After QC, we ran our illumina short reads through a program called Novoplasty v.4.3.1. for mitochondrial genome assembly.

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 --mail-user=zehnp1jr@cmich.edu

#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.

This will be the .fasta file from NOVOPLasty.

Next you will want to take annotations from MITOS2 and manually check gene boundaries in Artemis

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