Culture-independent detection and characterisation of Mycobacterium tuberculosis and M. africanum in sputum samples using shotgun metagenomics on a benchtop sequencer

Abstract

Tuberculosis remains a major global health problem. Laboratory diagnostic methods that allow effective, early detection of cases are central to management of tuberculosis in the individual patient and in the community. Since the 1880s, laboratory diagnosis of tuberculosis has relied primarily on microscopy and culture. However, microscopy fails to provide species- or lineage-level identification and culture-based workflows for diagnosis of tuberculosis remain complex, expensive, slow, technically demanding and poorly able to handle mixed infections. We therefore explored the potential of shotgun metagenomics, sequencing of DNA from samples without culture or target-specific amplification or capture, to detect and characterise strains from the Mycobacterium tuberculosis complex in smear-positive sputum samples obtained from The Gambia in West Africa. Eight smear- and culture-positive sputum samples were investigated using a differential-lysis protocol followed by a kit-based DNA extraction method, with sequencing performed on a benchtop sequencing instrument, the Illumina MiSeq. The number of sequence reads in each sputum-derived metagenome ranged from 989,442 to 2,818,238. The proportion of reads in each metagenome mapping against the human genome ranged from 20% to 99%. We were able to detect sequences from the M. tuberculosis complex in all eight samples, with coverage of the H37Rv reference genome ranging from 0.002X to 0.7X. By analysing the distribution of large sequence polymorphisms (deletions and the locations of the insertion element IS6110) and single nucleotide polymorphisms (SNPs), we were able to assign seven of eight metagenome-derived genomes to a species and lineage within the M. tuberculosis complex. Two metagenome-derived mycobacterial genomes were assigned to M. africanum, a species largely confined to West Africa; the others that could be assigned belonged to lineages T, H or LAM within the clade of “modern” M. tuberculosis strains. We have provided proof of principle that shotgun metagenomics can be used to detect and characterise M. tuberculosis sequences from sputum samples without culture or target-specific amplification or capture, using an accessible benchtop-sequencing platform, the Illumina MiSeq, and relatively simple DNA extraction, sequencing and bioinformatics protocols. In our hands, sputum metagenomics does not yet deliver sufficient depth of coverage to allow sequence-based sensitivity testing; it remains to be determined whether improvements in DNA extraction protocols alone can deliver this or whether culture, capture or amplification steps will be required. Nonetheless, we can foresee a tipping point when a unified automated metagenomics-based workflow might start to compete with the plethora of methods currently in use in the diagnostic microbiology laboratory.

Next-Generation Sequencing for Pathogen Detection and Identification

“…Despite very low coverage of the pathogen genome (20-99% of the resulting reads per sample were derived from human DNA), these methods allowed for rapid detection of the organism and assignment to lineages, with implications for treatment. By comparison, culture-based methods would have taken weeks to months and more rapid M. tuberculosis tests, though providing results within hours, do not yield the full range of information that NGS did (Doughty et al, 2014). One caveat is that for this study, only smear-positive samples were used as a proof of concept.…”

“…Often, metagenomic sequencing directly from clinical samples does not yield the depth of coverage necessary for accurate, useful SNP-based analyses of bacterial pathogens (Doughty et al, 2014;Frey et al, 2014). In order to maximize the likelihood of detecting a novel agent, it is critical to maximize the output of the metagenome sequencing, in terms of breadth and depth of coverage.…”

Diversity and disease pathogenesis in Mycobacterium tuberculosis

“…Although beyond the scope of the current review, increasing evidence of stochastic behavior [73] as well as potential for epigenetic modifications, such as DNA methylation, to alter bacillary physiology [74], further supports the application of novel sampling methods and sequencing technologies [75] to catalog the full diversity of physiological states in clinical and experimental TB infection. The recent use of shotgun metagenomics to detect and characterize M. tuberculosis in clinical samples [76] might herald the widespread application of 'culture-free' techniques to this …”

Evaluation of the efficacy of two methods for direct extraction of DNA from Mycobacterium tuberculosis sputum

“…WGS has shown superiority over other bacterial typing methods and can be used to monitor disease transmission [3]. The long culture period hinders use of WGS as a diagnostic tool for TB [4]. The ideal situation would be to efficiently sequence directly from clinical specimens such as sputum.…”