TB-Genaly: A Multicenter Evaluated Whole Genome Sequencing Pipeline for Tuberculosis Drug Resistance Assessment and Transmission Analysis

Despite WGS enabling insights into TB drug resistance, transmission, and outbreaks, the pipelines for integrating resistance prediction with strain genetics and species identification are scarce, and they often fall short of WHO-recommended methods, hampered by data analysis barriers that hinder clinical use. We have established a free, function-rich, user-friendly, data confidentiality analysis pipeline for Mycobacterium tuberculosis (MTB) WGS data analysis that integrates drug-resistance prediction for 16 antituberculosis drugs, detection of variants, analysis of genetic relationships and MTB species identification. The sequencing data of 461 clinical isolates with phenotypic drug susceptibility test (pDST) results from Jilin Center for Disease Control and Prevention (Jilin CDC), Sichuan Center for Disease Control and Prevention (Sichuan CDC), Tianjin Center for Disease Control and Prevention (Tianjin CDC), and Hunan Center for Disease Control and Prevention (Hunan CDC) were used to evaluate the accuracy of TB-Genaly in predicting drug resistance. The results of multi-center validation showed that the sensitivity and specificity of drug resistance for rifampicin (95.8%, 92.6%), isoniazid (95.5%, 92.4%), and pyrazinamide (91.8%, 99.4%) were in line with expectations except ethambutol (87.3%, 92.7%). The sensitivity and specificity for second-line drug resistance were separately 87.8%-94.7% and 97.5%-98.0%, and for MDR-TB were 98.5% and 99.3%, respectively. TB-Genaly also analyzes the genetic relationships between multiple strains by reconstructing phylogenetic trees and calculating pairwise single nucleotide polymorphism (SNP) distances to identify genomic clusters. TB-Genaly is a multifunctional integrated pipeline that uses WGS raw data to accurately predict antituberculosis drug-resistance profiles, analyzing genetic relationships between multiple strains. TB-Genaly is a useful tool for guiding both treatment and epidemiological investigation.