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