 Hi, I'm Julia Giacchella and I'm studying medicinal and biological chemistry at the University of Edinburgh and Since I've completed bioinformatics internship. I basically fell in love with galaxy and I Created a tutorial on trajectory analysis using Monopole-3 And I'm going to show you now how it works So I am using human cell atlas dot use galaxy dot EU Because it's quite reliable when it comes to searching the tools But the tutorial should work fine on any galaxy you instance that you're using So you don't have to have another window open with the tutorial Obviously you can but it makes things easier when you click on this icon Which opens the tutorial mode and you can easily navigate to the tutorial you're doing in a moment, so We will be using data from case study series and You can just click on the tutorial and follow the instructions The easiest way to get data we'll be working on is to copy those two lines and then just go to upload data Click here and just paste it Then close the window and wait until your files are loaded Once it's loaded navigate to tutorial mode again and see what is the next step Normally those tools should be clickable, but if they are not just go to the search box and Type the name of the tool that you need to use So we are now extracting observations and variables from on data and If you don't want to type the name of the tool again, just click on the repeat arrow and Just change the parameters Now quickly rename the file so that you know what is what We are going to remove macrophages from our data set So the information about cell types is stored here and to check the number of this column You can look at this preview window and you can see that in our case. It's number 22. So keep that in mind Check which tool we are going to use next find it and Specify that we are going to filter out all macrophages from the column 22 we've just modified the file with cell Information that we are going to use for monocle free. So rename that file so that it's easy to track Alright, so back to the tutorial mode again Check the next step find the tool and now we are going to replace The title of one of the columns which stores gene names Because monocle would need to use the title called gene short name instead of the symbol So we can check that it's column number three and simply replace that title And this is the file that we are going to use as input data for a monocle free So let's rename the file so that it's easier to navigate afterwards Okay, so we've prepared information about cells and genes. So now let's move on to the expression matrix First we need to extract the full matrix from and data in the same way as we did with observations and variables We want to keep things tidy so again rename the file So let's get the genes and cells IDs from the files. We've already prepared as input files And we'll do that by cutting the columns out of the files All right, so getting cells IDs went fine But as you can see here Galaxy doesn't show the genes file that we want to get the genes IDs from So the reason for that may be incorrect format of the genes file So to change that just go to this file click on data types and change the data type to tabular Okay, cool. It helped. Now we can see the file we need So we just repeat the same steps as before a quick note about data types is Always do check if they are correct. And if you get any error Make sure that the data type of the file you're using is Compatible with the operation you want to perform We will now perform a series of steps to modify the unprocessed matrix By using genes and cells IDs from the files. We've already prepared We can now compare them to columns and rows in our unprocessed matrix and Select only those that corresponds to each other By this operation we added a column that is the same as the one we already have So the next step in the tutorial will show you how to remove it The genes IDs are currently in the first row So we need to transpose the matrix and only then we can perform the same operations as before We are now modifying the expression matrix Using genes IDs in the same way as we did before using cells IDs Once it's done the information we have in our cells file genes file and Expression matrix file is consistent. So we have now prepared all the files for monocle free So let's get started with monocle The workflow is quite straightforward and we will start with monocle free create tool and Now finally you can use those files that you have been preparing all that time Make sure that you choose the correct format of the file and in our case We are using TS file, but it's useful when you work with your own data in other formats The next step is preprocessing and we will accept the default settings Except dimensionality of the reduced space, which we will set to 250 The explanation why we use this value is in the tutorial So have a read through that But you are very welcome to play around this value so that you understand what it changes Next step is dimensionality reduction. So make sure you choose the preprocessed file And accept all the defaults Again, you can play around the dimensionality reduction methods All are also shown in the tutorial, but we are using you map because it gives the best results So now there is a blog of plotting in front of us because it would be nice to see the results of our work, right? But it's quite straightforward. Just go to plot cells then choose the file that you want to use for plotting and Change the cell attribute, which is the name of the column containing particular information about our results In our case that was cell type, genotype, batch and sex So you can always go back to the files We use at the beginning to check How those names are spelled to avoid any typos and errors? And actually you can stop here and switch this toggle bar from yes to no in the label cell groups Because otherwise you will get the labels on the plot and that won't be very Legible, which you will see in a moment Okay, so this is a cell types plot and You can see that the labels are very small and it would be better to have them next to the plot as a legend But let's just have a look at the all generated plots and change their names accordingly The full analysis of the appearance of the plots is in the main body of the tutorial So have a look at that to get more understanding of the biology behind The next step is clustering. So we will assign ourselves to clusters and partitions We will change the resolution value which corresponds to the granularity of the clusters and the r-value Which is related to partitions You can see the output here So almost all the cells were assigned to one partition what we wanted to obtain and Also the clusters correspond nicely with different cell types that we have in our data set So those are the genes that expression we are interested in the most So let's just copy and paste those genes and talk to them and this is how they look There are clearly some correlations that you can read about in the main tutorial text Speaking about the genes we can now use top markers tool to Generate a table with the genes most expressed in particular clusters or cell types there is the PDF file with a plot of the results and Also table which summarizes the results Now is the crucial part which is learning the trajectory graph Fairly simple. Make sure you choose the input file as the last CDS file generated by monocle Accept all the defaults click and execute and wait In the meantime, you can use another tool to plot the learned Projectory graph onto the cell types as you can see the trajectory graph is just a line going through the whole data set But we will need it to order the cells along this trajectory and to do so we will use order cells tool and visualize the output in pseudo time To order the cells monocle needs a starting point You can specify the starting point in different ways But we will use double negative cells as starting point and we know that they are stored in cell type Column, so let's just type in the dose values Finally, we can generate a plot in pseudo time just make sure that your input file is the output of order cells tool and Change the cell attribute to pseudo time So all the work you've done was to see this beautiful colorful plot that shows development of T cells I'll say that once more for more biological insight, please do have a look at the tutorial Now there is actually a lot of other things you can do to analyze this data more in depth But in Galaxy we will only use the tool for differential gene expression There is a bunch of information that you can use for downstream analysis But the aim of this tutorial was to show you the tools of a label in Galaxy to perform Trajectory analysis using monocle free So if you have any questions or comments, please do get in touch. I'll be very happy to hear from you So I hope you will enjoy working with Galaxy and monocle and I wish you happy coding