From basic Concepts to Advanced applications

Molecular Evolution & Phylogeny

By Ofir Cohen

The Bioinformatics UnitG.S. Wise Faculty of Life Science

Tel Aviv University, IsraelMay 2012

http://ibis.tau.ac.il/intro_bioinfo/phylogenyWorkshop/

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The Human Genome Project ("behind

the scene”) Venter et. al. , Science 292:1304-1351 (2001)

International Human Genome Sequencing Consortium, Nature, 409: 860-921 (2001)

The club resident J.D. Watson: Back2back with DJ. Venter -

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Genome Sequencing – Ongoing Revolution

The race is (still) on… The promise is huge…

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SRA(Sequence Read Archive)= raw seq. from next-generation machines

Trace=raw seq. from 90s machines)

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Darwin’s teachings–Tree-like evolution

Introduction – The tree concept

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Darwin’s teachings– common descent Introduction – The tree concept

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Common Descent – Modern evidence

Introduction – The tree concept

"The unity of life is no less remarkable than its diversity" "The unity of life is no less remarkable than its diversity" THEODOSIUS DOBZHANSK

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Mathematicians developed tools to analyze Trees

Adapted from Huson et al. 2008

 connected graph without cycles is a tree. Not a tree! (cycle) Rooted binary treeTree

Part of the wider field of graph theory

Bridges of Königsberg

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What is a Phylogenetic Tree? Phylogenetic tree:

(hypothetical) historical pattern of evolutionary relationships among organisms

Introduction – The tree concept

Homo

Bos

Mus

Rattus0.011

0.025

0.012

0.011

Gallus

0.038

0.066

0.01

Root

Node

Leaf

Branch

(Greek: phylon = race and genetic = birth)

sps

Horizontal branch length –proportional to evolutionary distances (unit = substitution / site)

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Molecular evidence of HIV transmission in a

criminal case

Introduction - Anecdotes

Metzker, Michael L. et al. (2002) Proc. Natl. Acad. Sci. USA 99, 14292-14297

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Criminal investigation

August 1994 a nurse tests negative for HIV. breaks off a messy 10 year affair with a doctor. Three weeks later the doctor gives his ex-mistress a vitamin B-12 shot

In January 1995, the nurse tests positive for both HIV and hepatitis C.

The doctor’s office records from the day are missing (but eventually found). The doctor had withdrawn blood samples from a known HIV patient and a known hepatitis C patient

the same day as the vitamin B-12 shot. The nurse had never had contact with either patient

Introduction - Anecdotes

Circumstantial evidence that the doctor injected blood from a patient of his into this ex-girlfriend….

How can this be proved using a phylogenetic approach?

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HIV – short background

Extreme heterogeneity Within each patient there are many different viral

strains ("quasi-species")

Introduction - Anecdotes

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History of the virus:

gp120(Gene tree)

PATIENT

VICTIM

CONTROLS

©2002 National Academy of Sciences, U.S.A.

Introduction - Anecdotes

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History of the virus:

RT (Gene tree)

VICTIM

PATIENT

Introduction - Anecdotes

Source sequences that are paraphyletic (other sequences are nested within them)

with respect to the recipient sequences provide evidence for the direction of transmission.

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Ernst Haeckel's Monophyletic tree of organisms, 1866

Reconstructing the tree of life

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Organisms classified into 2 domains: Eukaryotes including {plants, animals,

protists, fungi} Prkaryotes = Bacteria

Whittaker , 1969

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Reconstructing the Tree Of Life Carl Woese, 1977.  phylogenetic taxonomy of 16S ribosomal RNA

Critiques: Woese un-balanced the tree of life… (too much representation for microbial species)

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Phylogenetic analysis: Not only among organisms - Cancer

phylogenyA phylogeny of acute myeloid leukemia (AML) subtypes

Riester et al. 2010Liu et al. 2009

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Phylogenetic analysis: Not only in biology – Language evolution

Russell and Atkinson. 2003

Researchers learn the evolution of languages by treating them like genomes.

Instead of COGs (gene families), analyze COGNATES (words families)

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Reading Trees: Which tree is more accurate?

Reading Trees

Haeckel’s pedigree of man

Human "on top" – wrong!

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Rooted vs. Un-rooted treesRooted vs. Un-rooted treesRooted vs. Un-rooted treesRooted vs. Un-rooted trees

human

mousefugu

Drosophila

root

edge

internal nodeleaf

human

mouse

fuguDrosophila

root

edge

internal node (ancestor)

leaf

time

Reading Trees

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Gorilla gorilla(Gorilla)

Homo sapiens (human)

Pan troglodytes (Chimpanzee)

Gallus gallus (chicken)

How do we root a tree? Reading Trees

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Rooting based on a priori knowledge: Using Outgroup

Human

Chimp

Chicken

Gorilla

Human ChimpChicken Gorilla

Reading Trees

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Comparative Genomics – "All life is one"

Compare homologues sequences – Multiple Sequence Alignments

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Orthologs

speciation

ancestor

descendant 2 (e.g., dog)descendant 1 (e.g., human)

Orthologs will typically have the same or similar function in the course of evolution.

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Paralogs

Duplication

Evolutionary innovation - lack of the original selective pressure upon one copy, this copy is free to mutate and acquire new functions.

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Alignment and phylogeny are mutually dependant

Inaccurate tree building

MSA

Sequence alignment

0.4

Phylogeny reconstruction

Unaligned sequences

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Part II: Tools

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Multiple sequence alignment (MSA)

Several advanced MSA programs are available.Today we will use two:

MAFFT – fast and relatively accurate PRANK – distinct from all other MSA programs because

of its correct treatment of insertions/deletions

Tools - Alignments

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MAFFT Web server (& download option):

http://mafft.cbrc.jp/alignment/server/index.html Efficiency-tuned variants

quick & dirty or slow but accurate

Nucleic Acids Research, 2002, Vol. 30, No. 14 3059-3066© 2002 Oxford University Press

MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform

Kazutaka Katoh, Kazuharu Misawa1, Kei-ichi Kuma and Takashi Miyata*

Tools - Alignments

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Choosing a MAFFT strategy

qu

ick &

dirty slow

bu

t accurate

Tools - Alignments

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MAFFT outputSaving the output Choose a format: Clustal, Fasta, or

click "Reformat" to convert to a selection of other formats

Save page as a text file

A colored view of the alignment

Tools - Alignments

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PRANKTools - Alignments

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Classical alignment errors for HIV env

Tools - Alignments

CLUSTALW PRANK

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PRANK Web server: http://www.ebi.ac.uk/goldman-srv/webPRANK/

Tools - Alignments

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PRANK output

If you need a different format – copy the results to the READSEQ sequence converter: http://www-bimas.cit.nih.gov/molbio/readseq/

Tools - Alignments

38381. Download the sequence files from the web-site

http://ibis.tau.ac.il/intro_bioinfo/phylogenyWorkshop/Open "fahA.fas" in Notepad/Browser – these are 65 protein sequences in FASTA format.

2. Run PRANK web serverhttp://www.ebi.ac.uk/goldman-srv/webPRANK/

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Trees Reconstruction Methods

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Phylogeny reconstructionDifferent approaches (algorithms / programs): Distance based methods (e.g. neighbor-joining, as in ClustalW)

Fast but inaccurate Maximum parsimony (e.g. MEGA) Maximum likelihood methods (e.g. phyML, RAxML)

Accurate but slower Bayesian methods (e.g. MrBayes)

Most accurate but very slow

ABCDE

Guide tree

A

DCB

E

MSA

Pairwise distance table

Tools - Trees

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PhyMLThe most widely used maximum likelihood (ML) program Web server (& download): http://www.atgc-montpellier.fr/phyml/

Tools - Trees

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RAxML Web server: http://phylobench.vital-it.ch/raxml-bb/ Similar maximum likelihood (ML) methodology as phyML, but much faster

Faster results with bootstrap

Tools - Trees

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Bootstrapping

Now we have a tree, but what is the reliability of this tree?

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BootstrapA. Generate pseudo-data sets by sampling N positions Do not change the number of sequences. Resample (100-1000 time). 12345 100

1 : ATCTG…A 2 : ATCTG…C3 : ACTTA…C 4 : ACCTA…T

12345 1001 : AATTT…T2 : AATTT…G3 : AACTT…T4 : AACTT…T 11244 x

12345 1001 : TTTAT…T2 : TAACC…G3 : TAACC…T4 : TGGGA…T 4 7789…x

12345 1001 : AGGTA…T2 : AGGAC…G3 : AAAAC…A4 : AAAGG…C 15578… x

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BootstrapB. Reconstruct a tree from each data set.

12345 1001 : AATTT…T2 : AATTT…G3 : AACTT…T4 : AACTT…T 11244 x

12345 1001 : TTTAT…T2 : TAACC…G3 : TAACC…T4 : TGGGA…T 4 7789…x

12345 1001 : AGGTA…T2 : AGGAC…G3 : AAAAC…A4 : AAAGG…C 15578… x

Sp1Sp2

Sp3Sp4

Sp1Sp2

Sp3Sp4

Sp1Sp2

Sp3Sp4

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C. compute the majority rule consensus.

Sp1Sp2

Sp3Sp4

Sp1Sp2

Sp3Sp4

Sp1Sp2

Sp3Sp4

Sp1Sp2

Sp3

Sp4

67%100%

In 67% of the data sets, the split between SP1+SP2 and the rest of the tree was found.

Bootstrap

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1. Give "fahA.prank.phylip" or "fahA.mafft.phylip" as input to the RAxML webserver (don't forget to tick "Protein sequences" and “Maximum likelihood search” and enter your email)

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FigTree: tree visualization and figure creation

http://tree.bio.ed.ac.uk/software/figtree/

Manipulate a node

Manipulate a clade

Manipulate a taxon

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1. In case tree are not ready yet… download tree from website

2. Open "fahA.prank.phylip_phyml_tree.txt" in FigTree http://tree.bio.ed.ac.uk/software/figtree/

3. Play around with the different options and make a pretty figure!

1. Find out how to color specific clades, as below

2. Try each of the three options under "Layout"

4. Export a figure in PDF format(File Export Graphic…)

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Final Questions…