Structural Annotation Of Genomes Research Articles

Comparative Genome Annotation.

Newly sequenced genomes are being added to the tree of life at an unprecedented fast pace. A large proportion of such new genomes are phylogenetically close to previously sequenced and annotated genomes. In other cases, whole clades of closely related species or strains ought to be annotated simultaneously. Often, in subsequent studies, differences between the closely related species or strains are in the focus of research when the shared gene structures prevail. We here review methods for comparative structural genome annotation. The reviewed methods include classical approaches such as the alignment of protein sequences or protein profiles against the genome and comparative gene prediction methods that exploit a genome alignment to annotate either a single target genome or all input genomes simultaneously. We discuss how the methods depend on the phylogenetic placement of genomes, give advice on the choice of methods, and examine the consistency between gene structure annotations in an example. Furthermore, we provide practical advice on genome annotation in general.

Welcome to the big leaves: Best practices for improving genome annotation in non-model plant genomes.

Robust standards to evaluate quality and completeness are lacking in eukaryotic structural genome annotation, as genome annotation software is developed using model organisms and typically lacks benchmarking to comprehensively evaluate the quality and accuracy of the final predictions. The annotation of plant genomes is particularly challenging due to their large sizes, abundant transposable elements, and variable ploidies. This study investigates the impact of genome quality, complexity, sequence read input, and method on protein-coding gene predictions. The impact of repeat masking, long-read and short-read inputs, and de novo and genome-guided protein evidence was examined in the context of the popular BRAKER and MAKER workflows for five plant genomes. The annotations were benchmarked for structural traits and sequence similarity. Benchmarks that reflect gene structures, reciprocal similarity search alignments, and mono-exonic/multi-exonic gene counts provide a more complete view of annotation accuracy. Transcripts derived from RNA-read alignments alone are not sufficient for genome annotation. Gene prediction workflows that combine evidence-based and ab initio approaches are recommended, and a combination of short and long reads can improve genome annotation. Adding protein evidence from de novo assemblies, genome-guided transcriptome assemblies, or full-length proteins from OrthoDB generates more putative false positives as implemented in the current workflows. Post-processing with functional and structural filters is highly recommended. While the annotation of non-model plant genomes remains complex, this study provides recommendations for inputs and methodological approaches. We discuss a set of best practices to generate an optimal plant genome annotation and present a more robust set of metrics to evaluate the resulting predictions.

Draft Genomes of Six Philippine Erwinia mallotivora Isolates: Comparative Genomics and Genome-Wide Analysis of Candidate Secreted Proteins.

Erwinia mallotivora is one of the most important bacterial pathogens of papaya and causes bacterial crown rot disease in the Philippines. In this paper, we present the draft genome sequences of six Philippine E. mallotivora isolates to provide insights into the genes involved in host-pathogen interactions and compare their genomes to other Erwinia species. The genomes were sequenced using Illumina Miseq platform. The draft whole-genome assemblies of the E. mallotivora isolates are composed of 36-64 contigs with N50 value ranging from 285 to 332 kbp and cover 96.2-100% of the estimated genome size. Structural genome annotation of these assemblies has predicted 4489-4749 protein-coding genes. Comparative genomic analysis using orthologous gene sets led to the identification of conserved genes within the genus and species-specific gene orthologous groups, which collectively provide a baseline for functional genomic studies to determine genes affecting virulence and host specificity. Secreted proteins of E. mallotivora were also predicted and characterized to unravel putative genes involved in plant-pathogen interactions. This study provides the first draft whole-genome sequences of Philippine isolates of E. mallotivora, thus expanding the genomic knowledge for this species in comparison with other members of the genus Erwinia.

Fruit Development in Ficus carica L.: Morphological and Genetic Approaches to Fig Buds for an Evolution From Monoecy Toward Dioecy.

The mechanism behind the bud evolution towards breba or main crop in Ficus carica L. is uncertain. Anatomical and genetic studies may put a light on the possible similarities/differences between the two types of fruits. For this reason, we collected complimentary data from anatomical, X-ray imaging, and genetic techniques. The RNA seq together with structural genome annotation allowed the prediction of 34,629 known genes and 938 novel protein-coding genes. Transcriptome analysis of genes during bud differentiation revealed differentially expressed genes in two fig varieties (Dottato and Petrelli) and in breba and main crop. We chose Dottato and Petrelli because the first variety does not require pollination to set main crop and the latter does; moreover, Petrelli yields many brebas whereas Dottato few. Of the 1,615 and 1,904 loci expressed in Dottato and Petrelli, specifically in breba or main crop, respectively, only 256 genes appeared to be transcripts in both varieties. The buds of the two fig varieties were observed under optical microscope and using 3D X-ray tomography, highlighting differences mainly related to the stage of development. The X-ray images of buds showed a great structural similarity between breba and main crop during the initial stages of development. Analysis at the microscope indicated that inflorescence differentiation of breba was split in two seasons whereas that of main crop started at the end of winter of season 2 and was completed within 2 to 3 months. The higher expression of floral homeotic protein AGAMOUS in breba with respect to main crop, since this protein is required for normal development of stamens and carpels in the flower, may indicate an original role of these fruits for staminate flowers production for pollination of the main crop, as profichi in the caprifig. Several genes related to auxin (auxin efflux carrier, auxin response factor, auxin binding protein, auxin responsive protein) and to GA synthesis (GA20ox) were highly expressed in brebas with respect to main crop for the development of this parthenocarpic fruit.

Peptimapper: proteogenomics workflow for the expert annotation of eukaryotic genomes

BackgroundAccurate structural annotation of genomes is still a challenge, despite the progress made over the past decade. The prediction of gene structure remains difficult, especially for eukaryotic species, and is often erroneous and incomplete. We used a proteogenomics strategy, taking advantage of the combination of proteomics datasets and bioinformatics tools, to identify novel protein coding-genes and splice isoforms, assign correct start sites, and validate predicted exons and genes.ResultsOur proteogenomics workflow, Peptimapper, was applied to the genome annotation of Ectocarpus sp., a key reference genome for both the brown algal lineage and stramenopiles. We generated proteomics data from various life cycle stages of Ectocarpus sp. strains and sub-cellular fractions using a shotgun approach. First, we directly generated peptide sequence tags (PSTs) from the proteomics data. Second, we mapped PSTs onto the translated genomic sequence. Closely located hits (i.e., PSTs locations on the genome) were then clustered to detect potential coding regions based on parameters optimized for the organism. Third, we evaluated each cluster and compared it to gene predictions from existing conventional genome annotation approaches. Finally, we integrated cluster locations into GFF files to use a genome viewer. We identified two potential novel genes, a ribosomal protein L22 and an aryl sulfotransferase and corrected the gene structure of a dihydrolipoamide acetyltransferase. We experimentally validated the results by RT-PCR and using transcriptomics data.ConclusionsPeptimapper is a complementary tool for the expert annotation of genomes. It is suitable for any organism and is distributed through a Docker image available on two public bioinformatics docker repositories: Docker Hub and BioShaDock. This workflow is also accessible through the Galaxy framework and for use by non-computer scientists at https://galaxy.protim.eu.Data are available via ProteomeXchange under identifier PXD010618.

Reannotation and extended community resources for the genome of the non-seed plant Physcomitrella patens provide insights into the evolution of plant gene structures and functions

BackgroundThe moss Physcomitrella patens as a model species provides an important reference for early-diverging lineages of plants and the release of the genome in 2008 opened the doors to genome-wide studies. The usability of a reference genome greatly depends on the quality of the annotation and the availability of centralized community resources. Therefore, in the light of accumulating evidence for missing genes, fragmentary gene structures, false annotations and a low rate of functional annotations on the original release, we decided to improve the moss genome annotation.ResultsHere, we report the complete moss genome re-annotation (designated V1.6) incorporating the increased transcript availability from a multitude of developmental stages and tissue types. We demonstrate the utility of the improved P. patens genome annotation for comparative genomics and new extensions to the cosmoss.org resource as a central repository for this plant “flagship” genome. The structural annotation of 32,275 protein-coding genes results in 8387 additional loci including 1456 loci with known protein domains or homologs in Plantae. This is the first release to include information on transcript isoforms, suggesting alternative splicing events for at least 10.8% of the loci. Furthermore, this release now also provides information on non-protein-coding loci. Functional annotations were improved regarding quality and coverage, resulting in 58% annotated loci (previously: 41%) that comprise also 7200 additional loci with GO annotations. Access and manual curation of the functional and structural genome annotation is provided via the http://www.cosmoss.org model organism database.ConclusionsComparative analysis of gene structure evolution along the green plant lineage provides novel insights, such as a comparatively high number of loci with 5’-UTR introns in the moss. Comparative analysis of functional annotations reveals expansions of moss house-keeping and metabolic genes and further possibly adaptive, lineage-specific expansions and gains including at least 13% orphan genes.

A Proteogenomic Survey of the Medicago truncatula Genome

Peptide sequencing by computational assignment of tandem mass spectra to a database of putative protein sequences provides an independent approach to confirming or refuting protein predictions based on large-scale DNA and RNA sequencing efforts. This use of mass spectrometrically-derived sequence data for testing and refining predicted gene models has been termed proteogenomics. We report herein the application of proteogenomic methodology to a database of 10.9 million tandem mass spectra collected over a period of two years from proteolytically generated peptides isolated from the model legume Medicago truncatula. These spectra were searched against a database of predicted M. truncatula protein sequences generated from public databases, in silico gene model predictions, and a whole-genome six-frame translation. This search identified 78,647 distinct peptide sequences, and a comparison with the publicly available proteome from the recently published M. truncatula genome supported translation of 9,843 existing gene models and identified 1,568 novel peptides suggesting corrections or additions to the current annotations. Each supporting and novel peptide was independently validated using mRNA-derived deep sequencing coverage and an overall correlation of 93% between the two data types was observed. We have additionally highlighted examples of several aspects of structural annotation for which tandem MS provides unique evidence not easily obtainable through typical DNA or RNA sequencing. Proteogenomic analysis is a valuable and unique source of information for the structural annotation of genomes and should be included in such efforts to ensure that the genome models used by biologists mirror as accurately as possible what is present in the cell.

The Proteogenomic Mapping Tool

BackgroundHigh-throughput mass spectrometry (MS) proteomics data is increasingly being used to complement traditional structural genome annotation methods. To keep pace with the high speed of experimental data generation and to aid in structural genome annotation, experimentally observed peptides need to be mapped back to their source genome location quickly and exactly. Previously, the tools to do this have been limited to custom scripts designed by individual research groups to analyze their own data, are generally not widely available, and do not scale well with large eukaryotic genomes.ResultsThe Proteogenomic Mapping Tool includes a Java implementation of the Aho-Corasick string searching algorithm which takes as input standardized file types and rapidly searches experimentally observed peptides against a given genome translated in all 6 reading frames for exact matches. The Java implementation allows the application to scale well with larger eukaryotic genomes while providing cross-platform functionality.ConclusionsThe Proteogenomic Mapping Tool provides a standalone application for mapping peptides back to their source genome on a number of operating system platforms with standard desktop computer hardware and executes very rapidly for a variety of datasets. Allowing the selection of different genetic codes for different organisms allows researchers to easily customize the tool to their own research interests and is recommended for anyone working to structurally annotate genomes using MS derived proteomics data.

Relationship of oxidative stress and endothelial dysfunction in sleep apnoea

The aim of the present study was to evaluate ischaemic reactive hyperaemia (IRH) in obstructive sleep apnoea (OSA) and its relationship with oxidative stress. We studied 69 consecutive patients referred to our Sleep Unit (Reina Sofia University Hospital, Cordoba, Spain). Patients with chronic diseases or those taking medication were excluded. IRH was assessed before and after polysomnography. Morning IRH and oxidative stress markers were compared between patients with (apnoea-hypopnoea index (AHI) ≥ 5) and without (AHI < 5) OSA. Measurements were repeated in 25 severe OSA patients after continuous positive airway pressure (CPAP) therapy. We included 46 OSA patients (mean ± sd AHI 49 ± 32.1) and 23 non-OSA subjects (AHI 3 ± 0.9). The OSA patients showed a significant worsening of morning IRH, and a significant increase in malondialdehyde and 8-hydroxydeoxyguanosine levels. Only the oxygen desaturation index independently explained morning IRH, while malondialdehyde levels showed a weak effect on IRH. In severe OSA patients, IRH improved significantly after CPAP treatment, as did malondialdehyde, 8-hydroxydeoxyguanosine and protein carbonyl levels. In OSA patients, endothelial dysfunction and oxidative stress were observed, and IRH worsened after sleep. The increase in oxidative stress was not associated with IRH, while intermittent hypoxia was strongly associated with IRH. In severe OSA patients, CPAP treatment improved oxidative stress and endothelial function.

Identification of transcriptional signals in Encephalitozoon cuniculi widespread among Microsporidia phylum: support for accurate structural genome annotation

BackgroundMicrosporidia are obligate intracellular eukaryotic parasites with genomes ranging in size from 2.3 Mbp to more than 20 Mbp. The extremely small (2.9 Mbp) and highly compact (~1 gene/kb) genome of the human parasite Encephalitozoon cuniculi has been fully sequenced. The aim of this study was to characterize noncoding motifs that could be involved in regulation of gene expression in E. cuniculi and to show whether these motifs are conserved among the phylum Microsporidia.ResultsTo identify such signals, 5' and 3'RACE-PCR experiments were performed on different E. cuniculi mRNAs. This analysis confirmed that transcription overrun occurs in E. cuniculi and may result from stochastic recognition of the AAUAAA polyadenylation signal. Such experiments also showed highly reduced 5'UTR's (<7 nts). Most of the E. cuniculi genes presented a CCC-like motif immediately upstream from the coding start. To characterize other signals involved in differential transcriptional regulation, we then focused our attention on the gene family coding for ribosomal proteins. An AAATTT-like signal was identified upstream from the CCC-like motif. In rare cases the cytosine triplet was shown to be substituted by a GGG-like motif. Comparative genomic studies confirmed that these different signals are also located upstream from genes encoding ribosomal proteins in other microsporidian species including Antonospora locustae, Enterocytozoon bieneusi, Anncaliia algerae (syn. Brachiola algerae) and Nosema ceranae. Based on these results a systematic analysis of the ~2000 E. cuniculi coding DNA sequences was then performed and brings to highlight that 364 translation initiation codons (18.29% of total CDSs) had been badly predicted.ConclusionWe identified various signals involved in the maturation of E. cuniculi mRNAs. Presence of such signals, in phylogenetically distant microsporidian species, suggests that a common regulatory mechanism exists among the microsporidia. Furthermore, 5'UTRs being strongly reduced, these signals can be used to ensure the accurate prediction of translation initiation codons for microsporidian genes and to improve microsporidian genome annotation.

Genome wide survey, discovery and evolution of repetitive elements in three Entamoeba species

BackgroundIdentification and mapping of repetitive elements is a key step for accurate gene prediction and overall structural annotation of genomes. During the assembly and annotation of three highly repetitive amoeba genomes, Entamoeba histolytica, Entamoeba dispar, and Entamoeba invadens, we performed comparative sequence analysis to identify and map all class I and class II transposable elements in their sequences.ResultsHere, we report the identification of two novel Entamoeba-specific repeats: ERE1 and ERE2; ERE1 is spread across the three genomes and associated with different repeats in a species-specific manner, while ERE2 is unique to E. histolytica. We also report the identification of two novel subfamilies of LINE and SINE retrotransposons in E. dispar and provide evidence for how the different LINE and SINE subfamilies evolved in these species. Additionally, we found a putative transposase-coding gene in E. histolytica and E. dispar related to the mariner transposon Hydargos from E. invadens. The distribution of transposable elements in these genomes is markedly skewed with a tendency of forming clusters. More than 70% of the three genomes have a repeat density below their corresponding average value indicating that transposable elements are not evenly distributed. We show that repeats and repeat-clusters are found at syntenic break points between E. histolytica and E. dispar and hence, could work as recombination hot spots promoting genome rearrangements.ConclusionThe mapping of all transposable elements found in these parasites shows that repeat coverage is up to three times higher than previously reported. LINE, ERE1 and mariner elements were present in the common ancestor to the three Entamoeba species while ERE2 was likely acquired by E. histolytica after its separation from E. dispar. We demonstrate that E. histolytica and E. dispar share their entire repertoire of LINE and SINE retrotransposons and that Eh_SINE3/Ed_SINE1 originated as a chimeric SINE from Eh/Ed_SINE2 and Eh_SINE1/Ed_SINE3. Our work shows that transposable elements are organized in clusters, frequently found at syntenic break points providing insights into their contribution to chromosome instability and therefore, to genomic variation and speciation in these parasites.

FunSiP: a modular and extensible classifier for the prediction of functional sites in DNA

Many problems in genome annotation are tackled by using a classification model to predict functional sites such as splice sites, translation start sites or stop codons. Locating the correct position of these sites remains one of the most important but also one of the most difficult issues in the structural annotation of genomes. Most of the software currently in use is written for a very specific problem, thereby limiting the possibilities for reuse. We developed a software platform that uses a very general approach towards the classification of functional sites in DNA sequences. The program uses an ab initio approach towards the identification of these sites, and extends SpliceMachine, a previously developed splice site predictor that shows state-of-the-art performance for both donor and acceptor splice site recognition in the human and Arabidopsis thaliana genome. The program is developed as a stand-alone Java application, and is available as GPLv3 open-source software. The program, source and documentation can be obtained from the 'Software' section at http://bioinformatics.psb.ugent.be/. Supplementary data is available at Bioinformatics online.

AgBase: a functional genomics resource for agriculture.

BackgroundMany agricultural species and their pathogens have sequenced genomes and more are in progress. Agricultural species provide food, fiber, xenotransplant tissues, biopharmaceuticals and biomedical models. Moreover, many agricultural microorganisms are human zoonoses. However, systems biology from functional genomics data is hindered in agricultural species because agricultural genome sequences have relatively poor structural and functional annotation and agricultural research communities are smaller with limited funding compared to many model organism communities.DescriptionTo facilitate systems biology in these traditionally agricultural species we have established "AgBase", a curated, web-accessible, public resource for structural and functional annotation of agricultural genomes. The AgBase database includes a suite of computational tools to use GO annotations. We use standardized nomenclature following the Human Genome Organization Gene Nomenclature guidelines and are currently functionally annotating chicken, cow and sheep gene products using the Gene Ontology (GO). The computational tools we have developed accept and batch process data derived from different public databases (with different accession codes), return all existing GO annotations, provide a list of products without GO annotation, identify potential orthologs, model functional genomics data using GO and assist proteomics analysis of ESTs and EST assemblies. Our journal database helps prevent redundant manual GO curation. We encourage and publicly acknowledge GO annotations from researchers and provide a service for researchers interested in GO and analysis of functional genomics data.ConclusionThe AgBase database is the first database dedicated to functional genomics and systems biology analysis for agriculturally important species and their pathogens. We use experimental data to improve structural annotation of genomes and to functionally characterize gene products. AgBase is also directly relevant for researchers in fields as diverse as agricultural production, cancer biology, biopharmaceuticals, human health and evolutionary biology. Moreover, the experimental methods and bioinformatics tools we provide are widely applicable to many other species including model organisms.