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A method for preparing unbiased miRNA sequencing libraries miR ACS

Award Information
Agency: Department of Health and Human Services
Branch: National Institutes of Health
Contract: 2R44HG007788-02A1
Agency Tracking Number: R44HG007788
Amount: $1,794,024.00
Phase: Phase II
Program: SBIR
Solicitation Topic Code: 172
Solicitation Number: PA15-269
Timeline
Solicitation Year: 2015
Award Year: 2016
Award Start Date (Proposal Award Date): 2016-09-28
Award End Date (Contract End Date): 2019-02-28
Small Business Information
2161 DELAWARE ST STE E
Santa Cruz, CA 95060-5790
United States
DUNS: 013494781
HUBZone Owned: No
Woman Owned: No
Socially and Economically Disadvantaged: No
Principal Investigator
 SERGEI KAZAKOV
 (831) 426-7700
 skazakov@somagenics.com
Business Contact
 SERGEI KAZAKOV
Phone: (831) 426-7700
Email: skazakov@somagenics.com
Research Institution
N/A
Abstract

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 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 andapos and andapos 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 andapos and andapos 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 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 HEALTH RELATEDNESS NARRATIVE
MicroRNAs miRNAs are promising candidates as biomarkers of cancer and other diseases that could allow
early diagnosis sub typing choice of targeted treatments and monitoring of the progress of therapy Although
next generation sequencing NGS is the method of choice for discovering and expression profiling of miRNAs
it underestimates the abundance of most miRNAs some by as much as fold because of sequence bias
The goal of this grant application is to eliminate this bias to allow discovery of new RNA species and
determination of the true abundance of RNAs in biological samples

* Information listed above is at the time of submission. *

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