In this study, RNA-seq and proteomics, two orthogonal high-throughput technologies, were used to search the Saccharomyces cerevisiae
genome for new genomic features. RNA-seq data were aligned to the genome with three successively stringent set of parameters for the STAR aligner (Dobin et al., 2013). The varying levels of stringency elucidated some complexities in the RNA-seq data, such as the presence of read alignments that mapped to multiple genomic locations. The RNA-seq alignments indicated the presence of RNA transcripts derived from regions of the genome without annotations (un-annotated regions) in the Saccharomyces
Genome Database (SGD). To ensure that all of the high-quality curated annotations within SGD were accounted for appropriately, these datasets were categorised as either Primary or Secondary Annotations. Annotations of genomic regions where the primary sequence produced a molecule (e.g. snoRNA or peptide) were designated as Primary. Annotations of regions where other types of activity were present (e.g. histone binding sites, double-strand break hotspots) were classified as Secondary. Only the Primary Annotations were used as boundaries for determining locations of un-annotated regions. Open reading frames (ORFs) were present in these un-annotated regions. Therefore, the regions were translated in six frames to build a database of all theoretical peptides. Proteomics tandem mass spectra were then searched against this peptide database to find the presence of any expressed ORFs within the un-annotated regions. Two preliminary target ORFs have been found to contain RNA-seq alignments and were detected by the proteomics analysis, evidence that their transcripts may have been present in the original sample. The next step would be to verify these two preliminary target regions in the experimental laboratory to determine if they are in fact expressed as peptides, and if so, what possible functions the peptides may have. Throughout this study, the Un-Annotated Region Pipeline (UAR-Pipeline) software was constructed to facilitate the analysis of un-annotated regions given a genome sequence, a set of genomic annotations, and RNA-seq data. In addition, a Quickload Site within the Integrated Genome Browser (Nicol et al., 2009) was created to store and effectively visualise un-annotated regions against RNA-seq alignments, annotations, and other tracks of information such as conservation. The vast majority of annotations contained within the Quickload Site are also hosted by SGD; therefore, the Site would serve as a new resource for the research community through anticipated public access.
|Date of Award||2018|
|Supervisor||Geoffrey Barton (Supervisor), Nicholas Schurch (Supervisor) & Pieta Schofield (Supervisor)|