Enhancing Single-Cell RNA Sequencing to Elucidate Host Cellular Response to Ebola Virus Infection

Abstract

Recent and current outbreaks of Ebola Virus Disease in West Africa and the Democratic Republic of Congo highlight the need to understand the disease and its pathophysiology. Single-Cell RNA sequencing (scRNA-seq) is a new development that can deeply characterize how the host’s different cell-types modulate their gene expression in response to infection. Recently, our lab used scRNA-seq to study how Ebola virus disease attacks host cell machinery, the first such study for a Risk Group 4 (RG-4) pathogen. ScRNA-seq was tested on blood samples from nonhuman primates infected with Ebola virus, but an adapter artifact from the scRNA library preparation over-amplified in many libraries, and reduced the quantity and quality of meaningful reads. In this study, I use CRISPR-Cas9 to target and degrade this artifact, leaving cDNAs from host genes intact. The CRISPR-Cas9 assays reduced the presence of adapter multimers 10-fold on average, with reductions reaching 37-fold, allowing us to sequence over 15 libraries that failed previously and generate thousands of additional transcriptomes. I then developed a “cell specific PCR,” a method aimed at deeply sequencing the transcriptome of a specific single cell. This method selectively amplifies transcriptomes of interest by employing PCR primers corresponding to a cell’s DNA barcode assigned during scRNA-seq preparation. Finally, I began developing models to study other viral hemorrhagic fevers in a lower containment setting. In all, this study plays a direct role in improving scRNA-seq data to study RG-4 pathogens. By recovering scRNA-libraries and amplifying transcriptomes of interest, we help uncover mechanisms used by different cell-types to respond to viral infection, paving the way for more effective vaccines, diagnostics, and antiviral medication to prevent and treat tragic outbreaks around the world.