Global Analysis of Transcription Start Sites in the New Ovine Reference Genome (Oar rambouillet v1.0)

Abstract

The overall aim of the Ovine FAANG project is to provide a comprehensive annotation of the new highly contiguous sheep reference genome sequence (Oar rambouillet v1.0). Mapping of transcription start sites (TSS) is a key first step in understanding transcript regulation and diversity. Using 56 tissue samples collected from the reference ewe Benz2616, we have performed a global analysis of TSS and TSS-Enhancer clusters using Cap Analysis Gene Expression (CAGE) sequencing. CAGE measures RNA expression by 5 cap-trapping and has been specifically designed to allow the characterization of TSS within promoters to single-nucleotide resolution. We have adapted an analysis pipeline that uses TagDust2 for clean-up and trimming, Bowtie2 for mapping, CAGEfightR for clustering, and the Integrative Genomics Viewer (IGV) for visualization. Mapping of CAGE tags indicated that the expression levels of CAGE tag clusters varied across tissues. Expression profiles across tissues were validated using corresponding polyA+ mRNA-Seq data from the same samples. After removal of CAGE tags with <10 read counts, 39.3% of TSS overlapped with 5 ends of 31,113 transcripts that had been previously annotated by NCBI (out of a total of 56,308 from the NCBI annotation). For 25,195 of the transcripts, previously annotated by NCBI, no TSS meeting stringent criteria were identified. A further 14.7% of TSS mapped to within 50 bp of annotated promoter regions. Intersecting these predicted TSS regions with annotated promoter regions (±50 bp) revealed 46% of the predicted TSS were "novel" and previously un-annotated. Using whole-genome bisulfite sequencing data from the same tissues, we were able to determine that a proportion of these "novel" TSS were

Genome-Wide Histone Modifications and CTCF Enrichment Predict Gene Expression in Sheep Macrophages

“…The annotation build for Oar_rambouillet_v1.0 included few immune tissues for gene prediction. Generation of experimental data to improve TSS annotation is one of the objectives defined by the Ovine FAANG Project and cap analysis gene expression (CAGE) data on 55 tissues and alveolar macrophages was recently published ( Salavati et al, 2020 ). CAGE is an excellent method to confirm function and location of promoters and enhancers ( Andersson et al, 2014 ) for validation of ChIP-seq ( Wood et al, 2020 ).…”

“…Reads were quality checked with FastQC software ( Andrews and Babraham, 2016 ) with attention to duplication rate ( Supplementary Data 15 ). Sequence reads were mapped to the unmasked Rambouillet sheep genome (Oar_rambouillet_v1.0, GCA_002742125.1, Worley, 2017 ; Salavati et al, 2020 ), that excludes the mitochondrial genome, with BWA v0.7.17 ( Li and Durbin, 2009 ) (see Supplementary Data 15 and Supplementary Data 4 supplementary results for additional mapping details). Reads were sorted and indexed with Picard v2.9.2 2 .…”

Characterizing Genetic Regulatory Elements in Ovine Tissues

“…The peaks common to three samples with the greatest NRF were determined for H3K4me3 (F1, M1, and M2 for liver; F2, M1, and M2 for spleen; and F1, M1, and M2 for cerebellum), H3K27ac (F1, M1, and M2 for liver; F2, M1, and M2 for spleen; and F1, M1, and M2 for cerebellum), H3K4me1 (F1, M1, and M2 for liver; F2, M1, and M2 for spleen; and F1, M1, and M2 for cerebellum), H3K27me3 (F1, M1, and M2 for liver; F2, M1, and M2 for spleen; and F2, M1, and M2 for cerebellum), and CTCF (F2, M1, and M2 for liver; F2, M1, and M2 for spleen; and F2, M1, and M2 for cerebellum). These consensus peaks were compared with transcription start site locations identified with CAGE assays from the ewe used to generate the reference genome using the deepTools computeMatrix function, and heatmaps were plotted with the plotHeatmap function ( Ramírez et al, 2014 ; Salavati et al, 2020 ). Furthermore, peaks were annotated with the GTF file from the reference genome Oar_rambouillet_v1.0 , and peaks were categorized as near a transcription start site (TSS) (+2 to −2 kb), exonic, intronic, near a transcription termination site (TTS) (+1 to −1 kb), and intergenic using the Homer annotatePeaks.pl function ( Heinz et al, 2010 ).…”

“…Significantly enriched known and de novo CTCF motifs were identified and compared with other species by implementing the findMotifs.pl script in HOMER ( Heinz et al, 2010 ). The proximity of annotated TSS generated from CAGE data to promoter chromatin states was examined with deepTools computeMatrix and plotHeatmap functions ( Supplementary Figure 7 ) ( Ramírez et al, 2014 ; Salavati et al, 2020 ).…”

“…Interestingly, half siblings (F1 and M1, F2 and M2) displayed a greater number of peaks in common with each other. The proximity of H3K4me3 peaks to TSS was investigated by comparing consensus H3K4me3 peaks and CAGE data generated by Salavati et al (2020). Not surprisingly, H3K4me3 peaks were detected on both sides of the TSS in the liver, spleen, and cerebellum tissues.…”