Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples

Abstract

Routine full characterization of Mycobacterium tuberculosis 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 M. tuberculosis 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). M. tuberculosis 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 Mycobacterium bovis 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 M. tuberculosis DNA in direct samples.

Determinación de la mutación S315T del gen katG en aislados resistentes a Isoniacida de Mycobacterium tuberculosis mediante PCR-RFLP

“…De igual manera, investigaciones dirigidas al estudio de la resistencia por secuenciamiento del genoma completo ayudarían en el futuro a dilucidar los mecanismos moleculares de resistencia a isoniacida u otros medicamentos en aislados de Mycobacterium tuberculosis circulantes en el Ecuador [45][46][47] . La PCR-RFLP resulta una alternativa confiable y rápida para el diagnóstico de la resistencia a isoniacida, mediante la detección de mutación S315T del gen katG, otros codones mutados del mismo gen y/o demás genes que producen resistencia, en comparación con el método convencional de las proporciones.…”

Peer Review #2 of "Rapid multi-locus sequence typing direct from uncorrected long reads using Krocus (v0.1)"

“…With rapidly falling costs, long read sequencing technologies, such as from Pacific Biosciences (PacBio) and OPford Nanopore Technologies (ONT), are beginning to be used for outbreak investigations (*aria et al 2017;Quick et al 2015) and for rapid clinical diagnostics (Votintseva et al 2017). Long read sequencers from OPford Nanopore can produce sequence reads in a matter of minutes and sequencers from PacBio can produce sequences in a number of hours compared to short read sequencing technologies which takes hours/days.…”

Real-Time Sequencing of Mycobacterium tuberculosis: Are We There Yet?

“…Such errors must be accounted for in the analysis, with only high-quality base calls retained. In the case of MinION, which Votintseva et al applied-for the first time ever reported-to Mycobacterium (14), the authors identified a systematic A-to-G error bias in 1D reads, which must be accounted for in the bioinformatic analysis.…”