workshop tips to align dna sequences and build phylogenetic trees

7/28/2019 Workshop Tips to align DNA sequences and build phylogenetic trees

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Tips to align DNA sequencesbuild phylogenetic treesA QCBS workshop - Annie ArchambaultJuly 2013


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Intro

Level and content: practical How to get sequences,

To align

To build a tree

Your expectations

Tomorrow: Your turn

Participants

Jorge Ramirez

Pedram Samani

Genevieve Guay

Annie Archamba

Nomie Blanchet

Ariane Pelletier


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Get sequences In GenBank: The database

Use the Nucleotide , the Gene , or the Populatio

https://www.ncbi.nlm.nih.gov/nuccore/advanced

https://www.ncbi.nlm.nih.gov/popset/
https://www.ncbi.nlm.nih.gov/nuccore/advancedhttps://www.ncbi.nlm.nih.gov/popset/https://www.ncbi.nlm.nih.gov/popset/https://www.ncbi.nlm.nih.gov/nuccore/advanced


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Get the sequences Use the Blast similarity search

http://blast.ncbi.nlm.nih.gov/Blast.cgi

Use automation if many similar searches

e.g. http://qcbs.ca/wiki/commandline_remote_blas
http://blast.ncbi.nlm.nih.gov/Blast.cgihttp://qcbs.ca/wiki/commandline_remote_blasthttp://qcbs.ca/wiki/commandline_remote_blasthttp://blast.ncbi.nlm.nih.gov/Blast.cgi


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Get the sequences

Alternative : In reference databases (Genbank is not error-freeorganisms

SILVA http://www.arb-silva.de : aligned ribosomal RNA (16S/1(23S/28S, LSU) gene sequences from the Bacteria,Archaea anEukaryota

RDP http://rdp.cme.msu.edu/ The Ribosomal Database ProjecBacterial and Archaeal 16S rRNA seq

Greengenes http://greengenes.lbl.gov 16S rRNA gene sequen

alignment UNITE http://unite.ut.ee/ reference records of ITS sequences f

Ectomycorrhizal (ECM) fungi

Barcode of Life Data System (BOLD) http://www.boldsystems.animal mitochondrial cytochrome c oxidase I (COI)

Protist Ribosomal Reference Database http://ssu-rrna.org/, uneukaryotes Small SubUnit rRNA (18S)
http://www.arb-silva.de/http://rdp.cme.msu.edu/http://greengenes.lbl.gov/http://unite.ut.ee/http://www.boldsystems.org/http://ssu-rrna.org/http://ssu-rrna.org/http://ssu-rrna.org/http://ssu-rrna.org/http://www.boldsystems.org/http://unite.ut.ee/http://greengenes.lbl.gov/http://rdp.cme.msu.edu/http://www.arb-silva.de/http://www.arb-silva.de/http://www.arb-silva.de/


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Practical steps for alignment an

Align sequences with automated programs

Test a few algorithms

Adjust alignment by eye - Controversial

Trim (or exclude) ends, and regions with aligment you don

Identify the best-fit model for your data

Build trees (typically ML or Bayesian)

Share data, codes and models

Account for uncertainty

Branch support (Bootstrap or Bayesian trees)

Inferring evolutionary forces (e.g. positive selection)


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>Protea_witzenbergiana Some description here

CGCGAGAAGTCCACTGAACCTTATCATTTAGAGGAA

TTCCGTAGGTGAACCTGCGGAAGGATCATTGTCGAT

CCCGCGAACACGTCGAACGGTGACC

>Protea_wentzeliana Different description hereCGCGAGAAGTCCACTGAACCTTATCATTTAGAGGAA


CCCGCGAACACGTCGAACGGTGACC

>Protea_vogtsiae Other heree

CGCGAGAAGTCCACTGAACCTTATCATTTAGAGGAA


CCCGCGAACACGTCGAACGGTGACCGGGGGGCGA

>Protea_witzenbergiana Some description here

----C-------GCGA--------GAAGTCCACTGAACCTTATCATTTAGAGGAAGGAGA

TAGGTGAACCTGCGGAAGGATCATTGTCGATGCCTG

GAACACGTC-G-AACGGT-GACC-

>Protea_wentzeliana Different description here

----C-------GCGA--------

GAAGTCCACTGAACCTTATCATTTAGAGGAAGGAGA


GAACACGTC-G-AACGGT-GACC-

>Protea_vogtsiae Other heree

----C-------GCGA--------

GAAGTCCACTGAACCTTATCATTTAGAGGAAGGAGA


GAACACGTC-G-AACGGT-GACC-GGGG-G-G-CGA-G-TG----------

Fasta format

All your sequences into one file, in

the fasta format A text file

Greater than sign

Sequence name + Description

Return

The sequence (with - or not)

Return


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Lets align!

A plethora of alignment algorithms

Very different calculation methods

Will try only five (listed on the wiki):

Clustal

Muscle

PRANK

SATe

FAST

JalView, SuiteMSA, BioEdit (viewer)

On 3 datasets

PR10_fabaceae_1

ITS_oxytropis_84se

Fungal_refseq_ITS_


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Why care about alignment?

To have a reliable tree!

Because your matrix will be public

http://treebase.org

http://datadryad.org/ 80$
http://treebase.org/http://datadryad.org/http://datadryad.org/http://treebase.org/http://treebase.org/


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Lets align!

Questions to ponder

Would you trust aalignments?

Does one algorithoutperform all thcircumstances?

Which algorithm likely to use for yo

projects?


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Lets align!

Clustal Muscle Prank SAT

AdvantageEasy, Usedeverywhere

Fast distanceestimation,progressivealignment,refinement byrestricted

partitioning.

Corrects forinsertions anddeletions. Goodfor codons

Co-estimatealignments andtrees. Runsrelatively fast.Divide-and-conquer

realignment.


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Lets align!Clustal Muscle Prank SAT

PR10_fabaceae_11seq = With longgaps + CDS

8240 bp. Does notlocate the cdsregions

8560 bp. Foundsimilar cds regionof a divergentgene

9470 bp. Lowconfidence in cdsregion of adivergent gene.

8370. Found thesimilar cds regionof the divergentgene.

ITS_oxytropis_84se

q = highlyconserved good goog good good

Fungal_refseq_ITS_301seq = Highlydiverging

1070 pb.Is not careful inaligning lowsimilarity areas.

1850 bp Alignstogether stretcheswith low similarity.Finds conservedregions in themiddle.

21630 bp. Findsconserved regionin the middle.

1420 bp. Alignstogether stretchewith low similarityFinds conservedregions in themiddle.


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Lets align!


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Phylogenetic trees - Basics

Branch = edge: a lineage through time

Node: branching of a lineage into two. Byspeciation or gene duplication

Internal node

Leaf (terminal node) = Tip (OUT)

Branch length: Typically nb substitution/site ; isoften not constant;

Outgroup: used to find the root

Topology: The branching pattern of the tree

Terminology

It is a drawing = can be re-rooted, branches swapped


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Phylogenetic trees - Basics

Useful in :

Gene duplication events Recombination or horizontal gene transfer

Variation of selective pressures andadaptive evolution

Divergence times between species

Origin of epidemics

Host-parasite cospeciation events

Genealogies of somatic cells in cancer


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Phylogenetic trees Yang, Z., and B. Rannala. 2012. Molecular phylogenetics: principles a

Nature Reviews Genetics 13: 303314.

Hall, Berry G. Phylogenetic Trees Made Easy: A How-To Manual, Third

Aris-Brosou, S., and X. Xia. 2008. Phylogenetic Analyses: A Toolbox Exptowards Bayesian Methods. International Journal of Plant Genomics

Roquet, C., W. Thuiller, and S. Lavergne. 2013. Building megaphyloge

macroecology: taking up the challenge. Ecography 36: 013026.

http://treethinkers.org/Workshops in applied phylogenetics

http://www.molecularevolution.org/ Software description and glossa
http://www.nature.com/nrg/journal/v13/n5/full/nrg3186.htmlhttp://www.hindawi.com/journals/ijpg/2008/683509/http://www.hindawi.com/journals/ijpg/2008/683509/http://onlinelibrary.wiley.com/doi/10.1111/j.1600-0587.2012.07773.x/abstracthttp://onlinelibrary.wiley.com/doi/10.1111/j.1600-0587.2012.07773.x/abstracthttp://treethinkers.org/http://www.molecularevolution.org/http://www.molecularevolution.org/http://treethinkers.org/http://onlinelibrary.wiley.com/doi/10.1111/j.1600-0587.2012.07773.x/abstracthttp://onlinelibrary.wiley.com/doi/10.1111/j.1600-0587.2012.07773.x/abstracthttp://www.hindawi.com/journals/ijpg/2008/683509/http://www.hindawi.com/journals/ijpg/2008/683509/http://www.nature.com/nrg/journal/v13/n5/full/nrg3186.html


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Building the treeDistance Parsimony Max. Likelihood Bayesi

Based on Distance matrix Informativecharacters

All characters All cha

Approach Clustering Which treeexplains the datawith leastevolutionarychanges?

What tree and valuesgive the highestlikelihood to thisalignment?

What isprobabdistribubased data?

Score for

choosingtrees

Steps: minimum

number ofchanges

Log likelihood. A

relative number,cannot compareacross alignments

Posterio

that thcorrecunders

Yang, Z., and B. Rannala. 2012. Molecular phylogenetics: principles and pracNature Reviews Genetics 13: 303314.

Hall, Berry G. Phylogenetic Trees Made Easy: A How-To Manual, Third Edition
http://www.nature.com/nrg/journal/v13/n5/full/nrg3186.htmlhttp://www.nature.com/nrg/journal/v13/n5/full/nrg3186.html


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When not touse

Withnumerouslong gaps

With highly divergentsequences

With a model not fitto your data

When yPriors aappropdata

Strength Quick Efficient, andgenerally reliable

Consistent, efficient.Can be used for testsof evolution

ConsistCan btests of

Weaknesses Sensitive to

gap and todivergentsequences

Calculation cannot

be improved,because no model ofsequence evolution

Computationally

demanding.Depends on modelof evolution.

Comp

demanPosterioprobabbe hig


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Programs -commercial PAUP*

Programs -free

MEGA MEGA5, TNT MEGA5, RAxML,GARLi,PAML, Hyphy

BEAST, PhycasBayesPBUCKyPhy

Kumar 1994 (cited 2459 times); 2001

(cited 6481 tiems); 2004 (11433 tiems);2007 (cited 19499 times); 2011 (cited6226 times

RAxML, Stamatakis

2006 . Cited 3536times

GARLi, Zwickl 2006Cited 1574 times

BEAST,

2007 C

MrBaye2003 Ctimes; Cited 2


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Distance

HugeFlo BigFl MediumFl

HugeFl

BigFl 2/12

MediumFl 3/12 3/12

SmallFl 412 4/12 3/12

TinyFl 5/12 5/12 4/12


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Parsimony

Only informative ch


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Max. likelihood; Bayesian; distance

Rates of substitution bnucleotides

More complex: 6

Rate variation among

e.g. codon positio gamma

Proportion of invarian

Model of sequence evolution Includes

Which model fits your data?

jModelTest

MEGA5


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Newick format

tree ML_tree = [&U](SmallFlower:0.0,TinyFlower:0.0937:0.086766,(HugeFlower:0.08978,Big):0.118638):0.228517);

tree ML_tree = [&U](SmallFlower:0.0,TinyFlower:0.0937:0.086766,(HugeFlower:0.08978,Big)[&"bootstrapproportion"=78.0"]:0.118638)[&"bootproportion"=87.0"]:0.228517);

The tree fileNewick forma

Branc

Branc


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Build trees, general steps

Test which model of sequence evolution fits

your data

Choose method

Distance : MEGA5 - Quick, notpublications

Parsimony : MEGA5 - Not very popular

Max. likelihood : GARli - Efficient

Max. likelihood : RAxMLVery largedatasets, e.g thousands taxa + hundredsgenes. Use CAT.

Bayesian: BEAST2.0To answer questionswith a range of probable trees

At your com

Download 2ailgnment htcontent/uploads/20TS.txt ; http://qcbs.cacontent/uploads/20trnL.txt

Analyze withRAxML on yo

Use BEAST
http://qcbs.ca/wp-content/uploads/2013/06/ProteaFaurea_ITS.txthttp://qcbs.ca/wp-content/uploads/2013/06/ProteaFaurea_ITS.txthttp://qcbs.ca/wp-content/uploads/2013/06/ProteaFaurea_trnL.txthttp://qcbs.ca/wp-content/uploads/2013/06/ProteaFaurea_trnL.txthttp://qcbs.ca/wp-content/uploads/2013/06/ProteaFaurea_trnL.txthttp://qcbs.ca/wp-content/uploads/2013/06/ProteaFaurea_trnL.txthttp://qcbs.ca/wp-content/uploads/2013/06/ProteaFaurea_trnL.txthttp://qcbs.ca/wp-content/uploads/2013/06/ProteaFaurea_trnL.txthttp://qcbs.ca/wp-content/uploads/2013/06/ProteaFaurea_trnL.txthttp://qcbs.ca/wp-content/uploads/2013/06/ProteaFaurea_trnL.txthttp://qcbs.ca/wp-content/uploads/2013/06/ProteaFaurea_trnL.txthttp://qcbs.ca/wp-content/uploads/2013/06/ProteaFaurea_ITS.txthttp://qcbs.ca/wp-content/uploads/2013/06/ProteaFaurea_ITS.txthttp://qcbs.ca/wp-content/uploads/2013/06/ProteaFaurea_ITS.txt


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BEAST 2.0

Start from one of the tutorial

Know the model of substitution (e.g.from jModelTest)

Read carefully the BEAST FAQ from theWiki

For any problem, search and browsethrough the BEAST forum first
http://www.beast2.org/wiki/index.php/FAQhttps://groups.google.com/forum/https://groups.google.com/forum/http://www.beast2.org/wiki/index.php/FAQ


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Branch support

From a large sets of tree (many hundreds) Bootstrap replicates

Bayesian analysis

Compute consensus tree

Strict = 100% of the trees

Majority = a % set by the user

Report on your best tree (e.g. dendropy)
http://pythonhosted.org/DendroPy/scripts/sumtrees.htmlhttp://pythonhosted.org/DendroPy/scripts/sumtrees.html


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Why so many programs?

Umbrella programs (commercial)

Geneious 400$

G l d ti


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General recommendations

Anisimova, et al. 2013. State-of the art methodologies dictatstandards for phylogenetic analysis. BMC Evolutionary Biolog

161.

Write a question that the analysis will answer

Justify the choice of methods, test alternatives

Account for uncertainty (branch support, confidence int

Share the data
http://www.biomedcentral.com/1471-2148/13/161/abstracthttp://www.biomedcentral.com/1471-2148/13/161/abstracthttp://www.biomedcentral.com/1471-2148/13/161/abstracthttp://www.biomedcentral.com/1471-2148/13/161/abstracthttp://www.biomedcentral.com/1471-2148/13/161/abstracthttp://www.biomedcentral.com/1471-2148/13/161/abstract


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End!

workshop tips to align dna sequences and build phylogenetic trees

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