Pan-Lineage Mycobacterium tuberculosis Reference Genome for Enhanced Molecular Diagnosis.

In Mycobacterium tuberculosis (MTB) control, whole genome sequencing-based molecular drug susceptibility testing (molDST-WGS) has emerged as a pivotal tool. However, the current reliance on a single-strain reference limits molDST-WGS's true potential. To address this, we introduce a new pan-lineage reference genome, "MtbRf". We assembled "unmapped" reads from 3,614 MTB genomes (751 L1; 881 L2; 1,700 L3; and 282 L4) into 35 shared, annotated contigs (54 CDSs). We constructed MtbRf through: 1) searching for contig homologs among genome database that precipitating results uniquely within Mycobacteria genus; 2) comparing genomes with H37Rv ("lift-over") to define 18 insertions; and 3) filling gaps in H37Rv with insertions. MtbRf adds 1.18% sequences to H37rv, salvaging >60% of previously unmapped reads. Transcriptomics confirmed gene-expression of new CDSs. The new variants provided a moderate DST predictive value (AUROC 0.60-0.75). MtbRf thus unveils previously hidden genomic information, and lays the foundation for lineage-specific molDST-WGS.