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 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 pgm(self):
q = QSetup(self,maxdil=16,debug=False,mindilvol=60)
self.e.addIdleProgram(q.idler)
t7in = [s.getsample() for s in self.srcs]
if "negative" in self.qpcrStages:
qpcrPrimers=["REF","MX","T7X","T7AX","T7BX","T7WX"]
q.addSamples(decklayout.SSDDIL,1,qpcrPrimers,save=False) # Negative controls
# Save RT product from first (uncleaved) round and then use it during 2nd (cleaved) round for ligation and qPCR measurements
self.rndNum=0
self.nextID=self.firstID
curPrefix=[inp['prefix'] for inp in self.inputs]
while self.rndNum<self.nrounds:
self.rndNum=self.rndNum+1
prefixOut=["A" if p=="W" else "B" if p=="A" else "W" if p=="B" else "BADPREFIX" for p in curPrefix]
if self.rndNum==1:
self.t7vol1=self.t7vol1a
else:
self.t7vol1=max(20,self.pmolesIn*1000/min([inp.conc.final for inp in t7in])) # New input volueme
r1=self.oneround(q,t7in,prefixOut,prefixIn=curPrefix,keepCleaved=False,rtvol=self.rtvol1,t7vol=self.t7vol1,cycles=self.pcrcycles1,pcrdil=self.pcrdil1,pcrvol=self.pcrvol1,dolig=self.allLig)
# pcrvol is set to have same diversity as input
for i in range(len(r1)):
r1[i].name="%s_%d_R%dU_%s"%(curPrefix[i],self.nextID,self.inputs[i]['round']+self.rndNum,self.inputs[i]['ligand'])
self.nextID+=1
r1[i].conc.final=r1[i].conc.stock*self.templateDilution
if self.rndNum>=self.nrounds:
logging.warning("Warning: ending on an uncleaved round")
break
self.rndNum=self.rndNum+1
print "prefixIn=",curPrefix
print "prefixOut=",prefixOut
self.t7vol2=max(20,self.pmolesIn*1000/min([inp.conc.final for inp in r1]))
r2=self.oneround(q,r1,prefixOut,prefixIn=curPrefix,keepCleaved=True,rtvol=self.rtvol2,t7vol=self.t7vol2,cycles=self.pcrcycles2,pcrdil=self.pcrdil2,pcrvol=self.pcrvol2,dolig=True)
# pcrvol is set to have same diversity as input = (self.t7vol2*self.templateDilution/rnagain*stopdil*rtdil*extdil*exodil*pcrdil)
for i in range(len(self.inputs)):
r2[i].name="%s_%d_R%dC_%s"%(prefixOut[i],self.nextID,self.inputs[i]['round']+self.rndNum,self.inputs[i]['ligand'])
self.nextID+=1
r2[i].conc.final=r2[i].conc.stock*self.templateDilution
t7in=r2
curPrefix=prefixOut
if "finalpcr" in self.qpcrStages:
for i in range(len(r2)):
q.addSamples(src=r2[i],needDil=r2[i].conc.stock/self.qConc,primers=["T7X","T7"+prefixOut[i]+"X"])
print "######### qPCR ###########"
#q.addReferences(mindil=4,nsteps=6,primers=["T7X","MX","T7AX"])
if self.qpcrWait:
worklist.userprompt('Continue to setup qPCR')
q.run()
def 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 pgm(self):
q = QSetup(self, maxdil=16, debug=False, mindilvol=60)
self.e.addIdleProgram(q.idler)
input = [s.getsample() for s in self.srcs]
# Save RT product from first (uncleaved) round and then use it during 2nd (cleaved) round for ligation and qPCR measurements
prefixIn = [inp['prefix'] for inp in self.inputs]
prefixOut = [
"A" if p == "W" else
"B" if p == "A" else "W" if p == "B" else "BADPREFIX"
for p in prefixIn
]
qpcrPrimers = ["REF", "MX", "T7X"]
if "W" in prefixIn + prefixOut:
qpcrPrimers += ["T7WX"]
if "A" in prefixIn + prefixOut:
qpcrPrimers += ["T7AX"]
if "B" in prefixIn + prefixOut:
qpcrPrimers += ["T7BX"]
q.addSamples(decklayout.SSDDIL, 1, qpcrPrimers,
save=False) # Negative controls
print "Starting new cleavage round, will add prefix: ", prefixOut
names = [i.name for i in input]
print "######## T7 ###########"
print "Inputs: (t7vol=%.2f)" % self.t7vol
for inp in input:
print " %s: %.1ful@%.1f nM, use %.1f ul (%.3f pmoles)" % (
inp.name, inp.volume, inp.conc.stock, self.t7vol /
inp.conc.dilutionneeded(), self.t7vol * inp.conc.final / 1000)
print "input[0]=", input[0]
needDil = max([inp.conc.final for inp in input]) * 1.0 / self.qConc
if self.directT7:
# Just add MT7 and possibly water to each well
mconc = reagents.getsample("MT7").conc.dilutionneeded()
for i in range(len(input)):
watervol = self.t7vol * (1 - 1 / mconc) - input[i].volume
if watervol > 0.1:
self.e.transfer(watervol,
decklayout.WATER,
input[i],
mix=(False, False))
self.e.transfer(self.t7vol / mconc,
reagents.getsample("MT7"),
input[i],
mix=(False, False))
assert (input[i].volume == self.t7vol)
rxs = input
else:
rxs = self.runT7Setup(
src=input,
vol=self.t7vol,
srcdil=[inp.conc.dilutionneeded() for inp in input])
print "input[0]=", input[0]
#for i in range(len(rxs)):
# q.addSamples(src=rxs],needDil=needDil,primers=["T7"+prefixIn[i]+"X","MX","T7X","REF"],names=["%s.T-"%names[i]])
self.runT7Pgm(dur=self.t7dur, vol=self.t7vol)
print "Template conc=%.1f nM, estimated RNA concentration in T7 reaction at %.0f nM" % (
self.tmplFinalConc, self.rnaConc)
print "######## Stop ###########"
self.e.lihahome()
print "Have %.1f ul before stop" % rxs[0].volume
preStopVolume = rxs[0].volume
self.addEDTA(tgt=rxs, finalconc=2) # Stop to 2mM EDTA final
stopDil = rxs[0].volume / preStopVolume
if self.saveRNA:
self.saveSamps(
src=rxs,
vol=5,
dil=2,
plate=decklayout.DILPLATE,
dilutant=reagents.getsample("TE8"),
mix=(False,
False)) # Save to check [RNA] on Qubit, bioanalyzer
stop = [
"A-Stop" if n == "A" else
"B-Stop" if n == "B" else "W-Stop" if n == "W" else "BADPREFIX"
for n in prefixOut
]
for i in range(len(rxs)):
rxs[i].name = rxs[i].name + "." + stop[i]
needDil = self.rnaConc / self.qConc / stopDil
#q.addSamples(src=rxs,needDil=needDil,primers=["T7AX","MX","T7X","REF"],names=["%s.stopped"%r.name for r in rxs])
print "######## RT Setup ###########"
rtDil = 4
hiTemp = 95
rtDur = 20
rxin = rxs
rxs = self.runRT(src=rxs,
vol=self.rtvol,
srcdil=rtDil,
heatInactivate=True,
hiTemp=hiTemp,
dur=rtDur,
incTemp=50,
stop=[reagents.getsample(s) for s in stop
]) # Heat inactivate also allows splint to fold
print "RT volume= ", [x.volume for x in rxs]
for r in rxin:
if r.volume > 20:
print "Have more T7 reaction remaining than needed: %s has %.1f ul" % (
r.name, r.volume)
needDil /= rtDil
rtPostDil = 5
if rtPostDil != 1:
self.diluteInPlace(tgt=rxs, dil=rtPostDil)
needDil /= rtPostDil
#q.addSamples(src=rxs,needDil=needDil,primers=["T7AX","MX","REF"],names=["%s.rt"%r.name for r in rxs])
print "######## Ligation setup ###########"
extdil = 5.0 / 4
reagents.getsample("MLigase").conc = Concentration(5)
extvol = 20
print "Extension volume=", extvol
rxs = self.runLig(rxs, vol=extvol)
print "Ligation volume= ", [x.volume for x in rxs]
needDil = needDil / extdil
extpostdil = 4
if extpostdil > 1:
print "Dilution after extension: %.2f" % extpostdil
self.diluteInPlace(tgt=rxs, dil=extpostdil)
needDil = needDil / extpostdil
if not self.doexo:
self.pcrdil = self.pcrdil / extpostdil
if self.saveDil is not None:
ext = self.saveSamps(
src=rxs,
vol=3,
dil=self.saveDil,
dilutant=reagents.getsample("TE8"),
tgt=[Sample("%s.ext" % n, decklayout.DILPLATE) for n in names],
mix=(False, True)) # Save cDNA product for subsequent NGS
for i in range(len(rxs)):
q.addSamples(src=[ext[i]],
needDil=needDil / self.saveDil,
primers=[
"T7" + prefixIn[i] + "X",
"T7" + prefixOut[i] + "X", "MX", "T7X", "REF"
],
names=["%s.ext" % names[i]])
else:
for i in range(len(rxs)):
q.addSamples(src=[rxs[i]],
needDil=needDil,
primers=[
"T7" + prefixIn[i] + "X",
"T7" + prefixOut[i] + "X", "MX", "T7X", "REF"
],
names=["%s.ext" % names[i]])
if self.doexo:
print "######## Exo ###########"
prevvol = rxs[0].volume
rxs = self.runExo(rxs, incTime=30, inPlace=True)
exoDil = rxs[0].volume / prevvol
needDil /= exoDil
needDil /= 7 # Anecdotal based on Ct's -- large components (MX) reduced by exo digestion
q.addSamples(src=rxs,
needDil=needDil,
primers=["T7AX", "T7BX", "MX", "T7X", "REF"],
names=["%s.exo" % names[i] for i in range(len(rxs))])
#exo=self.saveSamps(src=rxs,vol=10*exoDil,dil=2/exoDil,dilutant=reagents.getsample("TE8"),tgt=[Sample("%s.exo"%n,decklayout.SAMPLEPLATE) for n in names]) # Save cDNA product
else:
exoDil = 1
exo = []
if self.doampure:
print "######## Ampure Cleanup ###########"
ratio = 1.5
elutionVol = 30
cleanIn = ext + exo + user
needDil = needDil * cleanIn[0].volume / elutionVol
clean = self.runAmpure(src=cleanIn,
ratio=ratio,
elutionVol=elutionVol)
q.addSamples(src=[clean[i] for i in range(len(rxs))],
needDil=needDil,
primers=["T7AX", "MX", "T7X", "REF"])
rxs = rxs + clean # Use the cleaned products for PCR
totalDil = stopDil * rtDil * rtPostDil * extdil * extpostdil * exoDil
fracRetained = rxs[0].volume / (self.t7vol * totalDil)
print "Total dilution from T7 to Pre-pcr Product = %.2f*%.2f*%.2f*%.2f*%.2f*%.2f = %.2f, fraction retained=%.0f%%" % (
stopDil, rtDil, rtPostDil, extdil, extpostdil, exoDil, totalDil,
fracRetained * 100)
if self.dopcr:
print "######### PCR #############"
print "PCR Volume: %.1f, Dilution: %.1f, volumes available for PCR: [%s]" % (
self.pcrvol, self.pcrdil, ",".join(
["%.1f" % r.volume for r in rxs]))
maxSampleVolume = 100 # Maximum sample volume of each PCR reaction (thermocycler limit, and mixing limit)
initConc = needDil * self.qConc / self.pcrdil
if self.doexo:
initConc = initConc * 7 * self.cleavage # Back out 7x dilution in exo step, but only use cleaved as input conc
else:
initConc = initConc * self.cleavage # Only use cleaved as input conc
gain = pcrgain(initConc, 400, self.pcrcycles)
finalConc = initConc * gain
print "Estimated starting concentration in PCR = %.1f nM, running %d cycles -> %.0f nM\n" % (
needDil * self.qConc, self.pcrcycles, finalConc)
pcr = self.runPCR(src=rxs,
vol=self.pcrvol,
srcdil=self.pcrdil,
ncycles=self.pcrcycles,
primers=["T7%sX" % x for x in prefixOut],
usertime=self.usertime,
fastCycling=True)
needDil = finalConc / self.qConc
pcrpostdil = 2
if pcrpostdil > 1:
self.diluteInPlace(pcr, pcrpostdil)
needDil = needDil / pcrpostdil
print "Projected final concentration = %.0f nM (after %.1fx dilution)" % (
needDil * self.qConc, pcrpostdil)
for i in range(len(pcr)):
pcr[i].conc = Concentration(stock=finalConc / pcrpostdil,
final=None,
units='nM')
if self.pcrSave:
# Save samples at 1x
if self.savedilplate:
sv = self.saveSamps(
src=pcr[:len(rxs)],
vol=[x.volume - 16.4 for x in pcr[:len(rxs)]],
dil=1,
plate=decklayout.DILPLATE)
else:
sv = self.saveSamps(
src=pcr[:len(rxs)],
vol=[x.volume - 16.4 for x in pcr[:len(rxs)]],
dil=1,
plate=decklayout.EPPENDORFS)
# for i in range(len(pcr)):
# q.addSamples(pcr,needDil,["T7%sX"%prefixOut[i]])
processEff = 0.5 # Estimate of overall efficiency of process
print "Saved %.2f pmoles of product (%.0f ul @ %.1f nM)" % (
sv[0].volume * sv[0].conc.stock / 1000, sv[0].volume,
sv[0].conc.stock)
print "######### qPCR ###########"
#q.addReferences(mindil=4,nsteps=6,primers=["T7X","MX","T7AX"])
q.run(confirm=False)
def pgm(self):
q = QSetup(self, maxdil=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)
class Constrict(TRP):
# Mix constriction inputs, constrict, PCR, remove barcodes
pcreff = 1.98
def __init__(self, inputs, nmolecules, nconstrict, vol):
super(Constrict, self).__init__()
self.inputs = inputs
self.nmolecules = nmolecules
self.nconstrict = nconstrict
self.qconc = 20e-12 # Target qPCR concentration
self.qprimers = ["End"]
self.mix_conc = 100e-9 # Concentration of mixdown
self.con_dilvol = 100 # Volume to use for constriction dilutions
self.con_maxdilperstage = 100 / 3.0 # Maximum dilution/stage
self.con_pcr1vol = 100
self.con_pcr1inputvol = 2
self.con_pcr1tgtconc = self.qconc * 4 # Enough to take qPCR without dilutiojn
self.con_pcr2dil = 4
self.con_pcr2vol = 50
self.con_pcr2tgtconc = 10e-9
self.regen_predilvol = 100
self.regen_predil = 25
self.regen_dil = 25
self.regen_vol = 100
self.regen_cycles = 10
self.rsrc = [reagents.add("%s-%s-%s" % (inputs[i]['name'], inputs[i]['left'], inputs[i]['right']),
decklayout.SAMPLEPLATE,
well=inputs[i]['well'] if 'well' in inputs[i] else None,
conc=Concentration(stock=inputs[i]['bconc'], units="nM"),
initVol=vol, extraVol=0)
for i in range(len(inputs))]
self.q = None # Defined in pgm()
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 mix(self, inp, weights,mixvol=100,tgtconc=None,maxinpvol=20):
"""Mix given inputs according to weights (by moles -- use conc.stock of each input)"""
vol = [weights[i] *1.0 / inp[i].conc.stock for i in range(len(inp))]
scale = mixvol / sum(vol)
conc=sum([inp[i].conc.stock * scale * vol[i] for i in range(len(inp))]) / mixvol
if tgtconc is not None and conc>tgtconc:
scale*=tgtconc*1.0/conc
if max(vol)*scale<4.0:
scale=4.1/max(vol) # At least one input with 4ul input
vol = [x * scale for x in vol] # Mix to make planned total without water
for i in range(len(vol)):
# Check if this would require more than available of any input
newscale= min(maxinpvol,inp[i].volume-inp[i].plate.unusableVolume()-2)/vol[i]
if newscale<1:
vol = [x * 1.0 * newscale for x in vol]
if tgtconc is not None:
mixvol *= newscale # Maintain same target concentration by reducing total volume
if min(vol) < 4.0:
# Some components are too small; split mixing
lowvol=[i for i in range(len(inp)) if vol[i]<4.0]
highvol=[i for i in range(len(inp)) if i not in lowvol]
assert len(highvol)>0
assert len(lowvol)>0
lowtgtconc=sum([inp[i].conc.stock *1.0/ weights[i] for i in highvol])/len(highvol)*sum([weights[i] for i in lowvol])
print("Running premix of samples "+",".join(["%d"%ind for ind in lowvol])+" with target concentration of %.4f"%lowtgtconc)
mix1=self.mix([inp[i] for i in lowvol],[weights[i] for i in lowvol],tgtconc=lowtgtconc,mixvol=mixvol,maxinpvol=maxinpvol)
wt1=sum([weights[i] for i in lowvol])
mix2=self.mix([inp[i] for i in highvol]+[mix1],[weights[i] for i in highvol]+[wt1],tgtconc=tgtconc,mixvol=mixvol,maxinpvol=maxinpvol)
return mix2
print("Mixing into %.0ful with tgtconc of %s, dil=%.2f"%(mixvol,"None" if tgtconc is None else "%.4f"%tgtconc,mixvol/sum(vol)))
for i in range(len(inp)):
print("%-30.30s %6.3fnM wt=%5.2f v=%5.2ful"%(inp[i].name,inp[i].conc.stock,weights[i],vol[i]))
watervol = mixvol - sum(vol)
#print "Mixdown: vols=[", ",".join(["%.2f " % v for v in vol]), "], water=", watervol, ", total=", mixvol, " ul"
mixdown = Sample('mixdown', plate=decklayout.SAMPLEPLATE)
if watervol < -0.1:
print("Total mixdown is %.1f ul, more than planned %.0f ul" % (sum(vol), mixvol))
assert False
elif watervol >= 4.0: # Omit if too small
self.e.transfer(watervol, decklayout.WATER, mixdown, (False, False))
else:
pass
ordering=sorted(list(range(len(inp))),key=lambda i: vol[i],reverse=True)
for i in ordering:
inp[i].conc.final = inp[i].conc.stock * vol[i] / mixvol # Avoid warnings about concentrations not adding up
self.e.transfer(vol[i], inp[i], mixdown, (False, False))
self.e.shakeSamples([mixdown])
if not mixdown.wellMixed:
self.e.mix(mixdown)
mixdown.conc = Concentration(stock=sum([inp[i].conc.stock * vol[i] for i in range(len(inp))]) / mixvol,
final=None, units='nM')
print("Mix product, %s, is in well %s with %.1ful @ %.2f nM"%(mixdown.name,mixdown.plate.wellname(mixdown.well),mixdown.volume,mixdown.conc.stock))
print("----------")
return mixdown
def constrict(self, constrictin, conc):
"""Constrict sample with concentration given by conc (in M)"""
# noinspection PyPep8Naming
AN = 6.022e23
dil = conc * (self.con_pcr1inputvol * 1e-6) * AN / self.nmolecules
nstages = int(math.ceil(math.log(dil) / math.log(self.con_maxdilperstage)))
dilperstage = math.pow(dil, 1.0 / nstages)
print("Diluting by %.0fx in %.0f stages of %.1f" % (dil, nstages, dilperstage))
s = [decklayout.WATER] + [constrictin] * self.nconstrict + [decklayout.SSDDIL]
self.e.sanitize(3, 50) # Heavy sanitize
for j in range(nstages):
print("Stage ", j, ", conc=", conc)
if conc <= self.qconc * 1e-9:
self.q.addSamples(s, needDil=1.0, primers=self.qprimers, save=False)
s = self.runQPCRDIL(s, self.con_dilvol, dilperstage, dilPlate=True)
conc /= dilperstage
cycles = int(
math.log(self.con_pcr1tgtconc / conc * self.con_pcr1vol / self.con_pcr1inputvol) / math.log(self.pcreff) + 0.5)
pcr1finalconc = conc * self.con_pcr1inputvol / self.con_pcr1vol * self.pcreff ** cycles
print("Running %d cycle PCR1 -> %.1f pM" % (cycles, pcr1finalconc * 1e12))
s = s + [decklayout.WATER] # Extra control of just water added to PCR mix
pcr = self.runPCR(primers=None, src=s, vol=self.con_pcr1vol,
srcdil=self.con_pcr1vol * 1.0 / self.con_pcr1inputvol,
ncycles=cycles, master="MConstrict", kapa=True)
for p in pcr:
p.conc = Concentration(stock=pcr1finalconc * 1e9, final=pcr1finalconc / self.con_pcr2dil, units='nM')
self.e.addIdleProgram(self.q.idler) # Now that constriction is done, can start on qPCR setup
needDil = max(4, pcr1finalconc / self.qconc)
print("Running qPCR of PCR1 products using %.1fx dilution" % needDil)
self.q.addSamples(pcr, needDil=needDil, primers=self.qprimers, save=True)
pcr = pcr[1:-2] # Remove negative controls
cycles2 = int(math.log(self.con_pcr2tgtconc / pcr1finalconc * self.con_pcr2dil) / math.log(self.pcreff) + 0.5)
pcr2finalconc = pcr1finalconc / self.con_pcr2dil * self.pcreff ** cycles2
if cycles2 > 0:
print("Running %d cycle PCR2 -> %.1f nM" % (cycles2, pcr2finalconc * 1e9))
pcr2 = self.runPCR(primers="End", src=pcr, vol=self.con_pcr2vol, srcdil=self.con_pcr2dil,
ncycles=cycles2, master="MKapa", kapa=True)
self.q.addSamples(pcr2, needDil=pcr2finalconc / self.qconc, primers=self.qprimers, save=True)
for p in pcr2:
p.conc = Concentration(stock=pcr2finalconc * 1e9, units='nM')
self.e.waitpgm()
return pcr2
else:
return pcr
def regenerate(self, inp, prefix):
"""Regenerate T7 templates without barcodes with each of the given prefixes"""
print("Regen Predilute: %.1f nM by %.1fx to %.2f nM" % (
inp[0].conc.stock, self.regen_predil, inp[0].conc.stock / self.regen_predil))
d1 = self.runQPCRDIL(inp, self.regen_predilvol, self.regen_predil, dilPlate=True)
inconc = inp[0].conc.stock / self.regen_predil / self.regen_dil
print("Regen PCR: %.3f nM with %d cycles -> %.1f nM" % (
inconc, self.regen_cycles, inconc * self.pcreff ** self.regen_cycles))
res = self.runPCR(src=d1, srcdil=self.regen_dil, vol=self.regen_vol,
ncycles=self.regen_cycles,
primers=["T7%sX" % p for p in prefix], fastCycling=False, master="MKapa", kapa=True)
return res
def pgm(self):
q = QSetup(self,maxdil=16,debug=False,mindilvol=60)
self.e.addIdleProgram(q.idler)
input = [s.getsample() for s in self.srcs]
qpcrPrimers=["REF","MX","T7X","T7AX","T7BX","T7WX"]
q.addSamples(decklayout.SSDDIL,1,qpcrPrimers,save=False) # Negative controls
# Save RT product from first (uncleaved) round and then use it during 2nd (cleaved) round for ligation and qPCR measurements
prefixIn=[inp['prefix'] for inp in self.inputs]
prefixOut=["A" if p=="W" else "B" if p=="A" else "W" if p=="B" else "BADPREFIX" for p in prefixIn]
print "Starting new cleavage round, will add prefix: ",prefixOut
names=[i.name for i in input]
print "######## T7 ###########"
print "Inputs: (t7vol=%.2f)"%self.t7vol
for inp in input:
print " %s: %.1ful@%.1f nM, use %.1f ul (%.3f pmoles)"%(inp.name,inp.volume,inp.conc.stock,self.t7vol/inp.conc.dilutionneeded(), self.t7vol*inp.conc.final/1000)
print "input[0]=",input[0]
needDil = max([inp.conc.final for inp in input])*1.0/self.qConc
if self.directT7:
# Just add MT7 and possibly water to each well
mconc=reagents.getsample("MT7").conc.dilutionneeded()
for i in range(len(input)):
watervol=self.t7vol*(1-1/mconc)-input[i].volume
if watervol>0.1:
self.e.transfer(watervol,decklayout.WATER,input[i],mix=(False,False))
self.e.transfer(self.t7vol/mconc,reagents.getsample("MT7"),input[i],mix=(False,False))
assert(input[i].volume==self.t7vol)
rxs=input
else:
rxs = self.runT7Setup(src=input,vol=self.t7vol,srcdil=[inp.conc.dilutionneeded() for inp in input])
print "input[0]=",input[0]
#for i in range(len(rxs)):
# q.addSamples(src=rxs],needDil=needDil,primers=["T7"+prefixIn[i]+"X","MX","T7X","REF"],names=["%s.T-"%names[i]])
self.runT7Pgm(dur=self.t7dur,vol=self.t7vol)
print "Template conc=%.1f nM, estimated RNA concentration in T7 reaction at %.0f nM"%(self.tmplFinalConc,self.rnaConc)
print "######## Stop ###########"
#self.saveSamps(src=rxs,vol=5,dil=10,plate=decklayout.EPPENDORFS,dilutant=reagents.getsample("TE8"),mix=(False,False)) # Save to check [RNA] on Qubit, bioanalyzer
self.e.lihahome()
print "Have %.1f ul before stop"%rxs[0].volume
preStopVolume=rxs[0].volume
self.addEDTA(tgt=rxs,finalconc=2) # Stop to 2mM EDTA final
stop=[ "A-Stop" if n=="A" else "B-Stop" if n=="B" else "W-Stop" if n=="W" else "BADPREFIX" for n in prefixOut]
for i in range(len(rxs)):
rxs[i].name=rxs[i].name+"."+stop[i]
stopDil=rxs[0].volume/preStopVolume
needDil = self.rnaConc/self.qConc/stopDil
#q.addSamples(src=rxs,needDil=needDil,primers=["T7AX","MX","T7X","REF"],names=["%s.stopped"%r.name for r in rxs])
print "######## RT Setup ###########"
rtDil=4
hiTemp=95
rtDur=20
rxin=rxs
rxs=self.runRT(src=rxs,vol=self.rtvol,srcdil=rtDil,heatInactivate=True,hiTemp=hiTemp,dur=rtDur,incTemp=50,stop=[reagents.getsample(s) for s in stop]) # Heat inactivate also allows splint to fold
print "RT volume= ",[x.volume for x in rxs]
for r in rxin:
if r.volume>20:
print "Have more T7 reaction remaining than needed: %s has %.1f ul"%(r.name,r.volume)
needDil /= rtDil
rtPostDil=5
if rtPostDil!=1:
self.diluteInPlace(tgt=rxs,dil=rtPostDil)
needDil /= rtPostDil
#q.addSamples(src=rxs,needDil=needDil,primers=["T7AX","MX","REF"],names=["%s.rt"%r.name for r in rxs])
print "######## Ligation setup ###########"
extdil=5.0/4
reagents.getsample("MLigase").conc=Concentration(5)
extvol=20;
print "Extension volume=",extvol
rxs=self.runLig(rxs,vol=extvol)
print "Ligation volume= ",[x.volume for x in rxs]
needDil=needDil/extdil
extpostdil=4
if extpostdil>1:
print "Dilution after extension: %.2f"%extpostdil
self.diluteInPlace(tgt=rxs,dil=extpostdil)
needDil=needDil/extpostdil
if not self.doexo:
self.pcrdil=self.pcrdil/extpostdil
if self.saveDil is not None:
ext=self.saveSamps(src=rxs,vol=3,dil=self.saveDil,dilutant=reagents.getsample("TE8"),tgt=[Sample("%s.ext"%n,decklayout.DILPLATE) for n in names],mix=(False,True)) # Save cDNA product for subsequent NGS
for i in range(len(rxs)):
q.addSamples(src=[ext[i]],needDil=needDil/self.saveDil,primers=["T7"+prefixIn[i]+"X","T7"+prefixOut[i]+"X","MX","T7X","REF"],names=["%s.ext"%names[i]])
else:
for i in range(len(rxs)):
q.addSamples(src=[rxs[i]],needDil=needDil,primers=["T7"+prefixIn[i]+"X","T7"+prefixOut[i]+"X","MX","T7X","REF"],names=["%s.ext"%names[i]])
if self.doexo:
print "######## Exo ###########"
prevvol=rxs[0].volume
rxs=self.runExo(rxs,incTime=30,inPlace=True)
exoDil=rxs[0].volume/prevvol
needDil/=exoDil
needDil/=7 # Anecdotal based on Ct's -- large components (MX) reduced by exo digestion
q.addSamples(src=rxs,needDil=needDil,primers=["T7AX","T7BX","MX","T7X","REF"],names=["%s.exo"%names[i] for i in range(len(rxs))])
#exo=self.saveSamps(src=rxs,vol=10*exoDil,dil=2/exoDil,dilutant=reagents.getsample("TE8"),tgt=[Sample("%s.exo"%n,decklayout.SAMPLEPLATE) for n in names]) # Save cDNA product
else:
exoDil=1
exo=[]
if self.doampure:
print "######## Ampure Cleanup ###########"
ratio=1.5
elutionVol=30
cleanIn=ext+exo+user
needDil=needDil*cleanIn[0].volume/elutionVol
clean=self.runAmpure(src=cleanIn,ratio=ratio,elutionVol=elutionVol)
q.addSamples(src=[clean[i] for i in range(len(rxs))],needDil=needDil,primers=["T7AX","MX","T7X","REF"])
rxs=rxs+clean # Use the cleaned products for PCR
totalDil=stopDil*rtDil*rtPostDil*extdil*extpostdil*exoDil
fracRetained=rxs[0].volume/(self.t7vol*totalDil)
print "Total dilution from T7 to Pre-pcr Product = %.2f*%.2f*%.2f*%.2f*%.2f*%.2f = %.2f, fraction retained=%.0f%%"%(stopDil,rtDil,rtPostDil,extdil,extpostdil,exoDil,totalDil,fracRetained*100)
if self.dopcr:
print "######### PCR #############"
print "PCR Volume: %.1f, Dilution: %.1f, volumes available for PCR: [%s]"%(self.pcrvol, self.pcrdil,",".join(["%.1f"%r.volume for r in rxs]))
maxSampleVolume=100 # Maximum sample volume of each PCR reaction (thermocycler limit, and mixing limit)
initConc=needDil*self.qConc/self.pcrdil
if self.doexo:
initConc=initConc*7*self.cleavage # Back out 7x dilution in exo step, but only use cleaved as input conc
else:
initConc=initConc*self.cleavage # Only use cleaved as input conc
gain=pcrgain(initConc,400,self.pcrcycles)
finalConc=initConc*gain
print "Estimated starting concentration in PCR = %.1f nM, running %d cycles -> %.0f nM\n"%(needDil*self.qConc,self.pcrcycles,finalConc)
pcr=self.runPCR(src=rxs,vol=self.pcrvol,srcdil=self.pcrdil,ncycles=self.pcrcycles,primers=["T7%sX"%x for x in prefixOut],usertime=self.usertime,fastCycling=True)
needDil=finalConc/self.qConc
pcrpostdil=2
if pcrpostdil>1:
self.diluteInPlace(pcr,pcrpostdil)
needDil=needDil/pcrpostdil
print "Projected final concentration = %.0f nM (after %.1fx dilution)"%(needDil*self.qConc,pcrpostdil)
for i in range(len(pcr)):
pcr[i].conc=Concentration(stock=finalConc/pcrpostdil,final=None,units='nM')
if self.pcrSave:
# Save samples at 1x
if self.savedilplate:
sv=self.saveSamps(src=pcr[:len(rxs)],vol=[x.volume-16.4 for x in pcr[:len(rxs)]],dil=1,plate=decklayout.DILPLATE)
else:
sv=self.saveSamps(src=pcr[:len(rxs)],vol=[x.volume-16.4 for x in pcr[:len(rxs)]],dil=1,plate=decklayout.EPPENDORFS)
# for i in range(len(pcr)):
# q.addSamples(pcr,needDil,["T7%sX"%prefixOut[i]])
processEff=0.5 # Estimate of overall efficiency of process
print "Saved %.2f pmoles of product (%.0f ul @ %.1f nM)"%(sv[0].volume*sv[0].conc.stock/1000,sv[0].volume,sv[0].conc.stock)
print "######### qPCR ###########"
#q.addReferences(mindil=4,nsteps=6,primers=["T7X","MX","T7AX"])
#worklist.userprompt('Continue to setup qPCR')
q.run()
class Barcoding(TRP):
"""Barcode multiple samples, mix them"""
def __init__(self, inputs, pcr1inputconc=0.05, used=None,doqpcr=True,inputPlate=decklayout.SAMPLEPLATE):
super(Barcoding, self).__init__()
if used is None:
used = []
self.inputs = inputs
self.qconc = 50e-12 # Target qPCR concentration
self.qprimers = ["End"]
self.doqpcr=doqpcr
self.bc1_inputvol = 4 # ul of input samples
self.mix_conc = 100e-9 # Concentration of mixdown
self.pcr1inputconc = pcr1inputconc
for inp in inputs:
bc = "%s-%s" % (inp['left'], inp['right'])
if bc in used:
logging.error("Barcode %s is being reused for %s" % (bc, inp['name']))
used.append(bc)
for x in inputs:
if not reagents.isReagent(x['name']):
reagents.add(x['name'], inputPlate, well=x['well'] if 'well' in x else None,
conc=Concentration(stock=x['conc'], units="nM"),
initVol=self.bc1_inputvol, extraVol=0)
else:
r = reagents.getsample(x['name'])
if r.conc.stock != x['conc']:
logging.error('Input %s has conflicting concentrations set: %f and %f', x['name'], r.conc.stock,
x['conc'])
assert False
self.q = None # Defined in pgm()
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 mix(self, inp, weights, tgtdil=1.0, maxusefrac=0.75, prefix="Mix"):
"""Mix given inputs according to weights (by moles -- use conc.stock of each input)"""
print("Mix: tgtdil=%.2f, inp=" % tgtdil, ",".join(
["%s@%.2f" % (inp[i].name, weights[i]) for i in range(len(inp))]))
relvol = [weights[i] * 1.0 / inp[i].conc.stock for i in range(len(inp))]
stages=mixsplit(vols=relvol,samps=inp,avail=[(i.volume-15.0)*maxusefrac-1.4 for i in inp],minvol=4.0,minmix=100.0,maxmix=100.0,plate=decklayout.SAMPLEPLATE,debug=False,prefix=prefix)
for s in stages:
dest=s[0]
moles=0
for i in range(len(s[1])-1,-1,-1):
src=s[1][i]
vol=s[2][i]
self.e.transfer(vol,src,dest,(True,False))
if src.conc is not None:
moles+=src.conc.stock*vol
newconc=moles/sum(s[2])
print("Adjusting concentration of %s from %s to %.2f nM"%(dest.name,str(dest.conc) if dest.conc is not None else -1,newconc))
dest.conc=Concentration(newconc,newconc,units='nM')
print(s[0].name,'\n =',"\n + ".join(['%5.1fuL %5.2f %s %s'%(s[2][i],s[1][i].conc.stock if s[1][i].conc is not None else 0,s[1][i].conc.units if s[1][i].conc is not None else " ",s[1][i].name) for i in range(len(s[1]))]),"\n-> %5.1ful %5.2f %s %s"%(dest.volume,dest.conc.stock,dest.conc.units,dest.name))
print()
return stages[0][0]
def oldmix(self, inp, weights, tgtdil=1.0):
"""Mix given inputs according to weights (by moles -- use conc.stock of each input)"""
print("Mix: tgtdil=%.2f, inp=" % tgtdil, ",".join(
["%s@%.2f" % (inp[i].name, weights[i]) for i in range(len(inp))]))
mixvol = 100.0
if len(inp) == 1:
if tgtdil > 1.0:
vol = [mixvol / tgtdil]
if vol[0] < 4.0:
vol[0] = 4.0
else:
# Special case, just dilute 10x
vol = [mixvol / 10]
else:
relvol = [weights[i] * 1.0 / inp[i].conc.stock for i in range(len(inp))]
scale = mixvol / sum(relvol)
for i in range(len(inp)):
if relvol[i] * scale > inp[i].volume - 16.4:
scale = (inp[i].volume - 16.4) / relvol[i]
vol = [x * scale for x in relvol]
if min(vol) > 4.0 and tgtdil > 1.0:
scale = max(1.0 / tgtdil, 4.0 / min(vol))
print("Rescale volumes by %.2f to get closer to target dilution of %.2f" % (scale, tgtdil))
vol = [x * scale for x in vol]
print("Mix1: vols=[", ",".join(["%.3f" % v for v in vol]), "]")
if min(vol) < 4.0:
logging.info("Minimum volume into mixing would be only %.2f ul - staging..." % min(vol))
if max(vol) < 4.0:
# All volumes are low, just too much
# Split into 2 stages
sel = list(range(int(len(inp) / 2)))
nsel = list(range(int(len(inp) / 2), len(inp)))
else:
# Choose a splitting threshold
mindilution = 4.0 / min(vol)
thresh = np.median(vol)
while mixvol / sum([v for v in vol if v < thresh]) < mindilution:
thresh = thresh * 0.8
print("Using %.2f as threshold to split mixdown" % thresh)
sel = [i for i in range(len(inp)) if vol[i] < thresh]
nsel = [i for i in range(len(inp)) if vol[i] >= thresh]
print("Mixing ", ",".join([inp[i].name for i in sel]), " with vols [", ",".join(
["%.2f" % vol[i] for i in sel]), "] in separate stage.")
tgtdil = float(np.median([vol[i] for i in nsel]) / sum([vol[i] for i in sel]))
print("tgtdil=%.2f" % tgtdil)
mix1 = self.mix([inp[i] for i in sel], [weights[i] for i in sel], tgtdil)
mix2 = self.mix([inp[i] for i in nsel] + [mix1],
[weights[i] for i in nsel] + [sum([weights[i] for i in sel])])
return mix2
watervol = mixvol - sum(vol)
print("Mixdown: vols=[", ",".join(["%.2f " % v for v in vol]), "], water=", watervol, ", total=", mixvol, " ul")
mixdown = Sample('mixdown', plate=decklayout.SAMPLEPLATE)
if watervol < -0.1:
print("Total mixdown is %.1f ul, more than planned %.0f ul" % (sum(vol), mixvol))
assert False
elif watervol > 0.0:
self.e.transfer(watervol, decklayout.WATER, mixdown, (False, False))
else:
pass
for i in range(len(inp)):
inp[i].conc.final = inp[i].conc.stock * vol[i] / mixvol # Avoid warnings about concentrations not adding up
self.e.transfer(vol[i], inp[i], mixdown, (False, i == len(inp) - 1))
self.e.shakeSamples([mixdown])
mixdown.conc = Concentration(stock=sum([inp[i].conc.stock * vol[i] for i in range(len(inp))]) / mixvol,
final=None, units='nM')
print("Mixdown final concentration = %.1f nM" % mixdown.conc.stock)
return mixdown
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
class IDPrep(TRP):
# Barcode multiple samples
pcreff = 1.98
def __init__(self, inputs):
super(IDPrep, self).__init__()
self.inputs = inputs
self.qconc = 0.020 # Target qPCR concentration in nM
self.qprimers = ["End"]
self.bc1_inputvol = 2 # ul into PCR1
used = []
for inp in inputs:
bc = "%s-%s" % (inp['left'], inp['right'])
if bc in used:
logging.error("Barcode %s is being reused for %s" % (bc, inp['name']))
used.append(bc)
print("used=",used)
self.rsrc = [reagents.add("%s-%s-%s" % (inputs[i]['name'], inputs[i]['left'], inputs[i]['right']),
decklayout.SAMPLEPLATE,
well=inputs[i]['well'] if 'well' in inputs[i] else None,
conc=Concentration(stock=inputs[i]['conc'], units="nM"),
initVol=self.bc1_inputvol, extraVol=0)
for i in range(len(inputs))]
self.q = None # Defined in pgm()
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 idbarcoding(self, rsrc, left, right):
"""Perform barcoding of the given inputs; rsrsc,left,right should all be equal length"""
pcrcycles = [4] # Don't need 2nd PCR since this will go directly into constriction
#pcr1inputconc = 0.05 # PCR1 concentration final in reaction
pcr1inputdil = 10
pcr1vol = 30
pcr1postdil = 100.0 / pcr1vol
pcr2dil = 50
pcr2minvol = 50.0
samps = [s.getsample() for s in rsrc]
print("Inputs:")
for i in range(len(samps)):
print("%2s %-10s %8s-%-8s %.1f%s" % (
samps[i].plate.wellname(samps[i].well), self.inputs[i]['name'], left[i], right[i], samps[i].conc.stock,samps[i].conc.units))
# Compute pcr1inputconc such that lowest concentration input ends up with at least 30ul after dilution
pcr1inputconc=min([s.conc.stock*s.volume/30.0/pcr1inputdil for s in samps])
print("Diluting inputs so PCR1 final template conc = %.0f pM"%(pcr1inputconc*1000))
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
# Run first pass dilution where needed
for i in range(len(samps)):
# Dilute down to desired conc
dil = samps[i].conc.stock / pcr1inputconc / pcr1inputdil
dilvol = samps[i].volume * dil
if dilvol > 100.0:
logging.notice("Dilution of input %s (%.1f ul) by %.2f would require %.1f ul" % (
samps[i].name, samps[i].volume, dil, dilvol))
# Do a first pass dilution into 150ul, then remove enough so second dilution can go into 100ul
dil1 = 100.0 / samps[i].volume
self.diluteInPlace(tgt=[samps[i]], dil=dil1)
print("First pass dilution of %s by %.1f/%.1f (conc now %.3f nM)" % (samps[i].name, dil1, dil, samps[i].conc.stock))
dil /= dil1
# Make sure they are all mixed
self.e.shakeSamples(samps)
# Final dilution
for s in samps:
# Dilute down to desired conc
dil = s.conc.stock / 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>100:
toremove=s.volume-100.0/dil
print("Removing %.1f ul from %s to allow enough room for dilution"%(toremove,s.name))
self.e.dispose(toremove, s)
self.diluteInPlace(tgt=[s], dil=dil)
print("Diluting %s by %.1f" % (s.name, dil))
pcr1 = self.runPCR(src=samps, srcdil=pcr1inputdil, ncycles=pcrcycles[0], vol=pcr1vol,
primers=[[left[i], right[i]] for i in range(len(left))], usertime=0, fastCycling=False,
inPlace=False, master="MKapa", kapa=True)
pcr1finalconc = pcr1inputconc * self.pcreff ** pcrcycles[0]
print("PCR1 output concentration = %.3f 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')
self.q.addSamples(src=pcr1, needDil=pcr1finalconc / self.qconc, primers=self.qprimers, save=True,
nreplicates=1)
if len(pcrcycles) > 1:
# Second PCR with 235p/236p on mixture (use at least 4ul of prior)
pcr2 = self.runPCR(src=pcr1, srcdil=pcr2dil / pcr1postdil, vol=max(pcr2minvol, pcr2dil / pcr1postdil * 4),
ncycles=pcrcycles[1],
primers="End", fastCycling=False, master="MKapa", kapa=True)
pcr2finalconc = min(200, pcr1finalconc / (pcr2dil / pcr1postdil) * self.pcreff ** pcrcycles[1])
print("PCR2 final conc = %.1f nM" % pcr2finalconc)
d2 = min(4.0, 150.0 / max([p.volume for p in pcr2]))
if d2 > 1:
pcr2finalconc /= d2
print("Post-dilute PCR2 by %.1fx to %.3fnM" % (d2, pcr2finalconc))
self.diluteInPlace(tgt=pcr2, dil=d2)
self.e.shakeSamples(pcr2)
for x in pcr2:
x.conc = Concentration(stock=pcr2finalconc, units='nM')
self.q.addSamples(src=pcr2, needDil=pcr2finalconc / self.qconc, primers=self.qprimers, save=True,
nreplicates=2)
res = pcr2
else:
res = pcr1
return res
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()