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 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 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 logmeasure(self,tip,height,submerge,zmax,zadd,time):
# Time is the time in seconds of this measurement
sample=self.lastSample[tip]
if len(sample.extrainfo)>0:
elapsed=time-sample.extrainfo[0]
else:
elapsed=timedelta(0)
#print "%s: %f"%(sample.name,elapsed)
sample.extrainfo=[time] # Keep track of last measurement time of this sample in the extrainfo list
if sample.plate.location.zmax is not None:
curzmax=2100-sample.plate.location.zmax-390+TIPOFFSETS[tip-1]
if zmax!=curzmax:
logging.warning("ZMax for plate %s, tip %d at time of run was %.0f, currently at %.0f"%(sample.plate.name, tip, zmax, curzmax))
zmax=curzmax
prevol=sample.volume-sample.lastadd # Liquid height is measured before next op, whose volume effect has already been added to sample.volume
if height==-1:
vol=sample.plate.getliquidvolume((zadd+submerge)/10.0)
if vol is not None:
if prevol<vol and prevol!=0:
# The liquid measure failed, but based on the previous volume estimate, it was guaranteed to fail since the submerge depth would've been below bottom
# But if we don't know the volume (ie a prefilled tube -> prevol=0), then log this as a fail
h=" @[DEEP <%.1fmm:<%.1ful#%d]"%((zadd+submerge)/10.0,vol,tip)
#h=""
else:
h=" @[FAIL <%.1fmm:<%.1ful#%d]"%((zadd+submerge)/10,vol,tip)
else:
h=" @[FAIL <%.1f#%d]"%((zadd+submerge)/10.0,tip)
else:
vol=sample.plate.getliquidvolume((height+submerge-zmax)/10.0)
if vol is None:
h=" @[%.1fmm,%.1fmm#%d]"%((height-zmax)/10.0,submerge/10.0,tip)
else:
if prevol==0:
logging.notice("Got a liquid height measurement for a well that should be empty -- assuming it was prefilled")
sample.volume=vol+sample.lastadd
prevol=vol
expectHeight=sample.plate.getliquidheight(prevol)
errorHeight=(height+submerge-zmax)-expectHeight*10
h=" @[%.1fmm,%.1fmm:%.1ful#%d]"%((height-zmax)/10.0,submerge/10.0,vol,tip)
if abs(errorHeight)>1:
if abs(errorHeight)>4:
emphasize="*"*(min(10,int(abs(errorHeight))-3))
else:
emphasize=''
h=h+"{%sE=%d;%.1ful}"%(emphasize,errorHeight,vol-prevol)
sample.volume=sample.volume+(vol-prevol)
# Insert BEFORE last history entry since the liquid height is measured before aspirate/dispense
hsplit=sample.history.split(' ')
if elapsed.total_seconds()>=600: # Log elapsed time if more than 10 min
sample.history=" ".join(hsplit[:-1]+["(T%.0f)"%(elapsed.total_seconds()/60)]+[h]+hsplit[-1:])
else:
sample.history=" ".join(hsplit[:-1]+[h]+hsplit[-1:])
def beadAddElutant(self,src,elutant=None,elutionVol=30,eluteTime=60,returnPlate=True,temp=None):
if elutant is None:
elutant=decklayout.WATER
[src,elutionVol,elutant]=listify([src,elutionVol,elutant])
for i in range(len(src)):
if elutionVol[i]<30:
logging.warning("elution from beads of %s with %.1f ul < minimum of 30ul"%(src[i].name,elutionVol[i]))
self.e.moveplate(src[i].plate,"Home")
self.e.transfer(elutionVol[i]-src[i].volume,elutant[i],src[i],(False,True))
if temp is None:
for plate in set([s.plate for s in src]):
self.e.shake(plate,dur=eluteTime,returnPlate=returnPlate,force=True)
else:
self.e.shakeSamples(src,dur=30,returnPlate=False)
worklist.pyrun('PTC\\ptcsetpgm.py elute TEMP@%d,%d TEMP@25,2'%(temp,eluteTime))
self.e.runpgm("elute",eluteTime/60,False,elutionVol[0])
if returnPlate:
self.e.moveplate(src[0].plate,"Home")
def __init__(self,op,tip,vol,wellx,welly,rack,grid,pos,lc,std,volset,isMulti):
self.op=op
self.tip=tip
self.vol=vol
self.lc=lc
self.std=std
self.volset=volset
self.isMulti=isMulti
self.sample=getSample(wellx,welly,rack,grid,pos)
if lc=='Air' or lc[0:7]=='Blowout' or lc=='Dip':
if op=='dispense':
self.sample.addhistory(lc,vol,tip)
elif op=='aspirate':
self.sample.addhistory(lc,-vol,tip)
else:
logging.notice("LogEntry: bad op (%s) for LC %s"%(op,lc))
assert False
self.sample.lastadd=0
elif op=='dispense':
self.sample.addhistory("",vol,tip)
self.sample.lastadd=vol
elif op=='detect':
self.sample.addhistory("detect",0,tip)
self.sample.lastadd=0
elif op=='aspirate':
self.sample.addhistory("",-vol,tip)
self.sample.lastadd=-(vol+SURFACEREMOVE) # Extra for tip wetting
elif op[0:3]=='mix':
self.sample.addhistory(op,vol,tip)
self.sample.lastadd=0
else:
logging.warning("LogEntry: bad op: %s"%op)
assert False
if self.sample.volume+self.sample.lastadd<0 and self.sample.volume!=0:
self.sample.history=self.sample.history + ("{Emptied%.2f}"%(self.sample.volume+self.sample.lastadd))
self.sample.volume=0
else:
self.sample.volume+=self.sample.lastadd
def addSamples(self, src, needDil, primers, nreplicates=1, names=None, saveVol=None, saveDil=None, save=True):
"""Add sample(s) to list of qPCRs to do"""
# print "addSamples(%s)"%src
if not isinstance(src, list):
src = [src]
if save:
# saveVol is total amount (after dilution) to be immediately saved
if saveDil is None:
saveDil = min(needDil, self.MAXDIL)
if 1 < needDil / saveDil < 2:
saveDil = math.sqrt(needDil)
elif saveDil > needDil:
logging.warning("addSamples: saveDil=" + saveDil + ", but needDil is only " + needDil)
saveDil = needDil
if saveVol is None:
saveVol = max(self.MINDILVOL * 1.0 / saveDil, self.TGTINVOL)
if names is None:
tgt = [Sample(diluteName(src[i].name, saveDil), decklayout.DILPLATE) for i in range(len(src))]
else:
tgt = [Sample(diluteName(names[i], saveDil), decklayout.DILPLATE) for i in range(len(src))]
sv = tgt
for i in range(len(sv)):
# print "Save ",src[i]
svtmp = self.trp.runQPCRDIL(src=[src[i]], vol=saveVol * saveDil, srcdil=saveDil, tgt=[tgt[i]],
dilPlate=True, dilutant=self.dilutant)
sv[i] = svtmp[0]
else:
saveDil = 1
sv = src
needDil = needDil / saveDil
nstages = int(math.ceil(math.log(needDil) / math.log(self.MAXDIL)))
ndil = len(src) * (nstages + (1 if save else 0))
logging.notice("QPCR: %dQ/%dD [%s], dilution:%.1fx, primers: [%s]" % (
len(src) * len(primers) * nreplicates, ndil,
",".join([s.name for s in src]) if names is None else ",".join(names), needDil, ",".join(primers)))
for svi in range(len(sv)):
s = sv[svi]
if s.hasBeads:
prereqs = []
else:
j0 = self.jobq.addShake(sample=s, prereqs=[])
prereqs = [j0]
intermed = s
for i in range(nstages):
dil = math.pow(needDil, 1.0 / nstages)
# print "stage ",i,", needDil=",needDil,", dil=",dil
if i > 0:
vol = self.MAXDILVOL
else:
vol = min(self.MAXDILVOL * 1.0, max(self.MINDILVOL * 1.0, dil * self.TGTINVOL * 1.0))
if intermed.plate == decklayout.DILPLATE:
firstWell = intermed.well + 4 # Skip by 4 wells at a time to optimize multi-tip movements
else:
firstWell = 0
if not save and i == 0 and names is not None:
# Need to replace the name in this condition
dest = Sample(diluteName(names[svi], dil), decklayout.DILPLATE, firstWell=firstWell)
else:
dest = Sample(diluteName(intermed.name, dil), decklayout.DILPLATE, firstWell=firstWell)
# print "dest=",dest
j1 = self.jobq.addMultiTransfer(volume=vol * (dil - 1) / dil, src=self.dilutant, dest=dest, prereqs=[])
prereqs.append(j1)
j2 = self.jobq.addTransfer(volume=vol / dil, src=intermed, dest=dest, prereqs=prereqs)
# print "Dilution of %s was %.2f instead of %.2f (error=%.0f%%)"%(dest.name,(dil/(1+dil))/(1/dil),dil,((dil/(1+dil))/(1/dil)/dil-1)*100)
if dest.hasBeads:
prereqs = [j2]
else:
j3 = self.jobq.addShake(sample=dest, prereqs=[j2])
prereqs = [j3]
intermed = dest
self.dilProds = self.dilProds + [intermed]
self.primers = self.primers + [primers]
self.nreplicates = self.nreplicates + [nreplicates]
def setVolumes(self):
# Computed parameters
self.rnaConc = 8314 * self.tmplFinalConc / (self.tmplFinalConc +
55) * self.t7dur / 30
maxConc = 1000 * self.stopConc * 4 / 0.9
if maxConc < self.rnaConc:
logging.warning("Stop@%.1f uM limits usable RNA to %.0f/%.0f nM" %
(self.stopConc, maxConc, self.rnaConc))
self.rnaConc = min(maxConc, self.rnaConc)
stopConc = self.rnaConc * 0.9
rtConc = stopConc / self.rtDil
rtdilConc = [
rtConc / self.rtpostdil[i] for i in range(len(self.rounds))
]
ligConc = [
None if self.rounds[i] == 'U' else rtdilConc[i] / 1.25
for i in range(len(self.rounds))
]
ligdilConc = [
None if self.rounds[i] == 'U' else ligConc[i] / self.extpostdil[i]
for i in range(len(self.rounds))
]
pcrConc = [
rtConc / self.pcrdil[i] if self.rounds[i] == 'U' else ligConc[i] /
self.pcrdil[i] for i in range(len(self.rounds))
]
for i in range(len(self.rounds)):
if ligConc[i] is None:
print "R%d Concs(nM): RNA: %.0f, Stop: %.0f, RT: %.0f, RTDil: %.0f, PCRIn: %.0f" % (
i, self.rnaConc, stopConc, rtConc, rtdilConc[i],
pcrConc[i])
else:
print "R%d Concs(nM): RNA: %.0f, Stop: %.0f, RT: %.0f, RTDil: %.0f, Lig: %.0f, LigDil: %.0f, PCRIn: %.0f" % (
i, self.rnaConc, stopConc, rtConc, rtdilConc[i],
ligConc[i], ligdilConc[i], pcrConc[i])
self.pcrvol = [
self.pcrcopies * self.pmolesIn * 1000 / pcrConc[i] /
math.pow(self.enrich, (i + 1.0)) for i in range(len(self.rounds))
]
# Use at least 100ul so the evaporation of the saved sample that occurs during the run will be relatively small
self.pcrvol = [max(100, v) for v in self.pcrvol]
pcrExtra = [
1.4 * math.ceil(v / self.maxPCRVolume) for v in self.pcrvol
]
self.minligvol = [
self.pcrvol[i] / self.pcrdil[i] +
(pcrExtra[i] +
(4.4 if self.saveDil is not None else 5.4
if 'ext' in self.qpcrStages else 0) + 15.1) / self.extpostdil[i]
for i in range(len(self.pcrvol))
]
print "minligvol=[%s]" % (",".join(
["%.1f" % v for v in self.minligvol]))
# Compute RT volume
self.rtvol = [((self.minligvol[i] / 1.25 + 3.3) /
self.rtpostdil[i]) if self.rounds[i] == 'C' else
(self.pcrvol[i] * 1.0 / self.pcrdil[i] +
(pcrExtra[i] + 15.1 + 3.3) / self.rtpostdil[i])
for i in range(len(self.rounds))]
print "self.rtvol=", self.rtvol, ", rtSave=", self.rtSave
if self.rtSave:
self.rtvol = [
max(15.0 / self.rtpostdil[i], self.rtvol[i]) +
(self.rtSaveVol + 1.4) / self.rtpostdil[i]
for i in range(len(self.rtvol))
] # Extra for saves
elif "rt" in self.qpcrStages: # Take from save if rtSave is set
self.rtvol = [
max(15.0 / self.rtpostdil[i], self.rtvol[i]) +
5.4 / self.rtpostdil[i] for i in range(len(self.rtvol))
] # Extra for qPCR
#print "self.rtvol=",self.rtvol
self.rtvol = [max(v, 8.0) for v in self.rtvol] # Minimum volume
self.rtvol = [min(v, self.maxSampVolume)
for v in self.rtvol] # Maximum volume
self.t7extravols = (
(4 + 1.4) * 0.9 if 'stopped' in self.qpcrStages else 0) + (
(5 + 1.4) * 0.9 if self.saveRNA else
0) + 3.3 # 3.3 in case pipette mixing used
#print "self.t7extravols=%.1f ul\n"%self.t7extravols
#print "self.rtvol=%s ul\n"%(",".join(["%.1f "%x for x in self.rtvol]))
self.t7vol = [
max((15.1 + self.rtvol[i] / 4.0 + 1.4) * 0.9 + self.t7extravols,
self.pmolesIn * 1000 / self.tmplFinalConc /
math.pow(self.enrich, i * 1.0))
for i in range(len(self.rounds))
]
#print "self.t7vol=%s ul\n"%(",".join(["%.1f "%x for x in self.t7vol]))
self.t7vol = [
max(18.0, v) for v in self.t7vol
] # Make sure that there's enough to add at least 2ul of stop
self.t7vol = [min(self.maxSampVolume, v)
for v in self.t7vol] # Make sure no tubes overflow
if 'template' in self.qpcrStages:
self.t7vol[0] += 5.4
def addSamples(self,
src,
needDil,
primers,
nreplicates=1,
names=None,
saveVol=None,
saveDil=None,
save=True):
'Add sample(s) to list of qPCRs to do'
#print "addSamples(%s)"%src
if not isinstance(src, list):
src = [src]
if save:
# saveVol is total amount (after dilution) to be immediately saved
if saveDil is None:
saveDil = min(needDil, self.MAXDIL)
if needDil / saveDil > 1 and needDil / saveDil < 2:
saveDil = math.sqrt(needDil)
elif saveDil > needDil:
logging.warning("addSamples: saveDil=", saveDil,
", but needDil is only ", needDil)
saveDil = needDil
if saveVol is None:
saveVol = max(self.MINDILVOL * 1.0 / saveDil, self.TGTINVOL)
if names is None:
tgt = [
Sample(diluteName(src[i].name, saveDil),
decklayout.DILPLATE) for i in range(len(src))
]
else:
tgt = [
Sample(diluteName(names[i], saveDil), decklayout.DILPLATE)
for i in range(len(src))
]
sv = tgt
for i in range(len(sv)):
#print "Save ",src[i]
svtmp = self.trp.runQPCRDIL(src=[src[i]],
vol=saveVol * saveDil,
srcdil=saveDil,
tgt=[tgt[i]],
dilPlate=True,
dilutant=self.dilutant)
sv[i] = svtmp[0]
else:
saveDil = 1
sv = src
needDil = needDil / saveDil
nstages = int(math.ceil(math.log(needDil) / math.log(self.MAXDIL)))
ndil = len(src) * (nstages + (1 if save else 0))
logging.notice(
"QPCR: %dQ/%dD [%s], dilution:%.1fx, primers: [%s]" %
(len(src) * len(primers) * nreplicates, ndil,
",".join([s.name
for s in src]) if names is None else ",".join(names),
needDil, ",".join(primers)))
for svi in range(len(sv)):
s = sv[svi]
if s.hasBeads:
prereqs = []
else:
j0 = self.jobq.addShake(sample=s, prereqs=[])
prereqs = [j0]
intermed = s
for i in range(nstages):
dil = math.pow(needDil, 1.0 / nstages)
#print "stage ",i,", needDil=",needDil,", dil=",dil
if i > 0:
vol = self.MAXDILVOL
else:
vol = min(self.MAXDILVOL,
max(self.MINDILVOL, dil * self.TGTINVOL))
if intermed.plate == decklayout.DILPLATE:
firstWell = intermed.well + 4 # Skip by 4 wells at a time to optimize multi-tip movements
else:
firstWell = 0
if not save and i == 0 and names is not None:
# Need to replace the name in this condition
dest = Sample(diluteName(names[svi], dil),
decklayout.DILPLATE,
firstWell=firstWell)
else:
dest = Sample(diluteName(intermed.name, dil),
decklayout.DILPLATE,
firstWell=firstWell)
#print "dest=",dest
j1 = self.jobq.addMultiTransfer(volume=vol * (dil - 1) / dil,
src=self.dilutant,
dest=dest,
prereqs=[])
prereqs.append(j1)
j2 = self.jobq.addTransfer(volume=vol / dil,
src=intermed,
dest=dest,
prereqs=prereqs)
#print "Dilution of %s was %.2f instead of %.2f (error=%.0f%%)"%(dest.name,(dil/(1+dil))/(1/dil),dil,((dil/(1+dil))/(1/dil)/dil-1)*100)
if dest.hasBeads:
prereqs = [j2]
else:
j3 = self.jobq.addShake(sample=dest, prereqs=[j2])
prereqs = [j3]
intermed = dest
self.dilProds = self.dilProds + [intermed]
self.primers = self.primers + [primers]
self.nreplicates = self.nreplicates + [nreplicates]
def setVolumes(self):
# Computed parameters
# Observed data: 0.1nM@30min -> gain ~1000
self.rnaConc=8314.0*self.tmplFinalConc/(self.tmplFinalConc+55)*self.t7dur/30
if self.tmplFinalConc<1:
self.rnaConc*=6 # Kludge based on being off at 0.1nM template concentrations
self.rnaConc=max(40.0,self.rnaConc) # Another kludge
if isinstance(self.stopConc,list):
minStopConc=min(self.stopConc)
else:
minStopConc=self.stopConc
maxConc=1000*minStopConc*4/0.9
if maxConc<self.rnaConc:
logging.warning( "Stop@%.1f uM limits usable RNA to %.0f/%.0f nM"%(minStopConc,maxConc,self.rnaConc))
self.rnaConc=min(maxConc,self.rnaConc)
stopConc=self.rnaConc*0.9
rtConc=stopConc/self.rtDil
rtdilConc=[rtConc/self.rtpostdil[i] for i in range(len(self.rounds))]
ligConc=[None if self.rounds[i]=='U' else rtdilConc[i]/1.25 for i in range(len(self.rounds))]
ligdilConc=[None if self.rounds[i]=='U' else ligConc[i]/self.extpostdil[i] for i in range(len(self.rounds))]
pcrConc=[rtConc/self.pcrdil[i] if self.rounds[i]=='U' else ligConc[i]/self.pcrdil[i] for i in range(len(self.rounds))]
for i in range(len(self.rounds)):
if ligConc[i] is None:
print("R%d Concs(nM): RNA: %.0f, Stop: %.0f, RT: %.0f, RTDil: %.0f, PCRIn: %.0f"%(i, self.rnaConc, stopConc, rtConc, rtdilConc[i], pcrConc[i]))
else:
print("R%d Concs(nM): RNA: %.0f, Stop: %.0f, RT: %.0f, RTDil: %.0f, Lig: %.0f, LigDil: %.0f, PCRIn: %.0f"%(i, self.rnaConc, stopConc, rtConc, rtdilConc[i], ligConc[i], ligdilConc[i], pcrConc[i]))
self.pcrvol=[self.pcrcopies*self.pmolesIn*1000/pcrConc[i]/math.pow(self.enrich,(i+1.0)) for i in range(len(self.rounds))]
# Use at least 100ul so the evaporation of the saved sample that occurs during the run will be relatively small
self.pcrvol=[max(100,v) for v in self.pcrvol]
print("pcrvol=[%s]"%(",".join(["%.1f"%v for v in self.pcrvol])))
pcrExtra=[1.4*math.ceil(v*1.0/self.maxPCRVolume) for v in self.pcrvol]
print("pcrExtra=[%s]"%(",".join(["%.1f"%v for v in pcrExtra])))
self.minligvol=[(self.pcrvol[i]*1.0+pcrExtra[i])/self.pcrdil[i]+((4.4 if self.saveDil is not None else 5.4 if 'ext' in self.qpcrStages else 0)+15.1)/self.extpostdil[i] for i in range(len(self.pcrvol))]
print("minligvol=[%s]"%(",".join(["%.1f"%v for v in self.minligvol])))
# Compute RT volume
self.rtvol=[ ((self.minligvol[i]/1.25+3.3)/self.rtpostdil[i]) if self.rounds[i]!='U' else (self.pcrvol[i]*1.0/self.pcrdil[i]+(pcrExtra[i]+15.1+3.3)/self.rtpostdil[i]) for i in range(len(self.rounds))]
print("self.rtvol=",self.rtvol,", rtSave=",self.rtSave)
if self.rtSave:
self.rtvol=[max(15.0/self.rtpostdil[i],self.rtvol[i])+(self.rtSaveVol+1.4)/self.rtpostdil[i] for i in range(len(self.rtvol))] # Extra for saves
elif "rt" in self.qpcrStages: # Take from save if rtSave is set
self.rtvol=[max(15.0/self.rtpostdil[i],self.rtvol[i])+5.4/self.rtpostdil[i] for i in range(len(self.rtvol))] # Extra for qPCR
self.rtvol=[max(v,8.0) for v in self.rtvol] # Minimum volume
self.rtvol=[min(v,self.maxSampVolume) for v in self.rtvol] # Maximum volume
# print ("self.rtvol=",self.rtvol)
self.t7extravols=((4+1.4) if 'stopped' in self.qpcrStages else 0)+ ((self.saveRNAVolume+1.4) if self.saveRNA else 0) # + 3.3 # 3.3 in case pipette mixing used
#print "self.t7extravols=%.1f ul\n"%self.t7extravols
#print "self.rtvol=%s ul\n"%(",".join(["%.1f "%x for x in self.rtvol]))
# Compute dilutions due to tref additions
trefdil=[1 for _ in self.rnddef ]
for i in range(len(self.rnddef)-1):
rd=self.rnddef[i]
if 'tref' in rd:
tref = rd['tref'][0]
if tref is not None:
trefname = 'TRef%d' % tref
if not reagents.isReagent(trefname):
reagents.add(name=trefname, conc=10, extraVol=30, well="E4")
trefs = reagents.getsample(trefname)
trefdil[i+1]=(trefs.conc.dilutionneeded())/(trefs.conc.dilutionneeded()-1)
print("Extra dilution due to tref additions: ",trefdil)
self.t7vol=[max((15.1+self.rtvol[i]/4.0+1.4)+self.t7extravols,self.pmolesIn*1000.0/(self.tmplFinalConc if i==0 else 200*self.templateDilution)*trefdil[i]/math.pow(self.enrich,i*1.0)) for i in range(len(self.rounds))]
print("self.t7vol=%s ul\n"%(",".join(["%.1f "%x for x in self.t7vol])))
#self.t7vol=[max(18.0,v) for v in self.t7vol] # Make sure that there's enough to add at least 2ul of stop
if 'template' in self.qpcrStages:
self.t7vol[0]+=5.4
self.t7vol=[min(self.maxSampVolume,v) for v in self.t7vol] # Make sure no tubes overflow