This file gives a "quick start" overview of how the tools in the TASBE Characterization Workflow fit together and how to use each tool with "normal" data. For more detailed information about how to interpret and debug the results from the tools, see the document Understanding Tool Results.
The Characterization Workflow of the TASBE tools comprises three tools:
The general workflow for using these is as follows:
If you wish to follow along with the steps in this documentation, you can download the files used in this demo.
The page for creating a FACS machine looks like this:
The "FACS Machine Name" field is how the machine will be described in menus later, when you select it as the instrument that generated a piece of data. The "Notes" field is whatever other information you want to include for yourself.
The most critical part of the form is the channels, which list the set of fluorescence measurements that are available from this FACS machine Make sure each "Channel Name" is exactly as it will appear in the .fcs files generated by your FACS machine. If you get this wrong, then when you try to create color models or run experiments, you will get complaints that your channels cannot be found. Laser and filter information is not currently used in the analysis --- it is for your benefit only.
Once you've created your FACS machines, you can review them by using the "List all my FACS machines" page and clicking on the "View Details" link by a machine.
For a characterization experiment, the controls not only establish confirmation that the positive and negative behavior of the cells is able to be read correctly, but also provide a color models that is used for calibrating the analysis so that results can always be reported in standardized equivalent units.
To do this, you will need the following set of controls:
Creating a color model is done in two stages; first you use "Create a control set" to upload a collection of .fcs files for your controls.
On this page, start by choosing a name for this data set, adding any notes you want, along with the FACS Machine and channels that you want to use.
Below this, there are places to upload the blank and bead files, plus a file of constitutive fluorescent protein expression for each channel that you have chosen:
Finally, there is a table to put in as many three-fluorescence translation models as you have in your control set:
It doesn't matter which fluorescent protein goes in Channel 1 versus Channel 2: a translation model is built in both directions. However, you must have a pair of [Channel]/FITC-A for every other channel that you are using. If you do not, the analysis software will not be able to translate those channels into standard MEFL units.
Once you have created the control set, you will be taken to a settings page for analyzing it into a color model:
On this page, you just need to enter the name of the color model and how you want the channels displayed. There are also options for changing thresholds used in constructing the color model; unless you have a reason to do so, it is best to stick with the defaults.
When you hit the "Run color model" button, the system will process for a while, then eventually display your results:
This page includes a link for downloading a "CM.mat" file containing the Matlab/Octave data structures for the color model. When you load this .mat file, it will report many warnings, as the objects used on the server are converted into structures; you should ignore these warnings: the data inside is intact. The page also includes a collection of all of the graphs generated with the color model, which may be downloaded in either .png or PDF format:
Just as with FACS machines, you can return to your sets of control data using the "List all my control sets" page and return to your color models using the "List all my color models" page. You can also produce new models from the same set of controls using the "Create Color Model" link in the list of control sets.
Finally, to run analysis of a characterization experiment, you fill in three sections on the "Enter data" page.
First is the metadata for the experiment, including what construct you have used and the conditions for the experiment. Although these are all free-text fields, they are required in order to encourage good data curation. Note that the Device name will be used as part of file names later, so you should keep it terse.
In the next section of the form, you select which colors are being used for which parts of the characterization experiment:
At present, the software assumes that you are always characterizing a one-input and one-output device that is not affected directly by your inducer. There will eventually be options for characterization of other classes of devices. At present, if you are characterizing just an inducer, then select an unused channel for your "output".
The final section of the form is where you write the name of your inducer and upload all of the .fcs files holding your experimental data:
This table has one row for each induction level that you gathered data for, and one column for each replicate. The example shown has 11 induction levels for which one replicate has been gathered each. If it were in triplicate, then two presses of "Add another set of files" would add two more columns, into which more files could be added. At present, the form requires that there be no missing replicates; this will be generalized in the future.
Once you have entered a data-set, you will be taken to a page where you can enter the settings for running your analysis. The experiment name and results prefix are your choice of how you want graphs labelled and graph files named. Another setting that you may wish to change, if you have information, is the MEFL per plasmid, which sets a linear relation between observed fluorescence and expected number of plasmids in the cell. The other settings control the "binning" that will be used for dividing cells by CFP level and thresholds for excluding data. Unless you have reason to do otherwise, stay with the default.
Finally, when you hit "Run experiment analysis", the system will process for several minutes, depending on how much data you have uploaded, before taking you to a results page:
The results page shows the characterization analysis and a downloadable .mat file with the data. When you load this .mat file, it will report many warnings, as the objects used on the server are converted into structures; you should ignore these warnings: the data inside is intact. The pages also includes a collection of all of the graphs generated from the analysis of the characterization data. These graphs are mainly useful as controls, showing the calibration of each channel and the measured levels of per-cell expression noise.
These graphs are: