Definition of the yeast transcriptome using next-generation RNA sequencing
MetadataShow full item record
RNA sequencing (RNA-seq) is a high-throughput method by which the sequence of each RNA molecule in an organism can be determined. Because of its high-throughput nature and high accuracy, RNA-seq allows for the study of the transcriptome at base-pair resolution and for the discovery of novel transcripts and splice junctions. Although the transcriptome of Saccharomyces cerevisiae (baker’s yeast) has been previously studied, the use of RNA-seq allows for the characterization of novel and low abundance transcripts that are difficult to study using more traditional methods. A robust library preparation is necessary to take advantage of the strengths of RNA-seq. In order to find an appropriate library preparation, several protocols were prototyped and the results of these preparations were compared against similar expression microarray experiments. qPCR was used to validate the RNA abundance of a selection of genes with a known response to the treatment being used. After several iterations, a library preparation was found which reliably reproduced the microarray results while offering additional information such as the transcription start site and transcript architecture. However, this preparation did not preserve the directionality of the RNA transcripts. Strand direction information is useful when assigning sequenced reads to transcripts and when differentiating signal from noise. A new library preparation is being prototyped which will preserve the directionality of the transcripts, allowing for an additional dimension of information to be captured by the data. The yeast response to various perturbations including heat shock and sporulation will be characterized using this method. By preserving the directionality of the RNA transcripts and by studying yeast under several conditions, the yeast transcriptome will be defined at a higher resolution.