def gateBCellDataSet(fcs, *args, **kwargs):
ag.agconf.ag_verbose = False
no_clutter1 = ag.gateThreshold(fcs,
"no_clutter",
"FSC-A",
"FSC-H",
thresh=0,
orientation='vertical',
population="upper",
update=False)
no_clutter = ag.gateThreshold(fcs,
"no_clutter",
"FSC-A",
"FSC-H",
parentGate=no_clutter1,
thresh=250000,
orientation='vertical',
population="lower",
update=False)
date = ag.getFileName(ag.getParent(ag.getParent(fcs.filePath)))
plate = ag.getFileName(ag.getParent(fcs.filePath))
sampleName = ag.getFileName(fcs.filePath)
fileName = "plots/phase_II/BCell/singlets/" + date + "-" + plate + "-" + sampleName + "-singlets.png"
singlets = ag.gatePC(fcs,
"FSC-A",
"FSC-H",
"singlets",
center='density',
widthScale=4,
heightScale=4,
parentGate=no_clutter,
filePlot=fileName)
#singlets=ag.gatePC(fcs,"FSC-A", "FSC-H", "singlets",center='density',widthScale=4, heightScale=4, parentGate=no_clutter,update=False)
fcs.update(ag.AGgate(singlets, None, "FSC-A", "FSC-H", "singlets"),
QC=True,
xlim=[0, 250000],
ylim=[0, 500000])
#PBMCs
PBMCstep1 = ag.gateThreshold(fcs,
name="PBMCstep1",
xCol="FSC-A",
yCol="SSC-A",
parentGate=singlets,
orientation="horisontal",
thresh=70000,
population="upper",
update=False)
PBMCstep2 = ag.gateThreshold(fcs,
name="PBMCstep1",
xCol="FSC-A",
yCol="SSC-A",
parentGate=PBMCstep1,
orientation="horisontal",
thresh=180000,
population="lower",
update=False)
neutrofil_cutoff = ag.getHighestDensityPoint(fcs,
"FSC-A",
"SSC-A",
parentGate=PBMCstep2)[1]
PBMCstep3 = ag.gateThreshold(fcs,
name="PBMCstep1",
xCol="FSC-A",
yCol="SSC-A",
parentGate=singlets,
orientation="horisontal",
thresh=neutrofil_cutoff,
population="lower",
update=False)
start = ag.getHighestDensityPoint(fcs,
"FSC-A",
"SSC-A",
parentGate=PBMCstep3)[1]
if start > 20000:
start = 20000
PBMCstep4 = ag.shortestPathMatrix(fcs,
name="PBMCstep2",
xCol="FSC-A",
yCol="SSC-A",
maxStep=10,
boundaries=[start, neutrofil_cutoff],
sigma=2,
parentGate=singlets,
bins=50,
update=False)
fileName = "plots/phase_II/BCell/PBMCs/" + date + "-" + plate + "-" + sampleName + "-PBMCs.png"
PBMC = ag.gatePC(fcs,
name="PBMC",
xCol="FSC-A",
yCol="SSC-A",
center='centroid',
widthScale=4,
heightScale=3,
parentGate=PBMCstep4,
filePlot=fileName)
#PBMC=ag.gatePC(fcs,name="PBMC",xCol="FSC-A",yCol="SSC-A",center='centroid',widthScale=4, heightScale=3, parentGate=PBMCstep2,update=False)
fcs.update(ag.AGgate(PBMC, singlets, "FSC-A", "SSC-A", "PBMC"),
QC=True,
xlim=[0, 250000],
ylim=[0, 100000])
#CD45pos
mean, sigma, maxVal = ag.axisStats(fcs(), "CD34", PBMC())
mean, sigma = ag.halfNormalDistribution(fcs,
"CD34",
mean=mean,
parentGate=PBMC,
direction='left',
scale='bilog',
T=200)
ylim = ag.valleySeek(fcs,
"CD45",
parentGate=PBMC,
interval=[0, 2500],
sigma=1,
bins=1000)
xlim = ag.inverseBilogTransform([mean + 2 * abs(sigma)], 200)[0]
fileName = "plots/phase_II/BCell/CD45pos/" + date + "-" + plate + "-" + sampleName + "-CD45.png"
#CD45pos=ag.gateCorner(fcs,name="CD45pos",xCol="CD34",yCol="CD45",xThresh=xlim,yThresh=ylim,xOrientation="lower",yOrientation="upper", parentGate=PBMC,scale='bilog',T=200, filePlot=fileName)
CD45pos = ag.gateTiltedLine(fcs,
xCol="CD34",
yCol="CD45",
name="CD45pos",
parentGate=PBMC,
startPoint=(xlim, ylim),
theta=70,
endLimits=(None, None),
population='upper',
scale='bilog',
T=200,
filePlot=fileName)
fcs.update(ag.AGgate(CD45pos, PBMC, "CD34", "CD45", "CD45pos"),
QC=True,
xlim=[-4000, 250000],
ylim=[-2000, 70000],
scale='logish')
#CD34pos
CD45neg = ag.gateTiltedLine(fcs,
xCol="CD34",
yCol="CD45",
name="CD45pos",
parentGate=PBMC,
startPoint=(xlim, ylim),
theta=70,
endLimits=(None, None),
population='lower',
scale='bilog',
T=200,
filePlot=None)
#CD45neg=ag.gateCorner(fcs,name="CD45pos", xCol="CD34", yCol="CD45",xThresh=xlim,yThresh=ylim,xOrientation="lower",yOrientation="upper",Outer=True, parentGate=PBMC,scale='bilog', T=200,update=False)
ylim = ag.valleySeek(fcs,
"CD45",
parentGate=CD45neg,
interval=[500, 3500],
sigma=3,
bins=300,
scale='bilog',
T=200)
CD34step1 = ag.gateThreshold(fcs,
name="CD34step1",
xCol="CD45",
parentGate=CD45neg,
thresh=ylim,
scale='bilog',
T=200,
sigma=3,
population="lower",
update=False)
xlim = ag.valleySeek(fcs,
"CD34",
parentGate=CD34step1,
interval=[1500, 5000],
sigma=1,
bins=300,
scale='bilog',
T=200)
CD34pos = ag.gateThreshold(fcs,
name="CD34pos",
xCol="CD34",
yCol="CD45",
parentGate=CD34step1,
thresh=xlim,
population='upper',
scale='bilog',
T=200,
update=False)
fileName = "plots/phase_II/BCell/CD34pos/" + date + "-" + plate + "-" + sampleName + "-CD34.png"
ag.backGate(fcs,
"CD34",
"CD45",
background_population=PBMC,
population=CD34pos,
scale='bilog',
T=200,
markersize=0.5,
filePlot=fileName)
#CD45pos is not truly the parent of CD34, that would be PBMC.
#However The comparison is more robust to CD45
fcs.update(ag.AGgate(CD34pos, CD45pos, "CD34", "CD45", "CD34pos"),
QC=True,
xlim=[-2000, 250000],
ylim=[-2000, 70000],
scale='logish')
lim = ag.valleySeek(fcs,
xCol="CD19",
parentGate=CD45pos,
interval=[0, 4000],
bins=1000,
sigma=2,
scale='logish')
fileName = "plots/phase_II/BCell/CD19pos/" + date + "-" + plate + "-" + sampleName + "-CD19.png"
CD19pos = ag.gateThreshold(fcs,
"CD19pos",
"CD19",
"CD45",
parentGate=CD45pos,
thresh=lim,
scale='logish',
orientation='vertical',
population='upper',
filePlot=fileName)
#CD19pos=ag.gateThreshold(fcs,"CD19pos","CD19","CD45",parentGate=CD45pos,thresh=lim,scale='logish',orientation='vertical',population='upper')
fcs.update(ag.AGgate(CD19pos, CD45pos, "CD19", "CD45", "BCells"),
QC=True,
xlim=[-2000, 100000],
ylim=[1000, 100000],
scale='logish')
#We also add another BCell population, out of PBMCs
#That way we both have to more 'robust ratio' of CD19/CD45 and the 'truer ratio' but more noisy CD19/PBMC (that we can compare cross-panel to TCells)
fcs.update(ag.AGgate(CD19pos,
PBMC,
"CD19",
"CD45",
"ratio_BCellsPBMC",
RatioGate=True),
QC=False)
xlim = ag.valleySeek(fcs,
"IgD",
parentGate=CD19pos,
interval=[500, 2500],
bins=300,
sigma=2,
scale='logish',
T=200)
rightQuad = ag.gateThreshold(fcs,
"tmp",
"IgD",
"CD27",
thresh=xlim,
parentGate=CD19pos,
scale='logish',
T=200)
mean, sigma, maxval = ag.axisStats(fcs(),
"CD27",
vI=rightQuad(),
scale='logish',
T=200)
if maxval > 800: #Cover the case if upper cluster is more dense than lower
maxval = 0
mean, sigma = ag.halfNormalDistribution(fcs,
xCol='CD27',
mean=maxval,
direction='left',
parentGate=rightQuad,
scale='logish',
T=200)
ylim = ag.inverseLogishTransform([mean + 4 * abs(sigma)], 200)[0]
fileName = "plots/phase_II/BCell/quadgate/" + date + "-" + plate + "-" + sampleName + "-quadgate.png"
switchB, preSwitchB, naiveB, exhaustedB = ag.customQuadGate(
fcs, ['switchB', 'preSwitchB', 'naiveB', 'exhaustedB'],
"IgD",
"CD27",
threshList=[xlim, xlim, ylim, ylim],
parentGate=CD19pos,
scale='logish',
T=200,
filePlot=fileName)
fcs.update(ag.AGgate(switchB, CD19pos, "IgD", "CD27", "switchB"),
QC=True,
xlim=[-2000, 100000],
ylim=[-2000, 70000],
scale='logish')
fcs.update(ag.AGgate(preSwitchB, CD19pos, "IgD", "CD27", "preSwitchB"),
QC=False)
fcs.update(ag.AGgate(naiveB, CD19pos, "IgD", "CD27", "naiveB"), QC=False)
fcs.update(ag.AGgate(exhaustedB, CD19pos, "IgD", "CD27", "exhaustedB"),
QC=False)
lim = ag.valleySeek(fcs,
"IgA",
parentGate=switchB,
interval=[750, 2000],
bins=300,
sigma=1,
scale='logish',
T=200)
fileName = "plots/phase_II/BCell/IgApos/" + date + "-" + plate + "-" + sampleName + "-IgApos.png"
IgApos = ag.gateThreshold(fcs,
"IgAPos",
"IgA",
"CD19",
orientation='vertical',
parentGate=switchB,
thresh=lim,
scale='logish',
T=200,
filePlot=fileName)
#IgApos=ag.gateThreshold(fcs,"IgAPos", "IgA","CD19", orientation='vertical', parentGate=switchB, thresh=lim, scale='logish')
fcs.update(ag.AGgate(IgApos, switchB, "IgA", "CD19", "IgApos"),
QC=True,
xlim=[-2000, 100000],
ylim=[0, 100000],
scale='logish')
return fcs
def gateBCellDataSet(fcs, *args, **kwargs):
ag.agconf.ag_verbose = False
no_clutter = ag.gateThreshold(fcs,
"no_clutter",
"FSC-A",
"FSC-H",
thresh=250000,
orientation='vertical',
population="lower",
update=False)
date = ag.getFileName(ag.getParent(ag.getParent(fcs.filePath)))
plate = ag.getFileName(ag.getParent(fcs.filePath))
sampleName = ag.getFileName(fcs.filePath)
fileName = "plots/phase_I/BCell/singlets/" + date + "-" + plate + "-" + sampleName + "-singlets.png"
singlets = ag.gatePC(fcs,
"FSC-A",
"FSC-H",
"singlets",
center='density',
widthScale=4,
heightScale=4,
parentGate=no_clutter,
filePlot=fileName)
#singlets=ag.gatePC(fcs,"FSC-A", "FSC-H", "singlets",center='density',widthScale=4, heightScale=4, parentGate=no_clutter,update=False)
fcs.update(ag.AGgate(singlets, None, "FSC-A", "FSC-H", "singlets"),
QC=True,
xlim=[0, 250000],
ylim=[0, 500000])
PBMCstep1 = ag.gateThreshold(fcs,
name="PBMCstep1",
xCol="FSC-A",
yCol="SSC-A",
parentGate=singlets,
orientation="horisontal",
thresh=100000,
population="lower",
update=False)
start = ag.getHighestDensityPoint(fcs,
"FSC-A",
"SSC-A",
parentGate=PBMCstep1)[1]
if start > 20000:
start = 20000
PBMCstep2 = ag.shortestPathMatrix(fcs,
name="PBMCstep2",
xCol="FSC-A",
yCol="SSC-A",
maxStep=10,
boundaries=[start, 100000],
sigma=2,
parentGate=PBMCstep1,
bins=50,
update=False)
fileName = "plots/phase_I/BCell/PBMCs/" + date + "-" + plate + "-" + sampleName + "-PBMCs.png"
PBMC = ag.gatePC(fcs,
name="PBMC",
xCol="FSC-A",
yCol="SSC-A",
center='centroid',
widthScale=4,
heightScale=3,
parentGate=PBMCstep2,
filePlot=fileName)
#PBMC=ag.gatePC(fcs,name="PBMC",xCol="FSC-A",yCol="SSC-A",center='centroid',widthScale=4, heightScale=3, parentGate=PBMCstep2,update=False)
fcs.update(ag.AGgate(PBMC, singlets, "FSC-A", "SSC-A", "PBMC"),
QC=True,
xlim=[0, 250000],
ylim=[0, 100000])
mean, sigma, maxVal = ag.axisStats(fcs(), "CD34", PBMC())
mean, sigma = ag.halfNormalDistribution(fcs,
"CD34",
mean=mean,
parentGate=PBMC,
direction='left',
scale='linear')
ylim = ag.valleySeek(fcs,
"CD45",
parentGate=PBMC,
interval=[0, 2500],
sigma=1,
bins=1000)
xlim = mean + 4 * sigma
fileName = "plots/phase_I/BCell/CD45pos/" + date + "-" + plate + "-" + sampleName + "-CD45.png"
CD45pos = ag.gateCorner(fcs,
name="CD45pos",
xCol="CD34",
yCol="CD45",
xThresh=xlim,
yThresh=ylim,
xOrientation="lower",
yOrientation="upper",
parentGate=PBMC,
scale='logish',
filePlot=fileName)
#CD45pos=ag.gateCorner(fcs,name="CD45pos",xCol="CD34",yCol="CD45",xThresh=xlim,yThresh=ylim,xOrientation="lower",yOrientation="upper", parentGate=PBMC,scale='logish')
fcs.update(ag.AGgate(CD45pos, PBMC, "CD34", "CD45", "CD45pos"),
QC=True,
xlim=[-4000, 250000],
ylim=[-2000, 70000],
scale='logish')
CD45neg = ag.gateCorner(fcs,
name="CD45pos",
xCol="CD34",
yCol="CD45",
xThresh=xlim,
yThresh=ylim,
xOrientation="lower",
yOrientation="upper",
Outer=True,
parentGate=PBMC,
scale='logish',
update=False)
ylim = ag.valleySeek(fcs,
"CD45",
parentGate=CD45neg,
interval=[500, 3500],
sigma=1,
bins=1000,
scale='logish')
CD34step1 = ag.gateThreshold(fcs,
name="CD34step1",
xCol="CD45",
parentGate=CD45neg,
thresh=ylim,
scale='logish',
population="lower",
update=False)
xlim = ag.valleySeek(fcs,
"CD34",
parentGate=CD34step1,
interval=[1500, 5000],
sigma=3,
bins=300,
scale='logish')
fileName = "plots/phase_I/BCell/CD34pos/" + date + "-" + plate + "-" + sampleName + "-CD34.png"
CD34pos = ag.gateThreshold(fcs,
name="CD34pos",
xCol="CD34",
yCol="CD45",
parentGate=CD34step1,
thresh=xlim,
population='upper',
scale='logish',
update=False)
ag.backGate(fcs,
"CD34",
"CD45",
background_population=PBMC,
population=CD34pos,
scale='logish',
markersize=0.5,
filePlot=fileName)
#CD45pos is not truly the parent of CD34, that would be PBMC.
#However The comparison is more robust to CD45
fcs.update(ag.AGgate(CD34pos, CD45pos, "CD34", "CD45", "CD34pos"),
QC=True,
xlim=[-2000, 250000],
ylim=[-2000, 70000],
scale='logish')
lim = ag.valleySeek(fcs,
xCol="CD19",
parentGate=CD45pos,
interval=[0, 2000],
bins=1000,
sigma=2,
scale='logish')
fileName = "plots/phase_I/BCell/CD19pos/" + date + "-" + plate + "-" + sampleName + "-CD19.png"
CD19pos = ag.gateThreshold(fcs,
"CD19pos",
"CD19",
"CD45",
parentGate=CD45pos,
thresh=lim,
scale='logish',
orientation='vertical',
population='upper',
filePlot=fileName)
#CD19pos=ag.gateThreshold(fcs,"CD19pos","CD19","CD45",parentGate=CD45pos,thresh=lim,scale='logish',orientation='vertical',population='upper')
fcs.update(ag.AGgate(CD19pos, CD45pos, "CD19", "CD45", "BCells"),
QC=True,
xlim=[-2000, 100000],
ylim=[1000, 100000],
scale='logish')
#We also add another BCell population, out of PBMCs
#That way we both have to more 'robust ratio' of CD19/CD45 and the 'truer ratio' but more noisy CD19/PBMC (that we can compare cross-panel to TCells)
fcs.update(ag.AGgate(CD19pos,
PBMC,
"CD19",
"CD45",
"ratio_BCellsPBMC",
RatioGate=True),
QC=False)
xlim = ag.valleySeek(fcs,
"IgD",
parentGate=CD19pos,
interval=[500, 2500],
bins=300,
sigma=2,
scale='logish')
ylim = ag.valleySeek(fcs,
"CD27",
parentGate=CD19pos,
interval=[500, 1000],
bins=300,
sigma=2,
scale='logish')
fileName = "plots/phase_I/BCell/quadgate/" + date + "-" + plate + "-" + sampleName + "-quadgate.png"
solution = ag.variableQuadGate(fcs, ['', '', '', ''],
"IgD",
"CD27", [xlim, xlim, ylim, ylim],
testRange=[2000, 3000],
position='left',
parentGate=CD19pos,
scale='logish',
only_solution=True,
scoreThresh=0.6)
switchB, preSwitchB, naiveB, exhaustedB, solution = ag.variableQuadGate(
fcs, ['switchB', 'preSwitchB', 'naiveB', 'exhaustedB'],
"IgD",
"CD27",
solution,
testRange=[0, ylim],
position='right',
parentGate=CD19pos,
scale='logish',
filePlot=fileName)
#switchB, preSwitchB, naiveB, exhaustedB,solution = ag.variableQuadGate(fcs,['switchB','preSwitchB','naiveB','exhaustedB'], "IgD", "CD27", threshList=solution, testRange=[0,ylim], position='right', parentGate=CD19pos,scale='logish')
#switchB, preSwitchB, naiveB, exhaustedB,solution = ag.variableQuadGate(fcs(), "IgD", "CD27", solution, testRange=[0,ylim], position='right', vI=CD19pos,scale='logish', plot=False)
fcs.update(ag.AGgate(switchB, CD19pos, "IgD", "CD27", "switchB"),
QC=True,
xlim=[-2000, 100000],
ylim=[-2000, 70000],
scale='logish')
fcs.update(ag.AGgate(preSwitchB, CD19pos, "IgD", "CD27", "preSwitchB"),
QC=False)
fcs.update(ag.AGgate(naiveB, CD19pos, "IgD", "CD27", "naiveB"), QC=False)
fcs.update(ag.AGgate(exhaustedB, CD19pos, "IgD", "CD27", "exhaustedB"),
QC=False)
CD38thresh = gatePlasmablasts(fcs, switchB)
switchB = fcs("switchB")
fileName = "plots/phase_I/BCell/plasmablasts/" + date + "-" + plate + "-" + sampleName + "-plasmablasts.png"
plasmablasts = ag.gateCorner(fcs,
"plasmablasts",
"CD24",
"CD38",
1000,
CD38thresh,
"lower",
"upper",
parentGate=switchB,
scale='logish',
filePlot=fileName)
#plasmablasts=ag.gateCorner(fcs, "plasmablasts", "CD24", "CD38",1000,CD38thresh,"lower","upper", parentGate=switchB,scale='logish')
fcs.update(ag.AGgate(plasmablasts, switchB, "CD24", "CD38",
"plasmablasts"),
QC=True,
xlim=[-2000, 50000],
ylim=[-2000, 70000],
scale='logish')
xmean, xsigma, xmaxVal = ag.axisStats(fcs(), xCol="CD24", vI=naiveB())
ymean, ysigma, ymaxVal = ag.axisStats(fcs(), xCol="CD38", vI=naiveB())
naiveB = fcs("naiveB")
fileName = "plots/phase_I/BCell/transitionals/" + date + "-" + plate + "-" + sampleName + "-transitionals.png"
transitionals = ag.gateCorner(fcs,
"transitionals",
"CD24",
"CD38",
xThresh=xmaxVal,
yThresh=ymaxVal + 2000,
parentGate=naiveB,
scale='logish',
filePlot=fileName)
#transitionals=ag.gateCorner(fcs, "transitionals","CD24", "CD38",xThresh = xmaxVal, yThresh=ymaxVal+2000, parentGate=naiveB, scale='logish')
fcs.update(ag.AGgate(transitionals, naiveB, "CD24", "CD38",
"transitionals"),
QC=True,
xlim=[-2000, 40000],
ylim=[-2000, 40000],
scale='logish')
lim = ag.valleySeek(fcs,
"IgA",
parentGate=switchB,
interval=[750, 2000],
bins=300,
sigma=1,
scale='logish')
fileName = "plots/phase_I/BCell/IgApos/" + date + "-" + plate + "-" + sampleName + "-IgApos.png"
IgApos = ag.gateThreshold(fcs,
"IgAPos",
"IgA",
"CD19",
orientation='vertical',
parentGate=switchB,
thresh=lim,
scale='logish',
filePlot=fileName)
#IgApos=ag.gateThreshold(fcs,"IgAPos", "IgA","CD19", orientation='vertical', parentGate=switchB, thresh=lim, scale='logish')
fcs.update(ag.AGgate(IgApos, switchB, "IgA", "CD19", "IgApos"),
QC=True,
xlim=[-2000, 100000],
ylim=[0, 100000],
scale='logish')
return fcs
def gateBCellDataSet(fcs, *args, **kwargs):
ag.agconf.ag_verbose = False
no_clutter1 = ag.gateThreshold(fcs,
"no_clutter",
"FSC-A",
"FSC-H",
thresh=0,
orientation='vertical',
population="upper",
update=False)
no_clutter = ag.gateThreshold(fcs,
"no_clutter",
"FSC-A",
"FSC-H",
parentGate=no_clutter1,
thresh=250000,
orientation='vertical',
population="lower",
update=False)
date = ag.getFileName(ag.getParent(ag.getParent(fcs.filePath)))
plate = ag.getFileName(ag.getParent(fcs.filePath))
sampleName = ag.getFileName(fcs.filePath)
fileName = "plots/phase_II/BCell/singlets/" + date + "-" + plate + "-" + sampleName + "-singlets.png"
singlets = ag.gatePC(fcs,
"FSC-A",
"FSC-H",
"singlets",
center='density',
widthScale=4,
heightScale=4,
parentGate=no_clutter,
filePlot=fileName)
#singlets=ag.gatePC(fcs,"FSC-A", "FSC-H", "singlets",center='density',widthScale=4, heightScale=4, parentGate=no_clutter,update=False)
fcs.update(ag.AGgate(singlets, None, "FSC-A", "FSC-H", "singlets"),
QC=True,
xlim=[0, 250000],
ylim=[0, 500000])
#PBMCs
PBMCstep1 = ag.gateThreshold(fcs,
name="PBMCstep1",
xCol="FSC-A",
yCol="SSC-A",
parentGate=singlets,
orientation="horisontal",
thresh=70000,
population="upper",
update=False)
PBMCstep2 = ag.gateThreshold(fcs,
name="PBMCstep1",
xCol="FSC-A",
yCol="SSC-A",
parentGate=PBMCstep1,
orientation="horisontal",
thresh=180000,
population="lower",
update=False)
neutrofil_cutoff = ag.getHighestDensityPoint(fcs,
"FSC-A",
"SSC-A",
parentGate=PBMCstep2)[1]
PBMCstep3 = ag.gateThreshold(fcs,
name="PBMCstep1",
xCol="FSC-A",
yCol="SSC-A",
parentGate=singlets,
orientation="horisontal",
thresh=neutrofil_cutoff,
population="lower",
update=False)
start = ag.getHighestDensityPoint(fcs,
"FSC-A",
"SSC-A",
parentGate=PBMCstep3)[1]
if start > 20000:
start = 20000
PBMCstep4 = ag.shortestPathMatrix(fcs,
name="PBMCstep2",
xCol="FSC-A",
yCol="SSC-A",
maxStep=10,
boundaries=[start, neutrofil_cutoff],
sigma=2,
parentGate=singlets,
bins=50,
update=False)
fileName = "plots/phase_II/BCell/PBMCs/" + date + "-" + plate + "-" + sampleName + "-PBMCs.png"
PBMC = ag.gatePC(fcs,
name="PBMC",
xCol="FSC-A",
yCol="SSC-A",
center='centroid',
widthScale=4,
heightScale=3,
parentGate=PBMCstep4,
filePlot=fileName)
#PBMC=ag.gatePC(fcs,name="PBMC",xCol="FSC-A",yCol="SSC-A",center='centroid',widthScale=4, heightScale=3, parentGate=PBMCstep2,update=False)
fcs.update(ag.AGgate(PBMC, singlets, "FSC-A", "SSC-A", "PBMC"),
QC=True,
xlim=[0, 250000],
ylim=[0, 100000])
#CD45pos
mean, sigma, maxVal = ag.axisStats(fcs(), "CD34", PBMC())
mean, sigma = ag.halfNormalDistribution(fcs,
"CD34",
mean=mean,
parentGate=PBMC,
direction='left',
scale='bilog',
T=200)
ylim = ag.valleySeek(fcs,
"CD45",
parentGate=PBMC,
interval=[0, 2500],
sigma=1,
bins=1000)
xlim = ag.inverseBilogTransform([mean + 2 * abs(sigma)], 200)[0]
fileName = "plots/phase_II/BCell/CD45pos/" + date + "-" + plate + "-" + sampleName + "-CD45.png"
#CD45pos=ag.gateCorner(fcs,name="CD45pos",xCol="CD34",yCol="CD45",xThresh=xlim,yThresh=ylim,xOrientation="lower",yOrientation="upper", parentGate=PBMC,scale='bilog',T=200, filePlot=fileName)
CD45pos = ag.gateTiltedLine(fcs,
xCol="CD34",
yCol="CD45",
name="CD45pos",
parentGate=PBMC,
startPoint=(xlim, ylim),
theta=70,
endLimits=(None, None),
population='upper',
scale='bilog',
T=200,
filePlot=fileName)
fcs.update(ag.AGgate(CD45pos, PBMC, "CD34", "CD45", "CD45pos"),
QC=True,
xlim=[-4000, 250000],
ylim=[-2000, 70000],
scale='logish')
lim = ag.valleySeek(fcs,
xCol="CD19",
parentGate=CD45pos,
interval=[0, 4000],
bins=1000,
sigma=2,
scale='logish')
fileName = "plots/phase_II/BCell/CD19pos/" + date + "-" + plate + "-" + sampleName + "-CD19.png"
CD19pos = ag.gateThreshold(fcs,
"CD19pos",
"CD19",
"CD45",
parentGate=CD45pos,
thresh=lim,
scale='logish',
orientation='vertical',
population='upper',
filePlot=fileName)
#CD19pos=ag.gateThreshold(fcs,"CD19pos","CD19","CD45",parentGate=CD45pos,thresh=lim,scale='logish',orientation='vertical',population='upper')
fcs.update(ag.AGgate(CD19pos, CD45pos, "CD19", "CD45", "BCells"),
QC=True,
xlim=[-2000, 100000],
ylim=[1000, 100000],
scale='logish')
#We also add another BCell population, out of PBMCs
#That way we both have to more 'robust ratio' of CD19/CD45 and the 'truer ratio' but more noisy CD19/PBMC (that we can compare cross-panel to TCells)
fcs.update(ag.AGgate(CD19pos,
PBMC,
"CD19",
"CD45",
"ratio_BCellsPBMC",
RatioGate=True),
QC=False)
xlim = ag.valleySeek(fcs,
"IgD",
parentGate=CD19pos,
interval=[500, 2500],
bins=300,
sigma=2,
scale='logish',
T=200)
rightQuad = ag.gateThreshold(fcs,
"tmp",
"IgD",
"CD27",
thresh=xlim,
parentGate=CD19pos,
scale='logish',
T=200)
mean, sigma, maxval = ag.axisStats(fcs(),
"CD27",
vI=rightQuad(),
scale='logish',
T=200)
if maxval > 800: #Cover the case if upper cluster is more dense than lower
maxval = 0
mean, sigma = ag.halfNormalDistribution(fcs,
xCol='CD27',
mean=maxval,
direction='left',
parentGate=rightQuad,
scale='logish',
T=200)
ylim = ag.inverseLogishTransform([mean + 4 * abs(sigma)], 200)[0]
fileName = "plots/phase_II/BCell/quadgate/" + date + "-" + plate + "-" + sampleName + "-quadgate.png"
switchB, preSwitchB, naiveB, exhaustedB = ag.customQuadGate(
fcs, ['switchB', 'preSwitchB', 'naiveB', 'exhaustedB'],
"IgD",
"CD27",
threshList=[xlim, xlim, ylim, ylim],
parentGate=CD19pos,
scale='logish',
T=200,
filePlot=fileName)
#solution = ag.variableQuadGate(fcs,['','','',''], "IgD", "CD27", [xlim, xlim, ylim, ylim], testRange=[2000,3000], position='left', parentGate=CD19pos,scale='logish',T=200,only_solution=True, scoreThresh=0.6)
#switchB, preSwitchB, naiveB, exhaustedB,solution = ag.variableQuadGate(fcs, ['switchB', 'preSwitchB', 'naiveB', 'exhaustedB'], "IgD", "CD27", solution, testRange=[0,ylim], position='right', parentGate=CD19pos,scale='logish',T=200, filePlot=fileName)
#switchB, preSwitchB, naiveB, exhaustedB,solution = ag.variableQuadGate(fcs,['switchB','preSwitchB','naiveB','exhaustedB'], "IgD", "CD27", threshList=solution, testRange=[0,ylim], position='right', parentGate=CD19pos,scale='logish')
#switchB, preSwitchB, naiveB, exhaustedB,solution = ag.variableQuadGate(fcs(), "IgD", "CD27", solution, testRange=[0,ylim], position='right', vI=CD19pos,scale='logish', plot=False)
fcs.update(ag.AGgate(switchB, CD19pos, "IgD", "CD27", "switchB"),
QC=True,
xlim=[-2000, 100000],
ylim=[-2000, 70000],
scale='logish')
fcs.update(ag.AGgate(preSwitchB, CD19pos, "IgD", "CD27", "preSwitchB"),
QC=False)
fcs.update(ag.AGgate(naiveB, CD19pos, "IgD", "CD27", "naiveB"), QC=False)
fcs.update(ag.AGgate(exhaustedB, CD19pos, "IgD", "CD27", "exhaustedB"),
QC=False)
plasmablastStep1 = ag.gateThreshold(fcs,
"plasmatmp",
"CD24",
"CD38",
thresh=10000,
orientation='horisontal',
parentGate=switchB,
scale='logish',
T=200)
if not len(plasmablastStep1()) == 0:
xlim = max(ag.getGatedVector(fcs(), "CD24",
vI=plasmablastStep1())) + 50
if xlim <= 150 or xlim >= 1000:
xlim = 600
else:
xlim = 600
CD38thresh = gatePlasmablasts(fcs, switchB, xlim)
switchB = fcs("switchB")
fileName = "plots/phase_II/BCell/plasmablasts/" + date + "-" + plate + "-" + sampleName + "-plasmablasts.png"
plasmablasts = ag.gateCorner(fcs,
"plasmablasts",
"CD24",
"CD38",
xlim,
CD38thresh,
"lower",
"upper",
parentGate=switchB,
scale='logish',
T=200,
filePlot=fileName)
#plasmablasts=ag.gateCorner(fcs, "plasmablasts", "CD24", "CD38",1000,CD38thresh,"lower","upper", parentGate=switchB,scale='logish')
fcs.update(ag.AGgate(plasmablasts, switchB, "CD24", "CD38",
"plasmablasts"),
QC=True,
xlim=[-2000, 50000],
ylim=[-2000, 70000],
scale='logish')
xmean, xsigma, xmaxVal = ag.axisStats(fcs(), xCol="CD24", vI=naiveB())
ymean, ysigma, ymaxVal = ag.axisStats(fcs(), xCol="CD38", vI=naiveB())
naiveB = fcs("naiveB")
fileName = "plots/phase_II/BCell/transitionals/" + date + "-" + plate + "-" + sampleName + "-transitionals.png"
transitionals = ag.gateCorner(fcs,
"transitionals",
"CD24",
"CD38",
xThresh=xmaxVal,
yThresh=ymaxVal + 2000,
parentGate=naiveB,
scale='logish',
T=200,
filePlot=fileName)
#transitionals=ag.gateCorner(fcs, "transitionals","CD24", "CD38",xThresh = xmaxVal, yThresh=ymaxVal+2000, parentGate=naiveB, scale='logish')
fcs.update(ag.AGgate(transitionals, naiveB, "CD24", "CD38",
"transitionals"),
QC=True,
xlim=[-2000, 40000],
ylim=[-2000, 40000],
scale='logish')
lim = ag.valleySeek(fcs,
"IgA",
parentGate=switchB,
interval=[750, 2000],
bins=300,
sigma=1,
scale='logish',
T=200)
fileName = "plots/phase_II/BCell/IgApos/" + date + "-" + plate + "-" + sampleName + "-IgApos.png"
IgApos = ag.gateThreshold(fcs,
"IgAPos",
"IgA",
"CD19",
orientation='vertical',
parentGate=switchB,
thresh=lim,
scale='logish',
T=200,
filePlot=fileName)
#IgApos=ag.gateThreshold(fcs,"IgAPos", "IgA","CD19", orientation='vertical', parentGate=switchB, thresh=lim, scale='logish')
fcs.update(ag.AGgate(IgApos, switchB, "IgA", "CD19", "IgApos"),
QC=True,
xlim=[-2000, 100000],
ylim=[0, 100000],
scale='logish')
return fcs
