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Highly accurate small-RNA sequencing of single cells (RealSeq-SC)

Award Number: R44HG009863
ORGANIZATION: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 12/05/2019
PERIOD OF PERFORMANCE END DATE: 03/31/2023

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Highly accurate small-RNA sequencing of single cells (RealSeq-SC) - Abstract The goal of this grant application is to develop the first commercially available library preparation kit for profiling small RNAs from single cells using NGS methods. Single-cell analyses of mRNA have allowed the identification of crucial differences between cells that were otherwise considered identical. These findings have shown that there is intrinsic “noise” in the regulation of gene expression within a population of cells that plays an important role in determining cell fates. Unfortunately, there is currently a lack of information about the cell-to-cell variability of levels of microRNAs that as gene expression regulators may also play a critical role. Indeed, there is no commercially available library preparation kit for miRNAs and other small RNAs that can profile single cells. We propose to quantify miRNAs from single cells using an advanced, proprietary “low input single adapter and circularization” technology that allows sensitive and unbiased detection. The core single adapter and circularization technology, for higher input quantities, demonstrated unbiased detection of over 70% of all miRNAs in a benchmark Universal miRNA pool, compared to ~35% from the best competitor kit. We have further developed this technology for single-cell analysis by creating a novel “low input version” that retains the detection accuracy even at single- cell levels. Data from our Phase I studies show that this “low input adapter” minimizes dropout events (a critical and common problem in single cell analysis) by increasing the efficiency of miRNA detection. Another major obstacle for single-cell miRNA sequencing is formation of adapter-dimers lacking miRNA inserts during library preparation that critically reduces the amount of useful miRNA sequencing reads. We employ three separate strategies to dramatically reduce the presence of adapter-dimers in the library. Also, our protocol performs all steps from cell lysis to final purification of amplified libraries in a single tube to reduce loss of miRNA from single-cells and to reduce the possibility of contamination of single-cell samples by environmental RNA. In Phase I we demonstrated proof-of-principle by detecting small RNAs from single-cells for three different cell lines. In Phase II, we will further develop and optimize our technology to significantly increase sensitivity and detection accuracy of miRNAs and other small RNAs from single cells for commercial viability. We will also develop a kit for single-cell small RNA-seq library preparation (RealSeq-SC).


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: 2024 ( Subtotal = $0 )
2024	2020	REALSEQ BIOSCIENCES, INC	2125 DELAWARE AVE STE B	SANTA CRUZ	CA	95060	SANTA CRUZ	USA	Human Genome Research	000	4	11/16/2023	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2022 ( Subtotal = $0 )
2022	2020	REALSEQ BIOSCIENCES, INC.	2125 DELAWARE AVE STE B	SANTA CRUZ	CA	95060	SANTA CRUZ	USA	Human Genome Research	000	4	8/23/2022	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2020 ( Subtotal = $702,432 )
2020	2020	REALSEQ BIOSCIENCES, INC.	2161 DELAWARE AVE STE E	SANTA CRUZ	CA	95060	SANTA CRUZ	USA	Human Genome Research	005	4	6/4/2020	NON-COMPETING CONTINUATION	$765,766
2020	2019	REALSEQ BIOSCIENCES, INC.	2161 DELAWARE AVE STE E	SANTA CRUZ	CA	95060	SANTA CRUZ	USA	Human Genome Research	003	3	2/29/2020	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$1
2020	2019	REALSEQ BIOSCIENCES, INC.	2161 DELAWARE AVE STE E	SANTA CRUZ	CA	95060	SANTA CRUZ	USA	Human Genome Research	004	3	5/15/2020	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	-$63,334
2020	2019	REALSEQ BIOSCIENCES, INC.	2161 DELAWARE AVE STE E	SANTA CRUZ	CA	95060	SANTA CRUZ	USA	Human Genome Research	001	3	12/5/2019	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$948,988
2020	2019	SOMAGENICS, INC.	2161 DELAWARE ST STE E	SANTA CRUZ	CA	95060	SANTA CRUZ	USA	Human Genome Research	000	2	12/4/2019	COMPETING CONTINUATION	-$948,988
2020	2019	SOMAGENICS, INC.	2161 DELAWARE ST STE E	SANTA CRUZ	CA	95060	SANTA CRUZ	USA	Human Genome Research	002	2	2/29/2020	COMPETING CONTINUATION	-$1
														Subtotal = $702,432

Issue Date FY: 2019 ( Subtotal = $948,989 )
2019	2019	SOMAGENICS, INC.	2161 DELAWARE ST STE E	SANTA CRUZ	CA	95060	SANTA CRUZ	USA	Human Genome Research	000	2	9/20/2019	COMPETING CONTINUATION	$948,989
														Subtotal = $948,989

Grand Total All Awards = $1,651,421

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Highly accurate small-RNA sequencing of single cells (RealSeq-SC)

Award Number: R44HG009863

ORGANIZATION: NATIONAL HUMAN GENOME RESEARCH INSTITUTE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 12/05/2019

PERIOD OF PERFORMANCE END DATE: 03/31/2023

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