3. PAR gene tree analyses and between-sex pairwise distances
3.1 Make FASTA files for each exon
# Define directories and files
krakendir="kraken/bTaeGut1.pat.W.v2"
genes="ZF_PAR.genes.new.list"
home="/cfs/klemming/projects/supr/snic2020-2-25/user_data/hanna/sylvioidea_sexchromosome/supplementary_code_testing"
# Combine female and male PAR exon sequences from all species into a single file
cat samples_sex_sameline_ref.tsv | cut -f 3 | while read sp ; do
cat $krakendir/${sp}/${sp}.PAR.exonSeparate.female.fasta | sed "s/>/>${sp}_female_/"
done > allSp.PAR.exonSeparate.female.male.fasta
cat samples_sex_sameline_ref.tsv | cut -f 3 | while read sp ; do
cat $krakendir/${sp}/${sp}.PAR.exonSeparate.male.fasta | sed "s/>/>${sp}_male_/"
done >> allSp.PAR.exonSeparate.female.male.fasta
# Generate list of unique PAR exons
cat allSp.PAR.exonSeparate.female.male.fasta | grep ">" | sed 's/male_/\t/' | cut -f 2 | sort | uniq > allPARexons.list
# Function to format fasta files into single-line sequences
oneline_fasta() {
awk '/^>/ {printf("\n%s\n",$0);next;} { printf("%s",$0);} END {printf("\n");}' "$1"
}
# Separate fasta files by gene and save them
mkdir exonSeparate
cat allSp.PAR.exonSeparate.female.male.fasta | grep ">" | sed 's/male_/\t/' | cut -f 2 | sort | uniq | while read gene ; do
oneline_fasta allSp.PAR.exonSeparate.female.male.fasta | grep ${gene}$ -A 1 | grep -v "^--" | sed 's/SylAtr_1EV02922/SylAtr/' > exonSeparate/${gene}.fasta
done
3.2 Make alignments with PRANK
interactive -A naiss2024-5-340 --cores 7 --mem 50000 --tmp 100
salloc --cores=24 -t 2:00:00 -A naiss2024-5-340 -p main
mkdir exonSeparate/prank2
cd exonSeparate/prank2
module load bioinfo-tools
module load prank/170427
module load parallel
# Generate file list and align using prank
ls ../ | grep fasta$ > files
srun parallel -j 22 'prank -d=../{} -o={}.prank.aln -f=fasta -F ' :::: files
srun parallel -j 22 'prank -d=../{} -o={}.prank.F.aln -f=fasta +F ' :::: files
# Rename sequences in alignment files and make lists of files
mkdir IDfix
ls | grep fasta.prank.aln.best.fas | while read file ; do
cat $file | sed 's/male_/male\t/' | cut -f 1 > IDfix/${file}
done
# Create file list for each gene in order
cd IDfix
ls | grep aln | sed 's/_/\t/' | cut -f 1 | sort | uniq | while read gene ; do
ls | grep $gene | sed 's/_/\t/' | sed 's/.fasta.prank.aln.best.fas//' | cut -f 2 | sort -n | awk '{print "'"$gene"'""_"$1".fasta.prank.aln.best.fas"}' | tr '\r\n' ' '
echo
done > file_lists_wide.list
# Download catfasta2phyml.pl script here: https://github.com/nylander/catfasta2phyml
# Concatenate sequence files
mkdir concat
ls | grep aln | sed 's/_/\t/' | cut -f 1 | sort | uniq | while read gene ; do
cat file_lists_wide.list | grep $gene | while read line ; do
../../../catfasta2phyml.pl -f $line -c > concat/${gene}.fasta.prank.aln.best.fas
done
done
# Add zebra finch sequences to the alignment
mkdir mafft_ZF
mkdir TaeGut_sequences
mkdir renamed
cp concat/*fasta.prank.aln.best.fas mafft_ZF/
# Rename sequences and process zebra finch data
cd $home
cat PAR_genes_geneID_transID_geneName.list | while read name gene trans ; do
cp exonSeparate/prank2/IDfix/concat/${gene}.fasta.prank.aln.best.fas exonSeparate/prank2/IDfix/renamed/${name}.fasta.prank.aln.best.fas
done
cat PAR_genes_geneID_transID_geneName.list | while read name gene trans ; do
oneline_fasta ../data/external_raw/genome/longestTranscripts.exons.fa | grep $trans -A 1 | sed 's/>/>TaeGut_Z\t/' | cut -f 1 > exonSeparate/prank2/IDfix/TaeGut_sequences/${name}.TaeGut.fa
done
cat W_PAR_genes_geneID_transID_withGeneName.updated.list | while read name gene trans ; do
oneline_fasta ../data/external_raw/genome/longestTranscripts.exons.fa | grep $trans -A 1 | sed 's/>/>TaeGut_W\t/' | cut -f 1 >> exonSeparate/prank2/IDfix/TaeGut_sequences/${name}.TaeGut.fa
done
# Re-align using MAFFT
cd exonSeparate/prank2/IDfix
ls renamed/ | sed 's/.fasta.prank.aln.best.fas//' | while read gene ; do
mafft --reorder --add TaeGut_sequences/${gene}.TaeGut.fa --auto renamed/${gene}.fasta.prank.aln.best.fas > mafft_ZF/${gene}.realn.fasta
done
3.3 Trim alignments with trimAI
# Trim alignments with TrimAl
module load trimAl/1.4.1
cd mafft_ZF
ls | grep fasta$ | while read gene ; do trimal -in $gene -out ${gene}.auto.trim -automated1 ; done
# Species-specific trimming
# RAX - removing PanBia (would remove half the sequence if kept)
trimal -in RAX.realn.fasta.auto.trim -selectseqs { 2,3 } > RAX.realn.fasta.auto.selectseqs.trim
iqtree -s RAX.realn.fasta.auto.selectseqs.trim -m TEST -bb 1000 -alrt 1000
# uncharacterized3 - remove PanBia
trimal -in uncharacterized3.realn.fasta.auto.trim -selectseqs { 28,29 } > uncharacterized3.realn.fasta.auto.selectseqs.trim
# uncharacterized4 - remove PycBar (short) and AcrSch (bad alignment)
trimal -in uncharacterized4.realn.fasta.auto.trim -selectseqs { 0,1,4,5 } > uncharacterized4.realn.fasta.auto.selectseqs.trim
# ST8SIA3 - remove SylAtr (short)
trimal -in ST8SIA3.realn.fasta.auto.trim -selectseqs { 4,5 } > ST8SIA3.realn.fasta.auto.selectseqs.trim
ls | grep fasta$ | while read gene ; do trimal -in $gene.auto.selectseqs.trim -out ${gene}.auto.selectseqs.gt.0.8.trim -gt 0.8 ; done
module load bioinfo-tools
module load iqtree/1.5.3-omp
ls | grep fasta$ | while read gene ; do iqtree -s ${gene}.auto.selectseqs.gt.0.8.trim -m TEST -bb 1000 -alrt 1000 ; done
ls | grep fasta$ | while read gene ; do iqtree -s ${gene}.auto.gt.0.8.trim -m TEST -bb 1000 -alrt 1000 ; done