DNA microscopy for spatially resolved genomic analyses in intact tissue

从细胞生物学到临床病理学的生物医学研究和实践依赖于成像细胞和组织， 它们所表达的分子。然而，虽然测序现在被广泛用于基因组分析， 在大量样品和单细胞中的转录组和相互作用，它通常不能解决空间 组织这些档案。这导致基因组学和细胞生物学之间的差距不断扩大， 组织病理学因此，对收集空间分辨基因组学的方法存在巨大需求 数据不幸的是，即使是空间基因组学的最新技术进步仍然面临重大障碍 并且不能被广泛采用，因为它们要么需要昂贵的、专门的和缓慢的成像设备， 质量有限的数据或无法处理肿瘤等特异质样本。在这里，我们提出了一个全新的 方法- DNA显微镜-作为一种新的，可扩展的，具有成本效益的，细胞空间基因组学的通用方法， 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测序，并使用 推理算法DNA显微镜依赖于一种新的基于PCR的方法， 仅仅基于扩散定律的信息：原始样品中的两个分子越接近， 很可能它们在DNA显微镜反应中有联合产物。测序后， 通过计算这些信息恢复。在初步结果中，我们提供了端到端的演示 DNA显微镜恢复准确的图像，没有任何光学显微镜，没有任何事先 关于组织、细胞或其组织的知识。在这里，我们将发展，扩大和传播DNA 显微镜广泛的实用工具，特别是与临床病理学样本。我们将发展DNA显微镜 读出生物样品中转录物组的空间分布，并在不同细胞中验证它， 细胞系和原代细胞（Aim 1）。我们将扩展DNA显微镜用于整个转录组的空间分析 使用随机化和库引物策略进行分析，以及使用 靶向甲基化DNA胞嘧啶和特异性乙酰化组蛋白的抗体-寡核苷酸缀合物（Aim 2）。我们将最大限度地提高DNA显微镜的影响，通过调整它的空间转录分析的签名， 在2D组织切片和整个包埋（3D）组织中的全转录组（Aim 3），证明了成功的 在不同的组织中进行分析，包括脑和人类肿瘤。我们将广泛传播DNA显微镜， 研究或病理学实验室的用户（目标4）。我们使用的试剂和我们发明的方案 简单明了，我们将通过向其他实验室分发反应室， 软件，并进行推广。DNA显微镜不需要特殊或昂贵的设备， PCR协议可以很容易地在任何实验室采用，并将处理细胞，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性影响。

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.