RNA-seq Sample Preparation Kits Strongly Affect Transcriptome Profiles of a Gas-Fermenting Bacterium

Microbiol Spectr. 2022 Aug 31;10(4):e0230322. doi: 10.1128/spectrum.02303-22. Epub 2022 Jul 27.


Transcriptome analysis via RNA sequencing (RNA-seq) has become a standard technique employed across various biological fields of study. The rapid adoption of the RNA-seq approach has been mediated, in part, by the development of different commercial RNA-seq library preparation kits compatible with standard 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 specific group of organisms (e.g., eukaryotes versus prokaryotes) or genomic GC content. Therefore, the 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. This work reports a side-by-side comparison of RNA-seq data from Clostridium autoethanogenum obtained using three commercial rRNA removal and strand-specific library construction products of NuGEN Technologies, Qiagen, and Zymo Research and assesses their performance relative to published data. While all three vendors advertise their products as suitable for prokaryotes, we found significant differences in their performance regarding 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. IMPORTANCE RNA-seq is a powerful technique for transcriptome profiling while involving elaborate sample processing before library sequencing. We show that RNA-seq library preparation kits can strongly affect the outcome of an RNA-seq experiment. Although library preparation benefits from the availability of various commercial kits, choosing appropriate products for the specific samples can be challenging for new users or for users working with unconventional organisms. Evaluating the performance of different commercial products requires significant financial and time investments infeasible for most researchers. Therefore, users are often guided in their choice of kits by published data involving similar input samples. We conclude that important consideration should be given to selecting sample processing workflows for any given organism.

Keywords: RNA sequencing; RNA-seq; acetogen; transcriptome profiling; transcriptomics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteria
  • Gene Expression Profiling / methods
  • Gene Library
  • High-Throughput Nucleotide Sequencing* / methods
  • RNA-Seq
  • Sequence Analysis, RNA / methods
  • Specimen Handling
  • Transcriptome*