def finish(self):
self.e.lihahome()
worklist.userprompt("Process complete. Continue to turn off reagent cooler")
self.e.setreagenttemp(None)
#Sample.printallsamples("At completion")
hasError=False
for s in Sample.getAllOnPlate():
if s.volume<1.0 and s.conc is not None and not s.hasBeads:
print "ERROR: Insufficient volume for ", s," need at least ",1.0-s.volume," ul additional"
#hasError=True
elif s.volume<2.5 and s.conc is not None:
print "WARNING: Low final volume for ", s
elif s.volume>s.plate.maxVolume:
print "ERROR: Excess final volume (",s.volume,") for ",s,", maximum is ",s.plate.maxVolume
hasError=True
if hasError:
print "NO OUTPUT DUE TO ERRORS"
assert(False)
print "Wells used: samples: %d, dilutions: %d, qPCR: %d"%(Sample.numSamplesOnPlate(decklayout.SAMPLEPLATE),Sample.numSamplesOnPlate(decklayout.DILPLATE),Sample.numSamplesOnPlate(decklayout.QPCRPLATE))
# Save worklist to a file
#e.saveworklist("trp1.gwl")
(scriptname,ext)=os.path.splitext(sys.argv[0])
self.e.savegem(scriptname+".gem")
self.e.savesummary(scriptname+".txt")
Sample.savematlab(scriptname+".m")
def addReferences(self,mindil=1,nsteps=6,dstep=4,nreplicates=1,ref=None,primers=None):
'Add all needed references'
#print "addReferences(mindil=",mindil,", nsteps=",nsteps,", dstep=",dstep,", nrep=", nreplicates, ", ref=",ref,")"
# Make sure the ref reagent is loaded
if ref is None:
ref=reagents.getsample("QPCRREF")
if primers is None:
primers=self.allprimers()
dils=[1]
for i in range(nsteps):
needDil=mindil*math.pow(dstep,i)
srcDil=1
src=[ref]
for j in range(len(dils)):
if needDil/dils[j] <= self.MAXDIL:
srcDil=dils[j]
if srcDil==1:
src=[ref]
else:
srcname="%s.D%d"%(ref.name,srcDil)
src=[Sample.lookup(srcname)]
if src[0] is None:
src=[Sample(srcname,decklayout.DILPLATE)]
break
tmp=self.MINDILVOL
self.MINDILVOL=75 # Make sure there's enough for resuing dilutions
self.addSamples(src=src,needDil=needDil/srcDil,primers=primers,nreplicates=nreplicates,save=needDil/srcDil>self.MAXDIL,saveVol=75)
self.MINDILVOL=tmp
dils.append(needDil)
self.addSamples(src=[self.dilutant],needDil=1,primers=primers,nreplicates=nreplicates,save=False)
def finish(self):
self.e.lihahome()
worklist.userprompt("Process complete. Continue to turn off reagent cooler")
self.e.setreagenttemp(None)
#Sample.printallsamples("At completion")
hasError=False
for s in Sample.getAllOnPlate():
if s.volume<1.0 and s.conc is not None and not s.emptied:
logging.error("Insufficient volume for %s: need at least %.1f ul additional"%(s.name,1.0-s.volume),fatal=False)
#hasError=True
elif s.volume<2.5 and s.conc is not None and not s.emptied:
logging.warning("Low final volume for "+ s.name)
elif s.volume>s.plate.maxVolume:
logging.erorr("Excess final volume (%.1f) for %s: maximum is %.1f ul"%(s.volume,s.name,s.plate.maxVolume),fatal=False)
hasError=True
if hasError:
logging.error("NO OUTPUT DUE TO ERRORS")
print "Wells used: samples: %d, dilutions: %d, qPCR: %d"%(Sample.numSamplesOnPlate(decklayout.SAMPLEPLATE),Sample.numSamplesOnPlate(decklayout.DILPLATE),Sample.numSamplesOnPlate(decklayout.QPCRPLATE))
# Save worklist to a file
#e.saveworklist("trp1.gwl")
(scriptname,ext)=os.path.splitext(sys.argv[0])
self.e.savegem(scriptname+".gem")
self.e.savesummary(scriptname+".txt")
Sample.savematlab(scriptname+".m")
def runQPCR(self,src,vol,primers,nreplicates=1,enzName="EvaUSER"):
## QPCR setup
worklist.comment("runQPCR: primers=%s, source=%s"%([p for p in primers],[s.name for s in src]))
[src,vol,nreplicates]=listify([src,vol,nreplicates])
self.e.shakeSamples(src,returnPlate=True)
# Build a list of sets to be run
torun=[]
for repl in range(max(nreplicates)):
for p in primers:
for i in range(len(src)):
if nreplicates[i]<=repl:
continue
if repl==0:
sampname="%s.Q%s"%(src[i].name,p)
else:
sampname="%s.Q%s.%d"%(src[i].name,p,repl+1)
s=Sample(sampname,decklayout.QPCRPLATE)
torun=torun+[(src[i],s,p,vol[i])]
# Add enzyme
e=reagents.getsample(enzName)
v=[a[3]/e.conc.dilutionneeded() for a in torun]
t=[a[1] for a in torun]
self.e.multitransfer(v,e,t)
# Make the target have 'none' concentration so we can multiadd to it again
for s in t:
s.conc=None
# Fill the master mixes
dil={}
for p in primers:
mname="P-%s"%p
if not reagents.isReagent(mname):
reagents.add(name=mname,conc=4,extraVol=30)
mq=reagents.getsample(mname)
t=[a[1] for a in torun if a[2]==p]
v=[a[3]/mq.conc.dilutionneeded() for a in torun if a[2]==p]
assert(v>0)
self.e.multitransfer(v,mq,t,(False,False))
dil[p]=1.0/(1-1/e.conc.dilutionneeded()-1/mq.conc.dilutionneeded())
# Add the samples
self.e.sanitize() # In case we are aligned
for a in torun:
s=a[0]
t=a[1]
p=a[2]
v=a[3]/dil[p]
t.conc=None # Concentration of master mix is irrelevant now
self.e.transfer(v,s,t)
return [a[1] for a in torun]
def 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 getSample(wellx,welly,rack,grid,pos):
plate=Plate.lookup(grid,pos)
if plate is None:
plate=Plate(rack,grid,pos)
wellname="%c%d"%(ord('A')+welly-1,wellx)
well=plate.wellnumber(wellname)
s=Sample.lookupByWell(plate, well)
if s is None:
s=Sample("%s.%d.%d.%d" % (rack, grid, pos, well), plate, well)
if grid==3:
s.volume=20000 # Troughs
else:
s.volume=0
return s
def runT7Setup(self, theo, src, vol, srcdil, tgt=None, rlist=["MT7"]):
[theo, src, tgt, srcdil] = listify([theo, src, tgt, srcdil])
for i in range(len(src)):
if tgt[i] is None:
if theo[i]:
tgt[i] = Sample("%s.T+" % src[i].name,
decklayout.SAMPLEPLATE)
else:
tgt[i] = Sample("%s.T-" % src[i].name,
decklayout.SAMPLEPLATE)
worklist.comment("runT7: source=%s" % [str(s) for s in src])
rvols = [reagents.getsample(x).conc.volneeded(vol) for x in rlist]
rtotal = sum(rvols)
sourcevols = [vol * 1.0 / s for s in srcdil]
if any(theo):
theovols = [
(vol * 1.0 /
reagents.getsample("Theo").conc.dilutionneeded() if t else 0)
for t in theo
]
watervols = [
vol - theovols[i] - sourcevols[i] - rtotal
for i in range(len(src))
]
else:
watervols = [vol - sourcevols[i] - rtotal for i in range(len(src))]
if any([w < -1e-10 for w in watervols]):
print "runT7Setup: Negative amount of water required: ", watervols
assert False
if sum(watervols) > 0.01:
self.e.multitransfer(watervols, decklayout.WATER, tgt)
for ir in range(len(rlist)):
self.e.multitransfer([rvols[ir] for s in tgt],
reagents.getsample(rlist[ir]), tgt)
if any(theo):
self.e.multitransfer(
[tv for tv in theovols if tv > 0.01],
reagents.getsample("Theo"),
[tgt[i] for i in range(len(theovols)) if theovols[i] > 0],
ignoreContents=True)
for i in range(len(src)):
self.e.transfer(sourcevols[i], src[i], tgt[i])
self.e.shakeSamples(tgt, returnPlate=True)
for t in tgt:
t.ingredients['BIND'] = 1e-20 * sum(t.ingredients.values())
return tgt
def runQPCRDIL(self,
src,
vol,
srcdil,
tgt=None,
dilPlate=False,
pipMix=False,
dilutant=decklayout.SSDDIL):
[src, vol, srcdil] = listify([src, vol, srcdil])
vol = [float(v) for v in vol]
if tgt is None:
if dilPlate:
tgt = [
Sample(diluteName(src[i].name, srcdil[i]),
decklayout.DILPLATE) for i in range(len(src))
]
else:
tgt = [
Sample(diluteName(src[i].name, srcdil[i]),
decklayout.SAMPLEPLATE) for i in range(len(src))
]
srcvol = [vol[i] / srcdil[i] for i in range(len(vol))]
watervol = [vol[i] - srcvol[i] for i in range(len(vol))]
if len(watervol) > 4 and sum(watervol) > 800:
print "Could optimize distribution of ", len(
watervol), " moves of ", dilutant.name, ": vol=[", [
"%.1f" % w for w in watervol
], "]"
self.e.multitransfer(watervol, dilutant, tgt, (False, False))
self.e.shakeSamples(src, returnPlate=True)
for i in range(len(src)):
tgt[i].conc = None # Assume dilutant does not have a concentration of its own
# Check if we can align the tips here
if i < len(src) - 3 and tgt[i].well + 1 == tgt[
i + 1].well and tgt[i].well + 2 == tgt[
i +
2].well and tgt[i].well + 3 == tgt[i + 3].well and tgt[
i].well % 4 == 0 and self.e.cleanTips != 15:
#print "Aligning tips"
self.e.sanitize()
self.e.transfer(srcvol[i], src[i], tgt[i],
(not src[i].isMixed(), pipMix))
if tgt[i].conc != None:
tgt[i].conc.final = None # Final conc are meaningless now
return tgt
def runIncubation(self,
src=None,
vol=None,
srcdil=None,
tgt=None,
enzymes=None,
incTemp=37,
incTime=15,
hiTemp=None,
hiTime=0,
inPlace=False):
if len(enzymes) != 1:
print "ERROR: runIncubation only supports a single master mix"
assert False
if inPlace:
if tgt is not None:
print "ERROR: tgt specified for in-place incubation"
assert False
elif tgt is None:
tgt = [
Sample("%s.%s" % (src[i].name, enzymes[0].name),
decklayout.SAMPLEPLATE) for i in range(len(src))
]
if srcdil == None:
# Minimum dilution (no water)
srcdil = 1 / (1 -
sum([1 / e.conc.dilutionneeded() for e in enzymes]))
if vol is None and inPlace:
vol = [s.volume * srcdil for s in src]
[src, tgt, vol, srcdil] = listify([src, tgt, vol, srcdil])
# Adjust source dilution
for i in range(len(src)):
src[i].conc = Concentration(srcdil[i], 1)
if inPlace:
self.runRxInPlace(src, vol, enzymes[0], returnPlate=False)
tgt = src
else:
self.e.stage('User', enzymes, src, tgt, vol, destMix=False)
self.e.shakeSamples(tgt, returnPlate=False)
if hiTemp is None:
worklist.pyrun('PTC\\ptcsetpgm.py INC TEMP@%.0f,%.0f TEMP@25,30' %
(incTemp, incTime * 60))
else:
assert (hiTime > 0)
worklist.pyrun(
'PTC\\ptcsetpgm.py INC TEMP@%.0f,%.0f TEMP@%.0f,%.0f TEMP@25,30'
% (incTemp, incTime * 60, hiTemp, hiTime * 60))
self.e.runpgm("INC",
incTime + hiTime + 2,
False,
max(vol),
hotlidmode="TRACKING",
hotlidtemp=10)
return tgt
def beadSupernatant(self,
src,
tgt=None,
sepTime=None,
residualVolume=10,
plate=None):
if plate is None:
plate = decklayout.SAMPLEPLATE
if tgt is None:
tgt = []
for i in range(len(src)):
tgt.append(Sample("%s.SN" % src[i].name, plate))
[src, tgt] = listify([src, tgt])
if any([s.plate != src[0].plate for s in src]):
print "beadSupernatant: Attempt to magsep on multiple plates at the same time"
assert False
self.e.moveplate(src[0].plate, "Magnet") # Move to magnet
self.sepWait(src, sepTime)
for i in range(len(src)):
self.e.transfer(src[i].amountToRemove(residualVolume), src[i],
tgt[i],
(False, False)) # Transfer elution to new tube
self.e.moveplate(src[0].plate, "Home")
return tgt
def saveSamps(self,
src,
vol,
dil,
tgt=None,
dilutant=None,
plate=None,
mix=(True, False)):
[src, vol, dil] = listify([src, vol, dil])
if plate is None:
plate = decklayout.REAGENTPLATE
if tgt is None:
tgt = [
Sample(diluteName(src[i].name, dil[i]), plate)
for i in range(len(src))
]
if any([d != 1.0 for d in dil]):
if dilutant is None:
dilutant = decklayout.WATER
self.e.multitransfer(
[vol[i] * (dil[i] - 1) for i in range(len(vol))], dilutant,
tgt, (False, False))
self.e.shakeSamples(src, returnPlate=True)
for i in range(len(src)):
self.e.transfer(vol[i], src[i], tgt[i], mix)
tgt[i].conc = Concentration(1.0 / dil[i])
return tgt
def addReferences(self, mindil=1, nsteps=6, dstep=4, nreplicates=1, ref=None, primers=None):
"""Add all needed references"""
# print "addReferences(mindil=",mindil,", nsteps=",nsteps,", dstep=",dstep,", nrep=", nreplicates, ", ref=",ref,")"
# Make sure the ref reagent is loaded
if ref is None:
ref = reagents.getsample("QPCRREF")
if primers is None:
primers = self.allprimers()
dils = [1.0]
for i in range(nsteps):
needDil = mindil * math.pow(dstep, i)
srcDil = 1
src = [ref]
for j in range(len(dils)):
if needDil / dils[j] <= self.MAXDIL:
srcDil = dils[j]
if srcDil == 1:
src = [ref]
else:
srcname = "%s.D%d" % (ref.name, srcDil)
src = [Sample.lookup(srcname)]
if src[0] is None:
src = [Sample(srcname, decklayout.DILPLATE)]
break
tmp = self.MINDILVOL
self.MINDILVOL = 75 # Make sure there's enough for resuing dilutions
self.addSamples(src=src, needDil=needDil / srcDil, primers=primers, nreplicates=nreplicates,
save=needDil / srcDil > self.MAXDIL, saveVol=75)
self.MINDILVOL = tmp
dils.append(needDil)
self.addSamples(src=[self.dilutant], needDil=1, primers=primers, nreplicates=nreplicates, save=False)
def getsample(self):
if self.sample is None:
#print "Creating sample for reagent %s with %.1f ul"%(self.name,self.initVol)
self.sample=Sample(self.name,self.plate,self.preferredWell,self.conc,hasBeads=self.hasBeads,volume=self.initVol)
wellname=self.sample.plate.wellname(self.sample.well)
if self.preferredWell != None and self.preferredWell != wellname:
print "WARNING: %s moved from preferred well %s to %s\n"%(self.name,self.preferredWell,wellname)
return self.sample
def finish(self):
self.e.lihahome()
worklist.userprompt(
"Process complete. Continue to turn off reagent cooler")
self.e.setreagenttemp(None)
#Sample.printallsamples("At completion")
hasError = False
for s in Sample.getAllOnPlate():
if s.volume < 1.0 and s.conc is not None and not s.hasBeads:
print "ERROR: Insufficient volume for ", s, " need at least ", 1.0 - s.volume, " ul additional"
#hasError=True
elif s.volume < 2.5 and s.conc is not None:
print "WARNING: Low final volume for ", s
elif s.volume > s.plate.maxVolume:
print "ERROR: Excess final volume (", s.volume, ") for ", s, ", maximum is ", s.plate.maxVolume
hasError = True
if hasError:
print "NO OUTPUT DUE TO ERRORS"
assert (False)
print "Wells used: samples: %d, dilutions: %d, qPCR: %d" % (
Sample.numSamplesOnPlate(decklayout.SAMPLEPLATE),
Sample.numSamplesOnPlate(decklayout.DILPLATE),
Sample.numSamplesOnPlate(decklayout.QPCRPLATE))
# Save worklist to a file
#e.saveworklist("trp1.gwl")
(scriptname, ext) = os.path.splitext(sys.argv[0])
self.e.savegem(scriptname + ".gem")
self.e.savesummary(scriptname + ".txt")
Sample.savematlab(scriptname + ".m")
def execute(self,ops):
reagents=None
for iteration in range(2):
print "Iteration ",iteration+1
# Need to clear here
trp=TRP()
if iteration==0:
trp.addTemplates(["IN"],200) # Add a template with stock concentration 200nM
else:
reagents=Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume<0:
r.initvolume=-r.volume+10
Sample.clearall()
ops(self)
trp.finish()
def getsample(self):
if self.sample is None:
#print "Creating sample for reagent %s with %.1f ul"%(self.name,self.initVol)
self.sample = Sample(self.name,
self.plate,
self.preferredWell,
self.conc,
hasBeads=self.hasBeads,
volume=self.initVol,
extrainfo=self.extrainfo,
ingredients=self.ingredients)
wellname = self.sample.plate.wellname(self.sample.well)
if self.preferredWell != None and self.preferredWell != wellname:
logging.warning("%s moved from preferred well %s to %s\n" %
(self.name, self.preferredWell, wellname))
return self.sample
def runQPCR(self, src, vol, srcdil, primers=["A", "B"], nreplicates=1):
## QPCR setup
worklist.comment("runQPCR: primers=%s, source=%s" %
([p for p in primers], [s.name for s in src]))
[src, vol, srcdil,
nreplicates] = listify([src, vol, srcdil, nreplicates])
self.e.shakeSamples(src, returnPlate=True)
# Build a list of sets to be run
torun = []
for repl in range(max(nreplicates)):
for p in primers:
for i in range(len(src)):
if nreplicates[i] <= repl:
continue
if repl == 0:
sampname = "%s.Q%s" % (src[i].name, p)
else:
sampname = "%s.Q%s.%d" % (src[i].name, p, repl + 1)
s = Sample(sampname, decklayout.QPCRPLATE)
torun = torun + [(src[i], s, p, vol[i])]
# Fill the master mixes
dil = {}
for p in primers:
mname = "MQ%s" % p
if not reagents.isReagent(mname):
reagents.add(name=mname, conc=15.0 / 9.0, extraVol=30)
mq = reagents.getsample(mname)
t = [a[1] for a in torun if a[2] == p]
v = [a[3] / mq.conc.dilutionneeded() for a in torun if a[2] == p]
self.e.multitransfer(v, mq, t, (False, False))
dil[p] = 1.0 / (1 - 1 / mq.conc.dilutionneeded())
# Add the samples
self.e.sanitize() # In case we are aligned
for a in torun:
s = a[0]
t = a[1]
p = a[2]
v = a[3] / dil[p]
t.conc = None # Concentration of master mix is irrelevant now
self.e.transfer(v, s, t)
return [a[1] for a in torun]
def distribute(self,
src,
dil,
vol,
wells,
tgt=None,
dilutant=None,
plate=decklayout.SAMPLEPLATE):
if tgt is None:
tgt = [
Sample("%s.dist%d" % (src[0].name, j), plate)
for j in range(wells)
]
if dilutant is None:
dilutant = decklayout.WATER
self.e.multitransfer([vol * (dil - 1) for i in range(wells)], dilutant,
tgt)
self.e.multitransfer([vol for i in range(wells)], src[0], tgt)
return tgt
def run(self):
'Run the dilutions and QPCR setup'
# Setup qPCRs
#self.jobq.dump()
self.idler(100000)
self.trp.e.waitpgm(
) # May still need to wait for PTC to complete before able to do final jobs
self.idler(100000)
if self.jobq.len() > 0:
logging.error("Blocked jobs remain on queue:", fatal=False)
self.jobq.dump()
assert False
tgt1 = Sample("Barcoded.Mixdown1", decklayout.EPPENDORFS)
for i in range(1, len(self.dilProds)):
self.e.transfer(12.5 * 1.2 * 2 / 1.5,
self.dilProds[i],
tgt1,
mix=(False, False))
def runRTSetup(self, src, vol, srcdil, tgt=None, rtmaster=None):
if rtmaster is None:
rtmaster = reagents.getsample("MPosRT")
if tgt is None:
tgt = [
Sample(s.name + ".RT+", decklayout.SAMPLEPLATE) for s in src
]
[src, tgt, vol, srcdil] = listify([src, tgt, vol, srcdil])
# Adjust source dilution
for i in range(len(src)):
src[i].conc = Concentration(srcdil[i], 1)
self.e.stage('RTPos', [rtmaster], [src[i] for i in range(len(src))],
[tgt[i] for i in range(len(tgt))],
[vol[i] for i in range(len(vol))],
destMix=False)
#self.e.shakeSamples(tgt,returnPlate=True)
return tgt
def 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 printallsamples(fd):
Sample.printallsamples(fd=fd)
def logspeed(self,platename,speed):
logging.notice("logspeed(%s,%d)"%(platename,speed))
Sample.addallhistory("(S@%d)"%speed,onlyplate=platename)
def beadWash(self,
src,
washTgt=None,
sepTime=None,
residualVolume=10,
keepWash=False,
numWashes=2,
wash=None,
washVol=50,
keepFinal=False,
finalTgt=None,
keepVol=4.2,
keepDil=5):
# Perform washes
# If keepWash is true, retain all washes (combined)
# If keepFinal is true, take a sample of the final wash (diluted by keepDil)
if wash is None:
wash = decklayout.WATER
[src, wash] = listify([src, wash])
# Do all washes while on magnet
assert (len(set([s.plate for s in src])) == 1) # All on same plate
if keepWash:
if washTgt is None:
washTgt = []
for i in range(len(src)):
if s[i].volume - residualVolume + numWashes * (
washVol - residualVolume
) > decklayout.DILPLATE.maxVolume - 20:
print "Saving %.1f ul of wash in eppendorfs" % (
numWashes * washVol)
washTgt.append(
Sample("%s.Wash" % src[i].name,
decklayout.EPPENDORFS))
else:
washTgt.append(
Sample("%s.Wash" % src[i].name,
decklayout.DILPLATE))
if keepFinal:
if finalTgt is None:
finalTgt = []
for i in range(len(src)):
finalTgt.append(
Sample("%s.Final" % src[i].name, decklayout.DILPLATE))
if any([s.volume > residualVolume for s in src]):
# Separate and remove supernatant
self.e.moveplate(src[0].plate, "Magnet") # Move to magnet
self.sepWait(src, sepTime)
# Remove the supernatant
for i in range(len(src)):
if src[i].volume > residualVolume:
amt = src[i].amountToRemove(residualVolume)
if keepWash:
self.e.transfer(amt, src[i],
washTgt[i]) # Keep supernatants
washTgt[i].conc = None # Allow it to be reused
else:
self.e.dispose(amt, src[i]) # Discard supernatant
# Wash
for washnum in range(numWashes):
self.e.moveplate(src[0].plate, "Home")
if keepFinal and washnum == numWashes - 1:
'Retain sample of final'
for i in range(len(src)):
src[i].conc = None
self.e.transfer(washVol - src[i].volume,
wash[i],
src[i],
mix=(False, True)) # Add wash
self.e.shake(src[0].plate, returnPlate=True)
self.saveSamps(src=src,
tgt=finalTgt,
vol=keepVol,
dil=keepDil,
plate=decklayout.DILPLATE)
else:
for i in range(len(src)):
src[i].conc = None
self.e.transfer(
washVol - src[i].volume,
wash[i],
src[i],
mix=(False, False)
) # Add wash, no need to pipette mix since some heterogenity won't hurt here
self.e.shake(src[0].plate, returnPlate=False)
self.e.moveplate(src[0].plate, "Magnet") # Move to magnet
self.sepWait(src, sepTime)
for i in range(len(src)):
amt = src[i].amountToRemove(residualVolume)
if keepWash:
self.e.transfer(amt, src[i], washTgt[i]) # Remove wash
washTgt[i].conc = None # Allow it to be reused
else:
self.e.dispose(amt, src[i]) # Remove wash
self.e.moveplate(src[0].plate, "Home")
# Should only be residualVolume left with beads now
result = []
if keepWash:
result = result + washTgt
if keepFinal:
result = result + finalTgt
return result
from Experiment.experiment import Experiment
import math
from TRPLib.trp import TRP
reagents=None
input="BT409"
srcprefix="B"
prodprefix="A"
for iteration in range(2):
print "Iteration ",iteration+1
trp=TRP()
if iteration==0:
trp.addTemplates([input],200) # Add a template with stock concentration 200nM
else:
reagents=Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume<0:
r.initvolume=-r.volume+20
Sample.clearall()
# Round 1 (Keep uncleaved +theo)
t71=trp.runT7(theo=[False,True],src=[input,input],tgt=[],vol=[16,18],srcdil=4)
sv1t7=trp.saveSamps(src=t71,tgt=[],vol=10,dil=[4,4])
rt1=trp.runRT(pos=[True,True],src=t71,tgt=[],vol=[15,22],srcdil=2)
trp.diluteInPlace(tgt=rt1,dil=2)
sv1rt=trp.saveSamps(src=rt1,tgt=[],vol=15,dil=2)
pcr1=trp.runPCR(prefix=[srcprefix],src=rt1[1],tgt=[],vol=50,srcdil=4)
trp.diluteInPlace(tgt=pcr1,dil=3)
sv1pcr=trp.saveSamps(src=pcr1,tgt=["R1"],vol=125,dil=1)
def runPCR(self,
prefix,
src,
vol,
srcdil,
tgt=None,
ncycles=20,
suffix='S',
sepPrimers=True,
primerDil=4):
## PCR
[prefix, src, tgt, vol, srcdil,
suffix] = listify([prefix, src, tgt, vol, srcdil, suffix])
for i in range(len(tgt)):
if tgt[i] is None:
tgt[i] = Sample(
"%s.P%s%s" % (src[i].name, prefix[i], suffix[i]),
src[i].plate)
# Adjust source dilution
for i in range(len(src)):
src[i].conc = Concentration(srcdil[i], 1)
if sepPrimers:
sampvols = [vol[i] / srcdil[i] for i in range(len(src))]
mm = reagents.getsample("MPCR")
mmvols = [
vol[i] / mm.conc.dilutionneeded() for i in range(len(src))
]
for s in prefix + suffix:
if not reagents.isReagent(s):
reagents.add(name=s, conc=primerDil, extraVol=30)
sprefix = [reagents.getsample(p) for p in prefix]
ssuffix = [reagents.getsample(p) for p in suffix]
prefixvols = [
vol[i] / sprefix[i].conc.dilutionneeded()
for i in range(len(src))
]
suffixvols = [
vol[i] / ssuffix[i].conc.dilutionneeded()
for i in range(len(src))
]
watervols = [
vol[i] - mmvols[i] - prefixvols[i] - suffixvols[i] -
sampvols[i] for i in range(len(src))
]
print "water=", watervols, ", mm=", mmvols, ", prefix=", prefixvols, ", suffix=", suffixvols, ", samp=", sampvols
self.e.multitransfer(watervols, decklayout.WATER, tgt,
(False, False)) # Transfer water
self.e.multitransfer(mmvols, mm, tgt,
(False, False)) # PCR master mix
sprefixset = set(sprefix)
ssuffixset = set(ssuffix)
if len(sprefixset) < len(ssuffixset):
# Distribute sprefix first
for p in sprefixset:
self.e.multitransfer([
prefixvols[i]
for i in range(len(src)) if sprefix[i] == p
], p, [tgt[i] for i in range(len(src)) if sprefix[i] == p],
(False, False))
# Then individually add ssuffix
for i in range(len(src)):
self.e.transfer(suffixvols[i], ssuffix[i], tgt[i],
(False, False))
else:
# Distribute ssuffix first
for p in ssuffixset:
self.e.multitransfer([
suffixvols[i]
for i in range(len(src)) if ssuffix[i] == p
], p, [tgt[i] for i in range(len(src)) if ssuffix[i] == p],
(False, False))
# Then individually add sprefix
for i in range(len(src)):
self.e.transfer(prefixvols[i], sprefix[i], tgt[i],
(False, False))
# Now add templates
for i in range(len(src)):
self.e.transfer(sampvols[i], src[i], tgt[i], (False, False))
else:
primer = [prefix[i] + suffix[i] for i in range(len(prefix))]
print "primer=", primer
for up in set(primer):
s = "MPCR%s" % up
if not reagents.isReagent(s):
reagents.add(name=s, conc=4 / 3.0, extraVol=30)
self.e.stage(
'PCR%s' % up, [reagents.getsample("MPCR%s" % up)],
[src[i] for i in range(len(src)) if primer[i] == up],
[tgt[i] for i in range(len(tgt)) if primer[i] == up],
[vol[i] for i in range(len(vol)) if primer[i] == up],
destMix=False)
pgm = "PCR%d" % ncycles
self.e.shakeSamples(tgt, returnPlate=False)
# worklist.pyrun('PTC\\ptcsetpgm.py %s TEMP@95,120 TEMP@95,30 TEMP@55,30 TEMP@72,25 GOTO@2,%d TEMP@72,180 TEMP@16,2'%(pgm,ncycles-1))
worklist.pyrun(
'PTC\\ptcsetpgm.py %s TEMP@95,120 TEMP@95,10 TEMP@57,10 GOTO@2,%d TEMP@72,120 TEMP@25,2'
% (pgm, ncycles - 1))
self.e.runpgm(pgm,
4.80 + 1.55 * ncycles,
False,
max(vol),
hotlidmode="CONSTANT",
hotlidtemp=100)
return tgt
tmplqpcr=tmplqpcr+[srcprefix[i]+srcsuffix[i]]
ligmaster1=ligmaster1+["MLig"+ligprefix1[i]+stem1[i]]
pcr1=pcr1+[(srcprefix[i]+srcsuffix[i],ligprefix1[i]+srcsuffix[i])]
ligmaster2=ligmaster2+["MLig"+ligprefix2[i]+stem1[i]]
pcr2=pcr2+[(srcprefix[i]+srcsuffix[i],ligprefix2[i]+srcsuffix[i])]
print "srcs=",srcs
print "tmplqpcr=",tmplqpcr
print "ligmaster1=",ligmaster1
print "pcr1=",pcr1
print "ligmaster2=",ligmaster2
print "pcr2=",pcr2
# Create ligation master mix samples
for lm in set(ligmaster1+ligmaster2):
if Sample.lookup(lm)==None:
Sample(lm,Experiment.REAGENTPLATE,None,3)
for st in set(stop):
if Sample.lookup(st)==None:
Sample(st,Experiment.REAGENTPLATE,None,2)
for p in pcr1+pcr2:
for pm in p:
if Sample.lookup("MQ"+pm)==None:
Sample("MQ"+pm,Experiment.REAGENTPLATE,None,10.0/6)
for pm in tmplqpcr:
if Sample.lookup("MQ"+pm)==None:
Sample("MQ"+pm,Experiment.REAGENTPLATE,None,10.0/6)
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 addSamples(self,
src,
needDil,
primers,
nreplicates=1,
names=None,
saveVol=None,
saveDil=None,
save=True):
'Add sample(s) to list of qPCRs to do'
#print "addSamples(%s)"%src
if not isinstance(src, list):
src = [src]
if save:
# saveVol is total amount (after dilution) to be immediately saved
if saveDil is None:
saveDil = min(needDil, self.MAXDIL)
if needDil / saveDil > 1 and needDil / saveDil < 2:
saveDil = math.sqrt(needDil)
elif saveDil > needDil:
logging.warning("addSamples: saveDil=", saveDil,
", but needDil is only ", needDil)
saveDil = needDil
if saveVol is None:
saveVol = max(self.MINDILVOL * 1.0 / saveDil, self.TGTINVOL)
if names is None:
tgt = [
Sample(diluteName(src[i].name, saveDil),
decklayout.DILPLATE) for i in range(len(src))
]
else:
tgt = [
Sample(diluteName(names[i], saveDil), decklayout.DILPLATE)
for i in range(len(src))
]
sv = tgt
for i in range(len(sv)):
#print "Save ",src[i]
svtmp = self.trp.runQPCRDIL(src=[src[i]],
vol=saveVol * saveDil,
srcdil=saveDil,
tgt=[tgt[i]],
dilPlate=True,
dilutant=self.dilutant)
sv[i] = svtmp[0]
else:
saveDil = 1
sv = src
needDil = needDil / saveDil
nstages = int(math.ceil(math.log(needDil) / math.log(self.MAXDIL)))
ndil = len(src) * (nstages + (1 if save else 0))
logging.notice(
"QPCR: %dQ/%dD [%s], dilution:%.1fx, primers: [%s]" %
(len(src) * len(primers) * nreplicates, ndil,
",".join([s.name
for s in src]) if names is None else ",".join(names),
needDil, ",".join(primers)))
for svi in range(len(sv)):
s = sv[svi]
if s.hasBeads:
prereqs = []
else:
j0 = self.jobq.addShake(sample=s, prereqs=[])
prereqs = [j0]
intermed = s
for i in range(nstages):
dil = math.pow(needDil, 1.0 / nstages)
#print "stage ",i,", needDil=",needDil,", dil=",dil
if i > 0:
vol = self.MAXDILVOL
else:
vol = min(self.MAXDILVOL,
max(self.MINDILVOL, dil * self.TGTINVOL))
if intermed.plate == decklayout.DILPLATE:
firstWell = intermed.well + 4 # Skip by 4 wells at a time to optimize multi-tip movements
else:
firstWell = 0
if not save and i == 0 and names is not None:
# Need to replace the name in this condition
dest = Sample(diluteName(names[svi], dil),
decklayout.DILPLATE,
firstWell=firstWell)
else:
dest = Sample(diluteName(intermed.name, dil),
decklayout.DILPLATE,
firstWell=firstWell)
#print "dest=",dest
j1 = self.jobq.addMultiTransfer(volume=vol * (dil - 1) / dil,
src=self.dilutant,
dest=dest,
prereqs=[])
prereqs.append(j1)
j2 = self.jobq.addTransfer(volume=vol / dil,
src=intermed,
dest=dest,
prereqs=prereqs)
#print "Dilution of %s was %.2f instead of %.2f (error=%.0f%%)"%(dest.name,(dil/(1+dil))/(1/dil),dil,((dil/(1+dil))/(1/dil)/dil-1)*100)
if dest.hasBeads:
prereqs = [j2]
else:
j3 = self.jobq.addShake(sample=dest, prereqs=[j2])
prereqs = [j3]
intermed = dest
self.dilProds = self.dilProds + [intermed]
self.primers = self.primers + [primers]
self.nreplicates = self.nreplicates + [nreplicates]
def 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 runLig(self,
prefix=None,
src=None,
vol=None,
srcdil=None,
tgt=None,
master=None,
anneal=True,
ligtemp=25):
if master is None:
master = [
reagents.getsample("MLigAN7")
if p == 'A' else reagents.getsample("MLigBN7") for p in prefix
]
#Extension
[src, tgt, vol, srcdil,
master] = listify([src, tgt, vol, srcdil, master])
if tgt is None:
tgt = [
Sample("%s.%s" % (src[i].name, master[i].name),
decklayout.SAMPLEPLATE) for i in range(len(src))
]
# Need to check since an unused ligation master mix will not have a concentration
minsrcdil = 1 / (1 - 1 / master[0].conc.dilutionneeded() - 1 /
reagents.getsample("MLigase").conc.dilutionneeded())
for i in srcdil:
if i < minsrcdil:
print "runLig: srcdil=%.2f, but must be at least %.2f based on concentrations of master mixes" % (
i, minsrcdil)
assert (False)
# Adjust source dilution
for i in range(len(src)):
src[i].conc = Concentration(srcdil[i], 1)
i = 0
while i < len(tgt):
lasti = i + 1
while lasti < len(tgt) and master[i] == master[lasti]:
lasti = lasti + 1
self.e.stage('LigAnneal', [master[i]],
src[i:lasti],
tgt[i:lasti], [vol[j] / 1.5 for j in range(i, lasti)],
1.5,
destMix=False)
i = lasti
if anneal:
self.e.shakeSamples(tgt, returnPlate=False)
self.e.runpgm("TRPANN",
5,
False,
max(vol),
hotlidmode="CONSTANT",
hotlidtemp=100)
self.e.stage('Ligation', [reagents.getsample("MLigase")], [],
tgt,
vol,
destMix=False)
self.e.shakeSamples(tgt, returnPlate=False)
self.runLigPgm(max(vol), ligtemp)
return tgt
import math
from TRPLib.trp import TRP
reagents = None
input = "BT409"
srcprefix = "B"
prodprefix = "A"
for iteration in range(2):
print "Iteration ", iteration + 1
trp = TRP()
if iteration == 0:
trp.addTemplates([input],
200) # Add a template with stock concentration 200nM
else:
reagents = Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume < 0:
r.initvolume = -r.volume + 20
Sample.clearall()
# Round 1 (Keep uncleaved +theo)
t71 = trp.runT7(theo=[False, True],
src=[input, input],
tgt=[],
vol=[16, 18],
srcdil=4)
sv1t7 = trp.saveSamps(src=t71, tgt=[], vol=10, dil=[4, 4])
rt1 = trp.runRT(pos=[True, True], src=t71, tgt=[], vol=[15, 22], srcdil=2)
trp.diluteInPlace(tgt=rt1, dil=2)
sv1rt = trp.saveSamps(src=rt1, tgt=[], vol=15, dil=2)
srcs = srcs + [input[i]]
tmplqpcr = tmplqpcr + [srcprefix[i] + srcsuffix[i]]
ligmaster = ligmaster + ["MLig" + ligprefix[i] + stem1[i]]
pcr = pcr + [
(srcprefix[i] + srcsuffix[i], ligprefix[i] + srcsuffix[i])
]
stop = stop + ["MStp" + srcsuffix[i]]
#print srcs
#print tmplqpcr
#print ligmaster
#print pcr
# Create ligation master mix samples
for lm in set(ligmaster):
if Sample.lookup(lm) == None:
Sample(lm, Experiment.REAGENTPLATE, None, 3)
for st in set(stop):
if Sample.lookup(st) == None:
Sample(st, Experiment.REAGENTPLATE, None, 2)
for p in pcr:
for pm in p:
if Sample.lookup("MQ" + pm) == None:
Sample("MQ" + pm, Experiment.REAGENTPLATE, None, 10.0 / 6)
for pm in tmplqpcr:
if Sample.lookup("MQ" + pm) == None:
Sample("MQ" + pm, Experiment.REAGENTPLATE, None, 10.0 / 6)
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 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, []
