Publications by authors named "Thomas Derrien"

48 Publications

Long-read assembly of a Great Dane genome highlights the contribution of GC-rich sequence and mobile elements to canine genomes.

Proc Natl Acad Sci U S A 2021 Mar;118(11)

Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109;

Technological advances have allowed improvements in genome reference sequence assemblies. Here, we combined long- and short-read sequence resources to assemble the genome of a female Great Dane dog. This assembly has improved continuity compared to the existing Boxer-derived (CanFam3.1) reference genome. Annotation of the Great Dane assembly identified 22,182 protein-coding gene models and 7,049 long noncoding RNAs, including 49 protein-coding genes not present in the CanFam3.1 reference. The Great Dane assembly spans the majority of sequence gaps in the CanFam3.1 reference and illustrates that 2,151 gaps overlap the transcription start site of a predicted protein-coding gene. Moreover, a subset of the resolved gaps, which have an 80.95% median GC content, localize to transcription start sites and recombination hotspots more often than expected by chance, suggesting the stable canine recombinational landscape has shaped genome architecture. Alignment of the Great Dane and CanFam3.1 assemblies identified 16,834 deletions and 15,621 insertions, as well as 2,665 deletions and 3,493 insertions located on secondary contigs. These structural variants are dominated by retrotransposon insertion/deletion polymorphisms and include 16,221 dimorphic canine short interspersed elements (SINECs) and 1,121 dimorphic long interspersed element-1 sequences (LINE-1_Cfs). Analysis of sequences flanking the 3' end of LINE-1_Cfs (i.e., LINE-1_Cf 3'-transductions) suggests multiple retrotransposition-competent LINE-1_Cfs segregate among dog populations. Consistent with this conclusion, we demonstrate that a canine LINE-1_Cf element with intact open reading frames can retrotranspose its own RNA and that of a SINEC_Cf consensus sequence in cultured human cells, implicating ongoing retrotransposon activity as a driver of canine genetic variation.
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http://dx.doi.org/10.1073/pnas.2016274118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980453PMC
March 2021

Identification of common predisposing loci to hematopoietic cancers in four dog breeds.

PLoS Genet 2021 Apr 1;17(4):e1009395. Epub 2021 Apr 1.

Univ Rennes, CNRS, IGDR (Institut de Génétique et Développement de Rennes)-UMR6290, Rennes, France.

Histiocytic sarcoma (HS) is a rare but aggressive cancer in both humans and dogs. The spontaneous canine model, which has clinical, epidemiological, and histological similarities with human HS and specific breed predispositions, provides a unique opportunity to unravel the genetic basis of this cancer. In this study, we aimed to identify germline risk factors associated with the development of HS in canine-predisposed breeds. We used a methodology that combined several genome-wide association studies in a multi-breed and multi-cancer approach as well as targeted next-generation sequencing, and imputation We combined several dog breeds (Bernese mountain dogs, Rottweilers, flat-coated retrievers, and golden retrievers), and three hematopoietic cancers (HS, lymphoma, and mast cell tumor). Results showed that we not only refined the previously identified HS risk CDKN2A locus, but also identified new loci on canine chromosomes 2, 5, 14, and 20. Capture and targeted sequencing of specific loci suggested the existence of regulatory variants in non-coding regions and methylation mechanisms linked to risk haplotypes, which lead to strong cancer predisposition in specific dog breeds. We also showed that these canine cancer predisposing loci appeared to be due to the additive effect of several risk haplotypes involved in other hematopoietic cancers such as lymphoma or mast cell tumors as well. This illustrates the pleiotropic nature of these canine cancer loci as observed in human oncology, thereby reinforcing the interest of predisposed dog breeds to study cancer initiation and progression.
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http://dx.doi.org/10.1371/journal.pgen.1009395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016107PMC
April 2021

Circulating tumor DNA is detectable in canine histiocytic sarcoma, oral malignant melanoma, and multicentric lymphoma.

Sci Rep 2021 Jan 13;11(1):877. Epub 2021 Jan 13.

Univ Rennes, CNRS, IGDR (Institut de génétique et développement de Rennes) UMR6290, 35000, Rennes, France.

Circulating tumor DNA (ctDNA) has become an attractive biomarker in human oncology, and its use may be informative in canine cancer. Thus, we used droplet digital PCR or PCR for antigen receptor rearrangement, to explore tumor-specific point mutations, copy number alterations, and chromosomal rearrangements in the plasma of cancer-affected dogs. We detected ctDNA in 21/23 (91.3%) of histiocytic sarcoma (HS), 2/8 (25%) of oral melanoma, and 12/13 (92.3%) of lymphoma cases. The utility of ctDNA in diagnosing HS was explored in 133 dogs, including 49 with HS, and the screening of recurrent PTPN11 mutations in plasma had a specificity of 98.8% and a sensitivity between 42.8 and 77% according to the clinical presentation of HS. Sensitivity was greater in visceral forms and especially related to pulmonary location. Follow-up of four dogs by targeting lymphoma-specific antigen receptor rearrangement in plasma showed that minimal residual disease detection was concordant with clinical evaluation and treatment response. Thus, our study shows that ctDNA is detectable in the plasma of cancer-affected dogs and is a promising biomarker for diagnosis and clinical follow-up. ctDNA detection appears to be useful in comparative oncology research due to growing interest in the study of natural canine tumors and exploration of new therapies.
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http://dx.doi.org/10.1038/s41598-020-80332-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806858PMC
January 2021

An integrative atlas of chicken long non-coding genes and their annotations across 25 tissues.

Sci Rep 2020 11 24;10(1):20457. Epub 2020 Nov 24.

PEGASE UMR 1348, INRA, AGROCAMPUS OUEST, 35590, Saint-Gilles, France.

Long non-coding RNAs (LNC) regulate numerous biological processes. In contrast to human, the identification of LNC in farm species, like chicken, is still lacunar. We propose a catalogue of 52,075 chicken genes enriched in LNC ( http://www.fragencode.org/ ), built from the Ensembl reference extended using novel LNC modelled here from 364 RNA-seq and LNC from four public databases. The Ensembl reference grew from 4,643 to 30,084 LNC, of which 59% and 41% with expression ≥ 0.5 and ≥ 1 TPM respectively. Characterization of these LNC relatively to the closest protein coding genes (PCG) revealed that 79% of LNC are in intergenic regions, as in other species. Expression analysis across 25 tissues revealed an enrichment of co-expressed LNC:PCG pairs, suggesting co-regulation and/or co-function. As expected LNC were more tissue-specific than PCG (25% vs. 10%). Similarly to human, 16% of chicken LNC hosted one or more miRNA. We highlighted a new chicken LNC, hosting miR155, conserved in human, highly expressed in immune tissues like miR155, and correlated with immunity-related PCG in both species. Among LNC:PCG pairs tissue-specific in the same tissue, we revealed an enrichment of divergent pairs with the PCG coding transcription factors, as for example LHX5, HXD3 and TBX4, in both human and chicken.
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http://dx.doi.org/10.1038/s41598-020-77586-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686352PMC
November 2020

PTPN11 mutations in canine and human disseminated histiocytic sarcoma.

Int J Cancer 2020 09 8;147(6):1657-1665. Epub 2020 Apr 8.

Faculty of Medicine, CNRS-University of Rennes 1, UMR6290, Institute of Genetics and Development of Rennes, SFR Biosit, Rennes, France.

In humans, histiocytic sarcoma (HS) is an aggressive cancer involving histiocytes. Its rarity and heterogeneity explain that treatment remains a challenge. Sharing high clinical and histopathological similarities with human HS, the canine HS is conversely frequent in specific breeds and thus constitutes a unique spontaneous model for human HS to decipher the genetic bases and to explore therapeutic options. We identified sequence alterations in the MAPK pathway in at least 63.9% (71/111) of HS cases with mutually exclusive BRAF (0.9%; 1/111), KRAS (7.2%; 8/111) and PTPN11 (56.75%; 63/111) mutations concentrated at hotspots common to human cancers. Recurrent PTPN11 mutations are associated to visceral disseminated HS subtype in dogs, the most aggressive clinical presentation. We then identified PTPN11 mutations in 3/19 (15.7%) human HS patients. Thus, we propose PTPN11 mutations as key events for a specific subset of human and canine HS: the visceral disseminated form. Finally, by testing drugs targeting the MAPK pathway in eight canine HS cell lines, we identified a better anti-proliferation activity of MEK inhibitors than PTPN11 inhibitors in canine HS neoplastic cells. In combination, these results illustrate the relevance of naturally affected dogs in deciphering genetic mechanisms and selecting efficient targeted therapies for such rare and aggressive cancers in humans.
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http://dx.doi.org/10.1002/ijc.32991DOI Listing
September 2020

Multi-species annotation of transcriptome and chromatin structure in domesticated animals.

BMC Biol 2019 12 30;17(1):108. Epub 2019 Dec 30.

GenPhySE, Université de Toulouse, INRA, INPT, ENVT, Chemin de Borde Rouge, Castanet-Tolosan Cedex, F-31326, France.

Background: Comparative genomics studies are central in identifying the coding and non-coding elements associated with complex traits, and the functional annotation of genomes is a critical step to decipher the genotype-to-phenotype relationships in livestock animals. As part of the Functional Annotation of Animal Genomes (FAANG) action, the FR-AgENCODE project aimed to create reference functional maps of domesticated animals by profiling the landscape of transcription (RNA-seq), chromatin accessibility (ATAC-seq) and conformation (Hi-C) in species representing ruminants (cattle, goat), monogastrics (pig) and birds (chicken), using three target samples related to metabolism (liver) and immunity (CD4+ and CD8+ T cells).

Results: RNA-seq assays considerably extended the available catalog of annotated transcripts and identified differentially expressed genes with unknown function, including new syntenic lncRNAs. ATAC-seq highlighted an enrichment for transcription factor binding sites in differentially accessible regions of the chromatin. Comparative analyses revealed a core set of conserved regulatory regions across species. Topologically associating domains (TADs) and epigenetic A/B compartments annotated from Hi-C data were consistent with RNA-seq and ATAC-seq data. Multi-species comparisons showed that conserved TAD boundaries had stronger insulation properties than species-specific ones and that the genomic distribution of orthologous genes in A/B compartments was significantly conserved across species.

Conclusions: We report the first multi-species and multi-assay genome annotation results obtained by a FAANG project. Beyond the generation of reference annotations and the confirmation of previous findings on model animals, the integrative analysis of data from multiple assays and species sheds a new light on the multi-scale selective pressure shaping genome organization from birds to mammals. Overall, these results emphasize the value of FAANG for research on domesticated animals and reinforces the importance of future meta-analyses of the reference datasets being generated by this community on different species.
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http://dx.doi.org/10.1186/s12915-019-0726-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936065PMC
December 2019

Transcriptome profiling of mouse samples using nanopore sequencing of cDNA and RNA molecules.

Sci Rep 2019 10 17;9(1):14908. Epub 2019 Oct 17.

Genoscope, Institut de biologie François-Jacob, Commissariat a l'Energie Atomique (CEA), Université Paris-Saclay, F-91057, Evry, France.

Our vision of DNA transcription and splicing has changed dramatically with the introduction of short-read sequencing. These high-throughput sequencing technologies promised to unravel the complexity of any transcriptome. Generally gene expression levels are well-captured using these technologies, but there are still remaining caveats due to the limited read length and the fact that RNA molecules had to be reverse transcribed before sequencing. Oxford Nanopore Technologies has recently launched a portable sequencer which offers the possibility of sequencing long reads and most importantly RNA molecules. Here we generated a full mouse transcriptome from brain and liver using the Oxford Nanopore device. As a comparison, we sequenced RNA (RNA-Seq) and cDNA (cDNA-Seq) molecules using both long and short reads technologies and tested the TeloPrime preparation kit, dedicated to the enrichment of full-length transcripts. Using spike-in data, we confirmed that expression levels are efficiently captured by cDNA-Seq using short reads. More importantly, Oxford Nanopore RNA-Seq tends to be more efficient, while cDNA-Seq appears to be more biased. We further show that the cDNA library preparation of the Nanopore protocol induces read truncation for transcripts containing internal runs of T's. This bias is marked for runs of at least 15 T's, but is already detectable for runs of at least 9 T's and therefore concerns more than 20% of expressed transcripts in mouse brain and liver. Finally, we outline that bioinformatics challenges remain ahead for quantifying at the transcript level, especially when reads are not full-length. Accurate quantification of repeat-associated genes such as processed pseudogenes also remains difficult, and we show that current mapping protocols which map reads to the genome largely over-estimate their expression, at the expense of their parent gene.
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http://dx.doi.org/10.1038/s41598-019-51470-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6797730PMC
October 2019

Prognostic value of somatic focal amplifications on chromosome 30 in canine oral melanoma.

Vet Comp Oncol 2020 Jun 13;18(2):214-223. Epub 2019 Sep 13.

CNRS-University of Rennes 1, UMR6290, Institute of Genetics and Development of Rennes, Faculty of Medicine, Rennes, France.

Canine oral melanoma is the first malignancy of the oral cavity in dogs and is characterized by a local invasiveness and a high metastatic propensity. A better knowledge of genetic alterations is expected to improve management of this tumour. Copy number alterations are known characteristics of mucosal melanomas both in dogs and humans. The goal of this study was to explore the prognostic value of somatic focal amplifications on chromosomes (Canis Familiaris [CFA]) 10 and 30 in canine oral melanoma. The cohort included 73 dogs with oral melanoma confirmed by histology, removed surgically without adjuvant therapy and with a minimal follow-up of 6 months. Epidemiological, clinical and histological data were collected and quantitative-PCR were performed on formalin-fixed paraffin-embedded (FFPE) samples to identify specific focal amplifications. The 73 dogs included in the study had a median survival time of 220 days. Focal amplifications on CFA 10 and 30 were recurrent (49.3% and 50.7% of cases, respectively) and CFA 30 amplification was significantly associated with the amelanotic phenotype (P = .046) and high mitotic index (MI; P = .0039). CFA 30 amplification was also linked to poor prognosis (P = .0005). Other negative prognostic factors included gingiva location (P = .003), lymphadenomegaly (P = .026), tumour ulceration at diagnosis (P = .003), MI superior to 6 mitoses over 10 fields (P = .001) and amelanotic tumour (P = .029). In multivariate analyses using Cox proportional hazards regression, CFA 30 amplification (Hazard ratio [HR] = 2.08; P = .011), tumour location (HR = 2.20; P = .005) and histological pigmentation (HR = 1.87; P = .036) were significantly associated with shorter survival time. Focal amplification of CFA 30 is linked to an aggressive subset and constitutes a new prognostic factor.
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http://dx.doi.org/10.1111/vco.12536DOI Listing
June 2020

Genome-Wide Analysis of Long Non-Coding RNA Profiles in Canine Oral Melanomas.

Genes (Basel) 2019 06 23;10(6). Epub 2019 Jun 23.

University of Rennes, CNRS, IGDR-UMR 6290, F-35000 Rennes, France.

Mucosal melanomas (MM) are rare aggressive cancers in humans, and one of the most common forms of oral cancers in dogs. Similar biological and histological features are shared between MM in both species, making dogs a powerful model for comparative oncology studies of melanomas. Although exome sequencing recently identified recurrent coding mutations in canine MM, little is known about changes in non-coding gene expression, and more particularly, in canine long non-coding RNAs (lncRNAs), which are commonly dysregulated in human cancers. Here, we sampled a large cohort ( = 52) of canine normal/tumor oral MM from three predisposed breeds (poodles, Labrador retrievers, and golden retrievers), and used deep transcriptome sequencing to identify more than 400 differentially expressed (DE) lncRNAs. We further prioritized candidate lncRNAs by comparative genomic analysis to pinpoint 26 dog-human conserved DE lncRNAs, including , , and lncRNAs. Using unsupervised co-expression network analysis with coding genes, we inferred the potential functions of the DE lncRNAs, suggesting associations with cancer-related genes, cell cycle, and carbohydrate metabolism Gene Ontology (GO) terms. Finally, we exploited our multi-breed design to identify DE lncRNAs within breeds. This study provides a unique transcriptomic resource for studying oral melanoma in dogs, and highlights lncRNAs that may potentially be diagnostic or therapeutic targets for human and veterinary medicine.
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http://dx.doi.org/10.3390/genes10060477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6628375PMC
June 2019

Identification of a Missense Variant in Involved in Dilution of Phaeomelanin Leading to White or Cream Coat Color in Dogs.

Genes (Basel) 2019 05 21;10(5). Epub 2019 May 21.

Institut de Génétique et Développement de Rennes, CNRS-UMR6290, Université de Rennes1, 35000 Rennes, France.

White coat color in mammals has been selected several times during the domestication process. Numerous dog breeds are fixed for one form of white coat color that involves darkly pigmented skin. The genetic basis of this color, due to the absence of pigment in the hairs, was suggested to correspond to extreme dilution of the phaeomelanin, by both the expression of only phaeomelanin (locus E) and its extreme dilution (locus I). To go further, we performed genome-wide association studies (GWAS) using a multiple breed approach. The first GWAS, using 34 white dogs and 128 non-white dogs, including White Shepherds, Poodles, Cotons de Tulear and Bichons allowed us to identify two significantly associated loci on the locus E and a novel locus on chromosome 20. A second GWAS using 15 other breeds presenting extreme phaeomelanin dilution confirmed the position of locus I on the chromosome 20 (position 55 Mb = 6 × 10). Using whole-genome sequencing, we identified a missense variant in the first exon of , a gene recently identified to be involved in human, mouse and horse pigmentation. We confirmed the role of this variant in phaeomelanin dilution of numerous canine breeds, and the conserved role of in mammalian pigmentation.
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http://dx.doi.org/10.3390/genes10050386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562630PMC
May 2019

Dynamic DNA material with emergent locomotion behavior powered by artificial metabolism.

Sci Robot 2019 04;4(29)

Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853, USA.

Metabolism is a key process that makes life alive-the combination of anabolism and catabolism sustains life by a continuous flux of matter and energy. In other words, the materials comprising life are synthesized, assembled, dissipated, and decomposed autonomously in a controlled, hierarchical manner using biological processes. Although some biological approaches for creating dynamic materials have been reported, the construction of such materials by mimicking metabolism from scratch based on bioengineering has not yet been achieved. Various chemical approaches, especially dissipative assemblies, allow the construction of dynamic materials in a synthetic fashion, analogous to part of metabolism. Inspired by these approaches, here, we report a bottom-up construction of dynamic biomaterials powered by artificial metabolism, representing a combination of irreversible biosynthesis and dissipative assembly processes. An emergent locomotion behavior resembling a slime mold was programmed with this material by using an abstract design model similar to mechanical systems. Dynamic properties, such as autonomous pattern generation and continuous polarized regeneration, enabled locomotion along the designated tracks against a constant flow. Furthermore, an emergent racing behavior of two locomotive bodies was achieved by expanding the program. Other applications, including pathogen detection and hybrid nanomaterials, illustrated further potential use of this material. Dynamic biomaterials powered by artificial metabolism could provide a previously unexplored route to realize "artificial" biological systems with regenerating and self-sustaining characteristics.
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http://dx.doi.org/10.1126/scirobotics.aaw3512DOI Listing
April 2019

Structure and stimuli-responsiveness of all-DNA dendrimers: theory and experiment.

Nanoscale 2019 Jan;11(4):1604-1617

Institute for Theoretical Physics, TU Wien, Wiedner Hauptstraße 8-10, A-1040 Vienna, Austria.

We present a comprehensive theoretical and experimental study of the solution phase properties of a DNA-based family of nanoparticles - dendrimer-like DNA molecules (DL-DNA). These charged DNA dendrimers are novel macromolecular aggregates, which hold high promise in targeted self-assembly of soft matter systems in the bulk and at interfaces. To describe the behaviour of this family of dendrimers (with generations ranging from G1 to G7), we use a theoretical model in which base-pairs of a single DL-DNA molecule are modeled by charged monomers, whose interactions are chosen to mimic the equilibrium properties of DNA correctly. Experimental results on the sizes and conformations of DL-DNA are based on static and dynamic light scattering; and molecular dynamics simulations are employed to model the equilibrium properties of DL-DNA, which compare favorably to the findings from experiments while at the same time providing a host of additional information and insight into the molecular structure of the nanostructures. We also examine the salt-responsiveness of these macromolecules, finding that despite the strong screening of electrostatic interactions brought about by the added salt, the macromolecules shrink only slightly, their size robustness stemming from the high bending rigidity of the DNA-segments. The study of these charged dendrimer systems is an important field of research in the area of soft matter due to their potential role for various interdisciplinary applications, ranging from molecular cages and carriers for drug delivery in a living organism to the development of dendrimer- and dendron-based ultra-thin films in the area of nanotechnology. These findings are essential to determine if DL-DNA is a viable candidate for the experimental realization of cluster crystals in the bulk, a novel form of solid with multiple site occupancy.
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http://dx.doi.org/10.1039/c8nr05814hDOI Listing
January 2019

Characterisation and functional predictions of canine long non-coding RNAs.

Sci Rep 2018 09 7;8(1):13444. Epub 2018 Sep 7.

Univ Rennes, CNRS, IGDR (Institut de génétique et développement de Rennes) - UMR 6290, F-35000, Rennes, France.

Long non-coding RNAs (lncRNAs) are a family of heterogeneous RNAs that play major roles in multiple biological processes. We recently identified an extended repertoire of more than 10,000 lncRNAs of the domestic dog however, predicting their biological functionality remains challenging. In this study, we have characterised the expression profiles of 10,444 canine lncRNAs in 26 distinct tissue types, representing various anatomical systems. We showed that lncRNA expressions are mainly clustered by tissue type and we highlighted that 44% of canine lncRNAs are expressed in a tissue-specific manner. We further demonstrated that tissue-specificity correlates with specific families of canine transposable elements. In addition, we identified more than 900 conserved dog-human lncRNAs for which we show their overall reproducible expression patterns between dog and human through comparative transcriptomics. Finally, co-expression analyses of lncRNA and neighbouring protein-coding genes identified more than 3,400 canine lncRNAs, suggesting that functional roles of these lncRNAs act as regulatory elements. Altogether, this genomic and transcriptomic integrative study of lncRNAs constitutes a major resource to investigate genotype to phenotype relationships and biomedical research in the dog species.
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http://dx.doi.org/10.1038/s41598-018-31770-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128939PMC
September 2018

Assembly Dynamics of Plasmonic DNA-Capped Gold Nanoparticle Monolayers.

Langmuir 2018 12 7;34(49):14711-14720. Epub 2018 Jun 7.

The self-assembly of nanoparticles in aqueous solutions promises wide applications but requires the careful balance of many parameters not present in organic solvents. While the presence of long-range electrostatic interactions in aqueous solutions may complicate such assemblies, they provide additional parameters through which to control self-assembly. Here, with DNA-capped gold nanoparticles and through the variation of the ionic strength in aqueous solutions, we explored the influence of electrostatic interactions on the adsorption of negatively charged nanoparticles on a positively charged surface. Specifically, we studied the kinetics of nanoparticle adsorption from solution using the quartz crystal microbalance with dissipation (QCM-D). We also characterized the structure of the adsorbed monolayers employing a combination of grazing incidence small-angle X-ray scattering (GISAXS) and scanning electron microscopy. We discovered that adsorption kinetics and monolayer structure were under the control of the DNA ligand length, solution ionic strength, and salt species. We also precisely fit the kinetics to a modified Langmuir model, which converged to the simple Langmuir model at high ionic strengths of magnesium chloride. We demonstrated that increasing the ionic strength and decreasing the DNA ligand lengths increased the surface coverage while decreasing the nanoparticle-nanoparticle spacing. The DNA-capped nanoparticle system reported here provides a readily applicable platform for controlling nanoparticle self-assembly in aqueous solution. Finally, we employ this tunability to create a system with a tunable plasmonic response. Our kinetics studies of the assembly process and further characterizations undertaken will facilitate the construction of nanoparticle arrays with precise structure, and such control will aid in the design of future plasmonic and optoelectronic devices.
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http://dx.doi.org/10.1021/acs.langmuir.8b00484DOI Listing
December 2018

An exome sequencing based approach for genome-wide association studies in the dog.

Sci Rep 2017 Nov 15;7(1):15680. Epub 2017 Nov 15.

Institut de Génétique et Développement de Rennes, CNRS-URM6290, Université Rennes1, Rennes, France.

Genome-wide association studies (GWAS) are widely used to identify loci associated with phenotypic traits in the domestic dog that has emerged as a model for Mendelian and complex traits. However, a disadvantage of GWAS is that it always requires subsequent fine-mapping or sequencing to pinpoint causal mutations. Here, we performed whole exome sequencing (WES) and canine high-density (cHD) SNP genotyping of 28 dogs from 3 breeds to compare the SNP and linkage disequilibrium characteristics together with the power and mapping precision of exome-guided GWAS (EG-GWAS) versus cHD-based GWAS. Using simulated phenotypes, we showed that EG-GWAS has a higher power than cHD to detect associations within target regions and less power outside target regions, with power being influenced further by sample size and SNP density. We analyzed two real phenotypes (hair length and furnishing), that are fixed in certain breeds to characterize mapping precision of the known causal mutations. EG-GWAS identified the associated exonic and 3'UTR variants within the FGF5 and RSPO2 genes, respectively, with only a few samples per breed. In conclusion, we demonstrated that EG-GWAS can identify loci associated with Mendelian phenotypes both within and across breeds.
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http://dx.doi.org/10.1038/s41598-017-15947-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5688105PMC
November 2017

Discovery of Human-Similar Gene Fusions in Canine Cancers.

Cancer Res 2017 11 7;77(21):5721-5727. Epub 2017 Sep 7.

Univ. Rennes 1, CNRS, Institut de Geéneétique et de Deéveloppement de Rennes (IGDR), UMR6290, Rennes, France.

Canine cancers represent a tremendous natural resource due to their incidence and striking similarities to human cancers, sharing similar clinical and pathologic features as well as oncogenic events, including identical somatic mutations. Considering the importance of gene fusions as driver alterations, we explored their relevance in canine cancers. We focused on three distinct human-comparable canine cancers representing different tissues and embryonic origins. Through RNA-Seq, we discovered similar gene fusions as those found in their human counterparts: - in B-cell lymphoma, - in glioma, and - in dermatofibrosarcoma protuberans-like. We showed not only similar partner genes but also identical breakpoints leading to oncogene overexpression. This study demonstrates similar gene fusion partners and mechanisms in human-dog corresponding tumors and allows for selection of targeted therapies in preclinical and clinical trials with pet dogs prior to human trials, within the framework of personalized medicine. .
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http://dx.doi.org/10.1158/0008-5472.CAN-16-2691DOI Listing
November 2017

Rapid transcriptional plasticity of duplicated gene clusters enables a clonally reproducing aphid to colonise diverse plant species.

Genome Biol 2017 02 13;18(1):27. Epub 2017 Feb 13.

Earlham Institute, Norwich Research Park, Norwich, NR4 7UZ, UK.

Background: The prevailing paradigm of host-parasite evolution is that arms races lead to increasing specialisation via genetic adaptation. Insect herbivores are no exception and the majority have evolved to colonise a small number of closely related host species. Remarkably, the green peach aphid, Myzus persicae, colonises plant species across 40 families and single M. persicae clonal lineages can colonise distantly related plants. This remarkable ability makes M. persicae a highly destructive pest of many important crop species.

Results: To investigate the exceptional phenotypic plasticity of M. persicae, we sequenced the M. persicae genome and assessed how one clonal lineage responds to host plant species of different families. We show that genetically identical individuals are able to colonise distantly related host species through the differential regulation of genes belonging to aphid-expanded gene families. Multigene clusters collectively upregulate in single aphids within two days upon host switch. Furthermore, we demonstrate the functional significance of this rapid transcriptional change using RNA interference (RNAi)-mediated knock-down of genes belonging to the cathepsin B gene family. Knock-down of cathepsin B genes reduced aphid fitness, but only on the host that induced upregulation of these genes.

Conclusions: Previous research has focused on the role of genetic adaptation of parasites to their hosts. Here we show that the generalist aphid pest M. persicae is able to colonise diverse host plant species in the absence of genetic specialisation. This is achieved through rapid transcriptional plasticity of genes that have duplicated during aphid evolution.
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http://dx.doi.org/10.1186/s13059-016-1145-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5304397PMC
February 2017

Long noncoding RNA repertoire in chicken liver and adipose tissue.

Genet Sel Evol 2017 01 10;49(1). Epub 2017 Jan 10.

UMR PEGASE, INRA, 35042, Rennes, France.

Background: Improving functional annotation of the chicken genome is a key challenge in bridging the gap between genotype and phenotype. Among all transcribed regions, long noncoding RNAs (lncRNAs) are a major component of the transcriptome and its regulation, and whole-transcriptome sequencing (RNA-Seq) has greatly improved their identification and characterization. We performed an extensive profiling of the lncRNA transcriptome in the chicken liver and adipose tissue by RNA-Seq. We focused on these two tissues because of their importance in various economical traits for which energy storage and mobilization play key roles and also because of their high cell homogeneity. To predict lncRNAs, we used a recently developed tool called FEELnc, which also classifies them with respect to their distance and strand orientation to the closest protein-coding genes. Moreover, to confidently identify the genes/transcripts expressed in each tissue (a complex task for weakly expressed molecules such as lncRNAs), we probed a particularly large number of biological replicates (16 per tissue) compared to common multi-tissue studies with a larger set of tissues but less sampling.

Results: We predicted 2193 lncRNA genes, among which 1670 were robustly expressed across replicates in the liver and/or adipose tissue and which were classified into 1493 intergenic and 177 intragenic lncRNAs located between and within protein-coding genes, respectively. We observed similar structural features between chickens and mammals, with strong synteny conservation but without sequence conservation. As previously reported, we confirm that lncRNAs have a lower and more tissue-specific expression than mRNAs. Finally, we showed that adjacent lncRNA-mRNA genes in divergent orientation have a higher co-expression level when separated by less than 1 kb compared to more distant divergent pairs. Among these, we highlighted for the first time a novel lncRNA candidate involved in lipid metabolism, lnc_DHCR24, which is highly correlated with the DHCR24 gene that encodes a key enzyme of cholesterol biosynthesis.

Conclusions: We provide a comprehensive lncRNA repertoire in the chicken liver and adipose tissue, which shows interesting patterns of co-expression between mRNAs and lncRNAs. It contributes to improving the structural and functional annotation of the chicken genome and provides a basis for further studies on energy storage and mobilization traits in the chicken.
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http://dx.doi.org/10.1186/s12711-016-0275-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5225574PMC
January 2017

FEELnc: a tool for long non-coding RNA annotation and its application to the dog transcriptome.

Nucleic Acids Res 2017 05;45(8):e57

Institut Génétique et Développement de Rennes, CNRS, UMR6290, University Rennes1, Rennes, Cedex 35043, France.

Whole transcriptome sequencing (RNA-seq) has become a standard for cataloguing and monitoring RNA populations. One of the main bottlenecks, however, is to correctly identify the different classes of RNAs among the plethora of reconstructed transcripts, particularly those that will be translated (mRNAs) from the class of long non-coding RNAs (lncRNAs). Here, we present FEELnc (FlExible Extraction of LncRNAs), an alignment-free program that accurately annotates lncRNAs based on a Random Forest model trained with general features such as multi k-mer frequencies and relaxed open reading frames. Benchmarking versus five state-of-the-art tools shows that FEELnc achieves similar or better classification performance on GENCODE and NONCODE data sets. The program also provides specific modules that enable the user to fine-tune classification accuracy, to formalize the annotation of lncRNA classes and to identify lncRNAs even in the absence of a training set of non-coding RNAs. We used FEELnc on a real data set comprising 20 canine RNA-seq samples produced by the European LUPA consortium to substantially expand the canine genome annotation to include 10 374 novel lncRNAs and 58 640 mRNA transcripts. FEELnc moves beyond conventional coding potential classifiers by providing a standardized and complete solution for annotating lncRNAs and is freely available at https://github.com/tderrien/FEELnc.
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http://dx.doi.org/10.1093/nar/gkw1306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5416892PMC
May 2017

A Point Mutation in a lincRNA Upstream of GDNF Is Associated to a Canine Insensitivity to Pain: A Spontaneous Model for Human Sensory Neuropathies.

PLoS Genet 2016 Dec 29;12(12):e1006482. Epub 2016 Dec 29.

CNRS, UMR 6290, Institut de Génétique et Développement de Rennes, Rennes, France.

Human Hereditary Sensory Autonomic Neuropathies (HSANs) are characterized by insensitivity to pain, sometimes combined with self-mutilation. Strikingly, several sporting dog breeds are particularly affected by such neuropathies. Clinical signs appear in young puppies and consist of acral analgesia, with or without sudden intense licking, biting and severe self-mutilation of the feet, whereas proprioception, motor abilities and spinal reflexes remain intact. Through a Genome Wide Association Study (GWAS) with 24 affected and 30 unaffected sporting dogs using the Canine HD 170K SNP array (Illumina), we identified a 1.8 Mb homozygous locus on canine chromosome 4 (adj. p-val = 2.5x10-6). Targeted high-throughput sequencing of this locus in 4 affected and 4 unaffected dogs identified 478 variants. Only one variant perfectly segregated with the expected recessive inheritance in 300 sporting dogs of known clinical status, while it was never present in 900 unaffected dogs from 130 other breeds. This variant, located 90 kb upstream of the GDNF gene, a highly relevant neurotrophic factor candidate gene, lies in a long intergenic non-coding RNAs (lincRNA), GDNF-AS. Using human comparative genomic analysis, we observed that the canine variant maps onto an enhancer element. Quantitative RT-PCR of dorsal root ganglia RNAs of affected dogs showed a significant decrease of both GDNF mRNA and GDNF-AS expression levels (respectively 60% and 80%), as compared to unaffected dogs. We thus performed gel shift assays (EMSA) that reveal that the canine variant significantly alters the binding of regulatory elements. Altogether, these results allowed the identification in dogs of GDNF as a relevant candidate for human HSAN and insensitivity to pain, but also shed light on the regulation of GDNF transcription. Finally, such results allow proposing these sporting dog breeds as natural models for clinical trials with a double benefit for human and veterinary medicine.
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http://dx.doi.org/10.1371/journal.pgen.1006482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5198995PMC
December 2016

Erratum to: Bioinformatics Pipeline for Transcriptome Sequencing Analysis.

Methods Mol Biol 2017 ;1468:E1

CNRS UMR6290 Dog Genetic Team, 2 av du Pr. Léon Bernard, Rennes, 35043, France.

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http://dx.doi.org/10.1007/978-1-4939-4035-6_17DOI Listing
January 2017

Re-annotation, improved large-scale assembly and establishment of a catalogue of noncoding loci for the genome of the model brown alga Ectocarpus.

New Phytol 2017 Apr 21;214(1):219-232. Epub 2016 Nov 21.

Algal Genetics Group, CNRS, UMR 8227, Integrative Biology of Marine Models, Sorbonne Université, UPMC Univ Paris 06, Station Biologique de Roscoff, CS 90074, F-29688, Roscoff, France.

The genome of the filamentous brown alga Ectocarpus was the first to be completely sequenced from within the brown algal group and has served as a key reference genome both for this lineage and for the stramenopiles. We present a complete structural and functional reannotation of the Ectocarpus genome. The large-scale assembly of the Ectocarpus genome was significantly improved and genome-wide gene re-annotation using extensive RNA-seq data improved the structure of 11 108 existing protein-coding genes and added 2030 new loci. A genome-wide analysis of splicing isoforms identified an average of 1.6 transcripts per locus. A large number of previously undescribed noncoding genes were identified and annotated, including 717 loci that produce long noncoding RNAs. Conservation of lncRNAs between Ectocarpus and another brown alga, the kelp Saccharina japonica, suggests that at least a proportion of these loci serve a function. Finally, a large collection of single nucleotide polymorphism-based markers was developed for genetic analyses. These resources are available through an updated and improved genome database. This study significantly improves the utility of the Ectocarpus genome as a high-quality reference for the study of many important aspects of brown algal biology and as a reference for genomic analyses across the stramenopiles.
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http://dx.doi.org/10.1111/nph.14321DOI Listing
April 2017

Bioinformatics Pipeline for Transcriptome Sequencing Analysis.

Methods Mol Biol 2017 ;1468:201-19

CNRS UMR6290 Dog Genetic Team, 2 av du Pr. Léon Bernard, Rennes, 35043, France.

The development of High Throughput Sequencing (HTS) for RNA profiling (RNA-seq) has shed light on the diversity of transcriptomes. While RNA-seq is becoming a de facto standard for monitoring the population of expressed transcripts in a given condition at a specific time, processing the huge amount of data it generates requires dedicated bioinformatics programs. Here, we describe a standard bioinformatics protocol using state-of-the-art tools, the STAR mapper to align reads onto a reference genome, Cufflinks to reconstruct the transcriptome, and RSEM to quantify expression levels of genes and transcripts. We present the workflow using human transcriptome sequencing data from two biological replicates of the K562 cell line produced as part of the ENCODE3 project.
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http://dx.doi.org/10.1007/978-1-4939-4035-6_14DOI Listing
January 2018

DNA Microgels as a Platform for Cell-Free Protein Expression and Display.

Biomacromolecules 2016 06 17;17(6):2019-26. Epub 2016 May 17.

Department of Biological and Environmental Engineering, Cornell University , Ithaca, New York 14853, United States.

Protein expression and selection is an essential process in the modification of biological products. Expressed proteins are selected based on desired traits (phenotypes) from diverse gene libraries (genotypes), whose size may be limited due to the difficulties inherent in diverse cell preparation. In addition, not all genes can be expressed in cells, and linking genotype with phenotype further presents a great challenge in protein engineering. We present a DNA gel-based platform that demonstrates the versatility of two DNA microgel formats to address fundamental challenges of protein engineering, including high protein yield, isolation of gene sets, and protein display. We utilize microgels to show successful protein production and capture of a model protein, green fluorescent protein (GFP), which is further used to demonstrate a successful gene enrichment through fluorescence-activated cell sorting (FACS) of a mixed population of microgels containing the GFP gene. Through psoralen cross-linking of the hydrogels, we have synthesized DNA microgels capable of surviving denaturing conditions while still possessing the ability to produce protein. Lastly, we demonstrate a method of producing extremely high local gene concentrations of up to 32 000 gene repeats in hydrogels 1 to 2 μm in diameter. These DNA gels can serve as a novel cell-free platform for integrated protein expression and display, which can be applied toward more powerful, scalable protein engineering and cell-free synthetic biology with no physiological boundaries and limitations.
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http://dx.doi.org/10.1021/acs.biomac.6b00183DOI Listing
June 2016

Improved canine exome designs, featuring ncRNAs and increased coverage of protein coding genes.

Sci Rep 2015 Aug 3;5:12810. Epub 2015 Aug 3.

Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.

By limiting sequencing to those sequences transcribed as mRNA, whole exome sequencing is a cost-efficient technique often used in disease-association studies. We developed two target enrichment designs based on the recently released annotation of the canine genome: the exome-plus design and the exome-CDS design. The exome-plus design combines the exons of the CanFam 3.1 Ensembl annotation, more recently discovered protein-coding exons and a variety of non-coding RNA regions (microRNAs, long non-coding RNAs and antisense transcripts), leading to a total size of ≈ 152 Mb. The exome-CDS was designed as a subset of the exome-plus by omitting all 3' and 5' untranslated regions. This reduced the size of the exome-CDS to ≈ 71 Mb. To test the capturing performance, four exome-plus captures were sequenced on a NextSeq 500 with each capture containing four pre-capture pooled, barcoded samples. At an average sequencing depth of 68.3x, 80% of the regions and well over 90% of the targeted base pairs were completely covered at least 5 times with high reproducibility. Based on the performance of the exome-plus, we estimated the performance of the exome-CDS. Overall, these designs provide flexible solutions for a variety of research questions and are likely to be reliable tools in disease studies.
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http://dx.doi.org/10.1038/srep12810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522663PMC
August 2015

Identification of long non-coding RNAs in insects genomes.

Curr Opin Insect Sci 2015 Feb 13;7:37-44. Epub 2015 Jan 13.

CNRS, UMR 6290, Institut de Génétique et Développement de Rennes, Université de Rennes 1, 2 Avenue du Pr. Léon Bernard, 35000 Rennes, France.

The development of high throughput sequencing technologies (HTS) has allowed researchers to better assess the complexity and diversity of the transcriptome. Among the many classes of non-coding RNAs (ncRNAs) identified the last decade, long non-coding RNAs (lncRNAs) represent a diverse and numerous repertoire of important ncRNAs, reinforcing the view that they are of central importance to the cell machinery in all branches of life. Although lncRNAs have been involved in essential biological processes such as imprinting, gene regulation or dosage compensation especially in mammals, the repertoire of lncRNAs is poorly characterized for many non-model organisms. In this review, we first focus on what is known about experimentally validated lncRNAs in insects and then review bioinformatic methods to annotate lncRNAs in the genomes of hexapods.
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http://dx.doi.org/10.1016/j.cois.2015.01.003DOI Listing
February 2015

DNA materials: bridging nanotechnology and biotechnology.

Acc Chem Res 2014 Jun 2;47(6):1902-11. Epub 2014 Jun 2.

Department of Biological & Environmental Engineering, Cornell University , Ithaca, New York 14853, United States.

CONSPECTUS: In recent decades, DNA has taken on an assortment of diverse roles, not only as the central genetic molecule in biological systems but also as a generic material for nanoscale engineering. DNA possesses many exceptional properties, including its biological function, biocompatibility, molecular recognition ability, and nanoscale controllability. Taking advantage of these unique attributes, a variety of DNA materials have been created with properties derived both from the biological functions and from the structural characteristics of DNA molecules. These novel DNA materials provide a natural bridge between nanotechnology and biotechnology, leading to far-ranging real-world applications. In this Account, we describe our work on the design and construction of DNA materials. Based on the role of DNA in the construction, we categorize DNA materials into two classes: substrate and linker. As a substrate, DNA interfaces with enzymes in biochemical reactions, making use of molecular biology's "enzymatic toolkit". For example, employing DNA as a substrate, we utilized enzymatic ligation to prepare the first bulk hydrogel made entirely of DNA. Using this DNA hydrogel as a structural scaffold, we created a protein-producing DNA hydrogel via linking plasmid DNA onto the hydrogel matrix through enzymatic ligation. Furthermore, to fully make use of the advantages of both DNA materials and polymerase chain reaction (PCR), we prepared thermostable branched DNA that could remain intact even under denaturing conditions, allowing for their use as modular primers for PCR. Moreover, via enzymatic polymerization, we have recently constructed a physical DNA hydrogel with unique internal structure and mechanical properties. As a linker, we have used DNA to interface with other functional moieties, including gold nanoparticles, clay minerals, proteins, and lipids, allowing for hybrid materials with unique properties for desired applications. For example, we recently designed a DNA-protein conjugate as a universal adapter for protein detection. We further demonstrate a diverse assortment of applications for these DNA materials including diagnostics, protein production, controlled drug release systems, the exploration of life evolution, and plasmonics. Although DNA has shown great potential as both substrate and linker in the construction of DNA materials, it is still in the initial stages of becoming a well-established and widely used material. Important challenges include the ease of design and fabrication, scaling-up, and minimizing cost. We envision that DNA materials will continue to bridge the gap between nanotechnology and biotechnology and will ultimately be employed for many real-world applications.
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http://dx.doi.org/10.1021/ar5001082DOI Listing
June 2014