Single-Cell RNA Sequencing and Genomic Methods Applications in Molecular Genetics and Systems Biology

Abstract

Sequencing technologies have been inherently tied to the field of genetics ever since it became clear that nucleic acids, not proteins, hold the key to heritability. We have come a long way from the first tedious effort to determine the complete sequence of alanine tRNA molecule in Saccharomyces cerevisiae over several years published in 1965, to measuring gene expression of tens of thousands of genes in millions of individual cells from any tissue and organism in a few hours today, providing invaluable insights in both biomedical research and clinical care.

In this work, we take advantage of and improve upon the latest cutting-edge approaches in molecular genetics, using both short and long-read sequencing of DNA and RNA to explore biological questions about tissues ranging from individual mouse neurons to populations of human viruses. Specifically, firstly, we describe an approach to utilize single-cell RNA sequencing in a mouse model of autism to explore the gene expression changes after conditionally knocking out the Pten gene in neurons in vivo. Secondly, we contribute to the single-cell RNA sequencing computational methodology by discovering, characterizing, and subsequently alleviating the systematic bias in gene expression measurement secondary to the artifact of internal oligo(dT) priming. Lastly, we combine both short and long-read sequencing to show that the single polymerase encoded by human cytomegalovirus is not sufficient for its genome stability and, instead, the virus is largely dependent on the activity of translesion polymerases produced by human cells.