Daniel:Notebook/PosSequencing/2017-4-14
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Bead Loading Analysis (Started Tuesday)[edit]
Image Analysis (Started Wednesday)[edit]
Analysis of the images taken on Wednesday. Since there are only 6 and I'm not planning on doing this long, I did the analysis manually. Here's the pipeline, done in Powerpoint.
- Start with Dapi image and identify (by eye) the bright clusters; circled in red File:20170412-D1Emulsion-DAPI3-Analysis.png
- Use "change image function" to replace DAPI image with brightfield image. The change image function keeps the position and dimensions of the original
- Mark and count (in blue) the beads that appear in the same plane as the red circled dots but had no signal. These are confirmed no-calls. The green circled dots are "wild cards", and are possibly no-calls, although they might just be out of phase which lowers there signal File:20170412-D1Emulsion-BF3-Analysis.png
- With the counts, I calculated the percentage of empties. Since I can't tell whether any signal is a single or double, I only count empties. This fraction, I assume is the Poissonian result of loading. Therefore, using the Poisson distribution, I can estimate the average loading, and use the Poisson CDF to estimate the number of beads with 2 or more (1-P(2)). The results are recorded below.
Field # Dapi Stained Droplets # Non-Dapi Droplets Wild Cards (Droplet?) Loading Percentage Loading Pct with Wildcards Likely Doubles % Likely Doubles % (+WC) 1 8 9 14 47.1 25.8 2.7 0.4 2 5 4 9 55.6 27.8 4.9 0.5 3 10 9 25 52.6 22.7 4.0 0.2 4 11 5 23 68.8 28.2 11.3 0.5 5 12 35 10 25.5 21.1 0.3 0.2 6 22 15 24 59.5 36.1 6.3 1.1 Total 68 77 105 46.9 27.2 2.6 0.4