Reporting in FlowJo™

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Good day. Here I'm going to talk about batch reporting from the layout editor and the table editor. The layout editor is a window that you can display all of the graphical information about your analysis to illustrate either a gating tree that you've applied to samples, or the responses, or the phenotypes that you see in your assay. And then actually generate a report to some external file type, or even just report in a batch process to a new layout within FlowJo, all of the graph plots for all of the samples within a group in your workspace.

How do we do this? If you open a new layout, which you can do by clicking this plus button, you can make as many layouts as you want, it says drag populations and statistics here. If I follow that rule, I drag a population that has been gated on a sample here in my workspace, over into the layout editor, and it populates the layout editor with that graph plot. I can format this graph plot any way I want. There's right-click options to do things like show adjunct histograms, or show the gating tree ancestry to get to this population of interest. I can also double-click on the plot, that will bring me to these properties menus, called the graph definition window.

I can change the X and Y axis the type of plot; all the annotation information; how things are displayed; the fonts, colors, and sizes; a legend if I have multiple plots overlaid on top of each other; and format this report with the plots looking just the way I want them. I have already done that for a couple different reports. Here's one where I'm just looking at a couple of functional markers in CD8+ t-cells, and explain the ancestry down below. What I want to do is actually move through a whole bunch of samples and report this same set of plots on every sample. Then review those samples so I know that the gates that I've applied are good for all the samples in my workspace.

To do this, we need to actually turn on a few options here under these bands called iteration, and batch options. I've already formatted my basic layout, and layouts always start with an iteration by option of off. So it is just drag and drop any information you want to display on this page, but if I want to display this information about each sample in a group, I can turn the iterate by option from off, to sample, and that will give me a drop-down list of all of the samples contained within this group I have specified here. I can focus my report on a specific group of samples within the workspace here. I've got my all samples group that contains all of the samples in the workspace, including my comps and my controls. Then I've got a group I'm just calling all stain which just has 20 samples in it. That's my real experiment. That's my real test group.

Maybe I just want to report on the 20 samples in that all stain group. What I'm going to do is select the group I want to report on, select the iterate by option, and set it to sample. Then I can review these here in the workspace in the layout editor, or I can create a batch report, either to a new layout within FlowJo, or some other external report, such as a webpage, a PowerPoint file, or a pdf. Let's go ahead and generate a batch report here within FlowJo, and what it's going to do is make five columns. It is going to batch through this group of samples, down. Here I have sample one, sample two, if I look down below, sample three, so on and so forth. Then the next patient and the third patient, fourth patient, fifth patient, all in one giant report here within FlowJo.

You can zoom in and out here in the bottom left-hand corner, and I'll just toggle off the show page breaks here. We can see this is the batched report showing that view of 20 different samples here within a new layout. You can see that layout, it says basic gates, which was the name of the original layout, batched. That's generating a report just within FlowJo, but if I go back to basic gates, I can also generate a report to a webpage. In this case, I want to choose where the location of the save is going to occur. The destination is where that file will be saved. I'm going to put it on my desktop, and I'm going to generate the report again by clicking this create batch report cog-button.

It is now going to go through the group I have specified, all stain containing 20 samples in it, by sample in the order of the sample list here, and generate this image for each of those 20 samples. It is going to link it, in this case, through an html link, to my web-browser. If I go here, I can see that the images are being reported in the FlowJo web files. Here's my link, the html document, links those images and it brings it up in my web-browser. I can then review all of the gating and responses here in a nice high-quality web page format.

Alternatively to web page, you can also do a PowerPoint file, or a pdf file. This will show one sample per page when you are iterating by sample. If I go to a pdf, put it on my desktop, create the batch report, it is going to generate those images, and save the pdf wherever I specify. Now I can have a portable pdf document that I can share with my colleagues, or my boss, to show them the approach that I have taken for my analysis. It pulls it up automatically into my pdf viewer, and I can see the plots there. I can zoom in on them, they are nice high-quality images displaying all the information here. I've also chosen to display a header and a footer on my reports showing the date and time that I've reported this information, as well as the FlowJo version number if you need to include that in the reporting that you do.

So far, that is just reporting a single sample per page, iterating by sample, but what if I have a more complex layout, such as these overlaid histograms? In this case I actually have four examples displayed on each of these plots. All of them come from the same patient, but they were processed, each sample, under a different stimulation condition to illustrate the different responses that you see in the marker outputs that are end-points for this asset. In this case I can't actually iterate by sample to generate the report because iterating by sample will only display one sample per page. You can choose which one, but it'll only show one sample per page.

If I go back to off, where I originally set-up this report, it goes back to my original layout. Then what I'm going to do is what we actually call key-word iteration options to make a complex iteration process. In this case, when I select keyword iteration, I actually have to pick two keywords to make this work. First of all, we need to pick an iterate by keyword. The iterate by keyword has to be the same between all four samples that are displayed on this page. What is the same between all the samples displayed on this page? What I've entered is the patient ID, PID, key-word that is the same for each of these samples. The patient ID here is LD1.

Then I need a discriminating key-word that is different between the samples on this page. What's different between these four samples is actually the stim-condition that I've labeled with. The stim key-word is different, so now when I iterate by patient id and discriminate by the stim condition, instead of having a drop-down list of 20 individual samples, I end up with a drop-down list of five distinct patient ID’s. I can scroll through the different patients, showing four specimens per patient and all the data updates.

Then I can create this batch report to whatever external file type I want, place it on the location I want to put it on, and then create the batch report. That will now generate that key-word batched iteration option that generates my histogram profiles for each of the patients in the group I have specified there. That is one way to illustrate your analysis, and generate reports from within FlowJo that are the graphical elements showing how you approached your gating, in this case, and enumerating of the statistics that are your final end-points.

If you want to actually generate a statistical report, that is not done through the layout editor. The layout editor is for generating graphical reports, and reviewing all of the gating approach and analysis that you've taken in this workspace. If you want to generate a statistical report, that is taking all of the numbers from the gated populations, either frequency of parents or statistics like the median of a particular gated population in a particular channel. Then you want to generate what we call a table. Table reports are accessed from this little lattice work looking table editor button up in the static toolbar, also available, again, in the navigate band of actions under the FlowJo tab, and there's a hot-key called command-T.

What you do is create a table and drag the populations or statistics you want to enumerate into this table editor window, and then iterate over a group of samples, usually by sample because you want to get the numbers out for each individual sample. Create a table to a display or some sort of external file-type, such as a text file, Excel file, or a sql database file, saved in a specific location, such as my desktop.

If I generate this table, let's say to a display, it is going to generate the table right here in a window, underneath the table editor. There's my table, and I've done a couple of things here, actually, to make this table look a little prettier. I've added some visualization options called a heat-map. Which heat-maps, the distribution of your statistic, through the visualize tab by selecting a population or a statistic that I've brought into the table editor, and applying the heat-map feature, which creates this little icon next to it. Then I see the actual distribution in a color-coded format with blue being low numbers, and yellow being high numbers. This is uniquely formatting for each statistic in a column here.

When you actually push the button and report here, you'll notice that the layout of the table itself starts out where each row is a gated population and a statistic, along with a parameter if necessary if you are enumerating medians or geometric means. Each of these rows, in the original table will become a column in the output table. Each row in the output, is the gated information from an actual sample in the order of the sample list here in the workspace.

Again, I'm reporting on a group of samples, which I specify here. I'm going to look at my all stain group and I'm going to iterate by sample through those samples in that group and generate a report showing these statistics for each of the gated populations or statistics in a table of whatever type that I set here. If I create the table to display, I get this table in a formatted window. I can always select within that window, and copy and paste all of that information into a blank Microsoft Excel file. Then I have generated that report. I'm just going to new workbook and press paste here, and I get my table with all of the headers being the gated populations and statistics that actually defines that population and the number that I'm interested in. Then each of the rows being the samples in the group, in the order that they are displayed in the workspace samples pane.

It is as easy to get data by copy and paste from the visual display, as it is to actually write a file, such as a text file here, to my desktop. When I create that table and go to it, I actually get this text document, which I can then open with Excel and take a look at. It is the exact same format as if I had copied and pasted from my displayed table in a text file, that you can now move around and share with your colleagues, send it to them, or link it to them in some way.

It is really up to you on how you want to get these numbers out of FlowJo. Either a copy and paste from display, or actually writing an external file itself. If I want to do a different type of file, I can do, again, a csv, Excel, or html formatted image. If I do the html format, then I actually get the formatting with the heat-maps or other visual formatting that you can apply under the visualize tab from the table editor. IF you do any of the other options, like just an Excel document, a text, or csv document, you just get the wrong numbers out. It is up to you how you want to actually display that.

I can even report this table right here within a layout. Maybe I want to report all of the information here on my actual batch layout for these samples. Let's go ahead and go to my table editor, and instead of saying I want to push this to file, I'm going to select to current layout. When I create the table, it gives me a warning to say that the numbers that they push out are static. You need to re-report if you changed the gating approach here, but now you get this table right on top of your layout. You can actually generate a report with the table right there in the graphical sense.

Those are the two different types of basic reporting that you can do from FlowJo. Either a graphical report, which you set up in the layout editor and then iterate through a group of samples to produce some sort of external file, or a new report batched within FlowJo. You can go to your file tab, and export the image that you displayed here as various image types, whatever your needs are. Then the actual, statistical reports, which are generated from the table editor, where you create a table, you drag in the gated populations and statistics that you want to display in your report. Optionally add a visualize tool here, such as a heat-map, to see the distribution, and then create the table to some external file format or just display it here within FlowJo. Then you can actually get those numbers out and see what's going on in your analysis.

Again, graphical reports are generated from the layout editor. Table reports are generated from the table editor, but the basic functionality of reporting is very similar in that you want to specify a group of samples within the workspace here that contains all of the samples that you want to generate a report on. Choose an iterate by option, many times that is going to be sample within the table editor and the layout editor, but sometimes you can generate reports batched over key-words or panel. Then choose the type of report that you want to push out. Whether that be a file, such as a text file in the table editor, or if you're in the layout editor, again you can generate a web page, PowerPoint, or a pdf file here. Then the final action to generate the report is to click the create batch report cog-button. That will generate the actual file that you can then use externally, outside of FlowJo, or review the information within FlowJo, depending on your output type.

This is Tim with FlowJo, just saying good luck with your analysis, and I look forward to talking in more detail about these features in the series of videos that we're putting together right now. Thanks for your time.