Molecular, Cellular, and Tissue Characterization Unit

表征单元-摘要 表征股将监督收集施工所需的所有图像和组学数据 MP病变和CHIP标本的多参数分析。在一项技术 第一年的评估/整合阶段，大部分数据收集将在LSP中进行， 核心设施作为确保可重复性的手段。特性描述股将与分析小组合作， 该股建立一个强有力的信息处理系统，以保持数据和元数据的可靠性 采集和储存Atlas标本和QC/QA样本。元数据将包括 必须符合FAIR标准。解离技术如单细胞RNA测序， DNA测序和FACS/CyTOF将为Atlas构建提供最高维度的数据， 只在组织环境之外的细胞上。FFPE活检的高度多重成像将用于 收集关于细胞状态和形态以及所选基因表达的空间分辨数据， 基因型（使用锁式滚环扩增探针）。对于MP Atlas，FFPE的激光捕获 随后将通过WES评估突变状态，通过TCR-seg评估T细胞克隆性， 通过RNA-Seq.分析单元将使用多视图学习和相关方法， 整合得到的空间和组学数据，并在scRNAseq不 可能（即分析FFPE MP时）。中心制定的标准操作规程将 评估并分发给其他HTAN成员。 目的1将描述基因表达的变化，突变的获得和细胞-细胞 通过测序、流式细胞术和CyTOF分析异质性。基于挂锁的FISH将用于提供 mRNA表达和突变状态的单细胞分辨率。目标2将收集高维高 使用两种互补技术：t-CyCIF和DEI，从FFPE标本中获得的图像分辨率。 抗体将经过严格测试，QA/QC数据将与Atlas图像相关联。CHIP和MP的子集 将使用连续切片在3D中分析样品。AIM 3将从连续活检中收集时间序列数据 的CHIP患者，深度UMI测序和流式细胞术将用于 测定变体等位基因频率和克隆进展。目标4将实施质量保证政策 灵感来自于质量保证官和病理学家Sandro Scarlata在CLIA环境中的经验。 这将涉及SOP、常规复检、能力培训和统计质量控制。目标5将 评估PATCH中心成员、HTAN其他中心开发的新技术或改进技术 网络，合作调查人员和公司（见支持信）。这包括直接比较 t-CyCIF和DEI与多路离子束成像（MIBI）现已在HMS和扫描质谱 在苏黎世的Bodenmiller实验室实施细胞计数成像。

CHARACTERIZATION UNIT - SUMMARY ABSTRACT The Characterization Unit will oversee collection of all image and omic data needed for construction of multi-parametric Atlases of MP lesions and CHIP specimens. Following a technology assessment/consolidation phase in year 1 most data collection will take place in the LSP and established core facilities as a means to ensure reproducibility. The Characterization Unit will work with the Analysis Unit to establish a robust information processing system that maintains the reliability of data and metadata collection and storage for Atlas specimens and QC/QA samples. Metadata will include the elements needed to comply with FAIR standards. Dissociative technologies such as single cell-RNA sequencing, DNA sequencing, and FACS/CyTOF will provide the highest dimensional data for Atlas construction but only on cells outside of their tissue context. Highly multiplexed imaging of FFPE biopsies will be used to collect spatially resolved data on cell states and morphology as well as expression of selected genes and genotypes (using padlock-rolling circle amplification probes). For the MP Atlas, laser capture of FFPE specimens will be followed by assessment of mutational status by WES, T cell clonality by TCR-seg and RNA expression by RNA-Seq. The Analysis unit will use multi-view learning and related methods to integrate the resulting spatial and omic data and to deconvolute bulk RNA sequence when scRNAseq is not possible (i.e. when analyzing FFPE MPs). Standard operating protocols developed in the Center will be evaluated and disseminated to other HTAN members. Aim 1 will characterize changes in gene expression, acquisition of mutations and cell-cell heterogeneity by sequencing, flow cytometry and CyTOF. Padlock-based FISH will be used to provide single-cell resolution of mRNA expression and mutational status. Aim 2 will collect high dimensional high resolution of images from FFPE specimens at using two complementary techniques: t-CyCIF and DEI. Antibodies will be rigorously tested and QA/QC data linked to Atlas images. A sub-set of CHIP and MP samples will be analyzed in 3D using serial sections. Aim 3 will collect time-series data from serial biopsies of CHIP patients in a longitudinal cohort and deep UMI sequencing and flow cytometry will be used to assay variant allele frequency and clonal progression. Aim 4 will implement quality assurance policies inspired by experience of quality assurance officer and pathologist Sandro Santagata in a CLIA setting. This will involve SOPs, routine retesting, competency training and statistical quality control. Aim 5 will evaluate new or improved technologies developed by PATCH Center members, other Centers in the HTAN network, collaborating investigators and companies (see letters of support). This includes direct comparison of t-CyCIF and DEI with Multiplexed Ion Beam Imaging (MIBI) now available at HMS and Scanning Mass Cytometry Imaging implemented in the Bodenmiller Lab in Zurich.