By analyzing sequencing kinetics of clinical isolates from SMRT-sequencing, we were able to de novo assemble the full genomes and DNA methylomes of clinical isolates spanning seven major MTBC lineages, isolated from several continents. Comparing sequences of de novo assemblies, rather than reference-based variant calling revealed an IS6110 element inserted into and inactivating a methyltransferase that was missed by prior analysis of the same isolate with short reads.

We extend recent methods of DNA methylation heterogeneity analysis and describe “Intercellular mosaic methylation”, a form of prokaryotic DNA methylation heterogeneity distinct from established forms. Though rare overall, intercellular mosaic methylation is nearly ubiquitous in isolates of the globally successful Beijing sublineage suggesting that IMM may confer an adaptive advantage to the Beijing strains.

Integrative analysis revealed widespread promoter methylation in M. tuberculosis, upstream of numerous clinically important genes and operons, and evidence for DNA methylation directly influencing promoter strength.    Comparative methylomic analysis distilled 351 hypervariable sites across M. tuberculosis clinical strains, which we propose as potential sites of epigenomic-driven phenotypic variation. This dataset and our findings can provide the basis for future work delineating the roles of DNA adenine methylation in M. tuberculosis physiology and adaptive evolution by the greater research community.

Our latest manuscript is now published in BMC Genomics!

In the article, we report the assembly and finishing of the avirulent reference genome H37Ra genome from single-molecule, real-time (SMRT) sequencing. Our assembly reveals that the number of H37Ra-specific variants is less than half of what the Sanger-based H37Ra reference sequence indicates, undermining and, in some cases, invalidating the conclusions of several studies. PE_PPE family genes, which are intractable to commonly-used sequencing platforms because of their repetitive and GC-rich nature, are overrepresented in the set of genes in which all reported H37Ra-specific variants are contradicted. We discuss how our results change the picture of virulence attenuation and the power of SMRT sequencing for producing high-quality reference genomes.

Check it out!

Last week, seven of us had the privilege of representing our lab and SDSU at the American Society for Microbiology Conference on Tuberculosis 2017 in the Big Apple, where we presented research, fostered new connections, and strengthened old ones with the international TB community. We learned a tremendous amount about the physiology and genetics of Mtb, as well as several novel and exciting methodologies for future studies on this deadly pathogen. Many in attendance took interest in the seven posters we presented, particularly our exposition of the areas of the genome frequently unresolved by Illumina “Hole” genome sequencing, which we term “blind spots”, presented by Tal Shmaya. We look forward to continuing with each of these projects as they develop into publications, and presenting our future findings at more conferences in the coming months and years!

The genetic basis of virulence in Mycobacterium tuberculosis has been investigated through genome comparisons of its virulent (H37Rv) and attenuated (H37Ra) sister strains. Such analysis, however, relies heavily on the accuracy of the sequences. While the H37Rv reference genome has had several corrections to date, that of H37Ra is unmodified since its original publication. Here, we report the assembly and finishing of the H37Ra genome from single-molecule, real-time (SMRT) sequencing. Our assembly reveals that the number of H37Ra-specific variants is less than half of what the Sanger-based H37Ra reference sequence indicates, undermining and, in some cases, invalidating the conclusions of several studies. PE_PPE family genes, which are intractable to commonly-used sequencing platforms because of their repetitive and GC-rich nature, are overrepresented in the set of genes in which all reported H37Ra-specific variants are contradicted. We discuss how our results change the picture of virulence attenuation and the power of SMRT sequencing for producing high-quality reference genomes.

https://doi.org/10.1101/064840

The manuscript is under review by BMC Genomics.