Nov 09, 2023
Mitochondrial Genome Assembly and Annotation
- zehnpjr 1
- 1Central Michigan University
Protocol Citation: zehnpjr 2023. Mitochondrial Genome Assembly and Annotation. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3xeqqg25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it has worked for us in the past.
Created: June 09, 2023
Last Modified: November 09, 2023
Protocol Integer ID: 83144
Keywords: mitochondrial genome assembly, invertebrate mitochondrial genome, mitochondrial genome, assembling invertebrate, phylogenetic tree, genome, annotation
Funders Acknowledgements:
NSF
Grant ID: ANT-1043745
NSF
Grant ID: OPP-0132032
Disclaimer
I have used the protocol, and it has worked for me, however I am not guaranteeing that this protocol will work.
Abstract
This is a step by step protocol for assembling invertebrate mitochondrial genomes, annotating the genomes , publishing the genomes and then step by step protocol for making a phylogenetic tree using the data from the mitochondrial genomes
Troubleshooting
Novoplasty v4.3.1
After QC, we ran our illumina short reads through a program called Novoplasty v.4.3.1. for mitochondrial genome assembly.
Citation
LINK
Next you want to upload a seed and a reference file. The seed is where you would want the assembler to start assembling the program. I used the COI of an already published mitochondrial genome. Next you will want to upload the reference genome. I used the already published mitochondrial genome of the sea spider Achelia bituberculata.
Then you want to create a config.txt file for your assembly. It should loo like this:
Project:
-----------------------
Project name= A_M
Type= mito
Genome Range= 12000-22000
K-mer = 33
Max memory=
Extended log= 0
Save assembled reads= no
Seed Input= /mnt/home/zehnp1jrCMICH/NOVOPlasty/NOVOPlasty/Seed.fasta
Extend seed directly= no
Reference sequence= /mnt/home/zehnp1jrCMICH/NOVOPlasty/NOVOPlasty/reference.fasta
Variance detection=
Chloroplast sequence=
Dataset 1:
-----------------------
Read Length = 151
Insert size = 300
Platform= illumina
Single/Paired = PE
Combined reads=
Forward reads = /mnt/home/zehnp1jrCMICH/NOVOPlasty/NOVOPlasty/M_R1.fastq
Reverse reads = /mnt/home/zehnp1jrCMICH/NOVOPlasty/NOVOPlasty/M_R2.fastq
Heteroplasmy:
-----------------------
MAF =
HP exclude list =
PCR-free=
Optional:
-----------------------
Insert size auto= yes
Use Quality Scores= no
Then you will want to create a submission file (.sh file) it should look like this:
#!/bin/sh --login
#SBATCH --time=5:00:00
#SBATCH --nodes=1
#SBATCH --cpus-per-task=20
#SBATCH --mem=250G
#SBATCH [email protected]
#SBATCH --mail-type=FAIL,BEGIN,END
#SBATCH -J A_Mitochondrial
cd ${SLURM_SUBMIT_DIR}
perl NOVOPlasty4.3.1.pl -c config.txt
scontrol show job $SLURM_JOB_ID
Then you want to submit the job. If using a cluster its a good idea to submit as a job rather than run in the terminal as you will most likely run out of resources and your task will be terminated in the terminal.
Submit consensus file from Artemis to MITOS for Annotation.
Citation
LINK
This will be the .fasta file from NOVOPLasty.
Next you will want to take annotations from MITOS2 and manually check gene boundaries in Artemis
Citation
LINK
Ammothea_clausi_example.tbl 
Mitogenome_notes_aftermitos (1).txt Next I followed these steps for my submission to NCBI.
Phylogenetic tree building with protein coding gene data 
Phylogenetic_tree_notes.txt
Phylogenetic_tree_notes.txt