phs001463
Differential Transcription Start Site Usage in Brain-related Samples
RNA-Seq is an effective method to study the transcriptome, but specialized methods are required to identify the 5'' ends of transcripts. Several methods have been published for this specific purpose, but their relative merits have not been systematically analyzed. Here, we directly compare the performance of five such methods with cellular RNA as well as a novel spike-in RNA assay that provides a solution to issues in interpreting results arising from uncertainties in annotation or RNA processing. Using a single human RNA sample, we constructed and sequenced ten libraries with these methods and one standard RNA-Seq library as control. We find that the CAGE method performed best and that most of its unannotated peaks are supported by evidence from other genomic methods. We applied the CAGE method to eight brain-related samples and revealed sample-specific TSS usage as well as a transcriptome-wide pattern in TSS usage.