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. 2019 May 20;20(1):389.
doi: 10.1186/s12864-019-5782-2.

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

Camus Nimmo  1   2 , Liam P Shaw  3   4 , Ronan Doyle  5   6 , Rachel Williams  5 , Kayleen Brien  7 , Carrie Burgess  5 , Judith Breuer  5 , Francois Balloux  3 , Alexander S Pym  7
Affiliations

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

Camus Nimmo et al. BMC Genomics. .

Erratum in

Abstract

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 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.

Results: 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.

Conclusions: Culture-free M. tuberculosis 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.

Keywords: Drug-resistant tuberculosis; Heteroresistance; Mycobacterium tuberculosis; Sputum; Whole genome sequencing; Within-patient diversity.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Variation in total number of heterozygous alleles (HAs) identified across all 33 patients in sequences generated from sputum and MGIT depending on minimum supporting read count threshold
Fig. 2
Fig. 2
Number of heterozygous alleles (HAs) found in directly sequenced sputum only (sputum), MGIT (MGIT) only or in both samples (shared) by patient
See this image and copyright information in PMC

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