APA-Scan: Detection and Visualization of 3'-UTR APA with RNA-seq and 3'-end-seq Data
BackgroundThe eukaryotic genome is capable of producing multiple isoforms from a gene by alternative polyadenylation (APA) during pre-mRNA processing. APA in the 3-untranslated region (3-UTR) of mRNA produces transcripts with shorter or longer 3-UTR. Often, 3-UTR serves as a binding platform for microRNAs and RNA-binding proteins, which affect the fate of the mRNA transcript. Thus, 3-UTR APA is known to modulate translation and provides a mean to regulate gene expression at the post-transcriptional level. Current bioinformatics pipelines have limited capability in profiling 3-UTR APA events due to incomplete annotations and a low-resolution analyzing power: widely available bioinformatics pipelines do not reference actionable polyadenylation (cleavage) sites but simulate 3-UTR APA only using RNA-seq read coverage, causing false positive identifications. To overcome these limitations, we developed APA-Scan, a robust program that identifies 3-UTR APA events and visualizes the RNA-seq short-read coverage with gene annotations. MethodsAPA-Scan utilizes either predicted or experimentally validated actionable polyadenylation signals as a reference for polyadenylation sites and calculates the quantity of long and short 3-UTR transcripts in the RNA-seq data. APA-Scan works in three major steps: (i) calculate the read coverage of the 3-UTR regions of genes; (ii) identify the potential APA sites and evaluate the significance of the events among two biological conditions; (iii) graphical representation of user specific event with 3-UTR annotation and read coverage on the 3-UTR regions. APA-Scan is implemented in Python3. Source code and a comprehensive users manual are freely available at https://github.com/compbiolabucf/APA-Scan. ResultAPA-Scan was applied to both simulated and real RNA-seq datasets and compared with two widely used baselines DaPars and APAtrap. In simulation APA-Scan significantly improved the accuracy of 3-UTR APA identification compared to the other baselines. The performance of APA-Scan was also validated by 3-end-seq data and qPCR on mouse embryonic fibroblast cells. The experiments confirm that APA-Scan can detect unannotated 3 -UTR APA events and improve genome annotation. ConclusionAPA-Scan is a comprehensive computational pipeline to detect transcriptome-wide 3-UTR APA events. The pipeline integrates both RNA-seq and 3-end-seq data information and can efficiently identify the significant events with a high-resolution short reads coverage plots.