AbstractThe emergence of Mycobacterium tuberculosis strains with complex drug resistance profiles necessitates a rapid and extensive drug susceptibility test for comprehensive guidance of patient treatment. Here, we developed two targeted-sequencing workflows based on Illumina MiSeq and Nanopore MinION for the prediction of drug resistance in M. tuberculosis towards 12 anti-tuberculous agents.A total of 163 M. tuberculosis cultured isolates collected from Hong Kong and Ethiopia were subjected to a multiplex PCR for simultaneous amplification of 19 drug-resistance associated genetic regions. The amplicons were then barcoded and sequenced in parallel on MiSeq and MinION in respective batch sizes of 24 and 12 samples. Both platforms successfully sequenced all samples with average depths of coverage of 1,127× and 1,649× respectively. Utilizing a self-developed Web-based bioinformatics pipeline, Bacteriochek-TB, for variant analysis, we found that the MiSeq and MinION result could achieve 100% agreement if variants with an allele frequency of <40% reported by MinION were excluded. For drug resistance prediction, both workflows achieved an average sensitivity of 94.8% and specificity of 98.0% when compared with phenotypic drug susceptibility test. The turnaround times for the MiSeq and MinION workflows were 38 and 15 hours, facilitating the delivery of treatment guidance at least 17-18 days earlier than pDST respectively. The higher cost per sample on the MinION platform (US$71.56) versus the MiSeq platform (US$67.83) was attributed to differences in batching capabilities.Our study demonstrated the interchangeability of MiSeq and MinION sequencing workflows for generation of accurate and actionable results for the treatment of tuberculosis.ImportanceTB therapy involving different combinations of antibiotics have been introduced to address the issue of drug resistance. However, this practice has led to increasing numbers of M. tuberculosis with complex drug resistance profiles. Molecular assays for rapid and comprehensive drug resistance profiling of M. tuberculosis are lacking.Here, we described targeted-sequencing workflows based on Illumina MiSeq and Nanopore MinION for the detection of drug resistance mutations scattered across 19 genetic regions in M. tuberculosis. A bioinformatics pipeline was also developed to translate raw datasets into clinician-friendly reports that provide comprehensive genetic information for the prediction of drug resistance towards 12 antibiotics.This is the first study to evaluate and compare the uses of Illumina and Nanopore platforms for diagnosis of drug-resistant tuberculosis. Remarkably, our diagnostic strategy is compatible with different sequencing platforms that can be applied in diagnostic centres with different levels of throughput and financial support for TB diagnosis.
Early Detection of Drug Susceptibility Test for Mycobacterium Tuberculosis by Slide DST Method Using Two Media- Middle Brook 7H9 Broth and Human Blood Media
