NB: If there are any question, comments or bugs, feel free to ask. ;)

# Introduction

The slides of the introduction are available here:ancestral_sequence_reconstruction.pdf

In this practical, you will learn how to prepare files for CodeML, how to use it for reconstructing ancestral sequences and how to compute the isoelectric point of protein sequences. There are a few scripts you will need to download during the practical.

If you need to make these scripts executable, use chmod:

chmod +x script.py

### Tools used in this practical:

# Libraries for PythonBiopython: http://biopython.org/wiki/Main_Page

# Package for ancestral sequence reconstruction (and many other things)

CodeML from PAML: http://abacus.gene.ucl.ac.uk/software/paml.html

# Alignment tools

MAFFT L-INS-i: http://mafft.cbrc.jp/alignment/software/

Clustal-Omega: http://www.clustal.org/omega/

(Clustal-Omega is the new aligner from ClustalW team, but much faster and more accurate)

# Alignment visualisation

Jalview: http://www.jalview.org/

# Phylogenetics tools

PhyML: http://www.atgc-montpellier.fr/phyml/binaries.php

FastTree: http://www.microbesonline.org/fasttree/

# Tree visualisation

NJplot: http://doua.prabi.fr/software/njplot

This practical will focus on the lysozyme, an enzyme (EC 3.2.1.17) that damages bacterial cell walls. The Uniprot page is: http://www.uniprot.org/uniprot/P61626

They evolved differently in Primates:

### Step 1: Prepare alignment.

The quality of the ancestral reconstruction will heavily depend on the quality of the alignment and the tree topology (branch lengths are re-estimated during the reconstruction).Please download the sequence file: lysozyme_primates.seq

Make a multiple alignment, either with Mafft-L-INS-i or Clustal-Omega:

mafft-linsi lysozyme_primates.seq > lysozyme_primates.fasta

or

clustal-omega-1.2.0-macosx --in lysozyme_primates.seq --out lysozyme_primates.fasta

(Please have a look at the alignment in Jalview)

The format of the resulting alignment is FASTA. However, most phylogenetic softwares use PHYLIP format. So, you have to convert it into PHYLIP.

Download the script "convert_fasta2phylip.py" and execute it:

convert_fasta2phylip.py lysozyme_primates.fasta lysozyme_primates.phy

(If you are not familiar, have a look at the differences between the alignment in FASTA and PHYLIP formats).

### Step 2: Prepare alignment.

We can now generate a tree, either with PhyML (one of the most accurate tool) or FastTree (very fast and pretty accurate):phyml -i lysozyme_primates.phy -d aa -m JTT -c 4 -a e -b 0

mv lysozyme_primates.phy_phyml_tree.txt lysozyme_primates.tree

Option used:

-i = input file

-d aa: amino acid sequences

-m JTT: (substitution matrix). JTT works fine for most proteins, but other matrices (WAG, LG) can do slightly better.

-c 4: (numbers of categories for the gamma distribution)

-a e: (estimate alpha parameter for the gamma distribution)

-b 0: (we don't want boostrap, as this will cause trouble for further analyses in CodeML).

or run:

FastTree -nosupport lysozyme_primates.phy > lysozyme_primates.tree

Option used:

-nosupport:(we don't want boostrap, as this will cause trouble for further analyses in CodeML).

Finally, we could root the tree. Use NJplot and root it by the group containing the Marmoset sequence (Callithrix jacchus).

Save it as "lysozyme_primates_rooted.tree"

### Step 3: Run ancestral sequence reconstruction.

The ancestral sequence reconstruction is done by CodeML, from the PAML package.

It is launched with "codeml control_file.ctl"

You may have to copy the file "jones.dat" from the dat folder in the PAML package, or indicate its location.

The control file contains many parameters:

seqfile = lysozyme_primates.phy * sequence data
filename

treefile = lysozyme_primates_root.tree * tree structure
file name

outfile = lysozyme_primates.mlc * main result file
name

noisy = 9 * 0,1,2,3,9: how much rubbish on the screen

verbose = 2 * 0: concise; 1: detailed, 2: too much

runmode = 0 * 0: user tree; 1: semi-automatic; 2:
automatic

* 3: StepwiseAddition; (4,5):PerturbationNNI; -2: pairwise

seqtype = 2 * 1:codons; 2:AAs; 3:codons-->AAs

clock = 0 * 0:no clock, 1:clock; 2:local clock; 3:CombinedAnalysis

aaDist = 0 * 0:equal, +:geometric; -:linear,
1-6:G1974,Miyata,c,p,v,a

aaRatefile = ./jones.dat * only used for aa seqs with
model=empirical(_F)

* dayhoff.dat, jones.dat, wag.dat, mtmam.dat, or your own

model = 2

* models for codons:

* 0:one, 1:b, 2:2 or more dN/dS ratios for branches

* models for AAs or codon-translated AAs:

* 0:poisson, 1:proportional,
2:Empirical, 3:Empirical+F

* 6:FromCodon, 7:AAClasses, 8:REVaa_0, 9:REVaa(nr=189)

icode = 0 * 0:universal code; 1:mammalian mt; 2-10:see below

Mgene = 0 *
codon: 0:rates, 1:separate; 2:diff pi, 3:diff kapa, 4:all
diff

* AA: 0:rates, 1:separate

fix_alpha = 0 * 0: estimate gamma shape parameter; 1: fix
it at alpha

alpha = 0.5 * initial or fixed alpha, 0:infinity (constant rate)

Malpha = 1 * different alphas for genes

ncatG = 4 * # of categories in dG of NSsites models

getSE = 0 * 0: don't want them, 1: want S.E.s of estimates

RateAncestor
= 1 * (0,1,2): rates (alpha>0) or ancestral states (1 or 2)

Small_Diff = .5e-6

cleandata = 0 * remove sites with ambiguity data (1:yes, 0:no)?

method = 1 * Optimization method 0: simultaneous; 1: one branch a
time

*
Genetic codes: 0:universal, 1:mammalian mt., 2:yeast mt., 3:mold mt.,

*
4: invertebrate mt., 5: ciliate nuclear, 6: echinoderm mt.,

*
7: euplotid mt., 8: alternative yeast nu. 9: ascidian mt.,

*
10: blepharisma nu.

*
These codes correspond to transl_table 1 to 11 of GENEBANK.

Explanation of some parameters:

`runmode = 0 => We provide the tree.`

`clock = 0 => We don't set a molecular clock. We assume that the genes are evolving at different rate.`

`aaDist = 0 => We don't use the physicochemical properties of the amino acid.`

`aaRatefile = ./jones.dat => We use the JTT matrix. Other matrix could be used (WAG, etc...)`

`model = 2 => `

We use an empirical model (= substitutions matrix such as JTT).`fix_alpha = 0`

=> We estimated the alpha parameter of the gamma distribution.`alpha = 0.5 => We start the estimation from 0.5`

```
RateAncestor
= 1 =>
```

Force the estimation of ancestral states.`cleandata = 0 => `

Keep all ambigous data ("-", "X").Please have a look at the output.

CodeML will also write into many files, but only two are of interest here:

`lysozyme_primates.mlc => Contains many information on evolutionary rates.`

- rst => Contains ancestral states for sites and for nodes.

In the rst file, there is also how the tree has been annotated, under the line "tree with node labels for Rod Page's TreeView".

You can copy this tree into a file (i.e. "lysozyme_primates_annotated.tree"), open it with NJplot, and display the "bootstrap values".

We can see at which node correspond which ancestral sequence.

Questions:

- What is the estimated alpha parameter of the gamma curve?

- How many categories where used? And what are their frequencies?

- In Jalview, we observed at column 68 a mixture of E(Glu), Q(Gln) and R(Arg). But what was the most likely state of this position in the last common ancestor of all sequences? What are the probabilities of A(AlA), E(Glu), Q(Gln) and R(Arg)?

- What are the evolutionary events (amino acid substitutions) at the basis of the Hominoidea clade (Hylobates lar, Gorilla, Pan Paniscus, Homo sapiens)?

Now it is time to extract ancestral sequences and put them in a file. The rst file is quite difficult to parse, hopefully, each ancestral sequence start by "node".

Download the following script and execute it: parse_rst.py

./parse_rst.py rst

It displays ancestral sequences in FASTA format. Let's put them in a file:

./parse_rst.py rst > ancestral_sequences.fasta

### Part 4: Compute physico-chemical properties on ancestral sequences.

In Biopython, there is a function to compute the isoelectric point (pI):

analysed_protein = ProtParam.ProteinAnalysis(sequence)

pI = analysed_protein.isoelectric_point()

The following script will compute the pI for all sequences in a FASTA file: compute_pI.py

By launching it, we can retrieve the pI for modern primate lysozymes:

./compute_pI.py lysozyme_primates.fasta

And similarly for ancestral sequences:

./compute_pI.py ancestral_sequences.fasta

### Part 5: Map properties on tree.

We could easily map ancestral properties on the tree. The tree provided in rst contains nodes where bootstrap information is.We just need to change the values of these nodes by the corresponding pI.

Download the following script and execute it: map_on_tree.py

./map_on_tree.py ancestral_sequences.fasta lysozyme_primates_annotated.tree > lysozyme_primates_annotated_pI.tree

Have a look at both trees in a text editor.

You can load it in NJplot and see the different pI at the bootstrap place.

Alternatively, you can install FigTree: http://tree.bio.ed.ac.uk/software/figtree/

and load the tree.

In FigTree, set the following parameters:

Appearance->Colour by:label

Setup: Colours -> Scheme: Colour gradient

Tick gradient

Line Weight 4

Hi, this is a great tutorial! What is the advantage of using method=1 instead of 0 here? Is it generally better to use optimization with one branch at a time?

ReplyDeleteThanks!

Hi,

ReplyDeleteFor the reconstruction of ancestral amino acid sequences, I used "model = 2" to select an empirical model of amino acid substitution, which assumes that amino acids have different probabilities of replacement.

As substitution matrix, I select the JTT one (./jones.dat).

Hi again,

ReplyDeleteThank for your response. I was wondering about the method though, not the model. In the mean time I saw there is actually a note on the PAML manual were they say that method=1 selects the most recent optimization method branch by branch instead of simultaneous.

OK. Sorry, I made a mistake in my previous post. So yes, method = 1 is better.

ReplyDeleteNo problem! Thank you for your time / this blog is great for first time PAML users.

ReplyDeleteHi Romain,

ReplyDeleteI have a question on gaps. It seems PAML cannot treat gaps and so one will obtain an ancestral sequence which does not contain gaps. Is that a problem in general?

Also, is there a way to extract the probability of each site for the ancestral sequence from the output?

Thank you!

Hi,

Delete1) It is true that PAML doesn't know how to treat gaps. There are two options: either using cleandata=1, and CodeML will exclude any column with a gap. This is the safety way. Or using cleandata=0, and CodeML will perform ancestral sequence reconstruction on any sites. If there is a gap in a column, it will consider the most likely amino acid. For exemple, if you only have one sequence with a "W" and the rest is gap "-", CodeML will assign a "W" to all other nodes. But you could then use a parsimony approach to say if at this node there a gap "-" or not (binary matrix 0 or 1).

This is a similar approach used by FastML:

http://fastml.tau.ac.il/overview.php#IndelReconst

2) In the ouput (rst file), there are the probabilities for each of the 20 amino acids to be present at each node for each position. You will have to program a parser.

That makes sense, thank you! I have used this approach for a set of sequences and while it is clear how to extract the inferred sequences, I have a few questions on the output.

Delete1) How to extract the probabilities from the rst file. E.g.

If for the sixth site the rst lists this information:

site Freq Data:

6 1 TT-STT----STTSSTT-TTT-STTTTTTTSS---T-----: T(0.515) T(0.950) T(0.951) S(0.999) S(1.000) S(1.000) S(1.000) S(1.000) S(1.000) S(1.000) S(0.993) S(0.993) S(1.000) S(0.999) S(1.000) S(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(0.993) T(0.993) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000)

How do these numbers / letters need to be interpretated? Are those subsequent tentatives and the most likely amino acid? I can't seem to find this info on the PAML manual.

2) What do the numbers in branch changes correspond to? E.g.

Branch 1: 42..43

36 I 0.575 -> V 0.992

3) Finally at the end of the rst file I find that I obtain high accuracy for the reconstruction for a site for all ancestral sequences (ie > 85%) however the accuracy for the sequence is 0.000, is that something to be expected?

Thank you!

1) How to extract the probabilities from the rst file. E.g.

DeleteIf for the sixth site the rst lists this information:

site Freq Data:

6 1 TT-STT----STTSSTT-TTT-STTTTTTTSS---T-----: T(0.515) T(0.950) T(0.951) S(0.999) S(1.000) S(1.000) S(1.000) S(1.000) S(1.000) S(1.000) S(0.993) S(0.993) S(1.000) S(0.999) S(1.000) S(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(0.993) T(0.993) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000) T(1.000)

How do these numbers / letters need to be interpretated? Are those subsequent tentatives and the most likely amino acid? I can't seem to find this info on the PAML manual.

=> The first number is the colon in your alignment.

The "TT-STT----STTSSTT-TTT-STTTTTTTSS---T-----" is the data observed at this column 6 in your alignment.

The numbers correspond to the probabilities of the best amino acid at these nodes.

You have 41 sequences, so the numbering of ancestral sequences starts at number 42. So at position 6, you have a probability of 51.5% to observed a T at node 42.

Then, a prob of 95.0% to observe a T at node 43.

Then, a prob of 95.1% to observe a T at node 44.

Then, a prob of 99.9% to observe a S at node 45.

(The node of the tree are in the "tree with node labels for Rod Page's TreeView" at the begining of the rst file).

2) What do the numbers in branch changes correspond to? E.g.

Branch 1: 42..43

36 I 0.575 -> V 0.992

=> PAML will number branches and nodes.

In your example, branch 1 is linking node 42 and 43.

And you can observe that a position 36 in your alignment, the probability to have an isoleucine at node 42 is 57.6% (pretty low), and the probability to have a valine at node 43 is 99.2% (very high).

3) Finally at the end of the rst file I find that I obtain high accuracy for the reconstruction for a site for all ancestral sequences (ie > 85%) however the accuracy for the sequence is 0.000, is that something to be expected?

=> No, it is quite weird. To be honest, I cannot really explain that. Could you send me your files (alignment, codeml command, rst file) and I could investigate that.

Thank you for your help!! That makes perfect sense. It would be great if your blog was referenced by PAML as additional documentation! OK I will email separately the data etc in the next week or so.

DeleteHi Romain,

ReplyDeleteYour tutorial is quite useful for beginners like me. But for some reasons example files and scripts are unavailable. Maybe you know what happened to them on EMBL web-server?..

Thank you!