Comparison of ribosomal RNA removal methods for transcriptome sequencing workflows in teleost fish

Authors: Abernathy, Jason; Overturf, Kenneth - Ken

Submitted to: Journal of Animal Science and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/26/2015
Publication Date: 1/5/2016
Citation: Abernathy, J.W., Overturf, K.E. 2016. Comparison of ribosomal RNA removal methods for transcriptome sequencing workflows in teleost fish. Journal of Animal Science and Biotechnology. 27(1):60-65.

Interpretive Summary: As part of research to improve animal breeding and production, geneticists can use gene expression studies to measure similarities and differences among animals. Recent advances in biotechnology have revolutionized the way in which geneticists study gene expression through the use of high-throughput gene sequencing. Sample preparation is critical for gene sequencing and commercial companies have developed ‘kits’ for this purpose. However, these kits are almost always optimized and marketed for use in human health and biomedicine rather than for agricultural research. We assessed the efficacy of three of the most common commercial kits typically used in biomedical applications for use in a high-throughput gene expression workflow in a major aquaculture species, the rainbow trout. Our study showed that the kits could be used to analyze trout tissues with some variation in efficiency between the kits. The findings from this report can be used by other scientists engaged in gene expression work with fish. Our work will also alert animal scientists to the need of testing commercial kits for their value in specific agricultural applications.

Technical Abstract: 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. rRNAs are commonly removed from total RNA via either mRNA selection or rRNA depletion. 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). Total RNA was isolated from rainbow trout liver and muscle tissue samples (n=24) and rRNAs removed using one of the three kits. Samples were analyzed visually by electrophoresis on the Agilent Bioanalyzer and by Illumina RNA-seq, screening for Oncorhynchus cytoplasmic and mitochondrial rRNA contamination. Assessment of rRNA removal on the Bioanalyzer did not necessarily match the RNA-seq data and 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. We found that 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. While all three kits were able to remove a substantial portion of the rRNA from fish tissue, the Ribo-Zero™ Gold rRNA Removal Kit eliminated significantly more contaminating ribosomal RNAs than the others.