1. Reed A. Cartwright Department of Genetics University of Georgia
Barking up the wrong tree: gaps in current phylogenetic methodology and a dawg-gone good solution
Reed A. Cartwright
Department of Genetics
University of Georgia

2. RA Cartwright rac@uga.edu - http://scit.us/
Phylogenies
Phylogenies are not known. They are inferred.
The accuracy of the inference is dependent on the quality of the data and quality of the methodology.
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3. Estimating Phylogenies
Retrieve sequences from a database or a sequencer.
Align sequences.
Estimate the phylogeny from the alignment.
However, bad alignments give bad phylogenies.
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4. RA Cartwright rac@uga.edu - http://scit.us/
How do we know it works?
Intuition
Concordance
Using rare instances of know phylogenies.
Simulations
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5. Why Simulate Phylogenies?
Techniques are often based on certain models of evolution.
Simulating sequence evolution based on these models produces an ideal situation to test the techniques.
Using other models can test how robust a technique is.
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6. RA Cartwright rac@uga.edu - http://scit.us/
Testing Procedure
1. Start with a “known” tree.
3. Estimate the trees
of the simulated data.
2. Simulate sequence
sets based on the tree.
4. Compare estimated trees
to the original tree. A B C D
A AATTCTTTGAGTTAA
B AATTCTTTGAGTTAA
C AATTCTTAAAGTTAA
D AATTCTTAAAGTTAA A B C D A B C D
A AAAAGATAAAGCAAA--A
B GAAAGATAAAGCAAA--A
C GAAAGATAAAGAAAAACA
D GAAAGATAAAGAAAAACA
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7. Measuring the accuracy of estimates
What do you want to measure, topology or branch lengths?
Simply binary system: correct or wrong.
More flexible system: accuracy of clade estimation.
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8. RA Cartwright rac@uga.edu - http://scit.us/
Clade Estimation
Estimated Clade
Yes No
Actual Clade
True Positive
False Negative
False Positive
True Negative
Sensitivity = TP/(TP+FN)
Specificity = TN/(FP+TN)
Positive Predictive Accuracy = TP/(TP+FP)
Negative Predictive Accuracy = TN/(FN+TN)
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9. How to best combine different clades into one metric?
Look at each clade separately?
Lump all clades of the same size together?
Lump all clades of different sizes together?
How to do it?
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10. RA Cartwright rac@uga.edu - http://scit.us/
Alignment Techniques
The quality of a phylogeny depends on the quality of an alignment.
There are two different classes of alignment techniques.
Pairwise alignments
Multiple alignments
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11. RA Cartwright rac@uga.edu - http://scit.us/
Pairwise alignments
Pairwise alignments align pairs of sequences.
Typically use a spreadsheet like technique with penalties for mismatches and gaps.
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12. RA Cartwright rac@uga.edu - http://scit.us/
Multiple alignments
Align multiple sequences.
Cannot directly use the techniques used for pairwise alignments.
Typical implementations use a guide tree derived from sequence similarity scores of pairwise alignments.
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13. RA Cartwright rac@uga.edu - http://scit.us/
Alignment Models
The typical model of alignment is the affine gap model.
In this model the cost of a gap is a linear function of the size of a gap: C(L) = O+E(L).
This corresponds to a geometric-exponential model of gap sizes.
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14. RA Cartwright rac@uga.edu - http://scit.us/
Power-Law
The only problem with this is that indels do not obey this model.
Several studies and some theory in nucleic acids and proteins have found that indels sizes obey a power law.
The appropriate cost model for a power law is logarithmic: C(L) = O+E*Log(L).
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15. RA Cartwright rac@uga.edu - http://scit.us/
So what?
Only one program does logarithmic alignments, and it only works on protein pairs.
Monotone by Richard Mott.
Clustal uses the affine gap model.
Above that Clustal uses a simple evolutionary model to estimate a guide tree for aligning multiple sequences.
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16. RA Cartwright rac@uga.edu - http://scit.us/
Dealing with gaps
Sequences are typically aligned before they are phylogenized.
This is silly. We should estimate alignments at the same we estimate phylogenies.
For now we are stuck doing it in pieces, but must be wary of introducing biases into our phylogenies when aligning.
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17. RA Cartwright rac@uga.edu - http://scit.us/
Dealing with gaps
Gaps contain phylogenetic signal which is usually ignored by researchers.
Can look at how gaps influence phylogenetics?
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18. RA Cartwright rac@uga.edu - http://scit.us/
Dealing with gaps
To study how gaps can influence phylogenies we need a program that can simulate molecular evolution with indels.
However, existing programs model indel formation rather poorly if they do at all.
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19. Dawg is the solution to this gap
Dawg stands for “DNA Assembly With Gaps.”
A portable and robust program for simulating molecular evolution.
Development Website:
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20. RA Cartwright rac@uga.edu - http://scit.us/
Comparing Software
Feature
Seq-Gen
Evolver
Rose
Dawg
Indels
Yes
Indel Parameter Estimator
Recombination
Substitution
GTR
PAM
Rate Heterogeneity
Γ+I Γ
Input Format
Switch
File
Unix
Mac OS X
Win32
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21. RA Cartwright rac@uga.edu - http://scit.us/
Parameters
Tree phylogeny
TreeScale coefficient to scale branch lengths by
Sequence root sequences
Length length of generated root sequences
Rates rate of evolution of each root nucleotide
Model model of evolution: GTR|JC|K2P|K3P|HKY|F81|F84|TN
Freqs nucleotide (ACGT) frequencies
Params parameters for the model of evolution
Width block width for indels and recombination
Scale block position scales
Gamma coefficients of variance for rate heterogeneity
Alpha shape parameters
Iota proportions of invariant sites
GapModel models of indel formation: NB|PL|US
Lambda rates of indel formation
GapParams parameter for the indel model
Reps number of data sets to output
File output file
Format output format: Fasta|Nexus|Phylip|Clustal
GapSingleChar output gaps as a single character
GapPlus distinguish insertions from deletions in alignment
LowerCase output sequences in lowercase
Translate translate outputed sequences to amino acids
NexusCode text or file to include between datasets in Nexus format
Seed PRNG seed (integers)
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22. RA Cartwright rac@uga.edu - http://scit.us/
Sample Input File
# example.dawg
Tree = ((AY727331: ,AY727330: ): ,
(AY727327: ,AY727326: ): );
Model = "GTR"
Params = { , , ,
, , }
Freqs = { , , , }
Length = 300
Lambda =
GapModel = "NB"
GapParams = {1, }
Format = "Clustal"
File = "example.aln"
Seed = 1981
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23. RA Cartwright rac@uga.edu - http://scit.us/
CLUSTAL multiple sequence alignment (Created by DAWG Version 1.0.0)
AY TTCGAAAATATGTTAGTACTCAATATGAATTCTTTGAGTTAAAAAAGATAAAGCAAA--A
AY TTCGAAAATATGTTAGTACTCAATATGAATTCTTTGAGTTAAGAAAGATAAAGCAAA--A
AY TTCAAAAATATGCTAGGACTGAATATGAATTCTTAAAGTTAAGAAAGATAAAGAAAAACA
AY TTCAAAAATATGCTAGGACTGAATATGAATTCTTAAAGTTAAGAAAGATAAAGAAAAACA
AY ATACATAATGTGATTTCAATATTCCAATTACCTAACAATACGGCTATCAATTAAACGATT
AY ATACATAATGTGATTTCAATATTCCAATTACCTAACAATACGGCTATCAATTAAACGATT
AY GTACATAATGTAAA----TTATTGCAA AAAACGGCTAACAATTAGACGATT
AY GTACATAATGTAAA----TTATTGCAA AAAACGGCTAACAATTAGACGATT
AY TTAGGATTACACCGACAAATATTAGGCCGATATGAATTTAACATCATGTTGTATTTAGAT
AY TTAGGATTACACCGACAAATATTAGGCCGATATGAATTTACCATCATGTTGTATTTAGAT
AY TTAGGATTACGCTGACAAATATTAGGATGATATTAATTTA------TCTTGTATTTAGAT
AY TTAGGATTACGCTGACAAATATTAGGATGATATTAATTTA------TCTTGTATTTAGAT
AY GCTGTCTTTTATTAACATTCATCATTAAAT-TTGGAACCTTTTGCATTTAAGAAGTACAT
AY GCTGTCTTTTATTAACATTCATCATTAAAT-TTGGAACCTTTTGTATTTAAGAAGTACAT
AY GCTGTCTTTTATCAACATTCATCACTAGATATTGGAACCTATTGCATCTAAGAAGTACAT
AY GCTGTCTTTTATCAACATTCATCACTAGATATTGGAACCTATTGCATCTAAGAAGTACAT
AY GTTTAATAGTGTTTAAAA-TATATATGAAATTGATCATAAGGA---TCTATAAATGCGGT
AY GTTTAATAGTGTTTATAA-TATATATGAAATTGATCGTAAGGA---TCTATAAATGCAGT
AY GTTTAATAGGGTT-AAAACTATATATGAAGTCGATTATAAGGAATTTCTATAAATGTAGC
AY GTTTAATAGGGTT-AAAACTATATATGAAGTCGATTATAAGGAATTTCTATAAATGTAGC
AY TCTTCAATTTCTTG
AY TCTTCAATTTCTTG
AY TCTTCAATTTCCTA
AY TCTTCAATTTCCTA
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24. RA Cartwright rac@uga.edu - http://scit.us/
Estimating Indel Rate
Dawg would be of little benefit if biologists could not estimate parameters of indel formation from real data.
Dawg’s indel model allows such estimation, which is implemented in a Perl script, lambda.pl.
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25. RA Cartwright rac@uga.edu - http://scit.us/
Lambda.pl
Take an alignment and a phylogeny.
The number of unique gaps in this alignment is approximately distributed as a Poisson with mean (λLT)
λ = rate of indel formation
L = average sequence length
T = total branch length
Therefore the rate of indel formation can be estimated as λ = N/(LT)
N = number of unique gaps in alignment
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26. Example Usage: Confidence Interval of Indel Rate
I aligned the sequences of chloroplast trnK introns from two Hibiscus and two Prunus species.
Using Paup*, I estimated the phylogeny and substitution parameters.
Using lambda.pl, I estimated the indel formation parameters.
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27. RA Cartwright rac@uga.edu - http://scit.us/
Example Usage
From these estimated parameters of evolution, I constructed an input file for Dawg.
From the input file Dawg produced a thousand simulated sequence sets.
The rate of indel formation was estimated for each of the simulated sequences.
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28. RA Cartwright rac@uga.edu - http://scit.us/
Results
The estimated rate of indel formation was
Bootstrapping gave a 95% CI of to
Biologically this is 8 to 21 indels per 100 substitutions.
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29. RA Cartwright rac@uga.edu - http://scit.us/
Thanks
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