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A method for preparing unbiased miRNA sequencing libraries (miR-ACS)

Award Number: R44HG007788
ORGANIZATION: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 08/11/2014
PERIOD OF PERFORMANCE END DATE: 02/28/2019

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A method for preparing unbiased miRNA sequencing libraries (miR-ACS) - ABSTRACT This proposal addresses the problem of sequence bias in next-generation sequencing (NGS) of small RNAs such as microRNAs (miRNAs) as well as fragments of larger RNAs. Because dysregulation of miRNA expression has been implicated in cancer and other diseases, accurate expression profiling of all miRNA sequences is important for understanding miRNA biology and for development of new biomarkers and therapeutic targets. NGS is currently the most comprehensive approach for discovery and expression profiling of small RNA sequences. However, NGS expression profiling data underestimate the abundance of most miRNAs in a sample, some by as much as 10,000-fold. Knowledge of the true abundances in samples, and not just the relative changes between samples, is important for reliable identification of miRNAs as biomarkers or drug-target candidates. Other advantages of unbiased detection include the ability to discover novel RNAs and detect low-abundance RNAs that cannot be detected by current NGS methods, especially in samples with a low concentration of RNA. The source of bias in currently available methods of preparation RNA sequencing libraries for NGS is inefficient and sequence-dependent ligation of the two sequencing adapters to the sample RNAs. The major factors contributing to this ligation bias are intramolecular folding of the miRNAs and intermolecular folding between miRNAs and adapters, which affect the ability of the ligase to access and ligate the miRNA ends. Thus there is a need for new, more accurate methods, and most previous small RNA profiling experiments should be re-evaluated. To address these problems, we are developing a new approach, miR- ACS (miRNA-Adapter Circularization and Sequencing), for preparing unbiased sequencing libraries that is applicable to miRNAs and other small RNAs as well as small fragments of large RNAs used in general RNA- Seq. Key features of miR-ACS include (i) ligation of miRNAs with only a single combo adapter (CAD) that combines sequences of the standard 3'- and 5'-adapters used for Illumina sequencing, producing miRNA-CAD ligation products; (ii) circularization of the miRNA-CAD products; (iii) blocking of free CAD species that are not ligated to miRNAs; and (iv) RT-PCR amplification of the circular miRNA-CAD products to produce standard sequencing amplicons containing a single RNA-specific sequence insert flanked by the 5'- and 3'-adapter sequences. In Phase I, we have demonstrated the feasibility of the miR-ACS approach (proof-of-concept) by greatly reducing the miRNA sequencing bias in comparison to the best current library prep methods. In Phase II we will thoroughly optimize miR-ACS to maximize bias reduction and to allow sequencing of a larger variety of RNAs (up to 150 nt in size) with very low RNA inputs. In addition, we will streamline the protocol to facilitate its adoption by users and for commercial viability.


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: 2019 ( Subtotal = $0 )
2019	2017	SOMAGENICS, INC.	2161 DELAWARE ST STE E	SANTA CRUZ	CA	95060	SANTA CRUZ	USA	Human Genome Research	001	3	6/26/2019	NON-COMPETING CONTINUATION	-$2,231
2019	2016	SOMAGENICS, INC.	2161 DELAWARE ST STE E	SANTA CRUZ	CA	95060	SANTA CRUZ	USA	Human Genome Research	000	2	6/26/2019	COMPETING CONTINUATION	$2,231
														Subtotal = $0

Issue Date FY: 2017 ( Subtotal = $803,773 )
2017	2017	SOMAGENICS, INC.	2161 DELAWARE ST STE E	SANTA CRUZ	CA	95060	SANTA CRUZ	USA	Human Genome Research	000	3	9/1/2017	NON-COMPETING CONTINUATION	$803,773
														Subtotal = $803,773

Issue Date FY: 2016 ( Subtotal = $990,251 )
2016	2016	SOMAGENICS, INC	2161 DELAWARE AVE	SANTA CRUZ	CA	95060	SANTA CRUZ	USA	Human Genome Research	000	2	9/28/2016	COMPETING CONTINUATION	$990,251
														Subtotal = $990,251

Grand Total All Awards = $1,794,024

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A method for preparing unbiased miRNA sequencing libraries (miR-ACS)

Award Number: R44HG007788

ORGANIZATION: NATIONAL HUMAN GENOME RESEARCH INSTITUTE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 08/11/2014

PERIOD OF PERFORMANCE END DATE: 02/28/2019

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