While these methods are well suited for distinguishing sites that arose from sequencing and mapping errors, they can not account for truely crosslinked RNA, which however is not bound to the PAR-CLIP factor of interest.Ī recent study has shown that this so-called background binding is a major confounder in PAR-CLIP experimnents, despite of the stringent washing steps in the PAR-CLIP protocol that are supposed to select for interactions with the protein of interest. Transition rates in the randommodel are estimated from the transions that are not specific to the PAR-CLIP experiment,. Previously published PAR-CLIP methods predict PAR-CLIP binding regions from read clustering and pinpoint binding sites by comparing the transition rates to a random model. To the best to our knowledge this is the first quantitative model that estimates the binding probability by integrating the coverage and transition properties of the mock experiment. Last but not least, the pipeline offers a module for predicting PAR-CLIP binding sites by incorporating a measured mock PAR-CLIP experiment.
#ICLIP PROTOCOL TIME FULL#
While the predefined modules should make it possible to perform a full analysis, it can easily be extended by defining custom modules.
The mockinbird PAR-CLIP pipeline offers modules to performs data processing starting from the raw fastq file down to biological binding analyses.
The complexity in the protocol requires several uncommon steps in the data preparation and quality control, such as analyses of the quality and distribution of the PAR-CLIP specific mutations. The resolution is achieved by introducing a base-analogon that is misread by the reverse-transcriptase when crosslinked to a protein. PAR-CLIP is a protocol derived from the general CLIP-seq technique and allows predicting protein-RNA binding sites at nucleotide resolution.
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