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INTEC Prof: Carlos Vergara Castillo. GENÓMICA LAB 3 — MULTIPLE SEQUENCE ALIGNMENT ___________________________________________________- [Software needed: MEGA 7 – see the end of the lab for download link, and web access] In this lab we will learn how to align sets of nucleotide sequences from different organisms to identify their similarities and differences. Multiple sequence alignment (MSA) is an extremely powerful methodology underlying many bioinformatic analyses. It can be used in conjunction with a wide range of evolutionary and functional analyses to identify everything from evolutionary relationships, to functional motifs and interaction domains. Reliable MSAs identify homologous residues among related sequences, data which are absolutely essential for phylogenetic analyses and motif identification. There are many methods available to produce MSAs. Fortunately, producing a MSA is very easy. Unfortunately, it is often just as easy to produce a poor MSA as a good MSA, and a bad MSA can weaken or completely compromise your analysis. In this lab, you will become familiar with a few of the MSA applications available. Additionally, you should also come to understand that producing a good MSA is a learned skill, and very often requires more than a simple plug- and-play approach. While all good MSA applications will give you their ‘best’ solution, often, some manual adjustment and biological intuition will produce an even better alignment. There are two major classes of MSA: global alignments and local alignments. You need to be familiar with the difference between these two, and use the application that is most appropriate for your particular needs. The single biggest mistake made with MSAs is assuming there is one tool that works equally well for all jobs. For example, most people simply use Clustal for their MSAs. While Clustal is a powerful application, it is completely inappropriate for a large number of alignment problems. Box 1. Global and Local Alignments Global alignments are those in which the set of sequences to be aligned are similar across their entire length. It is important to recognize that while global alignments assume you can align the whole sequence, they do permit gaps and differences in lengths (i.e. when some sequence are much longer or shorter than others). Local alignments are those in which there is similarity only at particular sub-regions of the sequence. Use a local alignment, for example, when part of the sequence is conserved, yet the rest has diverged so much that no similarity remains. The figures below (adapted from the MAFFT site) nicely illustrate the differences between global and local alignment. In each figure, “X”s indicate alignable residues, “o”s indicate unalignable residues and “-”s indicate gaps, A global alignment would be appropriate in this case since the sequences are similar across their entire length (from one end to the other):
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Clustal Clustal is by far the most popular MSA application. It is a global alignment tool that can be run on nearly any computer platform. It is also available through a number of web servers,
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  • Fall '19
  • DNA, Sequence alignment, MAFFT

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