Plant Transcriptome Analysis with HISAT–StringTie–Ballgown and TopHat–Cufflinks Pipelines

The development of next-generation sequencing technology has led to a burst of data in a single assay. Management of a large dataset requires high demands on bioinformatic skills and computing resources. Here we present two popular pipelines for RNA-seq data analysis, using open-source software tools HISAT–StringTie–Ballgown and TopHat–Cufflinks. To meet the need of plant scientist, we describe in detail how to perform such comprehensive analysis beginning with raw RNA-seq reads and available reference genome. It allows biologists to align short reads to a reference genome, measure the transcript abundance, and analyze gene differential expression under two or more conditions. We also discuss other RNA-seq tools that are comparable or alternative to this protocol.