Daniel:Notebook/PosSequencing/2017-4-14: Difference between revisions
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>Djacobse (Created page with "=Bead Loading Analysis (Started Tuesday)= Back to Calendar ==Image Analysis (Started [[Daniel:N...") |
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[[Image:20170412-D1Emulsion-BF3-Analysis.png|500px]] | [[Image:20170412-D1Emulsion-BF3-Analysis.png|500px]] | ||
<li>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. | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#CC66FF;font-size:12pt;font-weight:bold" align="center" | |||
| width="85" height="40" | Field | |||
| width="120" | # Dapi Stained Droplets | |||
| width="100" | # Non-Dapi Droplets | |||
| width="85" | Wild Cards (Droplet?) | |||
| width="85" | Loading Percentage | |||
| width="100" | Loading Pct with Wildcards | |||
| width="85" | Likely Doubles % | |||
| width="100" | Likely Doubles % (+WC) | |||
|- style="font-size:12pt" | |||
| align="center" height="15" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 8 | |||
| align="center" align="center" valign="bottom" | 9 | |||
| align="center" align="center" valign="bottom" | 14 | |||
| align="center" align="center" valign="bottom" | 47.1 | |||
| align="center" align="center" valign="bottom" | 25.8 | |||
| align="center" align="center" valign="bottom" | 2.7 | |||
| align="center" align="center" valign="bottom" | 0.4 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| align="center" height="15" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 4 | |||
| align="center" align="center" valign="bottom" | 9 | |||
| align="center" align="center" valign="bottom" | 55.6 | |||
| align="center" align="center" valign="bottom" | 27.8 | |||
| align="center" align="center" valign="bottom" | 4.9 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
|- style="font-size:12pt" | |||
| align="center" height="15" valign="bottom" | 3 | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" align="center" valign="bottom" | 9 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 52.6 | |||
| align="center" align="center" valign="bottom" | 22.7 | |||
| align="center" align="center" valign="bottom" | 4.0 | |||
| align="center" align="center" valign="bottom" | 0.2 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| align="center" height="15" valign="bottom" | 4 | |||
| align="center" align="center" valign="bottom" | 11 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 23 | |||
| align="center" align="center" valign="bottom" | 68.8 | |||
| align="center" align="center" valign="bottom" | 28.2 | |||
| align="center" align="center" valign="bottom" | 11.3 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
|- style="font-size:12pt" | |||
| align="center" height="15" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 12 | |||
| align="center" align="center" valign="bottom" | 35 | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" align="center" valign="bottom" | 25.5 | |||
| align="center" align="center" valign="bottom" | 21.1 | |||
| align="center" align="center" valign="bottom" | 0.3 | |||
| align="center" align="center" valign="bottom" | 0.2 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| align="center" height="15" valign="bottom" | 6 | |||
| align="center" align="center" valign="bottom" | 22 | |||
| align="center" align="center" valign="bottom" | 15 | |||
| align="center" align="center" valign="bottom" | 24 | |||
| align="center" align="center" valign="bottom" | 59.5 | |||
| align="center" align="center" valign="bottom" | 36.1 | |||
| align="center" align="center" valign="bottom" | 6.3 | |||
| align="center" align="center" valign="bottom" | 1.1 | |||
|- style="font-size:12pt;font-weight:bold" | |||
| height="15" valign="bottom" | Total | |||
| align="center" align="center" valign="bottom" | 68 | |||
| align="center" align="center" valign="bottom" | 77 | |||
| align="center" align="center" valign="bottom" | 105 | |||
| align="center" align="center" valign="bottom" | 46.9 | |||
| align="center" align="center" valign="bottom" | 27.2 | |||
| align="center" align="center" valign="bottom" | 2.6 | |||
| align="center" align="center" valign="bottom" | 0.4 | |||
|} |
Revision as of 21:17, 14 April 2017
Bead Loading Analysis (Started Tuesday)
Image Analysis (Started Wednesday)
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