def runT7Setup(self, theo, src, vol, srcdil, tgt=None, rlist=["MT7"]):
[theo, src, tgt, srcdil] = listify([theo, src, tgt, srcdil])
for i in range(len(src)):
if tgt[i] is None:
if theo[i]:
tgt[i] = Sample("%s.T+" % src[i].name,
decklayout.SAMPLEPLATE)
else:
tgt[i] = Sample("%s.T-" % src[i].name,
decklayout.SAMPLEPLATE)
worklist.comment("runT7: source=%s" % [str(s) for s in src])
rvols = [reagents.getsample(x).conc.volneeded(vol) for x in rlist]
rtotal = sum(rvols)
sourcevols = [vol * 1.0 / s for s in srcdil]
if any(theo):
theovols = [
(vol * 1.0 /
reagents.getsample("Theo").conc.dilutionneeded() if t else 0)
for t in theo
]
watervols = [
vol - theovols[i] - sourcevols[i] - rtotal
for i in range(len(src))
]
else:
watervols = [vol - sourcevols[i] - rtotal for i in range(len(src))]
if any([w < -1e-10 for w in watervols]):
print "runT7Setup: Negative amount of water required: ", watervols
assert False
if sum(watervols) > 0.01:
self.e.multitransfer(watervols, decklayout.WATER, tgt)
for ir in range(len(rlist)):
self.e.multitransfer([rvols[ir] for s in tgt],
reagents.getsample(rlist[ir]), tgt)
if any(theo):
self.e.multitransfer(
[tv for tv in theovols if tv > 0.01],
reagents.getsample("Theo"),
[tgt[i] for i in range(len(theovols)) if theovols[i] > 0],
ignoreContents=True)
for i in range(len(src)):
self.e.transfer(sourcevols[i], src[i], tgt[i])
self.e.shakeSamples(tgt, returnPlate=True)
for t in tgt:
t.ingredients['BIND'] = 1e-20 * sum(t.ingredients.values())
return tgt
def runQPCRDIL(self,
src,
vol,
srcdil,
tgt=None,
dilPlate=False,
pipMix=False,
dilutant=decklayout.SSDDIL):
[src, vol, srcdil] = listify([src, vol, srcdil])
vol = [float(v) for v in vol]
if tgt is None:
if dilPlate:
tgt = [
Sample(diluteName(src[i].name, srcdil[i]),
decklayout.DILPLATE) for i in range(len(src))
]
else:
tgt = [
Sample(diluteName(src[i].name, srcdil[i]),
decklayout.SAMPLEPLATE) for i in range(len(src))
]
srcvol = [vol[i] / srcdil[i] for i in range(len(vol))]
watervol = [vol[i] - srcvol[i] for i in range(len(vol))]
if len(watervol) > 4 and sum(watervol) > 800:
print "Could optimize distribution of ", len(
watervol), " moves of ", dilutant.name, ": vol=[", [
"%.1f" % w for w in watervol
], "]"
self.e.multitransfer(watervol, dilutant, tgt, (False, False))
self.e.shakeSamples(src, returnPlate=True)
for i in range(len(src)):
tgt[i].conc = None # Assume dilutant does not have a concentration of its own
# Check if we can align the tips here
if i < len(src) - 3 and tgt[i].well + 1 == tgt[
i + 1].well and tgt[i].well + 2 == tgt[
i +
2].well and tgt[i].well + 3 == tgt[i + 3].well and tgt[
i].well % 4 == 0 and self.e.cleanTips != 15:
#print "Aligning tips"
self.e.sanitize()
self.e.transfer(srcvol[i], src[i], tgt[i],
(not src[i].isMixed(), pipMix))
if tgt[i].conc != None:
tgt[i].conc.final = None # Final conc are meaningless now
return tgt
def saveSamps(self,
src,
vol,
dil,
tgt=None,
dilutant=None,
plate=None,
mix=(True, False)):
[src, vol, dil] = listify([src, vol, dil])
if plate is None:
plate = decklayout.REAGENTPLATE
if tgt is None:
tgt = [
Sample(diluteName(src[i].name, dil[i]), plate)
for i in range(len(src))
]
if any([d != 1.0 for d in dil]):
if dilutant is None:
dilutant = decklayout.WATER
self.e.multitransfer(
[vol[i] * (dil[i] - 1) for i in range(len(vol))], dilutant,
tgt, (False, False))
self.e.shakeSamples(src, returnPlate=True)
for i in range(len(src)):
self.e.transfer(vol[i], src[i], tgt[i], mix)
tgt[i].conc = Concentration(1.0 / dil[i])
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:
print "ERROR: runIncubation only supports a single master mix"
assert False
if inPlace:
if tgt is not None:
print "ERROR: tgt specified for in-place incubation"
assert False
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('User', enzymes, src, tgt, vol, destMix=False)
self.e.shakeSamples(tgt, returnPlate=False)
if hiTemp is None:
worklist.pyrun('PTC\\ptcsetpgm.py INC TEMP@%.0f,%.0f TEMP@25,30' %
(incTemp, incTime * 60))
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))
self.e.runpgm("INC",
incTime + hiTime + 2,
False,
max(vol),
hotlidmode="TRACKING",
hotlidtemp=10)
return tgt
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]
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="%s.D%d"%(ref.name,srcDil)
src=[Sample.lookup(srcname)]
if src[0] is None:
src=[Sample(srcname,decklayout.DILPLATE)]
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 beadSupernatant(self,
src,
tgt=None,
sepTime=None,
residualVolume=10,
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]):
print "beadSupernatant: Attempt to magsep on multiple plates at the same time"
assert False
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 getsample(self):
if self.sample is None:
#print "Creating sample for reagent %s with %.1f ul"%(self.name,self.initVol)
self.sample=Sample(self.name,self.plate,self.preferredWell,self.conc,hasBeads=self.hasBeads,volume=self.initVol)
wellname=self.sample.plate.wellname(self.sample.well)
if self.preferredWell != None and self.preferredWell != wellname:
print "WARNING: %s moved from preferred well %s to %s\n"%(self.name,self.preferredWell,wellname)
return self.sample
def getsample(self):
if self.sample is None:
#print "Creating sample for reagent %s with %.1f ul"%(self.name,self.initVol)
self.sample = Sample(self.name,
self.plate,
self.preferredWell,
self.conc,
hasBeads=self.hasBeads,
volume=self.initVol,
extrainfo=self.extrainfo,
ingredients=self.ingredients)
wellname = self.sample.plate.wellname(self.sample.well)
if self.preferredWell != None and self.preferredWell != wellname:
logging.warning("%s moved from preferred well %s to %s\n" %
(self.name, self.preferredWell, wellname))
return self.sample
def runQPCR(self, src, vol, srcdil, primers=["A", "B"], nreplicates=1):
## QPCR setup
worklist.comment("runQPCR: primers=%s, source=%s" %
([p for p in primers], [s.name for s in src]))
[src, vol, srcdil,
nreplicates] = listify([src, vol, srcdil, nreplicates])
self.e.shakeSamples(src, returnPlate=True)
# Build a list of sets to be run
torun = []
for repl in range(max(nreplicates)):
for p in primers:
for i in range(len(src)):
if nreplicates[i] <= repl:
continue
if repl == 0:
sampname = "%s.Q%s" % (src[i].name, p)
else:
sampname = "%s.Q%s.%d" % (src[i].name, p, repl + 1)
s = Sample(sampname, decklayout.QPCRPLATE)
torun = torun + [(src[i], s, p, vol[i])]
# Fill the master mixes
dil = {}
for p in primers:
mname = "MQ%s" % p
if not reagents.isReagent(mname):
reagents.add(name=mname, conc=15.0 / 9.0, extraVol=30)
mq = reagents.getsample(mname)
t = [a[1] for a in torun if a[2] == p]
v = [a[3] / mq.conc.dilutionneeded() for a in torun if a[2] == p]
self.e.multitransfer(v, mq, t, (False, False))
dil[p] = 1.0 / (1 - 1 / mq.conc.dilutionneeded())
# Add the samples
self.e.sanitize() # In case we are aligned
for a in torun:
s = a[0]
t = a[1]
p = a[2]
v = a[3] / dil[p]
t.conc = None # Concentration of master mix is irrelevant now
self.e.transfer(v, s, t)
return [a[1] for a in torun]
def 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 runRTSetup(self, src, vol, srcdil, tgt=None, rtmaster=None):
if rtmaster is None:
rtmaster = reagents.getsample("MPosRT")
if tgt is None:
tgt = [
Sample(s.name + ".RT+", decklayout.SAMPLEPLATE) 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)
self.e.stage('RTPos', [rtmaster], [src[i] for i in range(len(src))],
[tgt[i] for i in range(len(tgt))],
[vol[i] for i in range(len(vol))],
destMix=False)
#self.e.shakeSamples(tgt,returnPlate=True)
return tgt
def distribute(self,
src,
dil,
vol,
wells,
tgt=None,
dilutant=None,
plate=decklayout.SAMPLEPLATE):
if tgt is None:
tgt = [
Sample("%s.dist%d" % (src[0].name, j), plate)
for j in range(wells)
]
if dilutant is None:
dilutant = decklayout.WATER
self.e.multitransfer([vol * (dil - 1) for i in range(wells)], dilutant,
tgt)
self.e.multitransfer([vol for i in range(wells)], src[0], tgt)
return tgt
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
for k in range(max(nreplicates)):
for i in range(len(input)):
if nreplicates[i] > k:
srcs = srcs + [input[i]]
tmplqpcr = [srcprefix + srcsuffix]
pcr = [srcprefix + srcsuffix, ligprefix + srcsuffix]
print "srcs=", srcs
print "tmplqpcr=", tmplqpcr
print "ligmaster=", ligmaster
print "pcr=", pcr
print "stop=", stop
# Create ligation master mix samples
if Sample.lookup(ligmaster) == None:
Sample(ligmaster, Experiment.REAGENTPLATE, None, 3)
if Sample.lookup(stop) == None:
Sample(stop, Experiment.REAGENTPLATE, None, 2)
for pm in pcr:
if Sample.lookup("MQ" + pm) == None:
Sample("MQ" + pm, Experiment.REAGENTPLATE, None, 10.0 / 6)
for pm in tmplqpcr:
if Sample.lookup("MQ" + pm) == None:
Sample("MQ" + pm, Experiment.REAGENTPLATE, None, 10.0 / 6)
reagents = None
for iteration in range(2):
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]
tmplqpcr = tmplqpcr + [srcprefix[i] + srcsuffix[i]]
ligmaster = ligmaster + ["MLig" + ligprefix[i] + stem1[i]]
pcr = pcr + [
(srcprefix[i] + srcsuffix[i], ligprefix[i] + srcsuffix[i])
]
stop = stop + ["MStp" + srcsuffix[i]]
#print srcs
#print tmplqpcr
#print ligmaster
#print pcr
# Create ligation master mix samples
for lm in set(ligmaster):
if Sample.lookup(lm) == None:
Sample(lm, Experiment.REAGENTPLATE, None, 3)
for st in set(stop):
if Sample.lookup(st) == None:
Sample(st, Experiment.REAGENTPLATE, None, 2)
for p in pcr:
for pm in p:
if Sample.lookup("MQ" + pm) == None:
Sample("MQ" + pm, Experiment.REAGENTPLATE, None, 10.0 / 6)
for pm in tmplqpcr:
if Sample.lookup("MQ" + pm) == None:
Sample("MQ" + pm, Experiment.REAGENTPLATE, None, 10.0 / 6)
reagents = None
def runLig(self,
prefix=None,
src=None,
vol=None,
srcdil=None,
tgt=None,
master=None,
anneal=True,
ligtemp=25):
if master is None:
master = [
reagents.getsample("MLigAN7")
if p == 'A' else reagents.getsample("MLigBN7") for p in prefix
]
#Extension
[src, tgt, vol, srcdil,
master] = listify([src, tgt, vol, srcdil, master])
if tgt is None:
tgt = [
Sample("%s.%s" % (src[i].name, master[i].name),
decklayout.SAMPLEPLATE) for i in range(len(src))
]
# Need to check since an unused ligation master mix will not have a concentration
minsrcdil = 1 / (1 - 1 / master[0].conc.dilutionneeded() - 1 /
reagents.getsample("MLigase").conc.dilutionneeded())
for i in srcdil:
if i < minsrcdil:
print "runLig: srcdil=%.2f, but must be at least %.2f based on concentrations of master mixes" % (
i, minsrcdil)
assert (False)
# Adjust source dilution
for i in range(len(src)):
src[i].conc = Concentration(srcdil[i], 1)
i = 0
while i < len(tgt):
lasti = i + 1
while lasti < len(tgt) and master[i] == master[lasti]:
lasti = lasti + 1
self.e.stage('LigAnneal', [master[i]],
src[i:lasti],
tgt[i:lasti], [vol[j] / 1.5 for j in range(i, lasti)],
1.5,
destMix=False)
i = lasti
if anneal:
self.e.shakeSamples(tgt, returnPlate=False)
self.e.runpgm("TRPANN",
5,
False,
max(vol),
hotlidmode="CONSTANT",
hotlidtemp=100)
self.e.stage('Ligation', [reagents.getsample("MLigase")], [],
tgt,
vol,
destMix=False)
self.e.shakeSamples(tgt, returnPlate=False)
self.runLigPgm(max(vol), ligtemp)
return tgt
def beadWash(self,
src,
washTgt=None,
sepTime=None,
residualVolume=10,
keepWash=False,
numWashes=2,
wash=None,
washVol=50,
keepFinal=False,
finalTgt=None,
keepVol=4.2,
keepDil=5):
# Perform washes
# If keepWash is true, retain all washes (combined)
# If keepFinal is true, take a sample of the final wash (diluted by keepDil)
if wash is None:
wash = decklayout.WATER
[src, wash] = listify([src, wash])
# Do all washes while on magnet
assert (len(set([s.plate for s in src])) == 1) # All on same plate
if keepWash:
if washTgt is None:
washTgt = []
for i in range(len(src)):
if s[i].volume - residualVolume + numWashes * (
washVol - residualVolume
) > decklayout.DILPLATE.maxVolume - 20:
print "Saving %.1f ul of wash in eppendorfs" % (
numWashes * washVol)
washTgt.append(
Sample("%s.Wash" % src[i].name,
decklayout.EPPENDORFS))
else:
washTgt.append(
Sample("%s.Wash" % src[i].name,
decklayout.DILPLATE))
if keepFinal:
if finalTgt is None:
finalTgt = []
for i in range(len(src)):
finalTgt.append(
Sample("%s.Final" % src[i].name, decklayout.DILPLATE))
if any([s.volume > residualVolume for s in src]):
# Separate and remove supernatant
self.e.moveplate(src[0].plate, "Magnet") # Move to magnet
self.sepWait(src, sepTime)
# Remove the supernatant
for i in range(len(src)):
if src[i].volume > residualVolume:
amt = src[i].amountToRemove(residualVolume)
if keepWash:
self.e.transfer(amt, src[i],
washTgt[i]) # Keep supernatants
washTgt[i].conc = None # Allow it to be reused
else:
self.e.dispose(amt, src[i]) # Discard supernatant
# Wash
for washnum in range(numWashes):
self.e.moveplate(src[0].plate, "Home")
if keepFinal and washnum == numWashes - 1:
'Retain sample of final'
for i in range(len(src)):
src[i].conc = None
self.e.transfer(washVol - src[i].volume,
wash[i],
src[i],
mix=(False, True)) # Add wash
self.e.shake(src[0].plate, returnPlate=True)
self.saveSamps(src=src,
tgt=finalTgt,
vol=keepVol,
dil=keepDil,
plate=decklayout.DILPLATE)
else:
for i in range(len(src)):
src[i].conc = None
self.e.transfer(
washVol - src[i].volume,
wash[i],
src[i],
mix=(False, False)
) # Add wash, no need to pipette mix since some heterogenity won't hurt here
self.e.shake(src[0].plate, returnPlate=False)
self.e.moveplate(src[0].plate, "Magnet") # Move to magnet
self.sepWait(src, sepTime)
for i in range(len(src)):
amt = src[i].amountToRemove(residualVolume)
if keepWash:
self.e.transfer(amt, src[i], washTgt[i]) # Remove wash
washTgt[i].conc = None # Allow it to be reused
else:
self.e.dispose(amt, src[i]) # Remove wash
self.e.moveplate(src[0].plate, "Home")
# Should only be residualVolume left with beads now
result = []
if keepWash:
result = result + washTgt
if keepFinal:
result = result + finalTgt
return result
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 runPCR(self,
prefix,
src,
vol,
srcdil,
tgt=None,
ncycles=20,
suffix='S',
sepPrimers=True,
primerDil=4):
## PCR
[prefix, src, tgt, vol, srcdil,
suffix] = listify([prefix, src, tgt, vol, srcdil, suffix])
for i in range(len(tgt)):
if tgt[i] is None:
tgt[i] = Sample(
"%s.P%s%s" % (src[i].name, prefix[i], suffix[i]),
src[i].plate)
# Adjust source dilution
for i in range(len(src)):
src[i].conc = Concentration(srcdil[i], 1)
if sepPrimers:
sampvols = [vol[i] / srcdil[i] for i in range(len(src))]
mm = reagents.getsample("MPCR")
mmvols = [
vol[i] / mm.conc.dilutionneeded() for i in range(len(src))
]
for s in prefix + suffix:
if not reagents.isReagent(s):
reagents.add(name=s, conc=primerDil, extraVol=30)
sprefix = [reagents.getsample(p) for p in prefix]
ssuffix = [reagents.getsample(p) for p in suffix]
prefixvols = [
vol[i] / sprefix[i].conc.dilutionneeded()
for i in range(len(src))
]
suffixvols = [
vol[i] / ssuffix[i].conc.dilutionneeded()
for i in range(len(src))
]
watervols = [
vol[i] - mmvols[i] - prefixvols[i] - suffixvols[i] -
sampvols[i] for i in range(len(src))
]
print "water=", watervols, ", mm=", mmvols, ", prefix=", prefixvols, ", suffix=", suffixvols, ", samp=", sampvols
self.e.multitransfer(watervols, decklayout.WATER, tgt,
(False, False)) # Transfer water
self.e.multitransfer(mmvols, mm, tgt,
(False, False)) # PCR master mix
sprefixset = set(sprefix)
ssuffixset = set(ssuffix)
if len(sprefixset) < len(ssuffixset):
# Distribute sprefix first
for p in sprefixset:
self.e.multitransfer([
prefixvols[i]
for i in range(len(src)) if sprefix[i] == p
], p, [tgt[i] for i in range(len(src)) if sprefix[i] == p],
(False, False))
# Then individually add ssuffix
for i in range(len(src)):
self.e.transfer(suffixvols[i], ssuffix[i], tgt[i],
(False, False))
else:
# Distribute ssuffix first
for p in ssuffixset:
self.e.multitransfer([
suffixvols[i]
for i in range(len(src)) if ssuffix[i] == p
], p, [tgt[i] for i in range(len(src)) if ssuffix[i] == p],
(False, False))
# Then individually add sprefix
for i in range(len(src)):
self.e.transfer(prefixvols[i], sprefix[i], tgt[i],
(False, False))
# Now add templates
for i in range(len(src)):
self.e.transfer(sampvols[i], src[i], tgt[i], (False, False))
else:
primer = [prefix[i] + suffix[i] for i in range(len(prefix))]
print "primer=", primer
for up in set(primer):
s = "MPCR%s" % up
if not reagents.isReagent(s):
reagents.add(name=s, conc=4 / 3.0, extraVol=30)
self.e.stage(
'PCR%s' % up, [reagents.getsample("MPCR%s" % up)],
[src[i] for i in range(len(src)) if primer[i] == up],
[tgt[i] for i in range(len(tgt)) if primer[i] == up],
[vol[i] for i in range(len(vol)) if primer[i] == up],
destMix=False)
pgm = "PCR%d" % ncycles
self.e.shakeSamples(tgt, returnPlate=False)
# worklist.pyrun('PTC\\ptcsetpgm.py %s TEMP@95,120 TEMP@95,30 TEMP@55,30 TEMP@72,25 GOTO@2,%d TEMP@72,180 TEMP@16,2'%(pgm,ncycles-1))
worklist.pyrun(
'PTC\\ptcsetpgm.py %s TEMP@95,120 TEMP@95,10 TEMP@57,10 GOTO@2,%d TEMP@72,120 TEMP@25,2'
% (pgm, ncycles - 1))
self.e.runpgm(pgm,
4.80 + 1.55 * ncycles,
False,
max(vol),
hotlidmode="CONSTANT",
hotlidtemp=100)
return tgt
