Methods Development for Targeted Nanopore Sequencing

This dissertation focuses on methods development for "third-generation" (long-read) sequencing technologies. With a focus on nanopore sequencing, this work discusses methods development and applications for targeted sequencing of select genomic loci. The methods described here make extensive use of the CRISPR/Cas9 system for target enrichment, adapting these tools to ligate sequencing adaptors at desired loci. We apply this strategy to look at numerous features salient to human neoplasia: DNA methylation, structural variation, point mutations, and chromatin accessibility. The methods are then applied to cell lines and primary patient tissue; and these genomic features are evaluated and compared

BET Bromodomain Inhibition as a Therapeutic Strategy to Target c-Myc

SummaryMYC contributes to the pathogenesis of a majority of human cancers, yet strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have targeted MYC transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative coactivator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain proteins as regulatory factors for c-Myc. BET inhibition by JQ1 downregulates MYC transcription, followed by genome-wide downregulation of Myc-dependent target genes. In experimental models of multiple myeloma, a Myc-dependent hematologic malignancy, JQ1 produces a potent antiproliferative effect associated with cell-cycle arrest and cellular senescence. Efficacy of JQ1 in three murine models of multiple myeloma establishes the therapeutic rationale for BET bromodomain inhibition in this disease and other malignancies characterized by pathologic activation of c-Myc.PaperFlic

Nanopore native RNA sequencing of a human poly(A) transcriptome

High-throughput complementary DNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and modifications are not retained. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies. Our study generated 9.9 million aligned sequence reads for the human cell line GM12878, using thirty MinION flow cells at six institutions. These native RNA reads had a median length of 771 bases, and a maximum aligned length of over 21,000 bases. Mitochondrial poly(A) reads provided an internal measure of read-length quality. We combined these long nanopore reads with higher accuracy short-reads and annotated GM12878 promoter regions to identify 33,984 plausible RNA isoforms. We describe strategies for assessing 3′ poly(A) tail length, base modifications and transcript haplotypes

Long read mitochondrial genome sequencing using Cas9-guided adaptor ligation

The mitochondrial genome (mtDNA) is an important source of disease-causing genetic variability, but existing sequencing methods limit understanding, precluding phased measurement of mutations and clear detection of large sporadic deletions. We adapted a method for amplification-free sequence enrichment using Cas9 cleavage to obtain full length nanopore reads of mtDNA. We then utilized the long reads to phase mutations in a patient with an mtDNA-linked syndrome and demonstrated that this method can map age-induced mtDNA deletions. We believe this method will offer deeper insight into our understanding of mtDNA variation

Reprogramming of Polycomb-Mediated Gene Silencing in Embryonic Stem Cells by the miR-290 Family and the Methyltransferase Ash1l

Members of the miR-290 family are the most abundantly expressed microRNAs (miRNAs) in mouse embryonic stem cells (ESCs). They regulate aspects of differentiation, pluripotency, and proliferation of ESCs, but the molecular program that they control has not been fully delineated. In the absence of Dicer, ESCs fail to express mature miR-290 miRNAs and have selective aberrant overexpression of Hoxa, Hoxb, Hoxc, and Hoxd genes essential for body plan patterning during embryogenesis, but they do not undergo a full differentiation program. Introduction of mature miR-291 into DCR−/− ESCs restores Hox gene silencing. This was attributed to the unexpected regulation of Polycomb-mediated gene targeting by miR-291. We identified the methyltransferase Ash1l as a pivotal target of miR-291 mediating this effect. Collectively, our data shed light on the role of Dicer in ESC homeostasis by revealing a facet of molecular regulation by the miR-290 family

INITIAL OPERATION OF THE LEDA BEAM-INDUCED FLUORESCENCE DIAGNOSTIC

A diagnostic based on beam-induced fluorescence has been developed and used to examine the expanded beam in the High-Energy Beam Transport (HEBT) section of the Low Energy Demonstration Accelerator (LEDA). The system consists of a camera, a gas injector, a spectrometer, and a control system. Gas is injected to provide a medium for the beam to excite, the camera captures the resulting image of the fluorescing gas, and the spectrometer measures the spectrum of the emitted light. EPICS was used to control the camera and acquire and store images. Data analysis is presently being performed offline. A Kodak DCS420m professional CCD camera is the primary component of the optical system. InterScience, Inc. modified the camera with the addition of a gain of 4000 image intensifier, thereby producing an intensified camera with a sensitivity of {approximately}0.5 milli-lux. Light is gathered with a 1 inch format, 16-160 mm, Computar zoom lens. This lens is attached to the camera via a Century Precision Optics relay lens. Images obtained using only hydrogen from the beam stop exhibited features not yet understood. Images with good signal-to-noise ratio were obtained with the injection of sufficient nitrogen to raise the HEBT pressure to 2-8x10{sup {minus}6} torr. Two strong nitrogen lines, believed to be of the first negative group of N{sub 2}{sup +}, were identified at 391 and 428 nm

Coronal Heating as Determined by the Solar Flare Frequency Distribution Obtained by Aggregating Case Studies

Flare frequency distributions represent a key approach to addressing one of the largest problems in solar and stellar physics: determining the mechanism that counter-intuitively heats coronae to temperatures that are orders of magnitude hotter than the corresponding photospheres. It is widely accepted that the magnetic field is responsible for the heating, but there are two competing mechanisms that could explain it: nanoflares or Alfv\'en waves. To date, neither can be directly observed. Nanoflares are, by definition, extremely small, but their aggregate energy release could represent a substantial heating mechanism, presuming they are sufficiently abundant. One way to test this presumption is via the flare frequency distribution, which describes how often flares of various energies occur. If the slope of the power law fitting the flare frequency distribution is above a critical threshold, $\alpha=2$ as established in prior literature, then there should be a sufficient abundance of nanoflares to explain coronal heating. We performed $>$600 case studies of solar flares, made possible by an unprecedented number of data analysts via three semesters of an undergraduate physics laboratory course. This allowed us to include two crucial, but nontrivial, analysis methods: pre-flare baseline subtraction and computation of the flare energy, which requires determining flare start and stop times. We aggregated the results of these analyses into a statistical study to determine that $\alpha = 1.63 \pm 0.03$. This is below the critical threshold, suggesting that Alfv\'en waves are an important driver of coronal heating.Comment: 1,002 authors, 14 pages, 4 figures, 3 tables, published by The Astrophysical Journal on 2023-05-09, volume 948, page 7