DNA microscopy for spatially resolved genomic analyses in intact tissue

DNA 显微镜用于完整组织的空间分辨基因组分析

• 批准号: 9756434
• 负责人: Feng Zhang
• 金额: $ 121.25万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-28 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9756434
• 关键词: 3-Dimensional; Adopted; Algorithms; Antibodies; Benchmarking; Biological; Biological Assay; Biology; Biomedical Research; Brain; Cell Culture Techniques; Cell Line; Cells; Cellular biology; Clinic; Clinical; Clinical Pathology; Communities; Complex; Computational Technique; Computer software; Computing Methodologies; Cytosine; DNA; DNA Library; Data; Devices; Diffusion; Disease; Ensure; Equipment; Face; Gene Targeting; Genes; Genome; Genomic approach; Genomics; Histology; Histones; Histopathology; Human; Image; Joints; Knowledge; Laws; Libraries; Ligation; Measurement; Measures; Methods; Microscope; Microscopy; Molecular Biology; Mus; Oligonucleotides; Optics; Pathology; Patients; Pattern; Physiological; Positioning Attribute; Protocols documentation; Publications; RNA; Randomized; Reaction; Reagent; Research; Resolution; Retina; Sampling; Science; Slide; Spatial Distribution; Special Equipment; Tissue imaging; Tissues; Transcript; Visit; base; cellular imaging; cost; cost effective; disease diagnosis; epigenomics; genomic data; imaging modality; interest; melanoma; microscopic imaging; molecular pathology; novel; novel strategies; online tutorial; outreach; pathology imaging; programs; sequencing platform; single-cell RNA sequencing; tool; transcriptome; transcriptomics; tumor

Biomedical research and practice, from cell biology to clinical pathology relies on imaging cells and tissues and the molecules they express. However, while sequencing is now used extensively to profile genomes, transcriptomes and interactions in bulk samples and single cells, it typically cannot resolve the spatial organization of these profiles. This leads to an ever-widening gap between genomics and cell biology and histopathology. There is thus an enormous need for methods that would collect spatially resolved genomics data. Unfortunately, even the most recent technological advances for spatial genomics still face major barriers and cannot be broadly adopted, as they either require costly, specialized and slow imaging equipment, yield data of limited quality or cannot handle idiosyncratic samples like tumors. Here, we propose a completely novel approach – DNA microscopy – as a new, scalable, cost-effective, general method for spatial genomics in cells, tissue sections, and whole tissues. DNA microscopy encodes spatial organization into a DNA library, sequences it using standard sequencing, and infers the relative position of RNA, DNA or other molecules using inference algorithms. DNA microscopy relies on a novel PCR-based approach that leads to encoded spatial information just based on the laws of diffusion: the closer two molecules were in the original sample, the more likely they are to have a joint product in the DNA microscopy reaction. Following sequencing, an image is recovered by computation on this information. In preliminary results we provide an end-to-end demonstration that DNA microscopy recovers accurate images, without any optical microscopy, and without any prior knowledge on tissues, cells or their organization. Here, we will develop, expand and disseminate DNA microscopy to a broad utility tool, especially with clinical pathology samples. We will develop DNA microscopy to read out the spatial distribution of sets of transcripts in a biological sample and validate it across diverse cell lines and primary cells (Aim 1). We will extend DNA microscopy for spatial profiling of whole transcriptome profiling with randomized and oligonucleotide-library priming strategies, and of epigenomic markers using antibody-oligonucleotide conjugates targeting methylated DNA cytosine and specific acetylated histones (Aim 2). We will maximize DNA microscopy's impact by adapting it for spatial transcript analysis of signatures and whole transcriptomes in 2D tissue sections and in whole mount (3D) tissue (Aim 3), demonstrating successful analysis in diverse tissues, including brain and human tumors. We will disseminate DNA microcopy broadly to users in research or pathology labs (Aim 4). The reagents we use and the protocols we invented are straightforward, and we will facilitate dissemination by distributing reaction chambers to other labs, releasing software, and conducting outreach. DNA microscopy does not require special or costly equipment, relies on PCR protocols that can be easily adopted in any lab, and will handle cells, tissue sections, and whole tissue, thus maximizing its transformative impact on science and the clinic.

生物医学的研究和实践，从细胞生物学到临床病理学，都依赖于成像细胞和组织以及 它们所表达的分子。然而，尽管测序现在被广泛用于描绘基因组， 大样本和单个细胞中的转录和相互作用，它通常不能解析空间 这些配置文件的组织。这导致基因组学和细胞生物学之间的差距越来越大， 组织病理学。因此，对收集空间分辨基因组学的方法有着巨大的需求 数据。不幸的是，即使是空间基因组学的最新技术进步也面临着重大障碍 而且不能被广泛采用，因为它们要么需要昂贵的、专门的和缓慢的成像设备，要么产生 数据质量有限或无法处理肿瘤等特殊样本。在这里，我们提出了一部完整的小说 作为一种新的、可扩展的、经济有效的、通用的细胞空间基因组学方法， 组织切片和整个组织。DNA显微镜将空间组织编码到DNA文库中， 使用标准测序对其进行排序，并使用 推理算法。DNA显微技术依赖于一种基于聚合酶链式反应的新方法，这种方法可以导致编码的空间 基于扩散定律的信息：原始样品中的两个分子越接近，就越多 他们很可能在DNA显微镜反应中有一个联合产物。在排序之后，图像是 通过对这些信息的计算而恢复的。在初步结果中，我们提供了端到端演示 DNA显微镜可以恢复准确的图像，不需要任何光学显微镜，也不需要任何事先 关于组织、细胞或其组织的知识。在这里，我们将开发、扩展和传播DNA 显微镜是一种广泛实用的工具，尤其是临床病理标本。我们将发展DNA显微镜 读出生物样本中转录组的空间分布并验证其在不同细胞中的分布 线条和原生细胞(目标1)。我们将把DNA显微技术扩展到整个转录组的空间图谱 使用随机和寡核苷酸文库启动策略的简档分析，以及使用 针对甲基化DNA胞嘧啶和特异性乙酰化组蛋白的抗体-寡核苷酸结合物(目的 2)。我们将最大限度地发挥DNA显微镜的影响，将其应用于签名和空间转录分析 2D组织切片和整体(3D)组织中的完整转录本(目标3)，证明成功 在不同的组织中进行分析，包括大脑和人类肿瘤。我们将广泛传播DNA显微拷贝到 研究或病理实验室的用户(目标4)。我们使用的试剂和我们发明的方案是 很简单，我们将通过向其他实验室分发反应室来促进传播，释放 软件，并进行外展。DNA显微镜不需要特殊或昂贵的设备，依赖于 可在任何实验室轻松采用的聚合酶链式反应协议，可处理细胞、组织切片和整个组织， 从而最大限度地发挥其对科学和临床的变革影响。