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<jats:title>ABSTRACT</jats:title> <jats:p> Routine full characterization of <jats:named-content content-type="genus-species">Mycobacterium tuberculosis</jats:named-content> 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 the point of care. We demonstrate a low-cost method of DNA extraction directly from patient samples for <jats:named-content content-type="genus-species">M. tuberculosis</jats:named-content> WGS. We initially evaluated the method by using the Illumina MiSeq sequencer (40 smear-positive respiratory samples obtained after routine clinical testing and 27 matched liquid cultures). <jats:named-content content-type="genus-species">M. tuberculosis</jats:named-content> was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction were obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. With Illumina MiSeq/MiniSeq, the workflow from patient sample to results can be completed in 44/16 h at a reagent cost of £96/£198 per sample. We then employed a nonspecific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured <jats:named-content content-type="genus-species">Mycobacterium bovis</jats:named-content> strain BCG DNA and to combined culture-negative sputum DNA and BCG DNA. For flow cell version R9.4, the estimated turnaround time from patient to identification of BCG, detection of pyrazinamide resistance, and phylogenetic placement was 7.5 h, with full susceptibility results 5 h later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of <jats:named-content content-type="genus-species">M. tuberculosis</jats:named-content> DNA in direct samples. </jats:p>

Original publication

DOI

10.1128/jcm.02483-16

Type

Journal article

Journal

Journal of Clinical Microbiology

Publisher

American Society for Microbiology

Publication Date

05/2017

Volume

55

Pages

1285 - 1298