个基因分型平台进行 spoligotyping以及检测核心基因组SNV、IS6110插入位点和43个用于可变数目串联重复序列（VNTR）的定制位点的系统发育。该方法通过KvarQ脚本实施，以预测MTBC谱系/亚谱系和潜在的抗菌药物耐药性。在本研究中，通过使用这些分离株特有的SNV进行中位连接网络分析，准确区分了7株具有相同VNTR图谱的Mtb分离株（JP01至JP07）。除了核心基因组SNV外，在其中一株分离株中还检测到一个额外的IS6110插入，作为支持性遗传信息。使用TGS-TB进行的计算机分析结果与使用传统分子基因分型方法获得的结果一致，这表明NGS短读长可提供多种基因型以区分多种Mtb菌株，尽管在TGS-TB网站上进行全基因分型需要更长的NGS读长（≥300碱基）。大多数可用的短读长（~100碱基）可用于基于核心基因组系统发育来区分分离株。TGS-TB为临床和流行病学调查提供了更准确、更具鉴别力的菌株分型；NGS菌株分型为Mtb暴发和监测提供了全基因分型解决方案。TGS-TB网站：https://gph.niid.go.jp/tgs-tb/ 。
