Optimized design of antisense oligomers for targeted rRNA depletion

Abstract

RNA sequencing (RNA-seq) is extensively used to quantify gene expression transcriptome-wide. Although often paired with polyadenylate (poly(A)) selection to enrich for messenger RNA (mRNA), many applications require alternate approaches to counteract the high proportion of ribosomal RNA (rRNA) in total RNA. Recently, digestion using RNaseH and antisense DNA oligomers tiling target rRNAs has emerged as an alternative to commercial rRNA depletion kits. Here, we present a streamlined, more economical RNaseH-mediated rRNA depletion with substantially lower up-front costs, using shorter antisense oligos only sparsely tiled along the target RNA in a 5-min digestion reaction. We introduce a novel Web tool, Oligo-ASST, that simplifies oligo design to target regions with optimal thermodynamic properties, and additionally can generate compact, common oligo pools that simultaneously target divergent RNAs, e.g. across different species. We demonstrate the efficacy of these strategies by generating rRNA-depletion oligos for Xenopus laevis and for zebrafish, which expresses two distinct versions of rRNAs during embryogenesis. The resulting RNA-seq libraries reduce rRNA to <5% of aligned reads, on par with poly(A) selection, and also reveal expression of many non-adenylated RNA species. Oligo-ASST is freely available at https://mtleelab.pitt.edu/oligo to design antisense oligos for any taxon or to target any abundant RNA for depletion.

Efficient and specific DNA oligonucleotide rRNA probe-based rRNA removal in Talaromyces marneffei

“…We thus aimed to implement and modify a similar method for fungi rRNA depletion. Among the ribosomes of eukaryotes, 5S, 5.8S, 18S and 28S are the most abundant rRNA transcripts, accounting for a rough 80% of the total RNA (Kraus et al 2019 ; Phelps et al 2021 ). In order to deplete most of the rRNAs from total RNA, we first identified the 5S, 5.8S, 18S and 28S, as well as ITS1 and ITS2 rRNA sequences based on draft genome from Illumina assemblies of Talaromyces marneffei PM1 strain (Woo et al 2011 ) using software RNAmmer (Lagesen et al 2007 ) and blasted against NCBI to confirm the rRNA sequences.…”

“…Ribosomal RNA sequences, including 5S, 5.8S, 18S, 28S, ITS1 and ITS2 rRNA, were searched in the whole genome using RNAmmer (Lagesen et al 2007 ). Similar to the previous method (Phelps et al 2021 ), non-overlapping complementary DNA probes of 80-nt were synthesised (TSINGKE, Beijing, China) and mixed, in which 18S and 28S rRNA with a final concentration of 1 μM, and ITS1, ITS2, 5S as well as 5.8S rRNA with a final concentration of 0.05 μM. The primer sequences are listed in Supplementary Table S2.…”

Decoding rRNA sequences for improved metagenomics of sylvatic mosquito species

“…They comprise at least 80% of the total cellular RNA population. In RNA-seq experiments, their depletion is a necessary step during library preparation where it is not possible to selectively enrich target signals (5). To achieve this, the most routinely used depletion protocols require knowledge of rRNA sequence of the species of interest.…”

“…To achieve this, the most routinely used depletion protocols require knowledge of rRNA sequence of the species of interest. These protocols involve hybridizing antisense oligos (probes or primers) to rRNA molecules followed by digestion by ribonucleases (5, 6) or removal by bead capture (7).…”

Correction to ‘Optimized design of antisense oligomers for targeted rRNA depletion’

“…The article ( 1 ) has been updated. The correction does not affect the results, discussion and conclusions presented in the article.…”