Genomic Sequencing from Sputum for Tuberculosis Disease Diagnosis, Lineage Determination and Drug Susceptibility Prediction

BackgroundUniversal access to drug susceptibility testing for newly diagnosed tuberculosis patients is recommended. Access to culture-based diagnostics remains limited and targeted molecular assays are vulnerable to emerging resistance conferring mutations. Improved sample preparation protocols for direct-from-sputum sequencing of Mycobacterium tuberculosis would accelerate access to comprehensive drug susceptibility testing and molecular typing. MethodsWe assessed a thermo-protection buffer-based direct-from-sample M. tuberculosis whole-genome sequencing protocol. We prospectively processed and analyzed 60 acid-fast bacilli smear-positive sputum samples from tuberculosis patients in India and Madagascar. A diversity of semi-quantitative smear positivity level samples were included. Sequencing was performed using Illumina and MinION (monoplex and multiplex) technologies. We measured the impact of bacterial inoculum and sequencing platforms on M. tuberculosis genomic mean read depth, drug susceptibility prediction performance and typing accuracy. ResultsM. tuberculosis was identified from 88% (Illumina), 89% (MinION-monoplex) and 83% (MinION-multiplex) of samples for which sufficient DNA could be extracted. The fraction of M. tuberculosis reads from MinION sequencing was lower than from Illumina, but monoplexing grade 3+ sputum samples on MinION produced higher read depth than Illumina (p<0.05) and MinION multiplex (p<0.01). No significant difference in overall sensitivity and specificity of drug susceptibility predictions was seen across these sequencing modalities or within each sequencing technology when stratified by smear grade. Lineage typing agreement percentages between direct and culture-based sequencing were 85% (MinION-monoplex), 88% (Illumina) and 100% (MinION-multiplex) ConclusionsM. tuberculosis direct-from-sample whole-genome sequencing remains challenging. Improved and affordable sample treatment protocols are needed prior to clinical deployment.