Dataset Information

Lineage-specific SNPs for genotyping of Mycobacterium tuberculosis clinical isolates.

ABSTRACT: Tuberculosis (TB) is a severe infectious disease worldwide. Genetic variation of the causative agent, Mycobacterium tuberculosis (MTB), determines the outcomes of infection and anti-TB treatment. Until recently, there has been no effective and convenient way for classifying clinical isolates based on the DNA sequences of the divergent lineages of MTB infecting human populations. Here, we identified single nucleotide polymorphisms (SNPs) of six representative strains from Taiwan by whole-genome sequencing and comparing the results to the sequence of the H37Rv reference strain. One hundred and ten SNPs, each unique to one of the six strains, were used to genotype 150 additional isolates by applying DNA mass spectrometry. Lineage-specific SNPs were identified that could distinguish the major lineages of the clinical isolates. A subset including 32 SNPs was found to be sufficient to type four major groups of MTB isolates in Taiwan (ancient Beijing, modern Beijing, East African-Indian, and Latin-American Mediterranean). However, there was high genetic homozygosity within the Euro-American lineage, which included spoligotype-classified Haarlem and T strains. By whole-genome sequencing of 12 representative Euro-American isolates, we identified multiple subtype-specific SNPs which allowed us to distinguish two major branches within the Euro-American lineage.

SUBMITTER: Dou HY

PROVIDER: S-EPMC5431204 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Lineage-specific SNPs for genotyping of Mycobacterium tuberculosis clinical isolates.

Dou Horng-Yunn HY Lin Chien-Hsing CH Chen Yih-Yuan YY Yang Shiu-Ju SJ Chang Jia-Ru JR Wu Keh-Ming KM Chen Ying-Tsong YT Chin Pei-Ju PJ Liu Yen-Ming YM Su Ih-Jen IJ Tsai Shih-Feng SF

Scientific reports 20170503 1

Tuberculosis (TB) is a severe infectious disease worldwide. Genetic variation of the causative agent, Mycobacterium tuberculosis (MTB), determines the outcomes of infection and anti-TB treatment. Until recently, there has been no effective and convenient way for classifying clinical isolates based on the DNA sequences of the divergent lineages of MTB infecting human populations. Here, we identified single nucleotide polymorphisms (SNPs) of six representative strains from Taiwan by whole-genome s ...[more]

PMID: 28469152

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Project description:BackgroundCombining different molecular typing methods for Mycobacterium tuberculosis complex (MTBC) can be a powerful tool for molecular epidemiology-based investigation of TB. However, the current standard method that provides high discriminatory power for such a combination, mycobacterial interspersed repetitive units-variable numbers of tandem repeats typing (MIRU-VNTR), is laborious, time-consuming and often too costly for many resource-limited laboratories. We aimed to evaluate a reduced set of loci for MIRU-VNTR typing in combination with spoligotyping and SNP-typing for routine molecular epidemiology of TB.MethodSpoligotyping and SNP-typing, in combination with the 15 loci MIRU-VNTR typing, were first used to type clinical MTBC isolates (n = 158) from Madagascar. A step by step reduction of MIRU-VNTR loci number was then performed according to the Hunter and Gaston Discriminatory Index (HGDI) and to the Principal component analysis (PCA) correlation with the spoligotype profiles to evaluate the discrimination power inside the generated spoligotype clusters. The 15 MIRU-VNTR was used as reference and SNP-typing was used to determine the main MTBC lineages.ResultsOf the 158 clinical isolates studied, the SNP-typing classified 23 into Lineage 1 (14.6%), 31 into Lineage 2 (19.6%), 23 into Lineage 3 (14.6%) and 81 into Lineage 4 strains (51.3%). 37 different spoligotypes profiles were obtained, 15 of which were unique and 20 in clusters. 15-loci MIRU-VNTR typing revealed 144 different genotypes: 132 isolates had a unique MIRU-VNTR profile and 27 isolates were grouped into 12 clusters. After a stepwise reduction of the MIRU-VNTR loci number within each main spoligotype families, three different sets composed of 5 customised MIRU-VNTR loci had a similar discrimination level to the reference 15 loci MIRU-VNTR in lineage 1, lineage 2 and lineage 3. For lineage 4, a set of 4 and 3 MIRU-VNTR loci were proposed to subtype the Harleem and LAM spoligotype families, respectively. For the T spoligotype family, a set of 5 MIRU-VNTR loci was proposed.ConclusionAccording to the lineages and the spoligotype families, the number of MIRU-VNTR loci can be reduced to get an optimal classification of MTBC. These customized sets of MIRU-VNTR loci reduce workload and save resources while maintaining optimal discriminatory power.

Project description:BACKGROUND: Multiplex ligation-dependent probe amplification (MLPA) is a powerful tool to identify genomic polymorphisms. We have previously developed a single nucleotide polymorphism (SNP) and large sequence polymorphisms (LSP)-based MLPA assay using a read out on a liquid bead array to screen for 47 genetic markers in the Mycobacterium tuberculosis genome. In our assay we obtain information regarding the Mycobacterium tuberculosis lineage and drug resistance simultaneously. Previously we called the presence or absence of a genotypic marker based on a threshold signal level. Here we present a more elaborate data analysis method to standardize and streamline the interpretation of data generated by MLPA. The new data analysis method also identifies intermediate signals in addition to classification of signals as positive and negative. Intermediate calls can be informative with respect to identifying the simultaneous presence of sensitive and resistant alleles or infection with multiple different Mycobacterium tuberculosis strains. RESULTS: To validate our analysis method 100 DNA isolates of Mycobacterium tuberculosis extracted from cultured patient material collected at the National TB Reference Laboratory of the National Center for Tuberculosis and Lung Diseases in Tbilisi, Republic of Georgia were tested by MLPA. The data generated were interpreted blindly and then compared to results obtained by reference methods. MLPA profiles containing intermediate calls are flagged for expert review whereas the majority of profiles, not containing intermediate calls, were called automatically. No intermediate signals were identified in 74/100 isolates and in the remaining 26 isolates at least one genetic marker produced an intermediate signal. CONCLUSION: Based on excellent agreement with the reference methods we conclude that the new data analysis method performed well. The streamlined data processing and standardized data interpretation allows the comparison of the Mycobacterium tuberculosis MLPA results between different experiments. All together this will facilitate the implementation of the MLPA assay in different settings.

Dataset Information

Lineage-specific SNPs for genotyping of Mycobacterium tuberculosis clinical isolates.

Publications

Lineage-specific SNPs for genotyping of Mycobacterium tuberculosis clinical isolates.

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