 Dear students, in this module, I'm going to continue on elaborating the pairwise sequence alignment. You already know that if you are given two sequences and you want to align them against each other or compare them with each other, so you can slide them across each other and maximize the number of matches between the two sequences. The gaps may be inserted to accommodate insertions or relations, and we're going to look at them in detail by an example in this module as well. Your objective is to maximize the number of matches between the source and the target sequence. So let's review the three differences, the three sources of differences that can be there in biological sequences. So the sequences can differ from each other in three different ways, substitutions, insertions, relations. So if there are substitutions, then essentially, as you can see here, C has been replaced by a G. So the sequence ACGA has become AGGA. This substitution is actually a mismatch because C does not match G. The rest of the three nucleotides, that is, A, GA, are matching very nicely. So there is only one difference between these two sequences, and this difference can be classified as a substitution. Next, let's take a look at insertions. So if you have a sequence ACGA and you're comparing it with another sequence ACCA, then you can guess that there is an extra C here and this is an insertion. So the two sequences will match very nicely except for this C in the middle. So this is an insertion. Now let's take a look at deletions. In case of deletions, if you have a sequence ACGA and you're comparing it with another sequence AGA, you can clearly see that this C cytosine has been deleted from this sequence. So there are these three sources of variations that can be there in the sequences. So if there are no change between nucleotides, for instance, A matches an A, then it is classified as a match. However, if there are insertions or deletions, then we can create a gap. Let's see, ACGA will actually be compared with this sequence AC dot GA. This dot will mean that there is an insertion that has happened. Let me clearly write that again for you. If you're looking at this sequence versus this sequence, let me write the bigger sequence on top, then you can see a dot here. So this is a gap. The third option that you can have is the substitution which will result in a mismatch. This was shown to you in the first example where ACGA versus AGGA will result in a mismatch in C and G. So these are the variations that you need to consider while aligning sequences. And now I'll come to optimal alignment. So let me give you an example what an optimal alignment is. So let's consider a sequence. This is a rather longer sentence than the next. The second sequence is this is a short sentence. So if we try to align these two sequences, then we have two possibilities as an alignment. In the first one, it will start like this is a, it will be a perfect match. Then O will match with an O and sentence will match with the sentence. For the rest, you can insert the gap dots. Of course, if you look carefully, then there are a couple of mismatches as well, SHRT. In the second case, the two alignments were the same till here. H was matched and so was R. E was matched as well. EN was matched. CE was matched. And the rest is all gaps and a few mismatches as well. So if I were to ask you which alignment is better, this one or this one, what will be your answer? So it actually depends. In my opinion, this alignment makes more sense. While this alignment does not make a lot of sense. If you are looking at the English language, but for the biological sequences, this may just be more meaningful. So we'll be studying this in the later modules as well in detail. Before we go on, please do not forget that the biological context is very important to decide the answer to this question about which alignment was better in this case. So in conclusion, the global and local pairwise alignments, they help you to compare either the entire sequences or portions within the sequences in an exact fashion. Indels lead to gaps in the alignments while substitutions may lead to mismatches.