Daniel:Notebook/ComboLock/2016-9-12
Positive Control Amplicon Test 2 (Started 8/22/2016)[edit]
Results[edit]
As a refresher, the table below shows the samples that were sequenced.
Sample Name | LatchX2 | LatchX3 | PCAmp1 | PCAmp3 | Expected Product |
X2-1X | X | X | Amp1 | ||
X3-50X | X | X | Amp3 | ||
X2 Flip | X | X | Amp3 | ||
X3 Flip | X | X | Amp1 |
idx33 | idx44 | idx41 | idx42 | |
Name | X2-1X | X3-50X | X2 Flip | X3 Flip |
Amp1 Counts | 141253 | 180 | 165 | 74436 |
Amp3 Counts | 29 | 72462 | 26111 | 12 |
% Off Target | 0.021 | 0.248 | 0.628 | 0.016 |
Ratio to Normal | 2.53 | 0.79 |
So in analyzing this, it looks like some off-target amplification occurs, even in the normal samples. This is actually probably due more to sequencing errors than off target binding, but I could look closer if I want. The "Flip" samples use the wrong latch, but as I realized later, the positive control templates only require a padlock to bind to them in order to amplify. The sequence does not require a latch. Therefore, the best comparison is to compare the number of off-target binding events in the "Flip" samples vs. the number of off-target events in the normal samples using that template. In the case of the Amp1 template (compare X1-1X against X3 Flip), this ratio is actually less than 1, meaning the wrong latch yielded less off-target binding events (by fraction) than the normal. In the case of Amp3 (compare X3-50X against X2 Flip) the wrong latch increased off-target events 2.5 fold, but the actual number is still quite low (.63% or 0.0063)