Journal
FRONTIERS IN IMMUNOLOGY
Volume 12, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fimmu.2021.727626
Keywords
Multiplexed tissue imaging; CODEX; single-cell analysis; normalization; unsupervised clustering; spatial analysis; cell-type identification; colon
Categories
Funding
- Cancer Research UK [C27165/A29073] Funding Source: Medline
- NCI NIH HHS [U2C CA233195, F32 CA233203, F99 CA212231, R33 CA183692, U2C CA233238, T32 CA196585] Funding Source: Medline
- NHGRI NIH HHS [U54 HG010426] Funding Source: Medline
- NHLBI NIH HHS [R01 HL128173, R01 HL120724] Funding Source: Medline
- NIAID NIH HHS [U01 AI101984, U19 AI057229, U19 AI135976, U01 AI140498, U19 AI100627, P01 AI131374] Funding Source: Medline
- NIDDK NIH HHS [UG3 DK114937] Funding Source: Medline
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This study introduces the method for generating single-cell multiplexed imaging datasets and discusses the impact of different normalization techniques and clustering algorithms on cell-type identification. The research found that finer cell-type granularity leads to lower labeling accuracy; unsupervised clustering is more accurate in identifying cell types than manual gating; Z-score normalization helps mitigate noise effects in single-cell multiplexed imaging.
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.
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