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Intracellular Amplification for Applications in Single-cell DNA Sequencing

Description
Single-cell DNA sequencing (scDNA-seq) can identify genetic differencesbetween individual cells and has broad applications in studying biology. For example, because scDNA-seq preserves haplotypes, it enables the addition of information about the fitness of different combinations of mutations into studies that quantify the fitness of individual mutations. However, it requires separating…

Single-cell DNA sequencing (scDNA-seq) can identify genetic differencesbetween individual cells and has broad applications in studying biology. For example, because scDNA-seq preserves haplotypes, it enables the addition of information about the fitness of different combinations of mutations into studies that quantify the fitness of individual mutations. However, it requires separating cells manually or using machinery, which is time-consuming and costly as every cell requires a separate reaction. Thus, most studies are limited to a few hundred cells, and scaling up is expensive and challenging. This problem also makes it difficult to multiplex samples or to study multiple sample types in the same experiment. To solve these problems, I introduce a novel method for sequencing DNA in heterogeneous cell populations by using the cell itself as a container for sequencing reactions, eliminating the need to isolate individual cells. The method involves diffusing DNA polymerase and barcoded primers into intact cells and amplifying its DNA Intracellularly. To ensure that DNA from each cell can be uniquely identified, I use combinatorial barcoding, which assigns a specific barcode to each cell using a unique combination of non-unique nucleotide block sequences. This allows for the pooling of cells, making the method multiplexable and enabling the analysis of dozens of samples containing thousands of cells. The method is flexible and allows for targeted sequencing of a region of interest and whole genome sequencing. I optimize the method for various organisms and applications so it can be made accessible to a wide range of research groups.
ContributorsCrossland, Parker Ella (Author) / Geiler-Samerotte, Kerry (Thesis advisor) / Wideman, Jeremy (Committee member) / Brettner, Leandra (Committee member) / Arizona State University (Publisher)
Created2024