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 mix(self, inp, weights,mixvol=100,tgtconc=None,maxinpvol=20):
"""Mix given inputs according to weights (by moles -- use conc.stock of each input)"""
vol = [weights[i] *1.0 / inp[i].conc.stock for i in range(len(inp))]
scale = mixvol / sum(vol)
conc=sum([inp[i].conc.stock * scale * vol[i] for i in range(len(inp))]) / mixvol
if tgtconc is not None and conc>tgtconc:
scale*=tgtconc*1.0/conc
if max(vol)*scale<4.0:
scale=4.1/max(vol) # At least one input with 4ul input
vol = [x * scale for x in vol] # Mix to make planned total without water
for i in range(len(vol)):
# Check if this would require more than available of any input
newscale= min(maxinpvol,inp[i].volume-inp[i].plate.unusableVolume()-2)/vol[i]
if newscale<1:
vol = [x * 1.0 * newscale for x in vol]
if tgtconc is not None:
mixvol *= newscale # Maintain same target concentration by reducing total volume
if min(vol) < 4.0:
# Some components are too small; split mixing
lowvol=[i for i in range(len(inp)) if vol[i]<4.0]
highvol=[i for i in range(len(inp)) if i not in lowvol]
assert len(highvol)>0
assert len(lowvol)>0
lowtgtconc=sum([inp[i].conc.stock *1.0/ weights[i] for i in highvol])/len(highvol)*sum([weights[i] for i in lowvol])
print("Running premix of samples "+",".join(["%d"%ind for ind in lowvol])+" with target concentration of %.4f"%lowtgtconc)
mix1=self.mix([inp[i] for i in lowvol],[weights[i] for i in lowvol],tgtconc=lowtgtconc,mixvol=mixvol,maxinpvol=maxinpvol)
wt1=sum([weights[i] for i in lowvol])
mix2=self.mix([inp[i] for i in highvol]+[mix1],[weights[i] for i in highvol]+[wt1],tgtconc=tgtconc,mixvol=mixvol,maxinpvol=maxinpvol)
return mix2
print("Mixing into %.0ful with tgtconc of %s, dil=%.2f"%(mixvol,"None" if tgtconc is None else "%.4f"%tgtconc,mixvol/sum(vol)))
for i in range(len(inp)):
print("%-30.30s %6.3fnM wt=%5.2f v=%5.2ful"%(inp[i].name,inp[i].conc.stock,weights[i],vol[i]))
watervol = mixvol - sum(vol)
#print "Mixdown: vols=[", ",".join(["%.2f " % v for v in vol]), "], water=", watervol, ", total=", mixvol, " ul"
mixdown = Sample('mixdown', plate=decklayout.SAMPLEPLATE)
if watervol < -0.1:
print("Total mixdown is %.1f ul, more than planned %.0f ul" % (sum(vol), mixvol))
assert False
elif watervol >= 4.0: # Omit if too small
self.e.transfer(watervol, decklayout.WATER, mixdown, (False, False))
else:
pass
ordering=sorted(list(range(len(inp))),key=lambda i: vol[i],reverse=True)
for i in ordering:
inp[i].conc.final = inp[i].conc.stock * vol[i] / mixvol # Avoid warnings about concentrations not adding up
self.e.transfer(vol[i], inp[i], mixdown, (False, False))
self.e.shakeSamples([mixdown])
if not mixdown.wellMixed:
self.e.mix(mixdown)
mixdown.conc = Concentration(stock=sum([inp[i].conc.stock * vol[i] for i in range(len(inp))]) / mixvol,
final=None, units='nM')
print("Mix product, %s, is in well %s with %.1ful @ %.2f nM"%(mixdown.name,mixdown.plate.wellname(mixdown.well),mixdown.volume,mixdown.conc.stock))
print("----------")
return mixdown
def oldmix(self, inp, weights, tgtdil=1.0):
"""Mix given inputs according to weights (by moles -- use conc.stock of each input)"""
print("Mix: tgtdil=%.2f, inp=" % tgtdil, ",".join(
["%s@%.2f" % (inp[i].name, weights[i]) for i in range(len(inp))]))
mixvol = 100.0
if len(inp) == 1:
if tgtdil > 1.0:
vol = [mixvol / tgtdil]
if vol[0] < 4.0:
vol[0] = 4.0
else:
# Special case, just dilute 10x
vol = [mixvol / 10]
else:
relvol = [weights[i] * 1.0 / inp[i].conc.stock for i in range(len(inp))]
scale = mixvol / sum(relvol)
for i in range(len(inp)):
if relvol[i] * scale > inp[i].volume - 16.4:
scale = (inp[i].volume - 16.4) / relvol[i]
vol = [x * scale for x in relvol]
if min(vol) > 4.0 and tgtdil > 1.0:
scale = max(1.0 / tgtdil, 4.0 / min(vol))
print("Rescale volumes by %.2f to get closer to target dilution of %.2f" % (scale, tgtdil))
vol = [x * scale for x in vol]
print("Mix1: vols=[", ",".join(["%.3f" % v for v in vol]), "]")
if min(vol) < 4.0:
logging.info("Minimum volume into mixing would be only %.2f ul - staging..." % min(vol))
if max(vol) < 4.0:
# All volumes are low, just too much
# Split into 2 stages
sel = list(range(int(len(inp) / 2)))
nsel = list(range(int(len(inp) / 2), len(inp)))
else:
# Choose a splitting threshold
mindilution = 4.0 / min(vol)
thresh = np.median(vol)
while mixvol / sum([v for v in vol if v < thresh]) < mindilution:
thresh = thresh * 0.8
print("Using %.2f as threshold to split mixdown" % thresh)
sel = [i for i in range(len(inp)) if vol[i] < thresh]
nsel = [i for i in range(len(inp)) if vol[i] >= thresh]
print("Mixing ", ",".join([inp[i].name for i in sel]), " with vols [", ",".join(
["%.2f" % vol[i] for i in sel]), "] in separate stage.")
tgtdil = float(np.median([vol[i] for i in nsel]) / sum([vol[i] for i in sel]))
print("tgtdil=%.2f" % tgtdil)
mix1 = self.mix([inp[i] for i in sel], [weights[i] for i in sel], tgtdil)
mix2 = self.mix([inp[i] for i in nsel] + [mix1],
[weights[i] for i in nsel] + [sum([weights[i] for i in sel])])
return mix2
watervol = mixvol - sum(vol)
print("Mixdown: vols=[", ",".join(["%.2f " % v for v in vol]), "], water=", watervol, ", total=", mixvol, " ul")
mixdown = Sample('mixdown', plate=decklayout.SAMPLEPLATE)
if watervol < -0.1:
print("Total mixdown is %.1f ul, more than planned %.0f ul" % (sum(vol), mixvol))
assert False
elif watervol > 0.0:
self.e.transfer(watervol, decklayout.WATER, mixdown, (False, False))
else:
pass
for i in range(len(inp)):
inp[i].conc.final = inp[i].conc.stock * vol[i] / mixvol # Avoid warnings about concentrations not adding up
self.e.transfer(vol[i], inp[i], mixdown, (False, i == len(inp) - 1))
self.e.shakeSamples([mixdown])
mixdown.conc = Concentration(stock=sum([inp[i].conc.stock * vol[i] for i in range(len(inp))]) / mixvol,
final=None, units='nM')
print("Mixdown final concentration = %.1f nM" % mixdown.conc.stock)
return mixdown
