Ab initio Identification of Transcription Start Sites in the Bovine Genome

Annotation of transcription start sites (TSS) is critical to our understanding of gene structure and regulation. Unfortunately, the bovine genome lacks thorough annotation of TSS. To improve the annotation of TSS in the bovine genome we therefore implemented RAMPAGE (RNA Annotation and Mapping of Promoters for the Analysis of Gene Expression), a 5’-transcript sequencing technique, to identify TSS in different cattle tissues. This technique uses template switching and CAP trapping for sequencing transcript ends, allowing for ab initio identification of TSS at base pair resolution, as well as quantification of the expression of their respective transcripts. From two steers, RNA was extracted from brain, liver, muscle and spleen, treated with DNase, and subjected to terminator digestion to remove ribosomal RNA and fragmented transcripts. A template-switching oligonucleotide was added by reverse transcription such that only 5’-complete cDNAs were amplifiable. The RNA was then pooled, subjected to CAP trapping, and PCR amplified. The purified library was sequenced in a single lane of a HiSeq2000 PE100, producing on average 15 million reads per tissue, ranging from 8.4 million to 23.5 million. Using the RNA-seq aligner STAR, we found that on average 53% of reads mapped uniquely to the bovine genome, ranging from 34% to 63.2%. These data were used to map TSS genome-wide, and compare the relative expression of transcripts produced from tissue-specific alternative TSS. Furthermore, the addition of these TSS annotations to the reference highlights putative regulatory regions warranting future analysis, and will facilitate further functional annotation of the bovine genome.