Dataset Information

Strategies for Accurate Cell Type Identification in CODEX Multiplexed Imaging Data.

ABSTRACT: Multiplexed imaging is a recently developed and powerful single-cell biology research tool. However, it presents new sources of technical noise that are distinct from other types of single-cell data, necessitating new practices for single-cell multiplexed imaging processing and analysis, particularly regarding cell-type identification. Here we created single-cell multiplexed imaging datasets by performing CODEX on four sections of the human colon (ascending, transverse, descending, and sigmoid) using a panel of 47 oligonucleotide-barcoded antibodies. After cell segmentation, we implemented five different normalization techniques crossed with four unsupervised clustering algorithms, resulting in 20 unique cell-type annotations for the same dataset. We generated two standard annotations: hand-gated cell types and cell types produced by over-clustering with spatial verification. We then compared these annotations at four levels of cell-type granularity. First, increasing cell-type granularity led to decreased labeling accuracy; therefore, subtle phenotype annotations should be avoided at the clustering step. Second, accuracy in cell-type identification varied more with normalization choice than with clustering algorithm. Third, unsupervised clustering better accounted for segmentation noise during cell-type annotation than hand-gating. Fourth, Z-score normalization was generally effective in mitigating the effects of noise from single-cell multiplexed imaging. Variation in cell-type identification will lead to significant differential spatial results such as cellular neighborhood analysis; consequently, we also make recommendations for accurately assigning cell-type labels to CODEX multiplexed imaging.

SUBMITTER: Hickey JW

PROVIDER: S-EPMC8415085 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Strategies for Accurate Cell Type Identification in CODEX Multiplexed Imaging Data.

Hickey John W JW Tan Yuqi Y Nolan Garry P GP Goltsev Yury Y

Frontiers in immunology 20210813

Multiplexed imaging is a recently developed and powerful single-cell biology research tool. However, it presents new sources of technical noise that are distinct from other types of single-cell data, necessitating new practices for single-cell multiplexed imaging processing and analysis, particularly regarding cell-type identification. Here we created single-cell multiplexed imaging datasets by performing CODEX on four sections of the human colon (ascending, transverse, descending, and sigmoid) ...[more]

PMID: 34484237

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Project description:MotivationThe advent of highly multiplexed in situ imaging cytometry assays has revolutionized the study of cellular systems, offering unparalleled detail in observing cellular activities and characteristics. These assays provide comprehensive insights by concurrently profiling the spatial distribution and molecular features of numerous cells. In navigating this complex data landscape, unsupervised machine learning techniques, particularly clustering algorithms, have become essential tools. They enable the identification and categorization of cell types and subsets based on their molecular characteristics. Despite their widespread adoption, most clustering algorithms in use were initially developed for cell suspension technologies, leading to a potential mismatch in application. There is a critical gap in the systematic evaluation of these methods, particularly in determining the properties that make them optimal for in situ imaging assays. Addressing this gap is vital for ensuring accurate, reliable analyses and fostering advancements in cellular biology research.ResultsIn our extensive investigation, we evaluated a range of similarity metrics, which are crucial in determining the relationships between cells during the clustering process. Our findings reveal substantial variations in clustering performance, contingent on the similarity metric employed. These variations underscore the importance of selecting appropriate metrics to ensure accurate cell type and subset identification. In response to these challenges, we introduce FuseSOM, a novel ensemble clustering algorithm that integrates hierarchical multiview learning of similarity metrics with self-organizing maps. Through a rigorous stratified subsampling analysis framework, we demonstrate that FuseSOM outperforms existing best-practice clustering methods specifically tailored for in situ imaging cytometry data. Our work not only provides critical insights into the performance of clustering algorithms in this novel context but also offers a robust solution, paving the way for more accurate and reliable in situ imaging cytometry data analysis.Availability and implementationThe FuseSOM R package is available on Bioconductor and is available under the GPL-3 license. All the codes for the analysis performed can be found at Github.

Dataset Information

Strategies for Accurate Cell Type Identification in CODEX Multiplexed Imaging Data.

Publications

Strategies for Accurate Cell Type Identification in CODEX Multiplexed Imaging Data.

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