ViCTree: an automated framework for taxonomic classification from protein sequences

Motivation: The increasing rate of submission of genetic sequences into public databases is providing a growing resource for classifying the organisms that these sequences represent. To aid viral classification, we have developed ViCTree, which automatically integrates the relevant sets of sequences in NCBI GenBank and transforms them into an interactive maximum likelihood phylogenetic tree that can be updated automatically. ViCTree incorporates ViCTreeView, which is a JavaScript-based visualisation tool that enables the tree to be explored interactively in the context of pairwise distance data. Results: To demonstrate utility, ViCTree was applied to subfamily Densovirinae of family Parvoviridae. This led to the identification of six new species of insect virus. Availability: ViCTree is open-source and can be run on any Linux- or Unix-based computer or cluster. A tutorial, the documentation and the source code are available under a GPL3 license, and can be accessed at http://bioinformatics.cvr.ac.uk/victree_web/

XenDB: Full length cDNA prediction and cross species mapping in Xenopus laevis

BACKGROUND: Research using the model system Xenopus laevis has provided critical insights into the mechanisms of early vertebrate development and cell biology. Large scale sequencing efforts have provided an increasingly important resource for researchers. To provide full advantage of the available sequence, we have analyzed 350,468 Xenopus laevis Expressed Sequence Tags (ESTs) both to identify full length protein encoding sequences and to develop a unique database system to support comparative approaches between X. laevis and other model systems. DESCRIPTION: Using a suffix array based clustering approach, we have identified 25,971 clusters and 40,877 singleton sequences. Generation of a consensus sequence for each cluster resulted in 31,353 tentative contig and 4,801 singleton sequences. Using both BLASTX and FASTY comparison to five model organisms and the NR protein database, more than 15,000 sequences are predicted to encode full length proteins and these have been matched to publicly available IMAGE clones when available. Each sequence has been compared to the KOG database and ~67% of the sequences have been assigned a putative functional category. Based on sequence homology to mouse and human, putative GO annotations have been determined. CONCLUSION: The results of the analysis have been stored in a publicly available database XenDB . A unique capability of the database is the ability to batch upload cross species queries to identify potential Xenopus homologues and their associated full length clones. Examples are provided including mapping of microarray results and application of 'in silico' analysis. The ability to quickly translate the results of various species into 'Xenopus-centric' information should greatly enhance comparative embryological approaches. Supplementary material can be found at

Diversity in parasitic nematode genomes: the microRNAs of Brugia pahangi and Haemonchus contortus are largely novel

<b>BACKGROUND:</b> MicroRNAs (miRNAs) play key roles in regulating post-transcriptional gene expression and are essential for development in the free-living nematode Caenorhabditis elegans and in higher organisms. Whether microRNAs are involved in regulating developmental programs of parasitic nematodes is currently unknown. Here we describe the the miRNA repertoire of two important parasitic nematodes as an essential first step in addressing this question. <b>RESULTS:</b> The small RNAs from larval and adult stages of two parasitic species, Brugia pahangi and Haemonchus contortus, were identified using deep-sequencing and bioinformatic approaches. Comparative analysis to known miRNA sequences reveals that the majority of these miRNAs are novel. Some novel miRNAs are abundantly expressed and display developmental regulation, suggesting important functional roles. Despite the lack of conservation in the miRNA repertoire, genomic positioning of certain miRNAs within or close to specific coding genes is remarkably conserved across diverse species, indicating selection for these associations. Endogenous small-interfering RNAs and Piwi-interacting (pi)RNAs, which regulate gene and transposon expression, were also identified. piRNAs are expressed in adult stage H. contortus, supporting a conserved role in germline maintenance in some parasitic nematodes. <b>CONCLUSIONS:</b> This in-depth comparative analysis of nematode miRNAs reveals the high level of divergence across species and identifies novel sequences potentially involved in development. Expression of novel miRNAs may reflect adaptations to different environments and lifestyles. Our findings provide a detailed foundation for further study of the evolution and function of miRNAs within nematodes and for identifying potential targets for intervention

TBestDB: a taxonomically broad database of expressed sequence tags (ESTs)

The TBestDB database contains ∼370 000 clustered expressed sequence tag (EST) sequences from 49 organisms, covering a taxonomically broad range of poorly studied, mainly unicellular eukaryotes, and includes experimental information, consensus sequences, gene annotations and metabolic pathway predictions. Most of these ESTs have been generated by the Protist EST Program, a collaboration among six Canadian research groups. EST sequences are read from trace files up to a minimum quality cut-off, vector and linker sequence is masked, and the ESTs are clustered using phrap. The resulting consensus sequences are automatically annotated by using the AutoFACT program. The datasets are automatically checked for clustering errors due to chimerism and potential cross-contamination between organisms, and suspect data are flagged in or removed from the database. Access to data deposited in TBestDB by individual users can be restricted to those users for a limited period. With this first report on TBestDB, we open the database to the research community for free processing, annotation, interspecies comparisons and GenBank submission of EST data generated in individual laboratories. For instructions on submission to TBestDB, contact [email protected]. The database can be queried at

Coordinated RNA-Seq and peptidomics identify neuropeptides and G-protein coupled receptors (GPCRs) in the large pine weevil Hylobius abietis, a major forestry pest

Hylobius abietis (Linnaeus), or large pine weevil (Coleoptera, Curculionidae), is a pest of European coniferous forests. In order to gain understanding of the functional physiology of this species, we have assembled a de novo transcriptome of H. abietis, from sequence data obtained by Next Generation Sequencing. In particular, we have identified genes encoding neuropeptides, peptide hormones and their putative G-protein coupled receptors (GPCRs) to gain insights into neuropeptide-modulated processes. The transcriptome was assembled de novo from pooled paired-end, sequence reads obtained from RNA from whole adults, gut and central nervous system tissue samples. Data analysis was performed on the transcripts obtained from the assembly including, annotation, gene ontology and functional assignment as well as transcriptome completeness assessment and KEGG pathway analysis. Pipelines were created using Bioinformatics tools and techniques for prediction and identification of neuropeptides and neuropeptide receptors. Peptidomic analysis was also carried out using a combination of MALDI-TOF as well as Q-Exactive Orbitrap mass spectrometry to confirm the identified neuropeptide. 41 putative neuropeptide families were identified in H. abietis, including Adipokinetic hormone (AKH), CAPA and DH31. Neuropeptide F, which has not been yet identified in the model beetle T. castaneum, was identified. Additionally, 24 putative neuropeptide and 9 leucine-rich repeat containing G protein coupled receptor-encoding transcripts were determined using both alignment as well as non-alignment methods. This information, submitted to the NCBI sequence read archive repository (SRA accession: SRP133355), can now be used to inform understanding of neuropeptide-modulated physiology and behaviour in H. abietis; and to develop specific neuropeptide-based tools for H. abietis control

OrthoSelect: a protocol for selecting orthologous groups in phylogenomics

Background: Phylogenetic studies using expressed sequence tags (EST) are becoming a standard approach to answer evolutionary questions. Such studies are usually based on large sets of newly generated, unannotated, and error-prone EST sequences from different species. A first crucial step in EST-based phylogeny reconstruction is to identify groups of orthologous sequences. From these data sets, appropriate target genes are selected, and redundant sequences are eliminated to obtain suitable sequence sets as input data for tree-reconstruction software. Generating such data sets manually can be very time consuming. Thus, software tools are needed that carry out these steps automatically. Results: We developed a flexible and user-friendly software pipeline, running on desktop machines or computer clusters, that constructs data sets for phylogenomic analyses. It automatically searches assembled EST sequences against databases of orthologous groups (OG), assigns ESTs to these predefined OGs, translates the sequences into proteins, eliminates redundant sequences assigned to the same OG, creates multiple sequence alignments of identified orthologous sequences and offers the possibility to further process this alignment in a last step by excluding potentially homoplastic sites and selecting sufficiently conserved parts. Our software pipeline can be used as it is, but it can also be adapted by integrating additional external programs. This makes the pipeline useful for non-bioinformaticians as well as to bioinformatic experts. The software pipeline is especially designed for ESTs, but it can also handle protein sequences. Conclusion: OrthoSelect is a tool that produces orthologous gene alignments from assembled ESTs. Our tests show that OrthoSelect detects orthologs in EST libraries with high accuracy. In the absence of a gold standard for orthology prediction, we compared predictions by OrthoSelect to a manually created and published phylogenomic data set. Our tool was not only able to rebuild the data set with a specificity of 98%, but it detected four percent more orthologous sequences. Furthermore, the results OrthoSelect produces are in absolut agreement with the results of other programs, but our tool offers a significant speedup and additional functionality, e.g. handling of ESTs, computing sequence alignments, and refining them. To our knowledge, there is currently no fully automated and freely available tool for this purpose. Thus, OrthoSelect is a valuable tool for researchers in the field of phylogenomics who deal with large quantities of EST sequences. OrthoSelect is written in Perl and runs on Linux/Mac OS X

Species Identification and Profiling of Complex Microbial Communities Using Shotgun Illumina Sequencing of 16S rRNA Amplicon Sequences

The high throughput and cost-effectiveness afforded by short-read sequencing technologies, in principle, enable researchers to perform 16S rRNA profiling of complex microbial communities at unprecedented depth and resolution. Existing Illumina sequencing protocols are, however, limited by the fraction of the 16S rRNA gene that is interrogated and therefore limit the resolution and quality of the profiling. To address this, we present the design of a novel protocol for shotgun Illumina sequencing of the bacterial 16S rRNA gene, optimized to capture more than 90% of sequences in the Greengenes database and with nearly twice the resolution of existing protocols. Using several in silico and experimental datasets, we demonstrate that despite the presence of multiple variable and conserved regions, the resulting shotgun sequences can be used to accurately quantify the diversity of complex microbial communities. The reconstruction of a significant fraction of the 16S rRNA gene also enabled high precision (>90%) in species-level identification thereby opening up potential application of this approach for clinical microbial characterization.Comment: 17 pages, 2 tables, 2 figures, supplementary materia

LotuS2: an ultrafast and highly accurate tool for amplicon sequencing analysis

Background: Amplicon sequencing is an established and cost-efficient method for profiling microbiomes. However, many available tools to process this data require both bioinformatics skills and high computational power to process big datasets. Furthermore, there are only few tools that allow for long read amplicon data analysis. To bridge this gap, we developed the LotuS2 (less OTU scripts 2) pipeline, enabling user-friendly, resource friendly, and versatile analysis of raw amplicon sequences.Results: In LotuS2, six different sequence clustering algorithms as well as extensive pre- and post-processing options allow for flexible data analysis by both experts, where parameters can be fully adjusted, and novices, where defaults are provided for different scenarios.We benchmarked three independent gut and soil datasets, where LotuS2 was on average 29 times faster compared to other pipelines, yet could better reproduce the alpha- and beta-diversity of technical replicate samples. Further benchmarking a mock community with known taxon composition showed that, compared to the other pipelines, LotuS2 recovered a higher fraction of correctly identified taxa and a higher fraction of reads assigned to true taxa (48% and 57% at species; 83% and 98% at genus level, respectively). At ASV/OTU level, precision and F-score were highest for LotuS2, as was the fraction of correctly reported 16S sequences.Conclusion: LotuS2 is a lightweight and user-friendly pipeline that is fast, precise, and streamlined, using extensive pre- and post-ASV/OTU clustering steps to further increase data quality. High data usage rates and reliability enable high-throughput microbiome analysis in minutes

Optimizing transcriptome analysis using short-read RNA-seq in Atlantic salmon

Målet med studiet var å sammenligne genome-guided assembly og de novo assembly av transcriptomdata, for å finne ut hvilken metode som burde anbefales. De novo assembly er mye mere krevende angående datalagringsplass og tidsbruk, og resultatet av denne metoden bør være signifikant bedre for å rettferdiggjøre bruk av metoden. Gjennom en rekke dataanalyser og optimaliseringssteg ble metodene sammenlignet på tre ulike vis; read mapping ratio til genom og transkripter, BLAST hits av transkripter til genom og proteinsekvenser, samt antall og ratio av uttrykte gener i genom-guided assembly og BLAST-hits til proteinsekvenser. De novo assembly utvidet annoteringen signifikant, i vårt tilfelle med mer enn 20,000 transkripter. Genome-guided assembly ga bedre mapping, men metoden gir ingen nye oppdagelser i sekvensene. De novo assembly anbefales for utvidet annotering av gener.The aim of the study was to compare genome-guided and de novo assembly of transcriptomic data, to find out which method to recommend. De novo assembly is much more demanding regarding computational storage and time, and the result of this method should be significantly better to justify using this method. Through a number of computational analysis and optimization steps, the methods were compared in three different matters; read mapping rate to genome and transcripts, BLAST hits of transcripts to the genome and protein database and number and ratio of expressed genes in genome-guided assembly and BLAST hits to protein database. De novo assembly extended the annotation significantly, in our case with more than 20,000 transcripts. Genome-guided assembly gave better mapping, but the method does not give novel discoveries. De novo assembly is recommended for an extended annotation of genes.M-BIA

The Porcelain Crab Transcriptome and PCAD, the Porcelain Crab Microarray and Sequence Database

BACKGROUND: With the emergence of a completed genome sequence of the freshwater crustacean Daphnia pulex, construction of genomic-scale sequence databases for additional crustacean sequences are important for comparative genomics and annotation. Porcelain crabs, genus Petrolisthes, have been powerful crustacean models for environmental and evolutionary physiology with respect to thermal adaptation and understanding responses of marine organisms to climate change. Here, we present a large-scale EST sequencing and cDNA microarray database project for the porcelain crab Petrolisthes cinctipes. METHODOLOGY/PRINCIPAL FINDINGS: A set of approximately 30K unique sequences (UniSeqs) representing approximately 19K clusters were generated from approximately 98K high quality ESTs from a set of tissue specific non-normalized and mixed-tissue normalized cDNA libraries from the porcelain crab Petrolisthes cinctipes. Homology for each UniSeq was assessed using BLAST, InterProScan, GO and KEGG database searches. Approximately 66% of the UniSeqs had homology in at least one of the databases. All EST and UniSeq sequences along with annotation results and coordinated cDNA microarray datasets have been made publicly accessible at the Porcelain Crab Array Database (PCAD), a feature-enriched version of the Stanford and Longhorn Array Databases. CONCLUSIONS/SIGNIFICANCE: The EST project presented here represents the third largest sequencing effort for any crustacean, and the largest effort for any crab species. Our assembly and clustering results suggest that our porcelain crab EST data set is equally diverse to the much larger EST set generated in the Daphnia pulex genome sequencing project, and thus will be an important resource to the Daphnia research community. Our homology results support the pancrustacea hypothesis and suggest that Malacostraca may be ancestral to Branchiopoda and Hexapoda. Our results also suggest that our cDNA microarrays cover as much of the transcriptome as can reasonably be captured in EST library sequencing approaches, and thus represent a rich resource for studies of environmental genomics