###### tags: `hybpiper` `reese` `phylogeny`

# Abronia HybSeq Phylogeny

[TOC]

## Sequence Data

### February 2021 NovaSeq

38 samples in: `/media/johnsonseq/2021-02-08_novaseq6000_fastq/Abronia`

### June 2021 NovaSeq

24 samples in: `/media/johnsonseq/2021-06-24_novaseq6000_fastq/Abronia`

## Trim Reads with FASTP
`cp fastp.sh fastp2.sh`

## HybPiper

To improve sequence recovery we made a new Angiosperms353 target file with sequences from Abronia. The new target file is located at:

`/scratch/joh97948/Abronia/angiosperms353.abronia.fasta`

Make a namelist (run from the reads directory):
`ls | cut -f 1 -d '.' | sort -u > ../hybpiper/new_namelist.txt`

while read name

do
/opt/apps/Software/HybPiper/reads_first.py --prefix $name --bwa -r ../reads/"$name".R*.paired.fastq -b /scratch/joh97948/Abronia/angiosperms353.abronia.fasta --cpu 50 --timeout 5000
python /opt/apps/Software/HybPiper/intronerate.py --prefix $name
python /opt/apps/Software/HybPiper/cleanup.py $name

done < new_namelist.txt

### Get seq lengths

Run from the `Abronia/hybpiper` directory

`python /opt/apps/Software/HybPiper/get_seq_lengths.py /scratch/joh97948/Abronia/angiosperms353.abronia.fasta namelist.txt DNA > abroniaseqlengths.txt`

### Get hybpiper stats
Run from the `Abronia/hybpiper` directory:

`python /opt/apps/Software/HybPiper/hybpiper_stats.py abroniaseqlengths.txt namelist.txt > hybpipstats.txt`

### Extract supercontig sequences

Run from the HybPiper output directory (`Abronia/hybpiper`), unaligned sequences go into the Sequences

`python /opt/apps/Software/HybPiper/retrieve_sequences.py /scratch/joh97948/Abronia/angiosperms353.abronia.fasta ../new_namelist.txt --fasta_dir /scratch/sherpric/Abronia/Sequences2 supercontig`

## Phylogeny

### MAFFT

Run this from the Sequences directory to remove the gene name from the sample IDs:
`parallel sed -i "s/-{}//g" {}_supercontig.fasta :::: genelist.txt`

Run this from the Abronia directory:

`parallel "mafft --preservecase --maxiterate 1000 --localpair Sequences2/{}_supercontig.fasta > MAFFT2/{}.mafft.fasta" :::: genelist.txt`
### Trimal

Run from the Abronia directory:

`parallel -j 10 --eta trimal -in MAFFT2/{}.mafft.fasta -out trimal2/{}.trimal.fasta -gt .5 :::: genelist.txt`

### IQTree
Run from the Abronia directory:
`parallel --eta -j 10 iqtree -s trimal2/{}.trimal.fasta -m MFP -B 1000 :::: genelist.txt `

### ASTRAL

Remove the gene names from the sequence names.
Run this from the trimal directory.

`parallel sed -i "s/-{}//g" {}.trimal.fasta.treefile :::: ../genelist.txt`

Go back to the Abronia directory.
Collapse gene trees so that they don't have branches with poor support:

`mkdir collapsed`

`parallel "nw_ed trimal/{}.trimal.fasta.treefile 'i & b<=30' o > collapsed/{}.collapsed.tre" :::: genelist.txt`

Put all of the gene trees into a single file
`cat collapsed/*.tre > abronia.genetrees.tre`

Run ASTRAL

Run from the Abronia directory
`java -jar /opt/apps/Software/ASTRAL/Astral/astral.5.6.3.jar -i abronia.genetrees.tre > abronia.astral.tre`

Change the names so that the species are listed and not just the accessions:

`python /scratch/joh97948/Abronia/treeLabelReplace.py abronia.astral.tre /scratch/joh97948/Abronia/abronia_label_replace.txt > abronia.astral.renamed.tre`

Download `abronia.astral.tre` to your computer and open in FigTree to see it!

### PhyParts

Reroot all of the gene trees using the Mirabilis outgroup sequences:

Make a directory for rerooted trees:

`mkdir reroot`

The `reroot_trees_mirabilis.py` script will reroot all gene tres based

`parallel "python reroot_trees_mirabilis.py collapsed2/{}.collapsed.tre > reroot/{}.rerooted.tre" :::: genelist.txt`

Some trees will be empty either because we didn't have sequences or we don't have any outgroup (and therefore cannot re-root the tree). Delete these:

`find reroot -size 0 -delete`

Also reroot the ASTRAL species tree:

`python reroot_trees_mirabilis.py abronia.astral.tre > abronia.astral.rerooted.tre`

Run PhyParts to calculate bipartitions on the rooted trees:

`mkdir phyparts; cd phyparts`

`java -jar /opt/apps/Software/phyparts/target/phyparts-0.0.1-SNAPSHOT-jar-with-dependencies.jar -a 1 -d ../reroot -m ../abronia.astral.rerooted.tre -v`

Download the phyparts output and the rerooted astral tree to make the pie charts (the script doesn't really work on a remote computer)

`python ~/phyloscripts/phypartspiecharts/phypartspiecharts.py --taxon_subst ../abronia_label_replace.csv abronia.astral.rerooted.tre out 339`