Obtenção de genomas completos de alta qualidade e a influência destes em análises genômicas: uma abordagem para Corynebacterium pseudotuberculosis

Abstract

Corynebacterium pseudotuberculosis belongs to the CMNR group (Corynebacterium, Mycobacterium, Nocardia, and Rhodococcus). This bacterium has its most significant relevance within veterinarian medicine, the causative agent of infections in ruminants. Among the diseases caused by C. pseudotuberculosis, we can highlight Caseous Lymphadenitis whose treatment methods are unsatisfactory. Therefore, it is necessary to identify the molecular bases of this pathogen for the development of new therapeutic and prophylactic approaches, such as vaccines. Thus, the comparative study genomics of C. pseudotuberculosis by our research group has been essential for understanding their mechanisms of virulence and pathogenicity. However, because of the old technology limitations and the unavailability of advanced computational methods at the time when several isolates were sequenced, incomplete genomes have been generated. Therefore, in this work, we use the chromosomal optical mapping, which uses restriction enzymes throughout the entire genome to assist in contigs scaffolding. Through this approach, aimed to generate high-quality genomic data, as reference sequences to provide greater precision and reliability for molecular structural and functional analysis of this species. So, 11 strains representing the biovar ovis and equi were sequenced in Ion Torrent PGMTM and re-sequenced in Illumina Hiseq 2500 platforms using paired-end library, and after submitting to the optical map technique. The scaffolding process of the contigs by the optical map information allowed to detect assembly errors and genomic inversions in six strains. Our results revealed that the nar cluster, which determinate the biovar type, was not present in 4 previously deposited strains. It was possible to identify a real chromosomal rearrangement flanked by transposases in C. pseudotuberculosis 162. These complete and accurate genomes were essential for the assembly of 50 genomes of C. pseudotuberculosis biovar ovis. When analyzing these genomes, it was possible to evidence the absence of the ~"cutinase like family gene" was revealed in C. pseudotuberculosis 1002 and 6 more mutant genome strains. Also, point nucleotide variants previously unreported in this species are described, which may show the influence of environmental pressures on the genome. The present work could contribute to the quality of the genomes and expand the molecular knowledge of C. pseudotuberculosis, updating information about the pan-genome of the species.