Portable sequencing of Mycobacterium tuberculosis for clinical and epidemiological applications.

With >1 million associated deaths in 2020, human tuberculosis (TB) caused by the bacteria Mycobacterium tuberculosis remains one of the deadliest infectious diseases. A plethora of genomic tools and bioinformatics pipelines have become available in recent years to assist the whole genome sequencing of M. tuberculosis. The Oxford Nanopore Technologies (ONT) portable sequencer is a promising platform for cost-effective application in clinics, including personalizing treatment through detection of drug resistance-associated mutations, or in the field, to assist epidemiological and transmission investigations. In this study, we performed a comparison of 10 clinical isolates with DNA sequenced on both long-read ONT and (gold standard) short-read Illumina HiSeq platforms. Our analysis demonstrates the robustness of the ONT variant calling for single nucleotide polymorphisms, despite the high error rate. Moreover, because of improved coverage in repetitive regions where short sequencing reads fail to align accurately, ONT data analysis can incorporate additional regions of the genome usually excluded (e.g. pe/ppe genes). The resulting extra resolution can improve the characterization of transmission clusters and dynamics based on inferring closely related isolates. High concordance in variants in loci associated with drug resistance supports its use for the rapid detection of resistant mutations. Overall, ONT sequencing is a promising tool for TB genomic investigations, particularly to inform clinical and surveillance decision-making to reduce the disease burden.

Genetic diversity of candidate loci linked to Mycobacterium tuberculosis resistance to bedaquiline, delamanid and pretomanid.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the deadliest infectious diseases worldwide. Multidrug and extensively drug-resistant strains are making disease control difficult, and exhausting treatment options. New anti-TB drugs bedaquiline (BDQ), delamanid (DLM) and pretomanid (PTM) have been approved for the treatment of multi-drug resistant TB, but there is increasing resistance to them. Nine genetic loci strongly linked to resistance have been identified (mmpR5, atpE, and pepQ for BDQ; ddn, fgd1, fbiA, fbiB, fbiC, and fbiD for DLM/PTM). Here we investigated the genetic diversity of these loci across >33,000 M. tuberculosis isolates. In addition, epistatic mutations in mmpL5-mmpS5 as well as variants in ndh, implicated for DLM/PTM resistance in M. smegmatis, were explored. Our analysis revealed 1,227 variants across the nine genes, with the majority (78%) present in isolates collected prior to the roll-out of BDQ and DLM/PTM. We identified phylogenetically-related mutations, which are unlikely to be resistance associated, but also high-impact variants such as frameshifts (e.g. in mmpR5, ddn) with likely functional effects, as well as non-synonymous mutations predominantly in MDR-/XDR-TB strains with predicted protein destabilising effects. Overall, our work provides a comprehensive mutational catalogue for BDQ and DLM/PTM associated genes, which will assist with establishing associations with phenotypic resistance; thereby, improving the understanding of the causative mechanisms of resistance for these drugs, leading to better treatment outcomes.

An integrated whole genome analysis of Mycobacterium tuberculosis reveals insights into relationship between its genome, transcriptome and methylome.

Human tuberculosis disease (TB), caused by Mycobacterium tuberculosis (Mtb), is a complex disease, with a spectrum of outcomes. Genomic, transcriptomic and methylation studies have revealed differences between Mtb lineages, likely to impact on transmission, virulence and drug resistance. However, so far no studies have integrated sequence-based genomic, transcriptomic and methylation characterisation across a common set of samples, which is critical to understand how DNA sequence and methylation affect RNA expression and, ultimately, Mtb pathogenesis. Here we perform such an integrated analysis across 22 M. tuberculosis clinical isolates, representing ancient (lineage 1) and modern (lineages 2 and 4) strains. The results confirm the presence of lineage-specific differential gene expression, linked to specific SNP-based expression quantitative trait loci: with 10 eQTLs involving SNPs in promoter regions or transcriptional start sites; and 12 involving potential functional impairment of transcriptional regulators. Methylation status was also found to have a role in transcription, with evidence of differential expression in 50 genes across lineage 4 samples. Lack of methylation was associated with three novel variants in mamA, likely to cause loss of function of this enzyme. Overall, our work shows the relationship of DNA sequence and methylation to RNA expression, and differences between ancient and modern lineages. Further studies are needed to verify the functional consequences of the identified mechanisms of gene expression regulation.