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Technical Note

Comparative Analysis of rRNA Removal Methods for RNA-Seq Differential Expression in Halophilic Archaea

1
Biology Department, Duke University, Durham, NC 27708, USA
2
University Program in Genetics and Genomics, Duke University, Durham, NC 27708, USA
3
New England Biolabs, Ipswich, MA 01938, USA
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Academic Editor: Hannu Myllykallio
Biomolecules 2022, 12(5), 682; https://doi.org/10.3390/biom12050682
Received: 14 April 2022 / Revised: 4 May 2022 / Accepted: 6 May 2022 / Published: 10 May 2022
(This article belongs to the Collection Archaea: Diversity, Metabolism and Molecular Biology)
Despite intense recent research interest in archaea, the scientific community has experienced a bottleneck in the study of genome-scale gene expression experiments by RNA-seq due to the lack of commercial and specifically designed rRNA depletion kits. The high rRNA:mRNA ratio (80–90%: ~10%) in prokaryotes hampers global transcriptomic analysis. Insufficient ribodepletion results in low sequence coverage of mRNA, and therefore, requires a substantially higher number of replicate samples and/or sequencing reads to achieve statistically reliable conclusions regarding the significance of differential gene expression between case and control samples. Here, we show that after the discontinuation of the previous version of RiboZero (Illumina, San Diego, CA, USA) that was useful in partially or completely depleting rRNA from archaea, archaeal transcriptomics studies have experienced a slowdown. To overcome this limitation, here, we analyze the efficiency for four different hybridization-based kits from three different commercial suppliers, each with two sets of sequence-specific probes to remove rRNA from four different species of halophilic archaea. We conclude that the key for transcriptomic success with the currently available tools is the probe-specificity for the rRNA sequence hybridization. With this paper, we provide insights into the archaeal community for selecting certain reagents and strategies over others depending on the archaeal species of interest. These methods yield improved RNA-seq sensitivity and enhanced detection of low abundance transcripts. View Full-Text
Keywords: archaea; RNAs-seq; rRNA removal; halophiles; transcriptomics archaea; RNAs-seq; rRNA removal; halophiles; transcriptomics
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MDPI and ACS Style

Pastor, M.M.; Sakrikar, S.; Rodriguez, D.N.; Schmid, A.K. Comparative Analysis of rRNA Removal Methods for RNA-Seq Differential Expression in Halophilic Archaea. Biomolecules 2022, 12, 682. https://doi.org/10.3390/biom12050682

AMA Style

Pastor MM, Sakrikar S, Rodriguez DN, Schmid AK. Comparative Analysis of rRNA Removal Methods for RNA-Seq Differential Expression in Halophilic Archaea. Biomolecules. 2022; 12(5):682. https://doi.org/10.3390/biom12050682

Chicago/Turabian Style

Pastor, Mar Martinez, Saaz Sakrikar, Deyra N. Rodriguez, and Amy K. Schmid. 2022. "Comparative Analysis of rRNA Removal Methods for RNA-Seq Differential Expression in Halophilic Archaea" Biomolecules 12, no. 5: 682. https://doi.org/10.3390/biom12050682

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