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Methods to study splicing from high-throughput RNA Sequencing data

The development of novel high-throughput sequencing (HTS) methods for RNA (RNA-Seq) has provided a very powerful mean to study splicing under multiple conditions at unprecedented depth. However, the complexity of the information to be analyzed has turned this into a challenging task. In the last few years, a plethora of tools have been developed, allowing researchers to process RNA-Seq data to study the expression of isoforms and splicing events, and their relative changes under different conditions. We provide an overview of the methods available to study splicing from short RNA-Seq data. We group the methods according to the different questions they address: 1) Assignment of the sequencing reads to their likely gene of origin. This is addressed by methods that map reads to the genome and/or to the available gene annotations. 2) Recovering the sequence of splicing events and isoforms. This is addressed by transcript reconstruction and de novo assembly methods. 3) Quantification of events and isoforms. Either after reconstructing transcripts or using an annotation, many methods estimate the expression level or the relative usage of isoforms and/or events. 4) Providing an isoform or event view of differential splicing or expression. These include methods that compare relative event/isoform abundance or isoform expression across two or more conditions. 5) Visualizing splicing regulation. Various tools facilitate the visualization of the RNA-Seq data in the context of alternative splicing. In this review, we do not describe the specific mathematical models behind each method. Our aim is rather to provide an overview that could serve as an entry point for users who need to decide on a suitable tool for a specific analysis. We also attempt to propose a classification of the tools according to the operations they do, to facilitate the comparison and choice of methods.

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