Votintseva, Antonina A., Bradley, Phelim, Pankhurst, Louise, Elias, Carlos del Ojo, Loose, Matthew, Nilgiriwala, Kayzad, Chatterjee, Anirvan, Smith, E. Grace, Sanderson, Nicolas, Walker, Timothy M., Morgan, Marcus R., Wyllie, David H., Walker, A. Sarah, Peto, Tim E.A., Crook, Derrick W. and Iqbal, Zamin
(2017)
Same-day diagnostic and surveillance data for tuberculosis via whole genome sequencing of direct respiratory samples.
Journal of Clinical Microbiology, 55
(5).
pp. 1285-1298.
ISSN 1098-660X
Full text not available from this repository.
Abstract
Routine full characterization of Mycobacterium tuberculosis (TB) is culture-based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near point of care.
We demonstrate a low-cost DNA extraction method for TB WGS direct from patient samples. We initially evaluated the method using the Illumina MiSeq sequencer (40 smear-positive respiratory samples, obtained after routine clinical testing, and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction was obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. Using an 70 Illumina MiSeq/MiniSeq the workflow from patient sample to results can be completed in 44/16 hours at a reagent cost of £96/£198 per sample.
We then employed a non-specific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis BCG strain (BCG), and to combined culture negative sputum DNA and BCG DNA. For flowcell version R9.4, the estimated turnaround time from patient to identification of BCG, detection of pyrazinamide resistance, and phylogenetic placement was 7.5 hours, with full susceptibility results 5 hours later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of the MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis in direct samples.
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