Next-generation RNA sequencing (RNA-seq) has been widely used to investigate alternative isoform regulations. Among them, alternative 3 : splice site (SS) and 5 : SS account for more than 30% of all alternative splicing (AS) events in higher eukaryotes. Recent studies have revealed that they play important roles in building complex organisms and have a critical impact on biological functions which could cause disease. Quite a few analytical methods have been developed to facilitate alternative 3 : SS and 5 : SS studies using RNA-seq data. However, these methods have various limitations and their performances may be further improved.