Troubleshooting‐Tutorial - Pas-Kapli/CoME-Tutorials GitHub Wiki

git clone https://github.com/Pas-Kapli/CoME-Tutorials.git
cd CoME-Tutorials

If you clone the repository, it will include all of the files for this tutorial. You do not need to do it for each exercise. Alternatively, you can download the necessary files and then extract them from the tar file.

wget https://raw.githubusercontent.com/Pas-Kapli/CoME-Tutorials/main/ex_1.tar.gz
tar -xzvf ex_1.tar.gz

You try to run the program using the code below, but you get an error message. (You may need to change the path to the mcmctree executable depending how you installed the program.)

mcmctree mcmctree.ctl

The wiki for mcmctree can be found at https://github.com/abacus-gene/paml/wiki/MCMCtree. Mcmctree is part of the paml package, the wiki for all of paml can be found here.

Determine the cause of the error and correct it to estimate a time calibrated tree.

A basic command for performing the delimitation is the following:

mptp --ml --multi --tree_file Gallotia.con.tre --output_file delimitation --outgroup Lacerta_lepida

"Gallotia.tre" is the input tree file and "Lacerta_lepida" is one of the outgroups included in the dataset.

You can download the input tree file with the following command in your working directory:

wget https://raw.githubusercontent.com/Pas-Kapli/CoME-Tutorials/main/ex_2/Gallotia.con.tre

If the analysis runs correctly we should see something like the following printed on the screen:

mptp 0.2.5_linux_x86_64, 15GB RAM, 8 cores
https://github.com/Pas-Kapli/mptp
Parsing tree file...
Loaded unrooted tree...
Converting to rooted tree...
Number of edges greater than minimum branch length: 93 / 128
Score Null Model: 267.532663
Best score for multi coalescent rate: 379.572833
LRT computed p-value: 0.000000
Writing delimitation file delimitation.txt ...
Number of delimited species: 10
Creating SVG delimitation file delimitation.svg ...
Done...

There should also be two output files in your home directory: delimitation.svg delimitation.txt

If that's not your outcome can you figure out what the problem might be?

wget https://raw.githubusercontent.com/Pas-Kapli/CoME-Tutorials/main/ex_3.tar.gz
tar -xzvf ex_3.tar.gz

Take a look at the list of files in sequenceFiles.txt and some of the individual sequence files. The script below is in the file example3.sh. It is supposed to combine the files and then align the sequences using muscle. The script does not produce your alignment, msa_align.fa. Note, you may have to change the path to muscle depending where you installed it.

#!/bin/bash

# This is included so you don't duplicate all the sequences
# if you run this script multiple times. 

rm -f sequenceToAlign.txt

for seq in $(cat sequenceFiles.txt)
do
        # Add sequence to file
        cat ${seq} >> sequenceToAlign.txt

done

sed -i 's/\t/\n/g' sequenceToAlign.txt

muscle -align sequenceToAlign.txt -output msa_align.fa &> outMuscle

Determine why the script does not produce the expected output.

wget https://raw.githubusercontent.com/Pas-Kapli/CoME-Tutorials/main/ex_4.tar.gz
tar -xzvf ex_4.tar.gz

You run the script example4.sh, shown below. You notice a warning message in outMuscle.

#!/bin/bash

# This is so the script can be run multiple times
# without needing to remove the sequence file 
# created previously.
rm -f sequenceToAlign.txt

for seq in $(cat sequenceFiles.txt)
do

        # Add sequence to file
        cat ${seq} >> sequenceToAlign.txt

done

# Formatting the sequences for muscle
sed -i 's/\t/\n/g' sequenceToAlign.txt

muscle -align sequenceToAlign.txt -output msa_align.fa &> outMuscle

Determine the cause of the warning message and how you would run the program without warnings.

You want to infer a phylogeny based on COI sequences for the genus Gallotia.

Download the relevant dataset with the following command:

mkdir exe5
cd exe5
wget https://raw.githubusercontent.com/Pas-Kapli/CoME-Tutorials/main/ex_5/Gallotia.COI.fasta

Try inferring a gene tree with the following raxml-ng command:

raxml-ng --search1 --msa Gallotia.COI.fasta --model GTR+G 

If the analysis runs correctly you should get the following output files in your working directory:

Gallotia.COI.raxml.bestModel Gallotia.COI.raxml.log Gallotia.COI.raxml.reduced.phy Gallotia.COI.raxml.bestTree Gallotia.COI.raxml.rba Gallotia.COI.raxml.startTree

If that's not the case can you identify what the problem is?

You want to run a bash script that executes a chain of other tools as follows:

1. It downloads a fasta file
2. It aligns the sequences
3. It runs a raxml phylogenetic inference
4. It performs a species delimitation with mPTP

Download the script with:

mkdir exe6
cd exe6
wget https://raw.githubusercontent.com/Pas-Kapli/CoME-Tutorials/main/ex_6/bash-pipe.sh

Make the script executable:

chmod u+x bash-pipe.sh

If the script runs successfully it should print:

Step1: Downloading data.. This alignment contains 65 sequences
Step2: Aligning sequences with mafft..
Step3: Running RAxML..
Step4: Running Species delimitation with mptp:
Final result: The number of delimited species is 10

Can you identify what went wrong?

wget https://raw.githubusercontent.com/Pas-Kapli/CoME-Tutorials/main/ex_7.tar.gz
tar -xzvf ex_7.tar.gz

You try to run the program using the code below, but you get an error message. Again, you may need to change the path to the mcmctree executable depending how you installed the program. The manuals for paml and mcmctree are listed in Exercise 1.

mcmctree mcmctree.ctl

Determine the cause of the error and correct it to estimate a time calibrated tree.

wget https://raw.githubusercontent.com/Pas-Kapli/CoME-Tutorials/main/ex_8.tar.gz
tar -xzvf ex_8.tar.gz

You run the script, whose contents are shown below, which combines all the sequences into one file and then runs raxml-ng. The program runs, but produces many warning messages.

#!/bin/bash

# This is so you can run the script multiple times
# with adding the sequences multiple times
rm -f msa.txt

for seqfile in $(cat sequenceFiles.txt)
do
	name=$(cat $seqfile | awk '{ print $1}')

	# Check to make sure sequence isn't empty
	if [[  $(cat $seqfile | awk '{print $2}' | grep [^-]) ]]; then

		seq=$(cat $seqfile | awk '{print $2}')

	fi

	# Add sequence to file
	echo ${name} >> msa.txt
	echo ${seq} >> msa.txt


done


raxml-ng --msa msa.txt --model JC --msa-format FASTA

Some of the warning messages reflect the variation in the data, while some of them are due to mistakes. What is the mistake?

You want to infer a phylogeny using this small alignment "seq-new.fasta"

mkdir exe9
cd exe9
wget https://raw.githubusercontent.com/Pas-Kapli/CoME-Tutorials/main/ex_9/seq-new.txt

Try inferring a gene-tree with the following command:

raxml-ng --search1 --msa seq-new.fasta --model GTR+G --force

If the analysis runs correctly you should get the following output files in your working directory: seq-new.raxml.bestModel seq-new.raxml.log seq-new.raxml.reduced.phy seq-new.raxml.bestTree seq-new.raxml.rba seq-new.raxml.startTree

If that's not the case can you identify what the problem is?