Ag100Pest / mitoPolishCLR

The Mitogenome Assembly Pipeline for CLR + Illumina Ag100Pest

Ag100pest CLR+Illumina mt genome pipeline

This approach builds upon the mitoVGP pipeline (https://github.com/gf777/mitoVGP) to assemble, polish, circularize, qv check, and annotate Ag100Pest mt genomes frm CLR + Illumina data.

Dependencies

mitoVGP

https://github.com/gf777/mitoVGP

Formenti, G., Rhie, A., Balacco, J. et al. Complete vertebrate mitogenomes reveal widespread repeats and gene duplications. Genome Biol 22, 120 (2021). https://doi.org/10.1186/s13059-021-02336-9

conda env create -f mitoVGP_conda_env_pacbio.yml --prefix /project/ag100pest/software/modified_mitoVGP/pacbio_mitoVGP

mitofinder

https://github.com/RemiAllio/MitoFinder

Allio, R., Schomaker-Bastos, A., Romiguier, J., Prosdocimi, F., Nabholz, B., & Delsuc, F. (2020) Mol Ecol Resour. 20, 892-905. (publication link)

Input data

• PB_list.txt: List of long reads
• I_list.txt: List of shotgun short reads
• <reference>.fasta: Mitochnodrial genome of a closely related species. In this case, Sitotroga cerealella mitochondrion, complete genome
• <reference>.gb: Mitochnodrial genbank of a closely related species. In this case, Sitotroga cerealella mitochondrion, complete genome

Mercury and Meryl

Basic Usage

Export software directory to your path. Add this to .bashrc to export PATH="/project/ag100pest/software/modified_mitoVGP/:$PATH"

Download reference.fasta and refererence.gb from a closely related species. get_ref.sh <MT ACCESSION ID>

Prepare expected inputs I_list.txt for Illumina Polishing and PB_list.txt with subreads.bam. Eg ls /project/ag100pest/Illumina_polishing/JAMU*{R1,R2}.fastq.bz2 > I_list.txt

submit_

Reference mt genomes

Reads are assembled if they align against the reference mitogenome, so best to have a complete assembly.
Find closely related mitogenomes at https://goat.genomehubs.org/ Pull fasta or gb with EDirect suite from NCBI esearch -db nucleotide -query “<Accession #>” | efetch -format fasta >out.fasta

Assembly

We use the mitoVGP approach to assemble, polish, and do preliminary trimming. Submit with sbatch submit_MTAsmPolishCLR.slurm which contains the shell script run_mitoVGP.sh. Currently this is hard-coded with species and parameters.

1. $mitoVGPdir/scripts/01_mtDNApipe 1. identifies MT-like reads by aligning long reads to reference sequence 2. assembles contigs with canu/1.8
2. $mitoVGPdir/scripts/02_blastMT identifies the best assembled mitocontig with blast
3. $mitoVGPdir/scripts/03_mitoPolish polishes mitocontig with two rounds of Arrow using the same reads used by Canu
4. $mitoVGPdir/scripts/04_map10x1 polishes mitocontig with short reads by aligning to the mitocontig
5. $mitoVGPdir/scripts/05_trimmer generates a trimmed version of the Canu mitocontig after short read polishing (map10x1). The resulting contig has 100 bp overlapping ends on both sides.

• If you need to span a long repetitive region, increase the mummer sensitivity -z 500 to -z 5000 (or more?)
• If the overlapping sequences at both ends of the contig are perfectly identical, then show-coords doesn't print BEGIN and END. The grep fails and kicks out an error message, "trimming failed; trimming with MitoFinder". Then we use the circularization approach of mitofinder, which uses a blast self-alignment to identify overlap instead. 6. Another round of polishing to clean up trimmed ends. This is not implemented yet. Skip if qv doesn't find "false kmers".

Annotation

MitoFinder. Annotations are found in <job_name>_MitoFinder_mitfi_Final_Results

Report summary

summary.sh prints results from mitofinder