Analysis of IS6110 insertion sites provide a glimpse into genome evolution of Mycobacterium tuberculosis

Abstract

Insertion sequence (IS) 6110 is found at multiple sites in the Mycobacterium tuberculosis genome and displays a high degree of polymorphism with respect to copy number and insertion sites. Therefore, IS6110 is considered to be a useful molecular marker for diagnosis and strain typing of M. tuberculosis. Generally IS6110 elements are identified using experimental methods, useful for analysis of a limited number of isolates. Since short read genome sequences generated using next-generation sequencing (NGS) platforms are available for a large number of isolates, a computational pipeline for identification of IS6110 elements from these datasets was developed. This study shows results from analysis of NGS data of 1377 M. tuberculosis isolates. These isolates represent all seven major global lineages of M. tuberculosis. Lineage specific copy number patterns and preferential insertion regions were observed. Intra-lineage differences were further analyzed for identifying spoligotype specific variations. Copy number distribution and preferential locations of IS6110 in different lineages imply independent evolution of IS6110, governed mainly through ancestral insertion, fitness (gene truncation, promoter activity) and recombinational loss of some copies. A phylogenetic tree based on IS6110 insertion data of different isolates was constructed in order to understand genome level variations of different markers across different lineages.

Exhaustive reconstruction of the CRISPR locus inMycobacterium tuberculosiscomplex using short reads

“…The third set of data comes from a collection of sequence reads archives (NCBI-SRA and EMBL-SRA) that has been retrieved from some state-of-the-art articles to represent the diversity of MTC lineages [20, 21]. This collection was completed by SRA queries on the NCBI search engine, with taxid values of 33894 and 78331, corresponding respectively to M. tuberculosis variant africanum and M. canettii organisms.…”

Unexpected diversity of CRISPR unveils some evolutionary patterns of repeated sequences inMycobacterium tuberculosis

“…This IS-mediated mechanism, that has been described in previous studies is a general mechanism, i.e. it happens independently of lineage and is the responsible of IS 6110 convergence of IS copy numbers (Roychowdhury, et al, 2015). The final result is the change from x to x-1 copies of IS 6110 , with the loss of all spacers between the two copies.…”

“…IS 6110 -RFLP was the golden standard to define epidemiological clusters at the end of the nineties and stayed so during around 20 years, until it was replaced by MIRU-VNTR 1 and more recently by Whole-Genome-Sequencing (Schurch, et al, 2010; Supply, et al, 2006; van Embden, et al, 1993; van Soolingen, et al, 2007) (for a recent review on evolution of TB molecular epidemiological methods, see also (Garcia De Viedma and Perez-Lago, 2018)). Previous results on IS 6110 insertion sites have shown that independent IS 6110 copy acquisition through transposition into hot-spots was a common mechanism explaining convergence in IS 6110 copy number in some of the MTBC sublineages (Dale, et al, 2003; Roychowdhury, et al, 2015). A recent paper on the micro- and macro-evolution of Lineage 2 of MTC in relation to IS 6110 transposition also stress the interest of such studies using WGS (Shitikov, et al, 2019).…”

Towards next generation diagnostics for tuberculosis: identification of novel molecular targets by large-scale comparative genomics

“…The insertion sequence IS6110 has been extensively used as a MTBC-specific marker since first described in 1990[8]. In addition, the IS6110 can be present in high copy numbers in some MTBC strains (from 0 to 27 copies)[9], causing the nucleic acid amplification tests (NAAT) targeting this sequence to achieve higher sensitivities for strains carrying several copies. However, the specificity of the IS6110 has been questioned since two decades ago[10–15] what, along with the fact that some strains lack this insertion sequence, can lead to an incorrect diagnosis[16,17].…”