Rapid Whole Genome Sequencing of M. tuberculosis directly from clinical samples. Direct WGS of MTB from Sputum

The rapid identification of antimicrobial resistance is essential for effective treatment of highly resistant Mycobacterium tuberculosis (M. tb). Whole genome sequencing provides comprehensive data on resistance mutations and strain typing for monitoring transmission, but unlike conventional molecular tests, this has only previously been achievable from cultured M. tb. Here we describe a method utilising biotinylated RNA baits, designed specifically for M. tb DNA to capture full M. tb genomes directly from infected sputum samples, allowing whole genome sequencing without the requirement of culture. This was carried out on 24 smear-positive sputum samples, collected from the UK and Lithuania where a matched culture sample was available, and two samplesthat had failed to grow in culture. M. tb sequencing data was obtained directly from all 24 smear-positive culture-positive sputa, of which 20 were high quality (>20x depth and >90% of genome covered). Results were compared with conventional molecular and culture-based methods, and high levels of concordance were observed between phenotypical resistance and predicted resistance based on genotype. High quality sequence data was obtained from one smear positive culture negative case. This study demonstrates for the first time, the successful and accurate sequencing of M. tb genomes directly from uncultured sputa. Identification of known resistance mutations within a week of sample receipt offers the prospect for personalised, rather than empirical, treatment of drug resistant tuberculosis, including the use of antimicrobial-sparing regimens, leading to improved outcomes.