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, []