####Pipeline for mitochondrial sequencing reads. Performs quality trimming and filtering, de novo assembly, and gene (protein-coding, tRNA, rRNA) annotations in GFF3 and FASTA format.

######Prerequisites:
Python 2.7
CAP3
ABySS
HMMER
FASTX_toolkit

####Add programs to path
Check to see if programs are in path.

For unix systems:
whereis cap3 abyss-pe fastq_quality_filter hmmbuild

If any are missing, edit environment file with nano ~/.bashrc and add an entry to the directory containing the program

export PATH=/path/to/program:$PATH

####Clone repository

git clone https://github.com/n-long/mito_assembly_annotation.git

####Create index for mitochondrial gene profiles (default is formatted for species closely related to Anolis carolinensis

hmmpress mito_bank.hmm

####Alternatively, create your own profiles from fasta sequences, one gene per file (can be single-gene sequence or multi-fasta formatted alignment of single genes from multiple species)

find . -maxdepth 1 -name "*.fa*" -exec hmmbuild {}_profile.hmm {} \; && cat *profile.hmm > mito_bank.hmm

tRNA/rRNA models are precompiled* in the mitfi/ subdirectory along with the Infernal executable (no PATH adding necessary), and will work for any species without calibration.

####Create symlinks to sequence reads inside mito_assembly_annotation directory

ln -s /path/to/*.fastq mito_assembly_annotation/

####Run mito_anno.py in mito_assembly_annotation directory (assumes sequences have been de-multiplexed and are free of barcodes/adaptors)

python mito_anno.py

Output filenames will match input files. Assembled mitochondrial genomes will end in contigs.fa, genes (including tRNA/rRNA) in _genes.fasta, and coordinates in .gff.

####*tRNA and rRNA databases come from the published MITOS (web server only) datasets

Bernt, M., Donath, A., Jühling, F., Externbrink, F., Florentz, C., Fritzsch, G., ... & Stadler, P. F. (2013). MITOS: Improved de novo metazoan mitochondrial genome annotation. Molecular phylogenetics and evolution, 69(2), 313-319.