MapRepeat: an approach for effective assembly of repetitive regions in prokaryotic genomes

The newest technologies for DNA sequencing have led to the determination of the primary structure of the genomes of organisms, mainly prokaryotes, with high efficiency and at lower costs. However, the presence of regions with repetitive sequences, in addition to the short reads produced by the Next-Generation Sequencing (NGS) platforms, created a lot of difficulty in reconstructing the original genome in silico. Thus, even today, genome assembly continues to be one of the major challenges in bioinformatics specifically when repetitive sequences are considered. In this paper, we present an approach to assemble repetitive regions in prokaryotic genomes. Our methodology enables (i) the identification of these regions through visual tools, (ii) the characterization of sequences on the extremities of gaps and (iii) the extraction of consensus sequences based on mapping of raw data to a reference genome. We also present a case study on the assembly of regions that encode ribosomal RNAs (rRNA) in the genome of Corynebacterium ulceransFRC11, in order to show the efficiency of the strategies presented here. The proposed methods and tools will help in finishing genome assemblies, besides reducing the running time and associated costs. Availability All scripts are available at http://github.com/dcbmariano/maprepea

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

Genome Sequence of Corynebacterium pseudotuberculosis MB20 bv. equi Isolated from a Pectoral Abscess of an Oldenburg Horse in California.

The genome of Corynebacterium pseudotuberculosis MB20 bv. equi was sequenced using the Ion Personal Genome Machine (PGM) platform, and showed a size of 2,363,089 bp, with 2,365 coding sequences and a GC content of 52.1%. These results will serve as a basis for further studies on the pathogenicity of C. pseudotuberculosis bv. equi

Analysis of quality raw data of second generation sequencers with Quality Assessment Software

<p>Abstract</p> <p>Background</p> <p>Second generation technologies have advantages over Sanger; however, they have resulted in new challenges for the genome construction process, especially because of the small size of the reads, despite the high degree of coverage. Independent of the program chosen for the construction process, DNA sequences are superimposed, based on identity, to extend the reads, generating contigs; mismatches indicate a lack of homology and are not included. This process improves our confidence in the sequences that are generated.</p> <p>Findings</p> <p>We developed Quality Assessment Software, with which one can review graphs showing the distribution of quality values from the sequencing reads. This software allow us to adopt more stringent quality standards for sequence data, based on quality-graph analysis and estimated coverage after applying the quality filter, providing acceptable sequence coverage for genome construction from short reads.</p> <p>Conclusions</p> <p>Quality filtering is a fundamental step in the process of constructing genomes, as it reduces the frequency of incorrect alignments that are caused by measuring errors, which can occur during the construction process due to the size of the reads, provoking misassemblies. Application of quality filters to sequence data, using the software Quality Assessment, along with graphing analyses, provided greater precision in the definition of cutoff parameters, which increased the accuracy of genome construction.</p

High-throughput sequencing of black pepper root transcriptome

Abstract\ud \ud Background\ud Black pepper (Piper nigrum L.) is one of the most popular spices in the world. It is used in cooking and the preservation of food and even has medicinal properties. Losses in production from disease are a major limitation in the culture of this crop. The major diseases are root rot and foot rot, which are results of root infection by Fusarium solani and Phytophtora capsici, respectively. Understanding the molecular interaction between the pathogens and the host’s root region is important for obtaining resistant cultivars by biotechnological breeding. Genetic and molecular data for this species, though, are limited. In this paper, RNA-Seq technology has been employed, for the first time, to describe the root transcriptome of black pepper.\ud \ud \ud Results\ud The root transcriptome of black pepper was sequenced by the NGS SOLiD platform and assembled using the multiple-k method. Blast2Go and orthoMCL methods were used to annotate 10338 unigenes. The 4472 predicted proteins showed about 52% homology with the Arabidopsis proteome. Two root proteomes identified 615 proteins, which seem to define the plant’s root pattern. Simple-sequence repeats were identified that may be useful in studies of genetic diversity and may have applications in biotechnology and ecology.\ud \ud \ud Conclusions\ud This dataset of 10338 unigenes is crucially important for the biotechnological breeding of black pepper and the ecogenomics of the Magnoliids, a major group of basal angiosperms.FAPESPAUFPAFINEPCAPE

Omics profiles used to evaluate the gene expression of Exiguobacterium antarcticum B7 during cold adaptation

Abstract\ud \ud Background\ud \ud Exiguobacterium antarcticum strain B7 is a Gram-positive psychrotrophic bacterial species isolated in Antarctica. Although this bacteria has been poorly studied, its genome has already been sequenced. Therefore, it is an appropriate model for the study of thermal adaptation. In the present study, we analyzed the transcriptomes and proteomes of E. antarcticum B7 grown at 0°C and 37°C by SOLiD RNA-Seq, Ion Torrent RNA-Seq and two-dimensional difference gel electrophoresis tandem mass spectrometry (2D-DIGE-MS/MS).\ud \ud \ud Results\ud We found expression of 2,058 transcripts in all replicates from both platforms and differential expression of 564 genes (absolute log2FC ≥1, P-value <0.001) comparing the two temperatures by RNA-Seq. A total of 73 spots were differentially expressed between the two temperatures on 2D-DIGE, 25 of which were identified by MS/MS. Some proteins exhibited patterns of dispersion in the gel that are characteristic of post-translational modifications.\ud \ud \ud Conclusions\ud Our findings suggest that the two sequencing platforms yielded similar results and that different omic approaches may be used to improve the understanding of gene expression. To adapt to low temperatures, E. antarcticum B7 expresses four of the six cold-shock proteins present in its genome. The cold-shock proteins were the most abundant in the bacterial proteome at 0°C. Some of the differentially expressed genes are required to preserve transcription and translation, while others encode proteins that contribute to the maintenance of the intracellular environment and appropriate protein folding. The results denote the complexity intrinsic to the adaptation of psychrotrophic organisms to cold environments and are based on two omic approaches. They also unveil the lifestyle of a bacterial species isolated in Antarctica.CNPqCAPESUFPAFINEPFAPEMIGFundação para a Ciência e a Tecnologia de Portugal (FCT

PIPS: Pathogenicity Island Prediction Software

The adaptability of pathogenic bacteria to hosts is influenced by the genomic plasticity of the bacteria, which can be increased by such mechanisms as horizontal gene transfer. Pathogenicity islands play a major role in this type of gene transfer because they are large, horizontally acquired regions that harbor clusters of virulence genes that mediate the adhesion, colonization, invasion, immune system evasion, and toxigenic properties of the acceptor organism. Currently, pathogenicity islands are mainly identified in silico based on various characteristic features: (1) deviations in codon usage, G+C content or dinucleotide frequency and (2) insertion sequences and/or tRNA genetic flanking regions together with transposase coding genes. Several computational techniques for identifying pathogenicity islands exist. However, most of these techniques are only directed at the detection of horizontally transferred genes and/or the absence of certain genomic regions of the pathogenic bacterium in closely related non-pathogenic species. Here, we present a novel software suite designed for the prediction of pathogenicity islands (pathogenicity island prediction software, or PIPS). In contrast to other existing tools, our approach is capable of utilizing multiple features for pathogenicity island detection in an integrative manner. We show that PIPS provides better accuracy than other available software packages. As an example, we used PIPS to study the veterinary pathogen Corynebacterium pseudotuberculosis, in which we identified seven putative pathogenicity islands

Bright light therapy versus physical exercise to prevent co-morbid depression and obesity in adolescents and young adults with attention-deficit/hyperactivity disorder: study protocol for a randomized controlled trial

Background: The risk for major depression and obesity is increased in adolescents and adults with attention-deficit / hyperactivity disorder (ADHD) and adolescent ADHD predicts adult depression and obesity. Non-pharmacological interventions to treat and prevent these co-morbidities are urgently needed. Bright light therapy (BLT) improves day– night rhythm and is an emerging therapy for major depression. Exercise intervention (EI) reduces obesity and improves depressive symptoms. To date, no randomized controlled trial (RCT) has been performed to establish feasibility and efficacy of these interventions targeting the prevention of co-morbid depression and obesity in ADHD. We hypothesize that the two manualized interventions in combination with mobile health-based monitoring and reinforcement will result in less depressive symptoms and obesity compared to treatment as usual in adolescents and young adults with ADHD. Methods: This trial is a prospective, pilot phase-IIa, parallel-group RCT with three arms (two add-on treatment groups [BLT, EI] and one treatment as usual [TAU] control group). The primary outcome variable is change in the Inventory of Depressive Symptomatology total score (observer-blinded assessment) between baseline and ten weeks of intervention. This variable is analyzed with a mixed model for repeated measures approach investigating the treatment effect with respect to all three groups. A total of 330 participants with ADHD, aged 14 – < 30 years, will be screened at the four study centers. To establish effect sizes, the sample size was planned at the liberal significance level of α = 0.10 (two-sided) and the power of 1-β = 80% in order to find medium effects. Secondary outcomes measures including change in obesity, ADHD symptoms, general psychopathology, health-related quality of life, neurocognitive function, chronotype, and physical fitness are explored after the end of the intervention and at the 12-week follow-up. This is the first pilot RCT on the use of BLT and EI in combination with mobile health-based monitoring and reinforcement targeting the prevention of co-morbid depression and obesity in adolescents and young adults with ADHD. If at least medium effects can be established with regard to the prevention of depressive symptoms and obesity, a larger scale confirmatory phase-III trial may be warranted.The trial is funded by the EU Framework Programme for Research and Innovation, Horizon 2020 (Project no. 667302). Funding period: January 2016–December 2020. This funding source had no role in the design of this study and will not have any role during its execution, analyses, interpretation of the data, or decision to submit results. Some local funds additionally contributed to carry out this study, especially for the preparation of the interventions: FBO research activity is by the Spanish Ministry of Economy and Competitiveness – MINECO (RYC-2011-09011) and by the University of Granada, Plan Propio de Investigación 2016, Excellence actions: Unit of Excellence on Exercise and Health (UCEES)

Evidence for Reductive Genome Evolution and Lateral Acquisition of Virulence Functions in Two Corynebacterium pseudotuberculosis Strains

Ruiz JC, D'Afonseca V, Silva A, et al. Evidence for Reductive Genome Evolution and Lateral Acquisition of Virulence Functions in Two Corynebacterium pseudotuberculosis Strains. PLoS ONE. 2011;6(4): e18551.Background: Corynebacterium pseudotuberculosis, a Gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity. Methodology and Findings: We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer. Conclusions: These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers CP001809 and CP001829