Whole-genome optical mapping reveals a mis-assembly between two rRNA operons of Corynebacterium pseudotuberculosis strain 1002

Mariano, Diego; Sousa, Thiago de Jesus; Pereira, Felipe Luiz; Aburjaile, Flávia Figueira; Barh, Debmalya; Rocha, Flávia Souza; Pinto, Anne Cybelle; Hassan, Syed Shah; Saraiva, Tessália Diniz Luerce; Dorella, Fernanda Alves; Carvalho, Alex F.; Leal, Carlos Augusto Gomes; Figueiredo, Henrique César Pereira; Silva, Artur M. S.; Ramos, Rommel Thiago Jucá; Azevedo, Vasco

doi:10.1186/s12864-016-2673-7

Cited by 12 publications

(12 citation statements)

References 32 publications

(40 reference statements)

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“…The second strategy involves long read (re)sequencing using either mate-pair libraries [ 35 ] or long-read techniques such as PacBio [ 36 ] and Nanopore [ 37 ]. The third strategy is based on genome maps [ 38 , 39 ]. These methodologies incur additional cost and require significant time investments.…”

Section: Introductionmentioning

confidence: 99%

Genomic repeats, misassembly and reannotation: a case study with long-read resequencing of Porphyromonas gingivalis reference strains

et al. 2018

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BackgroundWithout knowledge of their genomic sequences, it is impossible to make functional models of the bacteria that make up human and animal microbiota. Unfortunately, the vast majority of publicly available genomes are only working drafts, an incompleteness that causes numerous problems and constitutes a major obstacle to genotypic and phenotypic interpretation. In this work, we began with an example from the class Bacteroidia in the phylum Bacteroidetes, which is preponderant among human orodigestive microbiota. We successfully identify the genetic loci responsible for assembly breaks and misassemblies and demonstrate the importance and usefulness of long-read sequencing and curated reannotation.ResultsWe showed that the fragmentation in Bacteroidia draft genomes assembled from massively parallel sequencing linearly correlates with genomic repeats of the same or greater size than the reads. We also demonstrated that some of these repeats, especially the long ones, correspond to misassembled loci in three reference Porphyromonas gingivalis genomes marked as circularized (thus complete or finished). We prove that even at modest coverage (30X), long-read resequencing together with PCR contiguity verification (rrn operons and an integrative and conjugative element or ICE) can be used to identify and correct the wrongly combined or assembled regions. Finally, although time-consuming and labor-intensive, consistent manual biocuration of three P. gingivalis strains allowed us to compare and correct the existing genomic annotations, resulting in a more accurate interpretation of the genomic differences among these strains.ConclusionsIn this study, we demonstrate the usefulness and importance of long-read sequencing in verifying published genomes (even when complete) and generating assemblies for new bacterial strains/species with high genomic plasticity. We also show that when combined with biological validation processes and diligent biocurated annotation, this strategy helps reduce the propagation of errors in shared databases, thus limiting false conclusions based on incomplete or misleading information.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4429-4) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Genomic repeats, misassembly and reannotation: a case study with long-read resequencing of Porphyromonas gingivalis reference strains

et al. 2018

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“…One constraint on the performance of riboSeed is the quality of rRNA annotations in reference strains. Although it is impossible to concretely confirm in silico, we (and others [29]) have found several reference genomes during the course of this study that we suspect have collapsed rDNA repeats. We recommend using a tool such as 16Stimator [35] or rrnDB [42] to estimate number of 16S (and therefore rDNAs) prior to assembly, or stack to assess mapping depths 315 after running seed.…”

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confidence: 58%

“…CONTIGuator [12] uses contigs from a de novo assembly along with one or more reference sequences to generate a contig map and PCR primer sets to validate in the lab. Konnector [47] uses paired-end reads to make long reads to be used in a Bloom filter representation of a de Bruijn graph MapRepeat [28] uses a directed scaffolding method to fill in rDNA gaps, but limited to Ion Torrent reads, and affected by inversions between rDNAs [29] Pilon [49] compares mapping files to an assembly to correct mistakes and fill gaps GRabB [4] selectively assembles tandem rDNAs and mitochondria resolution of these difficult regions and provide a means to benefit from unexploited information in the SRA/ENA 25 short read archives.…”

Section: Introductionmentioning

confidence: 99%

riboSeed: leveraging prokaryotic genomic architecture to assemble across ribosomal regions

Waters

Abram

Brennan

et al. 2017

Preprint

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The vast majority of bacterial genome sequencing has been performed using Illumina short reads. Because of the inherent difficulty of resolving repeated regions with short reads alone, only ≈10% of sequencing projects have resulted in a closed genome. The most common repeated regions are those coding for ribosomal operons (rDNAs), which occur in a bacterial genome between 1 and 15 times, and are typically used as sequence markers to classify and identify bacteria. Here, we exploit conservation in the genomic context in which rDNAs occur across taxa to improve assembly of these regions relative to de novo sequencing by using the conserved nature of rDNAs across taxa and the uniqueness of their flanking regions within a genome. We describe a method to construct targeted pseudocontigs generated by iteratively assembling reads that map to a reference genome's rDNAs. These pseudocontigs are then used to more accurately assemble the newly-sequenced chromosome. We show that this method, implemented as riboSeed, correctly bridges across adjacent contigs in bacterial genome assembly and, when used in conjunction with other genome polishing tools, can assist in closure of a genome.

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“…The use of WGM technique has allowed the discover of mis-assemblies in bacterial genomes, such as a large inversion in Cp1002 recently published [18]. WGM has been considered a strategy of high accuracy to finish assemblies [18–21]. However, MapSolver is not capable to generate scaffolds.…”

Section: Resultsmentioning

confidence: 99%

SIMBA: a web tool for managing bacterial genome assembly generated by Ion PGM sequencing technology

et al. 2016

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BackgroundThe evolution of Next-Generation Sequencing (NGS) has considerably reduced the cost per sequenced-base, allowing a significant rise of sequencing projects, mainly in prokaryotes. However, the range of available NGS platforms requires different strategies and software to correctly assemble genomes. Different strategies are necessary to properly complete an assembly project, in addition to the installation or modification of various software. This requires users to have significant expertise in these software and command line scripting experience on Unix platforms, besides possessing the basic expertise on methodologies and techniques for genome assembly. These difficulties often delay the complete genome assembly projects.ResultsIn order to overcome this, we developed SIMBA (SImple Manager for Bacterial Assemblies), a freely available web tool that integrates several component tools for assembling and finishing bacterial genomes. SIMBA provides a friendly and intuitive user interface so bioinformaticians, even with low computational expertise, can work under a centralized administrative control system of assemblies managed by the assembly center head. SIMBA guides the users to execute assembly process through simple and interactive pages. SIMBA workflow was divided in three modules: (i) projects: allows a general vision of genome sequencing projects, in addition to data quality analysis and data format conversions; (ii) assemblies: allows de novo assemblies with the software Mira, Minia, Newbler and SPAdes, also assembly quality validations using QUAST software; and (iii) curation: presents methods to finishing assemblies through tools for scaffolding contigs and close gaps. We also presented a case study that validated the efficacy of SIMBA to manage bacterial assemblies projects sequenced using Ion Torrent PGM.ConclusionBesides to be a web tool for genome assembly, SIMBA is a complete genome assemblies project management system, which can be useful for managing of several projects in laboratories. SIMBA source code is available to download and install in local webservers at http://ufmg-simba.sourceforge.net.

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Whole-genome optical mapping reveals a mis-assembly between two rRNA operons of Corynebacterium pseudotuberculosis strain 1002

Cited by 12 publications

References 32 publications

Genomic repeats, misassembly and reannotation: a case study with long-read resequencing of Porphyromonas gingivalis reference strains

Genomic repeats, misassembly and reannotation: a case study with long-read resequencing of Porphyromonas gingivalis reference strains

riboSeed: leveraging prokaryotic genomic architecture to assemble across ribosomal regions

SIMBA: a web tool for managing bacterial genome assembly generated by Ion PGM sequencing technology

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