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

用于对带有修饰核苷酸的单个全长 RNA 链进行直接测序的统一纳米孔平台

基本信息

批准号：
10163247
负责人：
MARK A AKESON
金额：
$ 95.48万
依托单位：
UNIVERSITY OF CALIFORNIA SANTA CRUZ
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-06-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10163247
关键词：
7-methylguanosine Algorithms Bacterial Genome Benchmarking Bioinformatics Biology Cells Code Complementary DNA Complex Cytosine DNA Data Detection Documentation Eukaryotic Cell Faculty Genetic Genomics Grant Haplotypes Heterogeneous-Nuclear Ribonucleoproteins Human Human Genome In Vitro Individual Inosine Institutes International Length Malignant Neoplasms Maps Messenger RNA Methodology Methods Modification Nature Neurodegenerative Disorders Nuclear Nucleotides Performance Prokaryotic Cells Protein Isoforms Pseudouridine RNA RNA Splicing Reader Recovery Research Personnel Resolution Ribosomal RNA Role Sequence Analysis Single Nucleotide Polymorphism Technology Touch sensation Training Transcript Untranslated RNA Variant base embryonic stem cell experimental study genome-wide innovation insight knock-down member nanometer nanopore neural network algorithm recurrent neural network research and development sensor sequencing platform simulation statistics structural biology transcriptome sequencing voltage

项目摘要

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).

抽象的我们建议实施一个统一的平台来检测连续完整的RNA核苷酸修饰使用纳米孔直接 RNA 测序确定 RNA 链的长度。原则上，该技术可用于所有原核和真核细胞中的 RNA 分子类别。这是 RNA 的逻辑技术进步测序，因为当前标准，使用 Solexa 技术的合成测序需要将天然细胞 RNA 转化为短 (~300 nt) cDNA 扩增子。在此过程中，核苷酸修饰被擦除，完整 RNA 链的连续性丢失，从而妨碍 RNA 的准确定量同工型。纳米孔链测序克服了这些限制，因为约 2 纳米长的积分当完整的天然 RNA 链穿过孔时，传感器会触摸并识别它们上的每个碱基通过施加的电压。因此，实现了给定RNA链的端到端序列分析。在资助期间，我们将追求三个具体目标： 1) 建立 ONT direct 的基线绩效 RNA测序，并实施针对性改进； 2）实施发现和记录的方法天然 RNA 链上的核苷酸修饰； 3) 优化纳米孔技术用于 mRNA 分析同工型多样性。加州大学圣克鲁斯分校拥有独特的能力来开展该项目： i) 我们开创了纳米孔 RNA 链分析 (Akeson/Deamer)，并且最近证明了 Oxford Nanopore MinION 纳米孔测序仪可以解析单核苷酸变异和碱基修饰 16S rRNA 链（Akeson）。 ii) 我们的 RNA 中心有 14 名教职人员。该应用程序的共同研究者拥有以下方面的专业知识 RNA 剪接机制 (Sanford)，正常和异常 RNA 异构体的功能后果合成（Brooks）和RNA结构生物学（Ares）。 RNA 中心合作教师将提供建议核非编码 RNA 实验 (Carpenter, Kim) 和 H9 ES 细胞 (Salama)。 iii) 我们的基因组研究所在生物信息学方面获得国际认可，包括生物信息学方面的最新进展循环神经网络在人类基因组单倍型分析和全基因组纳米孔中的应用检测碱基修饰（Paten）。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Inflammation drives alternative first exon usage to regulate immune genes including a novel iron-regulated isoform of Aim2.

DOI：
10.7554/elife.69431
发表时间：
2021-05-28
期刊：
eLife
影响因子：
7.7
作者：
Robinson EK;Jagannatha P;Covarrubias S;Cattle M;Smaliy V;Safavi R;Shapleigh B;Abu-Shumays R;Jain M;Cloonan SM;Akeson M;Brooks AN;Carpenter S
通讯作者：
Carpenter S

Direct Nanopore Sequencing of Individual Full Length tRNA Strands.

DOI：
10.1021/acsnano.1c06488
发表时间：
2021-10-26
期刊：
ACS NANO
影响因子：
17.1
作者：
Thomas, Niki K.;Poodari, Vinay C.;Jain, Miten;Olsen, Hugh E.;Akeson, Mark;Abu-Shumays, Robin L.
通讯作者：
Abu-Shumays, Robin L.

Nanopore ReCappable sequencing maps SARS-CoV-2 5' capping sites and provides new insights into the structure of sgRNAs.

DOI：
10.1093/nar/gkac144
发表时间：
2022-04-08
期刊：
Nucleic acids research
影响因子：
14.9
作者：
Ugolini C;Mulroney L;Leger A;Castelli M;Criscuolo E;Williamson MK;Davidson AD;Almuqrin A;Giambruno R;Jain M;Frigè G;Olsen H;Tzertzinis G;Schildkraut I;Wulf MG;Corrêa IR;Ettwiller L;Clementi N;Clementi M;Mancini N;Birney E;Akeson M;Nicassio F;Matthews DA;Leonardi T
通讯作者：
Leonardi T

Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing.

DOI：
10.1261/rna.078703.121
发表时间：
2022-03
期刊：
RNA (New York, N.Y.)
影响因子：
0
作者：
Mulroney L;Wulf MG;Schildkraut I;Tzertzinis G;Buswell J;Jain M;Olsen H;Diekhans M;Corrêa IR Jr;Akeson M;Ettwiller L
通讯作者：
Ettwiller L