def addTemplates(self,names,stockconc,finalconc=None,units="nM",plate=decklayout.EPPENDORFS,looplengths=None,extraVol=30,wellnames=None,initVol=0):
'Add templates as "reagents", return the list of them'
if finalconc is None:
logging.warning("final concentration of template not specified, assuming 0.6x (should add to addTemplates() call")
[names,stockconc]=listify([names,stockconc])
finalconc=[0.6*x for x in stockconc]
else:
[names,stockconc,finalconc]=listify([names,stockconc,finalconc])
if len(set(names))!=len(names):
logging.error("addTemplates: template names must be unique")
r=[]
if looplengths is not None:
assert(len(names)==len(looplengths))
for i in range(len(names)):
if wellnames is None:
well=None
else:
well=wellnames[i]
if reagents.isReagent(names[i]):
r.append(reagents.lookup(names[i]))
elif looplengths is None:
r.append(reagents.add(names[i],plate=plate,conc=Concentration(stockconc[i],finalconc[i],units),extraVol=extraVol,well=well,initVol=initVol))
else:
r.append(reagents.add(names[i],plate=plate,conc=Concentration(stockconc[i],finalconc[i],units),extraVol=extraVol,extrainfo=looplengths[i],well=well,initVol=initVol))
return r
def addReferences(self,mindil=1,nsteps=6,dstep=4,nreplicates=1,ref=None,primers=None):
"""Add all needed references"""
#print "addReferences(mindil=",mindil,", nsteps=",nsteps,", dstep=",dstep,", nrep=", nreplicates, ", ref=",ref,")"
# Make sure the ref reagent is loaded
if ref is None:
ref=reagents.getsample("QPCRREF")
if primers is None:
primers=self.allprimers()
dils=[1.0]
for i in range(nsteps):
needDil=mindil*math.pow(dstep,i)
srcDil=1
src=[ref]
for j in range(len(dils)):
if needDil/dils[j] <= self.MAXDIL:
srcDil=dils[j]
if srcDil==1:
src=[ref]
else:
srcname=diluteName(ref.name,srcDil)
src=[Sample.lookup(srcname)]
if src[0] is None:
logging.error("%s not found"%srcname)
break
tmp=self.MINDILVOL
self.MINDILVOL=75 # Make sure there's enough for resuing dilutions
self.addSamples(src=src,needDil=needDil/srcDil,primers=primers,nreplicates=nreplicates,save=needDil/srcDil>self.MAXDIL,saveVol=75)
self.MINDILVOL=tmp
dils.append(needDil)
self.addSamples(src=[self.dilutant],needDil=1,primers=primers,nreplicates=nreplicates,save=False)
def runRxInPlace(self,src,vol,master,master2=None,master3=None,returnPlate=True,finalx=1.0):
'Run reaction on beads in given total volume'
[vol,src,master,master2,master3]=listify([vol,src,master,master2,master3])
mastervol=[vol[i]*finalx/master[i].conc.dilutionneeded() for i in range(len(vol))]
master2vol=[0 if master2[i] is None else vol[i]*finalx/master2[i].conc.dilutionneeded() for i in range(len(vol))]
master3vol=[0 if master3[i] is None else vol[i]*finalx/master3[i].conc.dilutionneeded() for i in range(len(vol))]
watervol=[vol[i]-src[i].volume-mastervol[i]-master2vol[i]-master3vol[i] for i in range(len(vol))]
if any([w < -0.02 for w in watervol]):
logging.error("runRxInPlace: negative amount of water needed: %.2f"%min(watervol))
for i in range(len(src)):
if watervol[i]+src[i].volume>=4.0 and watervol[i]>0.1:
self.e.transfer(watervol[i],decklayout.WATER,src[i],(False,False))
watervol[i]=0
for i in range(len(src)):
self.e.transfer(mastervol[i],master[i],src[i],(True,False))
for i in range(len(src)):
if master2vol[i]>0:
self.e.transfer(master2vol[i],master2[i],src[i],(True,False))
if master3vol[i]>0:
self.e.transfer(master3vol[i],master3[i],src[i],(True,False))
for i in range(len(src)):
if watervol[i]>=0.1:
self.e.transfer(watervol[i],decklayout.WATER,src[i],(False,False))
self.e.shakeSamples(src,returnPlate=returnPlate)
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 finish(self):
self.e.lihahome()
worklist.userprompt("Process complete. Continue to turn off reagent cooler")
self.e.setreagenttemp(None)
#Sample.printallsamples("At completion")
hasError=False
for s in Sample.getAllOnPlate():
if s.volume<1.0 and s.conc is not None and not s.emptied:
logging.error("Insufficient volume for %s: need at least %.1f ul additional"%(s.name,1.0-s.volume),fatal=False)
#hasError=True
elif s.volume<2.5 and s.conc is not None and not s.emptied:
logging.warning("Low final volume for "+ s.name)
elif s.volume>s.plate.maxVolume:
logging.erorr("Excess final volume (%.1f) for %s: maximum is %.1f ul"%(s.volume,s.name,s.plate.maxVolume),fatal=False)
hasError=True
if hasError:
logging.error("NO OUTPUT DUE TO ERRORS")
print "Wells used: samples: %d, dilutions: %d, qPCR: %d"%(Sample.numSamplesOnPlate(decklayout.SAMPLEPLATE),Sample.numSamplesOnPlate(decklayout.DILPLATE),Sample.numSamplesOnPlate(decklayout.QPCRPLATE))
# Save worklist to a file
#e.saveworklist("trp1.gwl")
(scriptname,ext)=os.path.splitext(sys.argv[0])
self.e.savegem(scriptname+".gem")
self.e.savesummary(scriptname+".txt")
Sample.savematlab(scriptname+".m")
def diluteInPlace(self,tgt,dil=None,finalvol=None):
# Dilute in place
# e.g.: trp.diluteInPlace(tgt=rt1,dil=2)
[tgt,dil,finalvol]=listify([tgt,dil,finalvol])
dilutant=decklayout.WATER
for i in range(len(tgt)):
if finalvol[i] is not None and dil[i] is None:
self.e.transfer(finalvol[i]-tgt[i].volume,dilutant,tgt[i],mix=(False,False))
elif finalvol[i] is None and dil[i] is not None:
self.e.transfer(tgt[i].volume*(dil[i]-1),dilutant,tgt[i],mix=(False,False))
else:
logging.error("diluteInPlace: cannot specify both dil and finalvol")
#print "after dilute, tgt[0]=",str(tgt[0]),",mixed=",tgt[0].isMixed()
return tgt # The name of the samples are unchanged -- the predilution names
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 TC to complete before able to do final jobs
self.idler(100000)
if self.jobq.len() > 0:
logging.error("Blocked jobs remain on queue:")
self.jobq.dump()
assert False
tgt1 = Sample("Barcoded.Mixdown1", decklayout.EPPENDORFS)
for i in range(1, len(self.dilProds)):
self.e.transfer(12.5 * 1.2 * 2 / 1.5, self.dilProds[i], tgt1, mix=(False, False))
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 runIncubation(self,src=None,vol=None,srcdil=None,tgt=None,enzymes=None,incTemp=37,incTime=15,hiTemp=None,hiTime=0,inPlace=False):
if len(enzymes)!=1:
logging.error("runIncubation only supports a single master mix")
if inPlace:
if tgt is not None:
logging.error("tgt specified for in-place incubation")
elif tgt is None:
tgt=[Sample("%s.%s"%(src[i].name,enzymes[0].name),decklayout.SAMPLEPLATE) for i in range(len(src))]
if srcdil==None:
# Minimum dilution (no water)
srcdil=1/(1-sum([1/e.conc.dilutionneeded() for e in enzymes]))
if vol is None and inPlace:
vol=[s.volume*srcdil for s in src]
[src,tgt,vol,srcdil]=listify([src,tgt,vol,srcdil])
# Adjust source dilution
for i in range(len(src)):
src[i].conc=Concentration(srcdil[i],1)
if inPlace:
self.runRxInPlace(src,vol,enzymes[0],returnPlate=False)
tgt=src
else:
self.e.stage('Incubation',enzymes,src,tgt,vol,destMix=False)
self.e.shakeSamples(tgt,returnPlate=(incTime is None))
if incTime is None:
print "Setup only of incubation with %s"%enzymes[0].name
else:
if hiTemp is None:
worklist.pyrun('PTC\\ptcsetpgm.py INC TEMP@%.0f,%.0f TEMP@25,30'%(incTemp,incTime*60))
print "Incubating at %dC for %d minutes without heat inactivation"%(incTemp, incTime)
hiTime=0
else:
assert(hiTime>0)
worklist.pyrun('PTC\\ptcsetpgm.py INC TEMP@%.0f,%.0f TEMP@%.0f,%.0f TEMP@25,30'%(incTemp,incTime*60,hiTemp,hiTime*60))
print "Incubating at %dC for %d minutes followed by heat inactivate at %dC for %d minutes"%(incTemp,incTime,hiTemp,hiTime)
self.e.runpgm("INC",incTime+hiTime+2,False,max(vol),hotlidmode="TRACKING",hotlidtemp=10)
return tgt
def beadSupernatant(self,src,tgt=None,sepTime=None,residualVolume=0.1,plate=None):
if plate is None:
plate=decklayout.SAMPLEPLATE
if tgt is None:
tgt=[]
for i in range(len(src)):
tgt.append(Sample("%s.SN"%src[i].name,plate))
[src,tgt]=listify([src,tgt])
if any([s.plate!=src[0].plate for s in src]):
logging.error("beadSupernatant: Attempt to magsep on multiple plates at the same time")
self.e.moveplate(src[0].plate,"Magnet") # Move to magnet
self.sepWait(src,sepTime)
for i in range(len(src)):
self.e.transfer(src[i].amountToRemove(residualVolume),src[i],tgt[i],(False,False)) # Transfer elution to new tube
self.e.moveplate(src[0].plate,"Home")
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
tgt1 = Sample("Barcoded.Mixdown1", decklayout.EPPENDORFS)
for i in range(1, len(self.dilProds)):
self.e.transfer(12.5 * 1.2 * 2 / 1.5,
self.dilProds[i],
tgt1,
mix=(False, False))
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 bindBeads(self,src,beads=None,beadConc=None,beadDil=None,bbuffer=None,incTime=60,addBuffer=False):
if beads is None:
beads=reagents.getsample("Dynabeads")
if bbuffer is None:
bbuffer=reagents.getsample("BeadBuffer")
[src,beads,bbuffer,beadConc,beadDil]=listify([src,beads,bbuffer,beadConc,beadDil])
for s in src:
if s.plate!=decklayout.SAMPLEPLATE:
logging.error( "runBeadCleanup: src "+s.name+" is not in sample plate.")
s.conc=None # Can't track concentration of beads
self.e.moveplate(src[0].plate,"Home") # Make sure we do this off the magnet
# Calculate volumes needed
beadDil=[beads[i].conc.stock/(beads[i].conc.final if beadConc[i] is None else beadConc[i]) if beadDil[i] is None else beadDil[i] for i in range(len(beads))]
if addBuffer:
totalvol=[s.volume/(1-1.0/beadDil-1.0/bbuffer[i].conc.dilutionneeded()) for s in src]
buffervol=[totalvol[i]/bbuffer[i].conc.dilutionneeded() for i in range(len(src))]
# Add binding buffer to bring to 1x (beads will already be in 1x, so don't need to provide for them)
for i in range(len(src)):
self.e.transfer(buffervol[i],bbuffer[i],src[i])
else:
buffervol=[0.0 for i in range(len(src))]
totalvol=[src[i].volume/(1-1.0/beadDil[i]) for i in range(len(src))]
beadvol=[totalvol[i]/beadDil[i] for i in range(len(totalvol))]
# Transfer the beads
for i in range(len(src)):
self.e.transfer(beadvol[i],beads[i],src[i],(True,False)) # Mix beads before
self.e.shakeSamples(src,dur=incTime,returnPlate=False)
def __init__(self, inputs):
super(IDPrep, self).__init__()
self.inputs = inputs
self.qconc = 0.020 # Target qPCR concentration in nM
self.qprimers = ["End"]
self.bc1_inputvol = 2 # ul into PCR1
used = []
for inp in inputs:
bc = "%s-%s" % (inp['left'], inp['right'])
if bc in used:
logging.error("Barcode %s is being reused for %s" % (bc, inp['name']))
used.append(bc)
print("used=",used)
self.rsrc = [reagents.add("%s-%s-%s" % (inputs[i]['name'], inputs[i]['left'], inputs[i]['right']),
decklayout.SAMPLEPLATE,
well=inputs[i]['well'] if 'well' in inputs[i] else None,
conc=Concentration(stock=inputs[i]['conc'], units="nM"),
initVol=self.bc1_inputvol, extraVol=0)
for i in range(len(inputs))]
self.q = None # Defined in pgm()
def pgm(self):
q = QSetup(self,maxdil=self.maxdilstep,debug=False,mindilvol=60)
self.e.addIdleProgram(q.idler)
if self.barcoding:
# Setup barcode primers for cleaved rounds only
self.bcprimers=[["BC-%s-R%d_T7"%(inp['ligand'],r+1) for inp in self.inputs] if self.rounds[r]=='C' else None for r in range(len(self.rounds))]
for bcp in self.bcprimers:
if bcp is not None:
for p in ["P-%s"%pp for pp in bcp]:
if not reagents.isReagent(p):
reagents.add(name=p,conc=4,extraVol=30,plate=decklayout.REAGENTPLATE,well="B2")
s=reagents.getsample(p) # Force allocation of a well
print("Adding %s to reagents at well %s"%(p,s.plate.wellname(s.well)))
print("BC primers=", self.bcprimers)
# Add any missing fields to inputs
for i in range(len(self.inputs)):
if 'ligand' not in self.inputs[i]:
self.inputs[i]['ligand']=None
if 'negligand' not in self.inputs[i]:
self.inputs[i]['negligand']=None
if 'round' not in self.inputs[i]:
self.inputs[i]['round']=None
if 'name' not in self.inputs[i]:
if self.inputs[i]['ligand'] is None:
self.inputs[i]['name']='%s_%d_R%d'%(self.inputs[i]['prefix'],self.inputs[i]['ID'],self.inputs[i]['round'])
else:
self.inputs[i]['name']='%s_%d_R%d_%s'%(self.inputs[i]['prefix'],self.inputs[i]['ID'],self.inputs[i]['round'],self.inputs[i]['ligand'])
# Add templates
if self.directT7:
self.srcs = self.addTemplates([inp['name'] for inp in self.inputs],stockconc=self.tmplFinalConc/self.templateDilution,finalconc=self.tmplFinalConc,plate=decklayout.SAMPLEPLATE,looplengths=[inp['looplength'] for inp in self.inputs],initVol=self.t7vol[0]*self.templateDilution,extraVol=0)
else:
self.srcs = self.addTemplates([inp['name'] for inp in self.inputs],stockconc=self.tmplFinalConc/self.templateDilution,finalconc=self.tmplFinalConc,plate=decklayout.DILPLATE,looplengths=[inp['looplength'] for inp in self.inputs],extraVol=15)
if self.dopcr:
# Reserve space for PCR products
pcrprods=[ [Sample("R%d-T%s"%(r,inp['ligand']),self.savePlate) for inp in self.inputs] for r in range(len(self.rounds))]
else:
pcrprods=None
t7in = [s.getsample() for s in self.srcs]
if "negative" in self.qpcrStages:
q.addSamples(decklayout.SSDDIL,1,self.allprimers,save=False) # Negative controls
if "reference" in self.qpcrStages:
q.addReferences(dstep=10,nsteps=5,primers=["WX","MX","T7X"] if self.useMX else ["WX","T7X"],ref=reagents.getsample("BT5310"),nreplicates=1)
# Save RT product from first (uncleaved) round and then use it during 2nd (cleaved) round for ligation and qPCR measurements
self.rndNum=0
self.nextID=self.firstID
curPrefix=[inp['prefix'] for inp in self.inputs]
r1=t7in
for roundType in self.rounds:
# Run a single round of roundType with r1 as input
# roundType is either "U" for uncleaved, or a new prefix for a cleaved round (with "T" being a T7 prepend)
# Set r1 to new output at end
if self.roundCallback is not None:
self.roundCallback(self,self.rndNum,roundType)
# Computed output prefix
if roundType=='U':
prefixOut=curPrefix
stop=["Unclvd" for _ in curPrefix]
else:
if roundType=='T':
stop=['T7%s'%p for p in curPrefix]
prefixOut=curPrefix
elif any([p==roundType for p in curPrefix]):
logging.error( "Round %d is a cleaved round but goes to %s without changing prefix"%(self.rndNum, roundType))
assert False
else:
prefixOut=[roundType for _ in curPrefix]
stop=prefixOut
# May be explicitly overridden
for i in range(len(self.inputs)):
if 'stop' in self.inputs[i]:
if isinstance(self.inputs[i]['stop'],list):
assert(len(self.inputs[i]['stop'])==len(self.rounds))
t=self.inputs[i]['stop'][self.rndNum]
else:
t=self.inputs[i]['stop']
if (roundType=='U') != (t=='U'):
print("Attempt to override round %d (type %s) with a input-specific round type of %s"%(self.rndNum, roundType, t))
assert False
if roundType!='U':
if t=='T':
stop[i]='T7%s'%curPrefix[i]
prefixOut[i]=curPrefix[i]
else:
stop[i]=t
prefixOut[i]=t
self.rndNum=self.rndNum+1
self.finalRound=self.rndNum==len(self.rounds)
db.pushStatus("%s%d"%(roundType,self.rndNum))
[r1,bc1]=self.oneround(q,r1,prefixOut=prefixOut,stop=stop,prefixIn=curPrefix,keepCleaved=(roundType!='U'),rtvol=self.rtvol[self.rndNum-1],t7vol=self.t7vol[self.rndNum-1],cycles=self.pcrcycles[self.rndNum-1],pcrdil=self.pcrdil[self.rndNum-1],pcrvol=self.pcrvol[self.rndNum-1],dolig=self.allLig or (roundType!='U'),pcrtgt=None if pcrprods is None else pcrprods[self.rndNum-1])
db.popStatus()
# Add TRefs specified in rnddefs
if 'tref' in self.rnddef[self.rndNum-1]:
tref=self.rnddef[self.rndNum-1]['tref']
assert len(tref)==len(r1)
for i in range(len(r1)):
if tref[i] is not None:
trefname='TRef%d'%tref[i]
print("Adding %s to %s"%(trefname,r1[i].name))
if not reagents.isReagent(trefname):
reagents.add(name=trefname,conc=10,extraVol=30,well="E4")
trefSamp=reagents.getsample(trefname)
oldConc=r1[i].conc.stock
oldUnits=r1[i].conc.units
oldVol=r1[i].volume
self.e.transfer(r1[i].volume/(trefSamp.conc.dilutionneeded()-1),trefSamp,r1[i],mix=(False,False)) # TODO: Check that these end up mixed
r1[i].conc=Concentration(stock=oldConc*oldVol/r1[i].volume, units=oldUnits) # Treat TRef as straight dilution
print("New conc=",r1[i].conc)
for i in range(len(r1)):
if self.inputs[i]['round'] is None:
r1[i].name="%s_%d"%(prefixOut[i],self.nextID)
else:
r1[i].name="%d_%s_R%d%c"%(self.nextID,prefixOut[i],self.inputs[i]['round']+self.rndNum,roundType)
if self.inputs[i]['ligand'] is not None:
r1[i].name="%s_%s"%(r1[i].name,self.inputs[i]['ligand'])
print("Used ID ", self.nextID," for ", r1[i].name,": ",r1[i])
self.nextID+=1
r1[i].conc.final=r1[i].conc.stock*self.templateDilution
for i in range(len(bc1)):
#print("Renaming",bc1[i].name)
pts=bc1[i].name.split(".")
bc1[i].name="%d_BC_R%d%c"%(self.nextID,self.inputs[i//2]['round']+self.rndNum,roundType)
if self.inputs[i//2]['ligand'] is not None:
bc1[i].name="%s_%s"%(bc1[i].name,self.inputs[i//2]['ligand'])
bc1[i].name+="_"+pts[-2]
print("Used ID ", self.nextID," for ", bc1[i].name,":",bc1[i])
self.nextID+=1
curPrefix=prefixOut
if "finalpcr" in self.qpcrStages:
for i in range(len(r1)):
if self.singlePrefix:
q.addSamples(src=r1[i],needDil=r1[i].conc.stock/self.qConc,primers=["T7X","MX"] if self.useMX else ["T7X"])
else:
# noinspection PyUnboundLocalVariable
q.addSamples(src=r1[i],needDil=r1[i].conc.stock/self.qConc,primers=["T7X",prefixOut[i]+"X"]+(["MX"] if self.useMX else []))
# Add TRefs if needed
for i in range(len(r1)):
if 'tref' in self.inputs[i]:
trefname='TRef%d'%self.inputs[i]['tref']
if not reagents.isReagent(trefname):
reagents.add(name=trefname,conc=10,extraVol=30)
tref=reagents.getsample(trefname)
self.e.transfer(r1[i].volume/(tref.conc.dilutionneeded()-1),tref,r1[i],mix=(False,False))
db.pushStatus('qPCR')
print("######### qPCR ########### %.0f min"%(clock.elapsed()/60))
self.allprimers=q.allprimers()
q.run(confirm=self.qpcrWait)
db.popStatus()
def pgm(self):
q = QSetup(self, maxdil=self.maxdilstep, debug=False, mindilvol=60)
self.e.addIdleProgram(q.idler)
if self.barcoding:
# Setup barcode primers for cleaved rounds only
self.bcprimers = [[
"BC-%s-R%d_T7" % (inp['ligand'], r + 1) for inp in self.inputs
] if self.rounds[r] == 'C' else None
for r in range(len(self.rounds))]
for bcp in self.bcprimers:
if bcp is not None:
for p in ["P-%s" % pp for pp in bcp]:
if not reagents.isReagent(p):
reagents.add(name=p,
conc=4,
extraVol=30,
plate=decklayout.REAGENTPLATE,
well="B2")
s = reagents.getsample(p) # Force allocation of a well
print "Adding %s to reagents at well %s" % (
p, s.plate.wellname(s.well))
print "BC primers=", self.bcprimers
# Add any missing fields to inputs
for i in range(len(self.inputs)):
if 'ligand' not in self.inputs[i]:
self.inputs[i]['ligand'] = None
if 'round' not in self.inputs[i]:
self.inputs[i]['round'] = None
if 'name' not in self.inputs[i]:
if self.inputs[i]['ligand'] is None:
self.inputs[i]['name'] = '%s_%d_R%d' % (
self.inputs[i]['prefix'], self.inputs[i]['ID'],
self.inputs[i]['round'])
else:
self.inputs[i]['name'] = '%s_%d_R%d_%s' % (
self.inputs[i]['prefix'], self.inputs[i]['ID'],
self.inputs[i]['round'], self.inputs[i]['ligand'])
# Add templates
if self.directT7:
self.srcs = self.addTemplates(
[inp['name'] for inp in self.inputs],
stockconc=self.tmplFinalConc / self.templateDilution,
finalconc=self.tmplFinalConc,
plate=decklayout.SAMPLEPLATE,
looplengths=[inp['looplength'] for inp in self.inputs],
initVol=self.t7vol[0] * self.templateDilution,
extraVol=0)
else:
self.srcs = self.addTemplates(
[inp['name'] for inp in self.inputs],
stockconc=self.tmplFinalConc / self.templateDilution,
finalconc=self.tmplFinalConc,
plate=decklayout.DILPLATE,
looplengths=[inp['looplength'] for inp in self.inputs],
extraVol=15)
t7in = [s.getsample() for s in self.srcs]
if "negative" in self.qpcrStages:
q.addSamples(decklayout.SSDDIL, 1, self.allprimers,
save=False) # Negative controls
if "reference" in self.qpcrStages:
q.addReferences(dstep=10,
nsteps=5,
primers=["T7WX", "MX", "T7X"],
ref=reagents.getsample("BT5310"),
nreplicates=1)
q.addReferences(dstep=10,
nsteps=5,
primers=["T7WX", "MX", "T7X"],
ref=reagents.getsample("BT5310"),
nreplicates=1)
# Save RT product from first (uncleaved) round and then use it during 2nd (cleaved) round for ligation and qPCR measurements
self.rndNum = 0
self.nextID = self.firstID
curPrefix = [inp['prefix'] for inp in self.inputs]
r1 = t7in
for roundType in self.rounds:
# Run a single round of roundType with r1 as input
# roundType is either "U" for uncleaved, or a new prefix for a cleaved round (with "T" being a T7 prepend)
# Set r1 to new output at end
# Computed output prefix
if roundType == 'U':
prefixOut = curPrefix
stop = ["Unclvd" for p in curPrefix]
else:
if roundType == 'T':
stop = ['T7%s' % p for p in curPrefix]
prefixOut = curPrefix
elif any([p == roundType for p in curPrefix]):
logging.error(
"Round %d is a cleaved round but goes to %s without changing prefix"
% (self.rndNum, roundType))
assert (False)
else:
prefixOut = [roundType for p in curPrefix]
stop = prefixOut
# May be explicitly overridden
for i in range(len(self.inputs)):
if 'stop' in self.inputs[i]:
if isinstance(self.inputs[i]['stop'], list):
assert (len(self.inputs[i]['stop']) == len(
self.rounds))
t = self.inputs[i]['stop'][self.rndNum]
else:
t = self.inputs[i]['stop']
if (roundType == 'U') != (t == 'U'):
print "Attempt to override round %d (type %s) with a input-specific round type of %s" % (
self.rndNum, roundType, t)
assert (False)
if roundType != 'U':
if t == 'T':
stop[i] = 'T7%s' % curPrefix[i]
prefixOut[i] = curPrefix[i]
else:
stop[i] = t
prefixOut[i] = t
self.rndNum = self.rndNum + 1
self.finalRound = self.rndNum == len(self.rounds)
r1 = self.oneround(q,
r1,
prefixOut=prefixOut,
stop=stop,
prefixIn=curPrefix,
keepCleaved=(roundType != 'U'),
rtvol=self.rtvol[self.rndNum - 1],
t7vol=self.t7vol[self.rndNum - 1],
cycles=self.pcrcycles[self.rndNum - 1],
pcrdil=self.pcrdil[self.rndNum - 1],
pcrvol=self.pcrvol[self.rndNum - 1],
dolig=self.allLig or (roundType != 'U'))
for i in range(len(r1)):
r1[i].name = "%s_%d" % (prefixOut[i], self.nextID)
if self.inputs[i]['round'] is not None:
r1[i].name = "%s_R%d%c" % (r1[i].name,
self.inputs[i]['round'] +
self.rndNum, roundType)
if self.inputs[i]['ligand'] is not None:
r1[i].name = "%s_%s" % (r1[i].name,
self.inputs[i]['ligand'])
print "Used ID ", self.nextID, " for ", r1[i].name, ": ", r1[i]
self.nextID += 1
r1[i].conc.final = r1[i].conc.stock * self.templateDilution
curPrefix = prefixOut
if "finalpcr" in self.qpcrStages:
for i in range(len(r1)):
if self.singlePrefix:
q.addSamples(src=r1[i],
needDil=r1[i].conc.stock / self.qConc,
primers=["T7X", "MX"])
else:
q.addSamples(src=r1[i],
needDil=r1[i].conc.stock / self.qConc,
primers=["T7X", prefixOut[i] + "X", "MX"])
print "######### qPCR ########### %.0f min" % (clock.elapsed() / 60)
self.allprimers = q.allprimers()
q.run(confirm=self.qpcrWait)
def idbarcoding(self, rsrc, left, right):
"""Perform barcoding of the given inputs; rsrsc,left,right should all be equal length"""
pcrcycles = [4] # Don't need 2nd PCR since this will go directly into constriction
#pcr1inputconc = 0.05 # PCR1 concentration final in reaction
pcr1inputdil = 10
pcr1vol = 30
pcr1postdil = 100.0 / pcr1vol
pcr2dil = 50
pcr2minvol = 50.0
samps = [s.getsample() for s in rsrc]
print("Inputs:")
for i in range(len(samps)):
print("%2s %-10s %8s-%-8s %.1f%s" % (
samps[i].plate.wellname(samps[i].well), self.inputs[i]['name'], left[i], right[i], samps[i].conc.stock,samps[i].conc.units))
# Compute pcr1inputconc such that lowest concentration input ends up with at least 30ul after dilution
pcr1inputconc=min([s.conc.stock*s.volume/30.0/pcr1inputdil for s in samps])
print("Diluting inputs so PCR1 final template conc = %.0f pM"%(pcr1inputconc*1000))
wellnum = 5
for s in left + right:
primer = "P-" + s
if not reagents.isReagent(primer):
reagents.add(primer, conc=Concentration(2.67, 0.4, 'uM'), extraVol=30, plate=decklayout.REAGENTPLATE,
well=decklayout.REAGENTPLATE.wellname(wellnum))
wellnum += 1
# Run first pass dilution where needed
for i in range(len(samps)):
# Dilute down to desired conc
dil = samps[i].conc.stock / pcr1inputconc / pcr1inputdil
dilvol = samps[i].volume * dil
if dilvol > 100.0:
logging.notice("Dilution of input %s (%.1f ul) by %.2f would require %.1f ul" % (
samps[i].name, samps[i].volume, dil, dilvol))
# Do a first pass dilution into 150ul, then remove enough so second dilution can go into 100ul
dil1 = 100.0 / samps[i].volume
self.diluteInPlace(tgt=[samps[i]], dil=dil1)
print("First pass dilution of %s by %.1f/%.1f (conc now %.3f nM)" % (samps[i].name, dil1, dil, samps[i].conc.stock))
dil /= dil1
# Make sure they are all mixed
self.e.shakeSamples(samps)
# Final dilution
for s in samps:
# Dilute down to desired conc
dil = s.conc.stock / pcr1inputconc / pcr1inputdil
if dil < 1.0:
logging.error("Input %s requires dilution of %.2f" % (s.name, dil))
elif dil > 1.0:
dilvol = s.volume * dil
if dilvol>100:
toremove=s.volume-100.0/dil
print("Removing %.1f ul from %s to allow enough room for dilution"%(toremove,s.name))
self.e.dispose(toremove, s)
self.diluteInPlace(tgt=[s], dil=dil)
print("Diluting %s by %.1f" % (s.name, dil))
pcr1 = self.runPCR(src=samps, srcdil=pcr1inputdil, ncycles=pcrcycles[0], vol=pcr1vol,
primers=[[left[i], right[i]] for i in range(len(left))], usertime=0, fastCycling=False,
inPlace=False, master="MKapa", kapa=True)
pcr1finalconc = pcr1inputconc * self.pcreff ** pcrcycles[0]
print("PCR1 output concentration = %.3f nM" % pcr1finalconc)
if pcr1postdil > 1:
pcr1finalconc /= pcr1postdil
print("Post dilute PCR1 by %.2fx to %.3f nM " % (pcr1postdil, pcr1finalconc))
self.diluteInPlace(tgt=pcr1, dil=pcr1postdil)
for x in pcr1:
x.conc = Concentration(stock=pcr1finalconc, units='nM')
self.q.addSamples(src=pcr1, needDil=pcr1finalconc / self.qconc, primers=self.qprimers, save=True,
nreplicates=1)
if len(pcrcycles) > 1:
# Second PCR with 235p/236p on mixture (use at least 4ul of prior)
pcr2 = self.runPCR(src=pcr1, srcdil=pcr2dil / pcr1postdil, vol=max(pcr2minvol, pcr2dil / pcr1postdil * 4),
ncycles=pcrcycles[1],
primers="End", fastCycling=False, master="MKapa", kapa=True)
pcr2finalconc = min(200, pcr1finalconc / (pcr2dil / pcr1postdil) * self.pcreff ** pcrcycles[1])
print("PCR2 final conc = %.1f nM" % pcr2finalconc)
d2 = min(4.0, 150.0 / max([p.volume for p in pcr2]))
if d2 > 1:
pcr2finalconc /= d2
print("Post-dilute PCR2 by %.1fx to %.3fnM" % (d2, pcr2finalconc))
self.diluteInPlace(tgt=pcr2, dil=d2)
self.e.shakeSamples(pcr2)
for x in pcr2:
x.conc = Concentration(stock=pcr2finalconc, units='nM')
self.q.addSamples(src=pcr2, needDil=pcr2finalconc / self.qconc, primers=self.qprimers, save=True,
nreplicates=2)
res = pcr2
else:
res = pcr1
return res