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=Bead Loading Analysis (Started [[Daniel:Notebook/PosSequencing/2017-4-11|Tuesday]])= [[Daniel:Notebook/PosSequencing|Back to Calendar]] ==Image Analysis (Started [[Daniel:Notebook/PosSequencing/2017-4-12|Wednesday]])== Analysis of the images taken on [[Daniel:Notebook/PosSequencing/2017-4-12|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. <ol> <li>Start with Dapi image and identify (by eye) the bright clusters; circled in red</li> [[Image:20170412-D1Emulsion-DAPI3-Analysis.png|500px]] <li>Use "change image function" to replace DAPI image with brightfield image. The change image function keeps the position and dimensions of the original</li> <li>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</li> [[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 |} [[Category:PosSeq]] [[Category:20170411]]
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