# How to make a robust amino acid phylogeny with adequate robustness support

This is the second part of this thread: Duplicated genes and genome fragmentation

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:

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...

• So what is the actual question here? How you can root the tree? Sep 11 '19 at 8:25
• 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__
Sep 11 '19 at 15:04
• 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__
Sep 11 '19 at 15:08
• 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__
Sep 11 '19 at 15:17
• 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. Sep 11 '19 at 15:43

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.

• The mod is asking for a Q&A, when I find out what this is, this will need to be done.
– M__
Sep 12 '19 at 12:52
• 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
Sep 13 '19 at 3:07
• 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
Sep 16 '19 at 23:48
• Hi @leah you can always post it as a separate question
– M__
Sep 17 '19 at 5:50
• 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
Oct 4 '19 at 20:17