A Simple, Cost-Effective, and Robust Method for rRNA Depletion in RNA-Sequencing Studies

Abstract

The profiling of gene expression by RNA sequencing (RNA-seq) has enabled powerful studies of global transcriptional patterns in all organisms, including bacteria. Because the vast majority of RNA in bacteria is rRNA, it is standard practice to deplete the rRNA from a total RNA sample such that the reads in an RNA-seq experiment derive predominantly from mRNA. One of the most commonly used commercial kits for rRNA depletion, the Ribo-Zero kit from Illumina, was recently discontinued abruptly and for an extended period of time. Here, we report the development of a simple, cost-effective, and robust method for depleting rRNA that can be easily implemented by any lab or facility. We first developed an algorithm for designing biotinylated oligonucleotides that will hybridize tightly and specifically to the 23S, 16S, and 5S rRNAs from any species of interest. Precipitation of these oligonucleotides bound to rRNA by magnetic streptavidin-coated beads then depletes rRNA from a complex, total RNA sample such that ∼75 to 80% of reads in a typical RNA-seq experiment derive from mRNA. Importantly, we demonstrate a high correlation of RNA abundance or fold change measurements in RNA-seq experiments between our method and the Ribo-Zero kit. Complete details on the methodology are provided, including open-source software for designing oligonucleotides optimized for any bacterial species or community of interest. IMPORTANCE The ability to examine global patterns of gene expression in microbes through RNA sequencing has fundamentally transformed microbiology. However, RNA-seq depends critically on the removal of rRNA from total RNA samples. Otherwise, rRNA would comprise upward of 90% of the reads in a typical RNA-seq experiment, limiting the reads coming from mRNA or requiring high total read depth. A commonly used kit for rRNA subtraction from Illumina was recently unavailable for an extended period of time, disrupting routine rRNA depletion. Here, we report the development of a “do-it-yourself” kit for rapid, cost-effective, and robust depletion of rRNA from total RNA. We present an algorithm for designing biotinylated oligonucleotides that will hybridize to the rRNAs from a target set of species. We then demonstrate that the designed oligonucleotides enable sufficient rRNA depletion to produce RNA-seq data with 75 to 80% of reads coming from mRNA. The methodology presented should enable RNA-seq studies on any species or metagenomic sample of interest.

Optimized design of antisense oligomers for targeted rRNA depletion

“…In the latter approach, DNA oligos complementary to rRNA facilitate their removal prior to library construction. These oligos can be biotinylated for magnetic bead affinity purification ( 6 , 22 , 23 ), commercialized for some taxa in the Ribo-Minus and the now discontinued Ribo-Zero Gold kits.…”

“…Antisense oligos can be used in conjunction with RNaseH to digest DNA–rRNA hybrids ( 5 , 24 ). Several studies in both mammals and bacteria have shown that RNaseH-mediated rRNA depletion is efficient, resulting in sequencing libraries with minimal rRNA derived reads ( 5 , 23–27 ). Commercial solutions have emerged for select taxa; however, the ease of this method allows it in principle to be readily adapted to any taxon, with the primary challenge being the design and acquisition of the 50 nt oligos that tile the specific rRNA sequences encoded in its transcriptome.…”

High Throughput Sequencing for the Detection and Characterization of RNA Viruses

“…DSN has been applied successfully for the sequencing of RNA viruses in complex matrices (Schuh et al, 2020 ; Zhou et al, 2020 ). For subtractive hybridization, unwanted rRNAs/cDNAs are hybridized to biotinylated DNA or Locked nucleic acid ( LNA ) probes and depleted with streptavidin beads (Briese et al, 2015 ; Culviner et al, 2020 ).…”

A synthetic RNA editing factor edits its target site in chloroplasts and bacteria

“…Editing e ciency was determined from the raw sequencing chromatograms using the EditR webserver available at http://baseeditr.com/. For RNA-seq analyses, 500 ng of DNase-treated RNA was rRNA-depleted 55 and then used for preparing sequencing libraries with the Illumina TruSeq Stranded total RNA library preparation kit as recommended by the manufacturer. The libraries were sequenced on an Illumina HiSeq4000 sequencer by Novogene.…”