Hands On

Background

Ok, so on the second day of our course you have assembled the mitogenome of your species with Hifiasm and Hicanu. Well, the contigs are the mitochondria, but they don’t represent a final mitochondrial sequences we usually find on databases, right? Firts: for some of you, hifiasm or hicanu is likely to have outputed more than one contig, and second, the contig size if much larger than a mitogenom usually is. True! This happens because of the circular nature of the molecule, and because of how assemblies work! Basically the overlaps of the circular molecule makes the assemblers confused and they end up concatenating the circular molecule many times!

So I have written a pipeline that can finish the assembly of your mitochondria for you! It basically blasts your assembled Pacbio HiFi contigs to close-related species mitogenome, it does a series of parsings to be sure you have a contig that is only the mitogenome and not a NUMT, they it will circularise the contig, cut it and anotate it with MitoFinder.

For more information on MitoHifi, have a look here: https://github.com/marcelauliano/MitoHiFi

Setting up the environment for running MitoHiFi

At your home directory, create a new directory to work on MitoHiFi and move to it:

mkdir ~/mitohifi_test_01/
cd ~/mitohifi_test_01/

After that, activate the mitohifi_env conda environment created in advance by your instructors that contains some dependencies needed to run MitoHiFi:

conda activate mitohifi_env

To run MitoHiFi, first you need a close-related mitochondria in fasta and genbank format. We have a script that can help you find this input. Giving the name of the species you are assembling, the script is going to look for the closest mitochondria it can find on NCBI. You can give the parameter -s to the script if you would like to restrict your mitochondria search for species within your given genus, but this means the script can download partial mitochondrial sequences. Otherwise, without -s, the script is going to search for complete mitochondrias only and as close as possible to your species on interest.

Before running the script, you need to export MitoHiFi’s directory to the PATH environment variable:

export PATH=$PATH:/home/ubuntu/Share/softwares/MitoHiFi/src/

Now you are ready to run the script to find the related mitogenome:

findMitoReference.py --species "Phalera bucephala" --email <your_email> --outfolder refData --min_length 15000

Where <your_email> should be replaced by your email (your personal/work email should work just fine)

findMitoReference.py output

This command will output a fasta (OQ830676.1.fasta) and a genbank (OQ830676.1.gb) file from the closely related mitogenome. When running MitoHiFi, you will use those files as input using, respectively, the -f and -g options.

Running MitoHiFi

Now let’s run MitoHiFi using an example dataset. Before that, we need to add MitoFinder (which is used by MitoHiFi to do the annotation step) to your PATH:

export PATH=$PATH:/home/ubuntu/Share/softwares/MitoFinder/

(Optional) You can test if mitofinder has been successfully added to your PATH by running the following command and checking if it returns a help message explaining how the mitofinder program is supposed to be run:

mitofinder -h

Now you need to create a symlink to the mitohifi script:

ln -s /home/ubuntu/Share/softwares/MitoHiFi/src/mitohifi.py

(Optional) You can test if mitohifi has been successfully set by running the help command:

python mitohifi.py -h

Copy the the test.fa file to your current directory. The test.fa is a multifasta file that contains 3 assembled contigs. It’s been generated in advance by your instructors.

PS: of course in the real world you assembly file will have a much higher number of contigs, but here we are working with a limited number for computational and time constraints.

cp /home/ubuntu/Share/MitoHiFi_data/test.fa .

Finally, run MitoHiFi for the contigs test dataset:

python mitohifi.py -c test.fa -f refData/OQ830676.1.fasta -g refData/OQ830676.1.gb -t 1 -o 5

The pipeline will probably take a few minutes to run. Once it’s done, it will output a message saying Pipeline finished!.

Questions:
1) What’s the meaning of each parameter used to run MitoHiFi? Hint: if in doubt, run python mitohifi.py -h and/or check the official MitoHiFi documentation at github.
2) Has MitoHiFi succeded creating final mitogenome fasta/genbank files? What are the names of those files?
3) Open the final mitogenome genbank file and answer: i) what’s the total length of the mitogenome?; ii) what’s the first gene in that mitogenome?
4) Go to BLAST webserver and do a blastn of the final mitogenome fasta file (final_mitogenome.fasta) against the Nucleotide collection (nr/nt). What’s the first hit you get from the alignment? What species does that hit come from? Click on the hit accession number hyperlink. You should be redirected to a new page. Go to the COMMENT section and answer: how was this sequence assembled?
5) Open the contigs_stats.tsv file and answer: is there another mitogenome assembly besides the final_mitogenome?

We can discuss the results together. =)