RNA‐Seq Library Construction Methods for Transcriptome Analysis

Abstract Next‐generation sequencing (NGS) technologies have revolutionized the study of genomics with an ever‐expanding list of applications. RNA‐Seq has emerged as a powerful method, applying transcriptome analysis to a wider range of organisms—most significantly, non‐model organisms lacking prior genomic sequencing. Whereas an initial concern of NGS datasets was the potential limitation of short read lengths, short read sequences have been successfully employed in creation of de novo transcriptome assemblies that allow for subsequent mapping of reads for expression analysis. Prior genomic sequence knowledge is no longer a requirement for identification of functional transcriptional elements and for global gene expression characterization. Significant cost reductions in generating RNA‐Seq data, and improvements in de novo assemblers, has allowed the analysis of transcriptomes in heretofore unsequenced plant species. These protocols describe standard methods for constructing RNA‐Seq libraries to be sequenced on Illumina sequencing platforms for comprehensive transcriptome analysis. © 2016 by John Wiley & Sons, Inc.