Enriched atlas of lncRNA and protein-coding genes for the GRCg7b chicken assembly and its functional annotation across 47 tissues.

Gene atlases for livestock are steadily improving thanks to new genome assemblies and new expression data improving the gene annotation. However, gene content varies across databases due to differences in RNA sequencing data and bioinformatics pipelines, especially for long non-coding RNAs (lncRNAs) which have higher tissue and developmental specificity and are harder to consistently identify compared to protein coding genes (PCGs). As done previously in 2020 for chicken assemblies galgal5 and GRCg6a, we provide a new gene atlas, lncRNA-enriched, for the latest GRCg7b chicken assembly, integrating "NCBI RefSeq", "EMBL-EBI Ensembl/GENCODE" reference annotations and other resources such as FAANG and NONCODE. As a result, the number of PCGs increases from 18,022 (RefSeq) and 17,007 (Ensembl) to 24,102, and that of lncRNAs from 5789 (RefSeq) and 11,944 (Ensembl) to 44,428. Using 1400 public RNA-seq transcriptome representing 47 tissues, we provided expression evidence for 35,257 (79%) lncRNAs and 22,468 (93%) PCGs, supporting the relevance of this atlas. Further characterization including tissue-specificity, sex-differential expression and gene configurations are provided. We also identified conserved miRNA-hosting genes with human counterparts, suggesting common function. The annotated atlas is available at gega.sigenae.org.

Cell specification and functional interactions in the pig blastocyst inferred from single-cell transcriptomics and uterine fluids proteomics.

The embryonic development of the pig comprises a long in utero pre- and peri-implantation development, which dramatically differs from mice and humans. During this peri-implantation period, a complex series of paracrine signals establishes an intimate dialogue between the embryo and the uterus. To better understand the biology of the pig blastocyst during this period, we generated a large dataset of single-cell RNAseq from early and hatched blastocysts, spheroid and ovoid conceptus and proteomic datasets from corresponding uterine fluids. Our results confirm the molecular specificity and functionality of the three main cell populations. We also discovered two previously unknown subpopulations of the trophectoderm, one characterised by the expression of LRP2, which could represent progenitor cells, and the other, expressing pro-apoptotic markers, which could correspond to the Rauber's layer. Our work provides new insights into the biology of these populations, their reciprocal functional interactions, and the molecular dialogue with the maternal uterine environment.

TAGADA: a scalable pipeline to improve genome annotations with RNA-seq data.

Genome annotation plays a crucial role in providing comprehensive catalog of genes and transcripts for a particular species. As research projects generate new transcriptome data worldwide, integrating this information into existing annotations becomes essential. However, most bioinformatics pipelines are limited in their ability to effectively and consistently update annotations using new RNA-seq data. Here we introduce TAGADA, an RNA-seq pipeline for Transcripts And Genes Assembly, Deconvolution, and Analysis. Given a genomic sequence, a reference annotation and RNA-seq reads, TAGADA enhances existing gene models by generating an improved annotation. It also computes expression values for both the reference and novel annotation, identifies long non-coding transcripts (lncRNAs), and provides a comprehensive quality control report. Developed using Nextflow DSL2, TAGADA offers user-friendly functionalities and ensures reproducibility across different computing platforms through its containerized environment. In this study, we demonstrate the efficacy of TAGADA using RNA-seq data from the GENE-SWiTCH project alongside chicken and pig genome annotations as references. Results indicate that TAGADA can substantially increase the number of annotated transcripts by approximately [Formula: see text] in these species. Furthermore, we illustrate how TAGADA can integrate Illumina NovaSeq short reads with PacBio Iso-Seq long reads, showcasing its versatility. TAGADA is available at github.com/FAANG/analysis-TAGADA.

Enhancer/gene relationships: Need for more reliable genome-wide reference sets.

Differences in cells' functions arise from differential activity of regulatory elements, including enhancers. Enhancers are cis-regulatory elements that cooperate with promoters through transcription factors to activate the expression of one or several genes by getting physically close to them in the 3D space of the nucleus. There is increasing evidence that genetic variants associated with common diseases are enriched in enhancers active in cell types relevant to these diseases. Identifying the enhancers associated with genes and conversely, the sets of genes activated by each enhancer (the so-called enhancer/gene or E/G relationships) across cell types, can help understanding the genetic mechanisms underlying human diseases. There are three broad approaches for the genome-wide identification of E/G relationships in a cell type: 1) genetic link methods or eQTL, 2) functional link methods based on 1D functional data such as open chromatin, histone mark or gene expression and 3) spatial link methods based on 3D data such as HiC. Since 1) and 3) are costly, the current strategy is to develop functional link methods and to use data from 1) and 3) as reference to evaluate them. However, there is still no consensus on the best functional link method to date, and method comparison remain seldom. Here, we compared the relative performances of three recent methods for the identification of enhancer-gene links, TargetFinder, Average-Rank, and the ABC model, using the three latest benchmarks from the field: a reference that combines 3D and eQTL data, called BENGI, and two genetic screening references, called CRiFF and CRiSPRi. Overall, none of the three methods performed best on the three references. CRiFF and CRISPRi reference sets are likely more reliable, but CRiFF is not genome-wide and CRiFF and CRISPRi are mostly available on the K562 cancer cell line. The BENGI reference set is genome-wide but likely contains many false positives. This study therefore calls for new reliable and genome-wide E/G reference data rather than new functional link E/G identification methods.

Major Reorganization of Chromosome Conformation During Muscle Development in Pig.

The spatial organization of the genome in the nucleus plays a crucial role in eukaryotic cell functions, yet little is known about chromatin structure variations during late fetal development in mammals. We performed in situ high-throughput chromosome conformation capture (Hi-C) sequencing of DNA from muscle samples of pig fetuses at two late stages of gestation. Comparative analysis of the resulting Hi-C interaction matrices between both groups showed widespread differences of different types. First, we discovered a complex landscape of stable and group-specific Topologically Associating Domains (TADs). Investigating the nuclear partition of the chromatin into transcriptionally active and inactive compartments, we observed a genome-wide fragmentation of these compartments between 90 and 110 days of gestation. Also, we identified and characterized the distribution of differential cis- and trans-pairwise interactions. In particular, trans-interactions at chromosome extremities revealed a mechanism of telomere clustering further confirmed by 3D Fluorescence in situ Hybridization (FISH). Altogether, we report major variations of the three-dimensional genome conformation during muscle development in pig, involving several levels of chromatin remodeling and structural regulation.

RNA-Seq Data for Reliable SNP Detection and Genotype Calling: Interest for Coding Variant Characterization and Cis-Regulation Analysis by Allele-Specific Expression in Livestock Species.

In addition to their common usages to study gene expression, RNA-seq data accumulated over the last 10 years are a yet-unexploited resource of SNPs in numerous individuals from different populations. SNP detection by RNA-seq is particularly interesting for livestock species since whole genome sequencing is expensive and exome sequencing tools are unavailable. These SNPs detected in expressed regions can be used to characterize variants affecting protein functions, and to study cis-regulated genes by analyzing allele-specific expression (ASE) in the tissue of interest. However, gene expression can be highly variable, and filters for SNP detection using the popular GATK toolkit are not yet standardized, making SNP detection and genotype calling by RNA-seq a challenging endeavor. We compared SNP calling results using GATK suggested filters, on two chicken populations for which both RNA-seq and DNA-seq data were available for the same samples of the same tissue. We showed, in expressed regions, a RNA-seq precision of 91% (SNPs detected by RNA-seq and shared by DNA-seq) and we characterized the remaining 9% of SNPs. We then studied the genotype (GT) obtained by RNA-seq and the impact of two factors (GT call-rate and read number per GT) on the concordance of GT with DNA-seq; we proposed thresholds for them leading to a 95% concordance. Applying these thresholds to 767 multi-tissue RNA-seq of 382 birds of 11 chicken populations, we found 9.5 M SNPs in total, of which ∼550,000 SNPs per tissue and population with a reliable GT (call rate ≥ 50%) and among them, ∼340,000 with a MAF ≥ 10%. We showed that such RNA-seq data from one tissue can be used to (i) detect SNPs with a strong predicted impact on proteins, despite their scarcity in each population (16,307 SIFT deleterious missenses and 590 stop-gained), (ii) study, on a large scale, cis-regulations of gene expression, with ∼81% of protein-coding and 68% of long non-coding genes (TPM ≥ 1) that can be analyzed for ASE, and with ∼29% of them that were cis-regulated, and (iii) analyze population genetic using such SNPs located in expressed regions. This work shows that RNA-seq data can be used with good confidence to detect SNPs and associated GT within various populations and used them for different analyses as GTEx studies.

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

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.

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

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.

Identification of a t(3;4)(p1.3;q1.5) translocation breakpoint in pigs using somatic cell hybrid mapping and high-resolution mate-pair sequencing.

Reciprocal translocations are the most frequently occurring constitutional structural rearrangements in mammalian genomes. In phenotypically normal pigs, an incidence of 1/200 is estimated for such rearrangements. Even if constitutional translocations do not necessarily induce defects and diseases, they are responsible for significant economic losses in domestic animals due to reproduction failures. Over the last 30 years, advances in molecular and cytogenetic technologies have led to major improvements in the resolution of the characterization of translocation events. Characterization of translocation breakpoints helps to decipher the mechanisms that lead to such rearrangements and the functions of the genes that are involved in the translocation. Here, we describe the fine characterization of a reciprocal translocation t(3;4) (p1.3;q1.5) detected in a pig line. The breakpoint was identified at the base-pair level using a positional cloning and chromosome walking strategy in somatic cell hybrids that were generated from an animal that carries this translocation. We show that this translocation occurs within the ADAMTSL4 gene and results in a loss of expression in homozygous carriers. In addition, by taking this translocation as a model, we used a whole-genome next-generation mate-pair sequencing approach on pooled individuals to evaluate this strategy for high-throughput screening of structural rearrangements.

Long noncoding RNA repertoire in chicken liver and adipose tissue.

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.

Bioinformatics Pipeline for Transcriptome Sequencing Analysis.

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.

Modeling of autosomal-dominant retinitis pigmentosa in Caenorhabditis elegans uncovers a nexus between global impaired functioning of certain splicing factors and cell type-specific apoptosis.

Retinitis pigmentosa (RP) is a rare genetic disease that causes gradual blindness through retinal degeneration. Intriguingly, seven of the 24 genes identified as responsible for the autosomal-dominant form (adRP) are ubiquitous spliceosome components whose impairment causes disease only in the retina. The fact that these proteins are essential in all organisms hampers genetic, genomic, and physiological studies, but we addressed these difficulties by using RNAi in Caenorhabditis elegans. Our study of worm phenotypes produced by RNAi of splicing-related adRP (s-adRP) genes functionally distinguishes between components of U4 and U5 snRNP complexes, because knockdown of U5 proteins produces a stronger phenotype. RNA-seq analyses of worms where s-adRP genes were partially inactivated by RNAi, revealed mild intron retention in developing animals but not in adults, suggesting a positive correlation between intron retention and transcriptional activity. Interestingly, RNAi of s-adRP genes produces an increase in the expression of atl-1 (homolog of human ATR), which is normally activated in response to replicative stress and certain DNA-damaging agents. The up-regulation of atl-1 correlates with the ectopic expression of the pro-apoptotic gene egl-1 and apoptosis in hypodermal cells, which produce the cuticle, but not in other cell types. Our model in C. elegans resembles s-adRP in two aspects: The phenotype caused by global knockdown of s-adRP genes is cell type-specific and associated with high transcriptional activity. Finally, along with a reduced production of mature transcripts, we propose a model in which the retina-specific cell death in s-adRP patients can be induced through genomic instability.

Coordinated international action to accelerate genome-to-phenome with FAANG, the Functional Annotation of Animal Genomes project.

We describe the organization of a nascent international effort, the Functional Annotation of Animal Genomes (FAANG) project, whose aim is to produce comprehensive maps of functional elements in the genomes of domesticated animal species.

Analysis of alternative splicing events in custom gene datasets by AStalavista.

Alternative splicing (AS) is a eukaryotic principle to derive more than one RNA product from transcribed genes by removing distinct subsets of introns from a premature polymer. We know today that this process is highly regulated and makes up a large part of the differences between species, cell types, and states. The key to compare AS across different genes or organisms is to tokenize the AS phenomenon into atomary units, so-called AS events. These events then usually are grouped by common patterns to investigate the underlying molecular mechanisms that drive their regulation. However, attempts to decompose loci with AS observations into events are often hampered by applying a limited set of a priori defined event patterns which are not capable to describe all AS configurations and therefore cannot decompose the phenomenon exhaustively. In this chapter, we describe working scenarios of AStalavista, a computational method that reports all AS events reflected by transcript annotations. We show how to practically employ AStalavista to study AS variation in complex transcriptomes, as characterized by the human GENCODE annotation. Our examples demonstrate how the inherent and universal AStalavista paradigm allows for an automatic delineation of AS events in custom gene datasets. Additionally, we sketch an example of an AStalavista use case including next-generation sequencing data (RNA-Seq) to enrich the landscape of discovered AS events.

An overview of gene expression dynamics during early ovarian folliculogenesis: specificity of follicular compartments and bi-directional dialog.

BACKGROUND: Successful early folliculogenesis is crucial for female reproductive function. It requires appropriate gene specific expression of the different types of ovarian cells at different developmental stages. To date, most gene expression studies on the ovary were conducted in rodents and did not distinguish the type of cell. In mono-ovulating species, few studies have addressed gene expression profiles and mainly concerned human oocytes. RESULTS: We used a laser capture microdissection method combined with RNA-seq technology to explore the transcriptome in oocytes and granulosa cells (GCs) during development of the sheep ovarian follicle. We first documented the expression profile of 15 349 genes, then focused on the 5 129 genes showing differential expression between oocytes and GCs. Enriched functional categories such as oocyte meiotic arrest and GC steroid synthesis reflect two distinct cell fates. We identified the implication of GC signal transduction pathways such as SHH, WNT and RHO GTPase. In addition, signaling pathways (VEGF, NOTCH, IGF1, etc.) and GC transzonal projections suggest the existence of complex cell-cell interactions. Finally, we highlighted several transcription regulators and specifically expressed genes that likely play an important role in early folliculogenesis. CONCLUSIONS: To our knowledge, this is the first comprehensive exploration of transcriptomes derived from in vivo oocytes and GCs at key stages in early follicular development in sheep. Collectively, our data advance our understanding of early folliculogenesis in mono-ovulating species and will be a valuable resource for unraveling human ovarian dysfunction such as premature ovarian failure (POF).

Landscape of transcription in human cells.

Eukaryotic cells make many types of primary and processed RNAs that are found either in specific subcellular compartments or throughout the cells. A complete catalogue of these RNAs is not yet available and their characteristic subcellular localizations are also poorly understood. Because RNA represents the direct output of the genetic information encoded by genomes and a significant proportion of a cell's regulatory capabilities are focused on its synthesis, processing, transport, modification and translation, the generation of such a catalogue is crucial for understanding genome function. Here we report evidence that three-quarters of the human genome is capable of being transcribed, as well as observations about the range and levels of expression, localization, processing fates, regulatory regions and modifications of almost all currently annotated and thousands of previously unannotated RNAs. These observations, taken together, prompt a redefinition of the concept of a gene.

Evidence for transcript networks composed of chimeric RNAs in human cells.

The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5' and 3' transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1) the non-random interconnections of genes involved, (2) the greater phylogenetic depth of the genes involved in many chimeric interactions, (3) the coordination of the expression of connected genes and (4) the close in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs. The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network.

Comprehensive polyadenylation site maps in yeast and human reveal pervasive alternative polyadenylation.

The emerging discoveries on the link between polyadenylation and disease states underline the need to fully characterize genome-wide polyadenylation states. Here, we report comprehensive maps of global polyadenylation events in human and yeast generated using refinements to the Direct RNA Sequencing technology. This direct approach provides a quantitative view of genome-wide polyadenylation states in a strand-specific manner and requires only attomole RNA quantities. The polyadenylation profiles revealed an abundance of unannotated polyadenylation sites, alternative polyadenylation patterns, and regulatory element-associated poly(A)(+) RNAs. We observed differences in sequence composition surrounding canonical and noncanonical human polyadenylation sites, suggesting novel noncoding RNA-specific polyadenylation mechanisms in humans. Furthermore, we observed the correlation level between sense and antisense transcripts to depend on gene expression levels, supporting the view that overlapping transcription from opposite strands may play a regulatory role. Our data provide a comprehensive view of the polyadenylation state and overlapping transcription.