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Massively Parallel Sequencing-Based Analyses of Genome and Protein Function

The advent of high-throughput DNA and RNA sequencing has made possible the assay of millions of nucleic acid molecules in parallel. This allows functional genomic elements to be identified from background in single-tube experiments. This dissertation discusses the development of two such functional screens as well as work implementing a third that was previously developed in my thesis laboratory.

Restriction-Associated DNA sequencing (RAD-Seq) is a complexity reduction sequencing method that allows the same subset of genomic sequence to be read across multiple samples. Differences in sample collection and data analysis allow manifold applications of RAD-Seq. Here we use RAD-Seq to identify mutant genes responsible for altered phenotypes in Caenorhabditis elegans and to identify hyper-invasive alleles in trout population admixtures.

Apart from acquiring genomic sequence data, massively-parallel sequencing can be used for counting applications that quantify activity across a large number of test molecules. This dissertation describes the development of a technique for simultaneously quantifying the activity of a restriction enzyme across all possible DNA substrates by linking digest of a sequenced genome to Illumina-sequencing in an unbiased fashion. Finally, a powerful approach to analyze transcriptional activation is described. This method quantifies output from millions of potential DNA transcriptional enhancers via RNA amplicon sequencing of covalently-linked randomer tags and is used in conjunction with RNA-Seq to provide a mechanistic view of hypoxic gene regulation in Drosophila.

This dissertation includes previously published, co-authored material

Identiferoai:union.ndltd.org:uoregon.edu/oai:scholarsbank.uoregon.edu:1794/19234
Date18 August 2015
CreatorsKamps-Hughes, Nicholas
ContributorsBowerman, Bruce
PublisherUniversity of Oregon
Source SetsUniversity of Oregon
Languageen_US
Detected LanguageEnglish
TypeElectronic Thesis or Dissertation
RightsAll Rights Reserved.

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