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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?

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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.

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    $\begingroup$ 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 ... $\endgroup$
    – K_081
    May 23, 2023 at 6:04
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    $\begingroup$ Thanks @M__ how about 15:00 hours GMT? $\endgroup$
    – K_081
    May 23, 2023 at 16:01
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    $\begingroup$ Sure @M__ . See you! $\endgroup$
    – K_081
    May 24, 2023 at 1:54
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    $\begingroup$ 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? $\endgroup$
    – K_081
    May 31, 2023 at 10:16
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    $\begingroup$ 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. $\endgroup$
    – M__
    May 31, 2023 at 10:48

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