Two Sets of Synthetic Spike-in Controls for Size Selection and Data Normalisation in small RNA-seq

There is an increasing interest in complementing standard RNA-seq experiments with small RNA expression data to obtain a comprehensive view of the transcriptome. Two aspects need to be considered when small RNA-seq (sRNA-seq) is used. First, sRNA-seq protocols are size selective and typically optimized for sequencing miRNAs. Second, given the dynamic composition of the small RNA fraction a reliable normalization strategy is needed to identify differentially expressed sRNAs. To address both issues we here present two sets of synthetic RNA spike-in controls for monitoring size selectivity and for performing normalization. Size spike-ins comprising 18 oligoribonucleotides (10–300 nucleotides) were designed and tested using controlled modifications to the size selectivity of sRNA-seq. As an applied example, the loss of RNA molecules <30 nucleotides during small RNA isolation from zebrafish oocytes is demonstrated. Normalization spike-ins comprising 19 oligoribonucleotides were designed to use as an external reference for DESeq normalization. Spike-in-based normalization improved the quantification of predetermined fold changes. Lastly, sRNA-seq on female and male zebrafish demonstrated that the higher miRNA content in males was correctly preserved after spike-in-based normalization.