Detection of Heteroresistance in Mycobacterium tuberculosis Using Nanopore-Based Amplicon Sequencing and Adaptive Sampling

Heteroresistance in Mycobacterium tuberculosis, which is the coexistence of drug-susceptible and drug-resistant subpopulations within the same host, poses a major obstacle to effective tuberculosis (TB) treatment and control. In this study, we optimized and compared two Nanopore sequencing-based enrichment strategies (amplicon sequencing and adaptive sampling) for heteroresistance detection in both clinical samples and synthetic model mixtures. We evaluated the performance of both approaches on cultured Mycobacterium tuberculosis isolates and direct sputum specimens, and assessed their detection limits using spiked mixtures of drug-resistant and drug-susceptible strains. Our results demonstrate that both enrichment strategies significantly increased sequencing depth at resistance-associated loci, enabling reliable detection of resistant variants present at approximately 1-5% frequency within complex sample matrices. These findings underscore the potential of nanopore sequencing as a scalable, sensitive, and portable tool for heteroresistance detection and for guiding individualized TB therapy, with important implications for antimicrobial stewardship and public health surveillance.