Citrus Settings (Cytobank)

A. 7. Events sampled:
1. Select cellular population: 2. Select markers: 3. Designate experimental groups: 4. Chose association models: Select the number of events
to be sampled from each file
Select cellular population Select all relevant phenotypic Assign FCS files to appropriate Up to three association models can
for analysis markers for clustering experimental groups be used for Citrus analysis (SAM, 8. Cluster size (%):
LASSO/GLMNET, and PAMR) The automatic setting for this is 5%,
but can be changed by the user
5. Cluster characterization:
Clusters can be characterized 9. Cross validation folds:
by cellular abundance or The automatic setting for this is 5,
median expression but can be changed by the user
6. Event sampling: 10. False discovery rate (%):
Events can be sampled The automatic setting for this is 1,
equally or proportionally but can be changed by the user

B. Number of model features Citrus Settings:

0 2 3 4 4 4 6 7 8 9 16 21 25 28 30
Number of characteristics
(populations or markers)
C. Events sampled per file: 9,141
Minimum cluster size: 5%
different between the groups Graph Features: Group Assignment: Random*
100

Cross Validation Error Rate

82218

Number of model features

cv.min model identifies 7 features that Ideally, a model should have low
82251

Feature False Discovery Rate

0 2 2 3 4 7 9 12 13 16 18 20 22 27 35
82246
Model Cross Validation Error Rate

cross validation error rate and

82257

predict differences between groups

100
Cross Validation Error Rate

cv.min
Feature False Discovery Rate 82258

low false discovery rate

82234
cv.min
cv.1se 82247

cv.1se
82238
75

Model Cross Validation Error Rate

82264

75
82248
82265
82262

Citrus defines three predictive cv.fdr.constrained 82256

82267
82252

models with varying stringency

50
82260

CD45
82268 82255 82250

(min, 1se, fdr.constrained) Citrus Settings:

82249
82261
82244 82266 0.93
50

1.72

Events sampled per file: 9,141

25
82263
82245
82259

82253 3.56
82243
82239

Minimum cluster size: 5%

5.02
82254 82236
82240
6.47

0
82227

Group Assignment: Correct

82221 82241
1.12 0.99 0.87 0.74 0.62 0.50 0.37 0.25 0.12 0.00
25

Regularization Threshold

Example of poorly predictive models,

with high cross validation rate and false Note: Despite poor model performance
discovery rate Citrus still identifies clusters
0

4.68 4.16 3.64 3.12 2.60 2.08 1.56 1.04 0.52 0.00

Regularization Threshold Citrus-defined radial hierarchy tree identifies 82238

significant features (from panel B)

D. Citrus Settings: E. Citrus Settings: F. 82258 82253

Events sampled per file: 5,000* Events sampled per file: 9,141 82241
82261

Minimum cluster size: 5% Minimum cluster size: 1%*

Group Assignment: Correct Group Assignment: Correct 82251
82223
Number of model features
82228
82077 82115

0 2 3 4 5 5 8 14 20 31 48 79 114 143
44971

Number of model features 82263

82062 82160

82158
82265
44956 82111 82130

0 1 2 5 5 5 6 7 7 9 15 17 20 24 27
82116

82255
82080 82202 82190
44988
100

82097 82204
44981 Cross Validation Error Rate 82139 82203 82207
82100

Feature False Discovery Rate

82200
44978 82216
100

82195

Cross Validation Error Rate cv.min

82206 82178

82229
82243
82186
82242

Feature False Discovery Rate 44986 44983 cv.1se 82228 82249 82221
82045
82247
cv.min cv.fdr.constrained
82227
82205
82162 82095 82050
82262
Model Cross Validation Error Rate

82163 82257
44993 82168

cv.1se 82267
82229
82240
82148

cv.fdr.constrained
75

82222 82247 82188 81951

82234 82147 82058
Model Cross Validation Error Rate

82262
82246
44984 82201

82259
82117
44997 82255
75

82239
82151
44992 82213

82268 82236 82232

82265 82211
82071 82177
82263
44994 82193 82258 82137
82252
44998 44985 82259 82235
82225 82267
82135 82210
82191
50

82264
82091 82192
44980 82254
82268 82136 82120

82256
82264
44999 44972 44964 82198

82266
82152
82230 82092
44996 82223

CD45
50

82181
81998 82266 82260
81950 82219

82252
81686 82131 82253
44990
82249
82090 82244 82150

CD45
82021
44995 82212
82184
82238
82196
82261
82165 82231
82256 82127

82017
0.41
25

82104 82251
82232
1.56
82164 82248 82217
82215 82067
44989

Model:
82250
44976 82159

44991 82129
82183
82185 82138
1.72 82250
82260
25

44979
2.59
82224 82236
82153 82241 82245
44952 82233
82170
3.36

Min
82101 82208
82220
82141 82189
44987 44982 82180 82199
3.89 82046 82194
82214 82179
82237
82209 82172

4.99 82243
82230
0

82161 82145
82173 82112
82155 82142

SE
82156 82169 82123

44973 44962
5.19
82257
82144 82102
82197
82134 82081
6.62
5.67 5.04 4.41 3.78 3.15 2.52 1.89 1.26 0.63 0.00
82087 82044
0

82149
6.48 82048

Constrained
82254
82176
44968

4.21 3.74 3.27 2.81 2.34 1.87 1.40 0.94 0.47 0.00 Regularization Threshold 81902
82089

Regularization Threshold 82239

Decreasing the minimum Decreasing the minimum cluster 82234

Decreasing the input cell number cluster size to 1% results in size to 1% creates a higher
This Citrus plot looks appropriate, but
results in 10% model error rate 10% model error rate density radial tree There are 7 model features
the model indicates it is not opimal
identfied by the min and SE models There are 25 model features
that predict differences between identfied by the constrained model
experimental groups that predict differences between
Citrus portrays data In a Citrus tree, all events are present in the experimental groups
as a radial tree, but central node, and subjected to further The hierarchical relationship
unlike SPADE all bifurcation. Each cell can be present in between parent and daughters
events are hierarchical. multiple nodes (unlike a SPADE tree). is revealed by subtle arrows.

Colored by Parameter Expression (CD45) Colored by significance models

G. 82238 CD45
2.02
H. 82238
Models have different
levels of strigency Model: Population:
2.97
82258 82253

Min
Background
82258 82253

K.
Parent clusters contain a mixture of cells
82261
4.14
82241
82261

that are subdivided between daughter clusters. SE

Cluster
Tree Geometry

82241
Original Radial

5.32

82223
82251
6.50 82223
82251
Constrained
(Default)

82228
82265 82263
82228
82265 82263 82255
82255
82229
82247
82229

82265
82262

82267
82247 82267
82262
82267
B6 Biased

82259
82259 82268 82236 82232
82268 82236 82232
82264
82256

CD64
82264 82266

CD45
82266 82256 82252
82249
82249 82252

82250
82260
82250

Model:
82260

82230 82243

82257
82230 82243
Min 82257

SE
82263 82251 82264 82265
82254
82254
82243
82239
Constrained 82234
82239

82234

CD45 CD45 CD64 CD64

I. 82268 J. 82268
IL10KO biased

82252 82266
CD4
(Manually generated)

82266 82267 82266 IL10KO biased 82267 B6 Biased

Lag3 CD8
Modifed Vertical
Tree Geometry

82260 82256 82264 82265 82260 82256 82264 82265

82243 82236 82260 82256

CD4

CD4

82230 82257 82252 82250 82259 82263 82251 82230 82257 82252 82250 82259 82263 82251

Lag3 Lag3 CD8 CD8

82239 82254 82243 82236 82249 82255 82262 82261 82241 <5%
* 82239 82254 82243 82236 82249 82255 82262 82261 82241

82234 82232 82228 82247 82258 82234 82232 82228 82247 82258
Citrus trees contain bifurcation Certain markers are discriminators
Model: points in which daughters between divergent daughters.
6.50 5.32 4.14 2.97 2.02 82223 82229 82253 82238
Min 82223 82229 82253 82238 diverge in their significance.
CD45 Expression SE
Constrained

A manually generated The Citrus tree only depicts nodes

vertical tree demonstrates that reach the minimum cluster size
parent-daughter relationships
and clarifies which
(here 5%), daughters with lower Figure 6
frequencies (e.g. in gray) are not shown.
clusters are subgroups. Kimball et al