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Daugharthy, Evan

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Daugharthy

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Daugharthy, Evan

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2014 - 20163

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Author's Publications: search.filters.isAuthorOfPublication.a076aa50-315b-416f-9032-b64cd649fa43

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    Towards Perfect Molecular Measurement
    (2016-09-16) Daugharthy, Evan; Yin, Peng; Church, George M.; Sorger, Peter; Klein, Allon
    The rapid pace of technological innovation has profoundly transformed life sciences research. Whereas less than 100 years ago researchers were often confined to descriptive and theoretical practices, modern scientists can perceive the inner workings of living systems using advanced measurement technology. This ability to acquire measurements of molecules enables the construction of mechanistic models of biological phenomena. Yet despite the power of existing measurement technologies, many aspects of life remain obscured. The things which we cannot observe or measure inspire continuing technology development efforts, such as the work presented in this dissertation. Here, we present a historical, empirical, and theoretical analysis of biology to determine from first principles the shape of future technologies. As a step in this progression, we developed fluorescent in situ sequencing (FISSEQ) of RNA molecules—enabling detection of the spatial organization of gene expression with single-molecule, single-nucleotide resolution. We demonstrate that RNA FISSEQ can be used on a wide array of samples for the investigation of cellular phenotype, gene regulation, and environment in situ, and examine RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. We describe improvements of the FISSEQ library construction method, such as for targeting certain species of RNA, for sequencing DNA in situ, and for detection of proteins and other biomolecules, and integration of FISSEQ with expansion microscopy (ExM) for resolution beyond the diffraction limit of light and enhanced sensitivity. We present two novel nucleic acid sequencing chemistries, a fully-automated FISSEQ robot, and our work building advanced software for processing and analyzing FISSEQ data. We describe our ongoing use of FISSEQ in the study of cancer, development, metabolism, and neuroscience. Finally, we speculate about massively multiplex molecular detection in vivo.
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    Nanoscale Imaging of RNA with Expansion Microscopy
    (2016) Chen, Fei; Wassie, Asmamaw T.; Cote, Allison J.; Sinha, Anubhav; Alon, Shahar; Asano, Shoh; Daugharthy, Evan; Chang, Jae-Byum; Marblestone, Adam; Church, George; Raj, Arjun; Boyden, Edward S.
    The ability to image RNA identity and location with nanoscale precision in intact tissues is of great interest for defining cell types and states in normal and pathological biological settings. Here, we present a strategy for expansion microscopy (ExM) of RNA. We developed a small molecule linker that enables RNA to be covalently attached to a swellable polyelectrolyte gel synthesized throughout a biological specimen. Then, post-expansion, fluorescent in situ hybridization (FISH) imaging of RNA can be performed with high yield and specificity, with single molecule precision, in both cultured cells and intact brain tissue. Expansion FISH (ExFISH) de-crowds RNAs and supports amplification of single molecule signals (i.e., via hybridization chain reaction (HCR)) as well as multiplexed RNA FISH readout. ExFISH thus enables super-resolution imaging of RNA structure and location with diffraction-limited microscopes in thick specimens, such as intact brain tissue and other tissues of importance to biology and medicine.
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    Publication
    Highly Multiplexed Subcellular RNA Sequencing in Situ
    (American Association for the Advancement of Science, 2014-03-21) Lee, Je Hyuk; Daugharthy, Evan; Scheiman, Jonathan; Kalhor, Reza; Ferrante, Thomas; Yang, Joyce; Terry, Richard; Jeanty, Sauveur; Li, Chao; Amamoto, Ryoji; Peters, Derek; Turczyk, Brian; Marblestone, Adam; Inverso, Samuel; Bernard, Amy; Mali, Prashant; Rios, Xavier; Aach, John; Church, George
    Understanding the spatial organization of gene expression with single nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked cDNA amplicons are sequenced within a biological sample. Using 30-base reads from 8,742 genes in situ, we examined RNA expression and localization in human primary fibroblasts using a simulated wound healing assay. FISSEQ is compatible with tissue sections and whole mount embryos, and reduces the limitations of optical resolution and noisy signals on single molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ.