Independent large scale duplications in multiple M. tuberculosis lineages overlapping the same genomic region

Mycobacterium tuberculosis, the causative agent of most human tuberculosis, infects one third of the world's population and kills an estimated 1.7 million people a year. With the world-wide emergence of drug resistance, and the finding of more functional genetic diversity than previously expected, there is a renewed interest in understanding the forces driving genome evolution of this important pathogen. Genetic diversity in M. tuberculosis is dominated by single nucleotide polymorphisms and small scale gene deletion, with little or no evidence for large scale genome rearrangements seen in other bacteria. Recently, a single report described a large scale genome duplication that was suggested to be specific to the Beijing lineage. We report here multiple independent large-scale duplications of the same genomic region of M. tuberculosis detected through whole-genome sequencing. The duplications occur in strains belonging to both M. tuberculosis lineage 2 and 4, and are thus not limited to Beijing strains. The duplications occur in both drug-resistant and drug susceptible strains. The duplicated regions also have substantially different boundaries in different strains, indicating different originating duplication events. We further identify a smaller segmental duplication of a different genomic region of a lab strain of H37Rv. The presence of multiple independent duplications of the same genomic region suggests either instability in this region, a selective advantage conferred by the duplication, or both. The identified duplications suggest that large-scale gene duplication may be more common in M. tuberculosis than previously considere

Primer locations for PCR Confirmation.

<p>A, B, C, and D are as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026038#pone-0026038-g003" target="_blank">Figure 3</a> and also described in text.</p

Summary of Large Scale Duplication Boundaries.

<p>*Numbers in parenthesis are nucleotide positions for the duplication boundaries derived from PCR amplification.</p>§<p>Genes in square brackets correspond to the partial duplication boundaries for T67 (L2 and R2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026038#pone-0026038-g003" target="_blank">Figure 3</a>) - see text for details.</p

Strains Displaying Duplications.

<p>Inh: Isoniazid,; Rif: Rifampin; Str: Streptomycin; Kan: Kanamycin; Eth: Ethionamide; Oflox: Ofloxaxcin; Tha: Tacrine; Emb: Ethambutol; Pza: Pyrazinimide; DS: Drug susceptible.</p

Schematic of duplication boundaries and junctions for M41.

<p>Genes within the tandem duplications are color coded. Genes in yellow are present in the upstream duplication copy, while genes in blue are present in the downstream copy.</p

PCR verification of tandem duplications.

<p>(A) Schematic representation of primer design strategy. The duplicated region is shown as a grey box, present as a single copy in the reference genome (top) and as a tandem duplication below. Primers A and B amplify the left flank and C and D amplify the right flank. Only when C and B and brought into close proximity by a tandem duplication can a product be generated using primers C and B. (B) 0.9% agarose gel loaded with 5 ul of PCR reactions using primers C and B specific for isolate M141 (top), M41 (middle) and cdc606 (bottom). Lane 1 contains the 2 log ladder (New England Biolabs, N3200). (C) 0.9% agarose gel loaded with 5 ul of PCR reactions using primers A and B (lanes 2–5) or C and D (lanes 7–10) specific for each isolate as indicated on the left. Lanes 1 and 6 contain the 2 log ladder (New England Biolabs, N3200).</p

Schematic of duplication boundaries and junctions for CDC606.

<p>Color coding is identical to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026038#pone-0026038-g005" target="_blank">Figure 5</a> with the exception that genes present in strain CDC606 but missing in the reference genome sequence for H37Rv are colored in red.</p

Coverage plots around duplication boundaries.

<p>Each plot shows the fold coverage for the alignment of the corresponding strain to H37Rv around the upstream (left column) or downstream (right column) duplication boundaries. Color coding for coverage plots is as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026038#pone-0026038-g002" target="_blank">Figure 2</a>. Genes in red are predicted repeats or transposable elements in H37Rv. Vertical green bars indicate the positions of PCR primers used (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026038#pone-0026038-t003" target="_blank">Table 3</a>).</p

Genes Involved in Nucleotide Metabolism and DNA repair enriched in the intersection of all duplicated regions.

<p>Genes Involved in Nucleotide Metabolism and DNA repair enriched in the intersection of all duplicated regions.</p