We propose to develop and commercialize high sensitivity and accuracy RNA sequencing kits suitable for gene expression measurements in limiting input samples, such as single cells. During Phase I, we proposed the concept of using molecular barcodes in the form of DNA sequence tags to label individual molecules (or copies) of RNA for gene expression analysis in single cells. The Phase I project proved to be highly productive, and feasibility was demonstrated. Counting the barcodes provides an absolute, digital quantitative measure of the number of transcripts expressed. In addition, the barcodes are shown to be useful in correcting for PCR bias, a common challenge in small samples requiring DNA amplification protocols. We also determined a low RNA representation efficiency of 0.1-3.8% overall yield in current single cell RNA sequencing sample preparation methods, and demonstrate that with our cDNA amplification and barcoding approach, more accurate measurements are obtained at significantly higher yields of ~22.5%. These remarkable results indicate that our method can significantly improve on current techniques which suffer from large losses and have inaccuracies introduced by amplification distortions. Because measurement of gene expression levels is important in life science research and also in many clinical settings, our proposed product if successful, would contribute greatly to advancements in these areas. For Phase II, we propose to extend the validated concept into a product development program to produce high efficiency, user-friendly single-cell RNA sequencing kits. Specifically, we will perform assay development to produce validated sets of primers for inclusion in these kits. We will also optimize and validate reaction mix formulations to enable the high efficiency sampling of low abundance RNA transcripts that are missed by other methods. Analysis tools for the upstream selection of custom primer sets or gene panels, and for the downstream analysis of sequencing data will also be developed.

Public Health Relevance

A molecular barcoding sequencing kit for highly efficient and accurate single cell gene expression analysis Narrative To better understand the human genome and the function of individual cells and their roles in health and disease, we will develop commercial sample preparation kits for massively parallel sequencing that enable highly sensitive and accurate gene expression analysis in single cells.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Small Business Innovation Research Grants (SBIR) - Phase II (R44)
Project #
5R44HG007130-03
Application #
8904694
Study Section
Special Emphasis Panel (ZRG1-IMST-J (15))
Program Officer
Smith, Michael
Project Start
2013-07-01
Project End
2016-07-31
Budget Start
2015-08-01
Budget End
2016-07-31
Support Year
3
Fiscal Year
2015
Total Cost
$626,600
Indirect Cost
Name
Cellular Research, Inc.
Department
Type
DUNS #
078299220
City
Palo Alto
State
CA
Country
United States
Zip Code
94304