RnaseIII and T4 Polynucleotide Kinase Sequence Biases and Solutions During RNA-Seq Library Construction

dc.contributor.utaustinauthoren_US
dc.contributor.utaustinauthorWilke, Claus O.en_US
dc.creatorLee, Changhoonen_US
dc.creatorHarris, R. Adronen_US
dc.creatorWall, Jason K.en_US
dc.creatorMayfield, R. Dayneen_US
dc.creatorWilke, Claus O.en_US
dc.date.accessioned2016-10-28T19:52:58Z
dc.date.available2016-10-28T19:52:58Z
dc.date.issued2013-07en_US
dc.description.abstractBackground: 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.en_US
dc.description.departmentWaggoner Center for Alcohol and Addiction Researchen_US
dc.description.sponsorshipNational Institute on Alcohol Abuse and Alcoholism (NIAAA) AA12404, AA019382, AA020926, AA016648en_US
dc.description.sponsorshipNational Institutes of Health (NIH) R01 GM088344en_US
dc.identifierdoi:10.15781/T2DR2PC0B
dc.identifier.citationLee, Changhoon, R. Adron Harris, Jason K. Wall, R. Dayne Mayfield, and Claus O. Wilke. "RNaseIII and T4 Polynucleotide Kinase sequence biases and solutions during RNA-seq library construction." Biology Direct, Vol. 8 (Jul., 2013): 16.en_US
dc.identifier.doi10.1186/1745-6150-8-16en_US
dc.identifier.issn1745-6150en_US
dc.identifier.urihttp://hdl.handle.net/2152/43320
dc.language.isoEnglishen_US
dc.relation.ispartofen_US
dc.relation.ispartofserialBiology Directen_US
dc.rightsAdministrative deposit of works to Texas ScholarWorks: This works author(s) is or was a University faculty member, student or staff member; this article is already available through open access or the publisher allows a PDF version of the article to be freely posted online. The library makes the deposit as a matter of fair use (for scholarly, educational, and research purposes), and to preserve the work and further secure public access to the works of the University.en_US
dc.rights.restrictionOpenen_US
dc.subjectrnaseiiien_US
dc.subjectt4pnken_US
dc.subjectsequence biasen_US
dc.subjectheat fragmentationen_US
dc.subjectoptikinaseen_US
dc.subjectrna-seqen_US
dc.subjectbiologyen_US
dc.titleRnaseIII and T4 Polynucleotide Kinase Sequence Biases and Solutions During RNA-Seq Library Constructionen_US
dc.typeArticleen_US

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