SPADE Settings (Cytobank)

A.
1. Define target number of nodes: 3. Select cellular population:
The automatic target number Select cellular population for
of nodes is 200, this can be analysis. Note: You cannot
user-defined select specific individual
samples or the number of
2. Select % of downsampling: events sampled
The automatic percent of 4. Select markers:
downsampled events is 10% Select all relevant
this can be user-defined phenotypic markers
for clustering

B. 200 Nodes C. 45 Nodes D. 45 Nodes E. 45 Nodes

(Representation #1) (Representation #2) (SPADE run #2)
5,217 cells
1 cell Cellular abundance
in each node is
expressed by
node size. 17,892 cells
22 cells
CD45
CD45 CD45
CD45 .97
1.41 1.41 17,892 cells
1.6
22 cells

In a SPADE tree, 6.59 6.59 19,447 cells 6.77

6.99
each cell is present 19 cells

in only a single node.

Marker intensity is The shape and orientation Sequential SPADE trees can look different
expressed by node color. of a SPADE tree is arbitrary between runs, in shape, node size,
and can be manually expression, and connections.
changed by the user.
F. # of markers used for clustering

Lineage Markers (10) All Markers (35)

Samples
G.
X-shift Informed Target Nodes

IL10KO #4
IL10KO #3
IL10KO #2
IL10KO #1
B6 #4
B6 #3
B6 #2

B6 #5
B6 #1

CD45
# of target nodes

89 nodes 45 nodes
13,442.92 cells 17,892 cells
1 cell 22 cells

CD19
Automatic Target Nodes (200)

CD4
Parameters

CD8
5,217 cells
1 cell
6,509.84 cells
1 cell

CD11c

Multiple nodes containing only 1 cell

can indicate tree overfragmentation.
NKp46
H.
CD11b+, CD64+

What are the cellular CD64

CD4+ phenotypes of the
CD11b+, CD64-
various clusters?
CD45-
CD45+ CD11b
CD19+
NKp46+
Phenotypes are defined
CD8+ by marker intensity How consistent are
How do SPADE trees from
replicate samples in node
different experimental
size and expression?
groups differ in node Figure 5
size and expression?
Kimball et al