Ribo-ODDR: Oligo Design pipeline for experiment-specific Depletion of Ribosomal RNAs in Ribo-seq

Abstract

Ribosome profiling (Ribo-seq) has revolutionized the study of RNA translation. The technique provides information on ribosome positions across all translated RNAs with nucleotide-resolution. However, several technical limitations restrict the sequencing depth of such experiments, the most common of which is the overabundance of ribosomal RNA (rRNA) fragments. Various strategies have been employed to tackle this issue, including the use of commercial rRNA depletion kits, however these may perform suboptimally. Here we show that the rRNA fragments generated via Riboseq vary significantly with differing experimental conditions, suggesting that a "one-size-fits-all" approach may result in inefficient rRNA depletion. In order to overcome this it is possible to use custom-designed biotinylated oligos complementary to the most abundant rRNA fragments, however currently no computational framework exists to aid the design of optimal oligos. We have developed Ribo-ODDR, an oligo design pipeline integrated with a user-friendly interface that assists in oligo selection for efficient experiment-specific rRNA depletion. Ribo-ODDR uses preliminary data to identify the most abundant rRNA fragments, and calculates the rRNA depletion efficiency of potential oligos. We show that Ribo-ODDR designed oligos lead to a significant increase in rRNA depletion, and increased sequencing depth as a result. Ribo-ODDR is freely accessible at https://github.com/fallerlab/Ribo-ODDR

Nuclease-mediated depletion biases in ribosome footprint profiling libraries

“…4), indicating that further species-specific and probably sample-specific optimization in either depletion protocols or depletion oligo design is required. Fortuitously, a recently published analytical framework (Ribo-ODDR) for designing depletion oligos has the potential for substantial enrichment of CDSmapping reads (Alkan et al 2020). Other groups have had success using double-strand specific DNase, combined with a high-temperature reannealing of the final DNA library that only leaves the most abundant sequences (the rRNA fragments) double-stranded (Chung et al 2015), or with targeted oligos against cDNA for the most abundant rRNA contaminants (Archer et al 2014(Archer et al , 2016, but these method have not to our knowledge been tested on a broad range of fragment lengths, nor with improved library preparation methods such as randomized unique molecular identifiers (McGlincy and Ingolia 2017).…”