ABSTRACT

Small RNA (sRNA) sequencing has been critical for our understanding of many cellular processes, including gene regulation. Nonetheless, the varying biochemical properties of sRNA, such as 5´ nucleotide modifications, make many sRNA subspecies incompatible with common protocols for sRNA sequencing. Here we describe 5XP-seq that outlines a novel strategy that captures a more complete picture of sRNA. By tagging 5´P sRNA during library preparation, 5XP-seq combines an open approach that includes all types of 5ʹ-terminal modifications (5´X), with a selective approach for 5-phosphorylated sRNA (5´P). We show that 5XP-seq not only enriches phosphorylated miRNA and piRNA but successfully discriminates these sRNA from all other sRNA species. We further demonstrate the importance of this strategy by successful inter-species validation of sRNAs that would have otherwise failed, including human to insect translation of several tRNA (tRFs) and rRNA (rRFs) fragments. By combining 5´ insensitive library strategies with 5´ sensitive tagging, we have successfully tackled an intrinsic bias in modern sRNA sequencing that will help us reveal the true complexity and the evolutionary significance of the sRNA world.

 抽象的

小 RNA (sRNA) 测序对于我们理解包括基因调控在内的许多细胞过程至关重要。尽管如此，sRNA 不同的生化特性（例如 5' 核苷酸修饰）使得许多 sRNA 亚种与 sRNA 测序的通用方案不兼容。在这里，我们描述了 5XP-seq，它概述了一种捕获更完整的 sRNA 图片的新颖策略。通过在文库制备过程中标记 5´P sRNA，5XP-seq 结合了包括所有类型 5´ 末端修饰 (5´X) 的开放方法和 5-磷酸化 sRNA (5´P) 的选择性方法。我们表明，5XP-seq 不仅富集了磷酸化 miRNA 和 piRNA，而且成功地将这些 sRNA 与所有其他 sRNA 种类区分开来。我们通过成功地对 sRNA 进行种间验证（包括几个 tRNA (tRFs) 和 rRNA (rRFs) 片段的人类到昆虫翻译）进一步证明了该策略的重要性，否则这些验证将失败。通过将 5' 不敏感文库策略与 5' 敏感标签相结合，我们成功解决了现代 sRNA 测序中的内在偏差，这将帮助我们揭示 sRNA 世界的真正复杂性和进化意义。