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

Award Number: R01HG010053
ORGANIZATION: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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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).


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2023 ( Subtotal = $0 )
2023	2021	UNIVERSITY OF CALIFORNIA SANTA CRUZ	1156 HIGH ST	SANTA CRUZ	CA	95064	SANTA CRUZ	USA	Human Genome Research	000	4	4/20/2023	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2021 ( Subtotal = $954,816 )
2021	2021	UNIVERSITY OF CALIFORNIA, SANTA CRUZ	1156 HIGH ST	SANTA CRUZ	CA	95064	SANTA CRUZ	USA	Human Genome Research	000	4	6/17/2021	NON-COMPETING CONTINUATION	$954,816
														Subtotal = $954,816

Issue Date FY: 2020 ( Subtotal = $954,816 )
2020	2020	UNIVERSITY OF CALIFORNIA, SANTA CRUZ	1156 HIGH ST	SANTA CRUZ	CA	95064	SANTA CRUZ	USA	Human Genome Research	000	3	5/12/2020	NON-COMPETING CONTINUATION	$954,816
														Subtotal = $954,816

Issue Date FY: 2019 ( Subtotal = $954,816 )
2019	2019	UNIVERSITY OF CALIFORNIA, SANTA CRUZ	1156 HIGH ST	SANTA CRUZ	CA	95064	SANTA CRUZ	USA	Human Genome Research	000	2	6/17/2019	NON-COMPETING CONTINUATION	$954,816
														Subtotal = $954,816

Issue Date FY: 2018 ( Subtotal = $954,816 )
2018	2018	UNIVERSITY OF CALIFORNIA, SANTA CRUZ	1156 HIGH ST	SANTA CRUZ	CA	95064	SANTA CRUZ	USA	Human Genome Research	000	1	5/30/2018	NEW	$954,816
														Subtotal = $954,816

Grand Total All Awards = $3,819,264

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

Award Number: R01HG010053

ORGANIZATION: NATIONAL HUMAN GENOME RESEARCH INSTITUTE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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