Terminator oligo blocking efficiently eliminates rRNA from Drosophila small RNA sequencing libraries

Abstract

A large number of methods are available to deplete ribosomal RNA reads from high-throughput RNA sequencing experiments. Such methods are critical for sequencing Drosophila small RNAs between 20 and 30 nucleotides because size selection is not typically sufficient to exclude the highly abundant class of 30 nucleotide 2S rRNA. Here we demonstrate that pre-annealing terminator oligos complimentary to Drosophila 2S rRNA prior to 5' adapter ligation and reverse transcription efficiently depletes 2S rRNA sequences from the sequencing reaction in a simple and inexpensive way. This depletion is highly specific and is achieved with minimal perturbation of miRNA and piRNA profiles.

Figure 1

Overall small RNA library preparation scheme with 2S block reaction.

Figure 2

Size distribution of small RNA reads from two experiments, with and without 2S block treatment. 2S filtered distributions were obtained after removal of 2S mapping sequences.

Figure 3

Comparison of Drosophila virilis ovary small RNA profiles between no block and 2S block treatments. A) log10-log10 miRNA profiles from two experiments, comparing no block and 2S block treatments, after 2S rRNA sequence filtering. Measurements are in Reads Per Million Mapped (RPMM) - where Mapped (M) is measured from the total number of reads mapping to the D. virilis genome. B) log10-log10 TE mapping small RNA profiles from two experiments, comparing non block and 2S block treatments. Measurements are the RPMM of non-2S RNA/non-miRNA mapping reads that map to each of the annotated Drosophila virilis TEs. C) miRNA profile similarity comparisons as measured by Pearson’s r. Values in parentheses are with highly expressed miRNAs excluded. Red indicates most similar pair among a set of comparisons. D) TE small RNA profile similarity. All results, block or no block, are highly similar.