Robust mapping of polyadenylated and non-polyadenylated RNA 3' ends at nucleotide resolution by 3'-end sequencing.
Robust mapping of polyadenylated and non-polyadenylated RNA 3' ends at nucleotide resolution by 3'-end sequencing.
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通过 3 末端测序，以核苷酸分辨率对聚腺苷酸化和非聚腺苷酸化 RNA 3 末端进行稳健定位。
DOI：
10.1016/j.ymeth.2019.05.016
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Chanfreau,GuillaumeF
中科院分区：
文献类型：
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作者：
Roy,KevinR;Chanfreau,GuillaumeF
3 '端poly(A)+测序是一种有效和经济的方法，用于全局测量mRNA水平和替代poly(A)位点的使用。常用的方法包括基于oligo(dT)19V逆转录（RT）的文库制备和高通量测序，使用以（dT）19结尾的定制引物。虽然大多数文库产品的第一个测序核苷酸反映了真核多聚(A)位点（pA），但很大一部分测序读取是由各种误引事件引起的。这可能导致错误的pA位点调用，从下游的几个核苷酸到上游的几千个碱基。虽然这些误启动事件可以通过提高退火严格程度（例如将温度从37 °C提高到42 °C）来减轻，但它们仍然持续存在于一个可观的水平（~ 10%），必须使用计算方法来防止人为调用。在这里，我们提出了一个生物信息学工作流程，用于精确绘制poly(a)+ 3 '末端和处理由oligo（dT）错误启动和样本多态性引起的工件。我们用三种不同的读映射程序（STAR、BWA和BBMap）测试了pA位点调用，并表明每种程序处理终端不匹配和软裁剪的方式对识别正确的pA位点有重大影响，而BWA需要最少的后处理来纠正伪影。我们演示了在模型真核生物中使用这种管道来绘制pA位点。并通过在文库准备（IVP-seq）之前使用体外聚腺苷化进一步将该技术应用于非聚腺苷化转录本。作为原理证明，我们发现一小部分trna含有CCU 3 ‘尾部，而不是典型的CCA尾部，并在全局上鉴定了由低效率剪接转录物产生的剪接中间体的3 ’末端。
3′-end poly(A)+ sequencing is an efficient and economical method for global measurement of mRNA levels and alternative poly(A) site usage. A common method involves oligo(dT)19V reverse-transcription (RT)-based library preparation and high-throughput sequencing with a custom primer ending in (dT)19. While the majority of library products have the first sequenced nucleotide reflect thebona fidepoly(A) site (pA), a substantial fraction of sequencing reads arise from various mis-priming events. These can result in incorrect pA site calls anywhere from several nucleotides downstream to several kilobases upstream from thebona fidepA site. While these mis-priming events can be mitigated by increasing annealing stringency (e.g. increasing temperature from 37 °C to 42 °C), they still persist at an appreciable level (∼10%) and computational methods must be used to prevent artifactual calls. Here we present a bioinformatics workflow for precise mapping of poly(A)+ 3′ ends and handling of artifacts due to oligo(dT) mis-priming and sample polymorphisms. We test pA site calling with three different read mapping programs (STAR, BWA, and BBMap), and show that the way in which each handles terminal mismatches and soft clipping has a substantial impact on identifying correct pA sites, with BWA requiring the least post-processing to correct artifacts. We demonstrate the use of this pipeline for mapping pA sites in the model eukaryoteS. cerevisiae, and further apply this technology to non-polyadenylated transcripts by employingin vitropolyadenylation prior to library prep (IVP-seq). As proof of principle, we show that a fraction of tRNAs harbor CCU 3′ tails instead of the canonical CCA tail, and globally identify 3′ ends of splicing intermediates arising from inefficiently spliced transcripts.