SMRT Genome Assembly Corrects Reference Errors, Resolving the Genetic Basis of Virulence in <i>Mycobacterium tuberculosis</i>

Elghraoui, Afif; Modlin, Samuel J; Valafar, Faramarz

doi:10.1101/064840

Cited by 3 publications

(3 citation statements)

References 51 publications

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“…PGAP is a thorough and extensive pipeline, making use of multiple gene-finding algorithms and comprehensive collections of a variety of databases. We applied each tool to genomes of two different species: Mycobacterium tuberculosis H37Ra(14) and Pseudomonas aeruginosa PAK(15).…”

Section: Methodsmentioning

confidence: 99%

Hybran: Hybrid Reference Transfer and ab initio Prokaryotic Genome Annotation

Elghraoui

Gunasekaran

Ramirez-Busby

et al. 2022

Preprint

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De novo assembly has become commonplace for microbial organisms, increasing the demand for reliable genome annotation. Ab initio annotation is not an ideal approach for closely related strains due to suboptimal matching of the short or hypervariable genomic features that reference-based annotation transfer can overcome through identification of conserved synteny. At the same time, reference-based annotation methods leave gaps in the annotation where structural variations introduce unique sequence. We present Hybran, a hybrid reference-based and ab initio prokaryotic genome annotation pipeline that transfers features from a curated reference annotation and supplements unannotated regions with ab initio predictions. It builds on existing tools to create initial annotations using both approaches, then compares and resolves them to produce the hybrid annotation. With this pipeline, full advantage is taken of the community's experimental efforts on reference strains to propagate as many known features as possible without sacrificing best-effort ab initio predictions for the remaining unannotated loci. Genome annotation performed in this way can facilitate comparative genomics and the investigation of evolutionary dynamics in microbial populations. Hybran is freely available at https://lpcdrp.gitlab.io/hybran

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Section: Methodsmentioning

confidence: 99%

Hybran: Hybrid Reference Transfer and ab initio Prokaryotic Genome Annotation

Elghraoui

Gunasekaran

Ramirez-Busby

et al. 2022

Preprint

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“…The DNA of all 333 isolates collected for long-read PacBio sequencing, including those from the WHO Supranational Reference Laboratories in Stockholm and Antwerp, and from NCBI's SRA database were extracted as described in a previous study 57 . The SMRT sequencing protocol was described previously 58,59 . 64 isolates were later re-sequenced due to low coverage.…”

Section: Dna Extraction and Sequencingmentioning

confidence: 99%

Rare alternative second line injectable drug resistance markers identified by gene-wise genome wide association in M. tuberculosis with unexplained resistance

Conkle-Gutierrez

Kim

Ramirez-Busby

et al. 2021

Preprint

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Point mutations in the rrs gene and eis promoter are known to confer resistance to second-line injectable drugs (SLIDs) amikacin (AMK), capreomycin (CAP), and kanamycin (KAN). While mutations in these canonical genes confer a majority of SLID-resistance, alternative mechanisms of resistance are not uncommon and threaten effective treatment decisions when using conventional molecular diagnostics. In total, 1184 clinical M. tuberculosis isolates from 7 countries were studied for genomic markers associated with phenotypic resistance. The markers rrs:A1401G and rrs:G1484T were associated with resistance to all three SLIDs, and three known markers in the eis promoter (eis:G-10A, eis:C-12T, and eis:C-14T) were similarly associated with kanamycin resistance (KAN-R). Among 325, 324, 270 AMK-R, CAP-R, and KAN-R isolates, 264 (81.2%), 250 (77.2%), and 249 (92.3%) harbored canonical mutations, respectively. Thirteen isolates harbored more than one canonical mutation. Canonical mutations did not account for 111 of the phenotypically resistant isolates. A gene-wise method identified three genes and promoters with mutations that, on aggregate, associated with unexplained resistance to at least one SLID. Our analysis associated whiB7 promoter mutations with KAN resistance, supporting clinical relevance for the previously demonstrated role of whiB7 overexpression in KAN resistance. We also provide evidence for the novel association of ppe51 (a gene previously associated with various antimicrobial compounds) with AMK resistance, and for the novel association of thrB with AMK and CAP resistance. The use of gene-wise association can provide additional insight, and therefore is recommended for identification of rare mechanisms of resistance when individual mutations carry insufficient statistical power.

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“…Polyketide and terpene metabolism mediate pathogenic processes unique to M. tuberculosis and are overrepresented in (Fig 9C). Polyketides are implicated heavily in virulence [101][102][103] , make up part of the outer lipid layer of the M. tuberculosis bacillus, the host-pathogen interface 104 , where they mediate processes to subvert mechanisms of host immunity 105 . Meanwhile, terpenes play an immunomodulatory role during early stages of M. tuberculosis infection and phagosomal maturation [106][107][108] .…”

Section: Novel Metabolic Annotations Fill Knowledge Gaps and May Imprmentioning

confidence: 99%

Structure-aware M. tuberculosis functional annotation uncloaks resistance, metabolic, and virulence genes

Modlin

Gunasekaran

et al. 2018

Preprint

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Each decade, billions are invested in Tuberculosis (TB) research to further characterize M. tuberculosis pathogenesis. Despite this investment, nearly half of the 4,031 M. tuberculosis protein-coding genes lack descriptive annotation in community databases, due largely to incomplete reconciliation with the literature and a lack of structure-based methods for functional inference. We coin the term "hypotheticome" as the set of genes in an organism without known function. For M. tuberculosis' hypotheticome, we compiled the set of genes lacking functional assignment in the most frequently used Mycobacteria annotation database through systematic, exhaustive manual literature curation and 3Dprotein structure-based inference, and reconciled these annotations with frequented functional databases, creating a comprehensive M. tuberculosis functional knowledge-base. In doing so, we also introduce standard usage of qualifying adjectives based on quantitative measures of certainty with the hope that this approach is adopted in choosing qualifiers for future functional assignments.Through these methods we functionally annotated 41.3% of the M. tuberculosis hypotheticome, and provide insight into its pathogenesis, antibiotic-resistance, and virulence. Processes implicated in the unique lifestyle of M. tuberculosis of long-term persistence and obligate pathogenesis in genotoxic host microenvironmentslipid metabolism, polyketide biosynthesis, and membrane transport and effluxwere overrepresented in our annotation. Our structural similarity approach unturned proteins that appear critical in host-interaction through apparent host mimicry, particularly involving the phagosome and vesicle-mediated transport, as well as putative structural analogs for highly mutable protein classes, including dozens of PE/PPE family proteins which are major players at the host-pathogen interface, and sixteen potential efflux pumps which are integral to M. tuberculosis drug tolerance. Hypotheses drawn from these proteins' function may help characterize the onset of latency and identify therapeutic targets. A unified annotation is essential for clear communication about M. tuberculosis. These improvements provide the most comprehensive M. tuberculosis genome annotation to date, and the approach presented can be applied to systematically annotate the genome of other organisms. We provide our novel annotations in General Feature Format with Enzyme Commission and Gene Ontology terms for integration into existing annotation frameworks.

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SMRT Genome Assembly Corrects Reference Errors, Resolving the Genetic Basis of Virulence in Mycobacterium tuberculosis

Cited by 3 publications

References 51 publications

Hybran: Hybrid Reference Transfer and ab initio Prokaryotic Genome Annotation

Hybran: Hybrid Reference Transfer and ab initio Prokaryotic Genome Annotation

Rare alternative second line injectable drug resistance markers identified by gene-wise genome wide association in M. tuberculosis with unexplained resistance

Structure-aware M. tuberculosis functional annotation uncloaks resistance, metabolic, and virulence genes

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