GSE200959
RNA-seq sample preparation kits strongly affect transcriptome profiles of a gas-fermenting bacterium
Transcriptome analysis via RNA sequencing (RNA-seq) has become a standard technique employed across a variety of biological fields of study. This rapid adoption of the RNA-seq approach has been mediated, in part, by the development of various commercial RNA-seq library preparation kits compatible with common next-generation sequencing (NGS) platforms. Generally, the essential steps of library preparation such as rRNA depletion and first-strand cDNA synthesis are tailored to a certain group of organisms (e.g. eukaryotes versus prokaryotes) or genomic GC content. Therefore, selection of appropriate commercial products is of crucial importance to capture the transcriptome of interest as closely to the native state as possible without introduction of technical bias. However, researchers rarely have the resources and time to test various commercial RNA-seq kits for their samples. In this work, we report a side-by-side comparison of RNA-seq data from the same Clostridium autoethanogenum input samples obtained using three commercial rRNA removal and strand-specific library construction products by NuGEN Technologies, Qiagen, and Zymo Research, and compare their performance with published data. While all three vendors advertise their products as suitable for prokaryotes, we found significant differences in their performance in terms of rRNA removal, strand-specificity, and, most importantly, transcript abundance distribution profiles. Notably, RNA-seq data obtained with Qiagen products were most similar to published data and delivered the best results in terms of library strandedness and transcript abundance distribution range. Our results highlight the importance of finding appropriate organism-specific workflows and library preparation products for RNA-seq studies. Overall design: RNA-seq of Clostridium autoethanogenum using NuGEN, Qiagen and Zymo library construction kits.