RNaseIII and T4 Polynucleotide Kinase sequence biases and solutions during RNA-seq library construction

Changhoon Lee, R. Adron Harris, Jason K. Wall, R. Dayne Mayfield, Claus O. Wilke

Research output: Contribution to journalArticlepeer-review

11 Scopus citations


Background: RNA-seq is a next generation sequencing method with a wide range of applications including single nucleotide polymorphism (SNP) detection, splice junction identification, and gene expression level measurement. However, the RNA-seq sequence data can be biased during library constructions resulting in incorrect data for SNP, splice junction, and gene expression studies. Here, we developed new library preparation methods to limit such biases.Results: A whole transcriptome library prepared for the SOLiD system displayed numerous read duplications (pile-ups) and gaps in known exons. The pile-ups and gaps of the whole transcriptome library caused a loss of SNP and splice junction information and reduced the quality of gene expression results. Further, we found clear sequence biases for both 5' and 3' end reads in the whole transcriptome library. To remove this bias, RNaseIII fragmentation was replaced with heat fragmentation. For adaptor ligation, T4 Polynucleotide Kinase (T4PNK) was used following heat fragmentation. However, its kinase and phosphatase activities introduced additional sequence biases. To minimize them, we used OptiKinase before T4PNK. Our study further revealed the specific target sequences of RNaseIII and T4PNK.Conclusions: Our results suggest that the heat fragmentation removed the RNaseIII sequence bias and significantly reduced the pile-ups and gaps. OptiKinase minimized the T4PNK sequence biases and removed most of the remaining pile-ups and gaps, thus maximizing the quality of RNA-seq data.Reviewers: This article was reviewed by Dr. A. Kolodziejczyk (nominated by Dr. Sarah Teichmann), Dr. Eugene Koonin, and Dr. Christoph Adami. For the full reviews, see the Reviewers' Comments section.

Original languageEnglish (US)
Article number16
JournalBiology Direct
Issue number1
StatePublished - Jul 4 2013
Externally publishedYes


  • Heat fragmentation
  • OptiKinase
  • RNA-seq
  • RNaseIII
  • Sequence bias
  • T4PNK

ASJC Scopus subject areas

  • Immunology
  • Ecology, Evolution, Behavior and Systematics
  • Modeling and Simulation
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • Applied Mathematics


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