def runLigPgm(self, vol, ligtemp, inactivate=True, inacttemp=65):
if inactivate:
pgm = "LIG15-%.0f" % ligtemp
worklist.pyrun(
'PTC\\ptcsetpgm.py %s TEMP@%.0f,900 TEMP@%.0f,600 TEMP@25,30' %
(pgm, ligtemp, inacttemp))
self.e.runpgm(pgm,
27,
False,
vol,
hotlidmode="TRACKING",
hotlidtemp=10)
elif ligtemp == 25:
worklist.comment('Ligation at room temp')
self.e.pause(15 * 60)
else:
pgm = "TRP%.0f-15" % ligtemp
worklist.pyrun('PTC\\ptcsetpgm.py %s TEMP@%.0f,900 TEMP@25,30' %
(pgm, ligtemp))
self.e.runpgm(pgm,
17,
False,
vol,
hotlidmode="TRACKING",
hotlidtemp=10)
def run(self,confirm=False,enzName="EvaUSER",waitForPTC=True):
"""Run the dilutions and QPCR setup"""
# Setup qPCRs
#self.jobq.dump()
self.idler(100000)
if waitForPTC:
self.trp.e.waitpgm() # May still need to wait for TC to complete before able to do final jobs
self.idler(100000)
worklist.flushQueue()
if self.jobq.len()>0:
logging.error( "Blocked jobs remain on queue:")
self.jobq.dump()
assert False
if len(self.dilProds)==0:
return
if confirm:
worklist.userprompt('Continue to setup qPCR')
worklist.comment('Starting qPCR setup')
self.trp.e.sanitize(force=True)
if all([allp in p for allp in self.allprimers() for p in self.primers]):
print("All samples use same qPCR primers")
# This allows the Eva to be distributed all at once
self.trp.runQPCR(src=self.dilProds,vol=self.volume,primers=self.allprimers(),nreplicates=self.nreplicates,enzName=enzName)
else:
for p in self.allprimers():
# Build list of relevant entries
ind=[ i for i in range(len(self.dilProds)) if p in self.primers[i]]
self.trp.runQPCR(src=[self.dilProds[i] for i in ind],vol=self.volume,primers=[p],nreplicates=[self.nreplicates[i] for i in ind],enzName=enzName)
def runQPCR(self,src,vol,primers,nreplicates=1,enzName="EvaUSER"):
## QPCR setup
worklist.comment("runQPCR: primers=%s, source=%s"%([p for p in primers],[s.name for s in src]))
[src,vol,nreplicates]=listify([src,vol,nreplicates])
self.e.shakeSamples(src,returnPlate=True)
# Build a list of sets to be run
torun=[]
for repl in range(max(nreplicates)):
for p in primers:
for i in range(len(src)):
if nreplicates[i]<=repl:
continue
if repl==0:
sampname="%s.Q%s"%(src[i].name,p)
else:
sampname="%s.Q%s.%d"%(src[i].name,p,repl+1)
s=Sample(sampname,decklayout.QPCRPLATE)
torun=torun+[(src[i],s,p,vol[i])]
# Add enzyme
e=reagents.getsample(enzName)
v=[a[3]/e.conc.dilutionneeded() for a in torun]
t=[a[1] for a in torun]
self.e.multitransfer(v,e,t)
# Make the target have 'none' concentration so we can multiadd to it again
for s in t:
s.conc=None
# Fill the master mixes
dil={}
for p in primers:
mname="P-%s"%p
if not reagents.isReagent(mname):
reagents.add(name=mname,conc=4,extraVol=30)
mq=reagents.getsample(mname)
t=[a[1] for a in torun if a[2]==p]
v=[a[3]/mq.conc.dilutionneeded() for a in torun if a[2]==p]
assert(v>0)
self.e.multitransfer(v,mq,t,(False,False))
dil[p]=1.0/(1-1/e.conc.dilutionneeded()-1/mq.conc.dilutionneeded())
# Add the samples
self.e.sanitize() # In case we are aligned
for a in torun:
s=a[0]
t=a[1]
p=a[2]
v=a[3]/dil[p]
t.conc=None # Concentration of master mix is irrelevant now
self.e.transfer(v,s,t)
return [a[1] for a in torun]
def runLigPgm(self,vol,ligtemp,inactivate=True,inacttemp=65):
if inactivate:
pgm="LIG15-%.0f"%ligtemp
worklist.pyrun('PTC\\ptcsetpgm.py %s TEMP@%.0f,900 TEMP@%.0f,600 TEMP@25,30'%(pgm,ligtemp,inacttemp))
self.e.runpgm(pgm,27,False,vol,hotlidmode="TRACKING",hotlidtemp=10)
elif ligtemp==25:
worklist.comment('Ligation at room temp')
self.e.pause(15*60)
else:
pgm="TRP%.0f-15"%ligtemp
worklist.pyrun('PTC\\ptcsetpgm.py %s TEMP@%.0f,900 TEMP@25,30'%(pgm,ligtemp))
self.e.runpgm(pgm,17,False,vol,hotlidmode="TRACKING",hotlidtemp=10)
def runT7Setup(self, theo, src, vol, srcdil, tgt=None, rlist=["MT7"]):
[theo, src, tgt, srcdil] = listify([theo, src, tgt, srcdil])
for i in range(len(src)):
if tgt[i] is None:
if theo[i]:
tgt[i] = Sample("%s.T+" % src[i].name,
decklayout.SAMPLEPLATE)
else:
tgt[i] = Sample("%s.T-" % src[i].name,
decklayout.SAMPLEPLATE)
worklist.comment("runT7: source=%s" % [str(s) for s in src])
rvols = [reagents.getsample(x).conc.volneeded(vol) for x in rlist]
rtotal = sum(rvols)
sourcevols = [vol * 1.0 / s for s in srcdil]
if any(theo):
theovols = [
(vol * 1.0 /
reagents.getsample("Theo").conc.dilutionneeded() if t else 0)
for t in theo
]
watervols = [
vol - theovols[i] - sourcevols[i] - rtotal
for i in range(len(src))
]
else:
watervols = [vol - sourcevols[i] - rtotal for i in range(len(src))]
if any([w < -1e-10 for w in watervols]):
print "runT7Setup: Negative amount of water required: ", watervols
assert False
if sum(watervols) > 0.01:
self.e.multitransfer(watervols, decklayout.WATER, tgt)
for ir in range(len(rlist)):
self.e.multitransfer([rvols[ir] for s in tgt],
reagents.getsample(rlist[ir]), tgt)
if any(theo):
self.e.multitransfer(
[tv for tv in theovols if tv > 0.01],
reagents.getsample("Theo"),
[tgt[i] for i in range(len(theovols)) if theovols[i] > 0],
ignoreContents=True)
for i in range(len(src)):
self.e.transfer(sourcevols[i], src[i], tgt[i])
self.e.shakeSamples(tgt, returnPlate=True)
for t in tgt:
t.ingredients['BIND'] = 1e-20 * sum(t.ingredients.values())
return tgt
def run(self):
'Run the dilutions and QPCR setup'
# Setup qPCRs
#self.jobq.dump()
self.idler(100000)
self.trp.e.waitpgm() # May still need to wait for PTC to complete before able to do final jobs
self.idler(100000)
if self.jobq.len()>0:
logging.error( "Blocked jobs remain on queue:",fatal=False)
self.jobq.dump()
assert False
worklist.comment('Starting qPCR setup')
for p in self.allprimers():
# Build list of relevant entries
ind=[ i for i in range(len(self.dilProds)) if p in self.primers[i]]
self.trp.runQPCR(src=[self.dilProds[i] for i in ind],vol=self.volume,primers=[p],nreplicates=[self.nreplicates[i] for i in ind])
def runT7Setup(self,src,vol,srcdil,ligands=None,tgt=None,rlist=["MT7"]):
if isinstance(ligands,bool):
if not ligands:
ligands=None
else:
assert('runT7Setup: ligands arg should be ligand samples or None, not True')
[ligands,src,tgt,srcdil]=listify([ligands,src,tgt,srcdil])
for i in range(len(src)):
if tgt[i] is None:
if ligands[i] is not None:
tgt[i]=Sample("%s.T+%s"%(src[i].name,ligands[i].name),decklayout.SAMPLEPLATE)
else:
tgt[i]=Sample("%s.T-"%src[i].name,decklayout.SAMPLEPLATE)
worklist.comment("runT7: source=%s"%[str(s) for s in src])
rvols=[reagents.getsample(x).conc.volneeded(vol) for x in rlist]
rtotal=sum(rvols)
sourcevols=[vol*1.0/s for s in srcdil]
ligandvols=[0 for s in srcdil]
watervols=[0 for s in srcdil]
for i in range(len(srcdil)):
if ligands[i] is not None:
ligandvols[i]=vol*1.0/ligands[i].conc.dilutionneeded()
watervols[i]=vol-ligandvols[i]-sourcevols[i]-rtotal
else:
watervols[i]=vol-sourcevols[i]-rtotal
if any([w<-.01 for w in watervols]):
logging.error("runT7Setup: Negative amount of water required: "+str(watervols))
if sum(watervols)>0.01:
self.e.multitransfer(watervols,decklayout.WATER,tgt)
for ir in range(len(rlist)):
self.e.multitransfer([rvols[ir] for s in tgt],reagents.getsample(rlist[ir]),tgt)
for i in range(len(ligands)):
if ligandvols[i] > 0.01:
self.e.transfer(ligandvols[i],ligands[i],tgt[i])
for i in range(len(src)):
self.e.transfer(sourcevols[i],src[i],tgt[i])
self.e.shakeSamples(tgt,returnPlate=True)
for t in tgt:
t.ingredients['BIND']=1e-20*sum(t.ingredients.values())
return tgt
def runQPCR(self, src, vol, srcdil, primers=["A", "B"], nreplicates=1):
## QPCR setup
worklist.comment("runQPCR: primers=%s, source=%s" %
([p for p in primers], [s.name for s in src]))
[src, vol, srcdil,
nreplicates] = listify([src, vol, srcdil, nreplicates])
self.e.shakeSamples(src, returnPlate=True)
# Build a list of sets to be run
torun = []
for repl in range(max(nreplicates)):
for p in primers:
for i in range(len(src)):
if nreplicates[i] <= repl:
continue
if repl == 0:
sampname = "%s.Q%s" % (src[i].name, p)
else:
sampname = "%s.Q%s.%d" % (src[i].name, p, repl + 1)
s = Sample(sampname, decklayout.QPCRPLATE)
torun = torun + [(src[i], s, p, vol[i])]
# Fill the master mixes
dil = {}
for p in primers:
mname = "MQ%s" % p
if not reagents.isReagent(mname):
reagents.add(name=mname, conc=15.0 / 9.0, extraVol=30)
mq = reagents.getsample(mname)
t = [a[1] for a in torun if a[2] == p]
v = [a[3] / mq.conc.dilutionneeded() for a in torun if a[2] == p]
self.e.multitransfer(v, mq, t, (False, False))
dil[p] = 1.0 / (1 - 1 / mq.conc.dilutionneeded())
# Add the samples
self.e.sanitize() # In case we are aligned
for a in torun:
s = a[0]
t = a[1]
p = a[2]
v = a[3] / dil[p]
t.conc = None # Concentration of master mix is irrelevant now
self.e.transfer(v, s, t)
return [a[1] for a in torun]
def pgm(self):
self.q = QSetup(self, maxdil=16, debug=False, mindilvol=100)
self.e.addIdleProgram(self.q.idler)
if self.doqpcr:
self.q.addReferences(dstep=10, primers=self.qprimers, ref=reagents.getsample("BT5310"))
print("### Barcoding #### (%.0f min)" % (clock.elapsed() / 60.0))
bcout = self.barcoding(names=[x['name'] for x in self.inputs], left=[x['left'] for x in self.inputs],
right=[x['right'] for x in self.inputs])
for i in range(len(self.inputs)):
x = self.inputs[i]
if 'bconc' in x and x['bconc'] is not None:
print("Resetting concentration of %s from expected %.1f to bconc setting of %.1f nM" % \
(x['name'], bcout[i].conc.stock, x['bconc']))
bcout[i].conc.stock = x['bconc']
print("### qPCR #### (%.0f min)" % (clock.elapsed() / 60.0))
self.q.run(confirm=False, enzName='EvaGreen')
print("### qPCR Done #### (%.0f min)" % (clock.elapsed() / 60.0))
print("### Final PCR Done #### (%.0f min)" % (clock.elapsed() / 60.0))
worklist.flushQueue()
if all(['bconc' in x and x['bconc'] is not None for x in self.inputs]):
print("### Mixdown #### (%.0f min)" % (clock.elapsed() / 60.0))
worklist.flushQueue()
worklist.comment('Start mixdown only at this point')
self.e.sanitize(force=True)
# mixdown = self.mix(bcout, [x['weight'] for x in self.inputs])
# Set default mix number to 1
for x in self.inputs:
if 'mix' not in x:
x['mix']=1
mixes=set([x['mix'] for x in self.inputs])
for m in mixes:
sel=[ i for i in range(len(self.inputs)) if self.inputs[i]['mix']==m]
print("sel=",sel)
mixdown = self.mix([bcout[i] for i in sel], [self.inputs[i]['weight'] for i in sel],prefix="Mix%d_"%m)
mixdown.name="Mix%d_Final"%m
# self.q.addSamples(mixdown, needDil=mixdown.conc.stock * 1e-9 / self.qconc, primers=self.qprimers,nreplicates=3)
else:
print("### Not doing mixdown as bconc not set in all inputs")
def run(self,confirm=False,enzName="EvaUSER"):
'Run the dilutions and QPCR setup'
# Setup qPCRs
#self.jobq.dump()
self.idler(100000)
self.trp.e.waitpgm() # May still need to wait for PTC to complete before able to do final jobs
self.idler(100000)
worklist.flushQueue()
if self.jobq.len()>0:
logging.error( "Blocked jobs remain on queue:",fatal=False)
self.jobq.dump()
assert False
if confirm:
worklist.userprompt('Continue to setup qPCR')
worklist.comment('Starting qPCR setup')
self.trp.e.sanitize(force=True)
for p in self.allprimers():
# Build list of relevant entries
ind=[ i for i in range(len(self.dilProds)) if p in self.primers[i]]
self.trp.runQPCR(src=[self.dilProds[i] for i in ind],vol=self.volume,primers=[p],nreplicates=[self.nreplicates[i] for i in ind],enzName=enzName)
def runT7Setup(self,theo,src,vol,srcdil,tgt=None,rlist=["MT7"]):
[theo,src,tgt,srcdil]=listify([theo,src,tgt,srcdil])
for i in range(len(src)):
if tgt[i] is None:
if theo[i]:
tgt[i]=Sample("%s.T+"%src[i].name,decklayout.SAMPLEPLATE)
else:
tgt[i]=Sample("%s.T-"%src[i].name,decklayout.SAMPLEPLATE)
worklist.comment("runT7: source=%s"%[str(s) for s in src])
rvols=[reagents.getsample(x).conc.volneeded(vol) for x in rlist]
rtotal=sum(rvols)
sourcevols=[vol*1.0/s for s in srcdil]
if any(theo):
theovols=[(vol*1.0/reagents.getsample("Theo").conc.dilutionneeded() if t else 0) for t in theo]
watervols=[vol-theovols[i]-sourcevols[i]-rtotal for i in range(len(src))]
else:
watervols=[vol-sourcevols[i]-rtotal for i in range(len(src))]
if any([w<-1e-10 for w in watervols]):
print "runT7Setup: Negative amount of water required: ",watervols
assert False
if sum(watervols)>0.01:
self.e.multitransfer(watervols,decklayout.WATER,tgt)
for ir in range(len(rlist)):
self.e.multitransfer([rvols[ir] for s in tgt],reagents.getsample(rlist[ir]),tgt)
if any(theo):
self.e.multitransfer([tv for tv in theovols if tv>0.01],reagents.getsample("Theo"),[tgt[i] for i in range(len(theovols)) if theovols[i]>0],ignoreContents=True)
for i in range(len(src)):
self.e.transfer(sourcevols[i],src[i],tgt[i])
self.e.shakeSamples(tgt,returnPlate=True)
for t in tgt:
t.ingredients['BIND']=1e-20*sum(t.ingredients.values())
return tgt
