SCAnDi: Single-cell and single molecule analysis for DNA identification

SCAnDi：用于 DNA 鉴定的单细胞和单分子分析

• 批准号: ES/Y010655/1
• 负责人: Iain Macaulay
• 金额: $ 65.23万
• 依托单位: Earlham Institute
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=ES%2FY010655%2F1
• 关键词: SCAnDi; Single; cell; single; molecule

Every nucleated cell in our bodies contains at least one copy of our entire genome, and thus has the potential to identify us. We shed cells wherever we go, often in a context specific manner: different types of contact will result in the transfer and mixture of different cell types. In samples derived from sexual assaults, the cells and DNA recovered will include mixtures of the victim(s) and attacker(s); other contacts including 'touch' DNA samples will contain cells and DNA from multiple contributors. Indeed, up to 45% of forensic samples within the UK criminal justice system contain human DNA from two or more individuals, often preventing successful search of these profiles against DNA databases.The aim of this project is to combine advances in single-cell isolation and analysis with established and novel approaches for DNA profiling. Typically when DNA is recovered from a mixed sample, cellular material is lysed and cellular and acellular DNA molecules are mixed, resulting in a complete loss of morphological information about the cell the DNA originated from. Obtaining cell-of-origin information could make a critical difference in deconvoluting these samples - especially higher order mixed samples - and ascribing a contextual narrative as to what cell the DNA came from, how it was transferred and by whom. Single-cell genomics has advanced rapidly over the last decade. We can now measure genetic diversity in individual cells, as well as molecular markers of cell "type" in addition to microscopic analysis of cell morphology. These technological advances have revolutionised studies in developmental and cancer biology but still remain largely unexplored in a forensic context.Laser Capture Microdissection (LCM) has previously been demonstrated to have applicability in deconvoluting cell mixtures, but advances in molecular biology, genomics, microfluidics and imaging cell sorting now offer the potential to isolate and profile individual cells. In a forensic setting this could enable: 1) obtaining single cells for DNA profiling; 2) the deconvolution of mixed DNA profiles - where DNA / cells from multiple individuals are present in a sample; 3) Linkage of specific cell types with DNA profiles (phenotype or epigenetic/transcriptomic markers); and 4) separation of cellular DNA from background DNA.Our multidisciplinary team will develop approaches for single-cell separation and subsequent single-cell DNA profiling using conventional and next-generation sequencing approaches, benchmarking the readout against gold standard profiling on bulk material. We will deliver proof-of-principle data from individual human cells, including artificial mixtures of single-source cells as well as post-coital mixtures. We will demonstrate the capability of single-cell profiling to distinguish individual donor information from complex mixtures. Furthermore, we will demonstrate the potential to assign cell type information to single-cell DNA profiles using imaging cell sorting, coupled with automated approaches for cell-type classification. These novel approaches need proof-of-principle data to test and demonstrate their ability to support forensic investigations, but we anticipate the application of this technology will complement traditional methods to considerably improve the reliability of such information as intelligence and as evidence in court. Critically, we will use this project as a platform for stakeholder engagement and outreach. We will establish a network of forensic practitioners, commercial suppliers, researchers and investigative and legal representatives and host two in-person workshops and additional bi-monthly meetings, to ensure the technical deliveries remain aligned with the needs of end-users. The project will also enable a framework for sample/data acquisition and transfer between our labs, enabling access to critical infrastructure and capability to support sustainable, longer-term community building.

我们体内的每个核细胞都包含至少一个我们整个基因组的副本，因此具有识别我们的潜力。我们通常以特定方式将细胞放在任何地方：不同类型的接触将导致不同细胞类型的转移和混合物。在源自性侵犯的样本中，回收的细胞和DNA将包括受害者和攻击者的混合物；包括“触摸” DNA样品在内的其他接触将包含来自多个贡献者的细胞和DNA。的确，英国刑事司法系统中多达45％的法医样本包含来自两个或更多个人的人DNA，通常会阻止成功地针对DNA数据库搜索这些概况。该项目的目的是结合单细胞隔离的进步和通过既定和新颖的DNA分析方法进行分析。通常，当从混合样品中回收DNA时，细胞材料会裂解，并且混合了细胞和细胞DNA分子，从而完全丢失了有关DNA起源于DNA的细胞的形态学信息。获取原始细胞信息可能会在对这些样品（尤其是高阶混合样品）的反价文中产生关键的差异，并将上下文叙述归因于DNA来自何种细胞，如何转移和由谁传递。在过去的十年中，单细胞基因组学迅速发展。现在，除了细胞形态的显微镜分析外，我们还可以测量单个细胞中的遗传多样性以及细胞“类型”的分子标记。这些技术进步已经彻底改变了发展和癌症生物学的研究，但在法医环境中仍然无法探索。捕捉微分辨率（LCM）先前已被证明具有适用性的细胞混合物，但是分子生物学，基因组学，微晶状体学和想象学和想象学，微素学和基因组学，基因组学，基因组学，微素学和想象力的进展现在，细胞分类提供了分离和剖析单个细胞的潜力。在法医环境中，这可以实现：1）获得单个细胞进行DNA分析； 2）混合DNA曲线的反卷积 - 样品中存在来自多个个体的DNA /细胞； 3）特定细胞类型与DNA谱（表型或表观遗传/转录组标记）的联系； 4）细胞DNA与背景DNA的分离。我们的多学科团队将使用常规和下一代测序方法开发用于单细胞分离和随后的单细胞DNA分析的方法，从而对批量材料的金标准分析进行基准测试。我们将提供来自单个人类细胞的原则数据，包括单源细胞的人造混合物以及后圆锥形的混合物。我们将展示单细胞分析的能力，以区分单个供体信息和复杂混合物的能力。此外，我们将展示使用成像细胞分类将细胞类型信息分配给单细胞DNA谱的潜力，并与自动方法进行分类的自动方法。这些新颖的方法需要原理数据来测试并证明其支持法医调查的能力，但是我们预计该技术的应用将补充传统方法，以大大提高智能和法庭证据等信息的可靠性。至关重要的是，我们将使用该项目作为利益相关者参与和推广的平台。我们将建立一个法医，商业供应商，研究人员以及调查和法律代表的网络，并举办两个面对面的研讨会和额外的双月会议，以确保技术交付与最终用户的需求保持一致。该项目还将为我们的实验室之间的样本/数据获取和转移提供一个框架，从而使关键基础设施和能力访问以支持可持续的长期社区建设。