Improvements to the HITS-CLIP protocol eliminate widespread mispriming artifacts

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


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Artifacts*
  • Binding Sites*
  • DNA Primers
  • Gene Expression
  • Gene Library
  • High-Throughput Nucleotide Sequencing* / methods
  • High-Throughput Nucleotide Sequencing* / standards
  • Immunoprecipitation* / methods
  • MicroRNAs / chemistry
  • MicroRNAs / genetics
  • MicroRNAs / metabolism
  • Protein Binding
  • RNA / chemistry
  • RNA / genetics*
  • RNA / metabolism*
  • RNA-Binding Proteins / metabolism*
  • Reverse Transcriptase Polymerase Chain Reaction / standards
  • Sensitivity and Specificity
  • Sequence Analysis, RNA


  • DNA Primers
  • MicroRNAs
  • RNA-Binding Proteins
  • RNA