小柯机器人

西班牙巴塞罗那科技园Eva Maria Novoa研究小组开发出了，使用末端捕获纳米孔cDNA测序对基因表达和尾部动力学进行转录组范围分析的工具Nano3P-seq。2022年12月19日出版的《自然-方法学》杂志发表了这项成果。

研究人员研发了纳米孔3′末端捕获测序（Nano3P-seq），这是一种依靠纳米孔cDNA测序的方法，以单次读取分辨率对RNA丰度、尾部组成和尾长动力学定量。通过基于模板切换的测序方案，Nano3P-seq可以从其3'末端对RNA分子进行测序，无论其聚腺苷酸化状态如何，而无需PCR扩增或连接RNA接头。研究证明了Nano3P-seq可对RNA丰度和尾部长度进行定量估计，并发现了多种RNA模式。研究发现在小鼠和斑马鱼模型中，除了mRNA和长非编码RNA还可以在16S线粒体核糖体RNA中检测到polyA尾巴。

此外，在脊椎动物胚胎发育过程中，mRNA尾巴长度在亚型特异性水平上受到动态调节，这与mRNA衰变相关。最后，研究人员证明了Nano3P-seq具有在不同长度polyA尾中检测非A碱基的能力，并揭示了它们在脊椎动物胚胎发生过程中的分布。总体而言，Nano3P-seq是一种用于准确估计单个读段中转录本水平、polyA长度和尾部组成异质性简单而稳健的方法，在编码和非编码转录组文库的制备中具有最小偏差。

据了解，RNA聚腺苷酸化在RNA成熟、命运决定和稳定性中起核心作用。在不同发育阶段polyA尾长会发送变化，这会影响mRNA的翻译效率和稳定性。

附：英文原文

Title: Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing

Author: Begik, Oguzhan, Diensthuber, Gregor, Liu, Huanle, Delgado-Tejedor, Anna, Kontur, Cassandra, Niazi, Adnan Muhammad, Valen, Eivind, Giraldez, Antonio J., Beaudoin, Jean-Denis, Mattick, John S., Novoa, Eva Maria

Issue&Volume: 2022-12-19

Abstract: RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3′ end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3′ end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide diversity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.

DOI: 10.1038/s41592-022-01714-w

Source: https://www.nature.com/articles/s41592-022-01714-w

Nature Methods：《自然—方法学》，创刊于2004年。隶属于施普林格·自然出版集团，最新IF：47.99
官方网址：https://www.nature.com/nmeth/
投稿链接：https://mts-nmeth.nature.com/cgi-bin/main.plex