qPCR
Make 1:1000 dilutions of all samples using 2 ml tubes
Add 16 ul of Illumina Library Quantification MasterMix to each well:
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
10 ul | KAPA SYBR FAST qPCR MM (2X) | 40 | 400 |
2 ul | Primer Premix (10X) | 40 | 80 |
4 ul | Ultra Pure Water | 40 | 160 |
16 ul | Total Volume | 40 | 640 |
Add 4 ul of template, pool, or standards to each well:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | LRII.5a | LRII.5a | LRII.5a |
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| NTC | NTC | NTC | ||
B | LRII.5b | LRII.5b | LRII.5b |
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| 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std | ||
C | locad2a | locad2a | locad2a |
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| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | ||
D | locad2b | locad2b | locad2b |
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| 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | ||
E | 0.05pM PhiX | 0.05pM PhiX | 0.05pM PhiX |
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| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | ||
F | 50pM PhiX | 50pM PhiX | 50pM PhiX |
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| 2 pM Std | 2 pM Std | 2 pM Std | ||
G |
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| 20 pM Std | 20 pM Std | 20 pM Std | |||||
H |
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Results:
I did 2 separate 1:1000 dilutions (a and b) of the pools. I also did one 1:1000 dilution and one original of our 50 pM PhiX. The results seems to corroborate the ratios I saw in the analysis.
03-04-22 Results:
Sample Name | Detector | Task | Ct | StdDev Ct | Qty | Mean Qty | |
|---|---|---|---|---|---|---|---|
LRII.5a | Standards | Unknown | 7.38 | 0.049 | 66.31 | 68.28 | 2.326 |
LRII.5a | Standards | Unknown | 7.35 | 0.049 | 67.68 | 68.28 | 2.326 |
LRII.5a | Standards | Unknown | 7.28 | 0.049 | 70.85 | 68.28 | 2.326 |
LRII.5b | Standards | Unknown | 7.26 | 0.080 | 71.85 | 76.53 | 4.132 |
LRII.5b | Standards | Unknown | 7.14 | 0.080 | 78.08 | 76.53 | 4.132 |
LRII.5b | Standards | Unknown | 7.11 | 0.080 | 79.67 | 76.53 | 4.132 |
PhiX0.05 | Standards | Unknown | 17.56 | 0.127 | 6.11E-02 | 6.45E-02 | 5.76E-03 |
PhiX0.05 | Standards | Unknown | 17.56 | 0.127 | 6.12E-02 | 6.45E-02 | 5.76E-03 |
PhiX0.05 | Standards | Unknown | 17.34 | 0.127 | 7.11E-02 | 6.45E-02 | 5.76E-03 |
PhiX50 | Standards | Unknown | 7.08 | 0.084 | 81.04 | 84.63 | 4.970 |
PhiX50 | Standards | Unknown | 7.06 | 0.084 | 82.55 | 84.63 | 4.970 |
PhiX50 | Standards | Unknown | 6.93 | 0.084 | 90.30 | 84.63 | 4.970 |
RMJan22-IIa | Standards | Unknown | 8.25 | 0.120 | 36.36 | 37.71 | 3.166 |
RMJan22-IIa | Standards | Unknown | 8.29 | 0.120 | 35.45 | 37.71 | 3.166 |
RMJan22-IIa | Standards | Unknown | 8.07 | 0.120 | 41.33 | 37.71 | 3.166 |
RMJan22-IIb | Standards | Unknown | 8.21 | 0.118 | 37.41 | 41.17 | 3.259 |
RMJan22-IIb | Standards | Unknown | 8.00 | 0.118 | 43.23 | 41.17 | 3.259 |
RMJan22-IIb | Standards | Unknown | 8.01 | 0.118 | 42.87 | 41.17 | 3.259 |
This averages out to:
Sample | Avg Qty | Std Deviation |
|---|---|---|
RMJan22-II | 39.44 | 3.441 |
LRII.5 | 72.41 | 5.425 |
PhiX 50 pM | 84.63 | 4.970 |
PhiX 0.05 pM | 6.45e-002 | 5.76e-003 |
PhiX avg original | 74.3 |
2-25-22 Results:
Sample Name | Detector | Task | Ct | StdDev Ct | Qty | Mean Qty | StdDev Qty |
|---|---|---|---|---|---|---|---|
LRII.5_Red_Pool | Unknown | Unknown | 9.31 | 0.605 | 16.65 | 12.97 | 5.204 |
LRII.5_Red_Pool | Unknown | Unknown | 10.17 | 0.605 | 9.29 | 12.97 | 5.204 |
LRII.5_Red_Pool | Unknown | Unknown | 5.7 | 2.734 | 196.45 | 106 | 74.13 |
RM_JAN22_II_Pool | Unknown | Unknown | 8.14 | 0.220 | 36.98 | 32.25 | 4.786 |
RM_JAN22_II_Pool | Unknown | Unknown | 8.34 | 0.220 | 32.36 | 32.25 | 4.786 |
RM_JAN22_II_Pool | Unknown | Unknown | 8.58 | 0.220 | 27.41 | 32.25 | 4.786 |
There was a clear outlier in the LRII.5 pool trio to which we should have paid more attention. The difference from previous values appears to explain the altered ratios we saw.
RM_JAN22_II: 2*(39.44/32.25)=2.45
LRII.5: 72.41/12.97=5.58
calculated RM/LR: 0.44
reads RM/LR: 508910/1412494=0.36
expected RM/LR: 2
reads PhiX/Samples: 709274/1921404 = 0.37
expected PhiX/Samples: 0.167
Calculated PhiX/Samples: