PROJECT SUMMARY/ABSTRACT: Single cell sequencing technologies have rapidly advanced over the past five years and have become essential tools in a broad array of fields, ranging from developmental biology to human genetics. One major advance was the development of high-throughput barcoding of RNA or DNA within droplets, which allowed for preparation of sequencing libraries as a pool rather than in a laborious 96 well plate format. This advance enabled sequencing analysis of thousands of cells in one batch. However, there has not been the same breakthrough in scale when sequencing both DNA and RNA from the same cell as established techniques involve sorting cells into plates or wells and ultimately separating DNA and RNA before library preparation. The separation of RNA from DNA is necessary because conventional PCR amplification requires a 95oC melting step, which degrades RNA. In this proposal, we will seek to optimize DNA amplification through recombination polymerase amplification (RPA), an isothermal form of PCR which can be performed simultaneously with reverse transcription of mRNA into cDNA. In this way, both RNA and DNA can be captured into barcoded, sequencing libraries within the same droplet. The technology could have impact in many different fields in which genotype-phenotype correlation is needed, such as characterizing genetic polymorphisms or genetic variants in cancer. In SA1, we will demonstrate proof-of-principle of same cell DNA amplicon and RNA transcriptome technology via isothermal RPA and reverse transcription. In SA2, we will determine the quantitative performance of the methodology in two use applications and test capability to genotype heterozygous alleles. If successful, this methodology could be widely adopted by laboratories in diverse fields to answer questions related to genotype-phenotype correlations.

PROJECT NARRATIVE: Understanding how our genes affect the behavior of individual cells is a fundamental question in many scientific fields ranging from human genetics to developmental biology to cancer biology. In this technology development grant, we will seek to vastly improve how to detect the identify of DNA variants while simultaneously determining their effects on RNA within the same cells. We hope this technology will facilitate more rapid discovery of the effects of genetic variants and cancer mutations to improve our understanding of the human condition and disease conditions.