SBIR-STTR Award

Sequencing is very powerful for identifying differences in genomic DNA that may regulate cell function and diseases, pre-dispose persons to certain diseases, or warn of adverse drug metabolism. It provides a basis for identifying differences in gene expression, though such applications have been limited and are problematic because each expressed gene can vary from a single copy per cell to 10's of thousands of copies. Further, its application to formalin fixed paraffin-embedded tissue (FFPE) is quite challenging. Millions of such samples, along with the corresponding treatment modalities and known clinical outcomes, are archived at clinical centers and hospitals. Millions of such samples have also been archived from in vivo studies of safety, metabolism, and animal models of disease. In addition, the utility of sequencing for routine experiments where array-based measurements are used today is limited by cost, and might also be limited by accuracy, sensitivity, and reproducibility of the sequencing data. This Phase I proposal develops and validates a new approach for addressing these challenges, ""Nuclease Probe Mediated Sequencing,"" provides a variation of candidate capture methods used to focus sequencing onto genes of interest, and addresses issues with array-based methods. The method proposed uses a lysis-only assay and either a variation of HTG's quantitative Nuclease Protection Assay (qNPA"") or ligation/exonuclease generated Padlock probes to generate DNA probes for sequencing by synthesis, combined with gene tagging and experiment tagging methods to permit pooling to provide cost reduction and higher sample throughput. , ,

Public Health Relevance:
This Phase I project will address challenges to using sequencing for measuring gene expression. The proposed method will enable researchers to use archived fixed tissue samples. It will also lower the cost per sample and increase the number of samples tested per sequencing run, while potentially increasing the accuracy, dynamic range and reproducibility of measurement, expanding the utility of sequencing to measure gene expression and providing a more cost effective and precise platform than currently provided by high density array-based measurement.

Thesaurus Terms:
Address;Animal Disease Models;Archives;Aspergillus Nuclease S1;Assay;Bioassay;Biologic Assays;Biological Assay;Body Tissues;Cell Function;Cell Process;Cell Physiology;Cells;Cellular Function;Cellular Physiology;Cellular Process;Circular Dna;Clinical;Closure By Ligation;Complementary Dna;Cytolysis;Dna;Dna Hybridization Probes;Dna Probes;Dna Sequence;Dna, Complementary;Data;Deoxyribonuclease S1;Deoxyribonucleic Acid;Development;Disease;Disorder;Endonuclease S-1;Endonuclease S1, Aspergillus;Evaluation;Exodeoxyribonuclease I;Exonuclease;Exonuclease 1;Exonuclease I;Formalin;Gel;Gene Expression;Gene Probes, Rna;Gene Products, Rna;Genes;Genomics;Hosp;Hela;Hela Cells;Hospitals;Intermediary Metabolism;Investigators;Ligation;Lysis;Metbl;Marketing;Measurement;Measures;Mediating;Messenger Rna;Metabolic Processes;Metabolism;Method Loinc Axis 6;Methodology;Methods;Micro Rna;Micrornas;Modality;Nuclease Protection Assays;Nuclease S-1;Oligo;Oligonucleotides;Outcome;Paraffin Embedding;Performance;Persons;Phase;Principal Investigator;Process;Programs (Pt);Programs [publication Type];Protocol;Protocols Documentation;Rna;Rna Probes;Rna, Messenger;Rna, Non-Polyadenylated;Reagent;Reproducibility;Research Personnel;Researchers;Ribonucleic Acid;Running;S1 Nuclease;Sbir;Sbirs (R43/44);Sampling;Small Business Innovation Research;Small Business Innovation Research Grant;Subcellular Process;Testing;Time;Tissue Sample;Tissues;Variant;Variation;Adduct;Base;Cdna;Cell Type;Comparative;Cost;Cost Effective;Density;Design;Designing;Disease/Disorder;Drug Metabolism;Experiment;Experimental Research;Experimental Study;In Vivo;Interest;Mrna;Mirna;New Approaches;Next Generation;Novel;Novel Approaches;Novel Strategies;Novel Strategy;Nuclease;Programs;Public Health Relevance;Research Study;Safety Study;Single Molecule;Tissue Fixing

Sequencing is very powerful for identifying differences in genomic DNA that may regulate cell function and diseases, pre-dispose persons to certain diseases, or warn of adverse drug metabolism. It provides a basis for identifying differences in gene expression, though such applications have been limited and are problematic because each expressed gene can vary from a single copy per cell to 10's of thousands of copies. Further, its application to formalin fixed paraffin-embedded tissue (FFPE) is quite challenging. Millions of such samples, along with the corresponding treatment modalities and known clinical outcomes, are archived at clinical centers and hospitals. Millions of such samples have also been archived from in vivo studies of safety, metabolism, and animal models of disease. In addition, the utility of sequencing for routine experiments where array-based measurements are used today is limited by cost, and might also be limited by accuracy, sensitivity, and reproducibility of the sequencing data. This Phase I proposal develops and validates a new approach for addressing these challenges, "Nuclease Probe Mediated Sequencing," provides a variation of candidate capture methods used to focus sequencing onto genes of interest, and addresses issues with array-based methods. The method proposed uses a lysis-only assay and either a variation of HTG's quantitative Nuclease Protection Assay (qNPA") or ligation/exonuclease generated Padlock probes to generate DNA probes for sequencing by synthesis, combined with gene tagging and experiment tagging methods to permit pooling to provide cost reduction and higher sample throughput.

Public Health Relevance:
This Phase I project will address challenges to using sequencing for measuring gene expression. The proposed method will enable researchers to use archived fixed tissue samples. It will also lower the cost per sample and increase the number of samples tested per sequencing run, while potentially increasing the accuracy, dynamic range and reproducibility of measurement, expanding the utility of sequencing to measure gene expression and providing a more cost effective and precise platform than currently provided by high density array-based measurement.

Public Health Relevance Statement:
Principal Investigator: Seligmann, Bruce E. Narrative This Phase I project will address challenges to using sequencing for measuring gene expression. The proposed method will enable researchers to use archived fixed tissue samples. It will also lower the cost per sample and increase the number of samples tested per sequencing run, while potentially increasing the accuracy, dynamic range and reproducibility of measurement, expanding the utility of sequencing to measure gene expression and providing a more cost effective and precise platform than currently provided by high density array-based measurement.

NIH Spending Category:
Bioengineering; Biotechnology; Genetics; Human Genome

Project Terms:
Address; adduct; Animal Disease Models; Archives; Aspergillus Nuclease S1; base; Biological Assay; Cell physiology; cell type; Cells; Circular DNA; Clinical; comparative; Complementary DNA; cost; cost effective; Cytolysis; Data; density; design; Development; Disease; DNA; DNA Probes; DNA Sequence; drug metabolism; Evaluation; Exodeoxyribonuclease I; Exonuclease; Formalin; Gel; Gene Expression; Gene Expression Profile; Genes; Genomics; Hela Cells; Hospitals; in vivo; interest; Ligation; Marketing; Measurement; Measures; Mediating; Messenger RNA; Metabolism; Methodology; Methods; MicroRNAs; Modality; next generation; novel; novel strategies; nuclease; Nuclease Protection Assays; Oligonucleotides; Outcome; Paraffin Embedding; Performance; Persons; Phase; Principal Investigator; Process; programs; Protocols documentation; public health relevance; Reagent; Reproducibility; Research Personnel; research study; RNA; RNA Probes; Running; safety study; Sampling; single molecule; Small Business Innovation Research Grant; Testing; Time; tissue fixing; Tissue Sample; Tissues; Variant