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Do proper .fcs flow-cytometry files already have the gating hierarchy, so one may calculate cell-counts without extra work?

https://github.com/whitews/FlowKit/

# setup some file paths for our data
base_dir = "data/8_color_data_set"

sample_path = os.path.join(base_dir, "fcs_files")
wsp_path = os.path.join(base_dir, "8_color_ICS.wsp")
session = fk.Session(sample_path)
session.import_flowjo_workspace(wsp_path)
# that's it, let's see what sample groups were loaded
session.get_sample_groups()
['default', 'All Samples', 'DEN']
# I happen to know all the "real" analysis is within the "DEN" group
sample_group = 'DEN'
# and the sample that are included in the group
session.get_group_sample_ids(sample_group)
['101_DEN084Y5_15_E03_009_clean.fcs',
 '101_DEN084Y5_15_E01_008_clean.fcs',
 '101_DEN084Y5_15_E05_010_clean.fcs']
# so what is the gating hierarchy for this group
print(session.get_gate_hierarchy(sample_group))
root
╰── Time
    ╰── Singlets
        ╰── aAmine-
            ╰── CD3+
                ├── CD4+
                │   ├── CD107a+
                │   ├── IFNg+
                │   ├── IL2+
                │   ╰── TNFa+
                ╰── CD8+
                    ├── CD107a+
                    ├── IFNg+
                    ├── IL2+
                    ╰── TNFa+
# looks good, let's go ahead and analyze them (using verbose mode to see each gate as it's processed)
session.analyze_samples(sample_group, verbose=True)
#### Processing gates for 3 samples (multiprocessing is enabled - 3 cpus) ####
101_DEN084Y5_15_E03_009_clean.fcs: processing gate Time
101_DEN084Y5_15_E03_009_clean.fcs: processing gate Singlets
101_DEN084Y5_15_E03_009_clean.fcs: processing gate aAmine-
101_DEN084Y5_15_E01_008_clean.fcs: processing gate Time
101_DEN084Y5_15_E01_008_clean.fcs: processing gate Singlets
101_DEN084Y5_15_E01_008_clean.fcs: processing gate aAmine-
101_DEN084Y5_15_E05_010_clean.fcs: processing gate Time
101_DEN084Y5_15_E03_009_clean.fcs: processing gate CD3+
101_DEN084Y5_15_E05_010_clean.fcs: processing gate Singlets
101_DEN084Y5_15_E05_010_clean.fcs: processing gate aAmine-
101_DEN084Y5_15_E03_009_clean.fcs: processing gate CD4+
101_DEN084Y5_15_E01_008_clean.fcs: processing gate CD3+
101_DEN084Y5_15_E01_008_clean.fcs: processing gate CD4+
101_DEN084Y5_15_E05_010_clean.fcs: processing gate CD3+
101_DEN084Y5_15_E03_009_clean.fcs: processing gate CD8+
101_DEN084Y5_15_E05_010_clean.fcs: processing gate CD4+
101_DEN084Y5_15_E01_008_clean.fcs: processing gate CD8+
101_DEN084Y5_15_E03_009_clean.fcs: processing gate CD107a+
101_DEN084Y5_15_E05_010_clean.fcs: processing gate CD8+
101_DEN084Y5_15_E01_008_clean.fcs: processing gate CD107a+
101_DEN084Y5_15_E03_009_clean.fcs: processing gate IFNg+
101_DEN084Y5_15_E05_010_clean.fcs: processing gate CD107a+
101_DEN084Y5_15_E01_008_clean.fcs: processing gate IFNg+
101_DEN084Y5_15_E03_009_clean.fcs: processing gate IL2+
101_DEN084Y5_15_E05_010_clean.fcs: processing gate IFNg+
101_DEN084Y5_15_E01_008_clean.fcs: processing gate IL2+
101_DEN084Y5_15_E03_009_clean.fcs: processing gate TNFa+
101_DEN084Y5_15_E05_010_clean.fcs: processing gate IL2+
101_DEN084Y5_15_E01_008_clean.fcs: processing gate TNFa+
101_DEN084Y5_15_E03_009_clean.fcs: processing gate CD107a+
101_DEN084Y5_15_E05_010_clean.fcs: processing gate TNFa+
101_DEN084Y5_15_E01_008_clean.fcs: processing gate CD107a+
101_DEN084Y5_15_E03_009_clean.fcs: processing gate IFNg+
101_DEN084Y5_15_E05_010_clean.fcs: processing gate CD107a+
101_DEN084Y5_15_E01_008_clean.fcs: processing gate IFNg+
101_DEN084Y5_15_E03_009_clean.fcs: processing gate IL2+
101_DEN084Y5_15_E05_010_clean.fcs: processing gate IFNg+
101_DEN084Y5_15_E01_008_clean.fcs: processing gate IL2+
101_DEN084Y5_15_E03_009_clean.fcs: processing gate TNFa+
101_DEN084Y5_15_E05_010_clean.fcs: processing gate IL2+
101_DEN084Y5_15_E01_008_clean.fcs: processing gate TNFa+
101_DEN084Y5_15_E05_010_clean.fcs: processing gate TNFa+
# and a look a the results
session.get_group_report(sample_group)
sample  gate_path   gate_name   gate_type   quadrant_parent parent  count   absolute_percent    relative_percent    level
0   101_DEN084Y5_15_E03_009_clean.fcs   (root,) Time    RectangleGate   None    None    283968  99.999648   99.999648   1
1   101_DEN084Y5_15_E03_009_clean.fcs   (root, Time)    Singlets    PolygonGate None    Time    236780  83.382341   83.382635   2
2   101_DEN084Y5_15_E03_009_clean.fcs   (root, Time, Singlets)  aAmine- PolygonGate None    Singlets    161823  56.986150   68.343188   3
3   101_DEN084Y5_15_E03_009_clean.fcs   (root, Time, Singlets, aAmine-) CD3+    PolygonGate None    aAmine- 132200  46.554377   81.694197   4
4   101_DEN084Y5_15_E03_009_clean.fcs   (root, Time, Singlets, aAmine-, CD3+)   CD4+    PolygonGate None    CD3+    81855   28.825330   61.917549   5
5   101_DEN084Y5_15_E03_009_clean.fcs   (root, Time, Singlets, aAmine-, CD3+)   CD8+    PolygonGate None    CD3+    46965   16.538777   35.525719   5
6   101_DEN084Y5_15_E03_009_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD4+) CD107a+ RectangleGate   None    CD4+    59  0.020777    0.072079    6
10  101_DEN084Y5_15_E03_009_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD8+) CD107a+ RectangleGate   None    CD8+    814 0.286651    1.733206    6
7   101_DEN084Y5_15_E03_009_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD4+) IFNg+   RectangleGate   None    CD4+    598 0.210586    0.730560    6
11  101_DEN084Y5_15_E03_009_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD8+) IFNg+   RectangleGate   None    CD8+    1224    0.431033    2.606196    6
8   101_DEN084Y5_15_E03_009_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD4+) IL2+    RectangleGate   None    CD4+    64  0.022538    0.078187    6
12  101_DEN084Y5_15_E03_009_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD8+) IL2+    RectangleGate   None    CD8+    127 0.044723    0.270414    6
9   101_DEN084Y5_15_E03_009_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD4+) TNFa+   RectangleGate   None    CD4+    288 0.101420    0.351842    6
13  101_DEN084Y5_15_E03_009_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD8+) TNFa+   RectangleGate   None    CD8+    821 0.289116    1.748110    6
0   101_DEN084Y5_15_E01_008_clean.fcs   (root,) Time    RectangleGate   None    None    290166  99.997932   99.997932   1
1   101_DEN084Y5_15_E01_008_clean.fcs   (root, Time)    Singlets    PolygonGate None    Time    239001  82.365287   82.366990   2
2   101_DEN084Y5_15_E01_008_clean.fcs   (root, Time, Singlets)  aAmine- PolygonGate None    Singlets    164655  56.743931   68.893017   3
3   101_DEN084Y5_15_E01_008_clean.fcs   (root, Time, Singlets, aAmine-) CD3+    PolygonGate None    aAmine- 133670  46.065782   81.181865   4
4   101_DEN084Y5_15_E01_008_clean.fcs   (root, Time, Singlets, aAmine-, CD3+)   CD4+    PolygonGate None    CD3+    82484   28.425899   61.707189   5
5   101_DEN084Y5_15_E01_008_clean.fcs   (root, Time, Singlets, aAmine-, CD3+)   CD8+    PolygonGate None    CD3+    47165   16.254153   35.284656   5
6   101_DEN084Y5_15_E01_008_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD4+) CD107a+ RectangleGate   None    CD4+    68  0.023434    0.082440    6
10  101_DEN084Y5_15_E01_008_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD8+) CD107a+ RectangleGate   None    CD8+    73  0.025157    0.154776    6
7   101_DEN084Y5_15_E01_008_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD4+) IFNg+   RectangleGate   None    CD4+    4   0.001378    0.004849    6
11  101_DEN084Y5_15_E01_008_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD8+) IFNg+   RectangleGate   None    CD8+    2   0.000689    0.004240    6
8   101_DEN084Y5_15_E01_008_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD4+) IL2+    RectangleGate   None    CD4+    6   0.002068    0.007274    6
12  101_DEN084Y5_15_E01_008_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD8+) IL2+    RectangleGate   None    CD8+    10  0.003446    0.021202    6
9   101_DEN084Y5_15_E01_008_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD4+) TNFa+   RectangleGate   None    CD4+    21  0.007237    0.025459    6
13  101_DEN084Y5_15_E01_008_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD8+) TNFa+   RectangleGate   None    CD8+    8   0.002757    0.016962    6
0   101_DEN084Y5_15_E05_010_clean.fcs   (root,) Time    RectangleGate   None    None    284846  99.844369   99.844369   1
1   101_DEN084Y5_15_E05_010_clean.fcs   (root, Time)    Singlets    PolygonGate None    Time    235331  82.488345   82.616923   2
2   101_DEN084Y5_15_E05_010_clean.fcs   (root, Time, Singlets)  aAmine- PolygonGate None    Singlets    160955  56.418031   68.395154   3
3   101_DEN084Y5_15_E05_010_clean.fcs   (root, Time, Singlets, aAmine-) CD3+    PolygonGate None    aAmine- 131305  46.025097   81.578702   4
4   101_DEN084Y5_15_E05_010_clean.fcs   (root, Time, Singlets, aAmine-, CD3+)   CD4+    PolygonGate None    CD3+    81135   28.439483   61.791249   5
5   101_DEN084Y5_15_E05_010_clean.fcs   (root, Time, Singlets, aAmine-, CD3+)   CD8+    PolygonGate None    CD3+    46546   16.315328   35.448764   5
6   101_DEN084Y5_15_E05_010_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD4+) CD107a+ RectangleGate   None    CD4+    70  0.024536    0.086276    6
10  101_DEN084Y5_15_E05_010_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD8+) CD107a+ RectangleGate   None    CD8+    306 0.107259    0.657414    6
7   101_DEN084Y5_15_E05_010_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD4+) IFNg+   RectangleGate   None    CD4+    9   0.003155    0.011093    6
11  101_DEN084Y5_15_E05_010_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD8+) IFNg+   RectangleGate   None    CD8+    348 0.121981    0.747647    6
8   101_DEN084Y5_15_E05_010_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD4+) IL2+    RectangleGate   None    CD4+    8   0.002804    0.009860    6
12  101_DEN084Y5_15_E05_010_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD8+) IL2+    RectangleGate   None    CD8+    65  0.022784    0.139647    6
9   101_DEN084Y5_15_E05_010_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD4+) TNFa+   RectangleGate   None    CD4+    28  0.009815    0.034510    6
13  101_DEN084Y5_15_E05_010_clean.fcs   (root, Time, Singlets, aAmine-, CD3+, CD8+) TNFa+   RectangleGate   None    CD8+    336 0.117775    0.721867    6
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The flow cytometry standard (FCS) file specification does not include gating information. There is a separate specification, GatingML, that does provide gating definitions, though few applications adhere strictly to that standard. As the author of FlowKit, we wanted to provide a tool for the Python bioinformatics community that reads and creates valid GatingML documents for FCS data.

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