Assessing Computational Steps for CLIP-Seq Data Analysis.

Qi Liu,Blair B Madison,Xue Zhong,Anil K Rustgi,Yu Shyr

doi:10.1155/2015/196082

Abstract

RNA-binding protein (RBP) is a key player in regulating gene expression at the posttranscriptional level. CLIP-Seq, with the ability to provide a genome-wide map of protein-RNA interactions, has been increasingly used to decipher RBP-mediated posttranscriptional regulation. Generating highly reliable binding sites from CLIP-Seq requires not only stringent library preparation but also considerable computational efforts. Here we presented a first systematic evaluation of major computational steps for identifying RBP binding sites from CLIP-Seq data, including preprocessing, the choice of control samples, peak normalization, and motif discovery. We found that avoiding PCR amplification artifacts, normalizing to input RNA or mRNAseq, and defining the background model from control samples can reduce the bias introduced by RNA abundance and improve the quality of detected binding sites. Our findings can serve as a general guideline for CLIP experiments design and the comprehensive analysis of CLIP-Seq data.

Highlights

RNA-binding proteins (RBPs) are the primary regulator of posttranscriptional gene expression [1]
Ranking peaks by the relative enrichment of distinct reads to RPKM obtained the highest correlation, ranging from 0.25 to 0.32. These results suggest that normalizing CLIP data to mRNAseq can improve the specificity when RBP targeting messenger RNA
Using Lin28b CLIP-Seq studies with input RNA or mRNAseq as control samples, we presented a systematic evaluation of different strategies to implement data preprocessing, peak normalization and ranking, and motif discovery for the analysis of CLIP-Seq data

Summary

Introduction

RNA-binding proteins (RBPs) are the primary regulator of posttranscriptional gene expression [1]. As soon as RNAs are transcribed, they are associated with RBPs to form ribonucleoprotein (RNP) complexes. A high resolution and precise map of protein-RNA interactions is essential for deciphering posttranscriptional regulation under various biological processes. CLIP (cross-linking and immunoprecipitation) is the main technology for studying protein-RNA interactions in vivo [2,3,4]. CLIP uses ultraviolet irradiation to form covalent crosslinks only at direct sites between RBP and RNAs in situ, followed by immunoprecipitation of the protein-RNA complex with an antibody specific to the RBP of interest. P value 9.07e − 10a 2.97e − 06d 1.07e − 13e 6.45e − 11f 9.79e − 15 8.42e − 09b 3.70e − 11g 1.00e − 14c 1.27e − 07 1.78e − 05 Caco. 1.00 1.00 1.00 1.00 1.00 1.00 1.00 and to obtain a comprehensive view of posttranscriptional regulatory networks

Methods

Results

Conclusion