def idler(self, t):
endTime = clock.elapsed() + t
if self.debug:
print("Idler(%.0f)" % t)
while clock.elapsed() < endTime:
j = self.jobq.getJob()
if j is None:
break
self.jobq.execJob(self.trp.e, j)
if self.debug:
print("Idler done with ", endTime - clock.elapsed(), " seconds remaining")
def pgm(self):
self.q = QSetup(self, maxdil=16, debug=False, mindilvol=60)
self.q.debug = True
self.q.addReferences(dstep=10, primers=self.qprimers, ref=reagents.getsample("BT5310"),nreplicates=2)
print("### Barcoding #### (%.0f min)" % (clock.elapsed() / 60.0))
self.idbarcoding(self.rsrc, left=[x['left'] for x in self.inputs],
right=[x['right'] for x in self.inputs])
print("### qPCR #### (%.0f min)" % (clock.elapsed() / 60.0))
self.q.run(confirm=False, enzName='EvaGreen')
def idler(self,t):
endTime=clock.elapsed()+t
if self.debug:
print "Idler(%.0f)"%t
while clock.elapsed()<endTime:
j=self.jobq.getJob()
if j is None:
break
self.jobq.execJob(self.trp.e,j)
if self.debug:
print "Idler done with ",endTime-clock.elapsed()," seconds remaining"
def idler(self,t):
endTime=clock.elapsed()+t
if self.debug:
print("Idler(%.0f)"%t)
njobs=0
while clock.elapsed()<endTime:
j=self.jobq.getJob()
if j is None:
break
self.jobq.execJob(self.trp.e,j)
njobs+=1
if self.debug:
print("Idler completed ",njobs," jobs with ",endTime-clock.elapsed()," seconds remaining")
def pgm(self):
self.q = QSetup(self, maxdil=16, debug=False, mindilvol=100)
self.e.addIdleProgram(self.q.idler)
if self.doqpcr:
self.q.addReferences(dstep=10, primers=self.qprimers, ref=reagents.getsample("BT5310"))
print("### Barcoding #### (%.0f min)" % (clock.elapsed() / 60.0))
bcout = self.barcoding(names=[x['name'] for x in self.inputs], left=[x['left'] for x in self.inputs],
right=[x['right'] for x in self.inputs])
for i in range(len(self.inputs)):
x = self.inputs[i]
if 'bconc' in x and x['bconc'] is not None:
print("Resetting concentration of %s from expected %.1f to bconc setting of %.1f nM" % \
(x['name'], bcout[i].conc.stock, x['bconc']))
bcout[i].conc.stock = x['bconc']
print("### qPCR #### (%.0f min)" % (clock.elapsed() / 60.0))
self.q.run(confirm=False, enzName='EvaGreen')
print("### qPCR Done #### (%.0f min)" % (clock.elapsed() / 60.0))
print("### Final PCR Done #### (%.0f min)" % (clock.elapsed() / 60.0))
worklist.flushQueue()
if all(['bconc' in x and x['bconc'] is not None for x in self.inputs]):
print("### Mixdown #### (%.0f min)" % (clock.elapsed() / 60.0))
worklist.flushQueue()
worklist.comment('Start mixdown only at this point')
self.e.sanitize(force=True)
# mixdown = self.mix(bcout, [x['weight'] for x in self.inputs])
# Set default mix number to 1
for x in self.inputs:
if 'mix' not in x:
x['mix']=1
mixes=set([x['mix'] for x in self.inputs])
for m in mixes:
sel=[ i for i in range(len(self.inputs)) if self.inputs[i]['mix']==m]
print("sel=",sel)
mixdown = self.mix([bcout[i] for i in sel], [self.inputs[i]['weight'] for i in sel],prefix="Mix%d_"%m)
mixdown.name="Mix%d_Final"%m
# self.q.addSamples(mixdown, needDil=mixdown.conc.stock * 1e-9 / self.qconc, primers=self.qprimers,nreplicates=3)
else:
print("### Not doing mixdown as bconc not set in all inputs")
def reset(self):
'Reset this experiment so we can generate it again after adjusting the reagent initial volumes and total time'
totalTime=clock.elapsed()
clock.reset(totalTime)
#print "After reset, elapsed=%d"%clock.elapsed()
worklist.reset()
self.e=Experiment()
self.e.setreagenttemp(6.0)
self.e.sanitize(3,50) # Heavy sanitize
reagents.reset()
Sample.clearall()
decklayout.initWellKnownSamples()
def reset(self):
'Reset this experiment so we can generate it again after adjusting the reagent initial volumes and total time'
totalTime = clock.elapsed()
clock.reset(totalTime)
#print "After reset, elapsed=%d"%clock.elapsed()
worklist.reset()
self.e = Experiment()
self.e.setreagenttemp(6.0)
self.e.sanitize(3, 50) # Heavy sanitize
reagents.reset()
Sample.clearall()
decklayout.initWellKnownSamples()
def pgm(self):
self.q = QSetup(self, maxdil=16, debug=False, mindilvol=60)
# Don't start idler (to minimize tip cross-contamination); last PCR allows plenty of time for doing dilutions without any effect on run time
# Will start after first constriction PCR is running
#self.q.debug = True
# self.e.addIdleProgram(self.q.idler)
self.q.addReferences(dstep=10, primers=self.qprimers, ref=reagents.getsample("BT5310"),nreplicates=2)
samps=[r.getsample() for r in self.rsrc]
for s in samps:
self.q.addSamples([s],needDil=max(10,s.conc.stock*1e-9/self.qconc),primers=self.qprimers)
print("### Mixdown #### (%.0f min)" % (clock.elapsed() / 60.0))
if len(samps)>1:
mixdown = self.mix(samps, [x['weight'] for x in self.inputs])
else:
mixdown=samps[0]
self.q.addSamples(mixdown, needDil=max(1.0,mixdown.conc.stock * 1e-9 / self.qconc), primers=self.qprimers)
print("Mixdown final concentration = %.0f pM" % (mixdown.conc.stock * 1000))
print("### Constriction #### (%.1f min)" % (clock.elapsed() / 60.0))
constricted = self.constrict(mixdown, mixdown.conc.stock * 1e-9)
print("### Regeneration #### (%.0f min)" % (clock.elapsed() / 60.0))
prefixes = set([x['left'][0] for x in self.inputs])
self.regenerate(constricted * len(prefixes), [p for p in prefixes for _ in constricted])
print("### qPCR #### (%.0f min)" % (clock.elapsed() / 60.0))
self.q.run(confirm=False, enzName='EvaGreen', waitForPTC=True)
print("### qPCR Done #### (%.0f min)" % (clock.elapsed() / 60.0))
worklist.userprompt("qPCR done -- only need to complete final PCR", 300)
self.e.waitpgm()
print("### Final PCR Done #### (%.0f min)" % (clock.elapsed() / 60.0))
def 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 pgm(self):
q = QSetup(self, maxdil=self.maxdilstep, debug=False, mindilvol=60)
self.e.addIdleProgram(q.idler)
if self.barcoding:
# Setup barcode primers for cleaved rounds only
self.bcprimers = [[
"BC-%s-R%d_T7" % (inp['ligand'], r + 1) for inp in self.inputs
] if self.rounds[r] == 'C' else None
for r in range(len(self.rounds))]
for bcp in self.bcprimers:
if bcp is not None:
for p in ["P-%s" % pp for pp in bcp]:
if not reagents.isReagent(p):
reagents.add(name=p,
conc=4,
extraVol=30,
plate=decklayout.REAGENTPLATE,
well="B2")
s = reagents.getsample(p) # Force allocation of a well
print "Adding %s to reagents at well %s" % (
p, s.plate.wellname(s.well))
print "BC primers=", self.bcprimers
# Add any missing fields to inputs
for i in range(len(self.inputs)):
if 'ligand' not in self.inputs[i]:
self.inputs[i]['ligand'] = None
if 'round' not in self.inputs[i]:
self.inputs[i]['round'] = None
if 'name' not in self.inputs[i]:
if self.inputs[i]['ligand'] is None:
self.inputs[i]['name'] = '%s_%d_R%d' % (
self.inputs[i]['prefix'], self.inputs[i]['ID'],
self.inputs[i]['round'])
else:
self.inputs[i]['name'] = '%s_%d_R%d_%s' % (
self.inputs[i]['prefix'], self.inputs[i]['ID'],
self.inputs[i]['round'], self.inputs[i]['ligand'])
# Add templates
if self.directT7:
self.srcs = self.addTemplates(
[inp['name'] for inp in self.inputs],
stockconc=self.tmplFinalConc / self.templateDilution,
finalconc=self.tmplFinalConc,
plate=decklayout.SAMPLEPLATE,
looplengths=[inp['looplength'] for inp in self.inputs],
initVol=self.t7vol[0] * self.templateDilution,
extraVol=0)
else:
self.srcs = self.addTemplates(
[inp['name'] for inp in self.inputs],
stockconc=self.tmplFinalConc / self.templateDilution,
finalconc=self.tmplFinalConc,
plate=decklayout.DILPLATE,
looplengths=[inp['looplength'] for inp in self.inputs],
extraVol=15)
t7in = [s.getsample() for s in self.srcs]
if "negative" in self.qpcrStages:
q.addSamples(decklayout.SSDDIL, 1, self.allprimers,
save=False) # Negative controls
if "reference" in self.qpcrStages:
q.addReferences(dstep=10,
nsteps=5,
primers=["T7WX", "MX", "T7X"],
ref=reagents.getsample("BT5310"),
nreplicates=1)
q.addReferences(dstep=10,
nsteps=5,
primers=["T7WX", "MX", "T7X"],
ref=reagents.getsample("BT5310"),
nreplicates=1)
# Save RT product from first (uncleaved) round and then use it during 2nd (cleaved) round for ligation and qPCR measurements
self.rndNum = 0
self.nextID = self.firstID
curPrefix = [inp['prefix'] for inp in self.inputs]
r1 = t7in
for roundType in self.rounds:
# Run a single round of roundType with r1 as input
# roundType is either "U" for uncleaved, or a new prefix for a cleaved round (with "T" being a T7 prepend)
# Set r1 to new output at end
# Computed output prefix
if roundType == 'U':
prefixOut = curPrefix
stop = ["Unclvd" for p in curPrefix]
else:
if roundType == 'T':
stop = ['T7%s' % p for p in curPrefix]
prefixOut = curPrefix
elif any([p == roundType for p in curPrefix]):
logging.error(
"Round %d is a cleaved round but goes to %s without changing prefix"
% (self.rndNum, roundType))
assert (False)
else:
prefixOut = [roundType for p in curPrefix]
stop = prefixOut
# May be explicitly overridden
for i in range(len(self.inputs)):
if 'stop' in self.inputs[i]:
if isinstance(self.inputs[i]['stop'], list):
assert (len(self.inputs[i]['stop']) == len(
self.rounds))
t = self.inputs[i]['stop'][self.rndNum]
else:
t = self.inputs[i]['stop']
if (roundType == 'U') != (t == 'U'):
print "Attempt to override round %d (type %s) with a input-specific round type of %s" % (
self.rndNum, roundType, t)
assert (False)
if roundType != 'U':
if t == 'T':
stop[i] = 'T7%s' % curPrefix[i]
prefixOut[i] = curPrefix[i]
else:
stop[i] = t
prefixOut[i] = t
self.rndNum = self.rndNum + 1
self.finalRound = self.rndNum == len(self.rounds)
r1 = self.oneround(q,
r1,
prefixOut=prefixOut,
stop=stop,
prefixIn=curPrefix,
keepCleaved=(roundType != 'U'),
rtvol=self.rtvol[self.rndNum - 1],
t7vol=self.t7vol[self.rndNum - 1],
cycles=self.pcrcycles[self.rndNum - 1],
pcrdil=self.pcrdil[self.rndNum - 1],
pcrvol=self.pcrvol[self.rndNum - 1],
dolig=self.allLig or (roundType != 'U'))
for i in range(len(r1)):
r1[i].name = "%s_%d" % (prefixOut[i], self.nextID)
if self.inputs[i]['round'] is not None:
r1[i].name = "%s_R%d%c" % (r1[i].name,
self.inputs[i]['round'] +
self.rndNum, roundType)
if self.inputs[i]['ligand'] is not None:
r1[i].name = "%s_%s" % (r1[i].name,
self.inputs[i]['ligand'])
print "Used ID ", self.nextID, " for ", r1[i].name, ": ", r1[i]
self.nextID += 1
r1[i].conc.final = r1[i].conc.stock * self.templateDilution
curPrefix = prefixOut
if "finalpcr" in self.qpcrStages:
for i in range(len(r1)):
if self.singlePrefix:
q.addSamples(src=r1[i],
needDil=r1[i].conc.stock / self.qConc,
primers=["T7X", "MX"])
else:
q.addSamples(src=r1[i],
needDil=r1[i].conc.stock / self.qConc,
primers=["T7X", prefixOut[i] + "X", "MX"])
print "######### qPCR ########### %.0f min" % (clock.elapsed() / 60)
self.allprimers = q.allprimers()
q.run(confirm=self.qpcrWait)
def barcoding(self, names, left, right):
"""Perform barcoding of the given inputs; rsrsc,left,right should all be equal length"""
pcrcycles = [5, 10]
pcr1inputdil = 10
pcr1vol = 30
pcr1postdil = 100.0 / pcr1vol
pcr2dil = 10*pcr1postdil
pcr2vol = 40.0
samps = [reagents.getsample(s) for s in names]
print("Inputs:")
for i in range(len(samps)):
print("%2s %-10s %8s-%-8s %s" % (
samps[i].plate.wellname(samps[i].well), self.inputs[i]['name'], left[i], right[i], str(samps[i].conc)))
wellnum = 5
for s in left + right:
primer = "P-" + s
if not reagents.isReagent(primer):
reagents.add(primer, conc=Concentration(2.67, 0.4, 'uM'), extraVol=30, plate=decklayout.REAGENTPLATE,
well=decklayout.REAGENTPLATE.wellname(wellnum))
wellnum += 1
for s in samps:
# Dilute down to desired conc
dil = s.conc.stock / self.pcr1inputconc / pcr1inputdil
if dil < 1.0:
logging.error("Input %s requires dilution of %.2f" % (s.name, dil))
elif dil > 1.0:
dilvol = s.volume * dil
if dilvol > 150.0:
maxdil = 150.0 / s.volume
logging.info(
"Dilution of input %s (%.1f ul) by %.2f would require %.1f ul -- only diluting by %.1fx" % (
s.name, s.volume, dil, dilvol, maxdil))
dil = maxdil
self.diluteInPlace(tgt=[s], dil=dil)
print("Diluting %s by %.1f" % (s.name, dil))
print("### PCR1 #### (%.0f min)" % (clock.elapsed() / 60.0))
pcr1 = self.runPCR(src=samps, srcdil=[s.conc.stock / self.pcr1inputconc for s in samps], ncycles=pcrcycles[0],
vol=pcr1vol,
primers=[[left[i], right[i]] for i in range(len(left))], usertime=30, fastCycling=False,
inPlace=False, master="MPCR1", kapa=False, annealTemp=57)
pcr1finalconc = self.pcr1inputconc * 2 ** pcrcycles[0]
print("PCR1 output concentration = %.1f nM" % pcr1finalconc)
if pcr1postdil > 1:
pcr1finalconc /= pcr1postdil
print("Post dilute PCR1 by %.2fx to %.3f nM " % (pcr1postdil, pcr1finalconc))
self.diluteInPlace(tgt=pcr1, dil=pcr1postdil)
for x in pcr1:
x.conc = Concentration(stock=pcr1finalconc, units='nM')
if len(pcrcycles) > 1:
# Second PCR with 235p/236p on mixture (use at least 4ul of prior)
print("### PCR2 #### (%.0f min)" % (clock.elapsed() / 60.0))
pcr2 = self.runPCR(src=pcr1, srcdil=pcr2dil / pcr1postdil, vol=pcr2vol, ncycles=pcrcycles[1],
primers=None, fastCycling=False, master="MPCR2", kapa=True, annealTemp=64)
pcr2finalconc = pcr1finalconc / (pcr2dil / pcr1postdil) * 2 ** pcrcycles[1]
print("PCR2 final conc = %.1f nM" % pcr2finalconc)
if pcr2finalconc > 200:
print("Capping at 200nM")
pcr2finalconc = 200
for x in pcr2:
x.conc = Concentration(stock=pcr2finalconc, units='nM')
if self.doqpcr:
self.q.addSamples(src=pcr2, needDil=pcr2finalconc * 1e-9 / self.qconc, primers=self.qprimers)
res = pcr2
else:
self.q.addSamples(src=pcr1, needDil=pcr1finalconc / (self.qconc * 1e9), primers=self.qprimers, save=True,
nreplicates=1)
res = pcr1
return res
def runPCR(self,primers,src,srcdil,vol=None,tgt=None,ncycles=20,usertime=None,fastCycling=False,inPlace=False,master="MTaq",annealTemp=None,kapa=False):
## PCR
if inPlace:
if vol!=None:
print "runPCR: cannot specify volume when using inPlace=True, srcdil and input volume determine reaction volume"
assert(False)
if tgt!=None:
print "runPCR: cannot specify tgt when using inPlace=True"
assert(False)
[primers,src,vol,srcdil]=listify([primers,src,vol,srcdil])
vol=[src[i].volume*srcdil[i] for i in range(len(src))]
tgt=src
else:
[primers,src,tgt,vol,srcdil]=listify([primers,src,tgt,vol,srcdil])
for i in range(len(tgt)):
if tgt[i] is None:
if isinstance(primers[i],list):
tgt[i]=Sample("%s.P%s"%(src[i].name,"+".join(primers[i])),src[i].plate)
else:
tgt[i]=Sample("%s.P%s"%(src[i].name,primers[i]),src[i].plate)
# Adjust source dilution
for i in range(len(src)):
src[i].conc=Concentration(srcdil[i],1)
logging.notice( "primer="+str(primers))
# Add reagent entries for any missing primers
if isinstance(primers[0],list):
allprimers=[x for y in primers for x in y]
else:
allprimers=primers
for up in set(allprimers):
s="P-%s"%up
if not reagents.isReagent(s):
reagents.add(name=s,conc=4,extraVol=30)
if isinstance(primers[0],list):
# Multiple primers
if inPlace:
assert len(primers[0])==2
self.runRxInPlace(src,vol,reagents.getsample(master),master2=[reagents.getsample("P-%s"%p[0]) for p in primers],master3=[reagents.getsample("P-%s"%p[1]) for p in primers],returnPlate=False)
else:
for i in range(len(primers)):
self.e.stage('PCR%d'%i,[reagents.getsample(master)]+[reagents.getsample("P-%s"%s) for s in primers[i]],src[i:i+1] ,tgt[i:i+1],vol[i:i+1],destMix=False)
#self.e.shakeSamples(tgt,returnPlate=False)
else:
# Single primer
if inPlace:
self.runRxInPlace(src,vol,reagents.getsample(master),master2=[reagents.getsample("P-%s"%p) for p in primers],returnPlate=False)
else:
for up in set(primers):
self.e.stage('PCR%s'%up,[reagents.getsample(master),reagents.getsample("P-%s"%up)],[src[i] for i in range(len(src)) if primers[i]==up],[tgt[i] for i in range(len(tgt)) if primers[i]==up],[vol[i] for i in range(len(vol)) if primers[i]==up],destMix=False)
#self.e.shakeSamples(tgt,returnPlate=False)
pgm="PCR%d"%ncycles
if usertime is None:
runTime=0
else:
runTime=usertime
if annealTemp is None:
annealTemp=60 if kapa else 57
meltTemp=98 if kapa else 95
hotTime=180 if kapa else 30
extTemp=72 if kapa else 68
if fastCycling:
cycling='TEMP@37,%d TEMP@95,%d TEMP@%.1f,10 TEMP@%.1f,10 TEMP @%.1f,1 GOTO@3,%d TEMP@%.1f,60 TEMP@25,2'%(1 if usertime is None else usertime*60,hotTime,meltTemp,annealTemp,extTemp,ncycles-1,extTemp)
runTime+=hotTime/60+2.8+1.65*ncycles
else:
cycling='TEMP@37,%d TEMP@95,%d TEMP@%.1f,30 TEMP@%.1f,30 TEMP@%.1f,30 GOTO@3,%d TEMP@%.1f,60 TEMP@25,2'%(1 if usertime is None else usertime*60,hotTime,meltTemp,annealTemp,extTemp,ncycles-1,extTemp)
runTime+=hotTime/60+2.8+3.0*ncycles
print "PCR volume=[",",".join(["%.1f"%t.volume for t in tgt]), "], srcdil=[",",".join(["%.1fx"%s for s in srcdil]),"], program: %s"%cycling
worklist.pyrun('PTC\\ptcsetpgm.py %s %s'%(pgm,cycling))
self.e.runpgm(pgm,runTime,False,max(vol),hotlidmode="CONSTANT",hotlidtemp=100)
# Mark samples as mixed (by thermal convection)
print "Marking samples as mixed (by thermal convection)"
for t in tgt:
t.wellMixed=True
t.lastMixed=clock.elapsed()
#self.e.shakeSamples(tgt,returnPlate=True)
return tgt
def oneround(self, q, inputs, prefixOut, stop, prefixIn, keepCleaved, t7vol, rtvol, pcrdil, cycles, pcrvol, dolig,pcrtgt=None):
primerSet=[set(["REF","T7X",prefixIn[i]+"X",prefixOut[i]+"X"]+(["MX"] if self.useMX else [])) for i in range(len(prefixIn))]
if self.extraQPCRPrimers is not None:
primerSet=[set(list(p) + self.extraQPCRPrimers) for p in primerSet]
print("primerSet=",primerSet)
if keepCleaved:
print("Starting new cleavage round, will add prefix: ",prefixOut)
assert dolig
else:
print("Starting new uncleaved round, will retain prefix: ",prefixIn)
print("stop=",stop,"prefixOut=",prefixOut,", prefixIn=",prefixIn,",t7vol=",round(t7vol,ndigits=2),",rtvol=",rtvol,",pcrdil=",pcrdil,",cycles=",cycles,",dolig=",dolig)
if self.rtCarryForward:
assert dolig
names=[i.name for i in inputs]
if self.rnaInput:
rxs=inputs
stopDil=1
else:
print("######## T7 ########### %.0f min"%(clock.elapsed()/60))
db.pushStatus("T7")
print("Inputs: (t7vol=%.2f)"%t7vol)
for inp in inputs:
if inp.conc.units=='nM':
print(" %s: %.1ful@%.1f %s, use %.1f ul (%.3f pmoles)"%(inp.name,inp.volume,inp.conc.stock,inp.conc.units,t7vol/inp.conc.dilutionneeded(), t7vol*inp.conc.final/1000))
else:
print(" %s: %.1ful@%.1f %s, use %.1f ul"%(inp.name,inp.volume,inp.conc.stock,inp.conc.units,t7vol/inp.conc.dilutionneeded()))
# inp.conc.final=inp.conc.stock*self.templateDilution
units=list(set([inp.conc.units for inp in inputs]))
if len(units)>1:
print("Inputs have inconsistent concentration units: ",units)
assert False
if units[0]=='nM':
needDil = max([inp.conc.stock for inp in inputs]) * 1.0 / self.qConc
else:
needDil = 100 / self.qConc # Assume 100nM
if self.directT7 and self.rndNum==1:
# Just add ligands and MT7 to each well
mconc=reagents.getsample("MT7").conc.dilutionneeded()
for i in range(len(inputs)):
watervol=t7vol*(1-1/mconc) - inputs[i].volume
if watervol<-0.1:
print("Negative amount of water (%.1f ul) needed for T7 setup"%watervol)
assert False
elif watervol>0.1:
self.e.transfer(watervol, decklayout.WATER, inputs[i], mix=(False, False))
self.e.transfer(t7vol / mconc, reagents.getsample("MT7"), inputs[i], mix=(False, False))
assert(abs(inputs[i].volume - t7vol) < 0.1)
# Add ligands last in case they crash out when they hit aqueous; this way, they'll be as dilute as possible
if keepCleaved:
for i in range(len(inputs)):
if self.inputs[i]['negligand'] is not None:
negligand=reagents.getsample(self.inputs[i]['negligand'])
self.e.transfer(t7vol / negligand.conc.dilutionneeded(), negligand, inputs[i], mix=(False, False))
names[i]+="+"
else:
for i in range(len(inputs)):
if self.inputs[i]['ligand'] is not None:
ligand=reagents.getsample(self.inputs[i]['ligand'])
self.e.transfer(t7vol / ligand.conc.dilutionneeded(), ligand, inputs[i], mix=(False, False))
names[i]+="+"
rxs=inputs
self.e.shakeSamples(inputs,returnPlate=True)
elif self.rndNum==len(self.rounds) and self.finalPlus and keepCleaved:
rxs = self.runT7Setup(ligands=[reagents.getsample(inp['ligand']) for inp in self.inputs],src=inputs, vol=t7vol, srcdil=[inp.conc.dilutionneeded() for inp in inputs])
for i in range(len(inputs)):
inp=inputs[i]
if self.inputs[i]['ligand'] is not None:
rxs += self.runT7Setup(ligands=[reagents.getsample(self.inputs[i]['ligand'])],src=[inp],vol=t7vol,srcdil=[inp.conc.dilutionneeded()])
prefixIn+=[prefixIn[i]]
prefixOut+=[prefixOut[i]]
stop+=[stop[i]]
primerSet+=[primerSet[i]]
names+=["%s+"%names[i]]
elif keepCleaved:
rxs = self.runT7Setup(ligands=[reagents.getsample(inp['negligand']) for inp in self.inputs], src=inputs, vol=t7vol, srcdil=[inp.conc.dilutionneeded() for inp in inputs])
else:
rxs = self.runT7Setup(ligands=[reagents.getsample(inp['ligand']) for inp in self.inputs], src=inputs, vol=t7vol, srcdil=[inp.conc.dilutionneeded() for inp in inputs])
if self.rndNum==1 and "template" in self.qpcrStages:
# Initial input
for i in range(len(rxs)):
q.addSamples(src=rxs[i],needDil=needDil,primers=primerSet[i],names=["%s.T"%names[i]])
self.runT7Pgm(dur=self.t7dur,vol=t7vol)
for i in range(len(rxs)):
rxs[i].name="%s.t7"%names[i]
self.e.lihahome()
print("Estimate usable RNA concentration in T7 reaction at %.0f nM"%self.rnaConc)
if self.rndNum==1:
worklist.userprompt("T7 Incubation Started",120)
self.e.waitpgm() # So elapsed time will be updated
db.popStatus()
if self.edtastop:
print("######## Stop ########### %.0f min"%(clock.elapsed()/60))
db.pushStatus("Stop")
print("Have %.1f ul before stop"%rxs[0].volume)
preStopVolume=rxs[0].volume
self.addEDTA(tgt=rxs,finalconc=2) # Stop to 2mM EDTA final
db.popStatus("Stop")
stopDil=rxs[0].volume/preStopVolume
else:
stopDil=1
if self.pauseAfterStop:
worklist.userprompt("Post EDTA pause")
if self.saveRNA:
self.saveSamps(src=rxs,vol=self.saveRNAVolume,dil=self.saveRNADilution,plate=self.savePlate,dilutant=reagents.getsample("TE8"),mix=(False,False)) # Save to check [RNA] on Qubit, bioanalyzer
needDil = self.rnaConc/self.qConc/stopDil
if "stopped" in self.qpcrStages:
for i in range(len(rxs)):
q.addSamples(src=rxs[i:i+1],needDil=needDil,primers=primerSet[i],names=["%s.stopped"%names[i]])
print("######## RT Setup ########### %.0f min"%(clock.elapsed()/60))
db.pushStatus("RT")
hiTemp=95
stop=["%s-Stop"%n for n in stop]
rt=self.runRT(src=rxs,vol=rtvol,srcdil=self.rtDil,heatInactivate=self.rtHI,hiTemp=hiTemp,dur=self.rtdur,incTemp=50,stop=[reagents.getsample(s) for s in stop],stopConc=self.stopConc) # Heat inactivate also allows splint to fold
rxs=rt
for i in range(len(rxs)):
if dolig and not self.singlePrefix:
rxs[i].name=names[i]+"."+prefixOut[i]+".rt"
else:
rxs[i].name=names[i]+".rt"
print("RT volume= [",",".join(["%.1f "%x.volume for x in rxs]),"]")
needDil /=self.rtDil
if self.rtpostdil[self.rndNum-1]>1:
print("Dilution after RT: %.2f"%self.rtpostdil[self.rndNum-1])
self.diluteInPlace(tgt=rxs,dil=self.rtpostdil[self.rndNum-1])
needDil=needDil/self.rtpostdil[self.rndNum-1]
# Discard extra volume of any sample that has more than current rt volume so that we can shake at high speed
for r in Sample.getAllOnPlate(rxs[0].plate):
if r not in rxs and r.volume>max(15+1.4,rxs[0].volume)+4:
remove=r.volume-(15+1.4)
oldvol=r.volume
if r.lastMixed is None:
r.lastMixed=clock.elapsed # Override since we don't care about mixing for disposal
self.e.dispose(remove,r)
print("Discarding some of %s to reduce volume from %.1f to %.1f to allow faster shaking"%(r.name,oldvol,r.volume))
print("RT volume= ",[x.volume for x in rxs])
self.e.shakeSamples(rxs)
if self.rtSave:
rtsv=self.saveSamps(src=rxs,vol=self.rtSaveVol,dil=self.rtSaveDil,plate=self.savePlate,dilutant=reagents.getsample("TE8"),mix=(False,False)) # Save to check RT product on gel (2x dil)
if "rt" in self.qpcrStages:
for i in range(len(rxs)):
q.addSamples(src=rtsv[i:i+1],needDil=needDil/2,primers=self.rtprimers[self.rndNum-1] if hasattr(self,'rtprimers') else primerSet[i],names=["%s.rt"%names[i]])
else:
if "rt" in self.qpcrStages:
for i in range(len(rxs)):
q.addSamples(src=rxs[i:i+1],needDil=needDil,primers=self.rtprimers[self.rndNum-1] if hasattr(self,'rtprimers') else primerSet[i],names=["%s.rt"%names[i]])
rtCarryForwardDil=10
rtCarryForwardVol=3.5
if self.rtCarryForward and not keepCleaved:
# Also include RT from a prior round from here on
for r in self.lastSaved:
newsamp=Sample("%s.samp"%r.name,decklayout.SAMPLEPLATE)
self.e.transfer(rxs[0].volume,r,newsamp,(False,False))
rxs.append(newsamp)
db.popStatus()
if dolig:
print("######## Ligation setup ########### %.0f min"%(clock.elapsed()/60))
db.pushStatus("Ligation")
extdil=5.0/4
reagents.getsample("MLigase").conc=Concentration(5)
if self.ligInPlace:
rxs=self.runLig(rxs,inPlace=True,srcdil=extdil,incTime=self.ligdur)
else:
rxs=self.runLig(rxs,inPlace=False,srcdil=extdil,vol=20,incTime=self.ligdur)
print("Ligation volume= ",[x.volume for x in rxs])
needDil=needDil/extdil
if self.extpostdil[self.rndNum-1]>1:
print("Dilution after extension: %.2f"%self.extpostdil[self.rndNum-1])
self.diluteInPlace(tgt=rxs,dil=self.extpostdil[self.rndNum-1])
needDil=needDil/self.extpostdil[self.rndNum-1]
pcrdil=pcrdil*1.0/self.extpostdil[self.rndNum-1]
if self.saveDil is not None:
ext=self.saveSamps(src=rxs,vol=3,dil=self.saveDil,dilutant=reagents.getsample("TE8"),tgt=[Sample("%s.ext"%n,self.savePlate) for n in names],mix=(False,True)) # Save cDNA product for subsequent NGS
if "ext" in self.qpcrStages:
for i in range(len(ext)):
# Make sure we don't take more than 2 more steps
maxdil=q.MAXDIL*q.MAXDIL
if needDil/self.saveDil>maxdil:
logging.notice( "Diluting ext by %.0fx instead of needed %.0f to save steps"%(maxdil,needDil/self.saveDil))
pset=primerSet[i]
if "extraQPCR" in self.inputs[i]:
pset.udpate(self.inputs[i]["extraQPCR"])
q.addSamples(src=[ext[i]],needDil=min(maxdil,needDil/self.saveDil),primers=pset,names=["%s.ext"%names[i]],save=False)
else:
if "ext" in self.qpcrStages:
print("needDil=",needDil)
for i in range(len(names)):
pset=primerSet[i]
if "extraQPCR" in self.inputs[i]:
pset.update(self.inputs[i]["extraQPCR"])
q.addSamples(src=[rxs[i]],needDil=needDil,primers=pset,names=["%s.ext"%names[i]])
isave=i+len(names)
if isave<len(rxs):
# samples restored
q.addSamples(src=[rxs[isave]],needDil=needDil/rtCarryForwardDil,primers=primerSet[isave])
db.popStatus()
else:
extdil=1
self.extpostdil[self.rndNum-1]=1
if self.rtpostdil[self.rndNum-1]>1:
pcrdil=pcrdil*1.0/self.rtpostdil[self.rndNum-1]
totalDil=stopDil*self.rtDil*self.rtpostdil[self.rndNum-1]*extdil*self.extpostdil[self.rndNum-1]
fracRetained=rxs[0].volume/(t7vol*totalDil)
print("Total dilution from T7 to Pre-pcr Product = %.2f*%.2f*%.2f*%.2f*%.2f = %.2f, fraction retained=%.0f%%"%(stopDil,self.rtDil,self.rtpostdil[self.rndNum-1],extdil,self.extpostdil[self.rndNum-1],totalDil,fracRetained*100))
if self.rtCarryForward and not keepCleaved:
# Remove the extra samples
assert(len(self.lastSaved)>0)
rxs=rxs[:len(rxs)-len(self.lastSaved)]
self.lastSaved=[]
if len(rxs)>len(inputs):
# Have extra samples due when self.finalPlus is True
rxs= rxs[0:len(inputs)] # Only keep -target products
prefixOut= prefixOut[0:len(inputs)]
prefixIn= prefixIn[0:len(inputs)]
stop= stop[0:len(inputs)]
if self.dopcr and not (keepCleaved and self.noPCRCleave):
print("######### PCR ############# %.0f min"%(clock.elapsed()/60))
db.pushStatus("PCR")
print("PCR Volume: %.1f, Dilution: %.1f, volumes available for PCR: [%s]"%(pcrvol, pcrdil,",".join(["%.1f"%r.volume for r in rxs])))
initConc=needDil*self.qConc/pcrdil
if keepCleaved:
initConc=initConc*self.cleavage # Only use cleaved as input conc
else:
initConc=initConc*(1-self.cleavage)
gain=pcrgain(initConc,400,cycles)
finalConc=min(200,initConc*gain)
print("Estimated starting concentration in PCR = %.1f nM, running %d cycles -> %.0f nM\n"%(needDil*self.qConc/pcrdil,cycles,finalConc))
nsplit=int(math.ceil(pcrvol*1.0/self.maxPCRVolume))
print("Split each PCR into %d reactions"%nsplit)
sampNeeded=pcrvol/pcrdil
if self.rtCarryForward and keepCleaved:
sampNeeded+=rtCarryForwardVol
if keepCleaved and self.rtCarryForward:
print("Saving %.1f ul of each pre-PCR sample" % rtCarryForwardVol)
self.lastSaved=[Sample("%s.sv"%x.name,self.savePlate) for x in rxs]
for i in range(len(rxs)):
# Save with rtCarryForwardDil dilution to reduce amount of RT consumed (will have Ct's 2-3 lower than others)
self.e.transfer(rtCarryForwardVol,rxs[i],self.lastSaved[i],(False,False))
self.e.transfer(rtCarryForwardVol*(rtCarryForwardDil-1),decklayout.WATER,self.lastSaved[i],(False,True)) # Use pipette mixing -- shaker mixing will be too slow
#print "NSplit=",nsplit,", PCR vol=",pcrvol/nsplit,", srcdil=",pcrdil,", input vol=",pcrvol/nsplit/pcrdil
minvol=min([r.volume for r in rxs])
maxpcrvol=(minvol-15-1.4*nsplit)*pcrdil
if maxpcrvol<pcrvol:
print("Reducing PCR volume from %.1ful to %.1ful due to limited input"%(pcrvol, maxpcrvol))
pcrvol=maxpcrvol
if keepCleaved:
master="MTaqC"
else:
master="MTaqU"
reagents.getsample(master) # Allocate for this before primers
if self.barcoding:
primers=self.bcprimers[self.rndNum-1]
if primers is not None and nsplit>1:
primers=primers*nsplit
else:
primers=None
if primers is None:
primers=[("T7%sX"%x).replace("T7T7","T7") for x in prefixOut]*nsplit
rnddef = self.rnddef[self.rndNum-1]
bcout=[]
if 'barcode' in rnddef:
# Add barcoding primers
assert len(rnddef['barcode'])==len(rxs)
dil=self.saveSamps(rxs,dil=50,vol=2,plate=decklayout.SAMPLEPLATE)
for i in range(len(rxs)):
dil[i].conc=Concentration(25,1)
for bc in rnddef['barcode'][i]:
tgt=Sample("%s.%s"%(rxs[i].name,bc),decklayout.SAMPLEPLATE)
bparts=bc.split("/")
for b in bparts:
if not reagents.isReagent("P-%s"%b):
reagents.add(name="P-%s"%b,conc=Concentration(2.67,0.4,'uM'),extraVol=30)
print("PCR-%s"%bc)
self.e.stage("PCR-%s"%bc,reagents=[reagents.getsample("MTaqBar"),reagents.getsample("P-%s"%bparts[0]),reagents.getsample("P-%s"%bparts[1])],samples=[tgt],sources=[dil[i] ],volume=50,destMix=False)
bcout.append(tgt)
print(tgt.name,"wellMixed=",tgt.wellMixed)
print("Running PCR with master=",master,", primers=",primers)
pcr=self.runPCR(src=rxs*nsplit,vol=pcrvol/nsplit,srcdil=pcrdil,ncycles=cycles,primers=primers,usertime=self.usertime if keepCleaved else None,fastCycling=False,inPlace=False,master=master,lowhi=self.lowhi,annealTemp=57)
if keepCleaved and self.regenPCRCycles is not None:
# Regenerate prefix
pcr2=self.runPCR(src=pcr,vol=self.regenPCRVolume,srcdil=self.regenPCRDilution,ncycles=self.regenPCRCycles,primers=None,usertime=None,fastCycling=False,inPlace=False,master="MTaqR",lowhi=self.lowhi,annealTemp=55)
# Add BT575p for 1 more cycle
for p in pcr2:
self.e.transfer(p.volume*0.5/10,reagents.getsample("Unclvd-Stop"),p,(False,False))
# One more cycle
cycling=' TEMP@95,30 TEMP@55,30 TEMP@68,30 TEMP@25,2'
thermocycler.setpgm('rfin',100,cycling)
self.e.runpgm("rfin",5.0,False,max([p.volume for p in pcr2]))
pcr=pcr2 # Use 2nd PCR as actual output
if len(pcr)<=len(names):
# Don't relabel if we've split
for i in range(len(pcr)):
pcr[i].name=names[i]+".pcr"
#print "Volume remaining in PCR input source: [",",".join(["%.1f"%r.volume for r in rxs]),"]"
needDil=finalConc/self.qConc
print("Projected final concentration = %.0f nM"%(needDil*self.qConc))
for i in range(len(pcr)):
pcr[i].conc=Concentration(stock=finalConc,final=None,units='nM')
db.popStatus()
if self.pcrSave:
# Save samples at 1x (move all contents -- can ignore warnings)
maxSaveVol=(100 if self.savedilplate else 1500)*1.0/nsplit
if self.finalRound and nsplit==1 and self.savedilplate and pcrtgt is None:
print("Skipping save of final PCR")
sv=pcr
else:
residual=2.4 # Amount to leave behind to avoid aspirating air
sv=self.saveSamps(src=pcr[:len(rxs)],vol=[min([maxSaveVol,x.volume-residual]) for x in pcr[:len(rxs)]],dil=1,plate=(self.savePlate if self.savedilplate else decklayout.EPPENDORFS),tgt=pcrtgt)
if nsplit>1:
# Combine split
for i in range(len(rxs),len(rxs)*nsplit):
self.e.transfer(min([maxSaveVol,pcr[i].volume-residual]),pcr[i],sv[i%len(sv)],mix=(False,False))
# Correct concentration (above would've assumed it was diluted)
for i in range(len(sv)):
sv[i].conc=pcr[i].conc
# Shake
self.e.shakeSamples(sv)
if "pcr" in self.qpcrStages:
for i in range(len(sv)):
q.addSamples(sv[i],needDil,primers=primerSet[i],names=["%s.pcr"%names[i]])
print("Have %.2f pmoles of product (%.0f ul @ %.1f nM)"%(sv[0].volume*sv[0].conc.stock/1000,sv[0].volume,sv[0].conc.stock))
# Save barcoded products too
if len(bcout)>0:
print("bcout=",",".join(str(b) for b in bcout))
print("mixed=",bcout[0].isMixed(),", wellMixed=",bcout[0].wellMixed)
bcsave=self.saveSamps(src=bcout,vol=[b.volume for b in bcout],dil=1,plate=self.savePlate,mix=(False,False))
if "bc" in self.qpcrStages:
print("Doing qPCR of barcoding: ",bcsave)
for i in range(len(bcsave)):
needDil=640
q.addSamples(src=bcsave[i],needDil=needDil,primers=["T7X","WX","ZX"]+(["MX"] if self.useMX else []),save=False)
else:
bcsave=[]
return sv, bcsave
else:
assert "pcr" not in self.qpcrStages ## Not implemented
return pcr[:len(rxs)], bcout
elif self.noPCRCleave:
print("Dilution instead of PCR: %.2f"%self.nopcrdil)
# Need to add enough t7prefix to compensate for all of the Stop primer currently present, regardless of whether it is for cleaved or uncleaved
# Will result in some short transcripts corresponding to the stop primers that are not used for cleaved product, producing just GGG_W_GTCTGC in the next round. These would be reverse-trancribed, but may compete for T7 yield
t7prefix=reagents.getsample("BT88")
dil=self.extpostdil[self.rndNum-1] # FIXME: Is this correct? Used to have a 'userDil' term
stopconc=1000.0/dil
bt88conc=t7prefix.conc.stock
relbt88=stopconc/bt88conc
print("Using EXT with %.0fnM of stop oligo as input to next T7, need %.2ful of BT88@%.0fnM per ul of sample"%(stopconc,relbt88,bt88conc))
for r in rxs:
vol=r.volume*relbt88
t7prefix.conc.final=t7prefix.conc.stock*vol/(r.volume+vol)
r.conc.final=r.conc.stock*r.volume/(r.volume+vol)
self.e.transfer(vol,t7prefix,r,mix=(False,False))
if self.nopcrdil>(1+relbt88):
self.diluteInPlace(tgt=rxs,dil=self.nopcrdil/(1.0+relbt88))
#needDil=needDil/self.nopcrdil # needDil not used subsequently
print("Dilution of EXT product: %.2fx * %.2fx = %2.fx\n"%(1+relbt88,self.nopcrdil/(1+relbt88),self.nopcrdil))
else:
print("Dilution of EXT product: %.2fx\n"%(1+relbt88))
return rxs, []
else:
return rxs, []
def pgm(self):
q = QSetup(self,maxdil=self.maxdilstep,debug=False,mindilvol=60)
self.e.addIdleProgram(q.idler)
if self.barcoding:
# Setup barcode primers for cleaved rounds only
self.bcprimers=[["BC-%s-R%d_T7"%(inp['ligand'],r+1) for inp in self.inputs] if self.rounds[r]=='C' else None for r in range(len(self.rounds))]
for bcp in self.bcprimers:
if bcp is not None:
for p in ["P-%s"%pp for pp in bcp]:
if not reagents.isReagent(p):
reagents.add(name=p,conc=4,extraVol=30,plate=decklayout.REAGENTPLATE,well="B2")
s=reagents.getsample(p) # Force allocation of a well
print("Adding %s to reagents at well %s"%(p,s.plate.wellname(s.well)))
print("BC primers=", self.bcprimers)
# Add any missing fields to inputs
for i in range(len(self.inputs)):
if 'ligand' not in self.inputs[i]:
self.inputs[i]['ligand']=None
if 'negligand' not in self.inputs[i]:
self.inputs[i]['negligand']=None
if 'round' not in self.inputs[i]:
self.inputs[i]['round']=None
if 'name' not in self.inputs[i]:
if self.inputs[i]['ligand'] is None:
self.inputs[i]['name']='%s_%d_R%d'%(self.inputs[i]['prefix'],self.inputs[i]['ID'],self.inputs[i]['round'])
else:
self.inputs[i]['name']='%s_%d_R%d_%s'%(self.inputs[i]['prefix'],self.inputs[i]['ID'],self.inputs[i]['round'],self.inputs[i]['ligand'])
# Add templates
if self.directT7:
self.srcs = self.addTemplates([inp['name'] for inp in self.inputs],stockconc=self.tmplFinalConc/self.templateDilution,finalconc=self.tmplFinalConc,plate=decklayout.SAMPLEPLATE,looplengths=[inp['looplength'] for inp in self.inputs],initVol=self.t7vol[0]*self.templateDilution,extraVol=0)
else:
self.srcs = self.addTemplates([inp['name'] for inp in self.inputs],stockconc=self.tmplFinalConc/self.templateDilution,finalconc=self.tmplFinalConc,plate=decklayout.DILPLATE,looplengths=[inp['looplength'] for inp in self.inputs],extraVol=15)
if self.dopcr:
# Reserve space for PCR products
pcrprods=[ [Sample("R%d-T%s"%(r,inp['ligand']),self.savePlate) for inp in self.inputs] for r in range(len(self.rounds))]
else:
pcrprods=None
t7in = [s.getsample() for s in self.srcs]
if "negative" in self.qpcrStages:
q.addSamples(decklayout.SSDDIL,1,self.allprimers,save=False) # Negative controls
if "reference" in self.qpcrStages:
q.addReferences(dstep=10,nsteps=5,primers=["WX","MX","T7X"] if self.useMX else ["WX","T7X"],ref=reagents.getsample("BT5310"),nreplicates=1)
# Save RT product from first (uncleaved) round and then use it during 2nd (cleaved) round for ligation and qPCR measurements
self.rndNum=0
self.nextID=self.firstID
curPrefix=[inp['prefix'] for inp in self.inputs]
r1=t7in
for roundType in self.rounds:
# Run a single round of roundType with r1 as input
# roundType is either "U" for uncleaved, or a new prefix for a cleaved round (with "T" being a T7 prepend)
# Set r1 to new output at end
if self.roundCallback is not None:
self.roundCallback(self,self.rndNum,roundType)
# Computed output prefix
if roundType=='U':
prefixOut=curPrefix
stop=["Unclvd" for _ in curPrefix]
else:
if roundType=='T':
stop=['T7%s'%p for p in curPrefix]
prefixOut=curPrefix
elif any([p==roundType for p in curPrefix]):
logging.error( "Round %d is a cleaved round but goes to %s without changing prefix"%(self.rndNum, roundType))
assert False
else:
prefixOut=[roundType for _ in curPrefix]
stop=prefixOut
# May be explicitly overridden
for i in range(len(self.inputs)):
if 'stop' in self.inputs[i]:
if isinstance(self.inputs[i]['stop'],list):
assert(len(self.inputs[i]['stop'])==len(self.rounds))
t=self.inputs[i]['stop'][self.rndNum]
else:
t=self.inputs[i]['stop']
if (roundType=='U') != (t=='U'):
print("Attempt to override round %d (type %s) with a input-specific round type of %s"%(self.rndNum, roundType, t))
assert False
if roundType!='U':
if t=='T':
stop[i]='T7%s'%curPrefix[i]
prefixOut[i]=curPrefix[i]
else:
stop[i]=t
prefixOut[i]=t
self.rndNum=self.rndNum+1
self.finalRound=self.rndNum==len(self.rounds)
db.pushStatus("%s%d"%(roundType,self.rndNum))
[r1,bc1]=self.oneround(q,r1,prefixOut=prefixOut,stop=stop,prefixIn=curPrefix,keepCleaved=(roundType!='U'),rtvol=self.rtvol[self.rndNum-1],t7vol=self.t7vol[self.rndNum-1],cycles=self.pcrcycles[self.rndNum-1],pcrdil=self.pcrdil[self.rndNum-1],pcrvol=self.pcrvol[self.rndNum-1],dolig=self.allLig or (roundType!='U'),pcrtgt=None if pcrprods is None else pcrprods[self.rndNum-1])
db.popStatus()
# Add TRefs specified in rnddefs
if 'tref' in self.rnddef[self.rndNum-1]:
tref=self.rnddef[self.rndNum-1]['tref']
assert len(tref)==len(r1)
for i in range(len(r1)):
if tref[i] is not None:
trefname='TRef%d'%tref[i]
print("Adding %s to %s"%(trefname,r1[i].name))
if not reagents.isReagent(trefname):
reagents.add(name=trefname,conc=10,extraVol=30,well="E4")
trefSamp=reagents.getsample(trefname)
oldConc=r1[i].conc.stock
oldUnits=r1[i].conc.units
oldVol=r1[i].volume
self.e.transfer(r1[i].volume/(trefSamp.conc.dilutionneeded()-1),trefSamp,r1[i],mix=(False,False)) # TODO: Check that these end up mixed
r1[i].conc=Concentration(stock=oldConc*oldVol/r1[i].volume, units=oldUnits) # Treat TRef as straight dilution
print("New conc=",r1[i].conc)
for i in range(len(r1)):
if self.inputs[i]['round'] is None:
r1[i].name="%s_%d"%(prefixOut[i],self.nextID)
else:
r1[i].name="%d_%s_R%d%c"%(self.nextID,prefixOut[i],self.inputs[i]['round']+self.rndNum,roundType)
if self.inputs[i]['ligand'] is not None:
r1[i].name="%s_%s"%(r1[i].name,self.inputs[i]['ligand'])
print("Used ID ", self.nextID," for ", r1[i].name,": ",r1[i])
self.nextID+=1
r1[i].conc.final=r1[i].conc.stock*self.templateDilution
for i in range(len(bc1)):
#print("Renaming",bc1[i].name)
pts=bc1[i].name.split(".")
bc1[i].name="%d_BC_R%d%c"%(self.nextID,self.inputs[i//2]['round']+self.rndNum,roundType)
if self.inputs[i//2]['ligand'] is not None:
bc1[i].name="%s_%s"%(bc1[i].name,self.inputs[i//2]['ligand'])
bc1[i].name+="_"+pts[-2]
print("Used ID ", self.nextID," for ", bc1[i].name,":",bc1[i])
self.nextID+=1
curPrefix=prefixOut
if "finalpcr" in self.qpcrStages:
for i in range(len(r1)):
if self.singlePrefix:
q.addSamples(src=r1[i],needDil=r1[i].conc.stock/self.qConc,primers=["T7X","MX"] if self.useMX else ["T7X"])
else:
# noinspection PyUnboundLocalVariable
q.addSamples(src=r1[i],needDil=r1[i].conc.stock/self.qConc,primers=["T7X",prefixOut[i]+"X"]+(["MX"] if self.useMX else []))
# Add TRefs if needed
for i in range(len(r1)):
if 'tref' in self.inputs[i]:
trefname='TRef%d'%self.inputs[i]['tref']
if not reagents.isReagent(trefname):
reagents.add(name=trefname,conc=10,extraVol=30)
tref=reagents.getsample(trefname)
self.e.transfer(r1[i].volume/(tref.conc.dilutionneeded()-1),tref,r1[i],mix=(False,False))
db.pushStatus('qPCR')
print("######### qPCR ########### %.0f min"%(clock.elapsed()/60))
self.allprimers=q.allprimers()
q.run(confirm=self.qpcrWait)
db.popStatus()