High-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP, also called CLIP-Seq) has been used to map global RNA–protein interactions. However, a critical caveat of HITS-CLIP results is that they contain nonlinear background noise––different extent of nonspecific interactions caused by individual transcript abundance––that has been inconsiderately normalized, resulting in sacrifice of sensitivity. To properly deconvolute RNA–protein interactions, we have implemented CLIPick, a flexible peak calling pipeline for analyzing HITS-CLIP data, which statistically determines the signal-to-noise ratio for each transcript based on the expression-dependent background simulation. Comprising of streamlined Python modules with an easy-to-use standalone graphical user interface, CLIPick robustly identifies significant peaks and quantitatively defines footprint regions within which RNA–protein interactions were occurred. CLIPick outperforms other peak callers in accuracy and sensitivity, selecting the largest number of peaks particularly in lowly expressed transcripts where such marginal signals are hard to discriminate. Specifically, the application of CLIPick to Argonaute (Ago) HITS-CLIP data were sensitive enough to uncover extended features of microRNA target sites, and these sites were experimentally validated. CLIPick enables to resolve critical interactions in a wide spectrum of transcript levels and extends the scope of HITS-CLIP analysis.
Bibliographical noteFunding Information:
Korean Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea [HI15C3137]. Korea University Future Research Grant. Conflict of interest statement. None declared.
© The Author(s) 2018.
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