• Rebecca E Colman, James M Schupp, Nathan D Hicks, David E Smith, Jordan L Buchhagen, Faramarz Valafar, Valeriu Crudu, Elena Romancenco, Ecaterina Noroc, Lynn Jackson, Donald G Catanzaro, Timothy C Rodwell, Antonino Catanzaro, Paul Keim, and David M Engelthaler.
• Translational Genomics Research Institute, Flagstaff, AZ, United States of America.
• Plos One. 2015 Jan 1; 10 (5): e0126626.

AbstractUndetected and untreated, low-levels of drug resistant (DR) subpopulations in clinical Mycobacterium tuberculosis (Mtb) infections may lead to development of DR-tuberculosis, potentially resulting in treatment failure. Current phenotypic DR susceptibility testing has a theoretical potential for 1% sensitivity, is not quantitative, and requires several weeks to complete. The use of "single molecule-overlapping reads" (SMOR) analysis with next generation DNA sequencing for determination of ultra-rare target alleles in complex mixtures provides increased sensitivity over standard DNA sequencing. Ligation free amplicon sequencing with SMOR analysis enables the detection of resistant allele subpopulations at ≥0.1% of the total Mtb population in near real-time analysis. We describe the method using standardized mixtures of DNA from resistant and susceptible Mtb isolates and the assay's performance for detecting ultra-rare DR subpopulations in DNA extracted directly from clinical sputum samples. SMOR analysis enables rapid near real-time detection and tracking of previously undetectable DR sub-populations in clinical samples allowing for the evaluation of the clinical relevance of low-level DR subpopulations. This will provide insights into interventions aimed at suppressing minor DR subpopulations before they become clinically significant.

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