IS-seq: a novel high throughput survey of in vivo IS6110 transposition in multiple Mycobacterium tuberculosis genomes

Abstract

Background: The insertion element IS6110 is one of the main sources of genomic variability in Mycobacterium tuberculosis, the etiological agent of human tuberculosis. Although IS 6110 has been used extensively as an epidemiological marker, the identification of the precise chromosomal insertion sites has been limited by technical challenges. Here, we present IS-seq, a novel method that combines high-throughput sequencing using Illumina technology with efficient combinatorial sample multiplexing to simultaneously probe 519 clinical isolates, identifying almost all the flanking regions of the element in a single experiment.

Results: We identified a total of 6,976 IS6110 flanking regions on the different isolates. When validated using reference strains, the method had 100% specificity and 98% positive predictive value. The insertions mapped to both coding and non-coding regions, and in some cases interrupted genes thought to be essential for virulence or in vitro growth. Strains were classified into families using insertion sites, and high agreement with previous studies was observed.

Conclusions: This high-throughput IS-seq method, which can also be used to map insertions in other organisms, extends previous surveys of in vivo interrupted loci and provides a baseline for probing the consequences of disruptions in M. tuberculosis strains.

Figure 1

Overview of IS-seq Method. Genomic DNA (gDNA) is randomly sheared and ligated to adapters containing 24 different barcodes (BC, purple circles). Amplification from the IS 6110 ends is done in a first PCR (PCR1) with primers that anneal on the IS 6110 (blue) and the adapter (green), incorporating additional BC (yellow circles) and the PE-sequencing primer (red). A second PCR (PCR2) is done to incorporate amplification primers (orange and light blue). Sequencing is carried out using regular PE-primers in an Illumina-GA II.

Figure 2

Genomic Distribution of IS6110Elements. The M. tuberculosis H37Rv circular genome is shown with outermost circles representing genes located on the positive (red) and negative (green) strands. Inner circle shows insertions in non-coding (purple) and coding (yellow) regions, with peak sizes scaled to indicate number of unique insertions per 200 bp window. Black lines indicate 2 kb regions with significantly more insertions than expected by a uniform distribution, brown boxes are 50Kb regions with significantly more insertions and blue boxes indicate regions of 50Kb with significantly less insertions.

Figure 3

Classification ofM. tuberculosisStrains Based on IS6110Insertions. An UPGMA tree was generated from the distance matrix obtained using Variation of Information metric for 504 strains. Colors represent the main lineage 4 families present in the study, using Beijing strains (lineage 2) as an outgroup. Reference sequenced bacterial genomes are indicated by numbers on the side: 1- TB str Haarlem, 2- TB H37Rv, 3- TB H37Ra, 4- TB CDC-1551, 5-TB-C, 6- TB KZN_4207, 7- TB_KZN_R596, 8- TB_KZN_V2475, 9- TB_KZN_1435, 10- TB_KZN_605, 11- TB_F11, 12- TB GM_1503, 13- TB 98-R604, 14- TB T85, 15- TB 210, 16- TB 02_1987 (see Methods).