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.hasBeads:
print "ERROR: Insufficient volume for ", s," need at least ",1.0-s.volume," ul additional"
#hasError=True
elif s.volume<2.5 and s.conc is not None:
print "WARNING: Low final volume for ", s
elif s.volume>s.plate.maxVolume:
print "ERROR: Excess final volume (",s.volume,") for ",s,", maximum is ",s.plate.maxVolume
hasError=True
if hasError:
print "NO OUTPUT DUE TO ERRORS"
assert(False)
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 pgm(self):
self.q = QSetup(self, maxdil=16, debug=False, mindilvol=60)
# Don't start idler (to minimize tip cross-contamination); last PCR allows plenty of time for doing dilutions without any effect on run time
# Will start after first constriction PCR is running
#self.q.debug = True
# self.e.addIdleProgram(self.q.idler)
self.q.addReferences(dstep=10, primers=self.qprimers, ref=reagents.getsample("BT5310"),nreplicates=2)
samps=[r.getsample() for r in self.rsrc]
for s in samps:
self.q.addSamples([s],needDil=max(10,s.conc.stock*1e-9/self.qconc),primers=self.qprimers)
print("### Mixdown #### (%.0f min)" % (clock.elapsed() / 60.0))
if len(samps)>1:
mixdown = self.mix(samps, [x['weight'] for x in self.inputs])
else:
mixdown=samps[0]
self.q.addSamples(mixdown, needDil=max(1.0,mixdown.conc.stock * 1e-9 / self.qconc), primers=self.qprimers)
print("Mixdown final concentration = %.0f pM" % (mixdown.conc.stock * 1000))
print("### Constriction #### (%.1f min)" % (clock.elapsed() / 60.0))
constricted = self.constrict(mixdown, mixdown.conc.stock * 1e-9)
print("### Regeneration #### (%.0f min)" % (clock.elapsed() / 60.0))
prefixes = set([x['left'][0] for x in self.inputs])
self.regenerate(constricted * len(prefixes), [p for p in prefixes for _ in constricted])
print("### qPCR #### (%.0f min)" % (clock.elapsed() / 60.0))
self.q.run(confirm=False, enzName='EvaGreen', waitForPTC=True)
print("### qPCR Done #### (%.0f min)" % (clock.elapsed() / 60.0))
worklist.userprompt("qPCR done -- only need to complete final PCR", 300)
self.e.waitpgm()
print("### Final PCR Done #### (%.0f min)" % (clock.elapsed() / 60.0))
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 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.hasBeads:
print "ERROR: Insufficient volume for ", s, " need at least ", 1.0 - s.volume, " ul additional"
#hasError=True
elif s.volume < 2.5 and s.conc is not None:
print "WARNING: Low final volume for ", s
elif s.volume > s.plate.maxVolume:
print "ERROR: Excess final volume (", s.volume, ") for ", s, ", maximum is ", s.plate.maxVolume
hasError = True
if hasError:
print "NO OUTPUT DUE TO ERRORS"
assert (False)
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 pgm(self):
q = QSetup(self,maxdil=16,debug=False,mindilvol=60)
self.e.addIdleProgram(q.idler)
t7in = [s.getsample() for s in self.srcs]
if "negative" in self.qpcrStages:
qpcrPrimers=["REF","MX","T7X","T7AX","T7BX","T7WX"]
q.addSamples(decklayout.SSDDIL,1,qpcrPrimers,save=False) # Negative controls
# 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]
while self.rndNum<self.nrounds:
self.rndNum=self.rndNum+1
prefixOut=["A" if p=="W" else "B" if p=="A" else "W" if p=="B" else "BADPREFIX" for p in curPrefix]
if self.rndNum==1:
self.t7vol1=self.t7vol1a
else:
self.t7vol1=max(20,self.pmolesIn*1000/min([inp.conc.final for inp in t7in])) # New input volueme
r1=self.oneround(q,t7in,prefixOut,prefixIn=curPrefix,keepCleaved=False,rtvol=self.rtvol1,t7vol=self.t7vol1,cycles=self.pcrcycles1,pcrdil=self.pcrdil1,pcrvol=self.pcrvol1,dolig=self.allLig)
# pcrvol is set to have same diversity as input
for i in range(len(r1)):
r1[i].name="%s_%d_R%dU_%s"%(curPrefix[i],self.nextID,self.inputs[i]['round']+self.rndNum,self.inputs[i]['ligand'])
self.nextID+=1
r1[i].conc.final=r1[i].conc.stock*self.templateDilution
if self.rndNum>=self.nrounds:
logging.warning("Warning: ending on an uncleaved round")
break
self.rndNum=self.rndNum+1
print "prefixIn=",curPrefix
print "prefixOut=",prefixOut
self.t7vol2=max(20,self.pmolesIn*1000/min([inp.conc.final for inp in r1]))
r2=self.oneround(q,r1,prefixOut,prefixIn=curPrefix,keepCleaved=True,rtvol=self.rtvol2,t7vol=self.t7vol2,cycles=self.pcrcycles2,pcrdil=self.pcrdil2,pcrvol=self.pcrvol2,dolig=True)
# pcrvol is set to have same diversity as input = (self.t7vol2*self.templateDilution/rnagain*stopdil*rtdil*extdil*exodil*pcrdil)
for i in range(len(self.inputs)):
r2[i].name="%s_%d_R%dC_%s"%(prefixOut[i],self.nextID,self.inputs[i]['round']+self.rndNum,self.inputs[i]['ligand'])
self.nextID+=1
r2[i].conc.final=r2[i].conc.stock*self.templateDilution
t7in=r2
curPrefix=prefixOut
if "finalpcr" in self.qpcrStages:
for i in range(len(r2)):
q.addSamples(src=r2[i],needDil=r2[i].conc.stock/self.qConc,primers=["T7X","T7"+prefixOut[i]+"X"])
print "######### qPCR ###########"
#q.addReferences(mindil=4,nsteps=6,primers=["T7X","MX","T7AX"])
if self.qpcrWait:
worklist.userprompt('Continue to setup qPCR')
q.run()
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:
print "Blocked jobs remain on queue:"
self.jobq.dump()
assert False
worklist.userprompt('Starting qPCR setup',timeout=5)
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,srcdil=10.0/4,primers=[p],nreplicates=[self.nreplicates[i] for i in ind])
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 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:
print "Blocked jobs remain on queue:"
self.jobq.dump()
assert False
worklist.userprompt('Starting qPCR setup', timeout=5)
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,
srcdil=10.0 / 4,
primers=[p],
nreplicates=[self.nreplicates[i] for i in ind])
def oneround(self, q, inputs, prefixOut, stop, prefixIn, keepCleaved, t7vol, rtvol, pcrdil, cycles, pcrvol, dolig,pcrtgt=None):
primerSet=[set(["REF","T7X",prefixIn[i]+"X",prefixOut[i]+"X"]+(["MX"] if self.useMX else [])) for i in range(len(prefixIn))]
if self.extraQPCRPrimers is not None:
primerSet=[set(list(p) + self.extraQPCRPrimers) for p in primerSet]
print("primerSet=",primerSet)
if keepCleaved:
print("Starting new cleavage round, will add prefix: ",prefixOut)
assert dolig
else:
print("Starting new uncleaved round, will retain prefix: ",prefixIn)
print("stop=",stop,"prefixOut=",prefixOut,", prefixIn=",prefixIn,",t7vol=",round(t7vol,ndigits=2),",rtvol=",rtvol,",pcrdil=",pcrdil,",cycles=",cycles,",dolig=",dolig)
if self.rtCarryForward:
assert dolig
names=[i.name for i in inputs]
if self.rnaInput:
rxs=inputs
stopDil=1
else:
print("######## T7 ########### %.0f min"%(clock.elapsed()/60))
db.pushStatus("T7")
print("Inputs: (t7vol=%.2f)"%t7vol)
for inp in inputs:
if inp.conc.units=='nM':
print(" %s: %.1ful@%.1f %s, use %.1f ul (%.3f pmoles)"%(inp.name,inp.volume,inp.conc.stock,inp.conc.units,t7vol/inp.conc.dilutionneeded(), t7vol*inp.conc.final/1000))
else:
print(" %s: %.1ful@%.1f %s, use %.1f ul"%(inp.name,inp.volume,inp.conc.stock,inp.conc.units,t7vol/inp.conc.dilutionneeded()))
# inp.conc.final=inp.conc.stock*self.templateDilution
units=list(set([inp.conc.units for inp in inputs]))
if len(units)>1:
print("Inputs have inconsistent concentration units: ",units)
assert False
if units[0]=='nM':
needDil = max([inp.conc.stock for inp in inputs]) * 1.0 / self.qConc
else:
needDil = 100 / self.qConc # Assume 100nM
if self.directT7 and self.rndNum==1:
# Just add ligands and MT7 to each well
mconc=reagents.getsample("MT7").conc.dilutionneeded()
for i in range(len(inputs)):
watervol=t7vol*(1-1/mconc) - inputs[i].volume
if watervol<-0.1:
print("Negative amount of water (%.1f ul) needed for T7 setup"%watervol)
assert False
elif watervol>0.1:
self.e.transfer(watervol, decklayout.WATER, inputs[i], mix=(False, False))
self.e.transfer(t7vol / mconc, reagents.getsample("MT7"), inputs[i], mix=(False, False))
assert(abs(inputs[i].volume - t7vol) < 0.1)
# Add ligands last in case they crash out when they hit aqueous; this way, they'll be as dilute as possible
if keepCleaved:
for i in range(len(inputs)):
if self.inputs[i]['negligand'] is not None:
negligand=reagents.getsample(self.inputs[i]['negligand'])
self.e.transfer(t7vol / negligand.conc.dilutionneeded(), negligand, inputs[i], mix=(False, False))
names[i]+="+"
else:
for i in range(len(inputs)):
if self.inputs[i]['ligand'] is not None:
ligand=reagents.getsample(self.inputs[i]['ligand'])
self.e.transfer(t7vol / ligand.conc.dilutionneeded(), ligand, inputs[i], mix=(False, False))
names[i]+="+"
rxs=inputs
self.e.shakeSamples(inputs,returnPlate=True)
elif self.rndNum==len(self.rounds) and self.finalPlus and keepCleaved:
rxs = self.runT7Setup(ligands=[reagents.getsample(inp['ligand']) for inp in self.inputs],src=inputs, vol=t7vol, srcdil=[inp.conc.dilutionneeded() for inp in inputs])
for i in range(len(inputs)):
inp=inputs[i]
if self.inputs[i]['ligand'] is not None:
rxs += self.runT7Setup(ligands=[reagents.getsample(self.inputs[i]['ligand'])],src=[inp],vol=t7vol,srcdil=[inp.conc.dilutionneeded()])
prefixIn+=[prefixIn[i]]
prefixOut+=[prefixOut[i]]
stop+=[stop[i]]
primerSet+=[primerSet[i]]
names+=["%s+"%names[i]]
elif keepCleaved:
rxs = self.runT7Setup(ligands=[reagents.getsample(inp['negligand']) for inp in self.inputs], src=inputs, vol=t7vol, srcdil=[inp.conc.dilutionneeded() for inp in inputs])
else:
rxs = self.runT7Setup(ligands=[reagents.getsample(inp['ligand']) for inp in self.inputs], src=inputs, vol=t7vol, srcdil=[inp.conc.dilutionneeded() for inp in inputs])
if self.rndNum==1 and "template" in self.qpcrStages:
# Initial input
for i in range(len(rxs)):
q.addSamples(src=rxs[i],needDil=needDil,primers=primerSet[i],names=["%s.T"%names[i]])
self.runT7Pgm(dur=self.t7dur,vol=t7vol)
for i in range(len(rxs)):
rxs[i].name="%s.t7"%names[i]
self.e.lihahome()
print("Estimate usable RNA concentration in T7 reaction at %.0f nM"%self.rnaConc)
if self.rndNum==1:
worklist.userprompt("T7 Incubation Started",120)
self.e.waitpgm() # So elapsed time will be updated
db.popStatus()
if self.edtastop:
print("######## Stop ########### %.0f min"%(clock.elapsed()/60))
db.pushStatus("Stop")
print("Have %.1f ul before stop"%rxs[0].volume)
preStopVolume=rxs[0].volume
self.addEDTA(tgt=rxs,finalconc=2) # Stop to 2mM EDTA final
db.popStatus("Stop")
stopDil=rxs[0].volume/preStopVolume
else:
stopDil=1
if self.pauseAfterStop:
worklist.userprompt("Post EDTA pause")
if self.saveRNA:
self.saveSamps(src=rxs,vol=self.saveRNAVolume,dil=self.saveRNADilution,plate=self.savePlate,dilutant=reagents.getsample("TE8"),mix=(False,False)) # Save to check [RNA] on Qubit, bioanalyzer
needDil = self.rnaConc/self.qConc/stopDil
if "stopped" in self.qpcrStages:
for i in range(len(rxs)):
q.addSamples(src=rxs[i:i+1],needDil=needDil,primers=primerSet[i],names=["%s.stopped"%names[i]])
print("######## RT Setup ########### %.0f min"%(clock.elapsed()/60))
db.pushStatus("RT")
hiTemp=95
stop=["%s-Stop"%n for n in stop]
rt=self.runRT(src=rxs,vol=rtvol,srcdil=self.rtDil,heatInactivate=self.rtHI,hiTemp=hiTemp,dur=self.rtdur,incTemp=50,stop=[reagents.getsample(s) for s in stop],stopConc=self.stopConc) # Heat inactivate also allows splint to fold
rxs=rt
for i in range(len(rxs)):
if dolig and not self.singlePrefix:
rxs[i].name=names[i]+"."+prefixOut[i]+".rt"
else:
rxs[i].name=names[i]+".rt"
print("RT volume= [",",".join(["%.1f "%x.volume for x in rxs]),"]")
needDil /=self.rtDil
if self.rtpostdil[self.rndNum-1]>1:
print("Dilution after RT: %.2f"%self.rtpostdil[self.rndNum-1])
self.diluteInPlace(tgt=rxs,dil=self.rtpostdil[self.rndNum-1])
needDil=needDil/self.rtpostdil[self.rndNum-1]
# Discard extra volume of any sample that has more than current rt volume so that we can shake at high speed
for r in Sample.getAllOnPlate(rxs[0].plate):
if r not in rxs and r.volume>max(15+1.4,rxs[0].volume)+4:
remove=r.volume-(15+1.4)
oldvol=r.volume
if r.lastMixed is None:
r.lastMixed=clock.elapsed # Override since we don't care about mixing for disposal
self.e.dispose(remove,r)
print("Discarding some of %s to reduce volume from %.1f to %.1f to allow faster shaking"%(r.name,oldvol,r.volume))
print("RT volume= ",[x.volume for x in rxs])
self.e.shakeSamples(rxs)
if self.rtSave:
rtsv=self.saveSamps(src=rxs,vol=self.rtSaveVol,dil=self.rtSaveDil,plate=self.savePlate,dilutant=reagents.getsample("TE8"),mix=(False,False)) # Save to check RT product on gel (2x dil)
if "rt" in self.qpcrStages:
for i in range(len(rxs)):
q.addSamples(src=rtsv[i:i+1],needDil=needDil/2,primers=self.rtprimers[self.rndNum-1] if hasattr(self,'rtprimers') else primerSet[i],names=["%s.rt"%names[i]])
else:
if "rt" in self.qpcrStages:
for i in range(len(rxs)):
q.addSamples(src=rxs[i:i+1],needDil=needDil,primers=self.rtprimers[self.rndNum-1] if hasattr(self,'rtprimers') else primerSet[i],names=["%s.rt"%names[i]])
rtCarryForwardDil=10
rtCarryForwardVol=3.5
if self.rtCarryForward and not keepCleaved:
# Also include RT from a prior round from here on
for r in self.lastSaved:
newsamp=Sample("%s.samp"%r.name,decklayout.SAMPLEPLATE)
self.e.transfer(rxs[0].volume,r,newsamp,(False,False))
rxs.append(newsamp)
db.popStatus()
if dolig:
print("######## Ligation setup ########### %.0f min"%(clock.elapsed()/60))
db.pushStatus("Ligation")
extdil=5.0/4
reagents.getsample("MLigase").conc=Concentration(5)
if self.ligInPlace:
rxs=self.runLig(rxs,inPlace=True,srcdil=extdil,incTime=self.ligdur)
else:
rxs=self.runLig(rxs,inPlace=False,srcdil=extdil,vol=20,incTime=self.ligdur)
print("Ligation volume= ",[x.volume for x in rxs])
needDil=needDil/extdil
if self.extpostdil[self.rndNum-1]>1:
print("Dilution after extension: %.2f"%self.extpostdil[self.rndNum-1])
self.diluteInPlace(tgt=rxs,dil=self.extpostdil[self.rndNum-1])
needDil=needDil/self.extpostdil[self.rndNum-1]
pcrdil=pcrdil*1.0/self.extpostdil[self.rndNum-1]
if self.saveDil is not None:
ext=self.saveSamps(src=rxs,vol=3,dil=self.saveDil,dilutant=reagents.getsample("TE8"),tgt=[Sample("%s.ext"%n,self.savePlate) for n in names],mix=(False,True)) # Save cDNA product for subsequent NGS
if "ext" in self.qpcrStages:
for i in range(len(ext)):
# Make sure we don't take more than 2 more steps
maxdil=q.MAXDIL*q.MAXDIL
if needDil/self.saveDil>maxdil:
logging.notice( "Diluting ext by %.0fx instead of needed %.0f to save steps"%(maxdil,needDil/self.saveDil))
pset=primerSet[i]
if "extraQPCR" in self.inputs[i]:
pset.udpate(self.inputs[i]["extraQPCR"])
q.addSamples(src=[ext[i]],needDil=min(maxdil,needDil/self.saveDil),primers=pset,names=["%s.ext"%names[i]],save=False)
else:
if "ext" in self.qpcrStages:
print("needDil=",needDil)
for i in range(len(names)):
pset=primerSet[i]
if "extraQPCR" in self.inputs[i]:
pset.update(self.inputs[i]["extraQPCR"])
q.addSamples(src=[rxs[i]],needDil=needDil,primers=pset,names=["%s.ext"%names[i]])
isave=i+len(names)
if isave<len(rxs):
# samples restored
q.addSamples(src=[rxs[isave]],needDil=needDil/rtCarryForwardDil,primers=primerSet[isave])
db.popStatus()
else:
extdil=1
self.extpostdil[self.rndNum-1]=1
if self.rtpostdil[self.rndNum-1]>1:
pcrdil=pcrdil*1.0/self.rtpostdil[self.rndNum-1]
totalDil=stopDil*self.rtDil*self.rtpostdil[self.rndNum-1]*extdil*self.extpostdil[self.rndNum-1]
fracRetained=rxs[0].volume/(t7vol*totalDil)
print("Total dilution from T7 to Pre-pcr Product = %.2f*%.2f*%.2f*%.2f*%.2f = %.2f, fraction retained=%.0f%%"%(stopDil,self.rtDil,self.rtpostdil[self.rndNum-1],extdil,self.extpostdil[self.rndNum-1],totalDil,fracRetained*100))
if self.rtCarryForward and not keepCleaved:
# Remove the extra samples
assert(len(self.lastSaved)>0)
rxs=rxs[:len(rxs)-len(self.lastSaved)]
self.lastSaved=[]
if len(rxs)>len(inputs):
# Have extra samples due when self.finalPlus is True
rxs= rxs[0:len(inputs)] # Only keep -target products
prefixOut= prefixOut[0:len(inputs)]
prefixIn= prefixIn[0:len(inputs)]
stop= stop[0:len(inputs)]
if self.dopcr and not (keepCleaved and self.noPCRCleave):
print("######### PCR ############# %.0f min"%(clock.elapsed()/60))
db.pushStatus("PCR")
print("PCR Volume: %.1f, Dilution: %.1f, volumes available for PCR: [%s]"%(pcrvol, pcrdil,",".join(["%.1f"%r.volume for r in rxs])))
initConc=needDil*self.qConc/pcrdil
if keepCleaved:
initConc=initConc*self.cleavage # Only use cleaved as input conc
else:
initConc=initConc*(1-self.cleavage)
gain=pcrgain(initConc,400,cycles)
finalConc=min(200,initConc*gain)
print("Estimated starting concentration in PCR = %.1f nM, running %d cycles -> %.0f nM\n"%(needDil*self.qConc/pcrdil,cycles,finalConc))
nsplit=int(math.ceil(pcrvol*1.0/self.maxPCRVolume))
print("Split each PCR into %d reactions"%nsplit)
sampNeeded=pcrvol/pcrdil
if self.rtCarryForward and keepCleaved:
sampNeeded+=rtCarryForwardVol
if keepCleaved and self.rtCarryForward:
print("Saving %.1f ul of each pre-PCR sample" % rtCarryForwardVol)
self.lastSaved=[Sample("%s.sv"%x.name,self.savePlate) for x in rxs]
for i in range(len(rxs)):
# Save with rtCarryForwardDil dilution to reduce amount of RT consumed (will have Ct's 2-3 lower than others)
self.e.transfer(rtCarryForwardVol,rxs[i],self.lastSaved[i],(False,False))
self.e.transfer(rtCarryForwardVol*(rtCarryForwardDil-1),decklayout.WATER,self.lastSaved[i],(False,True)) # Use pipette mixing -- shaker mixing will be too slow
#print "NSplit=",nsplit,", PCR vol=",pcrvol/nsplit,", srcdil=",pcrdil,", input vol=",pcrvol/nsplit/pcrdil
minvol=min([r.volume for r in rxs])
maxpcrvol=(minvol-15-1.4*nsplit)*pcrdil
if maxpcrvol<pcrvol:
print("Reducing PCR volume from %.1ful to %.1ful due to limited input"%(pcrvol, maxpcrvol))
pcrvol=maxpcrvol
if keepCleaved:
master="MTaqC"
else:
master="MTaqU"
reagents.getsample(master) # Allocate for this before primers
if self.barcoding:
primers=self.bcprimers[self.rndNum-1]
if primers is not None and nsplit>1:
primers=primers*nsplit
else:
primers=None
if primers is None:
primers=[("T7%sX"%x).replace("T7T7","T7") for x in prefixOut]*nsplit
rnddef = self.rnddef[self.rndNum-1]
bcout=[]
if 'barcode' in rnddef:
# Add barcoding primers
assert len(rnddef['barcode'])==len(rxs)
dil=self.saveSamps(rxs,dil=50,vol=2,plate=decklayout.SAMPLEPLATE)
for i in range(len(rxs)):
dil[i].conc=Concentration(25,1)
for bc in rnddef['barcode'][i]:
tgt=Sample("%s.%s"%(rxs[i].name,bc),decklayout.SAMPLEPLATE)
bparts=bc.split("/")
for b in bparts:
if not reagents.isReagent("P-%s"%b):
reagents.add(name="P-%s"%b,conc=Concentration(2.67,0.4,'uM'),extraVol=30)
print("PCR-%s"%bc)
self.e.stage("PCR-%s"%bc,reagents=[reagents.getsample("MTaqBar"),reagents.getsample("P-%s"%bparts[0]),reagents.getsample("P-%s"%bparts[1])],samples=[tgt],sources=[dil[i] ],volume=50,destMix=False)
bcout.append(tgt)
print(tgt.name,"wellMixed=",tgt.wellMixed)
print("Running PCR with master=",master,", primers=",primers)
pcr=self.runPCR(src=rxs*nsplit,vol=pcrvol/nsplit,srcdil=pcrdil,ncycles=cycles,primers=primers,usertime=self.usertime if keepCleaved else None,fastCycling=False,inPlace=False,master=master,lowhi=self.lowhi,annealTemp=57)
if keepCleaved and self.regenPCRCycles is not None:
# Regenerate prefix
pcr2=self.runPCR(src=pcr,vol=self.regenPCRVolume,srcdil=self.regenPCRDilution,ncycles=self.regenPCRCycles,primers=None,usertime=None,fastCycling=False,inPlace=False,master="MTaqR",lowhi=self.lowhi,annealTemp=55)
# Add BT575p for 1 more cycle
for p in pcr2:
self.e.transfer(p.volume*0.5/10,reagents.getsample("Unclvd-Stop"),p,(False,False))
# One more cycle
cycling=' TEMP@95,30 TEMP@55,30 TEMP@68,30 TEMP@25,2'
thermocycler.setpgm('rfin',100,cycling)
self.e.runpgm("rfin",5.0,False,max([p.volume for p in pcr2]))
pcr=pcr2 # Use 2nd PCR as actual output
if len(pcr)<=len(names):
# Don't relabel if we've split
for i in range(len(pcr)):
pcr[i].name=names[i]+".pcr"
#print "Volume remaining in PCR input source: [",",".join(["%.1f"%r.volume for r in rxs]),"]"
needDil=finalConc/self.qConc
print("Projected final concentration = %.0f nM"%(needDil*self.qConc))
for i in range(len(pcr)):
pcr[i].conc=Concentration(stock=finalConc,final=None,units='nM')
db.popStatus()
if self.pcrSave:
# Save samples at 1x (move all contents -- can ignore warnings)
maxSaveVol=(100 if self.savedilplate else 1500)*1.0/nsplit
if self.finalRound and nsplit==1 and self.savedilplate and pcrtgt is None:
print("Skipping save of final PCR")
sv=pcr
else:
residual=2.4 # Amount to leave behind to avoid aspirating air
sv=self.saveSamps(src=pcr[:len(rxs)],vol=[min([maxSaveVol,x.volume-residual]) for x in pcr[:len(rxs)]],dil=1,plate=(self.savePlate if self.savedilplate else decklayout.EPPENDORFS),tgt=pcrtgt)
if nsplit>1:
# Combine split
for i in range(len(rxs),len(rxs)*nsplit):
self.e.transfer(min([maxSaveVol,pcr[i].volume-residual]),pcr[i],sv[i%len(sv)],mix=(False,False))
# Correct concentration (above would've assumed it was diluted)
for i in range(len(sv)):
sv[i].conc=pcr[i].conc
# Shake
self.e.shakeSamples(sv)
if "pcr" in self.qpcrStages:
for i in range(len(sv)):
q.addSamples(sv[i],needDil,primers=primerSet[i],names=["%s.pcr"%names[i]])
print("Have %.2f pmoles of product (%.0f ul @ %.1f nM)"%(sv[0].volume*sv[0].conc.stock/1000,sv[0].volume,sv[0].conc.stock))
# Save barcoded products too
if len(bcout)>0:
print("bcout=",",".join(str(b) for b in bcout))
print("mixed=",bcout[0].isMixed(),", wellMixed=",bcout[0].wellMixed)
bcsave=self.saveSamps(src=bcout,vol=[b.volume for b in bcout],dil=1,plate=self.savePlate,mix=(False,False))
if "bc" in self.qpcrStages:
print("Doing qPCR of barcoding: ",bcsave)
for i in range(len(bcsave)):
needDil=640
q.addSamples(src=bcsave[i],needDil=needDil,primers=["T7X","WX","ZX"]+(["MX"] if self.useMX else []),save=False)
else:
bcsave=[]
return sv, bcsave
else:
assert "pcr" not in self.qpcrStages ## Not implemented
return pcr[:len(rxs)], bcout
elif self.noPCRCleave:
print("Dilution instead of PCR: %.2f"%self.nopcrdil)
# Need to add enough t7prefix to compensate for all of the Stop primer currently present, regardless of whether it is for cleaved or uncleaved
# Will result in some short transcripts corresponding to the stop primers that are not used for cleaved product, producing just GGG_W_GTCTGC in the next round. These would be reverse-trancribed, but may compete for T7 yield
t7prefix=reagents.getsample("BT88")
dil=self.extpostdil[self.rndNum-1] # FIXME: Is this correct? Used to have a 'userDil' term
stopconc=1000.0/dil
bt88conc=t7prefix.conc.stock
relbt88=stopconc/bt88conc
print("Using EXT with %.0fnM of stop oligo as input to next T7, need %.2ful of BT88@%.0fnM per ul of sample"%(stopconc,relbt88,bt88conc))
for r in rxs:
vol=r.volume*relbt88
t7prefix.conc.final=t7prefix.conc.stock*vol/(r.volume+vol)
r.conc.final=r.conc.stock*r.volume/(r.volume+vol)
self.e.transfer(vol,t7prefix,r,mix=(False,False))
if self.nopcrdil>(1+relbt88):
self.diluteInPlace(tgt=rxs,dil=self.nopcrdil/(1.0+relbt88))
#needDil=needDil/self.nopcrdil # needDil not used subsequently
print("Dilution of EXT product: %.2fx * %.2fx = %2.fx\n"%(1+relbt88,self.nopcrdil/(1+relbt88),self.nopcrdil))
else:
print("Dilution of EXT product: %.2fx\n"%(1+relbt88))
return rxs, []
else:
return rxs, []