 Here students, in this module we are going to continue on building our concept of multiple sequence alignment or simply MSA. So the multiple sequence alignment or MSA simply put helps us compare multiple biological sequences towards comparing them. Moreover, this comparison can help us to infer some very useful information regarding these sequences. For instance, you can find out consensus sequences between multiple sequences that is those portions within the sequence that are present in the other sequences as well and are very useful for some specific biological function. Moreover, the MSA can help you to construct the evolutionary relationships between these multiple sequences as in which sequence evolved from which other sequence and which sequence is the oldest one and which sequence is the latest result from the evolutionary process. Such a study is called phylogenetics or simply phylogeny. To apply MSA, you put multiple sequences against each other and you compare them and this can help you to identify secondary, tertiary or veterinary structures of the proteins as well. So this can be very useful if you want to study the protein function. This topic will be considered in the protein structure chapter. Moreover, as I just mentioned phylogeny can be extracted from such sequence comparisons as well and this topic will also be dealt in the upcoming chapters. Towards the MSA methodology or to understand how MSA works, if you remember, pairwise sequence alignment was simply a comparison between two biological sequences or two nucleotide sequences or two amino acid sequences. In MSA, you would want to do it for multiple such sequences. So therefore, you simply repeat the process of pairwise sequence alignment in a way that you end up aligning multiple sequences. So let's take a look at the process in detail in a step by step manner. So first of all, you find and list all the sequences to be compared and this can be done by simply finding their ID. Next, you compare these sequences that you already listed and you build pairwise alignments. So once you have performed pairwise alignments, then you move towards clustering. So clustering is essentially a strategy to make pairs of most similar sequences. That is, you perform clustering, you form pairs of sequences that are most similar to each other. In this way, if you have four sequences, for example, and you want to have a multiple sequence alignment, then two sequences that are closest will form the first pair and the other sequences that are closer or similar to each other will form another pair. So this process is called clustering. So next, using this clustering information, you create a hierarchy using binary trees. Or simply something like this. Next, you align the two best sequences and you keep them with you and you repeat the process for the next pair of best aligned sequences. So essentially, if you have two sequences, one and two given by A and C here, and you have aligned both of them and for the next B and D sequences, you have aligned them as well. So now you want to create an alignment between this pair and this pair. So you align the two pairs. Dear students, this is a very important point to remember. So in the first step, you have aligned sequences that are similar to each other, thereby creating pairs of sequences. And now you are aligning the pairs themselves. So in conclusion, multiple sequence alignment can help you compare multiple sequences in a go. And this process can help you to find out evolutionary relationship between sequences, also to predict secondary structures of proteins and moreover to create phylogenetic trees as well. An important thing to remember in this regard is that you need to consider only those sequences that are divergent or different from each other by less than 80%. So if you have two sequences that are similar to each other more than 80%, then you don't want to do that because this will create a bias towards these two sequences. And lastly, Clustall is a tool, it's an online software that can help you to perform multiple sequence alignment.