Multimodal RNA-seq using single-strand, double-strand, and circligase-based capture yields a refined and extended description of the C. elegans transcriptome. Caenorhabditis elegans

We have used a combination of three high-throughput RNA capture and sequencing methods to refine and augment the transcriptome map of a well studied genetic model, Caenorhabditis elegans. The three methods include a standard (non-directional) library preparation protocol relying on cDNA priming and foldback that has been used in several previous studies for transcriptome characterization in this species, and two directional protocols, one involving direct capture of single stranded RNA fragments and one involving circular-template PCR (circligase). We find that each RNA-seq approach shows specific limitations and biases, with the application of multiple methods providing a more complete map than was obtained from any single method. Of particular note in the analysis were substantial advantages of circligase-based and ssRNA-based capture for defining sequences and structures of the precise 5' ends (which were lost using the double strand cDNA capture method). Of the three methods, ssRNA capture was most effective in defining sequences to the polyA junction. Using datasets from a spectrum of C. elegans strains and stages and the UCSC Genome Browser, we provide a series of tools, which facilitate rapid visualization and assignment of gene structures. Overall design: single-strand-capture, double-strand-capture, and circligase-based RNA-seq

本研究结合三种高通量RNA捕获与测序方法，对经典遗传模式生物秀丽隐杆线虫（Caenorhabditis elegans）的转录组图谱进行优化与扩充。三种方法具体包括：一种基于互补DNA（cDNA）引物延伸与回折的标准（非定向）文库制备方案，该方案曾被多项既往研究用于该物种的转录组特征分析；以及两种定向文库制备方案，其一为单链RNA（ssRNA）片段直接捕获法，其二为环形模板PCR（circligase）技术。研究发现，每种RNA测序方法均存在特定的局限性与偏好性，联合使用多种方法可获得比单一方法更完整的转录组图谱。分析中尤为值得关注的是，基于circligase与单链RNA的捕获方法在精准界定5'端序列与结构方面具有显著优势——而双链互补DNA捕获法会丢失此类信息。三种方法中，单链RNA捕获法在界定多聚腺苷酸（polyA）连接位点附近序列方面效果最佳。本研究依托涵盖多种秀丽隐杆线虫品系与发育阶段的数据集，以及UCSC基因组浏览器（UCSC Genome Browser），开发了一系列可快速实现基因结构可视化与注释的工具。整体实验设计：单链捕获、双链捕获及基于circligase的RNA测序