FridrichMethod Version Compatibility
Reference examples tested with: CATALYST 1.26+, FlowSOM 2.10+, flowCore 2.14+; Rphenograph (GitHub: JinmiaoChenLab/Rphenograph).
Before using code patterns, verify installed versions match. If versions differ:
- R:
packageVersion('<pkg>')then?function_nameto verify parameters
Rphenograph is GitHub-only (remotes::install_github('JinmiaoChenLab/Rphenograph')) and returns a list - membership is igraph::membership(out[[2]]), not a vector. Adapt rather than retrying.
Clustering and Phenotyping
"Cluster my cytometry data to find cell types" -> Discover populations in high-dimensional data without gates, then annotate them by marker expression.
- R:
CATALYST::cluster()(wraps FlowSOM + ConsensusClusterPlus) - the field default - R:
FlowSOM::FlowSOM()directly, orRphenograph()for graph-based clustering
The Single Most Important Modern Insight -- Cluster on Type Markers; the Embedding Is for Looking, Not Measuring
Two rules carry most of the correctness here. First, the type-vs-state distinction: LINEAGE/type markers (CD3, CD4, CD8, CD19) DEFINE clusters; functional/STATE markers (phospho-epitopes, cytokines, Ki-67, activation markers) must be WITHHELD from clustering and tested within clusters instead (the DA/DS framework, Nowicka 2017 F1000Res 6:748). Clustering on state markers splits "activated CD4" from "resting CD4" and confounds abundance with activation - a classic, silent design error. Second, t-SNE/UMAP embeddings do NOT preserve inter-cluster distances, cluster sizes, or densities (the apparent "UMAP preserves global structure" edge over tSNE is largely an initialization artifact - Kobak & Linderman 2021 Nat Biotechnol 39:156). Define populations by clustering in the HIGH-DIMENSIONAL space and COLOR the embedding by cluster; never gate on the embedding or read biology off blob distances.
Algorithm Taxonomy
| Algorithm | Citation | Mechanism | Speed | Rare-pop | Determinism |
|---|---|---|---|---|---|
| FlowSOM | Van Gassen 2015 Cytometry A 87:636 | SOM grid -> MST (viz) -> consensus metaclustering | fastest | good if grid over-provisioned | stochastic; seed-controllable |
| PhenoGraph | Levine 2015 Cell 162:184 | kNN graph (Jaccard) + Louvain | moderate | strong (no preset k) | seed-fragile (>40% reassignment reported) |
| X-shift | Samusik 2016 Nat Methods 13:493 | weighted kNN density + auto cluster # | slow | excellent | more deterministic |
| flowMeans | Aghaeepour 2011 Cytometry A 79:6 | k-means multi-cluster + change-point k | fast | moderate | stochastic |
Benchmark: Weber & Robinson 2016 Cytometry A 89:1084 tested 18 methods - FlowSOM (with metaclustering) was a top performer AND by far fastest, hence the field default; but its accuracy depends on supplying the right number of metaclusters.
Why Over-Provision the Grid, Then Metacluster
Set the SOM grid (e.g. 10x10 = 100 nodes) MUCH larger than the number of populations expected, then metacluster down. The asymmetry: metaclustering can MERGE over-fine nodes into a real population, but can NEVER SPLIT a node that erroneously fused two cell types. Too coarse commits the unrecoverable error; too fine commits only the recoverable one. So over-cluster, then merge by hand off the median heatmap.
CATALYST Clustering Pipeline
Goal: Cluster on type markers and prepare for annotation.
Approach: prepData builds the SCE (panel marker_class flags type vs state); cluster() wraps FlowSOM+ConsensusClusterPlus. Defaults xdim=ydim=10, maxK=20 (the metacluster cap people forget); set seed on the function.
library(CATALYST)
sce <- prepData(fs, panel, md, transform = TRUE, cofactor = 5) # cofactor 5 = CyTOF; ~150 for fluorescence
sce <- cluster(sce, features = 'type', # type markers only
xdim = 10, ydim = 10, maxK = 20, seed = 42) # maxK caps metaclusters at 20 by default
plotExprHeatmap(sce, features = 'type', by = 'cluster_id', k = 'meta20', scale = 'last')
PhenoGraph (graph-based alternative)
Goal: Cluster with a kNN graph when a data-driven cluster count is wanted.
Approach: Rphenograph on the type-marker matrix (cells x markers); extract membership from the list.
library(Rphenograph)
type_expr <- t(assay(sce, 'exprs')[rowData(sce)$marker_class == 'type', ])
out <- Rphenograph(type_expr, k = 30) # only knob: k (neighbors)
sce$phenograph <- factor(igraph::membership(out[[2]])) # list -> membership, not a vector
Dimensionality Reduction (visualization only) and Annotation
Goal: Visualize structure and assign cell-type labels.
Approach: runDR subsamples per sample (cells=); color by cluster, never gate on it. Annotate from the median heatmap, then mergeClusters with a curated table.
sce <- runDR(sce, dr = 'UMAP', features = 'type', cells = 2000) # subsampled embedding
plotDR(sce, 'UMAP', color_by = 'meta20')
merging <- data.frame(old_cluster = 1:20,
new_cluster = c('CD4 T','CD4 T','CD8 T', '...')) # curated from the heatmap
sce <- mergeClusters(sce, k = 'meta20', table = merging, id = 'annotated')
Per-Method Failure Modes
Clustering on state markers
Trigger: activation/phospho markers in the clustering feature set. Mechanism: state contaminates lineage identity. Symptom: "activated" and "resting" versions of a type split as separate clusters. Fix: cluster on type only; test state markers within clusters (differential-analysis).
Seed-dependent "novel populations"
Trigger: a population that appears at one seed and vanishes at another. Mechanism: FlowSOM init / Louvain are stochastic. Symptom: non-reproducible clusters. Fix: set + report the seed; check multi-seed stability; treat unstable clusters as hypotheses.
Reading biology off the embedding
Trigger: "cluster A is closer to B than C." Mechanism: UMAP/tSNE distances are non-metric. Symptom: false developmental/relatedness claims. Fix: quantify in marker space; embedding for display only.
Clustering uncompensated/untransformed data
Trigger: raw linear input to FlowSOM. Mechanism: spillover + scale dominate Euclidean distance. Symptom: clusters track intensity, not biology. Fix: compensate + transform first.
Quantitative Thresholds
| Threshold | Source | Rationale |
|---|---|---|
| over-provision grid (10x10) >> expected pops | Van Gassen 2015 | metacluster can merge, never split |
| maxK = 20 default | CATALYST | metacluster cap; raise if expecting more |
| FlowSOM needs correct K | Weber & Robinson 2016 | accuracy depends on metacluster number |
| use median (not mean) per cluster | Bendall 2011 Science 332:687 | robust to doublet/spillover contamination |
Common Errors
| Error / symptom | Cause | Solution |
|---|---|---|
| clustering uses scatter/Time/state | features not restricted |
features='type' / colsToUse= lineage markers |
| Rphenograph result unusable | it returns a list | igraph::membership(out[[2]]) |
set.seed doesn't make FlowSOM reproducible |
internal reseeding | pass seed= to cluster() |
| only 20 clusters no matter what | maxK default |
raise maxK |
References
- Van Gassen 2015 Cytometry A 87(7):636-645 — FlowSOM.
- Levine 2015 Cell 162(1):184-197 — PhenoGraph.
- Samusik 2016 Nat Methods 13(6):493-496 — X-shift.
- Weber & Robinson 2016 Cytometry A 89(12):1084-1096 — clustering benchmark (FlowSOM top + fastest).
- Nowicka 2017 F1000Research 6:748 — CyTOF workflow; type-vs-state markers.
- Kobak & Linderman 2021 Nat Biotechnol 39:156-157 — embedding initialization artifact.
- Bendall 2011 Science 332(6030):687-696 — arcsinh-median analysis of CyTOF data.
Related Skills
- compensation-transformation - Compensate/transform before clustering
- gating-analysis - Supervised alternative; needed for rare populations
- differential-analysis - Test abundance/state of clusters between conditions
- cytometry-qc - Cluster only QC-passed events
- single-cell/clustering - Leiden/Louvain on scRNA-seq (shared graph-clustering ideas)
- imaging-mass-cytometry/phenotyping - Same CATALYST/FlowSOM conventions for imaging