A first complete catalog of highly expressed genes in eight chicken tissues reveals uncharacterized gene families specific for the chicken testis.

Based on next-generation sequencing, we established a repertoire of differentially overexpressed genes (DoEGs) in eight adult chicken tissues: the testis, brain, lung, liver, kidney, muscle, heart, and intestine. With 4,499 DoEGs, the testis had the highest number and proportion of DoEGs compared with the seven somatic tissues. The testis DoEG set included the highest proportion of long noncoding RNAs (lncRNAs; 1,851, representing 32% of the lncRNA genes in the whole genome) and the highest proportion of protein-coding genes (2,648, representing 14.7% of the protein-coding genes in the whole genome). The main significantly enriched Gene Ontology terms related to the protein-coding genes were "reproductive process," "tubulin binding," and "microtubule cytoskeleton." Using real-time quantitative reverse transcription-polymerase chain reaction, we confirmed the overexpression of genes that encode proteins already described in chicken sperm [such as calcium binding tyrosine phosphorylation regulated (CABYR), spermatogenesis associated 18 (SPATA18), and CDK5 regulatory subunit associated protein (CDK5RAP2)] but whose testis origin had not been previously confirmed. Moreover, we demonstrated the overexpression of vertebrate orthologs of testis genes not yet described in the adult chicken testis [such as NIMA related kinase 2 (NEK2), adenylate kinase 7 (AK7), and CCNE2]. Using clustering according to primary sequence homology, we found that 1,737 of the 2,648 (67%) testis protein-coding genes were unique genes. This proportion was significantly higher than the somatic tissues except muscle. We clustered the other 911 testis protein-coding genes into 495 families, from which 47 had all paralogs overexpressed in the testis. Among these 47 testis-specific families, eight contained uncharacterized duplicated paralogs without orthologs in other metazoans except birds: these families are thus specific for chickens/birds.NEW & NOTEWORTHY Comparative next-generation sequencing analysis of eight chicken tissues showed that the testis has highest proportion of long noncoding RNA and protein-coding genes of the whole genome. We identified new genes in the chicken testis, including orthologs of known mammalian testicular genes. We also identified 47 gene families in which all the members were overexpressed, if not exclusive, in the testis. Eight families, organized in duplication clusters, were unknown, without orthologs in metazoans except birds, and are thus specific for chickens/birds.

Seasonal remodeling of the progenitor pool and its distribution in the ewe mediobasal hypothalamus.

Recent studies have reported the presence of adult neurogenesis in the arcuate nucleus periventricular space (pvARH) and in the median eminence (ME), two structures involved in reproductive function. In sheep, a seasonal mammal, decreasing daylight in autumn induces a higher neurogenic activity in these two structures. However, the different types of neural stem and progenitor cells (NSCs/NPCs) that populate the arcuate nucleus and median eminence, as well as their location, have not been evaluated. Here, using semi-automatic image analyzing processes, we identified and quantified the different populations of NSCs/NPCs, showing that, during short days, higher densities of [SOX2 +] cells are found in pvARH and ME. In the pvARH, higher densities of astrocytic and oligodendrocitic progenitors mainly contribute to these variations. The different populations of NSCs/NPCs were mapped according to their position relative to the third ventricle and their proximity to the vasculature. We showed that [SOX2 +] cells extended deeper into the hypothalamic parenchyma during short days. Similarly, [SOX2 +] cells were found further from the vasculature in the pvARH and the ME, at this time of year, indicating the existence of migratory signals. The expression levels of neuregulin transcripts (NRGs), whose proteins are known to stimulate proliferation and adult neurogenesis and to regulate progenitor migration, as well as the expression levels of ERBB mRNAs, cognate receptors for NRGs, were assessed. We showed that mRNA expression changed seasonally in pvARH and ME, suggesting that the ErbB-NRG system is potentially involved in the photoperiodic regulation of neurogenesis in seasonal adult mammals.

Expanding duplication of the testis PHD Finger Protein 7 (PHF7) gene in the chicken genome.

Gene duplications increase genetic and phenotypic diversity and occur in complex genomic regions that are still difficult to sequence and assemble. PHD Finger Protein 7 (PHF7) acts during spermiogenesis for histone-to-histone protamine exchange and is a determinant of male fertility in Drosophila and the mouse. We aimed to explore and characterise in the chicken genome the expanding family of the numerous orthologues of the unique mouse Phf7 gene (highly expressed in the testis), observing the fact that this information is unclear and/or variable according to the versions of databases. We validated nine primer pairs by in silico PCR for their use in screening the chicken bacterial artificial chromosome (BAC) library to produce BAC-derived probes to detect and localise PHF7-like loci by fluorescence in situ hybridisation (FISH). We selected nine BAC that highlighted nine chromosomal regions for a total of 10 distinct PHF7-like loci on five Gallus gallus chromosomes: Chr1 (three loci), Chr2 (two loci), Chr12 (one locus), Chr19 (one locus) and ChrZ (three loci). We sequenced the corresponding BAC by using high-performance PacBio technology. After assembly, we performed annotation with the FGENESH program: there were a total of 116 peptides, including 39 PHF7-like proteins identified by BLASTP. These proteins share a common exon-intron core structure of 8-11 exons. Phylogeny revealed that the duplications occurred first between chromosomal regions and then inside each region. There are other duplicated genes in the identified BAC sequences, suggesting that these genomic regions exhibit a high rate of tandem duplication. We showed that the PHF7 gene, which is highly expressed in the rooster testis, is a highly duplicated gene family in the chicken genome, and this phenomenon probably concerns other bird species.

Combined Multiplexed Phage Display, High-Throughput Sequencing, and Functional Assays as a Platform for Identifying Modulatory VHHs Targeting the FSHR.

Developing modulatory antibodies against G protein-coupled receptors is challenging. In this study, we targeted the follicle-stimulating hormone receptor (FSHR), a significant regulator of reproduction, with variable domains of heavy chain-only antibodies (VHHs). We built two immune VHH libraries and submitted them to multiplexed phage display approaches. We used next-generation sequencing to identify 34 clusters of specifically enriched sequences that were functionally assessed in a primary screen based on a cAMP response element (CRE)-dependent reporter gene assay. In this assay, 23 VHHs displayed negative or positive modulation of FSH-induced responses, suggesting a high success rate of the multiplexed strategy. We then focused on the largest cluster identified (i.e., PRC1) that displayed positive modulation of FSH action. We demonstrated that PRC1 specifically binds to the human FSHR and human FSHR/FSH complex while potentiating FSH-induced cAMP production and Gs recruitment. We conclude that the improved selection strategy reported here is effective for rapidly identifying functionally active VHHs and could be adapted to target other challenging membrane receptors. This study also led to the identification of PRC1, the first potential positive modulator VHH reported for the human FSHR.

Seasonal vascular plasticity in the mediobasal hypothalamus of the adult ewe.

Sheep, like most seasonal mammals, exhibit a cyclic adaptive reproductive physiology that allows ewes to give birth to their progeny during the spring when environmental conditions are favorable to their survival. This process relies on the detection of day length (or photoperiod) and is associated with profound changes in cellular plasticity and gene expression in the hypothalamic-pituitary-gonadal axis, mechanisms that are suggested to participate in the seasonal adaptation of neuroendocrine circuits. Recently, pituitary vascular growth has been proposed as a seasonally regulated process in which the vascular endothelial growth factor A (VEGFA), a well-known angiogenic cytokine, is suspected to play a crucial role. However, whether this mechanism is restricted to the pituitary gland or also occurs in the mediobasal hypothalamus (MBH), a crucial contributor to the control of the reproductive function, remains unexplored. Using newly developed image analysis tools, we showed that the arcuate nucleus (ARH) of the MBH exhibits an enhanced vascular density during the long photoperiod or non-breeding season, associated with higher expression of VEGFA. In the median eminence (ME), a structure connecting the MBH to the pituitary gland, higher VEGFA, kinase insert domain receptor (KDR/VEGFR2) and plasmalemma vesicle-associated protein (PLVAP) gene expressions were detected during the long photoperiod. We also found that VEGFA and its receptor, VEGFR2, are expressed by neurons and tanycytes in both the ARH and ME. Altogether, these data show variations in the MBH vasculature according to seasons potentially through a VEGFA-dependent pathway, paving the way for future studies aiming to decipher the role of these changes in the hypothalamic control of seasonal reproduction.

Variations in genome size between wild and domesticated lineages of fowls belonging to the Gallus gallus species.

Efforts to elucidate the causes of biological differences between wild fowls and their domesticated relatives, the chicken, have to date mainly focused on the identification of single nucleotide mutations. Other types of genomic variations have however been demonstrated to be important in avian evolution and associated to variations in phenotype. They include several types of sequences duplicated in tandem that can vary in their repetition number. Here we report on genome size differences between the red jungle fowl and several domestic chicken breeds and selected lines. Sequences duplicated in tandem such as rDNA, telomere repeats, satellite DNA and segmental duplications were found to have been significantly re-shaped during domestication and subsequently by human-mediated selection. We discuss the extent to which changes in genome organization that occurred during domestication agree with the hypothesis that domesticated animal genomes have been shaped by evolutionary forces aiming to adapt them to anthropized environments.

Sequence properties of certain GC rich avian genes, their origins and absence from genome assemblies: case studies.

BACKGROUND: More and more eukaryotic genomes are sequenced and assembled, most of them presented as a complete model in which missing chromosomal regions are filled by Ns and where a few chromosomes may be lacking. Avian genomes often contain sequences with high GC content, which has been hypothesized to be at the origin of many missing sequences in these genomes. We investigated features of these missing sequences to discover why some may not have been integrated into genomic libraries and/or sequenced. RESULTS: The sequences of five red jungle fowl cDNA models with high GC content were used as queries to search publicly available datasets of Illumina and Pacbio sequencing reads. These were used to reconstruct the leptin, TNFα, MRPL52, PCP2 and PET100 genes, all of which are absent from the red jungle fowl genome model. These gene sequences displayed elevated GC contents, had intron sizes that were sometimes larger than non-avian orthologues, and had non-coding regions that contained numerous tandem and inverted repeat sequences with motifs able to assemble into stable G-quadruplexes and intrastrand dyadic structures. Our results suggest that Illumina technology was unable to sequence the non-coding regions of these genes. On the other hand, PacBio technology was able to sequence these regions, but with dramatically lower efficiency than would typically be expected. CONCLUSIONS: High GC content was not the principal reason why numerous GC-rich regions of avian genomes are missing from genome assembly models. Instead, it is the presence of tandem repeats containing motifs capable of assembling into very stable secondary structures that is likely responsible.

Relative effects of location relative to the corpus luteum and lactation on the transcriptome of the bovine oviduct epithelium.

BACKGROUND: Lactation and associated metabolic stresses during the post-partum period have been shown to impair fertility in dairy cows. The oviduct plays key roles in embryo development and the establishment of pregnancy in cattle. The aim of this study was to investigate the effects of lactation and location relative to the corpus luteum (CL) on the transcriptome of the bovine oviduct epithelium. RESULTS: An original animal model was used. At 60 days post-partum, Holstein lactating (n = 4) and non-lactating (i.e. never milked after calving; n = 5) cows, as well as control nulliparous heifers (n = 5), were slaughtered on Day 3 following induced estrus, and epithelial samples from the oviductal ampulla and isthmus ipsilateral and contralateral to the corpus luteum (CL) were recovered for RNA sequencing. In the oviduct ipsilateral to the CL, differentially expressed genes (DEGs) were identified between heifers compared with both postpartum cow groups. However, only 15 DEGs were identified between post-partum lactating and non-lactating cows in the ipsilateral isthmus and none were identified in the ipsilateral ampulla. In contrast, 192 and 2583 DEGs were identified between ipsilateral and contralateral ampulla and isthmus, respectively. In both regions, more DEGs were identified between ipsilateral and contralateral oviducts in non-lactating cows and heifers than in lactating cows. Functional annotation of the DEGs associated with comparisons between metabolic groups highlighted a number of over-represented biological functions and cell pathways including immune response and cholesterol/steroid biosynthesis. CONCLUSIONS: Gene expression in the oviduct epithelium, particularly in the isthmus, was more affected by the location relative to the CL than by lactation at Day 3 post-estrus. Furthermore, the effect of the proximity to the CL was modulated by the metabolic status of the cow.

Perturbation of mRNP biogenesis reveals a dynamic landscape of the Rrp6-dependent surveillance machinery trafficking along the yeast genome.

Eukaryotic cells have evolved a nuclear quality control (QC) system to monitor the co-transcriptional mRNA processing and packaging reactions that lead to the formation of export-competent ribonucleoprotein particles (mRNPs). Aberrant mRNPs that fail to pass the QC steps are retained in the nucleus and eliminated by the exonuclease activity of Rrp6. It is still unclear how the surveillance system is precisely coordinated both physically and functionally with the transcription machinery to detect the faulty events that may arise at each step of transcript elongation and mRNP formation. To dissect the QC mechanism, we previously implemented a powerful assay based on global perturbation of mRNP biogenesis in yeast by the bacterial Rho helicase. By monitoring model genes, we have shown that the QC process is coordinated by Nrd1, a component of the NNS complex (Nrd1-Nab3-Sen1) involved in termination, processing and decay of ncRNAs which is recruited by the CTD of RNAP II. Here, we have extended our investigations by analyzing the QC behaviour over the whole yeast genome. We performed high-throughput RNA sequencing (RNA-seq) to survey a large collection of mRNPs whose biogenesis is affected by Rho action and which can be rescued upon Rrp6 depletion. This genome-wide perspective was extended by generating high-resolution binding landscapes (ChIP-seq) of QC components along the yeast chromosomes before and after perturbation of mRNP biogenesis. Our results show that perturbation of mRNP biogenesis redistributes the QC components over the genome with a significant hijacking of Nrd1 and Nab3 from genomic loci producing ncRNAs to Rho-affected protein-coding genes, triggering termination and processing defects of ncRNAs.

But where did the centromeres go in the chicken genome models?

The chicken genome was the third vertebrate to be sequenced. To date, its sequence and feature annotations are used as the reference for avian models in genome sequencing projects developed on birds and other Sauropsida species, and in genetic studies of domesticated birds of economic and evolutionary biology interest. Therefore, an accurate description of this genome model is important to a wide number of scientists. Here, we review the location and features of a very basic element, the centromeres of chromosomes in the galGal5 genome model. Centromeres are elements that are not determined by their DNA sequence but by their epigenetic status, in particular by the accumulation of the histone-like protein CENP-A. Comparison of data from several public sources (primarily marker probes flanking centromeres using fluorescent in situ hybridization done on giant lampbrush chromosomes and CENP-A ChIP-seq datasets) with galGal5 annotations revealed that centromeres are likely inappropriately mapped in 9 of the 16 galGal5 chromosome models in which they are described. Analysis of karyology data confirmed that the location of the main CENP-A peaks in chromosomes is the best means of locating the centromeres in 25 galGal5 chromosome models, the majority of which (16) are fully sequenced and assembled. This data re-analysis reaffirms that several sources of information should be examined to produce accurate genome annotations, particularly for basic structures such as centromeres that are epigenetically determined.

Mariner Transposons Contain a Silencer: Possible Role of the Polycomb Repressive Complex 2.

Transposable elements are driving forces for establishing genetic innovations such as transcriptional regulatory networks in eukaryotic genomes. Here, we describe a silencer situated in the last 300 bp of the Mos1 transposase open reading frame (ORF) which functions in vertebrate and arthropod cells. Functional silencers are also found at similar locations within three other animal mariner elements, i.e. IS630-Tc1-mariner (ITm) DD34D elements, Himar1, Hsmar1 and Mcmar1. These silencers are able to impact eukaryotic promoters monitoring strong, moderate or low expression as well as those of mariner elements located upstream of the transposase ORF. We report that the silencing involves at least two transcription factors (TFs) that are conserved within animal species, NFAT-5 and Alx1. These cooperatively act with YY1 to trigger the silencing activity. Four other housekeeping transcription factors (TFs), neuron restrictive silencer factor (NRSF), GAGA factor (GAF) and GTGT factor (GTF), were also found to have binding sites within mariner silencers but their impact in modulating the silencer activity remains to be further specified. Interestingly, an NRSF binding site was found to overlap a 30 bp motif coding a highly conserved PHxxYSPDLAPxD peptide in mariner transposases. We also present experimental evidence that silencing is mainly achieved by co-opting the host Polycomb Repressive Complex 2 pathway. However, we observe that when PRC2 is impaired another host silencing pathway potentially takes over to maintain weak silencer activity. Mariner silencers harbour features of Polycomb Response Elements, which are probably a way for mariner elements to self-repress their transcription and mobility in somatic and germinal cells when the required TFs are expressed. At the evolutionary scale, mariner elements, through their exaptation, might have been a source of silencers playing a role in the chromatin configuration in eukaryotic genomes.

An Assessment of Fixed and Native Chromatin Preparation Methods to Study Histone Post-Translational Modifications at a Whole Genome Scale in Skeletal Muscle Tissue.

BACKGROUND: Genomic loci associated with histone marks are typically analyzed by immunoprecipitation of the chromatin followed by quantitative-PCR (ChIP-qPCR) or high throughput sequencing (ChIP-seq). Chromatin can be either cross-linked (X-ChIP) or used in the native state (N-ChIP). Cross-linking of DNA and proteins helps stabilizing their interactions before analysis. Despite X-ChIP is the most commonly used method, muscle tissue fixation is known to be relatively inefficient. Moreover, no protocol described a simple and reliable preparation of skeletal muscle chromatin of sufficient quality for subsequent high-throughput sequencing. Here we aimed to set-up and compare both chromatin preparation methods for a genome-wide analysis of H3K27me3, a broad-peak histone mark, using chicken P. major muscle tissue. RESULTS: Fixed and unfixed chromatin were prepared from chicken muscle tissues (Pectoralis major). Chromatin fixation, shearing by sonication or digestion and immunoprecipitation performed equivalently. High-quality Illumina reads were obtained (q30 > 93%). The bioinformatic analysis of the data was performed using epic, a tool based on SICER, and MACS2. Forty millions of reads were analyzed for both X-ChIP-seq and N-ChIP-seq experiments. Surprisingly, H3K27me3 X-ChIP-seq analysis led to the identification of only 2000 enriched regions compared to about 15,000 regions identified in the case of N-ChIP-seq. N-ChIP-seq peaks were more consistent between replicates compared to X-ChIP-seq. Higher N-ChIP-seq enrichments were confirmed by ChIP-qPCR at the PAX5 and SOX2 loci known to be enriched for H3K27me3 in myotubes and at the loci of common regions of enrichment identified in this study. CONCLUSIONS: Our findings suggest that the preparation of muscle chromatin for ChIP-seq in cross-linked conditions can compromise the systematic analysis of broad histone marks. Therefore, native chromatin preparation should be preferred to cross-linking when a ChIP experiment has to be performed on skeletal muscle tissue, particularly when a broad source signal is considered.

DensityMap: a genome viewer for illustrating the densities of features.

BACKGROUND: Several tools are available for visualizing genomic data. Some, such as Gbrowse and Jbrowse, are very efficient for small genomic regions, but they are not suitable for entire genomes. Others, like Phenogram and CViT, can be used to visualise whole genomes, but are not designed to display very dense genomic features (eg: interspersed repeats). We have therefore developed DensityMap, a lightweight Perl program that can display the densities of several features (genes, ncRNA, cpg, etc.) along chromosomes on the scale of the whole genome. A critical advantage of DensityMap is that it uses GFF annotation files directly to compute the densities of features without needing additional information from the user. The resulting picture is readily configurable, and the colour scales used can be customized for a best fit to the data plotted. RESULTS: DensityMap runs on Linux architecture with few requirements so that users can easily and quickly visualize the distributions and densities of genomic features for an entire genome. The input is GFF3-formated data representing chromosomes (linkage groups or pseudomolecules) and sets of features which are used to calculate representations in density maps. In practise, DensityMap uses a tilling window to compute the density of one or more features and the number of bases covered by these features along chromosomes. The densities are represented by colour scales that can be customized to highlight critical points. DensityMap can compare the distributions of features; it calculates several chromosomal density maps in a single image, each of which describes a different genomic feature. It can also use the genome nucleotide sequence to compute and plot a density map of the GC content along chromosomes. CONCLUSIONS: DensityMap is a compact, easily-used tool for displaying the distribution and density of all types of genomic features within a genome. It is flexible enough to visualize the densities of several types of features in a single representation. The images produced are readily configurable and their SVG format ensures that they can be edited.

Deep landscape update of dispersed and tandem repeats in the genome model of the red jungle fowl, Gallus gallus, using a series of de novo investigating tools.

BACKGROUND: The program RepeatMasker and the database Repbase-ISB are part of the most widely used strategy for annotating repeats in animal genomes. They have been used to show that avian genomes have a lower repeat content (8-12 %) than the sequenced genomes of many vertebrate species (30-55 %). However, the efficiency of such a library-based strategies is dependent on the quality and completeness of the sequences in the database that is used. An alternative to these library based methods are methods that identify repeats de novo. These alternative methods have existed for a least a decade and may be more powerful than the library based methods. We have used an annotation strategy involving several complementary de novo tools to determine the repeat content of the model genome galGal4 (1.04 Gbp), including identifying simple sequence repeats (SSRs), tandem repeats and transposable elements (TEs). RESULTS: We annotated over one Gbp. of the galGal4 genome and showed that it is composed of approximately 19 % SSRs and TEs repeats. Furthermore, we estimate that the actual genome of the red jungle fowl contains about 31-35 % repeats. We find that library-based methods tend to overestimate TE diversity. These results have a major impact on the current understanding of repeats distributions throughout chromosomes in the red jungle fowl. CONCLUSIONS: Our results are a proof of concept of the reliability of using de novo tools to annotate repeats in large animal genomes. They have also revealed issues that will need to be resolved in order to develop gold-standard methodologies for annotating repeats in eukaryote genomes.

A survey of transposable element classification systems--a call for a fundamental update to meet the challenge of their diversity and complexity.

The increase of publicly available sequencing data has allowed for rapid progress in our understanding of genome composition. As new information becomes available we should constantly be updating and reanalyzing existing and newly acquired data. In this report we focus on transposable elements (TEs) which make up a significant portion of nearly all sequenced genomes. Our ability to accurately identify and classify these sequences is critical to understanding their impact on host genomes. At the same time, as we demonstrate in this report, problems with existing classification schemes have led to significant misunderstandings of the evolution of both TE sequences and their host genomes. In a pioneering publication Finnegan (1989) proposed classifying all TE sequences into two classes based on transposition mechanisms and structural features: the retrotransposons (class I) and the DNA transposons (class II). We have retraced how ideas regarding TE classification and annotation in both prokaryotic and eukaryotic scientific communities have changed over time. This has led us to observe that: (1) a number of TEs have convergent structural features and/or transposition mechanisms that have led to misleading conclusions regarding their classification, (2) the evolution of TEs is similar to that of viruses by having several unrelated origins, (3) there might be at least 8 classes and 12 orders of TEs including 10 novel orders. In an effort to address these classification issues we propose: (1) the outline of a universal TE classification, (2) a set of methods and classification rules that could be used by all scientific communities involved in the study of TEs, and (3) a 5-year schedule for the establishment of an International Committee for Taxonomy of Transposable Elements (ICTTE).

Evolutionary relationships of iridoviruses and divergence of ascoviruses from invertebrate iridoviruses in the superfamily Megavirales.

The family Iridoviridae of the superfamily Megavirales currently consists of five genera. Three of these, Lymphocystivirus, Megalocytivirus and Ranavirus, are composed of species that infect vertebrates, and the other two, Chloriridovirus and Iridovirus, contain species that infect invertebrates. Until recently, the lack of genomic sequence data limited investigation of the evolutionary relationships between the invertebrate iridoviruses (IIVs) and vertebrate iridoviruses (VIVs), as well as the relationship of these viruses to those of the closely related family Ascoviridae, which only contains species that infect insects. To help clarify the phylogenetic relationships of these viruses, we recently published the annotated genome sequences of five additional IIV isolates. Here, using classical approaches of phylogeny via maximum likelihood, a Bayesian approach, and resolution of a core protein tree, we demonstrate that the invertebrate and vertebrate IV species constitute two lineages that diverged early during the evolution of the family Iridoviridae, before the emergence of the four IIV clades, previously referred to as Chloriridoviruses, Polyiridoviruses, Oligoiridoviruses and Crustaceoiridoviruses. In addition, we provide evidence that species of the family Ascoviridae have a more recent origin than most iridoviruses, emerging just before the differentiation between the Oligoiridoviruses and Crustaceoiridovirus clades. Our results also suggest that after emergence, based on their molecular clock, the ascoviruses evolved more quickly than their closest iridovirus relatives.

An improved genome of the model marine alga Ostreococcus tauri unfolds by assessing Illumina de novo assemblies.

BACKGROUND: Cost effective next generation sequencing technologies now enable the production of genomic datasets for many novel planktonic eukaryotes, representing an understudied reservoir of genetic diversity. O. tauri is the smallest free-living photosynthetic eukaryote known to date, a coccoid green alga that was first isolated in 1995 in a lagoon by the Mediterranean sea. Its simple features, ease of culture and the sequencing of its 13 Mb haploid nuclear genome have promoted this microalga as a new model organism for cell biology. Here, we investigated the quality of genome assemblies of Illumina GAIIx 75 bp paired-end reads from Ostreococcus tauri, thereby also improving the existing assembly and showing the genome to be stably maintained in culture. RESULTS: The 3 assemblers used, ABySS, CLCBio and Velvet, produced 95% complete genomes in 1402 to 2080 scaffolds with a very low rate of misassembly. Reciprocally, these assemblies improved the original genome assembly by filling in 930 gaps. Combined with additional analysis of raw reads and PCR sequencing effort, 1194 gaps have been solved in total adding up to 460 kb of sequence. Mapping of RNAseq Illumina data on this updated genome led to a twofold reduction in the proportion of multi-exon protein coding genes, representing 19% of the total 7699 protein coding genes. The comparison of the DNA extracted in 2001 and 2009 revealed the fixation of 8 single nucleotide substitutions and 2 deletions during the approximately 6000 generations in the lab. The deletions either knocked out or truncated two predicted transmembrane proteins, including a glutamate-receptor like gene. CONCLUSION: High coverage (>80 fold) paired-end Illumina sequencing enables a high quality 95% complete genome assembly of a compact ~13 Mb haploid eukaryote. This genome sequence has remained stable for 6000 generations of lab culture.

Genome sequence of a crustacean iridovirus, IIV31, isolated from the pill bug, Armadillidium vulgare.

Members of the family Iridoviridae are animal viruses that infect only invertebrates and poikilothermic vertebrates. The invertebrate iridovirus 31 (IIV31) was originally isolated from adult pill bugs, Armadillidium vulgare (class Crustacea, order Isopoda, suborder Oniscidea), found in southern California on the campus of the University of California, Riverside, USA. IIV31 virions are icosahedral, have a diameter of about 135 nm, and contain a dsDNA genome 220.222 kbp in length, with 35.09 mol % G+C content and 203 ORFs. Here, we describe the complete genome sequence of this virus and its annotation. This is the eighth genome sequence of an IIV reported.

Complete genome sequence of invertebrate iridovirus IIV-25 isolated from a blackfly larva.

Members of the family Iridoviridae are animal viruses that infect only invertebrates and poikilothermic vertebrates. Invertebrate iridovirus 25 (IIV-25) was originally isolated from the larva of a blackfly (Simulium spp., order Diptera) found in the Ystwyth river near Aberystwyth, Wales. IIV-25 virions are icosahedral, have a diameter of ~130 nm, and contain a dsDNA genome of 204.8 kbp, with a G+C content of 30.32 %, that codes for 177 proteins. Here, we describe the complete genome sequence of this virus and its annotation. This is the fifth genome sequence of an invertebrate iridovirus reported.

Complete genome sequence of invertebrate iridovirus IIV30 isolated from the corn earworm, Helicoverpa zea.

Members of the family Iridoviridae are animal viruses that infect only invertebrates and poikilothermic vertebrates. The invertebrate iridovirus 30 (IIV30) was originally isolated from a larva of the corn earworm, Helicoverpa zea (order lepidoptera, Family Noctuidae) in western Australia. The IIV30 virions are icosahedral, have a diameter of about 130nm, and contain a dsDNA genome of 198.5kbp with 28.11% in GC content and 177 coding sequences. Here we describe its complete genome sequence and annotate the genes for which we could assign a putative function. This is the sixth genome sequence of an invertebrate iridovirus reported.