def addTemplates(self,names,stockconc,finalconc=None,units="nM",plate=decklayout.EPPENDORFS,looplengths=None,extraVol=30,wellnames=None,initVol=0): 'Add templates as "reagents", return the list of them' if finalconc is None: logging.warning("final concentration of template not specified, assuming 0.6x (should add to addTemplates() call") [names,stockconc]=listify([names,stockconc]) finalconc=[0.6*x for x in stockconc] else: [names,stockconc,finalconc]=listify([names,stockconc,finalconc]) if len(set(names))!=len(names): logging.error("addTemplates: template names must be unique") r=[] if looplengths is not None: assert(len(names)==len(looplengths)) for i in range(len(names)): if wellnames is None: well=None else: well=wellnames[i] if reagents.isReagent(names[i]): r.append(reagents.lookup(names[i])) elif looplengths is None: r.append(reagents.add(names[i],plate=plate,conc=Concentration(stockconc[i],finalconc[i],units),extraVol=extraVol,well=well,initVol=initVol)) else: r.append(reagents.add(names[i],plate=plate,conc=Concentration(stockconc[i],finalconc[i],units),extraVol=extraVol,extrainfo=looplengths[i],well=well,initVol=initVol)) return r
def __init__(self, inputs, pcr1inputconc=0.05, used=None,doqpcr=True,inputPlate=decklayout.SAMPLEPLATE): super(Barcoding, self).__init__() if used is None: used = [] self.inputs = inputs self.qconc = 50e-12 # Target qPCR concentration self.qprimers = ["End"] self.doqpcr=doqpcr self.bc1_inputvol = 4 # ul of input samples self.mix_conc = 100e-9 # Concentration of mixdown self.pcr1inputconc = pcr1inputconc for inp in inputs: bc = "%s-%s" % (inp['left'], inp['right']) if bc in used: logging.error("Barcode %s is being reused for %s" % (bc, inp['name'])) used.append(bc) for x in inputs: if not reagents.isReagent(x['name']): reagents.add(x['name'], inputPlate, well=x['well'] if 'well' in x else None, conc=Concentration(stock=x['conc'], units="nM"), initVol=self.bc1_inputvol, extraVol=0) else: r = reagents.getsample(x['name']) if r.conc.stock != x['conc']: logging.error('Input %s has conflicting concentrations set: %f and %f', x['name'], r.conc.stock, x['conc']) assert False self.q = None # Defined in pgm()
def 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 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 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
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 idbarcoding(self, rsrc, left, right): """Perform barcoding of the given inputs; rsrsc,left,right should all be equal length""" pcrcycles = [4] # Don't need 2nd PCR since this will go directly into constriction #pcr1inputconc = 0.05 # PCR1 concentration final in reaction pcr1inputdil = 10 pcr1vol = 30 pcr1postdil = 100.0 / pcr1vol pcr2dil = 50 pcr2minvol = 50.0 samps = [s.getsample() for s in rsrc] print("Inputs:") for i in range(len(samps)): print("%2s %-10s %8s-%-8s %.1f%s" % ( samps[i].plate.wellname(samps[i].well), self.inputs[i]['name'], left[i], right[i], samps[i].conc.stock,samps[i].conc.units)) # Compute pcr1inputconc such that lowest concentration input ends up with at least 30ul after dilution pcr1inputconc=min([s.conc.stock*s.volume/30.0/pcr1inputdil for s in samps]) print("Diluting inputs so PCR1 final template conc = %.0f pM"%(pcr1inputconc*1000)) wellnum = 5 for s in left + right: primer = "P-" + s if not reagents.isReagent(primer): reagents.add(primer, conc=Concentration(2.67, 0.4, 'uM'), extraVol=30, plate=decklayout.REAGENTPLATE, well=decklayout.REAGENTPLATE.wellname(wellnum)) wellnum += 1 # Run first pass dilution where needed for i in range(len(samps)): # Dilute down to desired conc dil = samps[i].conc.stock / pcr1inputconc / pcr1inputdil dilvol = samps[i].volume * dil if dilvol > 100.0: logging.notice("Dilution of input %s (%.1f ul) by %.2f would require %.1f ul" % ( samps[i].name, samps[i].volume, dil, dilvol)) # Do a first pass dilution into 150ul, then remove enough so second dilution can go into 100ul dil1 = 100.0 / samps[i].volume self.diluteInPlace(tgt=[samps[i]], dil=dil1) print("First pass dilution of %s by %.1f/%.1f (conc now %.3f nM)" % (samps[i].name, dil1, dil, samps[i].conc.stock)) dil /= dil1 # Make sure they are all mixed self.e.shakeSamples(samps) # Final dilution for s in samps: # Dilute down to desired conc dil = s.conc.stock / pcr1inputconc / pcr1inputdil if dil < 1.0: logging.error("Input %s requires dilution of %.2f" % (s.name, dil)) elif dil > 1.0: dilvol = s.volume * dil if dilvol>100: toremove=s.volume-100.0/dil print("Removing %.1f ul from %s to allow enough room for dilution"%(toremove,s.name)) self.e.dispose(toremove, s) self.diluteInPlace(tgt=[s], dil=dil) print("Diluting %s by %.1f" % (s.name, dil)) pcr1 = self.runPCR(src=samps, srcdil=pcr1inputdil, ncycles=pcrcycles[0], vol=pcr1vol, primers=[[left[i], right[i]] for i in range(len(left))], usertime=0, fastCycling=False, inPlace=False, master="MKapa", kapa=True) pcr1finalconc = pcr1inputconc * self.pcreff ** pcrcycles[0] print("PCR1 output concentration = %.3f nM" % pcr1finalconc) if pcr1postdil > 1: pcr1finalconc /= pcr1postdil print("Post dilute PCR1 by %.2fx to %.3f nM " % (pcr1postdil, pcr1finalconc)) self.diluteInPlace(tgt=pcr1, dil=pcr1postdil) for x in pcr1: x.conc = Concentration(stock=pcr1finalconc, units='nM') self.q.addSamples(src=pcr1, needDil=pcr1finalconc / self.qconc, primers=self.qprimers, save=True, nreplicates=1) if len(pcrcycles) > 1: # Second PCR with 235p/236p on mixture (use at least 4ul of prior) pcr2 = self.runPCR(src=pcr1, srcdil=pcr2dil / pcr1postdil, vol=max(pcr2minvol, pcr2dil / pcr1postdil * 4), ncycles=pcrcycles[1], primers="End", fastCycling=False, master="MKapa", kapa=True) pcr2finalconc = min(200, pcr1finalconc / (pcr2dil / pcr1postdil) * self.pcreff ** pcrcycles[1]) print("PCR2 final conc = %.1f nM" % pcr2finalconc) d2 = min(4.0, 150.0 / max([p.volume for p in pcr2])) if d2 > 1: pcr2finalconc /= d2 print("Post-dilute PCR2 by %.1fx to %.3fnM" % (d2, pcr2finalconc)) self.diluteInPlace(tgt=pcr2, dil=d2) self.e.shakeSamples(pcr2) for x in pcr2: x.conc = Concentration(stock=pcr2finalconc, units='nM') self.q.addSamples(src=pcr2, needDil=pcr2finalconc / self.qconc, primers=self.qprimers, save=True, nreplicates=2) res = pcr2 else: res = pcr1 return res
def 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()
def setVolumes(self): # Computed parameters # Observed data: 0.1nM@30min -> gain ~1000 self.rnaConc=8314.0*self.tmplFinalConc/(self.tmplFinalConc+55)*self.t7dur/30 if self.tmplFinalConc<1: self.rnaConc*=6 # Kludge based on being off at 0.1nM template concentrations self.rnaConc=max(40.0,self.rnaConc) # Another kludge if isinstance(self.stopConc,list): minStopConc=min(self.stopConc) else: minStopConc=self.stopConc maxConc=1000*minStopConc*4/0.9 if maxConc<self.rnaConc: logging.warning( "Stop@%.1f uM limits usable RNA to %.0f/%.0f nM"%(minStopConc,maxConc,self.rnaConc)) self.rnaConc=min(maxConc,self.rnaConc) stopConc=self.rnaConc*0.9 rtConc=stopConc/self.rtDil rtdilConc=[rtConc/self.rtpostdil[i] for i in range(len(self.rounds))] ligConc=[None if self.rounds[i]=='U' else rtdilConc[i]/1.25 for i in range(len(self.rounds))] ligdilConc=[None if self.rounds[i]=='U' else ligConc[i]/self.extpostdil[i] for i in range(len(self.rounds))] pcrConc=[rtConc/self.pcrdil[i] if self.rounds[i]=='U' else ligConc[i]/self.pcrdil[i] for i in range(len(self.rounds))] for i in range(len(self.rounds)): if ligConc[i] is None: print("R%d Concs(nM): RNA: %.0f, Stop: %.0f, RT: %.0f, RTDil: %.0f, PCRIn: %.0f"%(i, self.rnaConc, stopConc, rtConc, rtdilConc[i], pcrConc[i])) else: print("R%d Concs(nM): RNA: %.0f, Stop: %.0f, RT: %.0f, RTDil: %.0f, Lig: %.0f, LigDil: %.0f, PCRIn: %.0f"%(i, self.rnaConc, stopConc, rtConc, rtdilConc[i], ligConc[i], ligdilConc[i], pcrConc[i])) self.pcrvol=[self.pcrcopies*self.pmolesIn*1000/pcrConc[i]/math.pow(self.enrich,(i+1.0)) for i in range(len(self.rounds))] # Use at least 100ul so the evaporation of the saved sample that occurs during the run will be relatively small self.pcrvol=[max(100,v) for v in self.pcrvol] print("pcrvol=[%s]"%(",".join(["%.1f"%v for v in self.pcrvol]))) pcrExtra=[1.4*math.ceil(v*1.0/self.maxPCRVolume) for v in self.pcrvol] print("pcrExtra=[%s]"%(",".join(["%.1f"%v for v in pcrExtra]))) self.minligvol=[(self.pcrvol[i]*1.0+pcrExtra[i])/self.pcrdil[i]+((4.4 if self.saveDil is not None else 5.4 if 'ext' in self.qpcrStages else 0)+15.1)/self.extpostdil[i] for i in range(len(self.pcrvol))] print("minligvol=[%s]"%(",".join(["%.1f"%v for v in self.minligvol]))) # Compute RT volume self.rtvol=[ ((self.minligvol[i]/1.25+3.3)/self.rtpostdil[i]) if self.rounds[i]!='U' else (self.pcrvol[i]*1.0/self.pcrdil[i]+(pcrExtra[i]+15.1+3.3)/self.rtpostdil[i]) for i in range(len(self.rounds))] print("self.rtvol=",self.rtvol,", rtSave=",self.rtSave) if self.rtSave: self.rtvol=[max(15.0/self.rtpostdil[i],self.rtvol[i])+(self.rtSaveVol+1.4)/self.rtpostdil[i] for i in range(len(self.rtvol))] # Extra for saves elif "rt" in self.qpcrStages: # Take from save if rtSave is set self.rtvol=[max(15.0/self.rtpostdil[i],self.rtvol[i])+5.4/self.rtpostdil[i] for i in range(len(self.rtvol))] # Extra for qPCR self.rtvol=[max(v,8.0) for v in self.rtvol] # Minimum volume self.rtvol=[min(v,self.maxSampVolume) for v in self.rtvol] # Maximum volume # print ("self.rtvol=",self.rtvol) self.t7extravols=((4+1.4) if 'stopped' in self.qpcrStages else 0)+ ((self.saveRNAVolume+1.4) if self.saveRNA else 0) # + 3.3 # 3.3 in case pipette mixing used #print "self.t7extravols=%.1f ul\n"%self.t7extravols #print "self.rtvol=%s ul\n"%(",".join(["%.1f "%x for x in self.rtvol])) # Compute dilutions due to tref additions trefdil=[1 for _ in self.rnddef ] for i in range(len(self.rnddef)-1): rd=self.rnddef[i] if 'tref' in rd: tref = rd['tref'][0] if tref is not None: trefname = 'TRef%d' % tref if not reagents.isReagent(trefname): reagents.add(name=trefname, conc=10, extraVol=30, well="E4") trefs = reagents.getsample(trefname) trefdil[i+1]=(trefs.conc.dilutionneeded())/(trefs.conc.dilutionneeded()-1) print("Extra dilution due to tref additions: ",trefdil) self.t7vol=[max((15.1+self.rtvol[i]/4.0+1.4)+self.t7extravols,self.pmolesIn*1000.0/(self.tmplFinalConc if i==0 else 200*self.templateDilution)*trefdil[i]/math.pow(self.enrich,i*1.0)) for i in range(len(self.rounds))] print("self.t7vol=%s ul\n"%(",".join(["%.1f "%x for x in self.t7vol]))) #self.t7vol=[max(18.0,v) for v in self.t7vol] # Make sure that there's enough to add at least 2ul of stop if 'template' in self.qpcrStages: self.t7vol[0]+=5.4 self.t7vol=[min(self.maxSampVolume,v) for v in self.t7vol] # Make sure no tubes overflow
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