Construção de Filogenias Moleculares

Transcrição

1 TP Basic Topic 2 Construção de Filogenias Moleculares

2 Steps to Construct a Molecular Phylogeny Method Choose gene(s) of interest Example YFG-sp1 Identify homologs YFG-sp6 YFG-sp2 YFG-sp1 YFG-sp3 YFG-sp4 YFG-sp5 Align sequences Calculate gene tree YFG Your Favourite Gene

3 Sequence Alignment method of sequence arranging to identify regions of similarity variable vs. conserved regions identify functional motifs form the basis of database search methods are the starting point for evolutionary studies

4 Sequence Alignment method of sequence arranging to identify regions of similarity Conserved region (high similarity) Variable region (low similarity)

5 Sequence Alignment Global alignment: tries to align the whole of one sequence to the whole of another Local alignment: looks for high-scoring matches between any part of two sequences generates similarity islands (sub-alignments) of high-scoring matches

6 The Problem of Sequence Alignment 9 matches 2 mismatches 8 not aligned ( gap ) Instead, we migth have written the sequences: 10 matches 1 mismatches 8 not aligned ( gap ) Total score DNA = x(matches) y(mismatches) z(gaps)

7 Multiple Hits Specie 1 Specie 2 TTGCTAATGCT ATGCAAATGCG TTGCTAATGCC ATGCTAATGCG ATGCTAATGCC

8 Models of DNA Sequence Evolution: why? To infer the actual number of substitutions from the observed number of differences distance calculation between sequences phylogenetic inference topology and branch lengths The standard set consists of 56 different models: Jukes and Cantor s one parameter model (JC) Kimura s two parameter model (K2)

9 Models of DNA Sequence Evolution The simplest substitution model One simgle overall substitution rate Distinction between transitions and transversions Two different substitution rates doi: /nrg3186

10 Phylogenetic Trees Illustrate the evolutionary relationships among genes and/or organisms intuitive way to infer relationships reconstruction of the evolutionary history inference of hypothetical common ancestors facilitate the testing of hypothesis Use phylogenies to test hypothesis about biologically important questions

11 Sequence Alignment and Phylogenetic Trees The changes between two sequences can be used to understand the evolutionary relatedness between these sequences

12 Phylogenetic Trees: Building Methods mathematical/statistical methods for inferring the tree topology as well as the lengths of the branches in the tree Method Characteristics Pros Cons Neighbor-Joining Distances the original data is transformed into a distance matrix; gives only one possible tree fastest method information is lost Maximum Parsimony Maximum Likelihood Characters use the raw data; possible trees are compared and each is given a score the best tree is the one with the smallest number of subst. uses all data (more accurate); allows statistical tests of explicit hypothesis uses only the informative sites; doesn t make use of evolutionary models computational intensive

13 Bootstrapping A method to randomly resample columns from the alignment (with replacement) to create a new dataset of the same size as the original alignment.

14 Bootstrapping Evaluates how often a particular branch in a tree is present in exactly the same topology It is not a test of tree accuracy It only gives information about the stability/reliability of the topology (given the data) High bootstrap values give confidence to the tree topology, not as being the real tree but as being a robust/stable one

15 The Eukaryotic rdna The interest in rdna comes from 3 important properties: codifies for ribosomal RNA virtually present in all cellular organisms share a common evolutionary history (homologues) rdna sequences are highly conserved different regions have different rates of nucleotide substitution (molecular chronometers) 18S 5.8S 26S ITS1 ITS2 conserved = potentially suited for phylogenies of genera or higher level taxa variable = potentially suited for phylogenies of species or lower level taxa

16 Sessão Prática 1) Recolha de sequências nucleotídicas referentes à região ITS1-5.8S-ITS2 de várias espécies de fungos a. Copiar simultaneamente todos os números de acesso indicados na tabela 2 para o campo de pesquisa da base de dados GenBank ( b. Descarregar os resultados através do campo Send to e guardar o ficheiro em formata FASTA com o nome: ITS_GEM1516_14Abril2016.fasta

17 Sessão Prática 2) Abrir o programa MEGA7 e criar um novo alinhamento a. Selecionar Create a new alignment b. Depois, ir ao menu Edit e selecionar Insert sequence from file

18 Sessão Prática a. Importar o ficheiro ITS_GEM1516_14Abril2016.fasta b. Com todas as sequências selecionadas iniciar o alinhamento com MUSCLE c. Cortar o alinhamento de forma a obter apenas a região ITS1-5.8S-ITS2 (manter entre as posições ) Q1) Existe alguma região no alinhamento que pareça ser mais conservada em relação a outras regiões? Porquê?

19 Sessão Prática d. Exportar o alinhamento resultante para formato MEGA 3) Calcular o modelo de evolução nucleotídica que melhor se adequa aos dados d. Selecionar o modo Partial deletion com 90% de cutoff para lidar com os gaps resultantes do alinhamento

20 Sessão Prática 4) Agora, vamos construir uma árvore filogenética utilizando o método de Maximum Likelihood com 100 replicados de bootstrapping

21 Sessão Prática a. Definir o modelo de evolução nucleotídica que mais se adequa aos dados (e que foi inferido anteriormente). b. Com os mesmos parâmetros...

22 Sessão Prática c. Depois de apresentada a árvore, forçar a raíz (outgroup) na linhagem de Taphrina deformans. Q3) Qual é o nível de confiança que se pode ter na topologia desta árvore, em relação aos grupos principais? Porquê? Q4) Comparar esta árvore com outra árvore filogenética do Reino Fungi (e.g. Fungal phylogeny.pdf na página da disciplina). Ambas as árvores representam o Filo Ascomicota de forma semelhante?

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