Pierre-François Roux

Analysis of Allele-Specific Expression in Mouse Liver by RNA-Seq: A Comparison With Cis -eQTL Identified Using Genetic Linkage

We report an analysis of allele-specific expression (ASE) and parent-of-origin expression in adult mouse liver using next generation sequencing (RNA-Seq) of reciprocal crosses of heterozygous F1 mice from the parental strains C57BL/6J and DBA/2J. We found a 60% overlap between genes exhibiting ASE and putative cis-acting expression quantitative trait loci (cis-eQTL) identified in an intercross between the same strains. We discuss the various biological and technical factors that contribute to the differences. We also identify genes exhibiting parental imprinting and complex expression patterns. Our study demonstrates the importance of biological replicates to limit the number of false positives with RNA-Seq data.

Re-Sequencing Data for Refining Candidate Genes and Polymorphisms in QTL Regions Affecting Adiposity in Chicken

In this study, we propose an approach aiming at fine-mapping adiposity QTL in chicken, integrating whole genome re-sequencing data. First, two QTL regions for adiposity were identified by performing a classical linkage analysis on 1362 offspring in 11 sire families obtained by crossing two meat-type chicken lines divergently selected for abdominal fat weight. Those regions, located on chromosome 7 and 19, contained a total of 77 and 84 genes, respectively. Then, SNPs and indels in these regions were identified by re-sequencing sires. Considering issues related to polymorphism annotations for regulatory regions, we focused on the 120 and 104 polymorphisms having an impact on protein sequence, and located in coding regions of 35 and 42 genes situated in the two QTL regions. Subsequently, a filter was applied on SNPs considering their potential impact on the protein function based on conservation criteria. For the two regions, we identified 42 and 34 functional polymorphisms carried by 18 and 24 genes, and likely to deeply impact protein, including 3 coding indels and 4 nonsense SNPs. Finally, using gene functional annotation, a short list of 17 and 4 polymorphisms in 6 and 4 functional genes has been defined. Even if we cannot exclude that the causal polymorphisms may be located in regulatory regions, this strategy gives a complete overview of the candidate polymorphisms in coding regions and prioritize them on conservation- and functional-based arguments.

Genome-Wide Characterization of RNA Editing in Chicken Embryos Reveals Common Features among Vertebrates

RNA editing results in a post-transcriptional nucleotide change in the RNA sequence that creates an alternative nucleotide not present in the DNA sequence. This leads to a diversification of transcription products with potential functional consequences. Two nucleotide substitutions are mainly described in animals, from adenosine to inosine (A-to-I) and from cytidine to uridine (C-to-U). This phenomenon is described in more details in mammals, notably since the availability of next generation sequencing technologies allowing whole genome screening of RNA-DNA differences. The number of studies recording RNA editing in other vertebrates like chicken is still limited. We chose to use high throughput sequencing technologies to search for RNA editing in chicken, and to extend the knowledge of its conservation among vertebrates. We performed sequencing of RNA and DNA from 8 embryos. Being aware of common pitfalls inherent to sequence analyses that lead to false positive discovery, we stringently filtered our datasets and found fewer than 40 reliable candidates. Conservation of particular sites of RNA editing was attested by the presence of 3 edited sites previously detected in mammals. We then characterized editing levels for selected candidates in several tissues and at different time points, from 4.5 days of embryonic development to adults, and observed a clear tissue-specificity and a gradual increase of editing level with time. By characterizing the RNA editing landscape in chicken, our results highlight the extent of evolutionary conservation of this phenomenon within vertebrates, attest to its tissue and stage specificity and provide support of the absence of non A-to-I events from the chicken transcriptome.

Combined QTL and Selective Sweep Mappings with Coding SNP Annotation and cis-eQTL Analysis Revealed PARK2 and JAG2 as New Candidate Genes for Adiposity Regulation

Very few causal genes have been identified by quantitative trait loci (QTL) mapping because of the large size of QTL, and most of them were identified thanks to functional links already known with the targeted phenotype. Here, we propose to combine selection signature detection, coding SNP annotation, and cis-expression QTL analyses to identify potential causal genes underlying QTL identified in divergent line designs. As a model, we chose experimental chicken lines divergently selected for only one trait, the abdominal fat weight, in which several QTL were previously mapped. Using new haplotype-based statistics exploiting the very high SNP density generated through whole-genome resequencing, we found 129 significant selective sweeps. Most of the QTL colocalized with at least one sweep, which markedly narrowed candidate region size. Some of those sweeps contained only one gene, therefore making them strong positional causal candidates with no presupposed function. We then focused on two of these QTL/sweeps. The absence of nonsynonymous SNPs in their coding regions strongly suggests the existence of causal mutations acting in cis on their expression, confirmed by cis-eQTL identification using either allele-specific expression or genetic mapping analyses. Additional expression analyses of those two genes in the chicken and mice contrasted for adiposity reinforces their link with this phenotype. This study shows for the first time the interest of combining selective sweeps mapping, coding SNP annotation and cis-eQTL analyses for identifying causative genes for a complex trait, in the context of divergent lines selected for this specific trait. Moreover, it highlights two genes, JAG2 and PARK2, as new potential negative and positive key regulators of adiposity in chicken and mice.

The Extent of mRNA Editing Is Limited in Chicken Liver and Adipose, but Impacted by Tissular Context, Genotype, Age, and Feeding as Exemplified with a Conserved Edited Site in COG3.

RNA editing is a posttranscriptional process leading to differences between genomic DNA and transcript sequences, potentially enhancing transcriptome diversity. With recent advances in high-throughput sequencing, many efforts have been made to describe mRNA editing at the transcriptome scale, especially in mammals, yielding contradictory conclusions regarding the extent of this phenomenon. We show, by detailed description of the 25 studies focusing so far on mRNA editing at the whole-transcriptome scale, that systematic sequencing artifacts are considered in most studies whereas biological replication is often neglected and multi-alignment not properly evaluated, which ultimately impairs the legitimacy of results. We recently developed a rigorous strategy to identify mRNA editing using mRNA and genomic DNA sequencing, taking into account sequencing and mapping artifacts, and biological replicates. We applied this method to screen for mRNA editing in liver and white adipose tissue from eight chickens and confirm the small extent of mRNA recoding in this species. Among the 25 unique edited sites identified, three events were previously described in mammals, attesting that this phenomenon is conserved throughout evolution. Deeper investigations on five sites revealed the impact of tissular context, genotype, age, feeding conditions, and sex on mRNA editing levels. More specifically, this analysis highlighted that the editing level at the site located on COG3 was strongly regulated by four of these factors. By comprehensively characterizing the mRNA editing landscape in chickens, our results highlight how this phenomenon is limited and suggest regulation of editing levels by various genetic and environmental factors.

Transcriptomes of whole blood and PBMC in chickens

Global transcriptome analysis of chicken whole blood to discover biomarkers of different phenotypes or physiological disorders has never been investigated so far. Whole blood provides significant advantages, allowing large scale and non-invasive sampling. However, generation of gene expression data from the blood of non-mammalian species remains a challenge, notably due to the nucleated red blood cells, hindering the use of well-established protocols. The aim of this study was to analyze the relevance of using whole blood cells (WB) to find biomarkers, instead of Peripheral Blood Mononuclear Cells (PBMC), usually chosen for immune challenges. RNA sources from WB and PBMC was characterized by microarray analysis. Our results show that the quality and quantity of RNA obtained from WB was suitable for further analyses, although the quality was lower than that from PBMC. The transcriptome profiling comparison revealed that the majority of genes were expressed in both WB and PBMC. Hemoglobin subunits were the major transcripts in WB, whereas the most enriched biological process was related to protein catabolic process. Most of the over-represented transcripts in PBMC were implicated in functions specific to thrombocytes, like coagulation and platelet activation, probably due to the large proportion of this nucleated cell type in chicken PBMC. Functions related to B and T cells and to other immune functions were also enriched in the PBMC subset. We conclude that WB is more suitable for large scale immunity oriented studies and other biological processes that have been poorly investigated so far.

Comparison of whole-genome (13X) and capture (87X) resequencing methods for SNP and genotype callings

The number of polymorphisms identified with next-generation sequencing approaches depends directly on the sequencing depth and therefore on the experimental cost. Although higher levels of depth ensure more sensitive and more specific SNP calls, economic constraints limit the increase of depth for whole-genome resequencing (WGS). For this reason, capture resequencing is used for studies focusing on only some specific regions of the genome. However, several biases in capture resequencing are known to have a negative impact on the sensitivity of SNP detection. Within this framework, the aim of this study was to compare the accuracy of WGS and capture resequencing on SNP detection and genotype calling, which differ in terms of both sequencing depth and biases. Indeed, we have evaluated the SNP calling and genotyping accuracy in a WGS dataset (13X) and in a capture resequencing dataset (87X) performed on 11 individuals. The percentage of SNPs not identified due to a sevenfold sequencing depth decrease was estimated at 7.8% using a down-sampling procedure on the capture sequencing dataset. A comparison of the 87X capture sequencing dataset with the WGS dataset revealed that capture-related biases were leading with the loss of 5.2% of SNPs detected with WGS. Nevertheless, when considering the SNPs detected by both approaches, capture sequencing appears to achieve far better SNP genotyping, with about 4.4% of the WGS genotypes that can be considered as erroneous and even 10% focusing on heterozygous genotypes. In conclusion, WGS and capture deep sequencing can be considered equivalent strategies for SNP detection, as the rate of SNPs not identified because of a low sequencing depth in the former is quite similar to SNPs missed because of method biases of the latter. On the other hand, capture deep sequencing clearly appears more adapted for studies requiring great accuracy in genotyping.