Spatial Targeted and Whole Transcriptome Sequencing via Microfluidic Barcoding

Abstract

Nearly every biological process is influenced, in some part, by the environment in which it occurs. Understanding this association could elucidate the mechanisms that drive spatially-dependent processes, such as those related to development or cancer. Methods that explore the relationship between the genome and transcriptome do exist, but they neglect consideration of native spatial organization. A reliable way to spatially explore the relationship between gene expression and the genome could be applicable to cancer research, specifically intratumoral heterogeneity. We seek to establish a protocol that enables the spatial co-profiling of targeted mutations and transcriptomics, providing us with adequate gDNA and cDNA libraries for further analysis. Using this method, we aim to amplify target mutations and multiple genes via multiplex in-situ PCR, creating spatial maps that trace mutant amplicons and cDNA to their location of origin within the tissue structure. Our model was validated using the MDA-MB-231 human TNBC cell line, measuring the quality of cDNA and gDNA libraries to determine success. Results varied across primer concentration and composition, fixation methods, and PCR protocols, and ultimately demonstrated successful in-situ PCR and spatial barcoding, but emphasized the need for further optimization to reduce diffusion and improve spatial accuracy. This technology could be expanded to characterize mutational profiles in tumors across cancer types, which has vast applications in the world of cancer therapeutics.