Christelle Robert

Exome Sequencing: Current and Future Perspectives

The falling cost of DNA sequencing has made the technology af fordable to many research groups, enabling researchers to link genomic variants to observed phenotypes in a range of species. This review focusses on whole exome sequencing and its applications in humans and other species. The exome has traditionally been defined to consist of only the protein coding portion of the genome; a region where mutations are likely to affect protein structure and function. There are several commercial kits available for exome sequencing in a number of species and, owing to the highly conserved nature of exons, many of these can be applied to other closely related species. The data set produced from exome sequencing is many times smaller than that of whole genome sequencing, making it more easily manageable and the analysis less complex. Exome sequencing for disease gene discovery in humans is well established and has been used successfully to identify mutations that are causative of complex and rare diseases. Exome sequencing has also been used in a number of domesticated and companion species. The successful application of exome sequencing to crops has yielded results that may be used in selective breeding to improve production in these species, and there is potential for exome sequenc- ing to provide similar advances in livestock species that have not yet been realised.

Errors in RNA-Seq quantification affect genes of relevance to human disease

BackgroundRNA-Seq has emerged as the standard for measuring gene expression and is an important technique often used in studies of human disease. Gene expression quantification involves comparison of the sequenced reads to a known genomic or transcriptomic reference. The accuracy of that quantification relies on there being enough unique information in the reads to enable bioinformatics tools to accurately assign the reads to the correct gene.ResultsWe apply 12 common methods to estimate gene expression from RNA-Seq data and show that there are hundreds of genes whose expression is underestimated by one or more of those methods. Many of these genes have been implicated in human disease, and we describe their roles. We go on to propose a two-stage analysis of RNA-Seq data in which multi-mapped or ambiguous reads can instead be uniquely assigned to groups of genes. We apply this method to a recently published mouse cancer study, and demonstrate that we can extract relevant biological signal from data that would otherwise have been discarded.ConclusionsFor hundreds of genes in the human genome, RNA-Seq is unable to measure expression accurately. These genes are enriched for gene families, and many of them have been implicated in human disease. We show that it is possible to use data that may otherwise have been discarded to measure group-level expression, and that such data contains biologically relevant information.

Identification of low-confidence regions in the pig reference genome (Sscrofa10.2)

Many applications of high throughput sequencing rely on the availability of an accurate reference genome. Variant calling often produces large data sets that cannot be realistically validated and which may contain large numbers of false-positives. Errors in the reference assembly increase the number of false-positives. While resources are available to aid in the filtering of variants from human data, for other species these do not yet exist and strict filtering techniques must be employed which are more likely to exclude true-positives. This work assesses the accuracy of the pig reference genome (Sscrofa10.2) using whole genome sequencing reads from the Duroc sow whose genome the assembly was based on. Indicators of structural variation including high regional coverage, unexpected insert sizes, improper pairing and homozygous variants were used to identify low quality (LQ) regions of the assembly. Low coverage (LC) regions were also identified and analyzed separately. The LQ regions covered 13.85% of the genome, the LC regions covered 26.6% of the genome and combined (LQLC) they covered 33.07% of the genome. Over half of dbSNP variants were located in the LQLC regions. Of copy number variable regions identified in a previous study, 86.3% were located in the LQLC regions. The regions were also enriched for gene predictions from RNA-seq data with 42.98% falling in the LQLC regions. Excluding variants in the LQ, LC, or LQLC from future analyses will help reduce the number of false-positive variant calls. Researchers using WGS data should be aware that the current pig reference genome does not give an accurate representation of the copy number of alleles in the original Duroc sow’s genome.

Macrophages.com: an on-line community resource for innate immunity research.

Macrophages play a major role in tissue remodelling during development, wound healing and tissue homeostasis, and are central to innate immunity and to the pathology of tissue injury and inflammation. Given this fundamental role in many aspects of biological function, an enormous wealth of information has accumulated on these fascinating cells in the literature and other public repositories. With the escalation of genome-scale data derived from macrophages and related haematopoietic cell types, there is a growing need for an integrated resource that seeks to compile, organise and analyse our collective knowledge of macrophage biology. Here we describe a community-driven web-based resource, macrophages.com that aims to provide a portal onto various types of Omics data to facilitate comparative genomic studies, promoter and transcriptional network analyses, models of macrophage pathways together with other information on these cells. To this end, the website combines public and in-house analyses of expression data with pre-analysed views of co-expressed genes as supported by the network analysis tool BioLayout Express(3D), as well as providing access to maps of pathways active in macrophages. Macrophages.com also provides access to an extensive image library of macrophages in adult/embryonic tissue sections prepared from normal and transgenic mice. In addition, the site links to the Human Protein Atlas database so as to provide direct access to protein expression patterns in human macrophages. Finally, an integrated gene-centric portal provides the tools for rapid promoter analysis studies based on a comprehensive set of CAGE-derived transcription start site (TSS) sequences in human and mouse genomes as generated by the Functional Annotation of Mammalian genomes (FANTOM) projects initiated by the RIKEN Omics Science Center. Our aim is to continue to grow the macrophages.com resource using publicly available data, as well as in-house generated knowledge. In so doing we aim to provide a user-friendly community website and a community portal for access to comprehensive sets of macrophage-related data.

Genome-Wide Identification of HrpL-Regulated Genes in the Necrotrophic Phytopathogen Dickeya dadantii 3937

Background Dickeya dadantii is a necrotrophic pathogen causing disease in many plants. Previous studies have demonstrated that the type III secretion system (T3SS) of D. dadantii is required for full virulence. HrpL is an alternative sigma factor that binds to the hrp box promoter sequence of T3SS genes to up-regulate their expression. Methodology/Principal Findings To explore the inventory of HrpL-regulated genes of D. dadantii 3937 (3937), transcriptome profiles of wild-type 3937 and a hrpL mutant grown in a T3SS-inducing medium were examined. Using a cut-off value of 1.5, significant differential expression was observed in sixty-three genes, which are involved in various cellular functions such as type III secretion, chemotaxis, metabolism, regulation, and stress response. A hidden Markov model (HMM) was used to predict candidate hrp box binding sites in the intergenic regions of 3937, including the promoter regions of HrpL-regulated genes identified in the microarray assay. In contrast to biotrophic phytopathgens such as Pseudomonas syringae, among the HrpL up-regulated genes in 3937 only those within the T3SS were found to contain a hrp box sequence. Moreover, direct binding of purified HrpL protein to the hrp box was demonstrated for hrp box-containing DNA fragments of hrpA and hrpN using the electrophoretic mobility shift assay (EMSA). In this study, a putative T3SS effector DspA/E was also identified as a HrpL-upregulated gene, and shown to be translocated into plant cells in a T3SS-dependent manner. Conclusion/Significances We provide the genome-wide study of HrpL-regulated genes in a necrotrophic phytopathogen (D. dadantii 3937) through a combination of transcriptomics and bioinformatics, which led to identification of several effectors. Our study indicates the extent of differences for T3SS effector protein inventory requirements between necrotrophic and biotrophic pathogens, and may allow the development of different strategies for disease control for these different groups of pathogens.

Design and development of exome capture sequencing for the domestic pig (Sus scrofa)

BackgroundThe domestic pig (Sus scrofa) is both an important livestock species and a model for biomedical research. Exome sequencing has accelerated identification of protein-coding variants underlying phenotypic traits in human and mouse. We aimed to develop and validate a similar resource for the pig.ResultsWe developed probe sets to capture pig exonic sequences based upon the current Ensembl pig gene annotation supplemented with mapped expressed sequence tags (ESTs) and demonstrated proof-of-principle capture and sequencing of the pig exome in 96 pigs, encompassing 24 capture experiments. For most of the samples at least 10x sequence coverage was achieved for more than 90% of the target bases. Bioinformatic analysis of the data revealed over 236,000 high confidence predicted SNPs and over 28,000 predicted indels.ConclusionsWe have achieved coverage statistics similar to those seen with commercially available human and mouse exome kits. Exome capture in pigs provides a tool to identify coding region variation associated with production traits, including loss of function mutations which may explain embryonic and neonatal losses, and to improve genomic assemblies in the vicinity of protein coding genes in the pig.

Low coverage sequencing of two Asian elephant ( Elephas maximus ) genomes

BackgroundThere are three species of elephant that exist, the Asian elephant (Elephas maximus) and two species of African elephant (Loxodonta africana and Loxodonta cyclotis). The populations of all three species are dwindling, and are under threat due to factors, such as habitat destruction and ivory hunting. The species differ in many respects, including in their morphology and response to disease. The availability of elephant genome sequence data from all three elephant species will complement studies of behaviour, genetic diversity, evolution and disease resistance.FindingsWe present low-coverage Illumina sequence data from two Asian elephants, representing approximately 5X and 2.5X coverage respectively. Both raw and aligned data are available, using the African elephant (L. africana) genome as a reference.ConclusionsThe data presented here are an important addition to the available genetic and genomic information on Asian and African elephants.

Identification and annotation of conserved promoters and macrophage-expressed genes in the pig genome.

BackgroundThe FANTOM5 consortium used Cap Analysis of Gene Expression (CAGE) tag sequencing to produce a comprehensive atlas of promoters and enhancers within the human and mouse genomes. We reasoned that the mapping of these regulatory elements to the pig genome could provide useful annotation and evidence to support assignment of orthology.ResultsFor human transcription start sites (TSS) associated with annotated human-mouse orthologs, 17% mapped to the pig genome but not to the mouse, 10% mapped only to the mouse, and 55% mapped to both pig and mouse. Around 17% did not map to either species. The mapping percentages were lower where there was not clear orthology relationship, but in every case, mapping to pig was greater than to mouse, and the degree of homology was also greater. Combined mapping of mouse and human CAGE-defined promoters identified at least one putative conserved TSS for >16,000 protein-coding genes. About 54% of the predicted locations of regulatory elements in the pig genome were supported by CAGE and/or RNA-Seq analysis from pig macrophages.ConclusionsComparative mapping of promoters and enhancers from humans and mice can provide useful preliminary annotation of other animal genomes. The data also confirm extensive gain and loss of regulatory elements between species, and the likelihood that pigs provide a better model than mice for human gene regulation and function.

The incredible complexity of RNA splicing.

Alternative splice isoforms are common and important and have been shown to impact many human diseases. A new study by Nellore et al. offers a comprehensive study of splice junctions in humans by re-analyzing over 21,500 public human RNA sequencing datasets.