def pgm(self):
q = QSetup(self, maxdil=16, debug=False, mindilvol=60)
self.e.addIdleProgram(q.idler)
input = [s.getsample() for s in self.srcs]
# Save RT product from first (uncleaved) round and then use it during 2nd (cleaved) round for ligation and qPCR measurements
prefixIn = [inp['prefix'] for inp in self.inputs]
prefixOut = [
"A" if p == "W" else
"B" if p == "A" else "W" if p == "B" else "BADPREFIX"
for p in prefixIn
]
qpcrPrimers = ["REF", "MX", "T7X"]
if "W" in prefixIn + prefixOut:
qpcrPrimers += ["T7WX"]
if "A" in prefixIn + prefixOut:
qpcrPrimers += ["T7AX"]
if "B" in prefixIn + prefixOut:
qpcrPrimers += ["T7BX"]
q.addSamples(decklayout.SSDDIL, 1, qpcrPrimers,
save=False) # Negative controls
print "Starting new cleavage round, will add prefix: ", prefixOut
names = [i.name for i in input]
print "######## T7 ###########"
print "Inputs: (t7vol=%.2f)" % self.t7vol
for inp in input:
print " %s: %.1ful@%.1f nM, use %.1f ul (%.3f pmoles)" % (
inp.name, inp.volume, inp.conc.stock, self.t7vol /
inp.conc.dilutionneeded(), self.t7vol * inp.conc.final / 1000)
print "input[0]=", input[0]
needDil = max([inp.conc.final for inp in input]) * 1.0 / self.qConc
if self.directT7:
# Just add MT7 and possibly water to each well
mconc = reagents.getsample("MT7").conc.dilutionneeded()
for i in range(len(input)):
watervol = self.t7vol * (1 - 1 / mconc) - input[i].volume
if watervol > 0.1:
self.e.transfer(watervol,
decklayout.WATER,
input[i],
mix=(False, False))
self.e.transfer(self.t7vol / mconc,
reagents.getsample("MT7"),
input[i],
mix=(False, False))
assert (input[i].volume == self.t7vol)
rxs = input
else:
rxs = self.runT7Setup(
src=input,
vol=self.t7vol,
srcdil=[inp.conc.dilutionneeded() for inp in input])
print "input[0]=", input[0]
#for i in range(len(rxs)):
# q.addSamples(src=rxs],needDil=needDil,primers=["T7"+prefixIn[i]+"X","MX","T7X","REF"],names=["%s.T-"%names[i]])
self.runT7Pgm(dur=self.t7dur, vol=self.t7vol)
print "Template conc=%.1f nM, estimated RNA concentration in T7 reaction at %.0f nM" % (
self.tmplFinalConc, self.rnaConc)
print "######## Stop ###########"
self.e.lihahome()
print "Have %.1f ul before stop" % rxs[0].volume
preStopVolume = rxs[0].volume
self.addEDTA(tgt=rxs, finalconc=2) # Stop to 2mM EDTA final
stopDil = rxs[0].volume / preStopVolume
if self.saveRNA:
self.saveSamps(
src=rxs,
vol=5,
dil=2,
plate=decklayout.DILPLATE,
dilutant=reagents.getsample("TE8"),
mix=(False,
False)) # Save to check [RNA] on Qubit, bioanalyzer
stop = [
"A-Stop" if n == "A" else
"B-Stop" if n == "B" else "W-Stop" if n == "W" else "BADPREFIX"
for n in prefixOut
]
for i in range(len(rxs)):
rxs[i].name = rxs[i].name + "." + stop[i]
needDil = self.rnaConc / self.qConc / stopDil
#q.addSamples(src=rxs,needDil=needDil,primers=["T7AX","MX","T7X","REF"],names=["%s.stopped"%r.name for r in rxs])
print "######## RT Setup ###########"
rtDil = 4
hiTemp = 95
rtDur = 20
rxin = rxs
rxs = self.runRT(src=rxs,
vol=self.rtvol,
srcdil=rtDil,
heatInactivate=True,
hiTemp=hiTemp,
dur=rtDur,
incTemp=50,
stop=[reagents.getsample(s) for s in stop
]) # Heat inactivate also allows splint to fold
print "RT volume= ", [x.volume for x in rxs]
for r in rxin:
if r.volume > 20:
print "Have more T7 reaction remaining than needed: %s has %.1f ul" % (
r.name, r.volume)
needDil /= rtDil
rtPostDil = 5
if rtPostDil != 1:
self.diluteInPlace(tgt=rxs, dil=rtPostDil)
needDil /= rtPostDil
#q.addSamples(src=rxs,needDil=needDil,primers=["T7AX","MX","REF"],names=["%s.rt"%r.name for r in rxs])
print "######## Ligation setup ###########"
extdil = 5.0 / 4
reagents.getsample("MLigase").conc = Concentration(5)
extvol = 20
print "Extension volume=", extvol
rxs = self.runLig(rxs, vol=extvol)
print "Ligation volume= ", [x.volume for x in rxs]
needDil = needDil / extdil
extpostdil = 4
if extpostdil > 1:
print "Dilution after extension: %.2f" % extpostdil
self.diluteInPlace(tgt=rxs, dil=extpostdil)
needDil = needDil / extpostdil
if not self.doexo:
self.pcrdil = self.pcrdil / extpostdil
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, decklayout.DILPLATE) for n in names],
mix=(False, True)) # Save cDNA product for subsequent NGS
for i in range(len(rxs)):
q.addSamples(src=[ext[i]],
needDil=needDil / self.saveDil,
primers=[
"T7" + prefixIn[i] + "X",
"T7" + prefixOut[i] + "X", "MX", "T7X", "REF"
],
names=["%s.ext" % names[i]])
else:
for i in range(len(rxs)):
q.addSamples(src=[rxs[i]],
needDil=needDil,
primers=[
"T7" + prefixIn[i] + "X",
"T7" + prefixOut[i] + "X", "MX", "T7X", "REF"
],
names=["%s.ext" % names[i]])
if self.doexo:
print "######## Exo ###########"
prevvol = rxs[0].volume
rxs = self.runExo(rxs, incTime=30, inPlace=True)
exoDil = rxs[0].volume / prevvol
needDil /= exoDil
needDil /= 7 # Anecdotal based on Ct's -- large components (MX) reduced by exo digestion
q.addSamples(src=rxs,
needDil=needDil,
primers=["T7AX", "T7BX", "MX", "T7X", "REF"],
names=["%s.exo" % names[i] for i in range(len(rxs))])
#exo=self.saveSamps(src=rxs,vol=10*exoDil,dil=2/exoDil,dilutant=reagents.getsample("TE8"),tgt=[Sample("%s.exo"%n,decklayout.SAMPLEPLATE) for n in names]) # Save cDNA product
else:
exoDil = 1
exo = []
if self.doampure:
print "######## Ampure Cleanup ###########"
ratio = 1.5
elutionVol = 30
cleanIn = ext + exo + user
needDil = needDil * cleanIn[0].volume / elutionVol
clean = self.runAmpure(src=cleanIn,
ratio=ratio,
elutionVol=elutionVol)
q.addSamples(src=[clean[i] for i in range(len(rxs))],
needDil=needDil,
primers=["T7AX", "MX", "T7X", "REF"])
rxs = rxs + clean # Use the cleaned products for PCR
totalDil = stopDil * rtDil * rtPostDil * extdil * extpostdil * exoDil
fracRetained = rxs[0].volume / (self.t7vol * totalDil)
print "Total dilution from T7 to Pre-pcr Product = %.2f*%.2f*%.2f*%.2f*%.2f*%.2f = %.2f, fraction retained=%.0f%%" % (
stopDil, rtDil, rtPostDil, extdil, extpostdil, exoDil, totalDil,
fracRetained * 100)
if self.dopcr:
print "######### PCR #############"
print "PCR Volume: %.1f, Dilution: %.1f, volumes available for PCR: [%s]" % (
self.pcrvol, self.pcrdil, ",".join(
["%.1f" % r.volume for r in rxs]))
maxSampleVolume = 100 # Maximum sample volume of each PCR reaction (thermocycler limit, and mixing limit)
initConc = needDil * self.qConc / self.pcrdil
if self.doexo:
initConc = initConc * 7 * self.cleavage # Back out 7x dilution in exo step, but only use cleaved as input conc
else:
initConc = initConc * self.cleavage # Only use cleaved as input conc
gain = pcrgain(initConc, 400, self.pcrcycles)
finalConc = initConc * gain
print "Estimated starting concentration in PCR = %.1f nM, running %d cycles -> %.0f nM\n" % (
needDil * self.qConc, self.pcrcycles, finalConc)
pcr = self.runPCR(src=rxs,
vol=self.pcrvol,
srcdil=self.pcrdil,
ncycles=self.pcrcycles,
primers=["T7%sX" % x for x in prefixOut],
usertime=self.usertime,
fastCycling=True)
needDil = finalConc / self.qConc
pcrpostdil = 2
if pcrpostdil > 1:
self.diluteInPlace(pcr, pcrpostdil)
needDil = needDil / pcrpostdil
print "Projected final concentration = %.0f nM (after %.1fx dilution)" % (
needDil * self.qConc, pcrpostdil)
for i in range(len(pcr)):
pcr[i].conc = Concentration(stock=finalConc / pcrpostdil,
final=None,
units='nM')
if self.pcrSave:
# Save samples at 1x
if self.savedilplate:
sv = self.saveSamps(
src=pcr[:len(rxs)],
vol=[x.volume - 16.4 for x in pcr[:len(rxs)]],
dil=1,
plate=decklayout.DILPLATE)
else:
sv = self.saveSamps(
src=pcr[:len(rxs)],
vol=[x.volume - 16.4 for x in pcr[:len(rxs)]],
dil=1,
plate=decklayout.EPPENDORFS)
# for i in range(len(pcr)):
# q.addSamples(pcr,needDil,["T7%sX"%prefixOut[i]])
processEff = 0.5 # Estimate of overall efficiency of process
print "Saved %.2f pmoles of product (%.0f ul @ %.1f nM)" % (
sv[0].volume * sv[0].conc.stock / 1000, sv[0].volume,
sv[0].conc.stock)
print "######### qPCR ###########"
#q.addReferences(mindil=4,nsteps=6,primers=["T7X","MX","T7AX"])
q.run(confirm=False)
def pgm(self):
q = QSetup(self,maxdil=16,debug=False,mindilvol=60)
self.e.addIdleProgram(q.idler)
input = [s.getsample() for s in self.srcs]
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
prefixIn=[inp['prefix'] for inp in self.inputs]
prefixOut=["A" if p=="W" else "B" if p=="A" else "W" if p=="B" else "BADPREFIX" for p in prefixIn]
print "Starting new cleavage round, will add prefix: ",prefixOut
names=[i.name for i in input]
print "######## T7 ###########"
print "Inputs: (t7vol=%.2f)"%self.t7vol
for inp in input:
print " %s: %.1ful@%.1f nM, use %.1f ul (%.3f pmoles)"%(inp.name,inp.volume,inp.conc.stock,self.t7vol/inp.conc.dilutionneeded(), self.t7vol*inp.conc.final/1000)
print "input[0]=",input[0]
needDil = max([inp.conc.final for inp in input])*1.0/self.qConc
if self.directT7:
# Just add MT7 and possibly water to each well
mconc=reagents.getsample("MT7").conc.dilutionneeded()
for i in range(len(input)):
watervol=self.t7vol*(1-1/mconc)-input[i].volume
if watervol>0.1:
self.e.transfer(watervol,decklayout.WATER,input[i],mix=(False,False))
self.e.transfer(self.t7vol/mconc,reagents.getsample("MT7"),input[i],mix=(False,False))
assert(input[i].volume==self.t7vol)
rxs=input
else:
rxs = self.runT7Setup(src=input,vol=self.t7vol,srcdil=[inp.conc.dilutionneeded() for inp in input])
print "input[0]=",input[0]
#for i in range(len(rxs)):
# q.addSamples(src=rxs],needDil=needDil,primers=["T7"+prefixIn[i]+"X","MX","T7X","REF"],names=["%s.T-"%names[i]])
self.runT7Pgm(dur=self.t7dur,vol=self.t7vol)
print "Template conc=%.1f nM, estimated RNA concentration in T7 reaction at %.0f nM"%(self.tmplFinalConc,self.rnaConc)
print "######## Stop ###########"
#self.saveSamps(src=rxs,vol=5,dil=10,plate=decklayout.EPPENDORFS,dilutant=reagents.getsample("TE8"),mix=(False,False)) # Save to check [RNA] on Qubit, bioanalyzer
self.e.lihahome()
print "Have %.1f ul before stop"%rxs[0].volume
preStopVolume=rxs[0].volume
self.addEDTA(tgt=rxs,finalconc=2) # Stop to 2mM EDTA final
stop=[ "A-Stop" if n=="A" else "B-Stop" if n=="B" else "W-Stop" if n=="W" else "BADPREFIX" for n in prefixOut]
for i in range(len(rxs)):
rxs[i].name=rxs[i].name+"."+stop[i]
stopDil=rxs[0].volume/preStopVolume
needDil = self.rnaConc/self.qConc/stopDil
#q.addSamples(src=rxs,needDil=needDil,primers=["T7AX","MX","T7X","REF"],names=["%s.stopped"%r.name for r in rxs])
print "######## RT Setup ###########"
rtDil=4
hiTemp=95
rtDur=20
rxin=rxs
rxs=self.runRT(src=rxs,vol=self.rtvol,srcdil=rtDil,heatInactivate=True,hiTemp=hiTemp,dur=rtDur,incTemp=50,stop=[reagents.getsample(s) for s in stop]) # Heat inactivate also allows splint to fold
print "RT volume= ",[x.volume for x in rxs]
for r in rxin:
if r.volume>20:
print "Have more T7 reaction remaining than needed: %s has %.1f ul"%(r.name,r.volume)
needDil /= rtDil
rtPostDil=5
if rtPostDil!=1:
self.diluteInPlace(tgt=rxs,dil=rtPostDil)
needDil /= rtPostDil
#q.addSamples(src=rxs,needDil=needDil,primers=["T7AX","MX","REF"],names=["%s.rt"%r.name for r in rxs])
print "######## Ligation setup ###########"
extdil=5.0/4
reagents.getsample("MLigase").conc=Concentration(5)
extvol=20;
print "Extension volume=",extvol
rxs=self.runLig(rxs,vol=extvol)
print "Ligation volume= ",[x.volume for x in rxs]
needDil=needDil/extdil
extpostdil=4
if extpostdil>1:
print "Dilution after extension: %.2f"%extpostdil
self.diluteInPlace(tgt=rxs,dil=extpostdil)
needDil=needDil/extpostdil
if not self.doexo:
self.pcrdil=self.pcrdil/extpostdil
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,decklayout.DILPLATE) for n in names],mix=(False,True)) # Save cDNA product for subsequent NGS
for i in range(len(rxs)):
q.addSamples(src=[ext[i]],needDil=needDil/self.saveDil,primers=["T7"+prefixIn[i]+"X","T7"+prefixOut[i]+"X","MX","T7X","REF"],names=["%s.ext"%names[i]])
else:
for i in range(len(rxs)):
q.addSamples(src=[rxs[i]],needDil=needDil,primers=["T7"+prefixIn[i]+"X","T7"+prefixOut[i]+"X","MX","T7X","REF"],names=["%s.ext"%names[i]])
if self.doexo:
print "######## Exo ###########"
prevvol=rxs[0].volume
rxs=self.runExo(rxs,incTime=30,inPlace=True)
exoDil=rxs[0].volume/prevvol
needDil/=exoDil
needDil/=7 # Anecdotal based on Ct's -- large components (MX) reduced by exo digestion
q.addSamples(src=rxs,needDil=needDil,primers=["T7AX","T7BX","MX","T7X","REF"],names=["%s.exo"%names[i] for i in range(len(rxs))])
#exo=self.saveSamps(src=rxs,vol=10*exoDil,dil=2/exoDil,dilutant=reagents.getsample("TE8"),tgt=[Sample("%s.exo"%n,decklayout.SAMPLEPLATE) for n in names]) # Save cDNA product
else:
exoDil=1
exo=[]
if self.doampure:
print "######## Ampure Cleanup ###########"
ratio=1.5
elutionVol=30
cleanIn=ext+exo+user
needDil=needDil*cleanIn[0].volume/elutionVol
clean=self.runAmpure(src=cleanIn,ratio=ratio,elutionVol=elutionVol)
q.addSamples(src=[clean[i] for i in range(len(rxs))],needDil=needDil,primers=["T7AX","MX","T7X","REF"])
rxs=rxs+clean # Use the cleaned products for PCR
totalDil=stopDil*rtDil*rtPostDil*extdil*extpostdil*exoDil
fracRetained=rxs[0].volume/(self.t7vol*totalDil)
print "Total dilution from T7 to Pre-pcr Product = %.2f*%.2f*%.2f*%.2f*%.2f*%.2f = %.2f, fraction retained=%.0f%%"%(stopDil,rtDil,rtPostDil,extdil,extpostdil,exoDil,totalDil,fracRetained*100)
if self.dopcr:
print "######### PCR #############"
print "PCR Volume: %.1f, Dilution: %.1f, volumes available for PCR: [%s]"%(self.pcrvol, self.pcrdil,",".join(["%.1f"%r.volume for r in rxs]))
maxSampleVolume=100 # Maximum sample volume of each PCR reaction (thermocycler limit, and mixing limit)
initConc=needDil*self.qConc/self.pcrdil
if self.doexo:
initConc=initConc*7*self.cleavage # Back out 7x dilution in exo step, but only use cleaved as input conc
else:
initConc=initConc*self.cleavage # Only use cleaved as input conc
gain=pcrgain(initConc,400,self.pcrcycles)
finalConc=initConc*gain
print "Estimated starting concentration in PCR = %.1f nM, running %d cycles -> %.0f nM\n"%(needDil*self.qConc,self.pcrcycles,finalConc)
pcr=self.runPCR(src=rxs,vol=self.pcrvol,srcdil=self.pcrdil,ncycles=self.pcrcycles,primers=["T7%sX"%x for x in prefixOut],usertime=self.usertime,fastCycling=True)
needDil=finalConc/self.qConc
pcrpostdil=2
if pcrpostdil>1:
self.diluteInPlace(pcr,pcrpostdil)
needDil=needDil/pcrpostdil
print "Projected final concentration = %.0f nM (after %.1fx dilution)"%(needDil*self.qConc,pcrpostdil)
for i in range(len(pcr)):
pcr[i].conc=Concentration(stock=finalConc/pcrpostdil,final=None,units='nM')
if self.pcrSave:
# Save samples at 1x
if self.savedilplate:
sv=self.saveSamps(src=pcr[:len(rxs)],vol=[x.volume-16.4 for x in pcr[:len(rxs)]],dil=1,plate=decklayout.DILPLATE)
else:
sv=self.saveSamps(src=pcr[:len(rxs)],vol=[x.volume-16.4 for x in pcr[:len(rxs)]],dil=1,plate=decklayout.EPPENDORFS)
# for i in range(len(pcr)):
# q.addSamples(pcr,needDil,["T7%sX"%prefixOut[i]])
processEff=0.5 # Estimate of overall efficiency of process
print "Saved %.2f pmoles of product (%.0f ul @ %.1f nM)"%(sv[0].volume*sv[0].conc.stock/1000,sv[0].volume,sv[0].conc.stock)
print "######### qPCR ###########"
#q.addReferences(mindil=4,nsteps=6,primers=["T7X","MX","T7AX"])
#worklist.userprompt('Continue to setup qPCR')
q.run()
def oneround(self, q, input, prefixOut, stop, prefixIn, keepCleaved, t7vol,
rtvol, pcrdil, cycles, pcrvol, dolig):
primerSet = [
set(["MX", "REF", "T7X", prefixIn[i] + "X", prefixOut[i] + "X"])
for i in range(len(prefixIn))
]
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=", t7vol, ",rtvol=", rtvol, ",pcrdil=", pcrdil, ",cycles=", cycles, ",dolig=", dolig
if self.rtCarryForward:
assert (dolig)
names = [i.name for i in input]
if self.rnaInput:
rxs = input
stopDil = 1
else:
print "######## T7 ########### %.0f min" % (clock.elapsed() / 60)
print "Inputs: (t7vol=%.2f)" % t7vol
inconc = [inp.conc.final for inp in input]
for inp in input:
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)
needDil = max([inp.conc.stock
for inp in input]) * 1.0 / self.qConc
else:
print " %s: %.1ful@%.1f %s, use %.1f ul" % (
inp.name, inp.volume, inp.conc.stock, inp.conc.units,
t7vol / inp.conc.dilutionneeded())
needDil = 100 / self.qConc # Assume 100nM
# inp.conc.final=inp.conc.stock*self.templateDilution
if self.directT7 and self.rndNum == 1:
# Just add ligands and MT7 to each well
if not keepCleaved:
for i in range(len(input)):
if self.inputs[i]['ligand'] is not None:
ligand = reagents.getsample(
self.inputs[i]['ligand'])
self.e.transfer(t7vol /
ligand.conc.dilutionneeded(),
ligand,
input[i],
mix=(False, False))
names[i] += "+"
mconc = reagents.getsample("MT7").conc.dilutionneeded()
for i in range(len(input)):
watervol = t7vol * (1 - 1 / mconc) - input[i].volume
if watervol > 0.1:
self.e.transfer(watervol,
decklayout.WATER,
input[i],
mix=(False, False))
self.e.transfer(t7vol / mconc,
reagents.getsample("MT7"),
input[i],
mix=(False, False))
assert (abs(input[i].volume - t7vol) < 0.1)
rxs = input
elif self.rndNum == len(
self.rounds) and self.finalPlus and keepCleaved:
rxs = self.runT7Setup(
src=input,
vol=t7vol,
srcdil=[inp.conc.dilutionneeded() for inp in input])
for i in range(len(input)):
inp = input[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(
src=input,
vol=t7vol,
srcdil=[inp.conc.dilutionneeded() for inp in input])
else:
rxs = self.runT7Setup(
ligands=[
reagents.getsample(inp['ligand'])
for inp in self.inputs
],
src=input,
vol=t7vol,
srcdil=[inp.conc.dilutionneeded() for inp in input])
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]])
needDil = needDil * max(
[inp.conc.dilutionneeded() for inp in input])
self.runT7Pgm(dur=self.t7dur, vol=t7vol)
for i in range(len(rxs)):
rxs[i].name = "%s.t7" % names[i]
print "Estimate usable RNA concentration in T7 reaction at %.0f nM" % self.rnaConc
print "######## Stop ########### %.0f min" % (clock.elapsed() / 60)
self.e.lihahome()
print "Have %.1f ul before stop" % rxs[0].volume
preStopVolume = rxs[0].volume
self.addEDTA(tgt=rxs, finalconc=2) # Stop to 2mM EDTA final
stopDil = rxs[0].volume / preStopVolume
if self.saveRNA:
self.saveSamps(
src=rxs,
vol=5,
dil=self.saveRNADilution,
plate=decklayout.DILPLATE,
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)
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]
if self.rtSave:
rtsv = self.saveSamps(
src=rxs,
vol=self.rtSaveVol,
dil=self.rtSaveDil,
plate=decklayout.DILPLATE,
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)
if dolig:
print "######## Ligation setup ########### %.0f min" % (
clock.elapsed() / 60)
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, decklayout.DILPLATE)
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))
q.addSamples(src=[ext[i]],
needDil=min(maxdil,
needDil / self.saveDil),
primers=primerSet[i],
names=["%s.ext" % names[i]],
save=False)
else:
if "ext" in self.qpcrStages:
print "needDil=", needDil
for i in range(len(names)):
q.addSamples(src=[rxs[i]],
needDil=needDil,
primers=primerSet[i],
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])
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(input):
# Have extra samples due when self.finalPlus is True
rxs = rxs[0:len(input)] # Only keep -target products
prefixOut = prefixOut[0:len(input)]
prefixIn = prefixIn[0:len(input)]
stop = stop[0:len(input)]
if self.dopcr and not (keepCleaved and self.noPCRCleave):
print "######### PCR ############# %.0f min" % (clock.elapsed() /
60)
maxvol = max([r.volume for r in rxs])
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
minsrcdil = 1 / (1 - 1.0 / 3 - 1.0 / 4)
sampNeeded = pcrvol / pcrdil
if self.rtCarryForward and keepCleaved:
sampNeeded += rtCarryForwardVol
maxvol = max([r.volume for r in rxs])
minvol = min([r.volume for r in rxs])
if keepCleaved and self.rtCarryForward:
assert (len(rxs) == len(rtCarryForward))
print "Saving %.1f ul of each pre-PCR sample" % (
rtCarryForwardVol)
self.lastSaved = [
Sample("%s.sv" % x.name, decklayout.DILPLATE) 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"
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
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'
worklist.pyrun('PTC\\ptcsetpgm.py rfin %s' % (cycling))
self.e.runpgm("rfin",
5.0,
False,
max([p.volume for p in pcr2]),
hotlidmode="CONSTANT",
hotlidtemp=100)
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')
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:
print "Skipping save of final PCR"
sv = pcr
else:
sv = self.saveSamps(
src=pcr[:len(rxs)],
vol=[
min([maxSaveVol, x.volume]) for x in pcr[:len(rxs)]
],
dil=1,
plate=(decklayout.DILPLATE if self.savedilplate else
decklayout.EPPENDORFS),
atEnd=self.savePCRAtEnd)
if nsplit > 1:
# Combine split
for i in range(len(rxs), len(rxs) * nsplit):
self.e.transfer(min([maxSaveVol, pcr[i].volume]),
pcr[i],
sv[i % len(sv)],
mix=(False,
i >= len(rxs) * (nsplit - 1)))
# Correct concentration (above would've assumed it was diluted)
for i in range(len(sv)):
sv[i].conc = pcr[i].conc
if "pcr" in self.qpcrStages:
for i in range(len(sv)):
q.addSamples(sv[i],
needDil,
primers=primerSet[i],
names=["%s.pcr" % names[i]])
processEff = 0.5 # Estimate of overall efficiency of process
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)
return sv
else:
assert "pcr" not in self.qpcrStages ## Not implemented
return pcr[:len(rxs)]
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] * userDil
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
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
def oneround(self,q,input,prefixOut,prefixIn,keepCleaved,t7vol,rtvol,pcrdil,cycles,pcrvol,dolig):
if keepCleaved:
print "Starting new cleavage round, will add prefix: ",prefixOut
assert(dolig)
else:
print "Starting new uncleaved round, will retain prefix: ",prefixIn
if self.rtSave:
assert(dolig)
names=[i.name for i in input]
print "######## T7 ###########"
print "Inputs: (t7vol=%.2f)"%t7vol
inconc=[inp.conc.final for inp in input]
for inp in input:
print " %s: %.1ful@%.1f nM, use %.1f ul (%.3f pmoles)"%(inp.name,inp.volume,inp.conc.stock,t7vol/inp.conc.dilutionneeded(), t7vol*inp.conc.final/1000)
# inp.conc.final=inp.conc.stock*self.templateDilution
needDil = max([inp.conc.stock for inp in input])*1.0/self.qConc
if self.directT7 and self.rndNum==1:
# Just add ligands and MT7 to each well
for i in range(len(input)):
ligand=reagents.getsample(self.inputs[i]['ligand'])
self.e.transfer(t7vol/ligand.conc.dilutionneeded(),ligand,input[i],mix=(False,False))
mconc=reagents.getsample("MT7").conc.dilutionneeded()
for i in range(len(input)):
watervol=t7vol*(1-1/mconc)-input[i].volume
if watervol>0.1:
self.e.transfer(watervol,decklayout.WATER,input[i],mix=(False,False))
self.e.transfer(t7vol/mconc,reagents.getsample("MT7"),input[i],mix=(False,False))
assert(abs(input[i].volume-t7vol)<0.1)
rxs=input
elif self.rndNum==self.nrounds and self.finalPlus:
rxs = self.runT7Setup(src=input,vol=t7vol,srcdil=[inp.conc.dilutionneeded() for inp in input])
rxs += self.runT7Setup(ligands=[reagents.getsample(inp['ligand']) for inp in self.inputs],src=input,vol=t7vol,srcdil=[inp.conc.dilutionneeded() for inp in input])
prefixIn+=prefixIn
prefixOut+=prefixOut
names+=["%s+"%n for n in names]
elif keepCleaved:
rxs = self.runT7Setup(src=input,vol=t7vol,srcdil=[inp.conc.dilutionneeded() for inp in input])
else:
rxs = self.runT7Setup(ligands=[reagents.getsample(inp['ligand']) for inp in self.inputs],src=input,vol=t7vol,srcdil=[inp.conc.dilutionneeded() for inp in input])
for i in range(len(rxs)):
rxs[i].name="%s.rx"%names[i]
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=["T7X","REF","T7"+prefixIn[i]+"X"],names=["%s.T-"%names[i]])
needDil = needDil*max([inp.conc.dilutionneeded() for inp in input])
t7dur=30
self.runT7Pgm(dur=t7dur,vol=t7vol)
print "Estimate RNA concentration in T7 reaction at %.0f nM"%self.rnaConc
self.rnaConc=min(40,inconc)*t7dur*65/30
print "######## Stop ###########"
#self.saveSamps(src=rxs,vol=5,dil=10,plate=decklayout.EPPENDORFS,dilutant=reagents.getsample("TE8"),mix=(False,False)) # Save to check [RNA] on Qubit, bioanalyzer
self.e.lihahome()
print "Have %.1f ul before stop"%rxs[0].volume
preStopVolume=rxs[0].volume
self.addEDTA(tgt=rxs,finalconc=2) # Stop to 2mM EDTA final
stop=["Unclvd-Stop" if (not dolig) else "A-Stop" if n=="A" else "B-Stop" if n=="B" else "W-Stop" if n=="W" else "BADPREFIX" for n in prefixOut]
stopDil=rxs[0].volume/preStopVolume
needDil = self.rnaConc/self.qConc/stopDil
if "stopped" in self.qpcrStages:
q.addSamples(src=rxs,needDil=needDil,primers=["T7AX","MX","T7X","REF"],names=["%s.stopped"%r.name for r in rxs])
print "######## RT Setup ###########"
rtDil=4
hiTemp=95
rtDur=20
rxs=self.runRT(src=rxs,vol=rtvol,srcdil=rtDil,heatInactivate=True,hiTemp=hiTemp,dur=rtDur,incTemp=50,stop=[reagents.getsample(s) for s in stop]) # Heat inactivate also allows splint to fold
print "RT volume= ",[x.volume for x in rxs]
needDil /= rtDil
if "rt" in self.qpcrStages:
q.addSamples(src=rxs,needDil=needDil,primers=["T7AX","MX","REF"],names=["%s.rt"%r.name for r in rxs])
rtSaveDil=10
rtSaveVol=3.5
if self.rtSave 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)
if dolig:
print "######## Ligation setup ###########"
extdil=5.0/4
reagents.getsample("MLigase").conc=Concentration(5)
rxs=self.runLig(rxs,inPlace=True)
print "Ligation volume= ",[x.volume for x in rxs]
needDil=needDil/extdil
extpostdil=2
if extpostdil>1:
print "Dilution after extension: %.2f"%extpostdil
self.diluteInPlace(tgt=rxs,dil=extpostdil)
needDil=needDil/extpostdil
if not self.doexo:
pcrdil=pcrdil/extpostdil
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,decklayout.DILPLATE) 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))
q.addSamples(src=[ext[i]],needDil=min(maxdil,needDil/self.saveDil),primers=["T7"+prefixIn[i]+"X","T7"+prefixOut[i]+"X","MX","T7X","REF"],names=["%s.ext"%names[i]],save=False)
else:
if "ext" in self.qpcrStages:
for i in range(len(input)):
q.addSamples(src=[rxs[i]],needDil=needDil,primers=["T7"+prefixIn[i]+"X","T7"+prefixOut[i]+"X","MX","T7X","REF"],names=["%s.ext"%names[i]])
isave=i+len(input)
if isave<len(rxs):
# samples restored
q.addSamples(src=[rxs[isave]],needDil=needDil/rtSaveDil,primers=["T7"+rxs[isave].name[0]+"X","T7"+("B" if rxs[isave].name[0]=="A" else "W" if rxs[isave].name[0]=="B" else "A")+"X","MX","T7X","REF"])
if self.doexo:
print "######## Exo ###########"
prevvol=rxs[0].volume
rxs=self.runExo(rxs,incTime=30,inPlace=True)
exoDil=rxs[0].volume/prevvol
needDil/=exoDil
needDil/=7 # Anecdotal based on Ct's -- large components (MX) reduced by exo digestion
if "exo" in self.qpcrStages:
q.addSamples(src=[rxs[i] for i in self.trackIndices],needDil=needDil,primers=["T7AX","T7BX","MX","T7X","REF"],names=["%s.exo"%names[i] for i in self.trackIndices])
exo=self.saveSamps(src=rxs,vol=10*exoDil,dil=2/exoDil,dilutant=reagents.getsample("TE8"),tgt=[Sample("%s.exo"%n,decklayout.SAMPLEPLATE) for n in names]) # Save cDNA product
else:
exoDil=1
exo=[]
else:
extdil=1
extpostdil=1
exoDil=1
if self.doampure:
print "######## Ampure Cleanup ###########"
ratio=1.5
elutionVol=30
cleanIn=ext+exo+user
needDil=needDil*cleanIn[0].volume/elutionVol
clean=self.runAmpure(src=cleanIn,ratio=ratio,elutionVol=elutionVol)
if "ampure" in self.qpcrStages:
q.addSamples(src=[clean[i] for i in self.trackIndices],needDil=needDil,primers=["T7AX","MX","T7X","REF"])
rxs=rxs+clean # Use the cleaned products for PCR
totalDil=stopDil*rtDil*extdil*extpostdil*exoDil
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,rtDil,extdil,extpostdil,exoDil,totalDil,fracRetained*100)
if self.rtSave 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(input):
rxs=rxs[0:len(input)] # Only keep -target products
prefixOut=prefixOut[0:len(input)]
prefixIn=prefixIn[0:len(input)]
if self.dopcr:
print "######### PCR #############"
print "PCR Volume: %.1f, Dilution: %.1f, volumes available for PCR: [%s]"%(pcrvol, pcrdil,",".join(["%.1f"%r.volume for r in rxs]))
maxSampleVolume=100 # Maximum sample volume of each PCR reaction (thermocycler limit, and mixing limit)
initConc=needDil*self.qConc/pcrdil
if keepCleaved:
if self.doexo:
initConc=initConc*7*self.cleavage # Back out 7x dilution in exo step, but only use cleaved as input conc
else:
initConc=initConc*self.cleavage # Only use cleaved as input conc
else:
initConc=initConc*(1-self.cleavage)
gain=pcrgain(initConc,400,cycles)
finalConc=initConc*gain
print "Estimated starting concentration in PCR = %.1f nM, running %d cycles -> %.0f nM\n"%(needDil*self.qConc,cycles,finalConc)
nsplit=int(math.ceil(pcrvol*1.0/maxSampleVolume))
print "Split each PCR into %d reactions"%nsplit
srcdil=(1-1.0/3-1.0/4)
sampNeeded=pcrvol/pcrdil
if self.rtSave and keepCleaved:
sampNeeded+=rtSaveVol
maxvol=max([r.volume for r in rxs]);
minvol=min([r.volume for r in rxs]);
predil=min(75/maxvol,(40+1.4*nsplit)/(minvol-sampNeeded)) # Dilute to have 40ul left -- keeps enough sample to allow good mixing
if keepCleaved and self.rtSave and predil>rtSaveDil:
print "Reducing predil from %.1f to %.1f (rtSaveDil)"%(predil, rtSaveDil)
predil=rtSaveDil
if predil>1:
self.diluteInPlace(rxs,predil)
self.e.shakeSamples(rxs)
print "Pre-diluting by %.1fx into [%s] ul"%(predil,",".join(["%.1f"%r.volume for r in rxs]))
if keepCleaved and self.rtSave:
assert(len(rxs)==len(rtSave))
print "Saving %.1f ul of each pre-PCR sample (@%.1f*%.1f dilution)"%(rtSaveVol ,predil, rtSaveDil/predil)
self.lastSaved=[Sample("%s.sv"%x.name,decklayout.DILPLATE) for x in rxs]
for i in range(len(rxs)):
# Save with rtSaveDil dilution to reduce amount of RT consumed (will have Ct's 2-3 lower than others)
self.e.transfer(rtSaveVol*predil,rxs[i],self.lastSaved[i],(False,False))
self.e.transfer(rtSaveVol*(rtSaveDil/predil-1),decklayout.WATER,self.lastSaved[i],(False,True)) # Use pipette mixing -- shaker mixing will be too slow
pcr=self.runPCR(src=rxs*nsplit,vol=pcrvol/nsplit,srcdil=pcrdil*1.0/predil,ncycles=cycles,primers=["T7%sX"%x for x in (prefixOut if keepCleaved else prefixIn)]*nsplit,usertime=self.usertime if keepCleaved else None,fastCycling=True,inPlace=False)
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')
if self.pcrSave:
# Save samples at 1x (move all contents -- can ignore warnings)
if self.savedilplate:
sv=self.saveSamps(src=pcr[:len(rxs)],vol=[x.volume for x in pcr[:len(rxs)]],dil=1,plate=decklayout.DILPLATE,atEnd=True)
else:
sv=self.saveSamps(src=pcr[:len(rxs)],vol=[x.volume for x in pcr[:len(rxs)]],dil=1,plate=decklayout.EPPENDORFS)
if nsplit>1:
# Combine split
for i in range(len(rxs),len(rxs)*nsplit):
self.e.transfer(pcr[i].volume-16.4,pcr[i],sv[i%len(sv)],mix=(False,i>=len(rxs)*(nsplit-1)))
# Correct concentration (above would've assumed it was diluted)
for i in range(len(sv)):
sv[i].conc=pcr[i].conc
if "pcr" in self.qpcrStages:
for i in range(len(pcr)):
q.addSamples(pcr,needDil,["T7%sX"%prefixOut[i]])
processEff=0.5 # Estimate of overall efficiency of process
print "Saved %.2f pmoles of product (%.0f ul @ %.1f nM)"%(sv[0].volume*sv[0].conc.stock/1000,sv[0].volume,sv[0].conc.stock)
return sv
else:
return pcr[:len(rxs)]
else:
return rxs
