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1.  De Novo sequencing and transcriptome analysis for Tetramorium bicarinatum: a comprehensive venom gland transcriptome analysis from an ant species 
BMC Genomics  2014;15(1):987.
Arthropod venoms are invaluable sources of bioactive substances with biotechnological application. The limited availability of some venoms, such as those from ants, has restricted the knowledge about the composition and the potential that these biomolecules could represent. In order to provide a global insight on the transcripts expressed in the venom gland of the Brazilian ant species Tetramorium bicarinatum and to unveil the potential of its products, high-throughput approach using Illumina technology has been applied to analyze the genes expressed in active venom glands of this ant species.
A total of 212,371,758 pairs of quality-filtered, 100-base-pair Illumina reads were obtained. The de novo assemblies yielded 36,042 contigs for which 27,873 have at least one predicted ORF among which 59.77% produce significant hits in the available databases. The investigation of the reads mapping toxin class revealed a high diversification with the major part consistent with the classical hymenopteran venom protein signature represented by venom allergen (33.3%), followed by a diverse toxin-expression profile including several distinct isoforms of phospholipase A1 and A2, venom serine protease, hyaluronidase, protease inhibitor and secapin. Moreover, our results revealed for the first time the presence of toxin-like peptides that have been previously identified from unrelated venomous animals such as waprin-like (snakes) and agatoxins (spiders and conus).
The non-toxin transcripts were mainly represented by contigs involved in protein folding and translation, consistent with the protein-secretory function of the venom gland tissue. Finally, about 40% of the generated contigs have no hits in the databases with 25% of the predicted peptides bearing signal peptide emphasizing the potential of the investigation of these sequences as source of new molecules. Among these contigs, six putative novel peptides that show homologies with previously identified antimicrobial peptides were identified.
To the best of our knowledge, this work reports the first large-scale analysis of genes transcribed by the venomous gland of the ant species T. bicarinatum and helps with the identification of Hymenoptera toxin arsenal. In addition, results from this study demonstrate that de novo transcriptome assembly allows useful venom gene expression analysis in a species lacking a genome sequence database.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-987) contains supplementary material, which is available to authorized users.
PMCID: PMC4256838  PMID: 25407482
Tetramorium bicarinatum; Social hymenoptera; Ant; Venom glands; Venom toxins; Hymenopteran allergens; de novo assembly; New generation sequencing; Illumina technology
2.  DNA Methylation and Transcription in a Distal Region Upstream from the Bovine AlphaS1 Casein Gene after Once or Twice Daily Milking 
PLoS ONE  2014;9(11):e111556.
Once daily milking (ODM) induces a reduction in milk production when compared to twice daily milking (TDM). Unilateral ODM of one udder half and TDM of the other half, enables the study of underlying mechanisms independently of inter-individual variability (same genetic background) and of environmental factors. Our results show that in first-calf heifers three CpG, located 10 kb upstream from the CSN1S1 gene were methylated to 33, 34 and 28%, respectively, after TDM but these levels were higher after ODM, 38, 38 and 33%, respectively. These methylation levels were much lower than those observed in the mammary gland during pregnancy (57, 59 and 50%, respectively) or in the liver (74, 78 and 61%, respectively). The methylation level of a fourth CpG (CpG4), located close by (29% during TDM) was not altered after ODM. CpG4 methylation reached 39.7% and 59.5%, during pregnancy or in the liver, respectively. CpG4 is located within a weak STAT5 binding element, arranged in tandem with a second high affinity STAT5 element. STAT5 binding is only marginally modulated by CpG4 methylation, but it may be altered by the methylation levels of the three other CpG nearby. Our results therefore shed light on mechanisms that help to explain how milk production is almost, but not fully, restored when TDM is resumed (15.1±0.2 kg/day instead of 16.2±0.2 kg/day, p<0.01). The STAT5 elements are 100 bp away from a region transcribed in the antisense orientation, in the mammary gland during lactation, but not during pregnancy or in other reproductive organs (ovary or testes). We now need to clarify whether the transcription of this novel RNA is a consequence of STAT5 interacting with the CSN1S1 distal region, or whether it plays a role in the chromatin structure of this region.
PMCID: PMC4219721  PMID: 25369064
3.  Re-Sequencing Data for Refining Candidate Genes and Polymorphisms in QTL Regions Affecting Adiposity in Chicken 
PLoS ONE  2014;9(10):e111299.
In this study, we propose an approach aiming at fine-mapping adiposity QTL in chicken, integrating whole genome re-sequencing data. First, two QTL regions for adiposity were identified by performing a classical linkage analysis on 1362 offspring in 11 sire families obtained by crossing two meat-type chicken lines divergently selected for abdominal fat weight. Those regions, located on chromosome 7 and 19, contained a total of 77 and 84 genes, respectively. Then, SNPs and indels in these regions were identified by re-sequencing sires. Considering issues related to polymorphism annotations for regulatory regions, we focused on the 120 and 104 polymorphisms having an impact on protein sequence, and located in coding regions of 35 and 42 genes situated in the two QTL regions. Subsequently, a filter was applied on SNPs considering their potential impact on the protein function based on conservation criteria. For the two regions, we identified 42 and 34 functional polymorphisms carried by 18 and 24 genes, and likely to deeply impact protein, including 3 coding indels and 4 nonsense SNPs. Finally, using gene functional annotation, a short list of 17 and 4 polymorphisms in 6 and 4 functional genes has been defined. Even if we cannot exclude that the causal polymorphisms may be located in regulatory regions, this strategy gives a complete overview of the candidate polymorphisms in coding regions and prioritize them on conservation- and functional-based arguments.
PMCID: PMC4205046  PMID: 25333370
4.  Complete Genome Sequence of a Field Strain of Peste des Petits Ruminants Virus Isolated during 2010-2014 Epidemics in Senegal 
Genome Announcements  2014;2(5):e00772-14.
Peste des petits ruminants virus (PPRV) infection is expanding and results in regular epizootic activities in Africa, the Middle East, and Asia. Here, we report the complete genome sequence of a field strain of PPRV isolated in Senegal (SnDk11I13) in 2013.
PMCID: PMC4175193  PMID: 25291758
5.  jvenn: an interactive Venn diagram viewer 
BMC Bioinformatics  2014;15(1):293.
Venn diagrams are commonly used to display list comparison. In biology, they are widely used to show the differences between gene lists originating from different differential analyses, for instance. They thus allow the comparison between different experimental conditions or between different methods. However, when the number of input lists exceeds four, the diagram becomes difficult to read. Alternative layouts and dynamic display features can improve its use and its readability.
jvenn is a new JavaScript library. It processes lists and produces Venn diagrams. It handles up to six input lists and presents results using classical or Edwards-Venn layouts. User interactions can be controlled and customized. Finally, jvenn can easily be embeded in a web page, allowing to have dynamic Venn diagrams.
jvenn is an open source component for web environments helping scientists to analyze their data. The library package, which comes with full documentation and an example, is freely available at
PMCID: PMC4261873  PMID: 25176396
Venn; Edward-Venn; Vizualisation; jquery; JavaScript
6.  Identification of large intergenic non-coding RNAs in bovine muscle using next-generation transcriptomic sequencing 
BMC Genomics  2014;15(1):499.
The advent of large-scale gene expression technologies has helped to reveal in eukaryotic cells, the existence of thousands of non-coding transcripts, whose function and significance remain mostly poorly understood. Among these non-coding transcripts, long non-coding RNAs (lncRNAs) are the least well-studied but are emerging as key regulators of diverse cellular processes. In the present study, we performed a survey in bovine Longissimus thoraci of lincRNAs (long intergenic non-coding RNAs not overlapping protein-coding transcripts). To our knowledge, this represents the first such study in bovine muscle.
To identify lincRNAs, we used paired-end RNA sequencing (RNA-Seq) to explore the transcriptomes of Longissimus thoraci from nine Limousin bull calves. Approximately 14–45 million paired-end reads were obtained per library. A total of 30,548 different transcripts were identified. Using a computational pipeline, we defined a stringent set of 584 different lincRNAs with 418 lincRNAs found in all nine muscle samples. Bovine lincRNAs share characteristics seen in their mammalian counterparts: relatively short transcript and gene lengths, low exon number and significantly lower expression, compared to protein-encoding genes. As for the first time, our study identified lincRNAs from nine different samples from the same tissue, it is possible to analyse the inter-individual variability of the gene expression level of the identified lincRNAs. Interestingly, there was a significant difference when we compared the expression variation of the 418 lincRNAs with the 10,775 known selected protein-encoding genes found in all muscle samples. In addition, we found 2,083 pairs of lincRNA/protein-encoding genes showing a highly significant correlated expression. Fourteen lincRNAs were selected and 13 were validated by RT-PCR. Some of the lincRNAs expressed in muscle are located within quantitative trait loci for meat quality traits.
Our study provides a glimpse into the lincRNA content of bovine muscle and will facilitate future experimental studies to unravel the function of these molecules. It may prove useful to elucidate their effect on mechanisms underlying the genetic variability of meat quality traits. This catalog will complement the list of lincRNAs already discovered in cattle and therefore will help to better annotate the bovine genome.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-499) contains supplementary material, which is available to authorized users.
PMCID: PMC4073507  PMID: 24948191
Cattle; Muscle; RNA-Seq; Beef; Long non-coding RNA
7.  Gonad transcriptome analysis of pearl oyster Pinctada margaritifera: identification of potential sex differentiation and sex determining genes 
BMC Genomics  2014;15(1):491.
Black pearl farming is based on culture of the blacklip pearl oyster Pinctada margaritifera (Mollusca, lophotrochozoa), a protandrous hermaphrodite species. At first maturation, all individuals are males. The female sex appears progressively from two years old, which represents a limitation for broodstock conditioning for aquaculture production. In marine mollusks displaying hermaphroditic features, data on sexual determinism and differentiation, including the molecular sex determining cascade, are scarce. To increase genomic resources and identify the molecular mechanisms whereby gene expression may act in the sexual dimorphism of P. margaritifera, we performed gonad transcriptome analysis.
The gonad transcriptome of P. margaritifera was sequenced from several gonadic samples of males and females at different development stages, using a Next-Generation-Sequencing method and RNAseq technology. After Illumina sequencing, assembly and annotation, we obtained 70,147 contigs of which 62.2% shared homologies with existing protein sequences, and 9% showed functional annotation with Gene Ontology terms. Differential expression analysis identified 1,993 differentially expressed contigs between the different categories of gonads. Clustering methods of samples revealed that the sex explained most of the variation in gonad gene expression. K-means clustering of differentially expressed contigs showed 815 and 574 contigs were more expressed in male and female gonads, respectively. The analysis of these contigs revealed the presence of known specific genes coding for proteins involved in sex determinism and/or differentiation, such as dmrt and fem-1 like for males, or foxl2 and vitellogenin for females. The specific gene expression profiles of pmarg-fem1-like, pmarg-dmrt and pmarg-foxl2 in different reproductive stages (undetermined, sexual inversion and regression) suggest that these three genes are potentially involved in the sperm-oocyte switch in P. margaritifera.
The study provides a new transcriptomic tool to study reproduction in hermaphroditic marine mollusks. It identifies sex differentiation and potential sex determining genes in P. margaritifera, a protandrous hermaphrodite species.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-491) contains supplementary material, which is available to authorized users.
PMCID: PMC4082630  PMID: 24942841
Pinctada margaritifera; Gametogenesis; Transcriptome; Differential expression; Sex determinism
8.  The genome of the white-rot fungus Pycnoporus cinnabarinus: a basidiomycete model with a versatile arsenal for lignocellulosic biomass breakdown 
BMC Genomics  2014;15(1):486.
Saprophytic filamentous fungi are ubiquitous micro-organisms that play an essential role in photosynthetic carbon recycling. The wood-decayer Pycnoporus cinnabarinus is a model fungus for the study of plant cell wall decomposition and is used for a number of applications in green and white biotechnology.
The 33.6 megabase genome of P. cinnabarinus was sequenced and assembled, and the 10,442 predicted genes were functionally annotated using a phylogenomic procedure. In-depth analyses were carried out for the numerous enzyme families involved in lignocellulosic biomass breakdown, for protein secretion and glycosylation pathways, and for mating type. The P. cinnabarinus genome sequence revealed a consistent repertoire of genes shared with wood-decaying basidiomycetes. P. cinnabarinus is thus fully equipped with the classical families involved in cellulose and hemicellulose degradation, whereas its pectinolytic repertoire appears relatively limited. In addition, P. cinnabarinus possesses a complete versatile enzymatic arsenal for lignin breakdown. We identified several genes encoding members of the three ligninolytic peroxidase types, namely lignin peroxidase, manganese peroxidase and versatile peroxidase. Comparative genome analyses were performed in fungi displaying different nutritional strategies (white-rot and brown-rot modes of decay). P. cinnabarinus presents a typical distribution of all the specific families found in the white-rot life style. Growth profiling of P. cinnabarinus was performed on 35 carbon sources including simple and complex substrates to study substrate utilization and preferences. P. cinnabarinus grew faster on crude plant substrates than on pure, mono- or polysaccharide substrates. Finally, proteomic analyses were conducted from liquid and solid-state fermentation to analyze the composition of the secretomes corresponding to growth on different substrates. The distribution of lignocellulolytic enzymes in the secretomes was strongly dependent on growth conditions, especially for lytic polysaccharide mono-oxygenases.
With its available genome sequence, P. cinnabarinus is now an outstanding model system for the study of the enzyme machinery involved in the degradation or transformation of lignocellulosic biomass.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-486) contains supplementary material, which is available to authorized users.
PMCID: PMC4101180  PMID: 24942338
Pycnoporus cinnabarinus; Genome annotation; CAZy; Auxiliary activities; Oxidoreductase; White-rot fungi; Lignocellulose
9.  RNAbrowse: RNA-Seq De Novo Assembly Results Browser 
PLoS ONE  2014;9(5):e96821.
Transcriptome analysis based on a de novo assembly of next generation RNA sequences is now performed routinely in many laboratories. The generated results, including contig sequences, quantification figures, functional annotations and variation discovery outputs are usually bulky and quite diverse. This article presents a user oriented storage and visualisation environment permitting to explore the data in a top-down manner, going from general graphical views to all possible details. The software package is based on biomart, easy to install and populate with local data. The software package is available under the GNU General Public License (GPL) at
PMCID: PMC4019526  PMID: 24823498
10.  Design and Characterization of a 52K SNP Chip for Goats 
PLoS ONE  2014;9(1):e86227.
The success of Genome Wide Association Studies in the discovery of sequence variation linked to complex traits in humans has increased interest in high throughput SNP genotyping assays in livestock species. Primary goals are QTL detection and genomic selection. The purpose here was design of a 50–60,000 SNP chip for goats. The success of a moderate density SNP assay depends on reliable bioinformatic SNP detection procedures, the technological success rate of the SNP design, even spacing of SNPs on the genome and selection of Minor Allele Frequencies (MAF) suitable to use in diverse breeds. Through the federation of three SNP discovery projects consolidated as the International Goat Genome Consortium, we have identified approximately twelve million high quality SNP variants in the goat genome stored in a database together with their biological and technical characteristics. These SNPs were identified within and between six breeds (meat, milk and mixed): Alpine, Boer, Creole, Katjang, Saanen and Savanna, comprising a total of 97 animals. Whole genome and Reduced Representation Library sequences were aligned on >10 kb scaffolds of the de novo goat genome assembly. The 60,000 selected SNPs, evenly spaced on the goat genome, were submitted for oligo manufacturing (Illumina, Inc) and published in dbSNP along with flanking sequences and map position on goat assemblies (i.e. scaffolds and pseudo-chromosomes), sheep genome V2 and cattle UMD3.1 assembly. Ten breeds were then used to validate the SNP content and 52,295 loci could be successfully genotyped and used to generate a final cluster file. The combined strategy of using mainly whole genome Next Generation Sequencing and mapping on a contig genome assembly, complemented with Illumina design tools proved to be efficient in producing this GoatSNP50 chip. Advances in use of molecular markers are expected to accelerate goat genomic studies in coming years.
PMCID: PMC3899236  PMID: 24465974
11.  Novel Avian Coronavirus and Fulminating Disease in Guinea Fowl, France 
Emerging Infectious Diseases  2014;20(1):105-108.
For decades, French guinea fowl have been affected by fulminating enteritis of unclear origin. By using metagenomics, we identified a novel avian gammacoronavirus associated with this disease that is distantly related to turkey coronaviruses. Fatal respiratory diseases in humans have recently been caused by coronaviruses of animal origin.
PMCID: PMC3884723  PMID: 24377831
coronavirus; guinea fowl; metagenomics; next-generation sequencing; viruses; France; avian coronavirus; zoonoses; fulminating disease
12.  Detection of Haplotypes Associated with Prenatal Death in Dairy Cattle and Identification of Deleterious Mutations in GART, SHBG and SLC37A2 
PLoS ONE  2013;8(6):e65550.
The regular decrease of female fertility over time is a major concern in modern dairy cattle industry. Only half of this decrease is explained by indirect response to selection on milk production, suggesting the existence of other factors such as embryonic lethal genetic defects. Genomic regions harboring recessive deleterious mutations were detected in three dairy cattle breeds by identifying frequent haplotypes (>1%) showing a deficit in homozygotes among Illumina Bovine 50k Beadchip haplotyping data from the French genomic selection database (47,878 Holstein, 16,833 Montbéliarde, and 11,466 Normande animals). Thirty-four candidate haplotypes (p<10−4) including previously reported regions associated with Brachyspina, CVM, HH1, and HH3 in Holstein breed were identified. Haplotype length varied from 1 to 4.8 Mb and frequencies from 1.7 up to 9%. A significant negative effect on calving rate, consistent in heifers and in lactating cows, was observed for 9 of these haplotypes in matings between carrier bulls and daughters of carrier sires, confirming their association with embryonic lethal mutations. Eight regions were further investigated using whole genome sequencing data from heterozygous bull carriers and control animals (45 animals in total). Six strong candidate causative mutations including polymorphisms previously reported in FANCI (Brachyspina), SLC35A3 (CVM), APAF1 (HH1) and three novel mutations with very damaging effect on the protein structure, according to SIFT and Polyphen-2, were detected in GART, SHBG and SLC37A2 genes. In conclusion, this study reveals a yet hidden consequence of the important inbreeding rate observed in intensively selected and specialized cattle breeds. Counter-selection of these mutations and management of matings will have positive consequences on female fertility in dairy cattle.
PMCID: PMC3676330  PMID: 23762392
13.  Novel Insights into the Bovine Polled Phenotype and Horn Ontogenesis in Bovidae 
PLoS ONE  2013;8(5):e63512.
Despite massive research efforts, the molecular etiology of bovine polledness and the developmental pathways involved in horn ontogenesis are still poorly understood. In a recent article, we provided evidence for the existence of at least two different alleles at the Polled locus and identified candidate mutations for each of them. None of these mutations was located in known coding or regulatory regions, thus adding to the complexity of understanding the molecular basis of polledness. We confirm previous results here and exhaustively identify the causative mutation for the Celtic allele (PC) and four candidate mutations for the Friesian allele (PF). We describe a previously unreported eyelash-and-eyelid phenotype associated with regular polledness, and present unique histological and gene expression data on bovine horn bud differentiation in fetuses affected by three different horn defect syndromes, as well as in wild-type controls. We propose the ectopic expression of a lincRNA in PC/p horn buds as a probable cause of horn bud agenesis. In addition, we provide evidence for an involvement of OLIG2, FOXL2 and RXFP2 in horn bud differentiation, and draw a first link between bovine, ovine and caprine Polled loci. Our results represent a first and important step in understanding the genetic pathways and key process involved in horn bud differentiation in Bovidae.
PMCID: PMC3661542  PMID: 23717440
14.  Gene-based single nucleotide polymorphism discovery in bovine muscle using next-generation transcriptomic sequencing 
BMC Genomics  2013;14:307.
Genetic information based on molecular markers has increasingly being used in cattle breeding improvement programmes, as a mean to improve conventionally phenotypic selection. Advances in molecular genetics have led to the identification of several genetic markers associated with genes affecting economic traits. Until recently, the identification of the causative genetic variants involved in the phenotypes of interest has remained a difficult task. The advent of novel sequencing technologies now offers a new opportunity for the identification of such variants. Despite sequencing costs plummeting, sequencing whole-genomes or large targeted regions is still too expensive for most laboratories. A transcriptomic-based sequencing approach offers a cheaper alternative to identify a large number of polymorphisms and possibly to discover causative variants. In the present study, we performed a gene-based single nucleotide polymorphism (SNP) discovery analysis in bovine Longissimus thoraci, using RNA-Seq. To our knowledge, this represents the first study done in bovine muscle.
Messenger RNAs from Longissimus thoraci from three Limousin bull calves were subjected to high-throughput sequencing. Approximately 36–46 million paired-end reads were obtained per library. A total of 19,752 transcripts were identified and 34,376 different SNPs were detected. Fifty-five percent of the SNPs were found in coding regions and ~22% resulted in an amino acid change. Applying a very stringent SNP quality threshold, we detected 8,407 different high-confidence SNPs, 18% of which are non synonymous coding SNPs. To analyse the accuracy of RNA-Seq technology for SNP detection, 48 SNPs were selected for validation by genotyping. No discrepancies were observed when using the highest SNP probability threshold. To test the usefulness of the identified SNPs, the 48 selected SNPs were assessed by genotyping 93 bovine samples, representing mostly the nine major breeds used in France. Principal component analysis indicates a clear separation between the nine populations.
The RNA-Seq data and the collection of newly discovered coding SNPs improve the genomic resources available for cattle, especially for beef breeds. The large amount of variation present in genes expressed in Limousin Longissimus thoracis, especially the large number of non synonymous coding SNPs, may prove useful to study the mechanisms underlying the genetic variability of meat quality traits.
PMCID: PMC3751807  PMID: 23651547
Single Nucleotide Polymorphism; Cattle; Muscle; RNA-Seq; Beef; Non synonymous coding variants
15.  High-density linkage mapping in a pine tree reveals a genomic region associated with inbreeding depression and provides clues to the extent and distribution of meiotic recombination 
BMC Biology  2013;11:50.
The availability of a large expressed sequence tags (EST) resource and recent advances in high-throughput genotyping technology have made it possible to develop highly multiplexed SNP arrays for multi-objective genetic applications, including the construction of meiotic maps. Such approaches are particularly useful in species with a large genome size, precluding the use of whole-genome shotgun assembly with current technologies.
In this study, a 12 k-SNP genotyping array was developed for maritime pine from an extensive EST resource assembled into a unigene set. The offspring of three-generation outbred and inbred mapping pedigrees were then genotyped. The inbred pedigree consisted of a classical F2 population resulting from the selfing of a single inter-provenance (Landes x Corsica) hybrid tree, whereas the outbred pedigree (G2) resulted from a controlled cross of two intra-provenance (Landes x Landes) hybrid trees. This resulted in the generation of three linkage maps based on SNP markers: one from the parental genotype of the F2 population (1,131 markers in 1,708 centimorgan (cM)), and one for each parent of the G2 population (1,015 and 1,110 markers in 1,447 and 1,425 cM for the female and male parents, respectively). A comparison of segregation patterns in the progeny obtained from the two types of mating (inbreeding and outbreeding) led to the identification of a chromosomal region carrying an embryo viability locus with a semi-lethal allele. Following selfing and segregation, zygote mortality resulted in a deficit of Corsican homozygous genotypes in the F2 population. This dataset was also used to study the extent and distribution of meiotic recombination along the length of the chromosomes and the effect of sex and/or genetic background on recombination. The genetic background of trees in which meiotic recombination occurred was found to have a significant effect on the frequency of recombination. Furthermore, only a small proportion of the recombination hot- and cold-spots were common to all three genotypes, suggesting that the spatial pattern of recombination was genetically variable.
This study led to the development of classical genomic tools for this ecologically and economically important species. It also identified a chromosomal region bearing a semi-lethal recessive allele and demonstrated the genetic variability of recombination rate over the genome.
PMCID: PMC3660193  PMID: 23597128
Unigene; SNP array; Linkage mapping; Segregation distortion; Recombination; Maritime pine; Pinus pinaster
16.  Transcriptional profiling of bud dormancy induction and release in oak by next-generation sequencing 
BMC Genomics  2013;14:236.
In temperate regions, the time lag between vegetative bud burst and bud set determines the duration of the growing season of trees (i.e. the duration of wood biomass production). Dormancy, the period during which the plant is not growing, allows trees to avoid cold injury resulting from exposure to low temperatures. An understanding of the molecular machinery controlling the shift between these two phenological states is of key importance in the context of climatic change. The objective of this study was to identify genes upregulated during endo- and ecodormancy, the two main stages of bud dormancy. Sessile oak is a widely distributed European white oak species. A forcing test on young trees was first carried out to identify the period most likely to correspond to these two stages. Total RNA was then extracted from apical buds displaying endo- and ecodormancy. This RNA was used for the generation of cDNA libraries, and in-depth transcriptome characterization was performed with 454 FLX pyrosequencing technology.
Pyrosequencing produced a total of 495,915 reads. The data were cleaned, duplicated reads removed, and sequences were mapped onto the oak UniGene data. Digital gene expression analysis was performed, with both R statistics and the R-Bioconductor packages (edgeR and DESeq), on 6,471 contigs with read numbers ≥ 5 within any contigs. The number of sequences displaying significant differences in expression level (read abundance) between endo- and ecodormancy conditions ranged from 75 to 161, depending on the algorithm used. 13 genes displaying significant differences between conditions were selected for further analysis, and 11 of these genes, including those for glutathione-S-transferase (GST) and dehydrin xero2 (XERO2) were validated by quantitative PCR.
The identification and functional annotation of differentially expressed genes involved in the “response to abscisic acid”, “response to cold stress” and “response to oxidative stress” categories constitutes a major step towards characterization of the molecular network underlying vegetative bud dormancy, an important life history trait of long-lived organisms.
PMCID: PMC3639946  PMID: 23575249
17.  Field Monitoring of Avian Influenza Viruses: Whole-Genome Sequencing and Tracking of Neuraminidase Evolution Using 454 Pyrosequencing 
Journal of Clinical Microbiology  2012;50(9):2881-2887.
Adaptation of avian influenza viruses (AIVs) from waterfowl to domestic poultry with a deletion in the neuraminidase (NA) stalk has already been reported. The way the virus undergoes this evolution, however, is thus far unclear. We address this question using pyrosequencing of duck and turkey low-pathogenicity AIVs. Ducks and turkeys were sampled at the very beginning of an H6N1 outbreak, and turkeys were swabbed again 8 days later. NA stalk deletions were evidenced in turkeys by Sanger sequencing. To further investigate viral evolution, 454 pyrosequencing was performed: for each set of samples, up to 41,500 reads of ca. 400 bp were generated and aligned. Genetic polymorphisms between duck and turkey viruses were tracked on the whole genome. NA deletion was detected in less than 2% of reads in duck feces but in 100% of reads in turkey tracheal specimens collected at the same time. Further variations in length were observed in NA from turkeys 8 days later. Similarly, minority mutants emerged on the hemagglutinin (HA) gene, with substitutions mostly in the receptor binding site on the globular head. These critical changes suggest a strong evolutionary pressure in turkeys. The increasing performances of next-generation sequencing technologies should enable us to monitor the genomic diversity of avian influenza viruses and early emergence of potentially pathogenic variants within bird flocks. The present study, based on 454 pyrosequencing, suggests that NA deletion, an example of AIV adaptation from waterfowl to domestic poultry, occurs by selection rather than de novo emergence of viral mutants.
PMCID: PMC3421805  PMID: 22718944
18.  A 3.7 Mb Deletion Encompassing ZEB2 Causes a Novel Polled and Multisystemic Syndrome in the Progeny of a Somatic Mosaic Bull 
PLoS ONE  2012;7(11):e49084.
Polled and Multisystemic Syndrome (PMS) is a novel developmental disorder occurring in the progeny of a single bull. Its clinical spectrum includes polledness (complete agenesis of horns), facial dysmorphism, growth delay, chronic diarrhea, premature ovarian failure, and variable neurological and cardiac anomalies. PMS is also characterized by a deviation of the sex-ratio, suggesting male lethality during pregnancy. Using Mendelian error mapping and whole-genome sequencing, we identified a 3.7 Mb deletion on the paternal bovine chromosome 2 encompassing ARHGAP15, GTDC1 and ZEB2 genes. We then produced control and affected 90-day old fetuses to characterize this syndrome by histological and expression analyses. Compared to wild type individuals, affected animals showed a decreased expression of the three deleted genes. Based on a comparison with human Mowat-Wilson syndrome, we suggest that deletion of ZEB2, is responsible for most of the effects of the mutation. Finally sperm-FISH, embryo genotyping and analysis of reproduction records confirmed somatic mosaicism in the founder bull and male-specific lethality during the first third of gestation. In conclusion, we identified a novel locus involved in bovid horn ontogenesis and suggest that epithelial-to-mesenchymal transition plays a critical role in horn bud differentiation. We also provide new insights into the pathogenicity of ZEB2 loss of heterozygosity in bovine and humans and describe the first case of male-specific lethality associated with an autosomal locus in a non-murine mammalian species. This result sets PMS as a unique model to study sex-specific gene expression/regulation.
PMCID: PMC3494662  PMID: 23152852
19.  Fine mapping of complex traits in non-model species: using next generation sequencing and advanced intercross lines in Japanese quail 
BMC Genomics  2012;13:551.
As for other non-model species, genetic analyses in quail will benefit greatly from a higher marker density, now attainable thanks to the evolution of sequencing and genotyping technologies. Our objective was to obtain the first genome wide panel of Japanese quail SNP (Single Nucleotide Polymorphism) and to use it for the fine mapping of a QTL for a fear-related behaviour, namely tonic immobility, previously localized on Coturnix japonica chromosome 1. To this aim, two reduced representations of the genome were analysed through high-throughput 454 sequencing: AFLP (Amplified Fragment Length Polymorphism) fragments as representatives of genomic DNA, and EST (Expressed Sequence Tag) as representatives of the transcriptome.
The sequencing runs produced 399,189 and 1,106,762 sequence reads from cDNA and genomic fragments, respectively. They covered over 434 Mb of sequence in total and allowed us to detect 17,433 putative SNP. Among them, 384 were used to genotype two Advanced Intercross Lines (AIL) obtained from three quail lines differing for duration of tonic immobility. Despite the absence of genotyping for founder individuals in the analysis, the previously identified candidate region on chromosome 1 was refined and led to the identification of a candidate gene.
These data confirm the efficiency of transcript and AFLP-sequencing for SNP discovery in a non-model species, and its application to the fine mapping of a complex trait. Our results reveal a significant association of duration of tonic immobility with a genomic region comprising the DMD (dystrophin) gene. Further characterization of this candidate gene is needed to decipher its putative role in tonic immobility in Coturnix.
PMCID: PMC3534603  PMID: 23066875
Quail; Tonic immobility; Sequencing; AFLP; Transcripts; SNP; AIL
20.  NG6: Integrated next generation sequencing storage and processing environment 
BMC Genomics  2012;13:462.
Next generation sequencing platforms are now well implanted in sequencing centres and some laboratories. Upcoming smaller scale machines such as the 454 junior from Roche or the MiSeq from Illumina will increase the number of laboratories hosting a sequencer. In such a context, it is important to provide these teams with an easily manageable environment to store and process the produced reads.
We describe a user-friendly information system able to manage large sets of sequencing data. It includes, on one hand, a workflow environment already containing pipelines adapted to different input formats (sff, fasta, fastq and qseq), different sequencers (Roche 454, Illumina HiSeq) and various analyses (quality control, assembly, alignment, diversity studies,…) and, on the other hand, a secured web site giving access to the results. The connected user will be able to download raw and processed data and browse through the analysis result statistics. The provided workflows can easily be modified or extended and new ones can be added. Ergatis is used as a workflow building, running and monitoring system. The analyses can be run locally or in a cluster environment using Sun Grid Engine.
NG6 is a complete information system designed to answer the needs of a sequencing platform. It provides a user-friendly interface to process, store and download high-throughput sequencing data.
PMCID: PMC3444930  PMID: 22958229
21.  Genome Sequences of Three Leuconostoc citreum Strains, LBAE C10, LBAE C11, and LBAE E16, Isolated from Wheat Sourdoughs 
Journal of Bacteriology  2012;194(6):1610-1611.
Leuconostoc citreum is a key microorganism in fermented foods of plant origin. Here we report the draft genome sequence for three strains of Leuconostoc citreum, LBAE C10, LBAE C11, and LBAE E16, which have been isolated from traditional French wheat sourdoughs.
PMCID: PMC3294844  PMID: 22374948
22.  Genome Sequence of Weissella confusa LBAE C39-2, Isolated from a Wheat Sourdough 
Journal of Bacteriology  2012;194(6):1608-1609.
Weissella confusa is a rod-shaped heterofermentative lactic acid bacterium from the family of Leuconostocaceae. Here we report the draft genome sequence of the strain W. confusa LBAE C39-2 isolated from a traditional French wheat sourdough.
PMCID: PMC3294846  PMID: 22374947
23.  Metabolic adaptation to a high-fat diet is associated with a change in the gut microbiota 
Gut  2011;61(4):543-553.
The gut microbiota, which is considered a causal factor in metabolic diseases as shown best in animals, is under the dual influence of the host genome and nutritional environment. This study investigated whether the gut microbiota per se, aside from changes in genetic background and diet, could sign different metabolic phenotypes in mice.
The unique animal model of metabolic adaptation was used, whereby C57Bl/6 male mice fed a high-fat carbohydrate-free diet (HFD) became either diabetic (HFD diabetic, HFD-D) or resisted diabetes (HFD diabetes-resistant, HFD-DR). Pyrosequencing of the gut microbiota was carried out to profile the gut microbial community of different metabolic phenotypes. Inflammation, gut permeability, features of white adipose tissue, liver and skeletal muscle were studied. Furthermore, to modify the gut microbiota directly, an additional group of mice was given a gluco-oligosaccharide (GOS)-supplemented HFD (HFD+GOS).
Despite the mice having the same genetic background and nutritional status, a gut microbial profile specific to each metabolic phenotype was identified. The HFD-D gut microbial profile was associated with increased gut permeability linked to increased endotoxaemia and to a dramatic increase in cell number in the stroma vascular fraction from visceral white adipose tissue. Most of the physiological characteristics of the HFD-fed mice were modulated when gut microbiota was intentionally modified by GOS dietary fibres.
The gut microbiota is a signature of the metabolic phenotypes independent of differences in host genetic background and diet.
PMCID: PMC3292714  PMID: 22110050
Gut microbes pyrosequencing; metabolic heterogeneity; high-fat diet responsiveness; type 2 diabetes; bacterial translocation; intestinal barrier function; intestinal bacteria; bone marrow transplantation; diabetes mellitus; gastrointestinal physiology; diabetes mellitus; ANAL; diabetes mellitus; diabetes mellitus
24.  RNA-Seq reveals genotype-specific molecular responses to water deficit in eucalyptus 
BMC Genomics  2011;12:538.
In a context of climate change, phenotypic plasticity provides long-lived species, such as trees, with the means to adapt to environmental variations occurring within a single generation. In eucalyptus plantations, water availability is a key factor limiting productivity. However, the molecular mechanisms underlying the adaptation of eucalyptus to water shortage remain unclear. In this study, we compared the molecular responses of two commercial eucalyptus hybrids during the dry season. Both hybrids differ in productivity when grown under water deficit.
Pyrosequencing of RNA extracted from shoot apices provided extensive transcriptome coverage - a catalog of 129,993 unigenes (49,748 contigs and 80,245 singletons) was generated from 398 million base pairs, or 1.14 million reads. The pyrosequencing data enriched considerably existing Eucalyptus EST collections, adding 36,985 unigenes not previously represented. Digital analysis of read abundance in 14,460 contigs identified 1,280 that were differentially expressed between the two genotypes, 155 contigs showing differential expression between treatments (irrigated vs. non irrigated conditions during the dry season), and 274 contigs with significant genotype-by-treatment interaction. The more productive genotype displayed a larger set of genes responding to water stress. Moreover, stress signal transduction seemed to involve different pathways in the two genotypes, suggesting that water shortage induces distinct cellular stress cascades. Similarly, the response of functional proteins also varied widely between genotypes: the most productive genotype decreased expression of genes related to photosystem, transport and secondary metabolism, whereas genes related to primary metabolism and cell organisation were over-expressed.
For the most productive genotype, the ability to express a broader set of genes in response to water availability appears to be a key characteristic in the maintenance of biomass growth during the dry season. Its strategy may involve a decrease of photosynthetic activity during the dry season associated with resources reallocation through major changes in the expression of primary metabolism associated genes. Further efforts will be needed to assess the adaptive nature of the genes highlighted in this study.
PMCID: PMC3248028  PMID: 22047139
25.  SigReannot-mart: a query environment for expression microarray probe re-annotations 
Expression microarrays are commonly used to study transcriptomes. Most of the arrays are now based on oligo-nucleotide probes. Probe design being a tedious task, it often takes place once at the beginning of the project. The oligo set is then used for several years. During this time period, the knowledge gathered by the community on the genome and the transcriptome increases and gets more precise. Therefore re-annotating the set is essential to supply the biologists with up-to-date annotations. SigReannot-mart is a query environment populated with regularly updated annotations for different oligo sets. It stores the results of the SigReannot pipeline that has mainly been used on farm and aquaculture species. It permits easy extraction in different formats using filters. It is used to compare probe sets on different criteria, to choose the set for a given experiment to mix probe sets in order to create a new one.
Database URL:
PMCID: PMC3263592  PMID: 21930501

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