Daniel:Notebook/ComboLock/2016-9-12

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Positive Control Amplicon Test 2 (Started 8/22/2016)[edit]

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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)