The empirical approach to rRNA depletion for NGS

Ribosomal RNA (rRNA) depletion is a must for Next Generation Sequencing (NGS) studies to reduce the number of irrelevant reads. While commercially-available kits such as the Ribo-zero or the BioMag® SelectaPure mRNA System are popular for rRNA depletion, there are some experimental situations in which another approach is required.

Next Generation Sequencing Image - tebu-bio Epicentre-IlluminaIn a detailed ribosome profiling protocol published by Jonathan Weissman’s group, entitled Genome-wide annotation and quantitation of translation by ribosome profiling (PMID: 23821443), for example, the authors describe their method for evaluating mRNA translation rates using a ribosome footprinting approach.

Polysome Profile

Polysome Profile

Briefly, nuclease-treated RNA is subjected to genome-wide deep sequencing in such a way that ribosomes have protected fragments of 26-34 nucleotides. Clearly, with this protocol one could not deplete the sample of rRNA, because mRNA fragments associated with entire ribosomes (including rRNA) are isolated.

Following a PAGE purification of RNA fragments 26-34 nucleotides in length using this protocol, one can observe rRNA contamination of the sample accounting for as many as 70% of the reads. With only 30% of sequencing data usable, this is a major technical problem. The authors recommend then designing a subtraction mix of twenty purchase Biotin-TEG DNA oligonucleotides designed empirically based on the sequencing reads for efficient depletion of rRNA. Such biotinylated rRNA depletion probes can be used in combination with BioMag® Nuclease-Free Streptavidin Particles for efficient depletion of rRNA. Using this approach, users observe a near complete depletion of the targeted sequences.

A similar approach of custom designed rRNA depletion probese is being used for exotic species including gram positive and gram negative bacteria as highlighted in this post.

One technical note: The Weissman protocol recommends a Circularization step prior to rRNA depletion using CircLigase I. Epicentre-An Illumina Company now recommends the superior CircLigase II to replace this enzyme in all protocols.