This study presents a comprehensive pan-lineage reference genome, termed "MtbRf", derived from a wide array of Mycobacterium tuberculosis lineages. MtbRf seeks to bridge the current bioinformatics gap in molecular drug susceptibility testing (molDST-WGS) by providing a more inclusive and accurate genetic reference.
Mycobacterium tuberculosis (MTB) remains a major global health threat, with millions of infections and fatalities reported annually. The urgent need for efficient drug susceptibility testing (DST) is ever-growing, especially in the face of the rise of drug-resistant MTB strains. While whole-genome sequencing-based molecular drug susceptibility testing (molDST-WGS) offers a rapid and enriched biological perspective, its efficacy is currently limited by the reliance on a single-strain reference genome.  Our study introduces "MtbRf", a novel pan-lineage reference genome constructed from "unmapped" reads of 3,614 MTB genomes spanning various lineages. Through meticulous assembly, comparative analysis, and integration with the existing H37Rv reference, MtbRf adds 1.18% more sequences, capturing more than 60% of previously elusive reads. This groundbreaking effort also resulted in the identification of novel coding sequences (CDSs), whose expressions were authenticated through transcriptomics. Our preliminary evaluations suggest that MtbRf could potentially refine the predictive accuracy of molDST-WGS, especially for new anti-tuberculosis agents.  By unveiling previously hidden genomic details, MtbRf provides an invaluable tool for future research and diagnostic endeavors. Its development heralds a significant step towards a more comprehensive and effective approach to global MTB control. This study was a collaborative effort, leveraging the resources of both the "Genome Research for Asian Tuberculosis" (GReAT) project and the "Comprehensive Resistance Prediction for Tuberculosis: an International Consortium" (CRyPTIC), aiming to drive forward advancements in MTB research and management.