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Comparative whole-genome analysis of clinical isolates reveals characteristic architecture of Mycobacterium tuberculosis pangenome.

Periwal V, Patowary A, Vellarikkal SK, Gupta A, Singh M, Mittal A, Jeyapaul S, Chauhan RK, Singh AV, Singh PK, Garg P, Katoch VM, Katoch K, Chauhan DS, Sivasubbu S, Scaria V - PLoS ONE (2015)

Bottom Line: We identified 74 HGCs that were absent from reference strains H37Rv and H37Ra but were present in most of clinical isolates.The pangenome approach is a promising tool for studying strain specific genetic differences occurring within species.We also suggest that since selecting appropriate target genes for typing purposes requires the expected target gene be present in all isolates being typed, therefore estimating the core-component of the species becomes a subject of prime importance.

View Article: PubMed Central - PubMed

Affiliation: GN Ramachandran Knowledge Center for Genome Informatics, CSIR Institute of Genomics and Integrative Biology (CSIR-IGIB), Mall Road, Delhi-110007, India; Academy of Scientific & Innovative Research (AcSIR), 2, Rafi Marg, Anusandhan Bhawan, New Delhi 110001, India.

ABSTRACT
The tubercle complex consists of closely related mycobacterium species which appear to be variants of a single species. Comparative genome analysis of different strains could provide useful clues and insights into the genetic diversity of the species. We integrated genome assemblies of 96 strains from Mycobacterium tuberculosis complex (MTBC), which included 8 Indian clinical isolates sequenced and assembled in this study, to understand its pangenome architecture. We predicted genes for all the 96 strains and clustered their respective CDSs into homologous gene clusters (HGCs) to reveal a hard-core, soft-core and accessory genome component of MTBC. The hard-core (HGCs shared amongst 100% of the strains) was comprised of 2,066 gene clusters whereas the soft-core (HGCs shared amongst at least 95% of the strains) comprised of 3,374 gene clusters. The change in the core and accessory genome components when observed as a function of their size revealed that MTBC has an open pangenome. We identified 74 HGCs that were absent from reference strains H37Rv and H37Ra but were present in most of clinical isolates. We report PCR validation on 9 candidate genes depicting 7 genes completely absent from H37Rv and H37Ra whereas 2 genes shared partial homology with them accounting to probable insertion and deletion events. The pangenome approach is a promising tool for studying strain specific genetic differences occurring within species. We also suggest that since selecting appropriate target genes for typing purposes requires the expected target gene be present in all isolates being typed, therefore estimating the core-component of the species becomes a subject of prime importance.

No MeSH data available.


Related in: MedlinePlus

(A) PCR validation of the 10 candidate genes (subset of 74 clusters identified) in 8 Indian clinical isolates (OSDD strains) and reference strains ATCC H37Ra and ATCC H37Rv.Arrow heads indicate the variably sized products in ATCC H37Ra and ATCC H37Rv genomes. Gene 4 and Gene 8 shares partial homology with laboratory strains and showed the presence of a differently sized product in ATCC H37Rv and ATCC H37Ra. (B) The alignment of Gene 4 against reference genomes showed an insertion of 175bp sequence in Gene 4 of OSDD strains with respect to reference strains. (C) The alignment of Gene 8 against reference genomes showed a deletion of 1,352bp sequence in Gene 8 of OSDD strains.
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pone.0122979.g008: (A) PCR validation of the 10 candidate genes (subset of 74 clusters identified) in 8 Indian clinical isolates (OSDD strains) and reference strains ATCC H37Ra and ATCC H37Rv.Arrow heads indicate the variably sized products in ATCC H37Ra and ATCC H37Rv genomes. Gene 4 and Gene 8 shares partial homology with laboratory strains and showed the presence of a differently sized product in ATCC H37Rv and ATCC H37Ra. (B) The alignment of Gene 4 against reference genomes showed an insertion of 175bp sequence in Gene 4 of OSDD strains with respect to reference strains. (C) The alignment of Gene 8 against reference genomes showed a deletion of 1,352bp sequence in Gene 8 of OSDD strains.

Mentions: We randomly picked 9 candidate HGCs sequences (Fig 7, genes 1–9) from the 74 clusters which were present in most of the clinical isolates and employed a classical PCR approach to experimentally validate these putative genic loci. PCR validation was performed on genomic DNA of the eight Indian OSDD strains sequenced in this study and laboratory strains Mtb ATCC H37Ra and Mtb ATCC H37Rv procured from ATCC. We designed primers for the 8 available OSDD strains using primer BLAST [37]. A detailed account of the forward and reverse primers used for the validation and their expected product sizes are summarized in S2 Table. All primers provided amplification of genomic DNA in expected sizes, substantiating and validating the presence of these genes in the strains under consideration. The PCR result for these 9 gene sequences are shown in Fig 8. Out of the 9 genes which were PCR validated, 7 genes were absolutely absent from H37Rv and H37Ra whereas 2 genes (Gene 4 and gene 8) displayed appearance of differentially sized gene products (these 2 genes shared partial homology with H37Ra and H37Rv).


Comparative whole-genome analysis of clinical isolates reveals characteristic architecture of Mycobacterium tuberculosis pangenome.

Periwal V, Patowary A, Vellarikkal SK, Gupta A, Singh M, Mittal A, Jeyapaul S, Chauhan RK, Singh AV, Singh PK, Garg P, Katoch VM, Katoch K, Chauhan DS, Sivasubbu S, Scaria V - PLoS ONE (2015)

(A) PCR validation of the 10 candidate genes (subset of 74 clusters identified) in 8 Indian clinical isolates (OSDD strains) and reference strains ATCC H37Ra and ATCC H37Rv.Arrow heads indicate the variably sized products in ATCC H37Ra and ATCC H37Rv genomes. Gene 4 and Gene 8 shares partial homology with laboratory strains and showed the presence of a differently sized product in ATCC H37Rv and ATCC H37Ra. (B) The alignment of Gene 4 against reference genomes showed an insertion of 175bp sequence in Gene 4 of OSDD strains with respect to reference strains. (C) The alignment of Gene 8 against reference genomes showed a deletion of 1,352bp sequence in Gene 8 of OSDD strains.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390332&req=5

pone.0122979.g008: (A) PCR validation of the 10 candidate genes (subset of 74 clusters identified) in 8 Indian clinical isolates (OSDD strains) and reference strains ATCC H37Ra and ATCC H37Rv.Arrow heads indicate the variably sized products in ATCC H37Ra and ATCC H37Rv genomes. Gene 4 and Gene 8 shares partial homology with laboratory strains and showed the presence of a differently sized product in ATCC H37Rv and ATCC H37Ra. (B) The alignment of Gene 4 against reference genomes showed an insertion of 175bp sequence in Gene 4 of OSDD strains with respect to reference strains. (C) The alignment of Gene 8 against reference genomes showed a deletion of 1,352bp sequence in Gene 8 of OSDD strains.
Mentions: We randomly picked 9 candidate HGCs sequences (Fig 7, genes 1–9) from the 74 clusters which were present in most of the clinical isolates and employed a classical PCR approach to experimentally validate these putative genic loci. PCR validation was performed on genomic DNA of the eight Indian OSDD strains sequenced in this study and laboratory strains Mtb ATCC H37Ra and Mtb ATCC H37Rv procured from ATCC. We designed primers for the 8 available OSDD strains using primer BLAST [37]. A detailed account of the forward and reverse primers used for the validation and their expected product sizes are summarized in S2 Table. All primers provided amplification of genomic DNA in expected sizes, substantiating and validating the presence of these genes in the strains under consideration. The PCR result for these 9 gene sequences are shown in Fig 8. Out of the 9 genes which were PCR validated, 7 genes were absolutely absent from H37Rv and H37Ra whereas 2 genes (Gene 4 and gene 8) displayed appearance of differentially sized gene products (these 2 genes shared partial homology with H37Ra and H37Rv).

Bottom Line: We identified 74 HGCs that were absent from reference strains H37Rv and H37Ra but were present in most of clinical isolates.The pangenome approach is a promising tool for studying strain specific genetic differences occurring within species.We also suggest that since selecting appropriate target genes for typing purposes requires the expected target gene be present in all isolates being typed, therefore estimating the core-component of the species becomes a subject of prime importance.

View Article: PubMed Central - PubMed

Affiliation: GN Ramachandran Knowledge Center for Genome Informatics, CSIR Institute of Genomics and Integrative Biology (CSIR-IGIB), Mall Road, Delhi-110007, India; Academy of Scientific & Innovative Research (AcSIR), 2, Rafi Marg, Anusandhan Bhawan, New Delhi 110001, India.

ABSTRACT
The tubercle complex consists of closely related mycobacterium species which appear to be variants of a single species. Comparative genome analysis of different strains could provide useful clues and insights into the genetic diversity of the species. We integrated genome assemblies of 96 strains from Mycobacterium tuberculosis complex (MTBC), which included 8 Indian clinical isolates sequenced and assembled in this study, to understand its pangenome architecture. We predicted genes for all the 96 strains and clustered their respective CDSs into homologous gene clusters (HGCs) to reveal a hard-core, soft-core and accessory genome component of MTBC. The hard-core (HGCs shared amongst 100% of the strains) was comprised of 2,066 gene clusters whereas the soft-core (HGCs shared amongst at least 95% of the strains) comprised of 3,374 gene clusters. The change in the core and accessory genome components when observed as a function of their size revealed that MTBC has an open pangenome. We identified 74 HGCs that were absent from reference strains H37Rv and H37Ra but were present in most of clinical isolates. We report PCR validation on 9 candidate genes depicting 7 genes completely absent from H37Rv and H37Ra whereas 2 genes shared partial homology with them accounting to probable insertion and deletion events. The pangenome approach is a promising tool for studying strain specific genetic differences occurring within species. We also suggest that since selecting appropriate target genes for typing purposes requires the expected target gene be present in all isolates being typed, therefore estimating the core-component of the species becomes a subject of prime importance.

No MeSH data available.


Related in: MedlinePlus