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

Sekizuka T, Yamashita A, Murase Y, Iwamoto T, Mitarai S, Kato S, Kuroda M - PLoS ONE (2015)

Bottom Line: An additional IS6110 insertion was detected in one of those isolates as supportive genetic information in addition to core genomic SNVs.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.

View Article: PubMed Central - PubMed

Affiliation: Pathogen Genomics Center, National Institute of Infectious Diseases, Shinjyuku-ku, Tokyo, Japan.

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/.

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Related in: MedlinePlus

Schematic representation of the IS6110 insertion detection strategy.A) The IS6110 sequence (Acc.# X94955 and X94956)-positive short reads are collected (A1-2), rearranged (A3), trimmed (A4), subtracted (A5) and mapped to the Mtb H37Rv chromosome (NC_000962.3) [30] using BWA-SW mapping [29]. B) Typical read mapping profile for the detection of the IS6110 insertion site in both directions.
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pone.0142951.g005: Schematic representation of the IS6110 insertion detection strategy.A) The IS6110 sequence (Acc.# X94955 and X94956)-positive short reads are collected (A1-2), rearranged (A3), trimmed (A4), subtracted (A5) and mapped to the Mtb H37Rv chromosome (NC_000962.3) [30] using BWA-SW mapping [29]. B) Typical read mapping profile for the detection of the IS6110 insertion site in both directions.

Mentions: Although the core genome phylogeny database includes 78 references, it is too computationally intensive to determine the precise differences for only outbreak-related query isolates. Thus, query-specific SNV sites are simple datasets used to perform further epidemiological investigations. Among the tested seven isolates (four from the outbreak and three from related isolates with similar MIRU-VNTR loci [26]), 6 query-specific SNVs were extracted to investigate the molecular epidemiological markers to trace back the outbreak between patients. The differences based on these 6 SNV sites can be visualized through a median-joining network using PopART software (Fig 4). The MIRU-VNTR loci of the four outbreak isolates (JP03, JP04, JP06 and JP07) were investigated prior to NGS, indicating that three additional isolates (JP01, JP02 and JP05) were closely related to the four outbreak isolates. The MIRU-VNTR results did not show sufficient discrimination among the seven isolates ranging from 2004 to 2011. NGS and TGS-TB analyses were performed to identify the SNVs among those isolates. No unique SNVs were detected for the four outbreak isolates (JP03, JP04, JP06 and JP07), and the two past isolates (JP01 and JP02) with two SNVs difference could be one outbreak source or potential ancestors related to this outbreak. It has been reported that the estimated rate of change in DNA sequences was 0.3–0.5 single nucleotide polymorphisms per genome per year [9, 13, 17, 18]; thus, the two detected SNVs represent a reasonable nucleotide substitution rate between the outbreak (2010/2011) and past isolates (2004/2005). In contrast, JP05 was isolated at a close time point, with four outbreak isolates in December 2010, and three SNV sites were detected when compared with four outbreak isolates. One additional IS6110 insertion was detected in JP05 (Figs 4B and 5), strongly suggesting that JP05 did not appear to be involved in the outbreak. Such SNV networks facilitate the detection of epidemiological factors, and the obtained network is consistent with the field study (data not shown).


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

Sekizuka T, Yamashita A, Murase Y, Iwamoto T, Mitarai S, Kato S, Kuroda M - PLoS ONE (2015)

Schematic representation of the IS6110 insertion detection strategy.A) The IS6110 sequence (Acc.# X94955 and X94956)-positive short reads are collected (A1-2), rearranged (A3), trimmed (A4), subtracted (A5) and mapped to the Mtb H37Rv chromosome (NC_000962.3) [30] using BWA-SW mapping [29]. B) Typical read mapping profile for the detection of the IS6110 insertion site in both directions.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643978&req=5

pone.0142951.g005: Schematic representation of the IS6110 insertion detection strategy.A) The IS6110 sequence (Acc.# X94955 and X94956)-positive short reads are collected (A1-2), rearranged (A3), trimmed (A4), subtracted (A5) and mapped to the Mtb H37Rv chromosome (NC_000962.3) [30] using BWA-SW mapping [29]. B) Typical read mapping profile for the detection of the IS6110 insertion site in both directions.
Mentions: Although the core genome phylogeny database includes 78 references, it is too computationally intensive to determine the precise differences for only outbreak-related query isolates. Thus, query-specific SNV sites are simple datasets used to perform further epidemiological investigations. Among the tested seven isolates (four from the outbreak and three from related isolates with similar MIRU-VNTR loci [26]), 6 query-specific SNVs were extracted to investigate the molecular epidemiological markers to trace back the outbreak between patients. The differences based on these 6 SNV sites can be visualized through a median-joining network using PopART software (Fig 4). The MIRU-VNTR loci of the four outbreak isolates (JP03, JP04, JP06 and JP07) were investigated prior to NGS, indicating that three additional isolates (JP01, JP02 and JP05) were closely related to the four outbreak isolates. The MIRU-VNTR results did not show sufficient discrimination among the seven isolates ranging from 2004 to 2011. NGS and TGS-TB analyses were performed to identify the SNVs among those isolates. No unique SNVs were detected for the four outbreak isolates (JP03, JP04, JP06 and JP07), and the two past isolates (JP01 and JP02) with two SNVs difference could be one outbreak source or potential ancestors related to this outbreak. It has been reported that the estimated rate of change in DNA sequences was 0.3–0.5 single nucleotide polymorphisms per genome per year [9, 13, 17, 18]; thus, the two detected SNVs represent a reasonable nucleotide substitution rate between the outbreak (2010/2011) and past isolates (2004/2005). In contrast, JP05 was isolated at a close time point, with four outbreak isolates in December 2010, and three SNV sites were detected when compared with four outbreak isolates. One additional IS6110 insertion was detected in JP05 (Figs 4B and 5), strongly suggesting that JP05 did not appear to be involved in the outbreak. Such SNV networks facilitate the detection of epidemiological factors, and the obtained network is consistent with the field study (data not shown).

Bottom Line: An additional IS6110 insertion was detected in one of those isolates as supportive genetic information in addition to core genomic SNVs.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.

View Article: PubMed Central - PubMed

Affiliation: Pathogen Genomics Center, National Institute of Infectious Diseases, Shinjyuku-ku, Tokyo, Japan.

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/.

Show MeSH
Related in: MedlinePlus