A novel strand-specific RNA-sequencing protocol using dU-adaptor-assembled Tn5

SUMMARY. Strand-specific RNA-seq is a powerful tool for discovery of novel transcripts, annotation of genomes, and profiling of gene expression levels. Tn5 transposase has been successfully applied in massive-scale sequencing projects; in particular, Tn5 adaptor modification is used in epigenetics, genomic structure, and chromatin visualization. We developed a novel dU-adaptor-assembled Tn5-mediated strand-specific RNA-sequencing protocol and compared this method with the leading dUTP method in terms of experimental procedure and multiple quality metrics of the generated libraries. The results showed the dU-Tn5 method is easy to operate and generates a strand-specific RNA-seq library of comparable quality considering library complexity, strand-specificity, evenness, and continuity of annotated transcript coverage. We also evaluated the performance of the dU-Tn5 method in identifying nitrogen-responsive protein-coding genes and long non-coding RNAs in soybean roots. The results indicated that ~62-70% of differentially expressed genes (DEGs) detected from conventional libraries were also detected in dU-Tn5 libraries, indicating good agreement of our method with the current standard; moreover, their fold-changes were highly correlated (R 0.9). Thus, our method provides a promising “do-it-yourself” stranded RNA-seq procedure for gene expression profiling.

摘要。链特异性RNA测序是发现新转录本、注释基因组以及分析基因表达水平的有力工具。Tn5转座酶已成功应用于大规模测序项目；特别是，Tn5接头修饰被用于表观遗传学、基因组结构和染色质可视化。我们开发了一种新型的由含dU接头组装的Tn5介导的链特异性RNA测序方案，并在实验流程以及所构建文库的多项质量指标方面将该方法与领先的dUTP方法进行了比较。结果表明，dU - Tn5方法易于操作，并且就文库复杂性、链特异性、均一性以及注释转录本覆盖的连续性而言，所构建的链特异性RNA测序文库质量相当。我们还评估了dU - Tn5方法在鉴定大豆根中氮响应蛋白编码基因和长链非编码RNA方面的性能。结果显示，从常规文库中检测到的差异表达基因（DEGs）约有62 - 70%也能在dU - Tn5文库中被检测到，这表明我们的方法与现行标准具有良好的一致性；此外，它们的倍数变化高度相关（R = 0.9）。因此，我们的方法为基因表达谱分析提供了一种有前景的“自己动手”的链特异性RNA测序流程。