Abstract
Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis (Mtb), claims 1.2 million lives annually. The rise of drug-resistant TB (DR-TB) strains further exacerbates the situation. Recent work from our laboratory using Illumina short-read sequencing discovered a cluster of drug-resistant Mtb isolates associated with poor treatment outcomes. This study aims to utilise comparative genomic analysis to identify genomic structural variants present in these isolates and confirm their presence with long-read Oxford Nanopore Technology (ONT).
Comparative genomic analysis showed that these isolates harbour a large genomic deletion (LGD) of more than 5kb, comprising phospholipases gene cluster (plcABC) and PPE38 genes in DR-TB strains genomic deletion of ~5kb that has not been reported previously. However, Illumina short-read sequencing technology has limited capacity to resolve large structural variations, gene duplications or variations. Therefore, an ONT MINION sequencing platform was utilised to confirm this LGD, and confirm it is not a result of limitations of short-read Illumina sequencing. Following three days of sequencing, raw reads were processed for quality control and aligned to the M. tuberculosis H37Rv reference genome using the bioinformatics software, Geneious Prime.
ONT sequencing confirmed the presence of this 5kb LGD, which affect genes critical for Mtb virulence. PlcABC genes support pathogenesis, intracellular survival, and lipid metabolism, while PPE38 modulates immune evasion by downregulating MHC class I expression on macrophages. Interestingly, the presence of these deletions in DR-TB strains raises questions about their potential role in drug resistance. These findings contribute to the current understanding of TB pathogenesis and suggest a possible link between virulence factor deletions and drug resistance. Further research is needed to explore the functional consequences of these deletions, which may provide novel insights into DR-TB and inform new therapeutic strategies.