VenturiOne – The Basics of Flow Cytometry Data Analysis

 

Our rapid data analysis platform to process, interpret, and report on your FCS data files. Find out how to process multiple files, use our compensation wizard, autogating and more on VenturiOne.

 

VenturiOne – The Basics of Flow Cytometry Data Analysis

When you open the VenturiOne software, the application looks like this. At the top, you have an Office 2007 style menu system with ribbon tabs that take you through the options within the software. To the left of the application, by default, you have the playlist. And then, on the right of the application, you have the workspace. The application workspace is divided
into several tabs which are the plots tab, the results tab, the overlays tab, and the reports tab.

Starting with the plots tab, the plot workspace is split into two areas: the plots area and, below that, the previews area. We will see the function of these as we start to load a sample. So, what I’ll do now is just load a single sample and then take you through the analysis of that single sample. We can look at doing more complicated analysis after this. So, I can either select open list mode and point the system to where the file are, or if I have a folder with data in I can highlight my data files and drag them onto the playlist. VenturiOne will allow you to select up to 400 files to go into the playlist, but, let’s start off nice and simple by selecting a single file.

So, I will select open this mode and select a single file to look at. A file occupies a single line of the playlist. So, every file that is loaded in will occupy a separate line and you will see the name of the file listed in the list mode column. Next to this, is the workspace column and here a workspace can be loaded into the playlist, or you can create a new workspace. And next to that, is the compensation column and, again, compensation data can be loaded into the playlist or you can create new compensation settings, or read the compensation data from the original file. The FCS key column allows you to display extra information from within the file by selecting a keyword. For example, a sample identifier. Once selected, that keyword will be displayed for all the files within the playlist.

First thing to do when you have loaded a file, or a set of files, is to go to the parameters tab and turn off any parameters that you are not interested in. Some cytometers collect multiple versions of parameters and sometimes have parameters that you may not be interested in. So, if you uncheck these parameters in the parameters tab, this will simplify the view in the workspace and other areas of the software so you can concentrate on what you are interested in.

So, when you do load a file the system by default will create a forward scatter against side scatter plot. And it would also show you the preview plots of all the parameters against each other. So, here we can see the preview plots of all the ungated data within this file. And we also have the forwards inside scatter plot at the top.

If I am only interested in a particular population of cells, say the lymphocytes, I can use either the home tab or the analyse tab of the ribbon to select a region type to use and then click on the plot and drag the region over the population of interest. In this case, for an elliptical region, I can rotate and resize by dragging the region to a new position. When you click on a region in the plot, the previews underneath will update to show only those events within that region. So, if I click outside the region in the plot, the previews will now update to show all events, and then if I click back onto the region it will now show the events in that region. On the plot where the region has been drawn, if the region is highlighted, I can hover over the region name and move it elsewhere within the plot.

If you want to change the size of the font, you need to go to the analyse tab of the ribbon. The analyse tab controls everything to do with the plots area. At the end of the ribbon, there is a fonts button, and here is where you can increase or decrease the size of the font. Or you can leave it on the default settings which we’ll do here.

To add more plots that are gated on the region I’ve created around the lymphocytes, I can now use the preview plots. Double-clicking on a preview plot will add that single plot to the clots area above. So, if I double-click here, and then look to the top, my new plot is actually there. As a good visual aid, the plots are colored to represent the region that they are gated on. So, carry on picking plots that you may be interested in analysing further or adding more regions too.

But, if at any point you make a mistake, the system has infinite numbers of undo’s so you can either press the ctrl and z button or click the undo icon in the top left-hand corner. And this will take you back however many steps you need.

The previews tab can be used to control what you see within the previews area. If you have large numbers of parameters, the previews area can sometimes look very busy and show you a really large number of plots. If you want to zoom in and out of those plots, you can move the mouse over the preview area, press the ctrl button on the keyboard, and use the mouse scroller to actually zoom in and out like this. You can also use this on the plots area above as well. Alternatively, you can use the slider controls at the bottom right-hand side of the application: there’s a button for the plots, the previews, the overlays, and the galleries. Select the one you want to use, and then use the slider to adjust the size.

You can also make it simpler by reducing the number of parameters as we have already seen. You can also use these filter options in the previews tab of the ribbon. The fluorescence only button will display fluorescence plots only. The gate filter will show fluorescence plots that haven’t been used for gating. So, by using the fluorescence only option, we’ve reduced the size of the preview plot area. So, as you have seen already, we can add a single plot to the plots area by double-clicking on that plot, but we can also add a whole row of plots by selecting the plus sign at the end of the row.

We can add a column by clicking on the plus sign at the bottom of the column of plots that you’re interested in. We can also add a whole set of plots to the plot space by pressing either the upper triangle or lower triangle plus sign. So, however the preview plots are gated, those gated plots will be added to the plots area, allowing you to now add regions to them. To add a region to the plots, go to the analyse tab or the home tab. The analyse tab, as I said earlier, deals with the plots area. So, I’m going to add a quadrant region by selecting the quadrant region button and then clicking on the plots which we want to add the quadrant too.

If you want to add quadrant regions to a lot of plots at once, you need to highlight all the plots that you want to add the region too, select the quadrant region type, press the control button on the keyboard, and then click on one of the highlighted plots. And that will add a quadrant region to all the selected plots. You can then look at moving the quadrant regions on an individual basis if you need to.

If you want to add single parameter histograms, then you can add those by, again, making sure you’ve clicked on a region, and make sure the previews are gated on that region, and then add the single parameter histograms. And then we can add a linear region to a single parameter plots by highlighting all the single parameter plots, selecting the linear region, and then hold the ctrl button down, and click and drag the region out. By default, it sets all of the regions in the same position that you set the original, but you can go through and optimise each one as you need to.

By default, the system will show you one statistic per plot. You can choose which statistic is selected in the statistic tab of the ribbon. Only one of these values can be selected, and the plots will then update to show the statistic value that you have chosen. For example, here you might want to select the number of events, or the percentage total, or percent gated. To see all the results on all the regions, you can go to the results tab of the workspace. And here, it displays by default a flat list of all the results from all the regions. In the results tab, you have the option to sort and group the results which will make them easier to look at. You can select additional statistics from the ribbon tab and the result table will update as you do that. And you can also group the results by dragging a table heading into the gray area at the top of the results table.

Grouping by x and y parameter can often give you the best view of the statistics, especially when looking at a single file. Or, if you are looking at multiple files, you can group by the file name and that will probably give you the best view of the data. To remove a grouping just click on the item you want to remove press ctrl and then drag the item out of the column header block.

The next thing we can do is to create a report from the plots and the results that are displayed. We can send single or multiple plots which are of interest to a report. And to do this, we need to highlight any plots that you want to send and then right-click over them, then choose the send the selected plot to the report option. And this will send the plots to the report. The send selected plots stats to the report option will just send the stats from each of the plots to the report. And the send selected plots and stats to the report will send both the plots and the stats that you’ve selected to the report.

Alternatively, you can send the selected plots to an overlay. Each time you select to send plots to the report it will send the plots to a new page of the report and that’s to make sure the items you send do not get overwritten. So, potentially you will need to sort the organisation of the report out once you have sent the items.

Once you have hit send to report, go to the report tab of the workspace. And here you will find the plots that you actually sent to the report. You can move the plots around and resize the plots as much as you like. You can add header and footer information that will appear on each page. You can add text boxes and import statistics and import images all to create a customised report. If you send statistics with the plots to the reports, you will get a statistics box for each plot. You can move the plots and the statistics around as much as you like.

You can customise the statistics that you have sent. So, if you want to reduce the stats that are present on the report, you highlight the report table and go to the statistics tab of the ribbon, and take out any statistics that you are not interested in and they will be removed from that table. If you have multiple statistic tables selected on one page, you can apply those changes at the same time.

For a more in-depth look at how to create a report please refer to the reports training video.

To try out VenturiOne with a free 30 day trial, please click here.

 

 

 

 

cytobee fcs data file managementmultiple fcs file analysis flow cytometry data