UC Berkeley

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.