Journal article
Accurate Profiling of Gene Expression and Alternative Polyadenylation with Whole Transcriptome Termini Site Sequencing (WTTS-Seq)
Genetics (Austin), Vol.203(2), pp.683-697
06/2016
Handle:
https://hdl.handle.net/2376/100419
PMCID: PMC4896187
PMID: 27098915
Abstract
Construction of next-generation sequencing (NGS) libraries involves RNA manipulation, which often creates noisy, biased, and artifactual data that contribute to errors in transcriptome analysis. In this study, a total of 19 whole transcriptome termini site sequencing (WTTS-seq) and seven RNA sequencing (RNA-seq) libraries were prepared from Xenopus tropicalis adult and embryo samples to determine the most effective library preparation method to maximize transcriptomics investigation. We strongly suggest that appropriate primers/adaptors are designed to inhibit amplification detours and that PCR overamplification is minimized to maximize transcriptome coverage. Furthermore, genome annotation must be improved so that missing data can be recovered. In addition, a complete understanding of sequencing platforms is critical to limit the formation of false-positive results. Technically, the WTTS-seq method enriches both poly(A)+ RNA and complementary DNA, adds 5'- and 3'-adaptors in one step, pursues strand sequencing and mapping, and profiles both gene expression and alternative polyadenylation (APA). Although RNA-seq is cost prohibitive, tends to produce false-positive results, and fails to detect APA diversity and dynamics, its combination with WTTS-seq is necessary to validate transcriptome-wide APA.
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Details
- Title
- Accurate Profiling of Gene Expression and Alternative Polyadenylation with Whole Transcriptome Termini Site Sequencing (WTTS-Seq)
- Creators
- Xiang Zhou - Department of Animal Sciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-7620Rui Li - Department of Animal Sciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-7620Jennifer J Michal - Department of Animal Sciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-7620Xiao-Lin Wu - Department of Animal Sciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-7620Zhongzhen Liu - School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong KongHui Zhao - School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong KongYin Xia - School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong KongWeiwei Du - Laboratory for Biotechnology and Bioanalysis, Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-7520Mark R Wildung - Laboratory for Biotechnology and Bioanalysis, Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-7520Derek J Pouchnik - Laboratory for Biotechnology and Bioanalysis, Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-7520Richard M Harland - Department of Molecular and Cell Biology, University of California Berkeley, California 94720-3200Zhihua Jiang - Department of Animal Sciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-7620 jiangz@wsu.edu
- Publication Details
- Genetics (Austin), Vol.203(2), pp.683-697
- Academic Unit
- Animal Sciences, Department of
- Publisher
- United States
- Grant note
- R21 HD076845 / NICHD NIH HHS
- Identifiers
- 99900546697101842
- Language
- English
- Resource Type
- Journal article