<p>To finish you need bootstrap support using a model-based tree building algorhithm, I use RAxML, specifically raxmlHPC (easily downloadable and compiles on Linux and OSX). The codes are quite complicated and I've given them below. </p>

<p>A robust maximum likelihood tree is,</p>

<pre><code>raxmlHPC -m PROTGAMMAILG -n output.tre -o outgroup-p 10000 -s alignment_file.phy
</code></pre>

<p>To make a bootstrap tree,</p>

<pre><code>raxmlHPC -b 459 -m PROTGAMMAILG -n output.tre -o outgroup -p 10000 -s /path_to_alignment.phy -N 500
</code></pre>

<p>Make a consensus tree of the bootstraps,</p>

<pre><code>raxmlHPC -f b -m PROTGAMMAILG -n output.tre -o outgroup -p 10000 -t /path_to_master_tree(best_tree) -z path_to_bootfile
</code></pre>

<p>You require bootstrap support >80% and please repeat this with and without 5-2/5-3 (it still looks long)</p>

<p>You need a fairly robust desktop, dedicated for around 24 hours to complete one calculation. You have enough results to draw a conclusion.</p>

<p>Viewing the tree, FigTree is easy.</p>

<p><strong>Root</strong></p>

<p>Check the work of Cavalier-Smith, he heavily focused on basal protists to find a species which evolved, using external evidence, before any other member of your group. One of the ingroups may have sufficient evidence to say it evolved before the rest and that is your root, which you select as an outgroup. </p>

CC BY-SA 4.0
2019-09-12T12:52:03.673Z
The mod is asking for a Q&A, when I find out what this is, this will need to be done.
M__
CC BY-SA 4.0
2019-09-13T03:07:10.477Z
Thank your for the detailed instructions @Michael G. I will give it a try and let you know how it goes. And thank you for the heads up about the program taking a long time to complete the calculation... I was not expecting that!
Leah
CC BY-SA 4.0
2019-09-16T23:48:00.983Z
Hi @Michael G. Just a quick note to let you know that I am still working on the phylogenetic tree, but since I have no experience compiling, it is taking me a little longer than I expected. I will post the results as soon as possible.
Leah
CC BY-SA 4.0
2019-09-17T05:50:28.440Z
Hi @leah you can always post it as a separate question
M__
CC BY-SA 4.0
2019-09-19T00:15:49.383Z
That's a good idea @Michael G. Will do!
Leah
CC BY-SA 4.0
2019-10-04T20:17:21.277Z
Well, it has taken me some time but I have finally managed to install RAxML and FigTree in Linux (I could not compile RAxML on OSX). I took my alignment file, saved it in Phylip format and I tried to run the first command line to build the robust maximum likelihood tree (raxmlHPC -m PROTGAMMAILG -n output.tre -o outgroup-p 10000 -s ~/Desktop/alignment_file.phy), but I got the following error: Error, the outgroup name "outgroup-p" you specified can not be found in the alignment, exiting ....  and I don't know how to fix it. Could you please help me out? Thank you very much!!
Leah
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2019-10-04T22:21:55.240Z
Can you put this as a separate question please?
M__
CC BY-SA 4.0
2019-10-05T00:46:32.793Z
Certainly. The question is posted in this thread: https://bioinformatics.stackexchange.com/questions/10534/how-to-define-an-outgroup-to-build-a-robust-amino-acid-tree. Thank you @Michael G.
Leah
CC BY-SA 4.0
2019-09-12T12:50:25.510Z
2019-09-12T12:50:25.510Z
M__
<p>This is the second part of this thread:  <a href="https://bioinformatics.stackexchange.com/questions/9309/duplicated-genes-and-genome-fragmentation?noredirect=1#comment12855_9309">Duplicated genes and genome fragmentation</a></p>

<p>As @Michael G. suggested, I did a pseudo-annotation of sequences 5-2 and 5-3 (i.e., I joined both sequences into one) and I did the phylogenetic analysis again. Here is the tree that I obtained: </p>

<p><a href="https://i.sstatic.net/rXaLi.jpg" rel="nofollow noreferrer"><img src="https://i.sstatic.net/rXaLi.jpg" alt="enter image description here"></a></p>

<p>Both of my sequences are shown in a red rectangle. The length of the branch seems to have decreased a bit. Also, I looked at the branches near sequence 5-1. One of them is the original query sequence from species <strong><em>A</em></strong> and the other one is a hypothetical protein from the same species (probably related to the query sequence, but I couldn't find any domains because it is only a few amino acids long). I still need to find the root of the phylogeny but I am not sure how to do this... </p>

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2019-09-11T08:25:06.030Z
So what is the actual question here? How you can root the tree?
terdon
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2019-09-11T15:04:44.240Z
The question is how to make a robust amino acid phylogeny with adequate robustness support. This is a generic question that many people will want answering and something I know a lot about. In my opinion, this has been a successful investigation given that the final tree is not a "rough and ready tree", but a formal likelihood-based tree. I see this as the end of the investigation and final response
M__
CC BY-SA 4.0
2019-09-11T15:08:39.457Z
I just have not got around to answering at this moment. I hope this is ok and we'll know in future when a rolling investigation is in-play either we  section the questions to specific topics or find an alternative forum
M__
CC BY-SA 4.0
2019-09-11T15:17:48.800Z
So @leah for forum rules it might be better to delete this and ask again OR change the title and specifically what you need is state of the art tree building to finish and conclude your study. Amino phylogeny is complicated and not trivial so in my opinion is a valid question. The idea is that someone in years in the future can understand from this post how to address a specific question. Stack exchange aims to get 90% of traffic via Google questions. Thus the question format is off-topic.
M__
CC BY-SA 4.0
2019-09-11T15:43:29.323Z
Exactly, thanks @MichaelG.! I agree that you two have had a very interesting discussion, it's really a shame it happened here since this site is not a good place to store discussions. It would be really, really great if you could combine all of this into one comprehensive Q&A though since there is a lot of great info you've both come up with.
terdon
CC BY-SA 4.0
2019-09-11T16:56:43.100Z
Ok.. @leah I will try and set up a Q&A, I can't remember how to do this, but I'll give it a whirl. Essentially the site has a chat feature.
M__
CC BY-SA 4.0
2019-09-12T00:49:35.553Z
Thank you for pointing out the problem with this thread. As @Michael G. suggested, I changed the title so that it is better suited to the format of the forum. Hopefully these posts can be moved to the Q&A format so that the information is available for other people later on.  As for the question itself, I agree with Michael G. that we are almost done, and I am very much looking forward to his input to round things off and get some final conclusions on this investigation!  Again, many thanks for all your help, I have certainly learned a lot through these discussions.
Leah
CC BY-SA 4.0
2019-09-12T01:20:21.680Z
I cannot comment... which seems odd while I can write answers.
Could you share with me the data you are using for the tree? I am developing a software for phylogenetic tree generation. I'd like to compare the tree and see if it can help you.
You can also try yourself if you like: [https://www.dnaservic.es/](https://www.dnaservic.es/)
I find that the root it finds makes usually far more sense than with other algorithms.
juanjo75es
CC BY-SA 4.0
2019-09-13T03:09:27.780Z
Thank you @juanjo75es. I will give your software a try and I will let you know how it goes. I hope the results will helpwith the software you are working on.
Leah
<phylogenetics><phylogeny><duplications>
How to make a robust amino acid phylogeny with adequate robustness support
# How to make a robust amino acid phylogeny with adequate robustness support

(Asked by: username_0 on 2019-09-11)

This is the second part of this thread: [Duplicated genes and genome fragmentation](https://bioinformatics.stackexchange.com/questions/9309/duplicated-genes-and-genome-fragmentation?noredirect=1#comment12855_9309)

As @Michael G. suggested, I did a pseudo-annotation of sequences 5-2 and 5-3 (i.e., I joined both sequences into one) and I did the phylogenetic analysis again. Here is the tree that I obtained:

[![enter image description here](https://i.sstatic.net/rXaLi.jpg)](https://i.sstatic.net/rXaLi.jpg)

Both of my sequences are shown in a red rectangle. The length of the branch seems to have decreased a bit. Also, I looked at the branches near sequence 5-1. One of them is the original query sequence from species ***A*** and the other one is a hypothetical protein from the same species (probably related to the query sequence, but I couldn't find any domains because it is only a few amino acids long). I still need to find the root of the phylogeny but I am not sure how to do this...

**Tags:** phylogenetics, phylogeny, duplications
> **Comments:**
> **username_1**: So what is the actual question here? How you can root the tree?  
> **username_2**: The question is how to make a robust amino acid phylogeny with adequate robustness support. This is a generic question that many people will want answering and something I know a lot about. In my opinion, this has been a successful investigation given that the final tree is not a "rough and ready tree", but a formal likelihood-based tree. I see this as the end of the investigation and final response  
> **username_2**: I just have not got around to answering at this moment. I hope this is ok and we'll know in future when a rolling investigation is in-play either we  section the questions to specific topics or find an alternative forum  
> **username_2**: So @leah for forum rules it might be better to delete this and ask again OR change the title and specifically what you need is state of the art tree building to finish and conclude your study. Amino phylogeny is complicated and not trivial so in my opinion is a valid question. The idea is that someone in years in the future can understand from this post how to address a specific question. Stack exchange aims to get 90% of traffic via Google questions. Thus the question format is off-topic.  
> **username_1**: Exactly, thanks @MichaelG.! I agree that you two have had a very interesting discussion, it's really a shame it happened here since this site is not a good place to store discussions. It would be really, really great if you could combine all of this into one comprehensive Q&A though since there is a lot of great info you've both come up with.  
> **username_2**: Ok.. @leah I will try and set up a Q&A, I can't remember how to do this, but I'll give it a whirl. Essentially the site has a chat feature.  
> **username_0**: Thank you for pointing out the problem with this thread. As @Michael G. suggested, I changed the title so that it is better suited to the format of the forum. Hopefully these posts can be moved to the Q&A format so that the information is available for other people later on.  As for the question itself, I agree with Michael G. that we are almost done, and I am very much looking forward to his input to round things off and get some final conclusions on this investigation!  Again, many thanks for all your help, I have certainly learned a lot through these discussions.  
> **username_3**: I cannot comment... which seems odd while I can write answers. Could you share with me the data you are using for the tree? I am developing a software for phylogenetic tree generation. I'd like to compare the tree and see if it can help you. You can also try yourself if you like: [https://www.dnaservic.es/](https://www.dnaservic.es/) I find that the root it finds makes usually far more sense than with other algorithms.  
> **username_0**: Thank you @juanjo75es. I will give your software a try and I will let you know how it goes. I hope the results will helpwith the software you are working on.  

---

## 1 answers

### Answer by username_2 on 2019-09-12. Score: 1 | Accepted answer

To finish you need bootstrap support using a model-based tree building algorhithm, I use RAxML, specifically raxmlHPC (easily downloadable and compiles on Linux and OSX). The codes are quite complicated and I've given them below. 
A robust maximum likelihood tree is,
raxmlHPC -m PROTGAMMAILG -n output.tre -o outgroup-p 10000 -s alignment_file.phy

To make a bootstrap tree,
raxmlHPC -b 459 -m PROTGAMMAILG -n output.tre -o outgroup -p 10000 -s /path_to_alignment.phy -N 500

Make a consensus tree of the bootstraps,
raxmlHPC -f b -m PROTGAMMAILG -n output.tre -o outgroup -p 10000 -t /path_to_master_tree(best_tree) -z path_to_bootfile

You require bootstrap support >80% and please repeat this with and without 5-2/5-3 (it still looks long)
You need a fairly robust desktop, dedicated for around 24 hours to complete one calculation. You have enough results to draw a conclusion.
Viewing the tree, FigTree is easy.
Root
Check the work of Cavalier-Smith, he heavily focused on basal protists to find a species which evolved, using external evidence, before any other member of your group. One of the ingroups may have sufficient evidence to say it evolved before the rest and that is your root, which you select as an outgroup.

> **Comments:**
> **username_2**: The mod is asking for a Q&A, when I find out what this is, this will need to be done.  
> **username_0**: Thank your for the detailed instructions @Michael G. I will give it a try and let you know how it goes. And thank you for the heads up about the program taking a long time to complete the calculation... I was not expecting that!  
> **username_0**: Hi @Michael G. Just a quick note to let you know that I am still working on the phylogenetic tree, but since I have no experience compiling, it is taking me a little longer than I expected. I will post the results as soon as possible.  
> **username_2**: Hi @leah you can always post it as a separate question  
> **username_0**: That's a good idea @Michael G. Will do!  
> **username_0**: Well, it has taken me some time but I have finally managed to install RAxML and FigTree in Linux (I could not compile RAxML on OSX). I took my alignment file, saved it in Phylip format and I tried to run the first command line to build the robust maximum likelihood tree (raxmlHPC -m PROTGAMMAILG -n output.tre -o outgroup-p 10000 -s ~/Desktop/alignment_file.phy), but I got the following error: Error, the outgroup name "outgroup-p" you specified can not be found in the alignment, exiting ....  and I don't know how to fix it. Could you please help me out? Thank you very much!!  
> **username_2**: Can you put this as a separate question please?  
> **username_0**: Certainly. The question is posted in this thread: https://bioinformatics.stackexchange.com/questions/10534/how-to-define-an-outgroup-to-build-a-robust-amino-acid-tree. Thank you @Michael G.  

---

