Comparison of Ribosomal RNA Removal Methods for Transcriptome Sequencing Workflows in Teleost Fish

RNA sequencing (RNA-Seq) is becoming the standard for transcriptome analysis. Removal of contaminating ribosomal RNA (rRNA) is a priority in the preparation of libraries suitable for sequencing. These methods have been well documented in mammals but typically require some optimization for lower vertebrates. Three commercial kits, including Dynabeads mRNA Purification Kit, RiboMinus Eukaryote System v2, and Ribo-Zero Gold rRNA Removal Kit were examined for the ability to remove rRNAs from rainbow trout (Oncorhynchus mykiss) RNA isolations. Total RNA was isolated from liver and muscle tissue samples (n = 24) and rRNAs removed using one of the three kits. Samples were analyzed visually on the Agilent Bioanalyzer and by Illumina RNA-seq, screening for Oncorhynchus rRNAs. There were significant differences between the kits in regards to their ability to remove rRNA, ranging from 2.74% - 10.94% rRNA sequences left behind per kit on average. Using the Bioanalyzer to evaluate ribosomal contamination in rRNA-depleted samples for RNA-Seq was good for detecting samples with higher concentrations of rRNA (>5%), but not very accurate at lower levels. Although all three kits were able to remove a substantial portion of the rRNA from different fish tissues, the Ribo-Zero Gold rRNA Removal Kit eliminated significantly more contaminating ribosomal RNAs than the others.