Dataset Information

TGS-TB: Total Genotyping Solution for Mycobacterium tuberculosis Using Short-Read Whole-Genome Sequencing.

ABSTRACT: 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/.

SUBMITTER: Sekizuka T

PROVIDER: S-EPMC4643978 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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TGS-TB: Total Genotyping Solution for Mycobacterium tuberculosis Using Short-Read Whole-Genome Sequencing.

Sekizuka Tsuyoshi T Yamashita Akifumi A Murase Yoshiro Y Iwamoto Tomotada T Mitarai Satoshi S Kato Seiya S Kuroda Makoto M

PloS one 20151113 11

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 genotyp ...[more]

PMID: 26565975

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Project description:Cambodia has one of the highest tuberculosis (TB) incidence rates in the WHO Western Pacific region. Remarkably though, the prevalence of multidrug-resistant TB (MDR-TB) remains low. We explored the genetic diversity of Mycobacterium tuberculosis (MTB) circulating in this unique setting using whole-genome sequencing (WGS). From October 2017 until January 2018, we collected one hundred sputum specimens from consenting adults older than 21 years of age, newly diagnosed with bacteriologically confirmed TB in 3 districts of Phnom Penh and Takeo provinces of Cambodia before they commence on their TB treatment, where eighty MTB isolates were successfully cultured and sequenced. Majority of the isolates belonged to Lineage 1 (Indo-Oceanic) (69/80, 86.25%), followed by Lineage 2 (East Asian) (10/80, 12.5%) and Lineage 4 (Euro-American) (1/80, 1.25%). Phenotypic resistance to both streptomycin and isoniazid was found in 3 isolates (3/80, 3.75%), while mono-resistance to streptomycin and isoniazid was identical at 2.5% (N = 2 each). None of the isolates tested was resistant to either rifampicin or ethambutol. The specificities of genotypic prediction for resistance to all drugs tested were 100%, while the sensitivities of genotypic resistance predictions to isoniazid and streptomycin were lower at 40% (2/5) and 80% (4/5) respectively. We identified 8 clusters each comprising of two to five individuals all residing in the Takeo province, making up half (28/56, 50%) of all individuals sampled in the province, indicating the presence of multiple ongoing transmission events. All clustered isolates were of Lineage 1 and none are resistant to any of the drugs tested. This study while demonstrating the relevance and utility of WGS in predicting drug resistance and inference of disease transmission, highlights the need to increase the representation of genotype-phenotype TB data from low and middle income countries in Asia and Africa to improve the accuracies for prediction of drug resistance.

Project description:BACKGROUND: Understanding Mycobacterium tuberculosis (Mtb) transmission is essential to guide efficient tuberculosis control strategies. Traditional strain typing lacks sufficient discriminatory power to resolve large outbreaks. Here, we tested the potential of using next generation genome sequencing for identification of outbreak-related transmission chains. METHODS AND FINDINGS: During long-term (1997 to 2010) prospective population-based molecular epidemiological surveillance comprising a total of 2,301 patients, we identified a large outbreak caused by an Mtb strain of the Haarlem lineage. The main performance outcome measure of whole genome sequencing (WGS) analyses was the degree of correlation of the WGS analyses with contact tracing data and the spatio-temporal distribution of the outbreak cases. WGS analyses of the 86 isolates revealed 85 single nucleotide polymorphisms (SNPs), subdividing the outbreak into seven genome clusters (two to 24 isolates each), plus 36 unique SNP profiles. WGS results showed that the first outbreak isolates detected in 1997 were falsely clustered by classical genotyping. In 1998, one clone (termed "Hamburg clone") started expanding, apparently independently from differences in the social environment of early cases. Genome-based clustering patterns were in better accordance with contact tracing data and the geographical distribution of the cases than clustering patterns based on classical genotyping. A maximum of three SNPs were identified in eight confirmed human-to-human transmission chains, involving 31 patients. We estimated the Mtb genome evolutionary rate at 0.4 mutations per genome per year. This rate suggests that Mtb grows in its natural host with a doubling time of approximately 22 h (400 generations per year). Based on the genome variation discovered, emergence of the Hamburg clone was dated back to a period between 1993 and 1997, hence shortly before the discovery of the outbreak through epidemiological surveillance. CONCLUSIONS: Our findings suggest that WGS is superior to conventional genotyping for Mtb pathogen tracing and investigating micro-epidemics. WGS provides a measure of Mtb genome evolution over time in its natural host context.

Dataset Information

TGS-TB: Total Genotyping Solution for Mycobacterium tuberculosis Using Short-Read Whole-Genome Sequencing.

Publications

TGS-TB: Total Genotyping Solution for Mycobacterium tuberculosis Using Short-Read Whole-Genome Sequencing.

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