QuickIsoSeq for Isoform Quantification in Large-Scale RNA Sequencing

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RNA-sequencing (RNA-seq) is a powerful technology for transcriptome profiling. While most RNA-seq projects focus on gene-level quantification and analysis, there is growing evidence that most mammalian genes are alternatively spliced to generate different isoforms that can be subsequently translated to protein molecules with diverse or even opposing biological functions. Quantifying the expression levels of these isoforms is key to understanding the genes biological functions in healthy tissues and the progression of diseases. Among open source tools developed for isoform quantification, Salmon, Kallisto, and RSEM are recommended based upon previous systematic evaluation of these tools using both experimental and simulated RNA-seq datasets. However, isoform quantification in practical RNA-seq data analysis needs to deal with many QC issues, such as the abundance of rRNAs in mRNA-seq, the efficiency of globin RNA depletion in whole blood samples, and potential sample swapping. To overcome these practical challenges, QuickIsoSeq was developed for large-scale RNA-seq isoform quantification along with QC. In this chapter, we describe the pipeline and detailed the steps required to deploy and use it to analyze RNA-seq datasets in practice. The QuickIsoSeq package can be downloaded from https://github.com/shanrongzhao/QuickIsoSeq.

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Methods in Molecular Biology