Menzies Health Institute Queensland, Griffith University, Southport, Qld, Australia.
Menzies Health Institute Queensland, Griffith University, Southport, Qld, Australia; School of Pharmacy and Medical Science, Griffith University, Southport, Qld, Australia.
Pathology. 2024 Dec;56(7):1028-1035. doi: 10.1016/j.pathol.2024.06.004. Epub 2024 Aug 14.
The emergence of spatial profiling technologies in recent years has accelerated opportunities to profile in detail the molecular attributes of a wide range of tissue pathologies using archival specimens. However, tissue treatment for fixation and storage does not always support generation of high-quality genomic data. The purpose of this study was to investigate the impacts of Proteinase K (ProtK) treatment, as a way to increase target transcript exposure, on downstream sequencing data quality metrics for spatial transcriptomic data using formalin-fixed, paraffin-embedded samples. In a series of four independent assessments using different tissue types (nasal mucosa, tonsil, pancreas), varying concentrations of ProtK (ranging from 0.1 to 1 μg/mL) were used as part of the sample processing workflow to generate transcriptomic data using the Nanostring GeoMx DSP and Illumina NextSeq 2000 platforms. Use of higher concentrations of ProtK was generally found to increase total reads (2-4-fold). However, negative probe counts also tended to be increased (2-12-fold), resulting in reductions in the signal-to-noise ratio (10-70% lower) and the number of genes detected above background (50-80% lower). These effects were not seen in all tissues and impacts of tissue handling and processing, beyond ProtK treatment, on data quality metrics, also require consideration. Regardless, these observations highlight the need for careful consideration of a range of sample processing factors and benefits that may be achieved through the optimisation of sample processing workflows for specific tissues as a way to maximise the generation of quality data using spatial transcriptomic approaches.
近年来，空间分析技术的出现加速了机会，可以使用存档标本详细分析广泛的组织病理学的分子属性。然而，用于固定和储存的组织处理并不总是支持高质量基因组数据的生成。本研究的目的是研究蛋白酶 K（ProtK）处理对空间转录组数据下游测序数据质量指标的影响，ProtK 处理是一种增加靶转录本暴露的方法，用于使用福尔马林固定、石蜡包埋的样本进行空间转录组数据。在使用不同组织类型（鼻黏膜、扁桃体、胰腺）进行的四项独立评估中，使用不同浓度的 ProtK（0.1 至 1μg/mL 之间）作为样品处理工作流程的一部分，使用 Nanostring GeoMx DSP 和 Illumina NextSeq 2000 平台生成转录组数据。通常发现使用较高浓度的 ProtK 会增加总读数（2-4 倍）。然而，负探针计数也往往增加（2-12 倍），导致信号噪声比降低（降低 10-70%）和检测到的基因数量低于背景（降低 50-80%）。并非所有组织都观察到这些影响，超出 ProtK 处理的组织处理和处理对数据质量指标的影响也需要考虑。无论如何，这些观察结果强调了需要仔细考虑一系列样品处理因素，并通过优化特定组织的样品处理工作流程来实现可能获得的收益，以最大化使用空间转录组方法生成高质量数据。
