Molecular, Cellular, and Tissue Characterization Unit

表征单元-摘要摘要 特定股将监督建设所需的所有图像和光学数据的收集 多参数MP皮损图集和切片标本。遵循一项技术 第一年的评估/整合阶段大多数数据收集将在LSP进行，并已确定 核心设施作为确保重现性的一种手段。特定股将与分析一起工作 建立一个强大的信息处理系统，以保持数据和元数据的可靠性 收集和储存图集标本和QC/QA样品。元数据将包括元素 需要遵守公平的标准。解离技术，如单细胞RNA测序， DNA测序和FACS/CyTOF将为Atlas的构建提供最高维度的数据，但 仅存在于组织环境之外的细胞上。FFPE活检的高度多路成像将用于 收集有关细胞状态和形态以及选定基因和表达的空间分辨数据 基因分型(使用挂锁-滚圈扩增探针)。对于MP地图集，激光捕获FFPE 随后将用WES评估突变状态，用TCR-SEG和TCR-SEG评估T细胞克隆 RNA-Seq.分析股将使用多视角学习和相关方法 当scRNAseq不是时，整合得到空间和组学数据并解卷成块RNA序列 可能(即在分析FFPE MPS时)。在该中心制定的标准操作规程将是 评估并向其他Htan成员分发。 目标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、流式细胞仪和细胞周期图检测异质性。基于挂锁的鱼类将用于提供 单细胞分辨的mRNA表达和突变状态。目标2将收集高维高 使用两种互补技术：T-CyCIF和DEI对FFPE标本的图像进行分辨率分析。 抗体将经过严格的检测，QA/QC数据将与Atlas图像相关联。芯片和MP的子集 样品将使用连续切片进行3D分析。AIM 3将从连续活检中收集时间序列数据 CHIP患者的纵向队列和深度UMI测序和流式细胞术将用于 检测变异等位基因频率和克隆性进展。目标4将实施质量保证政策 灵感来自质量保证官员和病理学家桑德罗·桑塔加塔在CLIA环境中的经验。 这将涉及标准操作规程、例行重新测试、能力培训和统计质量控制。目标5将 评估由Htan的补丁中心成员、其他中心开发的新技术或改进的技术 网络、合作调查人员和公司(见支持函)。这包括直接比较 T-CyCIF和DEI的多路离子束成像(MIBI)现已在HMS和扫描质谱仪上提供 苏黎世博登米勒实验室实施的细胞学成像。

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.