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BMC Genomics. 2016 May 5;17:338. doi: 10.1186/s12864-016-2675-5.

Improvements to the HITS-CLIP protocol eliminate widespread mispriming artifacts.

Author information

1
Department of Medicine, University of Colorado Denver, Aurora, CO, USA. austin.gillen@ucdenver.edu.
2
Department of Medicine, University of Colorado Denver, Aurora, CO, USA.
3
Present Address: Department of Bioengineering, University of Washington, Seattle, WA, USA.
4
Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, CO, USA.
5
Department of Medicine, University of Colorado Denver, Aurora, CO, USA. peter.kabos@ucdenver.edu.

Abstract

BACKGROUND:

High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) allows for high resolution, genome-wide mapping of RNA-binding proteins. This methodology is frequently used to validate predicted targets of microRNA binding, as well as direct targets of other RNA-binding proteins. Hence, the accuracy and sensitivity of binding site identification is critical.

RESULTS:

We found that substantial mispriming during reverse transcription results in the overrepresentation of sequences complementary to the primer used for reverse transcription. Up to 45 % of peaks in publicly available HITS-CLIP libraries are attributable to this mispriming artifact, and the majority of libraries have detectable levels of mispriming. We also found that standard techniques for validating microRNA-target interactions fail to differentiate between artifactual peaks and physiologically relevant peaks.

CONCLUSIONS:

Here, we present a modification to the HITS-CLIP protocol that effectively eliminates this artifact and improves the sensitivity and complexity of resulting libraries.

KEYWORDS:

CLIP-seq; HITS-CLIP; PAR-CLIP; iCLIP; microRNA

PMID:
27150721
PMCID:
PMC4858895
DOI:
10.1186/s12864-016-2675-5
[Indexed for MEDLINE]
Free PMC Article

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