from Experiment.sample import Sample
from Experiment.experiment import Experiment
import math
from TRPLib.TRP import TRP
reagents=None
input="BT411"
ctl="L2b12Ctl"
srcprefix="A"
prodprefix="B"
for iteration in range(2):
print "Iteration ",iteration+1
trp=TRP()
if iteration==0:
trp.addTemplates([input,ctl],160) # Add a template with stock concentration 80nM
else:
reagents=Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume<0:
r.initvolume=-r.volume+20
Sample.clearall()
# Round 1 (Keep uncleaved +theo)
t71=trp.runT7(theo=[False,True,False],src=[input,input,ctl],tgt=[],vol=18,srcdil=80.0/24)
sv1t7=trp.saveSamps(src=t71,tgt=[],vol=10,dil=4)
rt1=trp.runRT(pos=[True,True,True ],src=t71,tgt=[],vol=22,srcdil=2)
trp.diluteInPlace(tgt=rt1,dil=2)
sv1rt=trp.saveSamps(src=rt1,tgt=[],vol=15,dil=2)
pcr1=trp.runPCR(prefix=[srcprefix],src=rt1[1],tgt=[],vol=50,srcdil=4)
if Sample.lookup("MQ"+pm)==None:
Sample("MQ"+pm,Experiment.REAGENTPLATE,None,10.0/6)
for pm in tmplqpcr:
if Sample.lookup("MQ"+pm)==None:
Sample("MQ"+pm,Experiment.REAGENTPLATE,None,10.0/6)
reagents=None
for iteration in range(2):
print "Iteration ",iteration+1
trp=TRP()
if iteration==0:
trp.addTemplates(input,stockconc=10.0/6.0,units="x",plate=Experiment.EPPENDORFS) # Add a template
else:
reagents=Sample.getAllOnPlate(Experiment.REAGENTPLATE)+Sample.getAllOnPlate(Experiment.EPPENDORFS)
for r in reagents:
if r.volume<=0:
r.initvolume=-r.volume+r.plate.unusableVolume
Sample.clearall()
t71=trp.runT7(theo=False,src=srcs,tgt=[],vol=10,srcdil=10.0/6,dur=15,stopmaster=stop)
#print t71
t71=trp.diluteInPlace(tgt=t71,dil=5)
# Dilute input samples enough to use in qPCR directly (should be 5000/(rnagain*2*5) = 20)
qpcrdil1=trp.runQPCRDIL(src=t71,tgt=[],vol=100,srcdil=20,dilPlate=False)
rt1=trp.runRT(pos=True,src=t71,tgt=[],vol=30,srcdil=2)
rt1=trp.diluteInPlace(tgt=rt1,dil=5)
print "PCR2 input conc=%.3g pM, PCR cycles=%.1f, End Conc=%.0f nM" % (
pcrinconc2 * 1e12, cycles2, pcrinconc2 * (math.pow(pcreff, cycles2)) * 1e9)
print "Diversity at PCR1 input in first round = %.1g molecules" % (
pcrinconc1 * (inputConc / tmplConc) * (pcrvol * 1e-6) * 6.022e23)
print "Diversity at PCR2 input in second round = %.1g molecules" % (
pcrinconc2 * (pcrvol * 1e-6) * 6.022e23)
for iteration in range(2):
print "Iteration ", iteration + 1
trp = TRP()
templates = [inname + "-" + srcprefix, inname + "-" + srcprefix + "-spike"]
if iteration == 0:
#rnastore=Sample("RNA Storage",Experiment.REAGENTPLATE,None,None)
trp.addTemplates(
templates, inputConc * 1e9
) # Add a template with stock concentration same as subsequent PCR products
trp.addTemplates(
["BT371"],
10) # Add a template with known concentration as reference
else:
reagents = Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume < 0:
r.initvolume = -r.volume + 20
Sample.clearall()
currprefix = srcprefix
if currprefix == 'A':
altprefix = 'B'
else:
if Sample.lookup("MQ" + pm) == None:
Sample("MQ" + pm, Experiment.REAGENTPLATE, None, 10.0 / 6)
for pm in tmplqpcr:
if Sample.lookup("MQ" + pm) == None:
Sample("MQ" + pm, Experiment.REAGENTPLATE, None, 10.0 / 6)
reagents = None
for iteration in range(2):
print "Iteration ", iteration + 1
trp = TRP()
if iteration == 0:
trp.addTemplates(input,
stockconc=10.0 / 6.0,
units="x",
plate=Experiment.EPPENDORFS) # Add a template
else:
reagents = Sample.getAllOnPlate(
Experiment.REAGENTPLATE) + Sample.getAllOnPlate(
Experiment.EPPENDORFS)
for r in reagents:
if r.volume <= 0:
r.initvolume = -r.volume + r.plate.unusableVolume
Sample.clearall()
t71 = trp.runT7(theo=False,
src=srcs,
tgt=[],
vol=10,
srcdil=10.0 / 6,
for i in range(len(input)):
if nreplicates[i] > k:
inputs = inputs + [input[i]]
srcprefixes = srcprefixes + [srcprefix[i]]
if srcprefix[i] == 'A':
prodprefixes = prodprefixes + ['B']
else:
prodprefixes = prodprefixes + ['A']
reagents = None
for iteration in range(2):
print "Iteration ", iteration + 1
trp = TRP()
if iteration == 0:
trp.addTemplates(input,
8) # Add a template with stock concentration 8nM
else:
reagents = Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume < 0:
r.initvolume = -r.volume + 20
Sample.clearall()
t71 = trp.runT7(theo=[False for i in inputs] + [True for i in inputs],
src=inputs + inputs,
tgt=[],
vol=10,
srcdil=80.0 / 24,
dur=15)
trp.diluteInPlace(tgt=t71, dil=2)
rt1 = trp.runRT(pos=True, src=t71, tgt=[], vol=5, srcdil=2)
ligconc=tmplConc*3.0/10.0*rnagain/ligdil # Expected concentration of ligation product here
pcrinconc=ligconc/4;
cycles=math.ceil(math.log(endconc/pcrinconc,pcreff)) # Amplify to end of exponential phase
print "PCR input conc=%.3g pM, PCR cycles=%.1f, End Conc=%.0f nM"%(pcrinconc*1e12,cycles,pcrinconc*(math.pow(pcreff,cycles))*1e9)
# Additional QPCR dilution
qdillig=10000/(ligdil*5)
t7dil=rnagain*2*25 # Back out expected RNA gain
qdilt7=10000/t7dil
print "QPCR 2nd dilution: Ligation products: %.1f, T7 products: %.1f"%(qdillig, qdilt7)
for iteration in range(2):
print "Iteration ",iteration+1
trp=TRP()
if iteration==0:
trp.addTemplates(inputs,tmplConc*1e9) # Add a template with stock concentration
else:
reagents=Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume<0:
r.initvolume=-r.volume+20
Sample.clearall()
# Round 1 (Keep cleaved -theo)
print "***** Round 1 T7 *****"
t71=trp.runT7New(theo=[False for i in inputs]+[True for i in inputs],src=inputs+inputs,tgt=[],vol=10,srcdil=10.0/3)
sv1t7=trp.saveSamps(src=t71,vol=3,dil=25,plate=trp.e.DILPLATE)
rt1=trp.runRT(pos=True,src=t71,tgt=[],vol=5,srcdil=2)
trp.diluteInPlace(tgt=rt1,dil=4)
import math
from TRPLib.TRP import TRP
from TRPLib.TRP import findsamps
import copy
reagents=None
inputs=[]
concs=[10,5,2.5,1.25]
for iteration in range(2):
print "Iteration ",iteration+1
trp=TRP()
if iteration==0:
for conc in concs:
input="DNA@%d"%conc
inputs.append(input)
trp.addTemplates([input],conc,finalconc=conc)
else:
reagents=Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume<0:
r.initvolume=-r.volume+20
Sample.clearall()
dilneeded=[c/.020 for c in concs]
qdil1=[min(40,d/2) for d in dilneeded] # 40x for first dilution
qdil2=[dilneeded[i]*1.0/qdil1[i] for i in range(len(qdil1))] # Whatever remains
qpcrdil1=trp.runQPCRDIL(src=inputs,tgt=[],vol=100,srcdil=qdil1,dilPlate=False) # First dilution before starting PCR (so the rest of the QPCR setup can be done while PCR is running)
qpcrdil2x1=trp.runQPCRDIL(src=qpcrdil1,tgt=[],vol=100,srcdil=qdil2)
# qpcrdil2x2=trp.runQPCRDIL(src=qpcrdil1[0:2],tgt=['x2a','x2b'],vol=100,srcdil=[d*2 for d in qdil2[0:2]])
qpcrdil2x4=trp.runQPCRDIL(src=qpcrdil1[0:2],tgt=['x4a','x4b'],vol=100,srcdil=[d*4 for d in qdil2[0:2]])
qpcrdil2x4b=trp.runQPCRDIL(src=qpcrdil2x1[0:2],tgt=['x4ab','x4bb'],vol=100,srcdil=[4 for d in qdil2[0:2]])
srcprefix = "A"
prodprefix = "B"
rnagain = 10
# Assumed DNA:RNA gain
endconc = 10e-9
# PCR end concentration (nM) (at end of exponential phase)
pcreff = 1.9
# Mean efficiency of PCR during exponential phase
tmplConc = endconc / 3
# nM
for iteration in range(2):
print "Iteration ", iteration + 1
trp = TRP()
if iteration == 0:
trp.addTemplates(inputs, tmplConc *
1e9) # Add a template with stock concentration
else:
reagents = Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume < 0:
r.initvolume = -r.volume + 20
Sample.clearall()
# Round 1 (Keep uncleaved +theo)
t71 = trp.runT7New(theo=True,
src=inputs,
tgt=["%s.T1+" % i for i in inputs],
vol=10,
srcdil=10.0 / 3,
dur=30)
trp.diluteInPlace(tgt=t71, dil=5)
cycles2=math.ceil(math.log(endconc/pcrinconc2,pcreff)) # Amplify to end of exponential phase
# Add an extra 2 cycles to be sure
cycles1+=2
cycles2+=2
print "PCR1 input conc=%.3g pM, PCR cycles=%.1f, End Conc=%.0f nM"%(pcrinconc1*1e12,cycles1,pcrinconc1*(math.pow(pcreff,cycles1))*1e9)
print "PCR2 input conc=%.3g pM, PCR cycles=%.1f, End Conc=%.0f nM"%(pcrinconc2*1e12,cycles2,pcrinconc2*(math.pow(pcreff,cycles2))*1e9)
for iteration in range(2):
print "Iteration ",iteration+1
trp=TRP()
input="IN-"+srcprefix
if iteration==0:
#rnastore=Sample("RNA Storage",Experiment.REAGENTPLATE,None,None)
trp.addTemplates([input],tmplConc*1e9) # Add a template with stock concentration same as subsequent PCR products
else:
reagents=Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume<0:
r.initvolume=-r.volume+20
Sample.clearall()
currprefix=srcprefix
if currprefix=='A':
altprefix='B'
else:
altprefix='A'
sv=[]
svligtype=[]
svdil=[]
for i in range(len(input)):
if nreplicates[i]>k:
inputs=inputs+[input[i]];
srcprefixes=srcprefixes+[srcprefix[i]];
if srcprefix[i]=='A':
prodprefixes=prodprefixes+['B'];
else:
prodprefixes=prodprefixes+['A'];
reagents=None
for iteration in range(2):
print "Iteration ",iteration+1
trp=TRP()
if iteration==0:
trp.addTemplates(input,8) # Add a template with stock concentration 8nM
else:
reagents=Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume<0:
r.initvolume=-r.volume+20
Sample.clearall()
t71=trp.runT7(theo=[False for i in inputs]+[True for i in inputs],src=inputs+inputs,tgt=[],vol=10,srcdil=80.0/24,dur=15)
trp.diluteInPlace(tgt=t71,dil=2)
rt1=trp.runRT(pos=True,src=t71,tgt=[],vol=5,srcdil=2)
trp.diluteInPlace(tgt=rt1,dil=4)
lig1=trp.runLig(prefix=[prodprefixes[i%len(prodprefixes)] for i in range(len(rt1))],src=rt1,tgt=[],vol=10,srcdil=3)
trp.diluteInPlace(tgt=lig1,dil=4)
# Dilute input samples
qpcrdil1=trp.runQPCRDIL(src=inputs,tgt=[],vol=100,srcdil=40,dilPlate=True)
cycles1+=6+2
cycles2+=3+2
print "PCR1 input conc=%.3g pM, PCR cycles=%.1f, End Conc=%.0f nM"%(pcrinconc1*1e12,cycles1,pcrinconc1*(math.pow(pcreff,cycles1))*1e9)
print "PCR2 input conc=%.3g pM, PCR cycles=%.1f, End Conc=%.0f nM"%(pcrinconc2*1e12,cycles2,pcrinconc2*(math.pow(pcreff,cycles2))*1e9)
print "Diversity at PCR1 input in first round = %.1g molecules"%(pcrinconc1*(inputConc/tmplConc)*(pcrvol*1e-6)*6.022e23)
print "Diversity at PCR2 input in second round = %.1g molecules"%(pcrinconc2*(pcrvol*1e-6)*6.022e23)
for iteration in range(2):
print "Iteration ",iteration+1
trp=TRP()
templates=[inname+"-"+srcprefix,inname+"-"+srcprefix+"-spike"]
if iteration==0:
#rnastore=Sample("RNA Storage",Experiment.REAGENTPLATE,None,None)
trp.addTemplates(templates,inputConc*1e9) # Add a template with stock concentration same as subsequent PCR products
trp.addTemplates(["BT371"],10) # Add a template with known concentration as reference
else:
reagents=Sample.getAllOnPlate(Experiment.REAGENTPLATE)
for r in reagents:
if r.volume<0:
r.initvolume=-r.volume+20
Sample.clearall()
currprefix=srcprefix
if currprefix=='A':
altprefix='B'
else:
altprefix='A'
ligsave1=[]
ligsave2=[]