Genetic diversity within diagnostic sputum samples is mirrored in the culture of Mycobacterium tuberculosis

Culturing Mycobacterium tuberculosis (MTB) from tuberculosis cases is the basis for many research and clinical applications. Paradoxically, it is assumed to impose a diversity bottleneck, which, if true, would entail unexplored consequences. The alternative, culture-free sequencing from diagnostic samples, is a promising but challenging approach both to obtain and analyse the MTB genome from the complex sample. This study obtains high-quality genomes of sputum-culture pairs from two different settings after developing a workflow for sequencing from sputum and a tailored bioinformatics pipeline. Our approach reveals that 88% of variants called in culture-free sequencing analysis are false positives due to supplementary alignments, mostly in enriched-sputa samples. Overall, contrary to the bottleneck dogma, we identify a 97% variant agreement within sputum-culture pairs, with a high correlation also in the variants frequency (0.98). Our findings extrapolate to all publicly available data, thus demonstrating that in most cases culture accurately mirrors clinical samples.