ReNoteWeb - Web platform for the improvement of assembly result and annotation of prokaryotic genomes.

The reduction in the cost of DNA sequencing and the total time to perform this process has resulted in a significant increase in the deposit of biological information in public databases such as the NCBI (National Center for Biotechnology Information). The production of large volumes of data per run has culminated in the need to develop algorithms capable of handling data with this new feature and assisting in analyses such as the assembly and annotation of prokaryotic genomes. Over the years, several pipelines and computational tools have been developed to automate this task and consequently reduce the total time to know the genetic content of a given organism, especially non-model organisms, collaborating with the identification of possible targets with biotechnological applicability. In the case of automatic annotation tools, the accuracy of the results is widely observed in the literature, however, this does not excludes the manual curation process, where the information inferred in the automatic process is verified and enriched by the curators. This task requires a time which is directly proportional to the number of gene products of the target organism under study. To assist in this process, we present the ReNoteWeb web tool, endowed with a simple and intuitive interface, to perform the assembly enhancement process, with the possibility of identifying the missing products in the original genomic sequence. In addition, ReNoteWeb is capable of performing the annotation process for all products, based on information obtained from highly accurate external databases. The engine responsible for performing the data processing was developed in JAVA and the web platform uses the resources of the Yii framework. The annotation produced by this platform aims to reduce the overall time in the manual curation process. Twenty-three organisms were used to validate the tool. The efficiency was verified by comparing the annotation of these same organisms available in the NCBI database and the annotation performed on the RAST platform. The tool is available at: http://biod.ufpa.br/renoteweb/.

Protocol to analyze the bacterial pangenome using PAN2HGENE software.

The PAN2HGENE is a computational tool that enables two main analyses. First, the tool can identify gene products absent from the original prokaryotic genome sequence. Second, it enables automated comparative analysis for both complete and draft genomes. All analyses are performed through a simple and intuitive graphical user interface without the need for extensive and complex command lines. For complete details on the use and execution of this protocol, please refer to Silva de Oliveira (2021).

PAN2HGENE-tool for comparative analysis and identifying new gene products.

Advances in next-generation sequencing (NGS) platforms have had a positive impact on biological research, leading to the development of numerous omics approaches, including genomics, transcriptomics, metagenomics, and pangenomics. These analyses provide insights into the gene contents of various organisms. However, to understand the evolutionary processes of these genes, comparative analysis, which is an important tool for annotation, is required. Using comparative analysis, it is possible to infer the functions of gene contents and identify orthologs and paralogous genes via their homology. Although several comparative analysis tools currently exist, most of them are limited to complete genomes. PAN2HGENE, a computational tool that allows identification of gene products missing from the original genome sequence, with automated comparative analysis for both complete and draft genomes, can be used to address this limitation. In this study, PAN2HGENE was used to identify new products, resulting in altering the alpha value behavior in the pangenome without altering the original genomic sequence. Our findings indicate that this tool represents an efficient alternative for comparative analysis, with a simple and intuitive graphical interface. The PAN2HGENE have been uploaded to SourceForge and are available via: https://sourceforge.net/projects/pan2hgene-software.

CODON-Software to manual curation of prokaryotic genomes.

Genome annotation conceptually consists of inferring and assigning biological information to gene products. Over the years, numerous pipelines and computational tools have been developed aiming to automate this task and assist researchers in gaining knowledge about target genes of study. However, even with these technological advances, manual annotation or manual curation is necessary, where the information attributed to the gene products is verified and enriched. Despite being called the gold standard process for depositing data in a biological database, the task of manual curation requires significant time and effort from researchers who sometimes have to parse through numerous products in various public databases. To assist with this problem, we present CODON, a tool for manual curation of genomic data, capable of performing the prediction and annotation process. This software makes use of a finite state machine in the prediction process and automatically annotates products based on information obtained from the Uniprot database. CODON is equipped with a simple and intuitive graphic interface that assists on manual curation, enabling the user to decide about the analysis based on information as to identity, length of the alignment, and name of the organism in which the product obtained a match. Further, visual analysis of all matches found in the database is possible, impacting significantly in the curation task considering that the user has at his disposal all the information available for a given product. An analysis performed on eleven organisms was used to test the efficiency of this tool by comparing the results of prediction and annotation through CODON to ones from the NCBI and RAST platforms.

NGSReadsTreatment - A Cuckoo Filter-based Tool for Removing Duplicate Reads in NGS Data.

The Next-Generation Sequencing (NGS) platforms provide a major approach to obtaining millions of short reads from samples. NGS has been used in a wide range of analyses, such as for determining genome sequences, analyzing evolutionary processes, identifying gene expression and resolving metagenomic analyses. Usually, the quality of NGS data impacts the final study conclusions. Moreover, quality assessment is generally considered the first step in data analyses to ensure the use of only reliable reads for further studies. In NGS platforms, the presence of duplicated reads (redundancy) that are usually introduced during library sequencing is a major issue. These might have a serious impact on research application, as redundancies in reads can lead to difficulties in subsequent analysis (e.g., de novo genome assembly). Herein, we present NGSReadsTreatment, a computational tool for the removal of duplicated reads in paired-end or single-end datasets. NGSReadsTreatment can handle reads from any platform with the same or different sequence lengths. Using the probabilistic structure Cuckoo Filter, the redundant reads are identified and removed by comparing the reads with themselves. Thus, no prerequisite is required beyond the set of reads. NGSReadsTreatment was compared with other redundancy removal tools in analyzing different sets of reads. The results demonstrated that NGSReadsTreatment was better than the other tools in both the amount of redundancies removed and the use of computational memory for all analyses performed. Available in https://sourceforge.net/projects/ngsreadstreatment/ .

ImproveAssembly - Tool for identifying new gene products and improving genome assembly.

The availability of biological information in public databases has increased exponentially. To ensure the accuracy of this information, researchers have adopted several methods and refinements to avoid the dissemination of incorrect information; for example, several automated tools are available for annotation processes. However, manual curation ensures and enriches biological information. Additionally, the genomic finishing process is complex, resulting in increased deposition of drafts genomes. This introduces bias in other omics analyses because incomplete genomic content is used. This is also observed for complete genomes. For example, genomes generated by reference assembly may not include new products in the new sequence or errors or bias can occur during the assembly process. Thus, we developed ImproveAssembly, a tool capable of identifying new products missing from genomic sequences, which can be used for complete and draft genomes. The identified products can improve the annotation of complete genomes and drafts while significantly reducing the bias when the information is used in other omics analyses.

Transcriptome analysis of Corynebacterium pseudotuberculosis biovar Equi in two conditions of the environmental stress.

Corynebacterium pseudotuberculosis has been widely studied in an effort to understand its biological evolution. Transcriptomics has revealed possible candidates for virulence and pathogenicity factors of strain 1002 (biovar Ovis). Because C. pseudotuberculosis is classified into two biovars, Ovis and Equi, it was interesting to assess the transcriptional profile of biovar Equi strain 258, the causative agent of ulcerative lymphangitis. The genome of this strain was re-sequenced; the reassembly was completed using optical mapping technology, and the sequence was subsequently re-annotated. Two growth conditions that occur during the host infection process were simulated for the transcriptome: the osmotic and acid medium. Genes that may be associated with the microorganism's resilience under unfavorable conditions were identified through RNAseq, including genes present in pathogenicity islands. The RT-qPCR was performed to confirm the results in biological triplicate for each condition for some genes. The results extend our knowledge of the factors associated with the spread and persistence of C. pseudotuberculosis during the infection process and suggest possible avenues for studies related to the development of vaccines, diagnosis, and therapies that might help minimize damage to agribusinesses.

Heat shock stress: Profile of differential expression in Corynebacterium pseudotuberculosis biovar Equi.

Transcriptome studies on Corynebacterium pseudotuberculosis have recently contributed to the understanding about this microorganism's survival mechanisms in various hostile conditions. The gene expression profile of the C. pseudotuberculosis strain 1002 (Ovis biovar), has revealed genes that are possible candidates responsible for its maintenance in adverse environments, such as those found in the host. In another strain of this bacterium, 258 (Equi biovar), a high temperature condition was simulated, in order to verify which genes are responsible for promoting the persistence of the bacterium in these conditions, since it tolerates temperatures higher than 40°C, despite being a mesophilic bacterium. It was possible to generate a list of genes using RNAseq technology that possibly contribute to the survival of the bacteria in this hostile environment. A total of 562 genes were considered as differentially expressed, then, after the fold-change cutoff, 113 were considered induced and 114 repressed, resulting in a total of 227 genes. Therefore, hypothetical proteins presented a fold change above 6, and genes characteristically in control for this type of stress, such as hspR, grpE, and dnaK, presented a fold change above 3. The clpB gene, a chaperone, drew attention due to presenting a fold change above 3 and located in a pathogenicity island. These genes may contribute towards efficient solutions to the effects caused by ulcerative lymphangitis in equines, thus attenuating the damage it causes to agribusiness.

Genomic analysis of four strains of Corynebacterium pseudotuberculosis bv. Equi isolated from horses showing distinct signs of infection.

The genomes of four strains (MB11, MB14, MB30, and MB66) of the species Corynebacterium pseudotuberculosis biovar equi were sequenced on the Ion Torrent PGM platform, completely assembled, and their gene content and structure were analyzed. The strains were isolated from horses with distinct signs of infection, including ulcerative lymphangitis, external abscesses on the chest, or internal abscesses on the liver, kidneys, and lungs. The average size of the genomes was 2.3 Mbp, with 2169 (Strain MB11) to 2235 (Strain MB14) predicted coding sequences (CDSs). An optical map of the MB11 strain generated using the KpnI restriction enzyme showed that the approach used to assemble the genome was satisfactory, producing good alignment between the sequence observed in vitro and that obtained in silico. In the resulting Neighbor-Joining dendrogram, the C. pseudotuberculosis strains sequenced in this study were clustered into a single clade supported by a high bootstrap value. The structural analysis showed that the genomes of the MB11 and MB14 strains were very similar, while the MB30 and MB66 strains had several inverted regions. The observed genomic characteristics were similar to those described for other strains of the same species, despite the number of inversions found. These genomes will serve as a basis for determining the relationship between the genotype of the pathogen and the type of infection that it causes.

The assembly and annotation of the complete Rufous-bellied thrush mitochondrial genome.

Among known bird species, oscines are one of the few groups that produce complex vocalizations due to vocal learning. One of the most conspicuous oscine passerines in southeastern South America is the Rufous-bellied Thrush, Turdus rufiventris. The complete mitochondrial genome of this species was sequenced with the Illumina HiSeq platform (Illumina Inc., San Diego, CA), assembled using MITObim software and annotated by MITOS web server and Artemis software. This mitogenome contained 16 669 bases, organized as 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, and a control region (d-loop). The sequencing of the Rufous-bellied Thrush mitochondrial genome is of particular interest for better understanding of population genetics and phylogeography of the Turdidae family.

Genome Sequence of Corynebacterium pseudotuberculosis Strain PA02 Isolated from an Ovine Host in the Amazon.

In this work, we report the complete genome sequence of Corynebacterium pseudotuberculosis strain PA02 isolated from an ovine host. The genome contains 2,328,435 bp, a 52.2% G+C content, 2,035 coding sequences, 12 rRNA operons, 45 tRNAs, and 14 predicted pseudogenes.

GapBlaster-A Graphical Gap Filler for Prokaryote Genomes.

The advent of NGS (Next Generation Sequencing) technologies has resulted in an exponential increase in the number of complete genomes available in biological databases. This advance has allowed the development of several computational tools enabling analyses of large amounts of data in each of the various steps, from processing and quality filtering to gap filling and manual curation. The tools developed for gap closure are very useful as they result in more complete genomes, which will influence downstream analyses of genomic plasticity and comparative genomics. However, the gap filling step remains a challenge for genome assembly, often requiring manual intervention. Here, we present GapBlaster, a graphical application to evaluate and close gaps. GapBlaster was developed via Java programming language. The software uses contigs obtained in the assembly of the genome to perform an alignment against a draft of the genome/scaffold, using BLAST or Mummer to close gaps. Then, all identified alignments of contigs that extend through the gaps in the draft sequence are presented to the user for further evaluation via the GapBlaster graphical interface. GapBlaster presents significant results compared to other similar software and has the advantage of offering a graphical interface for manual curation of the gaps. GapBlaster program, the user guide and the test datasets are freely available at https://sourceforge.net/projects/gapblaster2015/. It requires Sun JDK 8 and Blast or Mummer.

De novo assembly and characterization of the Trichuris trichiura adult worm transcriptome using Ion Torrent sequencing.

Infection with helminthic parasites, including the soil-transmitted helminth Trichuris trichiura (human whipworm), has been shown to modulate host immune responses and, consequently, to have an impact on the development and manifestation of chronic human inflammatory diseases. De novo derivation of helminth proteomes from sequencing of transcriptomes will provide valuable data to aid identification of parasite proteins that could be evaluated as potential immunotherapeutic molecules in near future. Herein, we characterized the transcriptome of the adult stage of the human whipworm T. trichiura, using next-generation sequencing technology and a de novo assembly strategy. Nearly 17.6 million high-quality clean reads were assembled into 6414 contiguous sequences, with an N50 of 1606bp. In total, 5673 protein-encoding sequences were confidentially identified in the T. trichiura adult worm transcriptome; of these, 1013 sequences represent potential newly discovered proteins for the species, most of which presenting orthologs already annotated in the related species T. suis. A number of transcripts representing probable novel non-coding transcripts for the species T. trichiura were also identified. Among the most abundant transcripts, we found sequences that code for proteins involved in lipid transport, such as vitellogenins, and several chitin-binding proteins. Through a cross-species expression analysis of gene orthologs shared by T. trichiura and the closely related parasites T. suis and T. muris it was possible to find twenty-six protein-encoding genes that are consistently highly expressed in the adult stages of the three helminth species. Additionally, twenty transcripts could be identified that code for proteins previously detected by mass spectrometry analysis of protein fractions of the whipworm somatic extract that present immunomodulatory activities. Five of these transcripts were amongst the most highly expressed protein-encoding sequences in the T. trichiura adult worm. Besides, orthologs of proteins demonstrated to have potent immunomodulatory properties in related parasitic helminths were also predicted from the T. trichiura de novo assembled transcriptome.

Draft Genome Sequences of Two Pathogenic Corynebacterial Species Isolated from Cows.

The species Corynebacterium renale, Corynebacterium pilosum, and Corynebacterium cystitidis were initially thought to be the same species C. renale, but with different immunological types. These bacteria are the causative agent of cystitis, urethritis and pyelonephritis and are found usually as constituents of the normal flora in the lower urogenital tract of cattle. Therefore, we present the draft genome sequences of two pathogenic Corynebacterium species: C. renale CIP 52.96 and C. pilosum CIP 103422. The genome sequences of these species have 2,322,762 bp with 2,218 protein encoding genes and 2,548,014 bp with 2,428 protein encoding genes, respectively. These genomes can help clarify the virulence mechanisms of these unknown bacteria and enable the development of more effective methods for control.

Complete Genome Sequence of Corynebacterium pseudotuberculosis Strain PA01, Isolated from Sheep in Pará, Brazil.

Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis disease. In this work, we present the first complete genome sequence of Corynebacterium pseudotuberculosis strain PA01, isolated in northern Brazil from an infected sheep. The genome length is 2,337,920 bp, and 2,003 coding sequences (CDS), 12 rRNAs, and 49 tRNAs were predicted.

GIPSy: Genomic island prediction software.

Bacteria are highly diverse organisms that are able to adapt to a broad range of environments and hosts due to their high genomic plasticity. Horizontal gene transfer plays a pivotal role in this genome plasticity and in evolution by leaps through the incorporation of large blocks of genome sequences, ordinarily known as genomic islands (GEIs). GEIs may harbor genes encoding virulence, metabolism, antibiotic resistance and symbiosis-related functions, namely pathogenicity islands (PAIs), metabolic islands (MIs), resistance islands (RIs) and symbiotic islands (SIs). Although many software for the prediction of GEIs exist, they only focus on PAI prediction and present other limitations, such as complicated installation and inconvenient user interfaces. Here, we present GIPSy, the genomic island prediction software, a standalone and user-friendly software for the prediction of GEIs, built on our previously developed pathogenicity island prediction software (PIPS). We also present four application cases in which we crosslink data from literature to PAIs, MIs, RIs and SIs predicted by GIPSy. Briefly, GIPSy correctly predicted the following previously described GEIs: 13 PAIs larger than 30kb in Escherichia coli CFT073; 1 MI for Burkholderia pseudomallei K96243, which seems to be a miscellaneous island; 1 RI of Acinetobacter baumannii AYE, named AbaR1; and, 1 SI of Mesorhizobium loti MAFF303099 presenting a mosaic structure. GIPSy is the first life-style-specific genomic island prediction software to perform analyses of PAIs, MIs, RIs and SIs, opening a door for a better understanding of bacterial genome plasticity and the adaptation to new traits.

Corynebacterium pseudotuberculosis RNA-seq data from abiotic stresses.

Corynebacterium pseudotuberculosis causes significant loss to goat and sheep farmers because it is the causal agent of the infectious disease caseous lymphadenitis, which may lead to outcomes ranging from skin injury to animal death (Ruiz et al., 2011) [1]. This bacterium was grown under osmotic (2 M), acid (pH) and heat (50 °C) stress and under control (Normal-BHI brain heart infusion) conditions, which simulate the conditions faced by the bacteria during the infectious process. Subsequently, cDNA of each condition was sequenced by the SOLiD3 Plus platform using the RNA-Seq technique [2], [3], [4]. The data produced was processed to evaluate the differential gene expression, which is helpful to understand the adaptation mechanisms during the infection in the host. The sequencing data of all conditions are available in the European Bioinformatics Institute (EBI) repository under accession number E-MTAB-2017.

The impact of quality filter for RNA-Seq.

BACKGROUND: With the emergence of large-scale sequencing platforms since 2005, there has been a great revolution regarding methods for decoding DNA sequences, which have also affected quantitative and qualitative gene expression analyses through the RNA-Sequencing technique. However, issues related to the amount of data required for the analyses have been considered because they affect the reliability of the experiments. Thus, RNA depletion during sample preparation may influence the results. Moreover, because data produced by these platforms show variations in quality, quality filters are often used to remove sequences likely to contain errors to increase the accuracy of the results. However, when reads of quality filters are removed, the expression profile in RNA-Seq experiments may be influenced. RESULT: The present study aimed to analyze the impact of different quality filter values for Corynebacterium pseudotuberculosis (sequenced by SOLiD platform), Microcystis aeruginosa and Kineococcus radiotolerans (sequenced by Illumina platform) RNA-Seq data. Although up to 47.9% of the reads produced by the SOLiD technology were removed after the QV20 quality filter is applied, and 15.85% were removed from K. radiotolerans data set using the QV30 filter, Illumina data showed the largest number of unique differentially expressed genes after applying the most stringent filter (QV30), with 69 genes. In contrast, for SOLiD, the acid stress condition with the QV20 filter yielded only 41 unique differentially expressed genes. Even for the highest quality M. aeruginosa data, the quality filter affected the expression profile. The most stringent quality filter generated a greater number of unique differentially expressed genes: 9 for high molecular weight dissolved organic matter condition and 12 for low P conditions. CONCLUSION: Even high-accuracy sequencing technologies are subject to the influence of quality filters when evaluating RNA-Seq data using the reference approach.

Draft Genome Sequences of Vibrio fluvialis Strains 560 and 539, Isolated from Environmental Samples.

Vibrio fluvialis is a halophilic bacterium found in many environments and is mainly associated with sporadic cases and outbreaks of gastroenteritis in humans. Here, we describe the genome sequences of environmental strains of V. fluvialis 560 (Vf560) and V. fluvialis 539 (Vf539) possessing a variant of the integrative and conjugative element (ICE) SXT for the first time in Brazil and South America.