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DNA sequencing is the process of determining the identities of the nucleotides that make up a molecule of DNA. The rapid pace of advancements in sequencing technologies in recent years have made it possible to simultaneously determine the sequences of hundreds of millions of short DNA fragments. The ability to perform sequencing with such high throughput has revolutionized the study of biological systems, but the types of questions that can be answered through sequencing-based experiments can be limited by the presence of different kinds of noise and biases in these experiments. One class of applications of high-throughput sequencing involves identifying genetic variation, such as finding rare mutations in the genomes of cancerous cells. In these applications, the sensitivity with which rare genetic variants can be detected is limited by the relatively high rate with which current DNA sequencing technologies incorrectly identify nucleotides. In the first half of this thesis, we present a method for dramatically reducing the rate at which these incorrect identifications occur. Our method, called circle sequencing, creates redundant copies of the sequence of each input molecule of DNA. This is accomplished by circularizing each DNA fragment and performing rolling circle amplification on these circles with a strand-displacing polymerase. The resulting products consist of several physically linked copies of the original sequence in each fragment. When these products are sequenced, this informational redundancy protects against random errors introduced during sequencing, allowing for highly accurate recovery of the original sequence of each input molecule. By eliminating the vast majority of incorrectly identified nucleotides from the resulting data, our method enables the sensitive detection of rare variants and opens up exciting new questions involving such variants to direct measurement by sequencing. An entirely different application of high-throughput sequencing is to selectively capture and sequence stretches of DNA or RNA that are participating in a process of interest within a cell. The accuracy of quantitative inferences made by this type of experiment can be severely impacted, however, by biases introduced during the experimental manipulations used to isolate biologically relevant fragments of DNA from cells. Ribosome profiling is an experimental technique that consists of sequencing short stretches of messenger RNAs that are protected from nuclease digestion by the presence of a bound ribosome. The resulting data represents millions of snapshots of the locations of actively translating ribosomes. In theory, these snapshots can be used to determine how long ribosomes take to translate each type of codon by quantifying how often ribosomes are observed positioned over that codon. In practice, different studies in yeast attempting to do this have reached contradictory and counterintuitive conclusions. In the second half of this thesis, we perform a large-scale comparative analysis of data from many different ribosome profiling experiments in order to resolve these contradictions. We identify a previously unappreciated source of systematic bias in a subset of these experiments. This bias prevents these experiments from accurately measuring ribosomes in proportion to how long they spend at each position in vivo. Understanding this bias provides insight into the true signatures of translation dynamics in yeast and offers important guidance for the future design and interpretation of sequencing-based approaches to measuring these dynamics.

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BACKGROUND: A method to increase accuracy and ease-of-use, while decreasing time and cost in deoxyribonucleic acid (DNA) sequence identification, is sought after. Helicase, which unwinds DNA, and avidin, which strongly attracts biotin for potential attraction of biotinylated DNA segments, were investigated for use in a novel DNA sequencing method. AIM: This study aimed to (1) purify bacteriophage T7 gene product 4 helicase and helicase-avidin fusion protein in a bacterial host and (2) characterize their functionality. METHODS: Helicase and helicase-avidin were cloned for purification from bacteria. Helicase-avidin was solubilised via urea denaturation/renaturation. DNA and biotin binding were assessed using Electrophoretic Mobility Shift Assays and biotinylated resins, respectively. RESULTS: (1) Helicase and helicase-avidin proteins were successfully purified. (2) Helicase protein was able to bind DNA and avidin protein strongly bound biotin. CONCLUSION: Helicase and helicase-avidin can be purified in a functional form from a bacterial host, thus supporting further investigation for DNA sequencing purposes.

During entry into stationary phase, Escherichia coli expresses many genes which impart cellular resistance to numerous environmental stresses such as oxidative or acid stress. Many ofthese genes are regulated by the alternative sigma factor, RpoS To identify additional genes regulated by RpoS, a phenotype independent genetic screen was previously employed (L. Wei Masters thesis). The identities ofthe ten most highly RpoSdependent fusions were determined by DNA sequencing and subsequent sequence analysis using the BLAST algorithm Three fusions map to genes previously known to be RpoSdependent while the remaining seven represent new members ofthe regulon The expression of many ofthe RpoS-dependent fusions remained growth phase dependent even in the rpoS background This suggests that other growth phase regulatory factors in addition to RpoS may coordinate stationary phase gene expression Upon sequencing the remaining rsd fusions, three mutants mapped to narY which is part ofthe narZYWV operon encoding the secondary nitrate reductase Z (NRZ) This operon was selected for further investigation since NRZ has been previously reported to be constitutively expressed Expression studies using promoter lacZ fusions and nitrate reductase assays reveal that NRZ is induced ten-fold at the onset ofstationary phase and twenty-fold in the presence of nitrate Like other rsd fusions, growth phase dependent expression was observed in an rpoS background indicating that other regulatory factors may be involvedn the regulation of NRZ Northern analyses using probes specific to NRZ confirmed that transcription of NRZ is indeed dependent on RpoS. These results suggest that RpoS mediated regulation of NRZ may be an important physiological adaptation to reduced oxygen levels during transition to stationary phase / Thesis / Master of Science (MSc)

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