A Unified Nanopore Platform for Direct Sequencing of Individual Full Length RNA Strands Bearing Modified Nucleotides

摘要 我们建议实现一个统一的平台来检测连续FULL上的RNA核苷酸修改 使用纳米孔直接RNA测序来确定RNA链的长度。原则上，这项技术可以用于所有人 原核和真核细胞中的RNA分子类别。对于RNA来说，这是一个合乎逻辑的技术进步 测序，因为当前的标准，使用Solexa技术的合成测序需要 将天然的细胞RNA转化为短的(~300个核苷酸)的cDNA扩增片段。在此过程中，核苷酸修饰 被擦除，并且完整的RNA链的连续性丢失，从而排除了对RNA的准确定量 异构体。纳米孔链测序克服了这些限制，因为~2纳米长的积分 当天然RNA链穿过毛孔时，传感器会接触并识别每个碱基 通过施加的电压。因此，实现了对给定RNA链的端到端序列分析。 在授权期内，我们将追求三个具体目标：1)建立ONT直接基准性能 RNA测序，并实施有针对性的改进；2)实施发现和记录方法 对天然RNA链的核苷酸修饰；以及3)优化用于mRNA分析的纳米孔技术 亚型多样性。 加州大学圣克鲁斯分校拥有独特的设备来承担这一项目： I)我们率先进行了纳米孔RNA链分析(Akeson/Deamer)，最近证明了牛津大学 纳米孔小离子纳米孔测序仪可以在单个核苷酸变异和碱基修饰 16S rRNA链(Akeson)。 Ii)我们的RNA中心有14名教职员工。这项申请的联合调查人员具有以下专业知识 RNA剪接的机制(Sanford)，正常和异常RNA异构体的功能后果 合成(布鲁克斯)，和RNA的结构生物学(阿瑞斯)。合作的RNA中心教职员工将就 核非编码RNA实验(Carpenter，Kim)和H9 ES细胞上的实验(Salama)。 三)我们的基因组研究所在生物信息学方面是国际公认的，包括最近在 回归神经网络在人类基因组单倍型和全基因组纳米孔中的应用 碱基修饰的检测(专利)。

ABSTRACT We propose to implement a unified platform for detecting RNA nucleotide modifications on contiguous full length RNA strands using nanopore direct RNA sequencing. In principle, this technology could be used for all classes of RNA molecules in prokaryotic and eukaryotic cells. It is a logical technical advance for RNA sequencing because the current standard, sequencing-by-synthesis using Solexa technology requires conversion of native cellular RNA into short (~300 nt) cDNA amplicons. In so doing, nucleotide modifications are erased and the continuity of intact RNA strands is lost, thus precluding accurate quantification of RNA isoforms. Nanopore strand sequencing overcomes these limitations because the ~2 nanometer-long integral sensor touches and identifies each base along intact native RNA strands as they are driven through the pore by an applied voltage. Thus, end-to-end sequence analysis of a given RNA strand is achieved. During the grant period, we will pursue three specific aims: 1) Establish baseline performance of ONT direct RNA sequencing, and implement targeted improvements; 2) Implement methods to discover and document nucleotide modifications on native RNA strands; and 3) Optimize nanopore technology for analysis of mRNA isoform diversity. UC Santa Cruz is uniquely equipped to undertake this project: i) We pioneered nanopore RNA strand analysis (Akeson/Deamer) and recently demonstrated that the Oxford Nanopore MinION nanopore sequencer can resolve single nucleotide variants and base modifications in single 16S rRNA strands (Akeson). ii) Our RNA Center includes fourteen faculty members. Co-investigators on this application have expertise in mechanisms of RNA splicing (Sanford), the functional consequences of normal and aberrant RNA isoform synthesis (Brooks), and the structural biology of RNA (Ares). Collaborating RNA Center faculty will advise on nuclear non-coding RNA experiments (Carpenter, Kim) and on H9 ES cells (Salama). iii) Our Genomics Institute is internationally recognized for bioinformatics, including recent advances in application of Recurrent Neural Networks to human genome haplotyping, and genome-wide nanopore detection of base modifications (Paten).