The genome and DNA puff sequences of the fungus fly, Sciara coprophila, and genome-wide methods for studying DNA replicationUrban, JohncreatorGerbi, SusanAdvisorLarschan, EricaReaderMark Welch, DavidReaderRaphael, BenjaminReaderJohnson, MarkReaderBrown University. Department of Molecular Biology, Cell Biology and Biochemistrysponsor201727, 383 p.born digitalThesis (Ph. D.)--Brown University, 2017thesesDNA replication initiates from any given origin at most once per cell cycle to ensure perfect duplication of the hereditary material. DNA re-replication occurs when an origin initiates more than once. This can lead to DNA damage, gene amplification, and genomic rearrangements, all hallmarks of cancer. Whereas it is difficult to study re-replication in cancer cells, the fungus fly Sciara coprophila is a tractable alternative where re-replication occurs in the DNA puffs of giant polytene chromosomes in larval salivary glands during normal development. The DNA sequence for only one of many DNA puffs was known, preventing identification of shared motifs. Progress has also been thwarted by the absence of a genome sequence, necessary for genomics approaches. In this thesis, I present the first draft of the Sciara coprophila genome sequence, assembled using Illumina short reads, long single-molecule reads from Pacific Biosciences and Oxford Nanopore, and optical maps from BioNano Genomics. Using the Oxford Nanopore MinION, I established protocols to obtain high quality, ultra-long reads that exceed 100 kb in some cases, which were important in assembling multi-megabase contigs. RNA sequencing of the transcriptomes of both sexes across the lifecycle facilitated annotation of genes hidden in the genome sequence. High throughput sequencing of the salivary gland genome throughout the DNA re-replication stages identified multiple DNA puff sequences, which were confirmed with qPCR, and in some cases mapped by Fluorescence in situ Hybridization (FISH) to corresponding DNA puffs. Mapping the origins where DNA re-replication begins is still needed to locate cis-regulatory elements. Toward this goal, I have refined the genome-wide origin mapping technique Nascent Strand sequencing (NS-seq) by inclusion of important controls and found that nucleosomes are phased around a subset of origins near G-quadruplex motifs in the human genome. Moreover, I describe my progress in developing new single-molecule, genome-wide origin mapping techniques. Overall, this thesis presents the genome and DNA puff sequences of Sciara coprophila as well as methods to map the re-replication origins, thereby establishing a foundation to unravel the mechanism of site-specific DNA re-replication.GenomicsDNA replicationNanopore sequencinggenome assemblysciara coprophilaDNA amplificationDNA puffsLambda exonucleaseIlluminaPacific BiosciencesOxford Nanopore TechnologiesBioNano GenomicsDNA sequencingRNA sequencingfungus flyultra long readsDNA modificationsorigins of replicationG-quadruplexesnucleosome phasingnew model systemsEnglishRPB2017061610.7301/Z0G15Z98In CopyrightCollection is open for research.dissertations