Single nucleotide polymorphism discovery from expressed sequence tags in the waterflea Daphnia magna

<p>Abstract</p> <p>Background</p> <p><it>Daphnia </it>(Crustacea: Cladocera) plays a central role in standing aquatic ecosystems, has a well known ecology and is widely used in population studies and environmental risk assessments. <it>Daphnia magna </it>is, especially in Europe, intensively used to study stress responses of natural populations to pollutants, climate change, and antagonistic interactions with predators and parasites, which have all been demonstrated to induce micro-evolutionary and adaptive responses. Although its ecology and evolutionary biology is intensively studied, little is known on the functional genomics underpinning of phenotypic responses to environmental stressors. The aim of the present study was to find genes expressed in presence of environmental stressors, and target such genes for single nucleotide polymorphic (SNP) marker development.</p> <p>Results</p> <p>We developed three expressed sequence tag (EST) libraries using clonal lineages of <it>D. magna </it>exposed to ecological stressors, namely fish predation, parasite infection and pesticide exposure. We used these newly developed ESTs and other <it>Daphnia </it>ESTs retrieved from NCBI GeneBank to mine for SNP markers targeting synonymous as well as non synonymous genetic variation. We validate the developed SNPs in six natural populations of <it>D. magna </it>distributed at regional scale.</p> <p>Conclusions</p> <p>A large proportion (47%) of the produced ESTs are <it>Daphnia </it>lineage specific genes, which are potentially involved in responses to environmental stress rather than to general cellular functions and metabolic activities, or reflect the arthropod's aquatic lifestyle. The characterization of genes expressed under stress and the validation of their SNPs for population genetic study is important for identifying ecologically responsive genes in <it>D. magna</it>.</p

Recommendations for whole genome sequencing in diagnostics for rare diseases

In 2016, guidelines for diagnostic Next Generation Sequencing (NGS) have been published by EuroGentest in order to assist laboratories in the implementation and accreditation of NGS in a diagnostic setting. These guidelines mainly focused on Whole Exome Sequencing (WES) and targeted (gene panels) sequencing detecting small germline variants (Single Nucleotide Variants (SNVs) and insertions/deletions (indels)). Since then, Whole Genome Sequencing (WGS) has been increasingly introduced in the diagnosis of rare diseases as WGS allows the simultaneous detection of SNVs, Structural Variants (SVs) and other types of variants such as repeat expansions. The use of WGS in diagnostics warrants the re-evaluation and update of previously published guidelines. This work was jointly initiated by EuroGentest and the Horizon2020 project Solve-RD. Statements from the 2016 guidelines have been reviewed in the context of WGS and updated where necessary. The aim of these recommendations is primarily to list the points to consider for clinical (laboratory) geneticists, bioinformaticians, and (non-)geneticists, to provide technical advice, aid clinical decision-making and the reporting of the results

Streptococcus agalactiae clones infecting humans were selected and fixed through the extensive use of tetracycline

Streptococcus agalactiae (Group B Streptococcus, GBS) is a commensal of the digestive and genitourinary tracts of humans that emerged as the leading cause of bacterial neonatal infections in Europe and North America during the 1960s. Due to the lack of epidemiological and genomic data, the reasons for this emergence are unknown. Here we show by comparative genome analysis and phylogenetic reconstruction of 229 isolates that the rise of human GBS infections corresponds to the selection and worldwide dissemination of only a few clones. The parallel expansion of the clones is preceded by the insertion of integrative and conjugative elements conferring tetracycline resistance (TcR). Thus, we propose that the use of tetracycline from 1948 onwards led in humans to the complete replacement of a diverse GBS population by only few TcR clones particularly well adapted to their host, causing the observed emergence of GBS diseases in neonates. \ua9 2014 Macmillan Publishers Limited. All rights reserved

Genomic variation in European Sea bass: from SNP discovery within ESTs to genome scan.

European sea bass (Dicentrarchus labrax) is an economically important marine species in European aquaculture. Althou gh sea bass population structure is well known, aquaculture does not benefit fr om selection programs, sea bass production being nearly completely based on wild-caught fishes reproducing in semi-controlled conditions. More knowl edge on the sea bass genome would help the breeding progress in this species, th e study of natural populations and their evolution as well as the management of fisheries. The generation of large collections of Expr essed Sequence Tags (ESTs) would provide genomic resources for discovering new genes and new markers, identifying intron-exon boundaries and studying genes expression profiles. In this thesis, efforts were concentrated on the di scovery of Single Nucleotide Polymorphisms (SNPs) within ESTs. SNPs are the most abundant source of variation in most eukaryotic and prokaryotic genomes and can have applications both in aquaculture and natural popula tions. Approximately 30,000 ESTs from 14 libraries of five sea bass individuals have been sequenced. This large EST collection was described and compared to a similar set of ESTs generated for gilthead sea bream (S parus aurata), another economically important marine species. The processin g of ESTs led to the generation of 17,716 and 18,198 sea bass and sea bream u nique sequences, of which less than a third were common to both species. Autom atic annotation indicated that more protein coding sequences were generated f or sea bass than for sea bream. This was further confirmed by the prediction of Open Reading Frames (ORFs) and by the GC content of sea bass and sea bream un ique sequences. Gene Ontology (GO) annotation showed that the same categories were represented for both species. Six SNP discovery tools were used on sea bass ESTs and their performance was assessed by validating around 10% of the SNP candidates. This analysis demonstrated that the selection of redundant candidate SNPs (mismatches detected at least twice in the ESTs) was a good mean of improving SNP di scovery performance. The selection of SNP candidates with a minimum allele frequency greater or equal to 0.3 further enhanced SNP discovery performance although reducing the number of SNP candidates. Finally the selection of SNP candidates detect ed by several tools and the exclusion of indels were also good means of reduci ng the number of false positive candidate SNPs. Transition SNP candidates appea red to be less reliable than transversion SNP candidates due to the presence of RNA editing sites in EST collections. High quality of EST assembly and of th e flanking regions of SNP candidates revealed to be essential for an efficient SNP discovery. These conclusions led to the development of a pipeline integrating the six tes ted SNP discovery tools. This efficient and easy to use pipeline allows the detection of SNPs in any EST dataset , the selection of SNP candidates according to redundancy and/or minimum allel e frequency and the comparison of SNP candidates according to SNP discovery tool. It has been used successfully on EST collections of the fishes Dicentra rchus labrax, Sparus aurata, Anguilla anguilla and the waterflea Daphnia magna. The use of the six SNP discovery tools identified 1,072 unique SNP candidates of which a subset was validated. A total of 360 SNPs were discovered in introns and ESTs, proving that resequencing the conti gs predicted to be polymorphic was an efficient way of discovering SNPs. The nucleotide diversity of sea bass was estimated to one SNP every 137 bp and was high er in introns than in ESTs. The Mendelian inheritance was checked on 17 SNPs polymorphic on the Venezia Fbis family used to produce sea bass linkage maps. Four of them did not follow Mendelian inheritance, suggesting the presence of null alleles. Finally, 22 wild sea bass populations were successfully genotyped at 49 SNPs. This set of SNPs sufficed to confirm the established sea bass popu lation structure, namely the differentiation of Atlantic and Mediterranean samp les. Adriatic samples were shown to be genetically distinct from Western and Eastern Mediterranean samples. Selection analyses pointed to a locus that could be under natural selection in the Atlantic Ocean. In conclusion, a bioinformatic approach to discover SNPs was proven to be very valuable. Meanwhile SNP genotyping technologi es have evolved, allowing the validation of SNP candidates on the samples to be investigated.Introduction Chapter 1: Transcriptome characterisation of Expressed Sequence Tags of European sea bass and gilthead sea bream Chapter 2A: Mining for Single Nucleotide Polymorphisms in Expressed Sequence Tags of European sea bass Chapter 2B: In silico discovery of SNPs in EST data: evaluation of tools and strategies Chapter 3: Integration of SNP discovery tools in a single pipeline for automated EST mining Chapter 4: Discovery and validation of SNPs in ESTs of European sea bass Chapter 5: Assessment of population structure and detection of selection by Single Nucleotide Polymorphisms (SNPs) in European sea bass General Discussion Scientific Summary Wettenschappelijke Samenvatting Popular Summary Populaire Samenvattingstatus: publishe

SynTView --- an interactive multi-view genome browser for next-generation comparative microorganism genomics.

International audienceBACKGROUND: Dynamic visualisation interfaces are required to explore the multiple microbial genome data now available, especially those obtained by high-throughput sequencing --- a.k.a. "Next-Generation Sequencing" (NGS) --- technologies; they would also be useful for "standard" annotated genomes whose chromosome organizations may be compared. Although various software systems are available, few offer an optimal combination of feature-rich capabilities, non-static user interfaces and multi-genome data handling. RESULTS: We developed SynTView, a comparative and interactive viewer for microbial genomes, designed to run as either a web-based tool (Flash technology) or a desktop application (AIR environment). The basis of the program is a generic genome browser with sub-maps holding information about genomic objects (annotations). The software is characterised by the presentation of syntenic organisations of microbial genomes and the visualisation of polymorphism data (typically Single Nucleotide Polymorphisms --- SNPs) along these genomes; these features are accessible to the user in an integrated way. A variety of specialised views are available and are all dynamically inter-connected (including linear and circular multi-genome representations, dot plots, phylogenetic profiles, SNP density maps, and more). SynTView is not linked to any particular database, allowing the user to plug his own data into the system seamlessly, and use external web services for added functionalities. SynTView has now been used in several genome sequencing projects to help biologists make sense out of huge data sets. CONCLUSIONS: The most important assets of SynTView are: (i) the interactivity due to the Flash technology; (ii) the capabilities for dynamic interaction between many specialised views; and (iii) the flexibility allowing various user data sets to be integrated. It can thus be used to investigate massive amounts of information efficiently at the chromosome level. This innovative approach to data exploration could not be achieved with most existing genome browsers, which are more static and/or do not offer multiple views of multiple genomes. Documentation, tutorials and demonstration sites are available at the URL: http://genopole.pasteur.fr/SynTView

Recommendations for whole genome sequencing in diagnostics for rare diseases

In 2016, guidelines for diagnostic Next Generation Sequencing (NGS) have been published by EuroGentest in order to assist laboratories in the implementation and accreditation of NGS in a diagnostic setting. These guidelines mainly focused on Whole Exome Sequencing (WES) and targeted (gene panels) sequencing detecting small germline variants (Single Nucleotide Variants (SNVs) and insertions/deletions (indels)). Since then, Whole Genome Sequencing (WGS) has been increasingly introduced in the diagnosis of rare diseases as WGS allows the simultaneous detection of SNVs, Structural Variants (SVs) and other types of variants such as repeat expansions. The use of WGS in diagnostics warrants the re-evaluation and update of previously published guidelines. This work was jointly initiated by EuroGentest and the Horizon2020 project Solve-RD. Statements from the 2016 guidelines have been reviewed in the context of WGS and updated where necessary. The aim of these recommendations is primarily to list the points to consider for clinical (laboratory) geneticists, bioinformaticians, and (non-)geneticists, to provide technical advice, aid clinical decision-making and the reporting of the results.This work was supported by the Solve-RD project (European Union’s Horizon 2020 grant agreement No 779257) to MS, SB, OR and CG, and the National Centre for medical genomics (www.ncmg.cz, CZ.02.1.01/0.0/0.0/16_026/000844, LM2018132 (MSMT.cz) and 00064203/6003 (MZCR.cz) to MM). OR received financial support from Illumina for implementing whole genome sequencing into clinical diagnostics (Ge-Med project

SPG20 mutation in three siblings with familial hereditary spastic paraplegia

Troyer syndrome (MIM#275900) is an autosomal recessive form of complicated hereditary spastic paraplegia. It is characterized by progressive lower extremity spasticity and weakness, dysarthria, distal amyotrophy, developmental delay, short stature, and subtle skeletal abnormalities. It is caused by deleterious mutations in the SPG20 gene, encoding spartin, on Chromosome 13q13. Until now, six unrelated families with a genetically confirmed diagnosis have been reported. Here we report the clinical findings in three brothers of a consanguineous Moroccan family, aged 24, 17, and 7 yr old, with spastic paraplegia, short stature, motor and cognitive delay, and severe intellectual disability. Targeted exon capture and sequencing showed a homozygous nonsense mutation in the SPG20 gene, c.1369C>T (p.Arg457*), in the three affected boys.status: publishe

Approaches to homozygosity mapping and exome sequencing for the identification of novel types of CDG

In the past decade, the identification of most genes involved in Congenital Disorders of Glycosylation (CDG) (type I) was achieved by a combination of biochemical, cell biological and glycobiological investigations. This has been truly successful for CDG-I, because the candidate genes could be selected on the basis of the homology of the synthetic pathway of the dolichol linked oligosaccharide in human and yeast. On the contrary, only a few CDG-II defects were elucidated, be it that some of the discoveries represent wonderful breakthroughs, like e.g, the identification of the COG defects. In general, many rare genetic defects have been identified by positional cloning. However, only a few types of CDG have effectively been elucidated by linkage analysis and so-called reverse genetics. The reason is that the families were relatively small and could—except for CDG-PMM2— not be pooled for analysis. Hence, a large number of CDG cases has long remained unsolved because the search for the culprit gene was very laborious, due to the heterogeneous phenotype and the myriad of candidate defects. This has changed when homozygosity mapping came of age, because it could be applied to small (consanguineous) families. Many novel CDG genes have been discovered in this way. But the best has yet to come: what we are currently witnessing, is an explosion of novel CDG defects, thanks to exome sequencing: seven novel types were published over a period of only two years. It is expected that exome sequencing will soon become a diagnostic tool, that will continuously uncover new facets of this fascinating group of diseases.Fil: Matthij, Gert. Katholikie Universiteit Leuven; BélgicaFil: Rymen, Daisy. Katholikie Universiteit Leuven; BélgicaFil: Bistue Millon, Maria Beatriz. Katholikie Universiteit Leuven; Bélgica. Universidad Nacional de Cordoba. Facultad de Medicina. Centro de Estudios de las Metabolopatías Congénitas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Souche, Erika. Katholikie Universiteit Leuven; BélgicaFil: Race, Valérie. Katholikie Universiteit Leuven; Bélgic