Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
IntroductionLeishmania RNA virus type 1 (LRV1) is an endosymbiont of some Leishmania (Vianna) species in South America. Presence of LRV1 in parasites exacerbates disease severity in animal models and humans, related to a disproportioned innate immune response, and is correlated with drug treatment failures in humans. Although the virus was identified decades ago, its genomic diversity has been overlooked until now.Methodology/Principles findingsWe subjected LRV1 strains from 19 L. (V.) guyanensis and one L. (V.) braziliensis isolates obtained from cutaneous leishmaniasis samples identified throughout French Guiana with next-generation sequencing and de novo sequence assembly. We generated and analyzed 24 unique LRV1 sequences over their full-length coding regions. Multiple alignment of these new sequences revealed variability (0.5%±23.5%) across the entire sequence except for highly conserved motifs within the 5' untranslated region. Phylogenetic analyses showed that viral genomes of L. (V.) guyanensis grouped into five distinct clusters. They further showed a species-dependent clustering between viral genomes of L. (V.) guyanensis and L. (V.) braziliensis, confirming a long-term co-evolutionary history. Noteworthy, we identified cases of multiple LRV1 infections in three of the 20 Leishmania isolates.Conclusions/SignificanceHere, we present the first-ever estimate of LRV1 genomic diversity that exists in Leishmania (V.) guyanensis parasites. Genetic characterization and phylogenetic analyses of these viruses has shed light on their evolutionary relationships. To our knowledge, this study is also the first to report cases of multiple LRV1 infections in some parasites. Finally, this work has made it possible to develop molecular tools for adequate identification and genotyping of LRV1 strains for diagnostic purposes. Given the suspected worsening role of LRV1 infection in the pathogenesis of human leishmaniasis, these data have a major impact from a clinical viewpoint and for the management of Leishmania-infected patients.
ISSN: 1935-2727

pasteur-01583523
https://hal-riip.archives-ouvertes.fr/pasteur-01583523
DOI : 10.1371/journal.pntd.0005764
PUBMED : 28715422

Éditeur(s) : HAL CCSD Wiley/Blackwell
Résumé : International audience
Microsatellites are widely used in population genetics to uncover recent evolutionary events. They are typically genotyped using capillary sequencer, which capacity is usually limited to 9, at most 12 loci for each run, and which analysis is a tedious task that is performed by hand. With the rise of next-generation sequencing (NGS), a much larger number of loci and individuals are available from sequencing: for example, on a single run of a GS Junior, 28 loci from 96 individuals are sequenced with a 30X cover. We have developed an algorithm to automatically and efficiently genotype microsatellites from a collection of reads sorted by individual (e.g. specific PCR amplifications of a locus or a collection of reads that encompass a locus of interest). As the sequencing and the PCR amplification introduce artefactual insertions or deletions, the set of reads from a single microsatellite allele shows several length variants. The algorithm infers, without alignment, the true unknown allele(s) of each individual from the observed distributions of microsatellites length of all individuals. MicNeSs, a python implementation of the algorithm, can be used to genotype any microsatellite locus from any organism and has been tested on 454 pyrosequencing data of several loci from fruit flies (a model species) and red deers (a nonmodel species). Without any parallelization, it automatically genotypes 22 loci from 441 individuals in 11 hours on a standard computer. The comparison of MicNeSs inferences to the standard method shows an excellent agreement, with some differences illustrating the pros and cons of both methods.
ISSN: 1755-098X

hal-01544754
https://hal.archives-ouvertes.fr/hal-01544754
DOI : 10.1111/1755-0998.12467

Éditeur(s) : HAL CCSD Wiley/Blackwell
Résumé : Whole genome sequencing is helping generate robust phylogenetic hypotheses for a range of taxonomic groups that were previously recalcitrant to classical molecular phylogenetic approaches. As a case study, we performed a shallow shotgun sequencing of eight species in the tropical tree family Chrysobalanaceae to retrieve large fragments of high-copy number DNA regions and test the potential of these regions for phylogeny reconstruction. We were able to assemble the nuclear ribosomal cluster (nrDNA), the complete plastid genome (ptDNA) and a large fraction of the mitochondrial genome (mtDNA) with approximately 1000x, 450x and 120x sequencing depth respectively. The phylogenetic tree obtained with ptDNA resolved five of the seven internal nodes. In contrast, the tree obtained with mtDNA and nrDNA data were largely unresolved. This study demonstrates that genome skimming is a cost-effective approach and shows potential in plant molecular systematics within Chrysobalanaceae and other under-studied groups.
ISSN: 1755-098X

hal-01204217
https://hal.archives-ouvertes.fr/hal-01204217
DOI : 10.1111/1755-0998.12246
PRODINRA : 283530

Éditeur(s) : HAL CCSD
Résumé : il s'agit d'un type de produit dont les métadonnées ne correspondent pas aux métadonnées attendues dans les autres types de produit : ACTIVITY_REPORT
Understanding genomic evolution in wild populations is of particular importance because evolution is a central process in our understanding of populations’ history and their future response to global changes. In addition to fundamental knowledge, the study of populations’ evolution has wide applications such as conservation/restoration genetics or epidemiology. Indeed, genetic diversity is the ‘fuel’ for populations’ evolution and adaptation to new conditions, and estimating the extent and the geographic structure of genetic diversity, as well as identifying the evolutionary forces behind (demography, gene flow, and adaptation) are thus major challenges in the current context of global changes. [br/]A particular attention is currently devoted to the process of genetic divergence in wild populations, with emphases on gene flow, local adaptation, and their interactions. Indeed, adaptation in wild populations is supported in many species by many kinds of experiments based on both quantitative phenotypic traits and molecular data. However, the processes governing populations’ evolution are poorly understood in the tropical rainforest of Amazonia, and the relative influence of neutral and adaptive processes are continuously discussed. [br/]I am a young scientist interested in the process of genomic divergence in wild (tree) populations. I aimed at understanding how evolution structures the genetic diversity and governs molecular divergence in wild populations, with particular emphases on gene flow and local adaptation. During five years as a young scientist, I worked on answering the following questions, focusing on two tree species occupying vulnerable areas: mainly [i]Eperua falcata[/i] in Amazonia and, to a lesser extent, [i]Abies alba[/i] in the Mediterranean area.[br/][u]Scientific questions:[/u][br/]- Which evolutionary drivers are responsible of the structure of genetic diversity in wild populations occupying wide areas? This question was addressed at different geographical scales: from large (regional) to very local (microgeographic) scales. [br/]- What is the relative influence of neutral (gene flow, inbreeding) and adaptive drivers in the process of genetic divergence?[br/]- Is microgeographic adaptation possible in spite of extensive gene flow between populations occupying the extreme boundaries of an ecological gradient? [br/]- What is the extent of natural selection in the (whole) genome of wild populations? [br/][br/]My research work is at the interface between three promising disciplines: Evolutionary genomics (i.e. the study of populations evolution and the evolutionary drivers behind), Bioinformatics (i.e. the treatment of next-generation data), and Bayesian modelling (i.e. the empirical calibration of complex mechanistic model). I intend to take advantage of the recent advances in sequencing and informatic technologies to bring up to date the field of ecological genetics and evolutionary biology, by developing modern research strategies to study the process of genomic divergence in wild populations.
https://hal.archives-ouvertes.fr/hal-01204218

hal-01204218
https://hal.archives-ouvertes.fr/hal-01204218
PRODINRA : 286326

Éditeur(s) : HAL CCSD
Résumé : International audience
BACKGROUND: Dairy working increases the prevalence of lower airway respiratory diseases, especially COPD and asthma. Epidemiological studies have reported that chronic inhalation of organic dusts released during specific daily tasks could represent a major risk factor for development of these pathologies in dairy workers. Knowledge on size, nature and biological activity of such organic dusts remain however limited. OBJECTIVE: To compare size distribution, microbial composition and cellular effects of dusts liberated by the spreading of straw bedding in five French dairy farms located in Brittany. RESULTS: Mechanized distribution of straw bedding generated a cloud of inhalable dusts in the five dairy farms' barns. Thoracic particles having a 3-7.5µm size constituted 58.9-68.3% of these dusts. Analyses of thoracic dusts by next generation sequencing showed that the microbial dust composition differed between the five French farms, although Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria represent more than 97.5% of the bacterial phyla detected in each sample. Several bacteria genera comprising of human pathogenic species, such as Pseudomonas, Staphylococcus, Thermoactinomyces or Saccharopolyspora were identified. Cladosporium and Alternaria fungal genera, which are potent environmental determinants of respiratory symptoms, were detected in dusts collected in the five farms and their levels reached 15.5-51.1% and 9-24.7% of assignable fungal sequences in each sample, respectively. Finally, all dust samples significantly and strongly increased the expression of the pro-inflammatory TNF-α, IL-1β, IL-6 and IL-8 cytokines at both mRNA and protein levels in human monocyte-derived macrophages. Their effects were dose-dependent and detectable from 1µg/ml. The intensity of the macrophage responses however differed according to the samples. CONCLUSIONS: Our results strengthen the hypothesis that organic dusts released during the distribution of straw bedding are mainly constituted of thoracic particles which are small enough to deposit on lower bronchial epithelium of dairy farmers and induce inflammation.
Environmental Research

hal-01579610
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01579610
DOI : 10.1016/j.envres.2017.07.025
PUBMED : 28735230

Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
Background: Researchers sorely need markers and approaches for biodiversity exploration (both specimen linked and metagenomics) using the full potential of next generation sequencing technologies (NGST). Currently, most studies rely on expensive multiple tagging, PCR primer universality and/or the use of few markers, sometimes with insufficient variability. Methodology/Principal Findings: We propose a novel approach for the isolation and sequencing of a universal, useful and popular marker across distant, non-model metazoans: the complete mitochondrial genome. It relies on the properties of metazoan mitogenomes for enrichment, on careful choice of the organisms to multiplex, as well as on the wide collection of accumulated mitochondrial reference datasets for post-sequencing sorting and identification instead of individual tagging. Multiple divergent organisms can be sequenced simultaneously, and their complete mitogenome obtained at a very low cost. We provide in silico testing of dataset assembly for a selected set of example datasets. Conclusions/Significance: This approach generates large mitogenome datasets. These sequences are useful for phylogenetics, molecular identification and molecular ecology studies, and are compatible with all existing projects or available datasets based on mitochondrial sequences, such as the Barcode of Life project. Our method can yield sequences both from identified samples and metagenomic samples. The use of the same datasets for both kinds of studies makes for a powerful approach, especially since the datasets have a high variability even at species level, and would be a useful complement to the less variable 18S rDNA currently prevailing in metagenomic studies.
ISSN: 1932-6203

hal-01544766
https://hal.archives-ouvertes.fr/hal-01544766
DOI : 10.1371/journal.pone.0051263

Éditeur(s) : HAL CCSD Society for Molecular Biology and Evolution
Résumé : International audience
Although heterokaryons have been reported in nature, multicellular organisms are generally assumed genetically homogeneous. Here, we investigate the case of arbuscular mycorrhizal fungi (AMF) that form symbiosis with plant roots. The growth advantages they confer to their hosts are of great potential benefit to sustainable agricultural practices. However, measuring genetic diversity for these coenocytes is a major challenge: Within the same cytoplasm, AMF contain thousands of nuclei and show extremely high levels of genetic variation for some loci. The extent and physical location of polymorphism within and between AMF genomes is unclear. We used two complementary strategies to estimate genetic diversity in AMF, investigating polymorphism both on a genome scale and in putative single copy loci. First, we used data from whole-genome pyrosequencing of four AMF isolates to describe genetic diversity, based on a conservative network-based clustering approach. AMF isolates showed marked differences in genome-wide diversity patterns in comparison to a panel of control fungal genomes. This clustering approach further allowed us to provide conservative estimates of Rhizophagus spp. genomes sizes. Second, we designed new putative single copy genomic markers, which we investigated by massive parallel amplicon sequencing for two Rhizophagus irregularis and one Rhizophagus sp. isolates. Most loci showed high polymorphism, with up to 103 alleles per marker. This polymorphism could be distributed within or between nuclei. However, we argue that the Rhizophagus isolates under study might be heterokaryotic, at least for the putative single copy markers we studied. Considering that genetic information is the main resource for identification of AMF, we suggest that special attention is warranted for the study of these ecologically important organisms.
ISSN: 1759-6653

Droits : http://creativecommons.org/licenses/by/
hal-01224215
http://hal.upmc.fr/hal-01224215
http://hal.upmc.fr/hal-01224215/document
http://hal.upmc.fr/hal-01224215/file/Genome%20Biol%20Evol-2015-Boon-505-21.pdf
DOI : 10.1093/gbe/evv002

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Background : Biological communities are normally composed of a few abundant and many rare species. This pattern is particularly prominent in microbial communities, in which most constituent taxa are usually extremely rare. Although abundant and rare subcommunities may present intrinsic characteristics that could be crucial for understanding community dynamics and ecosystem functioning, microbiologists normally do not differentiate between them. Here, we investigate abundant and rare subcommunities of marine microbial eukaryotes, a crucial group of organisms that remains among the least-explored biodiversity components of the biosphere. We surveyed surface waters of six separate coastal locations in Europe, independently considering the picoplankton, nanoplankton, and microplankton/mesoplankton organismal size fractions.Results : Deep Illumina sequencing of the 18S rRNA indicated that the abundant regional community was mostly structured by organismal size fraction, whereas the rare regional community was mainly structured by geographic origin. However, some abundant and rare taxa presented similar biogeography, pointing to spatiotemporal structure in the rare microeukaryote biosphere. Abundant and rare subcommunities presented regular proportions across samples, indicating similar species-abundance distributions despite taxonomic compositional variation. Several taxa were abundant in one location and rare in other locations, suggesting large oscillations in abundance. The substantial amount of metabolically active lineages found in the rare biosphere suggests that this subcommunity constitutes a diversity reservoir that can respond rapidly to environmental change.Conclusions : We propose that marine planktonic microeukaryote assemblages incorporate dynamic and metabolically active abundant and rare subcommunities, with contrasting structuring patterns but fairly regular proportions, across space and time.
ISSN: 0960-9822

hal-01149637
http://hal.upmc.fr/hal-01149637
http://hal.upmc.fr/hal-01149637/document
http://hal.upmc.fr/hal-01149637/file/Logares_et_al_CurrentBiol_accepted.pdf
DOI : 10.1016/j.cub.2014.02.050

Éditeur(s) : HAL CCSD Oxford University Press (OUP)
Résumé : International audience
DNA sequencing technology is becoming more accessible to a variety of researchers as costs continue to decline. As researchers begin to sequence novel transcriptomes, most of these data sets lack a reference genome and will have to rely on de novo assemblers. Making comparisons across assemblies can be difficult: each program has its strengths and weaknesses, and no tool exists to comparatively evaluate these data sets. We developed software in R, called Sequence Comparative Analysis using Networks (SCAN), to perform statistical comparisons between distinct assemblies. SCAN uses a reference data set to identify the most accurate de novo assembly and the ``good'' transcripts in the user's data. We tested SCAN on three publicly available transcriptomes, each assembled using three assembly programs. Moreover, we sequenced the transcriptome of the oomycete Achlya hypogyna and compared de novo assemblies from Velvet, ABySS, and the CLC Genomics Workbench assembly algorithms. One thousand one hundred twenty-eight of the CLC transcripts were statistically similar to the reference, compared with 49 of the Velvet transcripts and 937 of the ABySS transcripts. SCAN's strength is providing statistical support for transcript assemblies in a biological context. However, SCAN is designed to compare distinct node sets in networks, therefore it can also easily be extended to perform statistical comparisons on any network graph regardless of what the nodes represent.
ISSN: 0737-4038

hal-01544800
https://hal.archives-ouvertes.fr/hal-01544800
DOI : 10.1093/molbev/mst087

Éditeur(s) : HAL CCSD Taylor & Francis
Résumé : International audience
Abstract Indoor fungal contamination has been associated with a wide range of adverse health effects, including infectious diseases, toxic effects and allergies. The diversity of fungi contributes to the complex role that they play in indoor environments and human diseases. Molds have a major impact on public health, and can cause different consequences in hospitals, homes and workplaces. This review presents the methods used to assess fungal contamination in these various environments, and discusses advantages and disadvantages for each method in consideration with different health risks. Air, dust and surface sampling strategies are compared, as well as the limits of various methods are used to detect and quantify fungal particles and fungal compounds. In addition to conventional microscopic and culture approaches, more recent chemical, immunoassay and polymerase chain reaction (PCR)-based methods are described. This article also identifies common needs for future multidisciplinary research and development projects in this field, with specific interests on viable fungi and fungal fragment detections. The determination of fungal load and the detection of species in environmental samples greatly depend on the strategy of sampling and analysis. Quantitative PCR was found useful to identify associations between specific fungi and common diseases. The next-generation sequencing methods may afford new perspectives in this area.
ISSN: 1040-841X

hal-00874670
https://hal.archives-ouvertes.fr/hal-00874670
DOI : 10.3109/1040841X.2013.777687
PUBMED : 23586944

Éditeur(s) : HAL CCSD
Résumé : International audience
Background Orodental diseases include several clinically and genetically heterogeneous disorders that can present in isolation or as part of a genetic syndrome. Due to the vast number of genes implicated in these disorders, establishing a molecular diagnosis can be challenging. We aimed to develop a targeted next-generation sequencing (NGS) assay to diagnose mutations and potentially identify novel genes mutated in this group of disorders. Methods We designed an NGS gene panel that targets 585 known and candidate genes in orodental disease. We screened a cohort of 101 unrelated patients without a molecular diagnosis referred to the Reference Centre for Oro-Dental Manifestations of Rare Diseases, Strasbourg, France, for a variety of orodental disorders including isolated and syndromic amelogenesis imperfecta (AI), isolated and syndromic selective tooth agenesis (STHAG), isolated and syndromic dentinogenesis imperfecta, isolated dentin dysplasia, otodental dysplasia and primary failure of tooth eruption. Results We discovered 21 novel pathogenic variants and identified the causative mutation in 39 unrelated patients in known genes (overall diagnostic rate: 39%). Among the largest subcohorts of patients with isolated AI (50 unrelated patients) and isolated STHAG (21 unrelated patients), we had a definitive diagnosis in 14 (27%) and 15 cases (71%), respectively. Surprisingly, COL17A1 mutations accounted for the majority of autosomal-dominant AI cases. Conclusions We have developed a novel targeted NGS assay for the efficient molecular diagnosis of a wide variety of orodental diseases. Furthermore, our panel will contribute to better understanding the contribution of these genes to orodental disease. Trial registration numbers NCT01746121 and NCT02397824
Journal of Medical Genetics

hal-01272929
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01272929
DOI : 10.1136/jmedgenet-2015-103302
PUBMED : 26502894

Éditeur(s) : HAL CCSD Society for the Study of Reproduction
Résumé : International audience
Mammalian spermatogenesis is a complex and highly orchestrated combination of processes in which male germline proliferation and differentiation result in the production of mature spermatozoa. If recent genome-wide studies have contributed to the in-depth analysis of the male germline protein-encoding transcriptome, little effort has yet been devoted to the systematic identification of novel unannotated transcribed regions expressed during mammalian spermatogenesis. We report high-resolution expression profiling of male germ cells in rat, using next-generation sequencing technology and highly enriched testicular cell populations. Among 20 424 high-confidence transcripts reconstructed, we defined a stringent set of 1419 long multi-exonic unannotated transcripts expressed in the testis (testis-expressed unannotated transcripts [TUTs]). TUTs were divided into 7 groups with different expression patterns. Most TUTs share many of the characteristics of vertebrate long noncoding RNAs (lncRNAs). We also markedly reinforced the finding that TUTs and known lncRNAs accumulate during the meiotic and postmeiotic stages of spermatogenesis in mammals and that X-linked meiotic TUTs do not escape the silencing effects of meiotic sex chromosome inactivation. Importantly, we discovered that TUTs and known lncRNAs with a peak expression during meiosis define a distinct class of noncoding transcripts that exhibit exons twice as long as those of other transcripts. Our study provides new insights in transcriptional profiling of the male germline and represents a high-quality resource for novel loci expressed during spermatogenesis that significantly contributes to rat genome annotation.
ISSN: 0006-3363

hal-01063924
https://hal.archives-ouvertes.fr/hal-01063924
DOI : 10.1095/biolreprod.114.118166
PUBMED : 24740603

Éditeur(s) : HAL CCSD
Résumé : International audience
Clear cell renal cell carcinoma (ccRCC) has a poor prognosis with a 50% risk of metastases. Little is known about the phenotypic and molecular profiles of metastases regarding their corresponding primary tumors. This study aimed to screen phenotypic and genotypic differences between metastases and their corresponding primary tumors. We selected four cases with available frozen material. The histological, immunohistochemical (VEGFA, CD31, SMA, Ki67, p53, PAR-3), FISH (VHL gene), next-generation sequencing (VHL and c-MET genes), multiplex ligation-dependent probe amplification, and array-(comparative genomic hybridization) CGH analyses were realized. Metastases were nodal, hepatic (synchronous), adrenal, and pulmonary (metachronous). High-grade tumor cells were significantly more frequent in metastases (p = 0.019). Metastases and high-grade zones of primary tumors shared similar characteristics compared to low-grade zones: a lower microscopic vascular density (43.5 vs 382.5 vessels/mm(2); p = 0.0027), a higher expression of VEGF (73 vs 10%, p = 0.045), Ki67 (37.6 vs 8.3%; p = 0.011), and p53 (54 vs 10.6%; p = 0.081), and a cytoplasmic and membranous PAR-3 staining. Metastases exhibited more chromosomal imbalances than primary tumors in total (18.75 ± 6.8; p = 0.044) with more genomic gains (13.5 ± 7; p = 0.013). The loss of chromosome 9 and gain of Xq were found in both primary tumors and metastases but gains of loci or chromosomes 2p, 3q, 5, 8q, 12, and 20 were only found in metastases. The VHL gene status was similar in each tumor couple. Although metastases and primary tumors share common histological features, this study highlights chromosomal differences specific to metastases which could be involved in ccRCC metastatic evolution.
Virchows Archiv

hal-01560281
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01560281
DOI : 10.1007/s00428-017-2124-0
PUBMED : 28488172

Éditeur(s) : HAL CCSD Nature Publishing Group
Résumé : International audience
The etiology of congenital heart defect (CHD) combines environmental and genetic factors. So far, there were studies reporting on the screening of a single gene on unselected CHD or on familial cases selected for specific CHD types. Our goal was to systematically screen a proband of familial cases of CHD on a set of genetic tests to evaluate the prevalence of disease-causing variant identification. A systematic screening of GATA4, NKX2-5, ZIC3 and Multiplex ligation-dependent probe amplification (MLPA) P311 Kit was setup on the proband of 154 families with at least two cases of non-syndromic CHD. Additionally, ELN screening was performed on families with supravalvular arterial stenosis. Twenty-two variants were found, but segregation analysis confirmed unambiguously the causality of 16 variants: GATA4 (1 x), NKX2-5 (6 x), ZIC3 (3 x), MLPA (2 x) and ELN (4 x). Therefore, this approach was able to identify the causal variant in 10.4% of familial CHD cases. This study demonstrated the existence of a de novo variant even in familial CHD cases and the impact of CHD variants on adult cardiac condition even in the absence of CHD. This study showed that the systematic screening of genetic factors is useful in familial CHD cases with up to 10.4% elucidated cases. When successful, it drastically improved genetic counseling by discovering unaffected variant carriers who are at risk of transmitting their variant and are also exposed to develop cardiac complications during adulthood thus prompting long-term cardiac follow-up. This study provides an important baseline at dawning of the next-generation sequencing era.
ISSN: 1018-4813

hal-01404211
https://hal-univ-bourgogne.archives-ouvertes.fr/hal-01404211
DOI : 10.1038/ejhg.2015.105
PUBMED : 26014430