title: RNaseIII and T4 polynucleotide Kinase sequence biases and solutions during RNA-seq library construction.
abstract: 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.
authors.affiliation: Waggoner Center for Alcohol and Addiction Research, Section of Neurobiology and Institute for Cellular and Molecular Biology, The University of Texas at Austin, TX 78712, USA. ninolee00@utexas.edu
authors.forename: Changhoon
authors.initials: C
authors.lastname: Lee
authors.forename: R Adron
authors.initials: RA
authors.lastname: Harris
authors.forename: Jason K
authors.initials: JK
authors.lastname: Wall
authors.forename: R Dayne
authors.initials: RD
authors.lastname: Mayfield
authors.forename: Claus O
authors.initials: CO
authors.lastname: Wilke
mesh_terms: D015723:Gene Library; D006358:Hot Temperature; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000737:chemistry*; D043244:Ribonuclease III / Q000737:chemistry*; D017423:Sequence Analysis, RNA / Q000379:methods*; D059467:Transcriptome
chemical_list: D011116:Polynucleotide 5'-Hydroxyl-Kinase; D043244:Ribonuclease III