Whole genome sequencing Mycobacterium tuberculosis directly from sputum identifies more genetic diversity than sequencing from culture

Background: Repeated culture reduces within-sample Mycobacterium tuberculosis 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 M. tuberculosis whole genome sequences generated from 39 paired clinical samples. In one sample 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 culture. Results: DNA directly sequenced from sputum showed significantly more within-sample diversity than that from mycobacterial growth incubator tube culture. This was demonstrated by more variants present as heterozygous alleles (HAs) where both a variant and wild type allele were present within a given sample (p<0.001) and greater within-sample Shannon diversity (p<0.001). Seven genes with high within-sample diversity have previously been identified as targets for positive selection, highlighting their potential role in adaptation to survival within the host and under drug pressure. Resistance associated variants present as HAs occurred in six patients, and in four cases may have provided a genotypic explanation for phenotypic resistance. Conclusions: Culture-free M. tuberculosis whole genome sequencing detects more within-sample diversity and may allow detection of mycobacteria that are not actively replicating.