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

Abstract

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.

The chromatin landscape of the euryarchaeonHaloferax volcanii

“…KAS-seq pro- vides several advantages in the H. volcanii context. First, due to the absence of straightforward methods to deplete H. volcanii ribosomal RNA (rRNA) from RNA sequencing libraries analogous to polyA-selection in eukaryotes 47,48 , it enables measurement of transcriptional activity at much lower cost than deep RNA-seq experiments. Second, and most important, it measures actively engaged polymerase molecules, and thus unlike the steady-state transcript levels that conventional RNA-seq quantifies, it provides a means to quantify active transcriptional activity.…”

TbsP and TrmB jointly regulate gapII to influence cell development phenotypes in the archaeon Haloferax volcanii

“…For samples collected in 2021, ribosomal RNA was removed with the PanArchaea riboPOOL kit according to the manufacturer protocol (siTOOLs Biotech, Germany), and sequencing libraries were constructed with the NEBNext UltraII Directional RNA Library Preparation Kit (New England BioLabs, #E7760) as described previously (Pastor et al., 2022). The fragment size of all libraries was measured using an Agilent Bioanalyzer DNA 1000 chip and then pooled and sequenced on the NovaSeq6000 at the Center for Genomic and Computational Biology at Duke University (Durham, NC).…”

“…Resident in hypersaline evaporation ponds and waters such as the Dead Sea, this organism experiences dramatic fluctuations in nutrient availability, desiccation/rehydration cycles, and oxidative stress (Oren, 2008). Haloferax volcanii is a member of the hypersalineadapted archaea, or haloarchaea, of the phylum Euryarchaeota.…”

A sweet new set of inducible and constitutive promoters for haloarchaea

“…Next, to map candidates for new inducible promoters, we employed RNA-seq from mid-exponential cultures with or without each of the four inducers. Using a set of specific probes for H. volcanii (Supplementary Table 1) (Pastor et al, 2022), we obtained a ribosomal RNA depletion of 99.7%, from a total number of reads of 120,234,156. By comparing the relative fold change of transcripts per million scale, we obtained 58 genes from which mRNA levels were significantly upregulated (log2 fold change ≥ 1 and p-value ≤ 0.05) for xylose, 42 by maltose, 0 for IPTG, and 45 for arabinose.…”