Department of Animal Sciences and Center for Reproductive Biology, Washington State University, Pullman, WA 99164-7620.
Int J Biol Sci. 2018 Sep 7;14(12):1709-1714. doi: 10.7150/ijbs.27168. eCollection 2018.
Alternative polyadenylation is an essential RNA processing event that contributes significantly to regulation of transcriptome diversity and functional dynamics in both animals and plants. Here we review newly developed next generation sequencing methods for genome-wide profiling of alternative polyadenylation (APA) sites, bioinformatics pipelines for data processing and both wet and dry laboratory approaches for APA validation. The library construction methods LITE-Seq (Low-Input 3'-Terminal sequencing) and PAC-seq (PolyA Click sequencing) tag polyA cDNA, while BAT-seq (BArcoded, three-prime specific sequencing) and PAPERCLIP (oly() binding rotein-mediated mRNA 3'nd etrieval by rossinking mmunorecipitation) enrich polyA RNA. Interestingly, only WTTS-seq (Whole Transcriptome Termini Site sequencing) targets both polyA RNA and polyA cDNA. Varieties of bioinformatics pipelines are well established to pursue read quality control, mapping, clustering, characterization and pathway analysis. The RHAPA (RNase H alternative polyadenylation assay) and 3'RACE-seq (3' rapid amplification of cDNA end sequencing) methods directly validate APA sites, while WTSS-seq (whole transcriptome start site sequencing), RNA-seq (RNA sequencing) and public APA databases can serve as indirect validation methods. We hope that these tools, pipelines and resources trigger huge waves of interest in the research community to investigate APA events underlying physiological, pathological and psychological changes and thus understand the information transfer events from genome to phenome relevant to economically important traits in both animals and plants.
可变多聚腺苷酸化是一种重要的 RNA 加工事件，它在动物和植物中对转录组多样性和功能动态的调控有重要贡献。在这里，我们回顾了用于全基因组分析可变多聚腺苷酸化（APA）位点的新一代测序方法、用于数据处理的生物信息学流程以及 APA 验证的湿实验和干实验方法。LITE-Seq（低输入 3' 末端测序）和 PAC-seq（多聚 A 点击测序）标记 polyA cDNA，而 BAT-seq（BArcoded，3' 端特异性测序）和 PAPERCLIP（poly()结合蛋白介导的 mRNA 3'末端回收通过交联免疫沉淀）富集 polyA RNA。有趣的是，只有 WTTS-seq（全转录本末端位点测序）同时靶向 polyA RNA 和 polyA cDNA。已经建立了各种生物信息学流程来进行读质量控制、映射、聚类、特征分析和途径分析。RHAPA（RNase H 可变多聚腺苷酸化测定）和 3'RACE-seq（3' 快速扩增 cDNA 末端测序）方法直接验证 APA 位点，而 WTSS-seq（全转录本起始位点测序）、RNA-seq（RNA 测序）和公共 APA 数据库可作为间接验证方法。我们希望这些工具、流程和资源能激发研究界对研究生理、病理和心理变化背后的 APA 事件的巨大兴趣，从而理解与动物和植物中经济重要性状相关的从基因组到表型的信息传递事件。
