Xylella fastidiosa comparative genomic database is an information resource to explore the annotation, genomic features, and biology of different strains

The Xylella fastidiosa comparative genomic database is a scientific resource with the aim to provide a user-friendly interface for accessing high-quality manually curated genomic annotation and comparative sequence analysis, as well as for identifying and mapping prophage-like elements, a marked feature of Xylella genomes. Here we describe a database and tools for exploring the biology of this important plant pathogen. The hallmarks of this database are the high quality genomic annotation, the functional and comparative genomic analysis and the identification and mapping of prophage-like elements. It is available from web site http://www.xylella.lncc.br.

A transcriptomic analysis of striped catfish (Pangasianodon hypophthalmus) in response to salinity adaptation: De novo assembly, gene annotation and marker discovery

The striped catfish (Pangasianodon hypophthalmus) culture industry in the Mekong Delta in Vietnam has developed rapidly over the past decade. The culture industry now however, faces some significant challenges, especially related to climate change impacts notably from predicted extensive saltwater intrusion into many low topographical coastal provinces across the Mekong Delta. This problem highlights a need for development of culture stocks that can tolerate more saline culture environments as a response to expansion of saline water-intruded land. While a traditional artificial selection program can potentially address this need, understanding the genomic basis of salinity tolerance can assist development of more productive culture lines. The current study applied a transcriptomic approach using Ion PGM technology to generate expressed sequence tag (EST) resources from the intestine and swim bladder from striped catfish reared at a salinity level of 9 ppt which showed best growth performance. Total sequence data generated was 467.8 Mbp, consisting of 4,116,424 reads with an average length of 112 bp. De novo assembly was employed that generated 51,188 contigs, and allowed identification of 16,116 putative genes based on the GenBank non-redundant database. GO annotation, KEGG pathway mapping, and functional annotation of the EST sequences recovered with a wide diversity of biological functions and processes. In addition, more than 11,600 simple sequence repeats were also detected. This is the first comprehensive analysis of a striped catfish transcriptome, and provides a valuable genomic resource for future selective breeding programs and functional or evolutionary studies of genes that influence salinity tolerance in this important culture species.

Sequencing, annotation and comparative analysis of nine BACs of giant panda (Ailuropoda melanoleuca)

A 10-fold BAC library for giant panda was constructed and nine BACs were selected to generate finish sequences. These BACs could be used as a validation resource for the de novo assembly accuracy of the whole genome shotgun sequencing reads of giant panda newly generated by the Illumina GA sequencing technology. Complete sanger sequencing, assembly, annotation and comparative analysis were carried out on the selected BACs of a joint length 878 kb. Homologue search and de novo prediction methods were used to annotate genes and repeats. Twelve protein coding genes were predicted, seven of which could be functionally annotated. The seven genes have an average gene size of about 41 kb, an average coding size of about 1.2 kb and an average exon number of 6 per gene. Besides, seven tRNA genes were found. About 27 percent of the BAC sequence is composed of repeats. A phylogenetic tree was constructed using neighbor-join algorithm across five species, including giant panda, human, dog, cat and mouse, which reconfirms dog as the most related species to giant panda. Our results provide detailed sequence and structure information for new genes and repeats of giant panda, which will be helpful for further studies on the giant panda.

Design and implementation of bioinformatics tools for large scale genome annotation

The continuous increase of genome sequencing projects produced a huge amount of data in the last 10 years: currently more than 600 prokaryotic and 80 eukaryotic genomes are fully sequenced and publically available. However the sole sequencing process of a genome is able to determine just raw nucleotide sequences. This is only the first step of the genome annotation process that will deal with the issue of assigning biological information to each sequence. The annotation process is done at each different level of the biological information processing mechanism, from DNA to protein, and cannot be accomplished only by in vitro analysis procedures resulting extremely expensive and time consuming when applied at a this large scale level. Thus, in silico methods need to be used to accomplish the task. The aim of this work was the implementation of predictive computational methods to allow a fast, reliable, and automated annotation of genomes and proteins starting from aminoacidic sequences. The first part of the work was focused on the implementation of a new machine learning based method for the prediction of the subcellular localization of soluble eukaryotic proteins. The method is called BaCelLo, and was developed in 2006. The main peculiarity of the method is to be independent from biases present in the training dataset, which causes the over‐prediction of the most represented examples in all the other available predictors developed so far. This important result was achieved by a modification, made by myself, to the standard Support Vector Machine (SVM) algorithm with the creation of the so called Balanced SVM. BaCelLo is able to predict the most important subcellular localizations in eukaryotic cells and three, kingdom‐specific, predictors were implemented. In two extensive comparisons, carried out in 2006 and 2008, BaCelLo reported to outperform all the currently available state‐of‐the‐art methods for this prediction task. BaCelLo was subsequently used to completely annotate 5 eukaryotic genomes, by integrating it in a pipeline of predictors developed at the Bologna Biocomputing group by Dr. Pier Luigi Martelli and Dr. Piero Fariselli. An online database, called eSLDB, was developed by integrating, for each aminoacidic sequence extracted from the genome, the predicted subcellular localization merged with experimental and similarity‐based annotations. In the second part of the work a new, machine learning based, method was implemented for the prediction of GPI‐anchored proteins. Basically the method is able to efficiently predict from the raw aminoacidic sequence both the presence of the GPI‐anchor (by means of an SVM), and the position in the sequence of the post‐translational modification event, the so called ω‐site (by means of an Hidden Markov Model (HMM)). The method is called GPIPE and reported to greatly enhance the prediction performances of GPI‐anchored proteins over all the previously developed methods. GPIPE was able to predict up to 88% of the experimentally annotated GPI‐anchored proteins by maintaining a rate of false positive prediction as low as 0.1%. GPIPE was used to completely annotate 81 eukaryotic genomes, and more than 15000 putative GPI‐anchored proteins were predicted, 561 of which are found in H. sapiens. In average 1% of a proteome is predicted as GPI‐anchored. A statistical analysis was performed onto the composition of the regions surrounding the ω‐site that allowed the definition of specific aminoacidic abundances in the different considered regions. Furthermore the hypothesis that compositional biases are present among the four major eukaryotic kingdoms, proposed in literature, was tested and rejected. All the developed predictors and databases are freely available at: BaCelLo http://gpcr.biocomp.unibo.it/bacello eSLDB http://gpcr.biocomp.unibo.it/esldb GPIPE http://gpcr.biocomp.unibo.it/gpipe

The Mycoplasma conjunctivae genome sequencing, annotation and analysis

BACKGROUND: The mollicute Mycoplasma conjunctivae is the etiological agent leading to infectious keratoconjunctivitis (IKC) in domestic sheep and wild caprinae. Although this pathogen is relatively benign for domestic animals treated by antibiotics, it can lead wild animals to blindness and death. This is a major cause of death in the protected species in the Alps (e.g., Capra ibex, Rupicapra rupicapra). METHODS: The genome was sequenced using a combined technique of GS-FLX (454) and Sanger sequencing, and annotated by an automatic pipeline that we designed using several tools interconnected via PERL scripts. The resulting annotations are stored in a MySQL database. RESULTS: The annotated sequence is deposited in the EMBL database (FM864216) and uploaded into the mollicutes database MolliGen http://cbi.labri.fr/outils/molligen/ allowing for comparative genomics. CONCLUSION: We show that our automatic pipeline allows for annotating a complete mycoplasma genome and present several examples of analysis in search for biological targets (e.g., pathogenic proteins).

Relative stability of DNA as a generic criterion for promoter prediction: whole genome annotation of microbial genomes with varying nucleotide base composition

The rapid increase in genome sequence information has necessitated the annotation of their functional elements, particularly those occurring in the non-coding regions, in the genomic context. Promoter region is the key regulatory region, which enables the gene to be transcribed or repressed, but it is difficult to determine experimentally. Hence an in silico identification of promoters is crucial in order to guide experimental work and to pin point the key region that controls the transcription initiation of a gene. In this analysis, we demonstrate that while the promoter regions are in general less stable than the flanking regions, their average free energy varies depending on the GC composition of the flanking genomic sequence. We have therefore obtained a set of free energy threshold values, for genomic DNA with varying GC content and used them as generic criteria for predicting promoter regions in several microbial genomes, using an in-house developed tool `PromPredict'. On applying it to predict promoter regions corresponding to the 1144 and 612 experimentally validated TSSs in E. coli (50.8% GC) and B. subtilis (43.5% GC) sensitivity of 99% and 95% and precision values of 58% and 60%, respectively, were achieved. For the limited data set of 81 TSSs available for M. tuberculosis (65.6% GC) a sensitivity of 100% and precision of 49% was obtained.

Annotation and characterization of the Plasmodium vivax rhoptry neck protein 4 (PvRON4)

Background: The tight junction (TJ) is one of the most important structures established during merozoite invasion of host cells and a large amount of proteins stored in Toxoplasma and Plasmodium parasites’ apical organelles are involved in forming the TJ. Plasmodium falciparum and Toxoplasma gondii apical membrane antigen 1 (AMA-1) and rhoptry neck proteins (RONs) are the two main TJ components. It has been shown that RON4 plays an essential role during merozoite and sporozoite invasion to target cells. This study has focused on characterizing a novel Plasmodium vivax rhoptry protein, RON4, which is homologous to PfRON4 and PkRON4. Methods: The ron4 gene was re-annotated in the P. vivax genome using various bioinformatics tools and taking PfRON4 and PkRON4 amino acid sequences as templates. Gene synteny, as well as identity and similarity values between open reading frames (ORFs) belonging to the three species were assessed. The gene transcription of pvron4, and the expression and localization of the encoded protein were also determined in the VCG-1 strain by molecular and immunological studies. Nucleotide and amino acid sequences obtained for pvron4 in VCG-1 were compared to those from strains coming from different geographical areas. Results: PvRON4 is a 733 amino acid long protein, which is encoded by three exons, having similar transcription and translation patterns to those reported for its homologue, PfRON4. Sequencing PvRON4 from the VCG-1 strain and comparing it to P. vivax strains from different geographical locations has shown two conserved regions separated by a low complexity variable region, possibly acting as a “smokescreen”. PvRON4 contains a predicted signal sequence, a coiled-coil α-helical motif, two tandem repeats and six conserved cysteines towards the carboxyterminus and is a soluble protein lacking predicted transmembranal domains or a GPI anchor. Indirect immunofluorescence assays have shown that PvRON4 is expressed at the apical end of schizonts and co-localizes at the rhoptry neck with PvRON2.

PartsList: a web-based system for dynamically ranking protein folds based on disparate attributes, including whole-genome expression and interaction information

As the number of protein folds is quite limited, a mode of analysis that will be increasingly common in the future, especially with the advent of structural genomics, is to survey and re-survey the finite parts list of folds from an expanding number of perspectives. We have developed a new resource, called PartsList, that lets one dynamically perform these comparative fold surveys. It is available on the web at http://bioinfo.mbb.yale.edu/partslist and http://www.partslist.org. The system is based on the existing fold classifications and functions as a form of companion annotation for them, providing ‘global views’ of many already completed fold surveys. The central idea in the system is that of comparison through ranking; PartsList will rank the approximately 420 folds based on more than 180 attributes. These include: (i) occurrence in a number of completely sequenced genomes (e.g. it will show the most common folds in the worm versus yeast); (ii) occurrence in the structure databank (e.g. most common folds in the PDB); (iii) both absolute and relative gene expression information (e.g. most changing folds in expression over the cell cycle); (iv) protein–protein interactions, based on experimental data in yeast and comprehensive PDB surveys (e.g. most interacting fold); (v) sensitivity to inserted transposons; (vi) the number of functions associated with the fold (e.g. most multi-functional folds); (vii) amino acid composition (e.g. most Cys-rich folds); (viii) protein motions (e.g. most mobile folds); and (ix) the level of similarity based on a comprehensive set of structural alignments (e.g. most structurally variable folds). The integration of whole-genome expression and protein–protein interaction data with structural information is a particularly novel feature of our system. We provide three ways of visualizing the rankings: a profiler emphasizing the progression of high and low ranks across many pre-selected attributes, a dynamic comparer for custom comparisons and a numerical rankings correlator. These allow one to directly compare very different attributes of a fold (e.g. expression level, genome occurrence and maximum motion) in the uniform numerical format of ranks. This uniform framework, in turn, highlights the way that the frequency of many of the attributes falls off with approximate power-law behavior (i.e. according to V–b, for attribute value V and constant exponent b), with a few folds having large values and most having small values.

Comparative organization of cattle chromosome 5 revealed by comparative mapping by annotation and sequence similarity and radiation hybrid mapping

A whole genome cattle-hamster radiation hybrid cell panel was used to construct a map of 54 markers located on bovine chromosome 5 (BTA5). Of the 54 markers, 34 are microsatellites selected from the cattle linkage map and 20 are genes. Among the 20 mapped genes, 10 are new assignments that were made by using the comparative mapping by annotation and sequence similarity strategy. A LOD-3 radiation hybrid framework map consisting of 21 markers was constructed. The relatively low retention frequency of markers on this chromosome (19%) prevented unambiguous ordering of the other 33 markers. The length of the map is 398.7 cR, corresponding to a ratio of ≈2.8 cR5,000/cM. Type I genes were binned for comparison of gene order among cattle, humans, and mice. Multiple internal rearrangements within conserved syntenic groups were apparent upon comparison of gene order on BTA5 and HSA12 and HSA22. A similarly high number of rearrangements were observed between BTA5 and MMU6, MMU10, and MMU15. The detailed comparative map of BTA5 should facilitate identification of genes affecting economically important traits that have been mapped to this chromosome and should contribute to our understanding of mammalian chromosome evolution.

AnnoTALE: Bioinformatics tools for identification, annotation, and nomenclature of TALEs from Xanthomonas genomic sequences

Transcription activator-like effectors (TALEs) are virulence factors, produced by the bacterial plant-pathogen Xanthomonas, that function as gene activators inside plant cells. Although the contribution of individual TALEs to infectivity has been shown, the specific roles of most TALEs, and the overall TALE diversity in Xanthomonas spp. is not known. TALEs possess a highly repetitive DNA-binding domain, which is notoriously difficult to sequence. Here, we describe an improved method for characterizing TALE genes by the use of PacBio sequencing. We present 'AnnoTALE', a suite of applications for the analysis and annotation of TALE genes from Xanthomonas genomes, and for grouping similar TALEs into classes. Based on these classes, we propose a unified nomenclature for Xanthomonas TALEs that reveals similarities pointing to related functionalities. This new classification enables us to compare related TALEs and to identify base substitutions responsible for the evolution of TALE specificities. © 2016, Nature Publishing Group. All rights reserved.

Cognitive load and assembly tasks : effect of instructional formats on learning assembly procedures

The study investigated the effect on learning of four different instructional formats used to teach assembly procedures. Cognitive load and spatial information processing theories were used to generate the instructional material. The first group received a physical model to study, the second an isometric drawing, the third an isometric drawing plus a model and the fourth an orthographic drawing. Forty secondary school students were presented with the four different instructional formats and subsequently tested on an assembly task. The findings indicated that there may be evidence to argue that the model format which only required encoding of an already constructed three dimensional representation, caused less extraneous cognitive load compared to the isometric and the orthographic formats. No significant difference was found between the model and the isometric-plus-model formats on all measures because 80% of the students in the isometric-plus-model format chose to use the model format only. The model format also did not differ significantly from other groups in total time taken to complete the assembly, in number of correctly assembled pieces and in time spent on studying the tasks. However, the model group had significantly more correctly completed models and required fewer extra looks than the other groups.

Mitochondrial genome deletions and minicircles are common in lice (Insecta: Phthiraptera)

Background The gene composition, gene order and structure of the mitochondrial genome are remarkably stable across bilaterian animals. Lice (Insecta: Phthiraptera) are a major exception to this genomic stability in that the canonical single chromosome with 37 genes found in almost all other bilaterians has been lost in multiple lineages in favour of multiple, minicircular chromosomes with less than 37 genes on each chromosome. Results Minicircular mt genomes are found in six of the ten louse species examined to date and three types of minicircles were identified: heteroplasmic minicircles which coexist with full sized mt genomes (type 1); multigene chromosomes with short, simple control regions, we infer that the genome consists of several such chromosomes (type 2); and multiple, single to three gene chromosomes with large, complex control regions (type 3). Mapping minicircle types onto a phylogenetic tree of lice fails to show a pattern of their occurrence consistent with an evolutionary series of minicircle types. Analysis of the nuclear-encoded, mitochondrially-targetted genes inferred from the body louse, Pediculus, suggests that the loss of mitochondrial single-stranded binding protein (mtSSB) may be responsible for the presence of minicircles in at least species with the most derived type 3 minicircles (Pediculus, Damalinia). Conclusions Minicircular mt genomes are common in lice and appear to have arisen multiple times within the group. Life history adaptive explanations which attribute minicircular mt genomes in lice to the adoption of blood-feeding in the Anoplura are not supported by this expanded data set as minicircles are found in multiple non-blood feeding louse groups but are not found in the blood-feeding genus Heterodoxus. In contrast, a mechanist explanation based on the loss of mtSSB suggests that minicircles may be selectively favoured due to the incapacity of the mt replisome to synthesize long replicative products without mtSSB and thus the loss of this gene lead to the formation of minicircles in lice.

The genome organization and affinities of an Australian isolate of carrot mottle umbravirus

The genomic sequence of an Australian isolate of carrot mottle umbravirus (CMoV-A) was determined from cDNA generated from dsRNA. This provides the first data on the genome organization and phylogeny of an umbravirus. The 4201-nucleotide genome contains four major open reading frames (ORFs). Analysis suggests that ORF2 encodes an RNA-dependent RNA polymerase, that ORF4 encodes a movement protein, and that the virus has no coat protein gene. The functions of ORFs 1 and 3 remain unknown. ORF2 is probably translated following ribosomal frameshifting. ORFs 3 and 4 are probably translated from a subgenomic mRNA. Sequence comparisons showed CMoV-A to be closely related to pea enation mosaic RNA2 NA2), but also to have affinities with the Bromoviridae. These findings shed light on the relationships between the luteoviruses, PEMV, and the umbraviruses and on the relationships between the carmo-like viruses and the Bromoviridae.

How to sequence and annotate insect mitochondrial genomes for systematic and comparative genomics research

Over the past decade the mitochondrial (mt) genome has become the most widely used genomic resource available for systematic entomology. While the availability of other types of ‘–omics’ data – in particular transcriptomes – is increasing rapidly, mt genomes are still vastly cheaper to sequence and are far less demanding of high quality templates. Furthermore, almost all other ‘–omics’ approaches also sequence the mt genome, and so it can form a bridge between legacy and contemporary datasets. Mitochondrial genomes have now been sequenced for all insect orders, and in many instances representatives of each major lineage within orders (suborders, series or superfamilies depending on the group). They have also been applied to systematic questions at all taxonomic scales from resolving interordinal relationships (e.g. Cameron et al., 2009; Wan et al., 2012; Wang et al., 2012), through many intraordinal (e.g. Dowton et al., 2009; Timmermans et al., 2010; Zhao et al. 2013a) and family-level studies (e.g. Nelson et al., 2012; Zhao et al., 2013b) to population/biogeographic studies (e.g. Ma et al., 2012). Methodological issues around the use of mt genomes in insect phylogenetic analyses and the empirical results found to date have recently been reviewed by Cameron (2014), yet the technical aspects of sequencing and annotating mt genomes were not covered. Most papers which generate new mt genome report their methods in a simplified form which can be difficult to replicate without specific knowledge of the field. Published studies utilize a sufficiently wide range of approaches, usually without justification for the one chosen, that confusion about commonly used jargon such as ‘long PCR’ and ‘primer walking’ could be a serious barrier to entry. Furthermore, sequenced mt genomes have been annotated (gene locations defined) to wildly varying standards and improving data quality through consistent annotation procedures will benefit all downstream users of these datasets. The aims of this review are therefore to: 1. Describe in detail the various sequencing methods used on insect mt genomes; 2. Explore the strengths/weakness of different approaches; 3. Outline the procedures and software used for insect mt genome annotation, and; 4. Highlight quality control steps used for new annotations, and to improve the re-annotation of previously sequenced mt genomes used in systematic or comparative research.