Robust detection of point mutations involved in multidrug-resistant Mycobacterium tuberculosis in the presence of co-occurrent resistance markers

Tuberculosis disease is a major global public health concern and the growing prevalence of drug-resistant Mycobacterium tuberculosis is making disease control more difficult. However, the increasing application of whole-genome sequencing as a diagnostic tool is leading to the profiling of drug resistance to inform clinical practice and treatment decision making. Computational approaches for identifying established and novel resistance-conferring mutations in genomic data include genome-wide association study (GWAS) methodologies, tests for convergent evolution and machine learning techniques. These methods may be confounded by extensive co-occurrent resistance, where statistical models for a drug include unrelated mutations known to be causing resistance to other drugs. Here, we introduce a novel 'cannibalistic' elimination algorithm (Hungry, Hungry SNPos) that attempts to remove these co-occurrent resistant variants. Using an M. tuberculosis genomic dataset for the virulent Beijing strain-type (n = 3,574) with phenotypic resistance data across five drugs (isoniazid, rifampicin, ethambutol, pyrazinamide, and streptomycin), we demonstrate that this new approach is considerably more robust than traditional methods and detects resistance-associated variants too rare to be likely picked up by correlation-based techniques like GWAS. Author summary Tuberculosis is one of the deadliest infectious diseases, being responsible for more than one million deaths per year. The causing bacteria are becoming increasingly drug-resistant, which is hampering disease control. At the same time, an unprecedented amount of bacterial whole-genome sequencing is increasingly informing clinical practice. In order to detect the genetic alterations responsible for developing drug resistance and predict resistance status from genomic data, bio-statistical methods and machine learning models have been employed. However, due to strongly overlapping drug resistance phenotypes and genotypes in multidrug-resistant datasets, the results of these correlation-based approaches frequently also contain mutations related to resistance against other drugs. In the past, this issue has often been ignored or partially resolved by either restricting the input data or in post-analysis screening-with both strategies relying on prior information. Here we present a heuristic algorithm for finding resistance-associated variants and demonstrate that it is considerably more robust towards co-occurrent resistance compared to traditional techniques. The software is available at https://github.com/julibeg/HHS.