Whole genome sequencing reveals genetic heterogeneity and suggests the role of selective bottleneck in defining the population structure of Mycobacterium tuberculosis clinical isolates.. Heterogeneity

Background: Whole genome sequencing has revolutionised the interrogation of mycobacterial genomes. Recent studies have reported conflicting findings on the genomic stability of M. tuberculosis during the evolution of drug resistance. In an age where whole genome sequencing is increasingly relied upon for defining the structure of bacterial genomes, it is important to investigate the reliability of next-generation sequencing data where a variant is only present in a minor percentage of the sequencing reads. This study aimed to define a reliable cut-off for identification of low frequency sequence variants and to subsequently investigate genetic heterogeneity and the evolution of drug resistance in M. tuberculosis, where heterogeneity implies the presence of both wild type and mutant bases. Results: Using next-generation sequencing in combination with Sanger sequencing and statistical analysis we defined a cut off of 30% variant frequency for reliable Illumina sequencing read frequency, in samples with an average coverage of 137. Using this cut-off we demonstrated a high rate of genetic diversity between single colonies isolated from one population, showing that by using the current sequencing technology, single colonies are not a true reflection of the diversity within a whole population and vice versa. We further showed that numerous heterogeneous variants emerge and disappear during the evolution of isoniazid resistance within individual patients. Our findings allowed us to formulate a model for the selective bottleneck which occurs during the course of infection, acting as a genomic purification event. Conclusions: Our study demonstrated true levels of genetic diversity within an M. tuberculosis population and showed that genetic diversity may be re-defined when a selective pressure, such as drug exposure, is imposed on M. tuberculosis populations during the course of infection.