Whole genome sequencing <i>Mycobacterium tuberculosis</i> directly from sputum identifies more genetic diversity than sequencing from culture

<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>Repeated culture reduces within-sample <jats:italic>Mycobacterium tuberculosis</jats:italic> genetic diversity due to selection of clones suited to growth in culture and/or random loss of lineages, but it is not known to what extent omitting the culture step altogether alters genetic diversity. We compared <jats:italic>M. tuberculosis</jats:italic> whole genome sequences generated from 33 paired clinical samples using two methods. In one method DNA was extracted directly from sputum then enriched with custom-designed SureSelect (Agilent) oligonucleotide baits and in the other it was extracted from mycobacterial growth indicator tube (MGIT) culture.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>DNA directly sequenced from sputum showed significantly more within-sample diversity than that from MGIT culture (median 5.0 vs 4.5 heterozygous alleles per sample, p=0.04). Resistance associated variants present as HAs occurred in four patients, and in two cases may provide a genotypic explanation for phenotypic resistance.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>Culture-free <jats:italic>M. tuberculosis</jats:italic> whole genome sequencing detects more within-sample diversity than a leading culture-based method and may allow detection of mycobacteria that are not actively replicating.</jats:p></jats:sec>