^{Genomics and Molecular Biology}

New rapid and robust transcriptome-based methods for cellular characterization of the tumor microenvironment and biomarker discovery are required to improve prognosis and treatment of cancer and other diseases. However, challenges with current approaches for the above applications include high sample requirements, poor sensitivity, low dynamic range, and limited throughput. To address these limitations, we have developed the DriverMap™ targeted RNA expression profi ling assay using a genome-wide set of 19,000 validated primer pairs that leverages the sensitivity of multiplex RT-PCR with the throughput and digital readout depth of next-generation sequencing (NGS). Starting from just 10pg (single-cell) to 100ng (10,000 cells)

of total RNA is suffi cient to quantify over 5 orders of magnitude variation in gene expression levels with performance similar to conventional qRT-PCR. Further, the use of gene-specifi c primers enables direct analysis of total RNA isolate and obviates the need for globin and rRNA depletion from whole blood samples. And, using a subset of primers empirically selected from the DriverMap™ assay, we have developed a novel single-cell targeted RNA expression (scTRex) profi ling assay compatible with conventional oligo dT-molecular indexing, single- cell barcoding strategies. In this study, we present the performance of the assay to analyze the level of immune cell infi ltration in tumor samples, and identify active pathways in tumor, xenograft samples and cell lines treated with small molecules. Preliminary studies demonstrate the assay’s unparalleled specifi city and sensitivity resulting in better detection of low abundance mRNA transcripts as well as an improved cost-eff ectiveness for highthroughput clinical applications.