Open Access Highly Accessed Research article

Genetic heterogeneity revealed by sequence analysis of Mycobacterium tuberculosis isolates from extra-pulmonary tuberculosis patients

Sarbashis Das1, Tanmoy Roychowdhury1, Parameet Kumar2, Anil Kumar2, Priya Kalra2, Jitendra Singh3, Sarman Singh3, HK Prasad2* and Alok Bhattacharya14*

Author Affiliations

1 School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi, India

2 Department of Biotechnology, All India Institute of Medical Sciences, New Delhi, India

3 Division of Clinical Microbiology and Molecular Medicine, Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India

4 School of Life Sciences, Jawaharlal Nehru University, New Delhi, India

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BMC Genomics 2013, 14:404  doi:10.1186/1471-2164-14-404

Published: 17 June 2013

Abstract

Background

Tuberculosis remains a major public health problem. Clinical tuberculosis manifests often as pulmonary and occasionally as extra-pulmonary tuberculosis. The emergence of drug resistant tubercle bacilli and its association with HIV is a formidable challenge to curb the spread of tuberculosis. There have been concerted efforts by whole genome sequencing and bioinformatics analysis to identify genomic patterns and to establish a relationship between the genotype of the organism and clinical manifestation of tuberculosis. Extra-pulmonary TB constitutes 15–20 percent of the total clinical cases of tuberculosis reported among immunocompetent patients, whereas among HIV patients the incidence is more than 50 percent. Genomic analysis of M. tuberculosis isolates from extra pulmonary patients has not been explored.

Results

The genomic DNA of 5 extra-pulmonary clinical isolates of M. tuberculosis derived from cerebrospinal fluid, lymph node fine needle aspirates (FNAC) / biopsies, were sequenced. Next generation sequencing approach (NGS) was employed to identify Single Nucleotide Variations (SNVs) and computational methods used to predict their consequence on functional genes. Analysis of distribution of SNVs led to the finding that there are mixed genotypes in patient isolates and that many SNVs are likely to influence either gene function or their expression. Phylogenetic relationship between the isolates correlated with the origin of the isolates. In addition, insertion sites of IS elements were identified and their distribution revealed a variation in number and position of the element in the 5 extra-pulmonary isolates compared to the reference M. tuberculosis H37Rv strain.

Conclusions

The results suggest that NGS sequencing is able to identify small variations in genomes of M. tuberculosis isolates including changes in IS element insertion sites. Moreover, variations in isolates of M. tuberculosis from non-pulmonary sites were documented. The analysis of our results indicates genomic heterogeneity in the clinical isolates.

Keywords:
Extra-pulmonary Tuberculosis; Next-generation Sequencing; Genetic Heterogeneity; Single Nucleotide Variations; Insertion Elements; Phylogeny; Spoligotyping