<p>Bootstrapping is easy just use <strong>iqtree</strong></p>
<pre><code>iqtree -s file -m GTR+I+G -T 6 -B 1000 
</code></pre>
<p>This will parallelise across 6 CPUs and results in 1000 bootstraps, whilst GTR is the mutation model, which models invariant sites under a 4 category gamma distribution. This is a robust model.</p>
<p>The <code>file</code> is aligned fasta format (its all data pipeline so I forget if I've shifted the format) - although this can be changed to any input format</p>
<p>The result will be in the treefile and is a professional quality tree, complete with bootstraps.</p>
<p>The following up analysis is a Bayesian tree and is more complicated.</p>
<p><strong>Note</strong> You must resolve/delete gaps using iqtree because of the way the authors are calling gaps - this related to your question here - <a href="https://bioinformatics.stackexchange.com/questions/21042/how-to-use-gblocks-for-trimming-single-copy-gene-sequences">How to use Gblocks for trimming single-copy gene sequences?</a> . Basically columns with gaps should be deleted.</p>
<hr />
<p>Question, are you sure this is not a Bayesian analysis? It would make sense if it was a Bayesian analysis that is 100% robust (there are robustness scores there). This would make sense for this tree and this data. As far  I am aware iTOL is used for viewing and manipulating trees rather than creating them.</p>

CC BY-SA 4.0
2023-05-23T06:04:26.947Z
Thank you very much for the answer @M__ . Yes, iTOL is used for viewing trees and not creating them. The tree itself is created by Fasttree which is part of OrthoFinder, but the output is a text file. I did use this command and I got an error : File not found GTR .  And then I included `-st DNA` in the command but it still did not resolve the issue.  This is what I get -- WARNING: Number of command-line arguments differs from checkpoint
WARNING: Command-line differs from checkpoint!
Reading alignment file Metabacillus_Tree.fa ... Fasta format detected
...
K_081
CC BY-SA 4.0
2023-05-23T12:31:56.490Z
Hi @KirtiKulanthaivel the best thing to do arrange a chat? If ok you will need your data available and access to a terminal and understand bash commands such as `more` and/or have access to a text editor such as BBEdit. `GTR` is central to the model, it will not work without it. Also could you install `iqtree` via conda? If that is not clear I will explain. If ok please specify availability in GMT, early afternoon is preferred. It is better to do this interactively than via question/answer. I will ask questions and ask you to post a small sample of your data and output errors. Let me know.
M__
CC BY-SA 4.0
2023-05-23T12:38:10.543Z
The bioinformatics chat room is here: https://chat.stackexchange.com/rooms/58858/bioinformatics. .. can alternatively do this via question/answer - the key thing is `GTR` must be resolved. Something is not right for this error to be thrown and I don't know what it is. The theory as to why is complicated but getting it working is getting it working.
M__
CC BY-SA 4.0
2023-05-23T16:01:46.670Z
Thanks @M__ how about 15:00 hours GMT?
K_081
CC BY-SA 4.0
2023-05-23T17:09:02.490Z
@KirtiKulanthaivel my apologies, I am not around today, but tomorrow is fine. 15:00 hours cool.
M__
CC BY-SA 4.0
2023-05-24T01:54:38.707Z
Sure @M__ . See you!
K_081
CC BY-SA 4.0
2023-05-31T10:16:03.653Z
Hi @M__ ! Just wanted you to know that the IQTREE code worked but I had to increase the bootstrap replicates from 100 to 1000 when the terminal read this message - #replicates must be >= 1000
The tree came out well, thank you very much. By the way, when running iqtree, there is a small table that shows the Gap/Ambiguity, Composition and p-value for the aligned SNP files in question (for 11 genomes in my case). It says 0.00 under Gap/Ambiguity and p-value and 'failed' under composition, although the tree is made. Why is that so?
K_081
CC BY-SA 4.0
2023-05-31T10:48:36.107Z
Hi @KirtiKulanthaivel thanks for the feedback. The gap/ambiguity is good because you removed all "gaps" via trimal. The ambiguity, is the sequence is high quality - no ambiguities. The p-value of 'failed' I would need to look up. It sounds like it's attempting to estimate ambiguities/gaps according to a known distribution (e.g. Poison) but it can't because there aren't any. If it was a p-value <0.01 I suspect that would be a problem, 'failed' seems fine.
M__
CC BY-SA 4.0
2023-05-22T13:57:16.787Z
2023-05-22T14:03:10.553Z
2023-05-22T14:03:10.553Z
M__
<p>When we run the OrthoFinder analysis tool on a group of genomes to get the orthologues shared by them one of the output files include a folder named 'Species_Tree' that contains a text file named 'SpeciesTree_rooted.txt' . These are its contents (This is an example I have used for the question - 11 genomes of the <em>Metabacillus</em> genus) :</p>
<blockquote>
<p>((Mlacus:0.244776,Mmangrovi:0.284609)1:0.0277466,(((Mdong:0.0284157,Midriensis:0.0250137)1:0.0553715,Mindicus:0.0904605)1:0.148105,(Mfastidiosus:0.207986,(Mniabensis:0.142138,((Mcrassostreae:0.0124653,Mlitoralis:0.0139468)1:0.116616,(Mhalosaccharovorans:0.0408989,Mschmidt:0.0422379)1:0.0872204)1:0.0402912)1:0.0849362)1:0.0750784)1:0.0277466);</p>
</blockquote>
<p>I used iTOL to construct the tree using this file but I could only see the branch lengths and no way to add any bootstrap values if there had been any, to the tree as seen in most research papers - 99, 98, 100 etc. Any suggestions?</p>

<phylogenetics><phylogeny><taxonomy><orthofinder>
How to add bootstrap values to the phylogenetic tree generated by OrthoFinder?
# How to add bootstrap values to the phylogenetic tree generated by OrthoFinder?

(Asked by: username_0 on 2023-05-22)

When we run the OrthoFinder analysis tool on a group of genomes to get the orthologues shared by them one of the output files include a folder named 'Species\_Tree' that contains a text file named 'SpeciesTree\_rooted.txt' . These are its contents (This is an example I have used for the question - 11 genomes of the *Metabacillus* genus) :

> ((Mlacus:0.244776,Mmangrovi:0.284609)1:0.0277466,(((Mdong:0.0284157,Midriensis:0.0250137)1:0.0553715,Mindicus:0.0904605)1:0.148105,(Mfastidiosus:0.207986,(Mniabensis:0.142138,((Mcrassostreae:0.0124653,Mlitoralis:0.0139468)1:0.116616,(Mhalosaccharovorans:0.0408989,Mschmidt:0.0422379)1:0.0872204)1:0.0402912)1:0.0849362)1:0.0750784)1:0.0277466);

I used iTOL to construct the tree using this file but I could only see the branch lengths and no way to add any bootstrap values if there had been any, to the tree as seen in most research papers - 99, 98, 100 etc. Any suggestions?

**Tags:** phylogenetics, phylogeny, taxonomy, orthofinder

---

## 1 answers

### Answer by username_1 on 2023-05-22. Score: 1 | Accepted answer

Bootstrapping is easy just use iqtree
iqtree -s file -m GTR+I+G -T 6 -B 1000 

This will parallelise across 6 CPUs and results in 1000 bootstraps, whilst GTR is the mutation model, which models invariant sites under a 4 category gamma distribution. This is a robust model.
The file is aligned fasta format (its all data pipeline so I forget if I've shifted the format) - although this can be changed to any input format
The result will be in the treefile and is a professional quality tree, complete with bootstraps.
The following up analysis is a Bayesian tree and is more complicated.
Note You must resolve/delete gaps using iqtree because of the way the authors are calling gaps - this related to your question here - How to use Gblocks for trimming single-copy gene sequences? . Basically columns with gaps should be deleted.

Question, are you sure this is not a Bayesian analysis? It would make sense if it was a Bayesian analysis that is 100% robust (there are robustness scores there). This would make sense for this tree and this data. As far  I am aware iTOL is used for viewing and manipulating trees rather than creating them.

> **Comments:**
> **username_0**: Thank you very much for the answer @M__ . Yes, iTOL is used for viewing trees and not creating them. The tree itself is created by Fasttree which is part of OrthoFinder, but the output is a text file. I did use this command and I got an error : File not found GTR .  And then I included `-st DNA` in the command but it still did not resolve the issue.  This is what I get -- WARNING: Number of command-line arguments differs from checkpoint WARNING: Command-line differs from checkpoint! Reading alignment file Metabacillus_Tree.fa ... Fasta format detected ...  
> **username_1**: Hi @KirtiKulanthaivel the best thing to do arrange a chat? If ok you will need your data available and access to a terminal and understand bash commands such as `more` and/or have access to a text editor such as BBEdit. `GTR` is central to the model, it will not work without it. Also could you install `iqtree` via conda? If that is not clear I will explain. If ok please specify availability in GMT, early afternoon is preferred. It is better to do this interactively than via question/answer. I will ask questions and ask you to post a small sample of your data and output errors. Let me know.  
> **username_1**: The bioinformatics chat room is here: https://chat.stackexchange.com/rooms/58858/bioinformatics. .. can alternatively do this via question/answer - the key thing is `GTR` must be resolved. Something is not right for this error to be thrown and I don't know what it is. The theory as to why is complicated but getting it working is getting it working.  
> **username_0**: Thanks @M__ how about 15:00 hours GMT?  
> **username_1**: @KirtiKulanthaivel my apologies, I am not around today, but tomorrow is fine. 15:00 hours cool.  
> **username_0**: Sure @M__ . See you!  
> **username_0**: Hi @M__ ! Just wanted you to know that the IQTREE code worked but I had to increase the bootstrap replicates from 100 to 1000 when the terminal read this message - #replicates must be >= 1000 The tree came out well, thank you very much. By the way, when running iqtree, there is a small table that shows the Gap/Ambiguity, Composition and p-value for the aligned SNP files in question (for 11 genomes in my case). It says 0.00 under Gap/Ambiguity and p-value and 'failed' under composition, although the tree is made. Why is that so?  
> **username_1**: Hi @KirtiKulanthaivel thanks for the feedback. The gap/ambiguity is good because you removed all "gaps" via trimal. The ambiguity, is the sequence is high quality - no ambiguities. The p-value of 'failed' I would need to look up. It sounds like it's attempting to estimate ambiguities/gaps according to a known distribution (e.g. Poison) but it can't because there aren't any. If it was a p-value <0.01 I suspect that would be a problem, 'failed' seems fine.  

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