Dataset Information

Re-sequencing and optical mapping reveals misassemblies and real inversions on Corynebacterium pseudotuberculosis genomes.

ABSTRACT: The number of draft genomes deposited in Genbank from the National Center for Biotechnology Information (NCBI) is higher than the complete ones. Draft genomes are assemblies that contain fragments of misassembled regions (gaps). Such draft genomes present a hindrance to the complete understanding of the biology and evolution of the organism since they lack genomic information. To overcome this problem, strategies to improve the assembly process are developed continuously. Also, the greatest challenge to the assembly progress is the presence of repetitive DNA regions. This article highlights the use of optical mapping, to detect and correct assembly errors in Corynebacterium pseudotuberculosis. We also demonstrate that choosing a reference genome should be done with caution to avoid assembly errors and loss of genetic information.

SUBMITTER: Sousa TJ

PROVIDER: S-EPMC6841979 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Re-sequencing and optical mapping reveals misassemblies and real inversions on Corynebacterium pseudotuberculosis genomes.

Sousa Thiago de Jesus TJ Parise Doglas D Profeta Rodrigo R Parise Mariana Teixeira Dornelles MTD Gomide Anne Cybelle Pinto ACP Kato Rodrigo Bentos RB Pereira Felipe Luiz FL Figueiredo Henrique Cesar Pereira HCP Ramos Rommel R Brenig Bertram B Costa da Silva Artur Luiz da ALD Ghosh Preetam P Barh Debmalya D Góes-Neto Aristóteles A Azevedo Vasco V

Scientific reports 20191108 1

The number of draft genomes deposited in Genbank from the National Center for Biotechnology Information (NCBI) is higher than the complete ones. Draft genomes are assemblies that contain fragments of misassembled regions (gaps). Such draft genomes present a hindrance to the complete understanding of the biology and evolution of the organism since they lack genomic information. To overcome this problem, strategies to improve the assembly process are developed continuously. Also, the greatest chal ...[more]

PMID: 31705053

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Project description:Corynebacterium pseudotuberculosis (CPS) is an important bacterial animal pathogen. CPS causes chronic, debilitating and currently incurable infectious diseases affecting a wide range of livestock and wild herbivores including camelids worldwide. Belonging to the Corynebacterium diphtheriae complex, this pathogen can also infect humans. The classical characterization of CPS is typically based on the testing of nitrate reductase activity, separating the two biovars Equi and Ovis. However, more refined resolutions are required to unravel routes of infection. This was realized in our study by generating and analyzing whole genome sequencing (WGS) data. Using newly created core genome multilocus sequence typing (cgMLST) profiles we were the first to discover isolates grouping in a cluster adjacent to clusters formed by CPS biovar Equi isolates. This novel cluster includes CPS isolates from alpacas, llamas, camels and dromedaries, which are characterized by a lack of nitrate reductase activity as encountered in biovar Ovis. This is of special interest for molecular epidemiology. Nevertheless, these isolates bear the genes of the nitrate locus, which are characteristic of biovar Equi isolates. However, sequence analysis of the genes narG and narH of the nitrate locus revealed indels leading to frameshifts and inactivity of the enzymes involved in nitrate reduction. Interestingly, one CPS isolate originating from another lama with an insertion in the MFS transporter (narT) is adjacent to a cluster formed by ovine CPS isolates biovar Equi. Based on this knowledge, the combination of biochemical and PCR based molecular biological nitrate reductase detection can be used for a fast and uncomplicated classification of isolates in routine diagnostics in order to check the origin of camelid CPS isolates. Further analysis revealed that partial sequencing of the ABC transporter substrate binding protein (CP258_RS07935) is a powerful tool to assign the biovars and the novel genomovar.

Project description:UnlabelledNext-generation sequencing (NGS) technologies have increased the scalability, speed, and resolution of genomic sequencing and, thus, have revolutionized genomic studies. However, eukaryotic genome sequencing initiatives typically yield considerably fragmented genome assemblies. Here, we assessed various state-of-the-art sequencing and assembly strategies in order to produce a contiguous and complete eukaryotic genome assembly, focusing on the filamentous fungus Verticillium dahliae. Compared with Illumina-based assemblies of the V. dahliae genome, hybrid assemblies that also include PacBio-generated long reads establish superior contiguity. Intriguingly, provided that sufficient sequence depth is reached, assemblies solely based on PacBio reads outperform hybrid assemblies and even result in fully assembled chromosomes. Furthermore, the addition of optical map data allowed us to produce a gapless and complete V. dahliae genome assembly of the expected eight chromosomes from telomere to telomere. Consequently, we can now study genomic regions that were previously not assembled or poorly assembled, including regions that are populated by repetitive sequences, such as transposons, allowing us to fully appreciate an organism's biological complexity. Our data show that a combination of PacBio-generated long reads and optical mapping can be used to generate complete and gapless assemblies of fungal genomes.ImportanceStudying whole-genome sequences has become an important aspect of biological research. The advent of next-generation sequencing (NGS) technologies has nowadays brought genomic science within reach of most research laboratories, including those that study nonmodel organisms. However, most genome sequencing initiatives typically yield (highly) fragmented genome assemblies. Nevertheless, considerable relevant information related to genome structure and evolution is likely hidden in those nonassembled regions. Here, we investigated a diverse set of strategies to obtain gapless genome assemblies, using the genome of a typical ascomycete fungus as the template. Eventually, we were able to show that a combination of PacBio-generated long reads and optical mapping yields a gapless telomere-to-telomere genome assembly, allowing in-depth genome analyses to facilitate functional studies into an organism's biology.

Project description:BackgroundPan-genomic studies aim, for instance, at defining the core, dispensable and unique genes within a species. A pan-genomics study for vaccine design tries to assess the best candidates for a vaccine against a specific pathogen. In this context, rather than studying genes predicted to be exported in a single genome, with pan-genomics it is possible to study genes present in different strains within the same species, such as virulence factors. The target organism of this pan-genomic work here presented is Corynebacterium pseudotuberculosis, the etiologic agent of caseous lymphadenitis (CLA) in goat and sheep, which causes significant economic losses in those herds around the world. Currently, only a few antigens against CLA are known as being the basis of commercial and still ineffective vaccines. In this regard, the here presented work analyses, in silico, five C. pseudotuberculosis genomes and gathers data to predict common exported proteins in all five genomes. These candidates were also compared to two recent C. pseudotuberculosis in vitro exoproteome results.ResultsThe complete genome of five C. pseudotuberculosis strains (1002, C231, I19, FRC41 and PAT10) were submitted to pan-genomics analysis, yielding 306, 59 and 12 gene sets, respectively, representing the core, dispensable and unique in silico predicted exported pan-genomes. These sets bear 150 genes classified as secreted (SEC) and 227 as potentially surface exposed (PSE). Our findings suggest that the main C. pseudotuberculosis in vitro exoproteome could be greater, appended by a fraction of the 35 proteins formerly predicted as making part of the variant in vitro exoproteome. These genomes were manually curated for correct methionine initiation and redeposited with a total of 1885 homogenized genes.ConclusionsThe in silico prediction of exported proteins has allowed to define a list of putative vaccine candidate genes present in all five complete C. pseudotuberculosis genomes. Moreover, it has also been possible to define the in silico predicted dispensable and unique C. pseudotuberculosis exported proteins. These results provide in silico evidence to further guide experiments in the areas of vaccines, diagnosis and drugs. The work here presented is the first whole C. pseudotuberculosis in silico predicted pan-exoproteome completed till today.

Dataset Information

Re-sequencing and optical mapping reveals misassemblies and real inversions on Corynebacterium pseudotuberculosis genomes.

Publications

Re-sequencing and optical mapping reveals misassemblies and real inversions on Corynebacterium pseudotuberculosis genomes.

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