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 Millions of such samples have also been archived from in vivo studies of safety, metabolism, and animal models of disease.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.
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.
