Accurate Profiling of Gene Expression and Alternative Polyadenylation with Whole Transcriptome Termini Site Sequencing (WTTS-Seq).
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.
Department of Animal Sciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-7620.
Xiang
X
Zhou
Department of Animal Sciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-7620.
Rui
R
Li
Department of Animal Sciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-7620.
Jennifer J
JJ
Michal
Department of Animal Sciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-7620.
Xiao-Lin
XL
Wu
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong.
Zhongzhen
Z
Liu
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong.
Hui
H
Zhao
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong.
Yin
Y
Xia
Laboratory for Biotechnology and Bioanalysis, Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-7520.
Weiwei
W
Du
Laboratory for Biotechnology and Bioanalysis, Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-7520.
Mark R
MR
Wildung
Laboratory for Biotechnology and Bioanalysis, Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-7520.
Derek J
DJ
Pouchnik
Department of Molecular and Cell Biology, University of California Berkeley, California 94720-3200.
Richard M
RM
Harland
Department of Animal Sciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-7620 jiangz@wsu.edu.
Zhihua
0000-0003-1986-088X
Z
Jiang
D000818:Animals; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics; D012680:Sensitivity and Specificity; D017423:Sequence Analysis, RNA / Q000379:methods; D059467:Transcriptome; D014981:Xenopus
D012333:RNA, Messenger
3′-termini sequencing; Bayesian model; amplification detours; missing data; transcriptome distribution