Multiple FCS File Analysis with VenturiOne for your flow cytometry data
So, now we will look at how you can analyse a whole series of files. As before, you open the required files that you need. And, as you load the files into the playlist, they switch from grey to black and once they are black they are fully loaded into the playlist and are ready to be analysed. If you have an existing workspace, you can apply to that workspace. But, what we’ll do in this case, is start from scratch and show you how to create a workspace and apply that workspace to the whole playlist. If you have saved compensation settings, you can copy and paste those settings to the playlist which is what we’ll do in this case.
So, right-click on the compensation cell of the first file and choose replace compensation and pick the compensation file that you want from the list and open it. That has now applied to the compensation to this particular file. I can then right-click in the cell again and select copy then select all of the files in the playlist and right-click and paste the compensation to all the files in the list.
Now I will create the plots that I’m interested in. I choose the region type, click and drag the region over the population of interest and adjust that region as necessary. Then, I will add the fluorescence plots to the plots area which show the data from the region we’ve created. Then, I’m going to select the plots I want my analysis regions on and select the region type and press ctrl and click over the selected plot and adjust each region as necessary. To apply the workspace that I’ve created to other files, I can right-click on the workspace cell
of the file and copy and right-click and paste on the workspace of individual samples. Or, I can right-click and select apply to the playlist, and then that will apply the workspace from the current file to all the files in the playlist.
You can also save the workspace by selecting the save as option, naming the workspace, and pressing save, and then you can apply that to all files within the playlist. This then means that if, at a later date, you’ve got similar samples you don’t have to create a new workspace. You can open a workspace that you created earlier and use that for your new samples.
We will now move on to creating a report. We’ve seen how we can create a report by sending individual items from the workspace to the report. But, when you have multiple files, there are two ways in which the system can work. The system can work in playlist mode and this enables you to send any plot from any of the available files to a single report. Or, if you were dealing with panels or repeating sets of samples then the other mode is a group mode. And the group mode allows you to create one report and apply that report to separate groups, and each group being a panel or a set of samples.
A group can be anything from one to four hundred samples. Only plots from samples within the group can be added to the report in group mode. Whilst in playlist mode any plot from any sample in the playlist can be added to the report the mode you choose depends on what you’re trying to achieve.
So to create a playlist report, simply pick plots from any of the samples by double-clicking on the sample, and right-clicking on the plot you’re interested in. And then, choose to send plots to report. In the reports tab of the ribbon, you will see a report wizard button. This wizard will help you create a report. A playlist report allows you to pick the same plots from all samples and add them to a single report. And a group report will allow you to pick plots from within the group and then apply those options to the rest of the groups in the playlist.
So let’s start with a playlist report. So, I will select a playlist report and then the system looks at the plots that are present in the first file in the playlist and allows you to pick which plots you are interested in. The same plots from all files in the playlist will be used and you can click on the plot and see a preview of that plot on the right-hand side. Pick the plots that you were interested in, and then press the next button.
If you want to display statistics on the report, check the display statistics checkbox. Then you can choose to have the statistics displayed either per plot or per playlist. If you choose per playlist, all of the statistical boxes will be displayed at the end of the report. If you choose per plot, you have the choice of the statistics being displayed either to the right, to the left, below, or above each plot. So pick per plot and the preferred statistic position and then press the next button. Here you can select what statistics you want to appear by adding or removing statistic types from and to the right-hand side. Once you have what you require, press the next button.
The next option allows you to choose how many plots appear per page. The more plots you want the smaller they appear and, if you have statistics per plot selected, this will restrict how many plots per page you are allowed. In this case, four plots per page is what I’ll select, and then you can choose the display order of the plots. So, you can either display all plots from the same file together or you can display the same plots from all files in the playlist together.
Then you can choose how to separate your sets of reports. In this case, we’ve got display plots from the same file together selected so we can use a separator to separate the results from each file. We can either draw a line between each file or start each file on a new page. Now select finish and the report will be created. In this case, we have three plots per file and four plots displayed per page. So here is sample one, and then sample two, and then sample three, etc.
With the playlist report we saw how we can report from all samples within the playlist. If you want to split your playlist down into smaller groups, you can. You can do this by going to the playlist tab of the ribbon and enabling the user groups and then set the group size to the number required. For example, this would typically be from a panel that you run it in multiples. And you would set this number to the size of the panel. In this case, say we are going to have eight samples in a group, so I have set the group size to eight and I am going to create one report that is applicable to the first eight samples and the second eight samples. And the report that is generated will automatically be carried over to the second group. So, I will go to reports and as before choose the report wizard. This time the group report is selected and the group size of eight is already pre-entered for us. When we go to the plot selection, we can now select any plots from any of the eight samples within that first group. So, what I’m going to do here is select all the relevant plots from each of the eight files within the group. And then these plots will be sent to the report and shown all together. Almost done.
Then, as before, we can choose whether to display statistics and where they will appear. Then we can choose what statistics are displayed and choose how many plots per page and the separator that we want to use. Then press finish and the report is generated. Now we see that we have one report that incorporates all of the eight files in the first group. If you then click anywhere in the second group of samples, the entire report will be updated to show the results from that second group. This means you only have to set up the report once for one of the groups, and then you can use it for all subsequent groups in your playlist. To see all of the results at once, you can print the results to PDF and save it to a file.
So that’s the end of the multiple file analysis tutorial. We do have a little bit more to cover on reports but that will be covered under the custom fields tutorial, where we actually talk about creating custom fields rules and calculations for putting into the report.
To try out multiple FCS file analysis, download your free trial of VenturiOne here.