 Dear students, in this module, we are going to run through an example of multiple sequence alignment using the progressive approach and guide trees. So, to warm you up, the background of this module is that multiple sequence alignment is a very useful strategy to compare multiple biological sequences. Towards that, progressive alignment approaches are employed and they are used repeatedly so that we arrive at the best multiple sequence alignment. Also, doing that will take a lot of time and you must take notice of that and we will see later how we can overcome that. So, let's start with our example. So, here we have an example of the globin protein from multiple species. So, to start you up the distance between two sequences or simply the difference between two sequences is equal to the number of identical residues that is same or similar residues over the number of aligned residues in the pairwise alignment. So, you take this ratio and you subtract this from one and you arrive at the distance or difference between the two sequences. So, once you have the distance between all sequences then you can construct something called a distance matrix. The distance matrix is provided here for you and multiple types of globens are listed here. So, once you compute the distance between each one of these proteins you can fill up this matrix. As you can see this matrix is already filled up using this formula after doing pairwise sequence alignment. So, you will notice that HBA horse that is globin from the horse is very similar to globin from the humans or HBA human. So, HBA horse has a distance of just 0.13 from the HBA human. So, the distance is very small which essentially means that these two proteins are very similar. Remember that also HBA horse here is also not very far from HBA human as shown by this distance which is 0.17. However, of course 0.13 is smaller. So, these two proteins are much more similar while these two proteins are a little less similar to each other. In this way all of these distances that are listed in this distance matrix describe the difference between the corresponding sequences. So, using these distances we can create a sequential branching wherein HBA human and horse which had a distance of just 0.13 are the closest. This is followed by HBB horse and HBB human as you just saw and the entire tree is constructed like that. So, this is called sequential branching and you can construct this using the progressive alignment. Also, you can construct another form of tree that is the guide tree and in this since you already saw that HBB human and HBB horse are similar while HBA human and HBA horse are similar as well. So, if you look at this guide tree then this distance that is 0.65 plus 0.55 is almost equal to 0.13 in this case or 0.12 while HBB horse and HBB human have a distance of 0.17 which is equal to the sum of these two approximately. So, using these distances and constructing these portions within the guide tree you can eventually construct the entire guide tree as well. So, progressive alignment therefore is very useful in comparing multiple sequences and constructing the guide trees. So, the alignment results as shown here so you have the globin proteins listed here and their sequences shown here. The regions that are aligned as consensus sequences are shown here in red while the other portions are left out. So, this is the multiple sequence alignment result using progressive approach. So, in conclusion the multiple sequence alignment can be better performed using specific clustering approaches such as the guide tree and this can help you to better compare the sequences towards consensus sequences as well as their phylogeny. Clustall is a free tool that is available which you can use it is available online to perform all of this for you in a second like that.