TABLE 1.

Comparison of manual gating, unsupervised and supervised clustering methods.

	Manual gating	Unsupervised clustering methods	Supervised clustering methods
Ease of use	Easy and straight forward for biologist	Tool dependent, generally easy to apply. See Table 3	Tool dependent, generally requires more steps than unsupervised clustering methods
Reproducibility	Reproducible between data for same user	Majority of the tools allow for setting a “seed” enabaling the reproducibility of the results. See Table 3	Variable (tool dependent)
Time cost	Experience and sample size dependent	Tool dependent, see Table 3	Tool dependent, generaly high. See Table 4
Flexibility	High, depends on user manual setting	Moderate, users can only adjust some parameters	Low
Novel subpopulation detection	Yes	Yes (tool dependent)	No (can only detect previously defined clusters)
Subpopulation/cluster identification	Manual (based on gating strategy)	Manual (based on cluster marker expression)	Automated (based on training set)
# of subpopulations/clusters	Experiment dependent	Variable (some allow users input; some automatically optimize #) See Table 3	Fixed (based on training set)
Prior knowledge requirement	Gating Experience, Marker expression for cellular identification	None for clustering; knowledge of marker expression for cluster identification	Training dataset or marker matrix, familiarity with bioinformatics