| Info |
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The red Low Read II samples were renormalized via pooling and the new MC samples were added to that same pool at 1 ul per sample. This pool was then adjusted to 1nM. The pool of the repeated RMJan22 samples was also adjusted to 1 nM. 50 ul of Low Read was added to 100 ul RMJan22. However, when the LowRead pool was qPCRed, one replicate was much higher than the other 2. The 1:2 ratio might be off because of this. |
RNaseP Check of qPCR machine recalibration:
Recalibration looks good. The 5,000 copy samples all reported back as close to 5,000 copies and the 10,000 copy samples all reported back as roughly 10,000 copies with no real outliers. A snapshot is below:
Sample Name | Detector | Task | Ct | StdDev Ct | Qty | Mean Qty | StdDev Qty |
|---|---|---|---|---|---|---|---|
5K | RNase P | Unknown | 27.80 | 0.052 | 5170.54 | 4935.10 | 168.205 |
5K | RNase P | Unknown | 27.80 | 0.052 | 5164.36 | 4935.10 | 168.205 |
10K | RNase P | Unknown | 26.77 | 0.058 | 10193.14 | 9923.36 | 380.987 |
10K | RNase P | Unknown | 26.82 | 0.058 | 9863.28 | 9923.36 | 380.987 |
NTC | RNase P | NTC | Undetermined | ||||
NTC | RNase P | NTC | Undetermined | ||||
NTC | RNase P | NTC | Undetermined | ||||
NTC | RNase P | NTC | Undetermined | ||||
Standard1 | RNase P | Standard | 29.94 | 0.039 | 1250.00 | ||
Standard1 | RNase P | Standard | 29.92 | 0.039 | 1250.00 | ||
Standard1 | RNase P | Standard | 30.00 | 0.039 | 1250.00 | ||
Standard1 | RNase P | Standard | 30.00 | 0.039 | 1250.00 | ||
Standard2 | RNase P | Standard | 28.92 | 0.059 | 2500.00 | ||
Standard2 | RNase P | Standard | 28.87 | 0.059 | 2500.00 | ||
Standard2 | RNase P | Standard | 28.82 | 0.059 | 2500.00 | ||
Standard2 | RNase P | Standard | 28.78 | 0.059 | 2500.00 | ||
Standard3 | RNase P | Standard | 27.81 | 0.034 | 5000.00 | ||
Standard3 | RNase P | Standard | 27.82 | 0.034 | 5000.00 | ||
Standard3 | RNase P | Standard | 27.86 | 0.034 | 5000.00 | ||
Standard3 | RNase P | Standard | 27.88 | 0.034 | 5000.00 | ||
Standard4 | RNase P | Standard | 26.91 | 0.013 | 10000.00 | ||
Standard4 | RNase P | Standard | 26.93 | 0.013 | 10000.00 | ||
Standard4 | RNase P | Standard | 26.90 | 0.013 | 10000.00 | ||
Standard4 | RNase P | Standard | 26.89 | 0.013 | 10000.00 | ||
Standard5 | RNase P | Standard | 25.76 | 0.062 | 20000.00 | ||
Standard5 | RNase P | Standard | 25.69 | 0.062 | 20000.00 | ||
Standard5 | RNase P | Standard | 25.67 | 0.062 | 20000.00 | ||
Standard5 | RNase P | Standard | 25.61 | 0.062 | 20000.00 |
...
Returns: 15371232
Divided by 8: 1921404 assigned to samples.
Assigned/Total (*100) = percent assigned: ~73%
...
Divided by 8: 1412494
LRII + locad2: 1921404
Even if all the unassigned reads are from locad2, this does not fix the expected ration of 2lo:1LR.
[508910+(2630678-1921404)] = 1218184 total possible locad2 reads
...