Open Access Research article

Single nucleotide polymorphism (SNP) analysis used for the phylogeny of the Mycobacterium tuberculosis complex based on a pyrosequencing assay

Adriana Cabal1, Mark Strunk2, José Domínguez345, María Antonia Lezcano15, María Asunción Vitoria56, Miguel Ferrero7, Carlos Martín158, María José Iglesias58 and Sofía Samper11059*

Author Affiliations

1 IIS Aragón, Hopsital Universitario Miguel Servet, Zaragoza, Spain

2 IIS Aragón, CIBER de Enfermedades Hepáticas y Digestivas, Zaragoza, Spain

3 Institut d’Investigació Germans Trias i Pujol, Badalona, Spain

4 Universitat Autònoma de Barcelona, Barcelona, Spain

5 CIBER de Enfermedades Respiratorias, Madrid, Spain

6 Hospital Universitario Lozano Blesa, CIBER de Enfermedades Respiratorias, Zaragoza, Spain

7 Hospital San Jorge, Huesca, Spain

8 Universidad de Zaragoza, CIBER de Enfermedades Respiratorias, Zaragoza, Spain

9 Instituto Aragonés de Ciencias de la Salud, Zaragoza, Spain

10 Hospital Miguel Servet – IIS Aragón, Laboratorio de Investigación Molecular, P. Isabel la Catolica 1-3, Zaragoza 50009, Spain

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BMC Microbiology 2014, 14:21  doi:10.1186/1471-2180-14-21

Published: 3 February 2014



Different polymorphisms have been described as markers to classify the lineages of the Mycobacterium tuberculosis complex. The analysis of nine single nucleotide polymorphisms (SNPs) was used to describe seven SNPs cluster groups (SCGs). We attempted to classify those strains that could not been categorized into lineages by the genotyping methods used in the routine testing.


The M. tuberculosis complex isolates collected in 2010 in our region were analysed. A new method based on multiplex-PCRs and pyrosequencing to analyse these SNPs was designed. For the pyrosequencing assay nine SNPs that defined the seven SCGs were selected from the literature: 1977, 74092, 105139, 232574, 311613, 913274, 2460626, 3352929 and gyrA95. In addition, SNPs in katG463, mgtC182, Ag85C103 and RDRio deletion were detected.


This work has permitted to achieve a better classification of Aragonian strains into SCGs and in some cases, to assign strains to its certain lineage. Besides, the description of a new pattern shared by two isolates “SCG-6c” reinforces the interest of SNPs to follow the evolution of M. tuberculosis complex.

M. tuberculosis; SNP; Pyrosequencing; SCG; Lineages; Cluster