This tool offers data transformation options, optional automated gating of debris using flowDensity and conversion of FCS files to text.
Input files
This tool uses FCS files as input and files are processed serially. Users choose which transformation algorithm to apply, and whether to automatically gate cellular debris, and/or compensate the data.
Output files
Output is a table of fluorescence intensity values for each marker, as a text file, FCS3.0 or R Object (flowFrame).
Gating output
Automatically gated output includes a summary of data pre- and post-gating as well as density scatter plots pre- and post- gating for each marker pair. Please check to make sure the gating is doing what you think it should be doing.
Compensation will be implemented according to the spillover matrix included in the FCS files (if available).
Transformation
This tools currently supports 3 transformation algorithms: FCSTrans, arcsinh and logicle. The latter two are implemented using flowCore, FCSTrans is documented in the paper cited below. Transformation algorithms are NOT applied to FSC and SSC channels.
Automated Gating
Automated gating is implemented with flowDensity. Cellular debris removal uses gate coordinates calculated based on the density of the forward scatter channel only. The calculated gate is vertical and located at the largest value of either the 0.1 quantile of the FSC density or the lowest density between the first and second density peaks. Cells smaller than the threshold are removed.
Users should verify whether data benefit from being gated. We recommend evaluating the number of events and scatter plots pre- and post-gating.
Example
Output text file:
Forward Scatter Side Scatter Marker1 Marker2 Marker3 Marker4 ... 449 157 551 129 169 292 ... 894 1023 199 277 320 227 ... 262 73 437 69 0 146 ... 340 115 509 268 0 74 ... ... ... ... ... ... ... ...
Gating summary:
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== BEFORE GATING ==
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events parameters
203584 13
FSC-A FSC-H SSC-A SSC-H APC-A APC-H7-A FITC-A PerCP-Cy5-5-A V450-A V500-A PE-A PE-Cy7-A Time
Min. 0 78 0 0.0 0 0.0 0 0 0 0 0.0 0 0
1st Qu. 869 769 544 514.0 940 0.0 1023 826 978 1104 358.8 913 4182
Median 1234 1081 754 704.0 1750 31.0 1247 1614 1971 1505 1612.0 1791 8392
Mean 1304 1108 1002 863.6 1629 812.8 1329 1597 1694 1765 1376.0 1813 8536
3rd Qu. 1675 1474 1240 1087.0 2326 1573.0 1629 2565 2326 2640 2261.0 2782 12760
Max. 4095 4061 4095 4012.0 4096 4086.0 4094 4022 4091 4096 4093.0 4096 17740
=========================
== AFTER GATING ==
=========================
events parameters
171547 13
FSC-A FSC-H SSC-A SSC-H APC-A APC-H7-A FITC-A PerCP-Cy5-5-A V450-A V500-A PE-A PE-Cy7-A Time
Min. 403 219 1.0 4.0 0 0.0 0 0 0 0 0 0 0
1st Qu. 949 849 599.0 565.0 979 0.0 1063 943 1203 1119 444 1060 4182
Median 1348 1170 782.0 730.0 1809 0.0 1258 1717 2082 1493 1785 1883 8358
Mean 1353 1177 989.2 885.9 1642 779.4 1330 1678 1778 1753 1458 1873 8504
3rd Qu. 1670 1475 1204.0 1076.0 2322 1559.0 1591 2623 2345 2630 2289 2808 12700
Max. 2953 2406 4055.0 3329.0 4069 4080.0 3498 3868 3612 4076 4093 3764 17730
Gating plots:
