From 06954e0e4973eed3566167f04324babb88a4e82a Mon Sep 17 00:00:00 2001 From: IvisTang Date: Fri, 2 Oct 2026 03:32:03 +0800 Subject: [PATCH] 20261002 --- .envrc | 2 +- .gitignore | 4 + .vscode/settings.json | 3 +- .../phylogeny_reconstruction/06.snaq.jl | 2 +- 99.scripts_v2/01.prepare_sequences.sh | 34 +- 99.scripts_v2/02.orthofinder.sh | 19 +- 99.scripts_v2/03.gene_phylogeny.sh | 290 + 99.scripts_v2/04.taxa_phylogeny.sh | 174 + 99.scripts_v2/05.dsuite.sh | 143 + 99.scripts_v2/bucky | 1 + 99.scripts_v2/utils/asm_qc.sh | 22 + 99.scripts_v2/utils/bucky.pl | 1503 +++ 99.scripts_v2/utils/classify_frameshifts.py | 269 + 99.scripts_v2/utils/get_og_seqs.py | 148 + 99.scripts_v2/utils/mbblock.txt | 12 + 99.scripts_v2/utils/mrbayes.sh | 20 + 99.scripts_v2/utils/raxml.sh | 20 + 99.scripts_v2/utils/snaq.jl | 195 + 99.scripts_v2/utils/snaq_raw.jl | 19 + pixi.lock | 9989 +++-------------- pixi.toml | 27 +- 21 files changed, 4336 insertions(+), 8560 deletions(-) create mode 100644 99.scripts_v2/03.gene_phylogeny.sh create mode 100644 99.scripts_v2/04.taxa_phylogeny.sh create mode 100644 99.scripts_v2/05.dsuite.sh create mode 160000 99.scripts_v2/bucky create mode 100644 99.scripts_v2/utils/asm_qc.sh create mode 100755 99.scripts_v2/utils/bucky.pl create mode 100644 99.scripts_v2/utils/classify_frameshifts.py create mode 100644 99.scripts_v2/utils/get_og_seqs.py create mode 100644 99.scripts_v2/utils/mbblock.txt create mode 100644 99.scripts_v2/utils/mrbayes.sh create mode 100755 99.scripts_v2/utils/raxml.sh create mode 100644 99.scripts_v2/utils/snaq.jl create mode 100644 99.scripts_v2/utils/snaq_raw.jl diff --git a/.envrc b/.envrc index 07487e6..692165b 100644 --- a/.envrc +++ b/.envrc @@ -1,6 +1,6 @@ set +u export PROJECTHOME="/home/ywtang/project/biyelunwen" -export SCRIPTS="$PROJECTHOME/99.scripts" +export SCRIPTS="$PROJECTHOME/99.scripts_v2" export PUEUE_CONFIG_PATH="$PROJECTHOME/.pueue.yml" watch_file pixi.lock eval "$(pixi shell-hook)" diff --git a/.gitignore b/.gitignore index 2270739..7abd692 100644 --- a/.gitignore +++ b/.gitignore @@ -1,6 +1,7 @@ # pixi environments .pixi/* !.pixi/config.toml +**/__pycache__ .pueue/* 00.rawdata/* 01.reference/* @@ -24,3 +25,6 @@ run.status.json !*/_description.md tmp/ +13.gene_phylogeny/* +14.taxa_phylogeny/* +15.dsuite/* \ No newline at end of file diff --git a/.vscode/settings.json b/.vscode/settings.json index fe57298..4aa6f1d 100644 --- a/.vscode/settings.json +++ b/.vscode/settings.json @@ -1,5 +1,6 @@ { "julia.executablePath": "/home/ywtang/project/biyelunwen/.pixi/envs/default/bin/julia", "julia.enableCrashReporter": false, - "julia.enableTelemetry": false + "julia.enableTelemetry": false, + "python-envs.defaultEnvManager": "ms-python.python:system" } \ No newline at end of file diff --git a/99.scripts/workflow/phylogeny_reconstruction/06.snaq.jl b/99.scripts/workflow/phylogeny_reconstruction/06.snaq.jl index a58c822..9a58120 100644 --- a/99.scripts/workflow/phylogeny_reconstruction/06.snaq.jl +++ b/99.scripts/workflow/phylogeny_reconstruction/06.snaq.jl @@ -1,5 +1,5 @@ #! /usr/bin/env julia -## Installing Dependencies +# Installing Dependencies ## using Pkg ## Pkg.add("Distributed") ## Pkg.add("DataFrames") diff --git a/99.scripts_v2/01.prepare_sequences.sh b/99.scripts_v2/01.prepare_sequences.sh index a343af7..5d7d9dc 100644 --- a/99.scripts_v2/01.prepare_sequences.sh +++ b/99.scripts_v2/01.prepare_sequences.sh @@ -11,21 +11,22 @@ seqkit replace -k HG.map.txt -p "^(\S+).*$" -r "{kv}" GCA_030763125.1_ASM3076312 # MG & ZG use hapA genome grep -Fwf MG.target.txt MG.gff > MG.target.gff grep -Fwf ZG.target.txt ZG.gff > ZG.target.gff +cd .. # Extract CDS sequences from GFF3 files using gffread -pueue add -- gffread -g raw/HN.fa -x HN.cds.fa -y HN.pep.fa raw/HN.gff3 -pueue add -- gffread -g raw/HG.fa -x HG.cds.fa -y HG.pep.fa raw/Hippophae_gyantsensis.gff -pueue add -- gffread -g raw/HS.fa -x HS.cds.fa -y HS.pep.fa raw/HS.gff3 -pueue add -- gffread -g raw/YN.genome.fa -x YN.cds.fa -y YN.pep.fa raw/YN.gff3 -pueue add -- gffread -g raw/ZG.genome.fa -x ZG.cds.fa -y ZG.pep.fa raw/ZG.target.gff -pueue add -- gffread -g raw/ZJ.genome.fa -x ZJ.cds.fa -y ZJ.pep.fa raw/ZJ.gff -pueue add -- gffread -g raw/ZY.fa -x ZY.cds.fa -y ZY.pep.fa raw/ZY.gff3 -pueue add -- gffread -g raw/Hippophae_tibetana.genomic.fasta -x HT.cds.fa -y HT.pep.fa raw/Hippophae_tibetana.mRNA.gff -pueue add -- gffread -g raw/MG.genome.fasta -x MG.cds.fa -y MG.pep.fa raw/MG.target.gff +gffread -V -g raw/HN.fa -x HN.cds.fa -y HN.pep.fa raw/HN.gff3 +gffread -V -g raw/HG.fa -x HG.cds.fa -y HG.pep.fa raw/Hippophae_gyantsensis.gff +gffread -V -g raw/HS.fa -x HS.cds.fa -y HS.pep.fa raw/HS.gff3 +gffread -V -g raw/YN.genome.fa -x YN.cds.fa -y YN.pep.fa raw/YN.gff3 +gffread -V -g raw/ZG.genome.fa -x ZG.cds.fa -y ZG.pep.fa raw/ZG.target.gff +gffread -V -g raw/ZJ.genome.fa -x ZJ.cds.fa -y ZJ.pep.fa raw/ZJ.gff +gffread -V -g raw/ZY.fa -x ZY.cds.fa -y ZY.pep.fa raw/ZY.gff3 +gffread -V -g raw/Hippophae_tibetana.genomic.fasta -x HT.cds.fa -y HT.pep.fa raw/Hippophae_tibetana.mRNA.gff +gffread -V -g raw/MG.genome.fasta -x MG.cds.fa -y MG.pep.fa raw/MG.target.gff cp raw/EA.gff3.cds EA.cds.fa cp raw/EM.genome.cds.fasta EM.cds.fa -cp raw/EA.gff3.pep EA.pep.fa -cp raw/EM.pep.fasta EM.pep.fa +seqkit grep -s -v -r -p '[.*][A-Z]' raw/EA.gff3.pep > EA.pep.fa +seqkit grep -s -v -r -p '[.*][A-Z]' raw/EM.pep.fasta > EM.pep.fa mkdir pep mkdir cds @@ -33,8 +34,15 @@ mv ./*.cds.fa cds mv ./*.pep.fa pep cd pep +# 保留主要转录本 primary_transcript ./ last_dot - # EM基因命名特殊性，导致直接使用primary_transcript会出现问题，直接复制即可 cp EM.pep.fa primary_transcripts/EM.pep.fa - +# 移除终止密码子和序列描述 +cd primary_transcripts +for i in *.pep.fa; do + base=$(basename "$i" .pep.fa) + echo "Processing $base" + sed -i "s/\.$//g" "$i" + sed -i "/^>/ s/[[:space:]].*//" "$i" +done diff --git a/99.scripts_v2/02.orthofinder.sh b/99.scripts_v2/02.orthofinder.sh index fddb88c..7000f67 100644 --- a/99.scripts_v2/02.orthofinder.sh +++ b/99.scripts_v2/02.orthofinder.sh @@ -1,3 +1,7 @@ +#!/bin/bash + +set -euo pipefail + # prepare input files for orthofinder mkdir input_pep cp ../11.reference_v2/pep/primary_transcripts/*.pep.fa input_pep @@ -17,5 +21,18 @@ done pueue add -- orthofinder -t 12 -a 4 -f input_pep/core -o orthofinder_out # assign outgroups -pueue add -- orthofinder -t 12 -a 4 --assign input_pep/outgroup --core orthofinder_out/Results_Hippophae_only +pueue add -- orthofinder -t 12 -a 4 --assign input_pep/outgroup --core orthofinder_out/Results_Aug03 +ln -s orthofinder_out/Results_Aug03 Hippophae_only +ln -s orthofinder_out/Results_Aug03_1/ Hippophae_outgroup + +# get all cds into a file +echo -ne >all_cds.fa +for i in ../11.reference_v2/cds/*.cds.fa; do + base=$(basename "$i" .cds.fa) + echo "Processing $base" + sed "s/>/>${base}@/g" "$i" >>all_cds.fa +done + +# get og list and extract cds sequences for each og +pueue add -- python3 "$PROJECTHOME"/99.scripts_v2/utils/get_og_seqs.py -d Hippophae_outgroup/Single_Copy_Orthologue_Sequences -a all_cds.fa -o ogs_cds diff --git a/99.scripts_v2/03.gene_phylogeny.sh b/99.scripts_v2/03.gene_phylogeny.sh new file mode 100644 index 0000000..0480798 --- /dev/null +++ b/99.scripts_v2/03.gene_phylogeny.sh @@ -0,0 +1,290 @@ +#!/bin/bash + +set -euo pipefail + +ln -s ../12.orthofinder/ogs_cds/ . + +# parallel MACSE alignment for each orthologous group +mkdir alignments +echo -n >macse.cmds +for og_fasta in ogs_cds/*.fa; do + og_name=$(basename "$og_fasta" .fa) + out_stem="alignments/$og_name" + echo "macse -prog alignSequences -seq $og_fasta -out_AA ${out_stem}.pal -out_NT ${out_stem}.nal > ${out_stem}.log 2>&1" >>macse.cmds +done +pueue add -- xargs -t -P 12 -I cmd -a macse.cmds bash -c "cmd" + +# "!" --> "-" +rg -l "!" alignments/*.nal >fs.list +mkdir alignments_clean +for og_fasta in ogs_cds/*.fa; do + og_name=$(basename "$og_fasta" .fa) + out_stem="alignments/$og_name" + echo "Processing $og_name" + sed 's/!/-/g' "${out_stem}.nal" >"alignments_clean/${og_name}.nal" +done + +# classify frameshift loci by independent event count and carrier pattern +python3 "$PROJECTHOME"/99.scripts_v2/utils/classify_frameshifts.py \ + --fasta-list fs.list \ + --output-dir frameshift_summary + +# trim alignments with trimAl +mkdir -p alignments_trimmed +echo -n >trimal.cmds +for i in alignments_clean/*.nal; do + j=$(basename "$i" .nal) + out_dir="alignments_trimmed" + echo "trimal -in $i -out $out_dir/${j}.trimed.fa -automated1" >>trimal.cmds +done +pueue add -- xargs -t -P 12 -I cmd -a trimal.cmds bash -c "cmd" + +# retain MSAs at least 300 bp long +min_alignment_length=300 +mkdir -p alignments_filtered +printf "gene\talignment_length_bp\n" >alignments_shorter_than_300bp.tsv +printf "gene\tremoved_sequence\n" >trimal_removed_sequences.tsv +for i in alignments_trimmed/*.fa; do + [[ -e "$i" ]] || continue + j=$(basename "$i" .trimed.fa) + untrimmed_alignment="alignments_clean/${j}.nal" + filtered_alignment="alignments_filtered/${j}.fa" + if [[ ! -s "$untrimmed_alignment" ]]; then + printf "Missing non-empty pre-trim alignment: %s\n" "$untrimmed_alignment" >&2 + exit 1 + fi + + removed_sequences=() + mapfile -t removed_sequences < <( + comm -23 \ + <(awk '/^>/{sub(/^>/, ""); sub(/[[:space:]].*$/, ""); print}' \ + "$untrimmed_alignment" | sort -u) \ + <(awk '/^>/{sub(/^>/, ""); sub(/[[:space:]].*$/, ""); print}' \ + "$i" | sort -u) + ) + if ((${#removed_sequences[@]} > 0)); then + rm -f "$filtered_alignment" + for sequence in "${removed_sequences[@]}"; do + printf "%s\t%s\n" "$j" "$sequence" >>trimal_removed_sequences.tsv + done + continue + fi + + alignment_length=$(awk ' + /^>/ { + if (found_sequence) + exit + found_sequence = 1 + next + } + found_sequence { + gsub(/[[:space:]]/, "") + sequence_length += length($0) + } + END { print sequence_length + 0 } + ' "$i") + + if ((alignment_length >= min_alignment_length)); then + if [[ ! "$i" -ef "$filtered_alignment" ]]; then + ln -f "$i" "$filtered_alignment" + fi + else + rm -f "$filtered_alignment" + printf "%s\t%d\n" "$j" "$alignment_length" >>alignments_shorter_than_300bp.tsv + fi +done + +# raxml-ng for each filtered orthologous group +mkdir -p raxml +echo -n >raxml.cmds +for i in alignments_filtered/*.fa; do + [[ -e "$i" ]] || continue + j=$(basename "$i" .fa) + out_prefix="raxml/${j}/${j}" + mkdir -p "raxml/${j}" + echo "$SCRIPTS/utils/raxml.sh $i $out_prefix 1 ZJ" >>raxml.cmds +done +pueue add -- xargs -t -P 12 -I cmd -a raxml.cmds bash -c "cmd" + +# mrbayes for each filtered orthologous group +mkdir -p mrbayes +echo -n >mrbayes.cmds +for i in alignments_filtered/*.fa; do + [[ -e "$i" ]] || continue + j=$(basename "$i" .fa) + out_prefix="mrbayes/${j}/${j}" + mkdir -p "mrbayes/${j}" + echo "bash $SCRIPTS/utils/mrbayes.sh $i ${out_prefix}.nex" >>mrbayes.cmds +done +pueue add -- xargs -t -P 12 -I cmd -a mrbayes.cmds bash -c "cmd" + +# mbsum +mkdir -p mbsum +echo -n "" >mbsum.skip.log +echo -n "" >mbsum.cmds +for i in mrbayes/*/mrbayes.log; do + og_dir=$(dirname "$i") + base=$(basename "$og_dir") + ## skip Average standard deviation of split frequencies > 0.01 + dsf=$(awk '/Average standard deviation of split frequencies:/ {out=$7} END{print out+0}' "$i" 2>/dev/null) + if awk -v d="$dsf" -v min_dsf=0.01 'BEGIN{if (d >= min_dsf) exit 0; exit 1}'; then + echo "Skipping ${base} due to high DSF: ${dsf}" >>mbsum.skip.log + continue + else + echo "mbsum ${og_dir}/*.t -n 1000 -o mbsum/${base}.in >> $og_dir/mbsum.log 2>&1" >>mbsum.cmds + fi +done +export PATH=$PATH:$SCRIPTS/bucky/bin +pueue add -- xargs -t -P 12 -I cmd -a mbsum.cmds bash -c "cmd" + +######################################################################### +# # +# DEPRECATED SECTION: TreeShrink and FastTree-based filtering # +# # +######################################################################### + +# remove long branches with TreeShrink and FastTree +mkdir -p treeshrink +echo -n >fasttree.cmds +for i in alignments_trimmed/*.trimed.fa; do + j=$(basename "$i" .trimed.fa) + mkdir -p treeshrink/"${j}" + absolute_path=$(readlink -f "$i") + ln -s "$absolute_path" "treeshrink"/"${j}"/input.fasta + echo "FastTree -nt -gtr -quiet treeshrink/${j}/input.fasta > treeshrink/${j}/input.tree" >>fasttree.cmds +done +pueue add -- xargs -t -P 12 -I cmd -a fasttree.cmds bash -c "cmd" + +pueue add -- run_treeshrink.py -f -i treeshrink/ -t input.tree -a input.fasta + +# filter orthologous groups after TreeShrink based on minimum taxon number and minimum sequence length +min_taxon=11 +min_seq_length=300 +mkdir -p final_alignments +echo -n "" >treeshrink_filtered.list +for i in treeshrink/*/output.fasta; do + j=$(dirname "$i") + j=$(basename "$j") + seqlen=$(seqkit fx2tab -C ATCG "$i" | awk '{print $3}' | sort -n | head -n 1) + seqnum=$(grep -c ">" "$i") + if [[ $seqnum -eq $min_taxon && $seqlen -ge $min_seq_length ]]; then + #cp -l "$i" final_alignments/"${j}.fa" + echo -n "" + else + echo "$i" >>treeshrink_filtered.list + fi +done + +# summarize sequences removed by TreeShrink +mkdir -p treeshrink_summary +removed_sequences="treeshrink_summary/treeshrink_removed_sequences.tsv" +removed_by_species="treeshrink_summary/treeshrink_removed_by_species.tsv" +removed_group_summary="treeshrink_summary/treeshrink_removed_group_summary.tsv" +printf "OG\tspecies\n" >"$removed_sequences" +for output_fasta in treeshrink/*/output.fasta; do + og_name=$(basename "$(dirname "$output_fasta")") + input_fasta="$(dirname "$output_fasta")/input.fasta" + comm -23 \ + <(awk '/^>/{sub(/^>/, ""); sub(/[[:space:]].*$/, ""); print}' "$input_fasta" | sort -u) \ + <(awk '/^>/{sub(/^>/, ""); sub(/[[:space:]].*$/, ""); print}' "$output_fasta" | sort -u) | + while IFS= read -r species; do + printf "%s\t%s\n" "$og_name" "$species" + done >>"$removed_sequences" +done +printf "species\tfiltered_OG_count\n" >"$removed_by_species" +awk -F '\t' 'NR > 1 {count[$2]++} END {for (species in count) print species, count[species]}' \ + OFS='\t' "$removed_sequences" | sort -k2,2nr -k1,1 >>"$removed_by_species" +printf "group\tfiltered_OG_count\n" >"$removed_group_summary" +awk -F '\t' ' + NR > 1 { + if ($2 == "EA" || $2 == "EM" || $2 == "ZJ") + outgroup[$1] = 1 + else + ingroup[$1] = 1 + } + END { + for (og in outgroup) outgroup_count++ + for (og in ingroup) ingroup_count++ + print "outgroup_EA_EM_ZJ", outgroup_count + 0 + print "ingroup", ingroup_count + 0 + } +' OFS='\t' "$removed_sequences" >>"$removed_group_summary" + +# compare OGs containing frameshifts with OGs filtered by TreeShrink +fs_overlap_detail="treeshrink_summary/treeshrink_fs_overlap.tsv" +fs_overlap_summary="treeshrink_summary/treeshrink_fs_overlap_summary.tsv" +printf "OG\tcategory\n" >"$fs_overlap_detail" +awk -F '\t' ' + NR == FNR { + og = $0 + sub(/^.*\//, "", og) + sub(/\.nal$/, "", og) + frameshift[og] = 1 + next + } + FNR > 1 { + treeshrink[$1] = 1 + } + END { + for (og in frameshift) { + if (og in treeshrink) + print og, "overlap" + else + print og, "fs.list_only" + } + for (og in treeshrink) { + if (!(og in frameshift)) + print og, "TreeShrink_only" + } + } +' OFS='\t' fs.list "$removed_sequences" | sort -k2,2 -k1,1 >>"$fs_overlap_detail" + +printf "category\tOG_count\n" >"$fs_overlap_summary" +awk -F '\t' ' + NR > 1 { + count[$2]++ + } + END { + overlap = count["overlap"] + 0 + fs_only = count["fs.list_only"] + 0 + treeshrink_only = count["TreeShrink_only"] + 0 + print "fs.list_total", overlap + fs_only + print "TreeShrink_total", overlap + treeshrink_only + print "overlap", overlap + print "fs.list_only", fs_only + print "TreeShrink_only", treeshrink_only + } +' OFS='\t' "$fs_overlap_detail" >>"$fs_overlap_summary" + +# raxml-ng for each filtered orthologous group +mkdir -p raxml +echo -n >raxml.cmds +for i in final_alignments/*.fa; do + j=$(basename "$i" .fa) + out_prefix="raxml/${j}/${j}" + mkdir -p "raxml/${j}" + echo "$SCRIPTS/utils/raxml.sh $i $out_prefix 1 ZJ" >>raxml.cmds +done +pueue add -- xargs -t -P 12 -I cmd -a raxml.cmds bash -c "cmd" + +# mrbayes for each filtered orthologous group +mkdir -p mrbayes +echo -n >mrbayes.cmds +for i in final_alignments/*.fa; do + j=$(basename "$i" .fa) + out_prefix="mrbayes/${j}/${j}" + mkdir -p "mrbayes/${j}" + echo "bash $SCRIPTS/utils/mrbayes.sh $i ${out_prefix}.nex" >>mrbayes.cmds +done +pueue add -- xargs -t -P 12 -I cmd -a mrbayes.cmds bash -c "cmd" + +## treeshrink with per-gene +mkdir -p treeshrink_per_gene +echo -n >treeshrink_per_gene.cmds +for i in final_alignments/*.fa; do + j=$(basename "$i" .fa) + out_prefix="treeshrink_per_gene/${j}/${j}" + mkdir -p "treeshrink_per_gene/${j}" + echo "bash $SCRIPTS/utils/treeshrink.sh $i $out_prefix" >>treeshrink_per_gene.cmds +done +pueue add -- xargs -t -P 12 -I cmd -a treeshrink_per_gene.cmds bash -c "cmd" diff --git a/99.scripts_v2/04.taxa_phylogeny.sh b/99.scripts_v2/04.taxa_phylogeny.sh new file mode 100644 index 0000000..dc7151e --- /dev/null +++ b/99.scripts_v2/04.taxa_phylogeny.sh @@ -0,0 +1,174 @@ +#!/bin/bash + +set -euo pipefail +export PATH="$PATH:${SCRIPTS}/bucky/bin" + +# treeshrink +mkdir -p treeshrink +for i in ../13.gene_phylogeny/alignments_filtered/*.fa; do + j=$(basename "$i" .fa) + mkdir -p treeshrink/"${j}" + absolute_path=$(readlink -f "$i") + ln -s "$absolute_path" "treeshrink"/"${j}"/input.fasta + ln -s "$(readlink -f ../13.gene_phylogeny/raxml)"/"${j}/${j}".raxml.bestTree "treeshrink"/"${j}"/input.tree +done +pueue add -- run_treeshrink.py -f -i treeshrink/ -t input.tree -a input.fasta -m per-gene + +# treeshrink summary +mkdir -p treeshrink_summary +removed_sequences="treeshrink_summary/treeshrink_removed_sequences.tsv" +removed_by_species="treeshrink_summary/treeshrink_removed_by_species.tsv" +removed_group_summary="treeshrink_summary/treeshrink_removed_group_summary.tsv" +printf "OG\tspecies\n" >"$removed_sequences" +for output_fasta in treeshrink/*/output.fasta; do + og_name=$(basename "$(dirname "$output_fasta")") + input_fasta="$(dirname "$output_fasta")/input.fasta" + comm -23 \ + <(awk '/^>/{sub(/^>/, ""); sub(/[[:space:]].*$/, ""); print}' "$input_fasta" | sort -u) \ + <(awk '/^>/{sub(/^>/, ""); sub(/[[:space:]].*$/, ""); print}' "$output_fasta" | sort -u) | + while IFS= read -r species; do + printf "%s\t%s\n" "$og_name" "$species" + done >>"$removed_sequences" +done +printf "species\tfiltered_OG_count\n" >"$removed_by_species" +awk -F '\t' 'NR > 1 {count[$2]++} END {for (species in count) print species, count[species]}' \ + OFS='\t' "$removed_sequences" | sort -k2,2nr -k1,1 >>"$removed_by_species" +printf "group\tfiltered_OG_count\n" >"$removed_group_summary" +awk -F '\t' ' + NR > 1 { + if ($2 == "EA" || $2 == "EM" || $2 == "ZJ") + outgroup[$1] = 1 + else + ingroup[$1] = 1 + } + END { + for (og in outgroup) outgroup_count++ + for (og in ingroup) ingroup_count++ + print "outgroup_EA_EM_ZJ", outgroup_count + 0 + print "ingroup", ingroup_count + 0 + } +' OFS='\t' "$removed_sequences" >>"$removed_group_summary" + +# LBS filter +min_taxon=11 +echo -n "" >treeshrink_filtered.list +for i in treeshrink/*/output.fasta; do + j=$(dirname "$i") + j=$(basename "$j") + seqnum=$(grep -c ">" "$i") + if [[ $seqnum -ge $min_taxon ]]; then + echo -n "" + else + echo "$j" >>treeshrink_filtered.list + fi +done + +# data preparation +ln -s ../13.gene_phylogeny/raxml . +ln -s ../13.gene_phylogeny/mbsum . + +## +## 第一套，所有基因保留，仅移除treeshrink过滤掉的基因 +## + +mkdir -p all_genes +cd all_genes + +# aster coalescence tree +for file in ../raxml/*/*.support; do + gene=$(basename "$(dirname "$file")") + if ! grep -Fqx -- "$gene" ../treeshrink_filtered.list; then + cat "$file" + fi +done >all.ml.tre +mkdir -p species_tree +pueue add -- astral4 -t 6 -i all.ml.tre -o species_tree/coal.tre --root ZJ + +# bucky +mkdir bucky +for mbfile in ../mbsum/*.in; do + gene=$(basename "$mbfile" .in) + if ! grep -Fqx -- "$gene" ../treeshrink_filtered.list; then + cp "$mbfile" bucky/"$gene".in + fi +done +cd bucky +tar -cf input.mb.tar -- ./*.in +rm -- ./*.in +pueue add -- "$SCRIPTS"/utils/bucky.pl -a 1 -o bucky_1 -s input.mb.tar + +# SNaQ +mkdir snaq +cd snaq +## run snaq.jl with interactive mode +# julia snaq.jl +cd ../.. + +## +## 第二套，移除treeshrink过滤掉的基因和移码突变基因 +mkdir -p exclude_all_fs +cd exclude_all_fs +cut -f 1 ../../13.gene_phylogeny/frameshift_summary/frameshift_locus_classification.tsv | sed 1d > fs_exclude.list + +# aster coalescence tree +for file in ../raxml/*/*.support; do + gene=$(basename "$(dirname "$file")") + if ! grep -Fqx -- "$gene" ../treeshrink_filtered.list fs_exclude.list; then + cat "$file" + fi +done >all.ml.tre +mkdir -p species_tree +pueue add -- astral4 -t 6 -i all.ml.tre -o species_tree/coal.tre --root ZJ + +# bucky +mkdir bucky +for mbfile in ../mbsum/*.in; do + gene=$(basename "$mbfile" .in) + if ! grep -Fqx -- "$gene" ../treeshrink_filtered.list fs_exclude.list; then + cp "$mbfile" bucky/"$gene".in + fi +done +cd bucky +tar -cf input.mb.tar -- ./*.in +rm -- ./*.in +pueue add -- "$SCRIPTS"/utils/bucky.pl -a 1 -o bucky_1 -s input.mb.tar + +mkdir snaq +cd snaq +## run snaq.jl with interactive mode +# julia snaq.jl +cd ../.. + +## 第三套，移除严重移码突变基因和treeshrink过滤掉的基因 +mkdir -p exclude_severe_fs +cd exclude_severe_fs +grep -wv single_species_single_event ../../13.gene_phylogeny/frameshift_summary/frameshift_locus_classification.tsv | cut -f 1 | sed 1d > fs_exclude.list + +# aster coalescence tree +for file in ../raxml/*/*.support; do + gene=$(basename "$(dirname "$file")") + if ! grep -Fqx -- "$gene" ../treeshrink_filtered.list fs_exclude.list; then + cat "$file" + fi +done >all.ml.tre +mkdir -p species_tree +pueue add -- astral4 -t 6 -i all.ml.tre -o species_tree/coal.tre --root ZJ + +# bucky +mkdir bucky +for mbfile in ../mbsum/*.in; do + gene=$(basename "$mbfile" .in) + if ! grep -Fqx -- "$gene" ../treeshrink_filtered.list fs_exclude.list; then + cp "$mbfile" bucky/"$gene".in + fi +done +cd bucky +tar -cf input.mb.tar -- ./*.in +rm -- ./*.in +pueue add -- "$SCRIPTS"/utils/bucky.pl -a 1 -o bucky_1 -s input.mb.tar + +mkdir snaq +cd snaq +## run snaq.jl with interactive mode +# julia snaq.jl +cd ../.. diff --git a/99.scripts_v2/05.dsuite.sh b/99.scripts_v2/05.dsuite.sh new file mode 100644 index 0000000..a37cb8d --- /dev/null +++ b/99.scripts_v2/05.dsuite.sh @@ -0,0 +1,143 @@ +#!/bin/bash + +set -euo pipefail + +ln -sf ../11.reference_v2/raw . +cd raw +ln -sf Hippophae_tibetana.genomic.fasta HT.fa +ln -sf YN.genome.fa YN.fa +seqkit grep -f ZG.target.txt ZG.genome.fa > ZG.fa +ln -sf ZJ.genome.fa ZJ.fa +seqkit grep -f MG.target.txt MG.genome.fasta > MG.fa +ln -sf EM.genome.fasta EM.fa +cd .. + +bash "$SCRIPTS"/utils/asm_qc.sh raw/ZJ.fa \ + raw/EM.fa \ + raw/EA.fa \ + raw/HT.fa \ + raw/ZY.fa \ + raw/ZG.fa \ + raw/MG.fa \ + raw/YN.fa \ + raw/HG.fa \ + raw/HS.fa \ + raw/HN.fa > qc.tsvWDw + +## +## minimap2 比对：YN 为参考（ZG 含单倍型出局），asm5 档位（属内分歧 0.001-0.007，EA/EM 约 0.03 也覆盖） +## ZJ 已剔除（跨科，分歧 0.19）；YN 不做自比对 +## +REF=raw/YN.fa +mkdir -p paf +pueue add -- minimap2 -cx asm5 --cs=long -t 8 "$REF" raw/HG.fa '>' paf/HG.paf +pueue add -- minimap2 -cx asm5 --cs=long -t 8 "$REF" raw/HN.fa '>' paf/HN.paf +pueue add -- minimap2 -cx asm5 --cs=long -t 8 "$REF" raw/HS.fa '>' paf/HS.paf +pueue add -- minimap2 -cx asm5 --cs=long -t 8 "$REF" raw/HT.fa '>' paf/HT.paf +pueue add -- minimap2 -cx asm5 --cs=long -t 8 "$REF" raw/MG.fa '>' paf/MG.paf +pueue add -- minimap2 -cx asm5 --cs=long -t 8 "$REF" raw/ZG.fa '>' paf/ZG.paf +pueue add -- minimap2 -cx asm5 --cs=long -t 8 "$REF" raw/ZY.fa '>' paf/ZY.paf +pueue add -- minimap2 -cx asm5 --cs=long -t 8 "$REF" raw/EA.fa '>' paf/EA.paf +pueue add -- minimap2 -cx asm5 --cs=long -t 8 "$REF" raw/EM.fa '>' paf/EM.paf + +## 覆盖速览（pilot 判档用；asm5 若 EA/EM 覆盖塌，对这两个换 asm10 重跑） +for f in paf/*.paf; do + awk -v OFS='\t' '{alen += $11; qlen = $2} END {printf "%s\tqlen=%d\taligned=%d\tcov=%.2f%%\n", FILENAME, qlen, alen, alen / qlen * 100}' "$f" +done + +## YN 自比对：Dsuite 需要 YN 作样本（自比对产生全纯合参考基因型 VCF） +pueue add -- minimap2 -cx asm5 --cs=long -t 8 "$REF" raw/YN.fa '>' paf/YN.paf + +## +## PAF 过滤：只留 primary 链（tp:A:P）并按参考坐标排序（paftools call 要求排序输入） +## primary 链是一对一共线块的近似；如需严格双向最佳命中，在此之上再加过滤 +## +mkdir -p paf_primary +grep 'tp:A:P' paf/HG.paf | sort -k6,6 -k8,8n > paf_primary/HG.paf +grep 'tp:A:P' paf/HN.paf | sort -k6,6 -k8,8n > paf_primary/HN.paf +grep 'tp:A:P' paf/HS.paf | sort -k6,6 -k8,8n > paf_primary/HS.paf +grep 'tp:A:P' paf/HT.paf | sort -k6,6 -k8,8n > paf_primary/HT.paf +grep 'tp:A:P' paf/MG.paf | sort -k6,6 -k8,8n > paf_primary/MG.paf +grep 'tp:A:P' paf/ZG.paf | sort -k6,6 -k8,8n > paf_primary/ZG.paf +grep 'tp:A:P' paf/ZY.paf | sort -k6,6 -k8,8n > paf_primary/ZY.paf +grep 'tp:A:P' paf/YN.paf | sort -k6,6 -k8,8n > paf_primary/YN.paf +grep 'tp:A:P' paf/EA.paf | sort -k6,6 -k8,8n > paf_primary/EA.paf +grep 'tp:A:P' paf/EM.paf | sort -k6,6 -k8,8n > paf_primary/EM.paf + +## +## paftools call：每样本一个 VCF（-s 样本名；-l/-L 最小块长阈值，可调） +## +mkdir -p vcf +paftools.js call -s HG -l 5000 -L 10000 paf_primary/HG.paf > vcf/HG.vcf +paftools.js call -s HN -l 5000 -L 10000 paf_primary/HN.paf > vcf/HN.vcf +paftools.js call -s HS -l 5000 -L 10000 paf_primary/HS.paf > vcf/HS.vcf +paftools.js call -s HT -l 5000 -L 10000 paf_primary/HT.paf > vcf/HT.vcf +paftools.js call -s MG -l 5000 -L 10000 paf_primary/MG.paf > vcf/MG.vcf +paftools.js call -s ZG -l 5000 -L 10000 paf_primary/ZG.paf > vcf/ZG.vcf +paftools.js call -s ZY -l 5000 -L 10000 paf_primary/ZY.paf > vcf/ZY.vcf +paftools.js call -s YN -l 5000 -L 10000 paf_primary/YN.paf > vcf/YN.vcf +paftools.js call -s EA -l 5000 -L 10000 paf_primary/EA.paf > vcf/EA.vcf +paftools.js call -s EM -l 5000 -L 10000 paf_primary/EM.paf > vcf/EM.vcf + +bgzip -f vcf/HG.vcf && tabix -f -p vcf vcf/HG.vcf.gz +bgzip -f vcf/HN.vcf && tabix -f -p vcf vcf/HN.vcf.gz +bgzip -f vcf/HS.vcf && tabix -f -p vcf vcf/HS.vcf.gz +bgzip -f vcf/HT.vcf && tabix -f -p vcf vcf/HT.vcf.gz +bgzip -f vcf/MG.vcf && tabix -f -p vcf vcf/MG.vcf.gz +bgzip -f vcf/ZG.vcf && tabix -f -p vcf vcf/ZG.vcf.gz +bgzip -f vcf/ZY.vcf && tabix -f -p vcf vcf/ZY.vcf.gz +bgzip -f vcf/YN.vcf && tabix -f -p vcf vcf/YN.vcf.gz +bgzip -f vcf/EA.vcf && tabix -f -p vcf vcf/EA.vcf.gz +bgzip -f vcf/EM.vcf && tabix -f -p vcf vcf/EM.vcf.gz + +## +## 合并为 10 类群多样本 VCF +## +pueue add -- bcftools merge -O z -o vcf/dsuite.10taxa.vcf.gz vcf/HG.vcf.gz vcf/HN.vcf.gz vcf/HS.vcf.gz vcf/HT.vcf.gz vcf/MG.vcf.gz vcf/ZG.vcf.gz vcf/ZY.vcf.gz vcf/YN.vcf.gz vcf/EA.vcf.gz vcf/EM.vcf.gz +tabix -f -p vcf vcf/dsuite.10taxa.vcf.gz + +## +## 物种树：主数据集第二套（exclude_all_fs）ASTRAL 树剔除 ZJ +## TODO: 剪树工具任选（nw_prune / gotree / R ape），产物放 tree/dsuite.nwk +## +mkdir -p tree +# nw_prune ../14.taxa_phylogeny/exclude_all_fs/species_tree/coal.tre ZJ > tree/dsuite.nwk + +## +## SETS 文件：双外类群交叉验证，EA / EM 各标一次 Outgroup +## +mkdir -p sets dsuite +cat > sets/EA_out.txt <<'EOF' +HG HG +HN HN +HS HS +HT HT +MG MG +ZG ZG +ZY ZY +YN YN +EA Outgroup +EM EM +EOF +cat > sets/EM_out.txt <<'EOF' +HG HG +HN HN +HS HS +HT HT +MG MG +ZG ZG +ZY ZY +YN YN +EA EA +EM Outgroup +EOF + +## +## Dtrios + Fbranch（EA / EM 各跑一遍） +## +pueue add -- Dsuite Dtrios -t tree/dsuite.nwk -o dsuite/EA_out vcf/dsuite.10taxa.vcf.gz sets/EA_out.txt +pueue add -- Dsuite Dtrios -t tree/dsuite.nwk -o dsuite/EM_out vcf/dsuite.10taxa.vcf.gz sets/EM_out.txt +Dsuite Fbranch tree/dsuite.nwk dsuite/EA_out_tree.txt > dsuite/EA_out_fbranch.txt +Dsuite Fbranch tree/dsuite.nwk dsuite/EM_out_tree.txt > dsuite/EM_out_fbranch.txt +# dtools.py dsuite/EA_out_fbranch.txt tree/dsuite.nwk +# dtools.py dsuite/EM_out_fbranch.txt tree/dsuite.nwk \ No newline at end of file diff --git a/99.scripts_v2/bucky b/99.scripts_v2/bucky new file mode 160000 index 0000000..9228d7c --- /dev/null +++ b/99.scripts_v2/bucky @@ -0,0 +1 @@ +Subproject commit 9228d7c9446e619df6fd609c3f48d74472b50c96 diff --git a/99.scripts_v2/utils/asm_qc.sh b/99.scripts_v2/utils/asm_qc.sh new file mode 100644 index 0000000..71364f0 --- /dev/null +++ b/99.scripts_v2/utils/asm_qc.sh @@ -0,0 +1,22 @@ +#!/usr/bin/env bash +# 轻量组装 QC：contig 数 / 总长度 / N50 / N 占比 / GC% +# 用法: bash asm_qc.sh > qc.tsv +set -euo pipefail + +files=() +for p in "$@"; do + if [ -d "$p" ]; then + while IFS= read -r f; do files+=("$f"); done \ + < <(find "$p" -type f \( -name '*.fa' -o -name '*.fasta' -o -name '*.fna' -o -name '*.fa.gz' -o -name '*.fasta.gz' -o -name '*.fna.gz' \)) + else + files+=("$p") + fi +done + +seqkit stats -a -T -j 8 "${files[@]}" | awk 'BEGIN{OFS="\t"} +NR==1 {print $0, "N%"; next} +{ + # $5=sum_len, $19=sum_n（N 碱基数） + n = ($5 > 0) ? sprintf("%.4f", $19 / $5 * 100) : "NA" + print $0, n +}' diff --git a/99.scripts_v2/utils/bucky.pl b/99.scripts_v2/utils/bucky.pl new file mode 100755 index 0000000..fd24370 --- /dev/null +++ b/99.scripts_v2/utils/bucky.pl @@ -0,0 +1,1503 @@ +#!/usr/bin/perl +use strict; +use warnings; +use POSIX; +use IO::Select; +use IO::Socket; +use Digest::MD5; +use Getopt::Long; +use Cwd qw(abs_path); +use Fcntl qw(:flock SEEK_END); +use POSIX qw(ceil :sys_wait_h); +use File::Path qw(remove_tree); +use Time::HiRes qw(time usleep); + +# Get OS name +my $os_name = $^O; + +# Turn on autoflush +$|++; + +# Max number of forks to use +my $max_forks = get_free_cpus(); + +# Server port +my $port = 10003; + +# Stores executing machine hostnames +my @machines; +my %machines; + +# Path to text file containing computers to run on +my $machine_file_path; + +# MrBayes block which will be used for each run +my $mb_block; + +# Where this script is located +my $script_path = abs_path($0); + +# Directory script was called from +my $init_dir = abs_path("."); + +# Where the script was called from +my $initial_directory = $ENV{PWD}; + +# General script settings +my $no_forks; + +# Allow for reusing info from an old run +my $input_is_dir = 0; + +# Allow user to specify mbsum output as input for the script +my $input_is_mbsum = 0; + +# How the script was called +my $invocation = "perl bucky.pl @ARGV"; + +# Name of output directory +my $project_name = "bucky-".int(time()); +#my $project_name = "bucky-dir"; + +# BUCKy settings +my $alpha = 1; +my $ngen = 1000000; + +my @unlink; + +# Read commandline settings +GetOptions( + "no-forks" => \$no_forks, + "machine-file=s" => \$machine_file_path, + "alpha|a=s" => \$alpha, + "ngen|n=i" => \$ngen, + "port=i" => \$port, + "no-mbsum|s" => \$input_is_mbsum, + "n-threads|T=i" => \$max_forks, + "out-dir|o=s" => \$project_name, + "server-ip=s" => \&client, # for internal usage only + "help|h" => sub { print &help; exit(0); }, + "usage" => sub { print &usage; exit(0); }, +); + + +# Get paths to required executables +my $bucky = check_path_for_exec("bucky"); +my $mbsum = check_path_for_exec("mbsum") if (!$input_is_mbsum); + +# Check that BUCKy version >= 1.4.4 +check_bucky_version($bucky); + +my $archive = shift(@ARGV); + +# Some error checking +die "You must specify an archive file.\n\n", &usage if (!defined($archive)); +die "Could not locate '$archive', perhaps you made a typo.\n" if (!-e $archive); +die "Could not locate '$machine_file_path'.\n" if (defined($machine_file_path) && !-e $machine_file_path); +die "Invalid alpha for BUCKy specified, input must be a float or 'infinity'.\n" if ($alpha !~ /(^inf(inity)?)|(^\d+(\.\d+)?$)/i); + +# Input is a previous run directory, reuse information +$input_is_dir++ if (-d $archive); + +# Determine which machines we will run the analyses on +if (defined($machine_file_path)) { + + # Get list of machines + print "Fetching machine names listed in '$machine_file_path'...\n"; + open(my $machine_file, '<', $machine_file_path); + chomp(@machines = <$machine_file>); + close($machine_file); + + # Check that we can connect to specified machines + foreach my $index (0 .. $#machines) { + my $machine = $machines[$index]; + print " Testing connection to: $machine...\n"; + + # Attempt to ssh onto machine with a five second timeout + my $ssh_test = `timeout 5 ssh -v $machine exit 2>&1`; + + # Look for machine's IP in test connection + my $machine_ip; + if ($ssh_test =~ /Connecting to \S+ \[(\S+)\] port \d+\./s) { + $machine_ip = $1; + } + + # Could connect but passwordless login not enabled + if ($ssh_test =~ /Are you sure you want to continue connecting \(yes\/no\)/s) { + print " Connection to $machine failed, removing from list of useable machines (passwordless login not enabled).\n"; + splice(@machines, $index, 1); + } + # Successful connection + elsif (defined($machine_ip)) { + print " Connection to $machine [$machine_ip] successful.\n"; + $machines{$machine} = $machine_ip; + } + # Unsuccessful connection + else { + print " Connection to $machine failed, removing from list of useable machines.\n"; + splice(@machines, $index, 1); + } + } +} + +print "\nScript was called as follows:\n$invocation\n\n"; + +my $archive_root; +my $archive_root_no_ext; +if (!$input_is_dir) { + + # Clean run with no prior output + + # Extract name information from input file + + if ($input_is_mbsum) { + ($archive_root = $archive) =~ s/.*\/(.*)/$1/; + ($archive_root_no_ext = $archive) =~ s/(.*\/)?(.*)(\.tar(\.gz)?$)|(\.tgz$)/$2/; + } + else { + ($archive_root = $archive) =~ s/.*\/(.*)/$1/; + ($archive_root_no_ext = $archive) =~ s/(.*\/)?(.*)(\.mb\.tar(\.gz)?$)|(\.mb\.tgz$)/$2/; + } + die "Could not determine archive root name, did you specify the proper input?\n" if (!defined($archive_root_no_ext)); + + # Initialize working directory + # Remove conditional eventually + mkdir($project_name) || die "Could not create '$project_name'$!.\n" if (!-e $project_name); + + my $archive_abs_path = abs_path($archive); + # Remove conditional eventually + run_cmd("ln -s $archive_abs_path $project_name/$archive_root") if (! -e "$project_name/$archive_root"); +} +else { + + # Prior output available, set relevant variables + + $project_name = $archive; + my @contents = glob("$project_name/*"); + + # Determine the archive name by looking for a symlink + my $found_name = 0; + foreach my $file (@contents) { + if (-l $file) { + $file =~ s/\Q$project_name\E\///; + $archive = $file; + $found_name = 1; + } + } + die "Could not locate archive in '$project_name'.\n" if (!$found_name); + + # Extract name information from input file +# ($archive_root = $archive) =~ s/.*\/(.*)/$1/; +# ($archive_root_no_ext = $archive) =~ s/(.*\/)?(.*)(\.mb\.tar(\.gz)?$)|(\.mb\.tgz$)/$2/; + if ($input_is_mbsum) { + ($archive_root = $archive) =~ s/.*\/(.*)/$1/; + ($archive_root_no_ext = $archive) =~ s/(.*\/)?(.*)(\.tar(\.gz)?$)|(\.tgz$)/$2/; + } + else { + ($archive_root = $archive) =~ s/.*\/(.*)/$1/; + ($archive_root_no_ext = $archive) =~ s/(.*\/)?(.*)(\.mb\.tar(\.gz)?$)|(\.mb\.tgz$)/$2/; + } + die "Could not determine archive root name, is your input file properly named?\n" if (!defined($archive_root_no_ext)); +} + +# The name of the output archive +my $mbsum_archive = "$archive_root_no_ext.mbsum.tar.gz"; +my $bucky_archive = "$archive_root_no_ext.BUCKy.tar"; +my $quartet_output = "$archive_root_no_ext.CFs.csv"; + +$mbsum_archive = $archive if ($input_is_mbsum); + +chdir($project_name); + +# Change how Ctrl+C is interpreted to allow for clean up +$SIG{'INT'} = 'INT_handler'; + +# Define and initialize directories +my $mb_out_dir = "mb-out/"; +my $mb_sum_dir = "mb-sum/"; + +mkdir($mb_out_dir) or die "Could not create '$mb_out_dir': $!.\n" if (!-e $mb_out_dir); +mkdir($mb_sum_dir) or die "Could not create '$mb_sum_dir': $!.\n" if (!-e $mb_sum_dir); + +# Check if completed genes from a previous run exist +my %complete_quartets; +if (-e $bucky_archive && -e $quartet_output) { + print "Archive containing completed quartets found for this dataset found in '$bucky_archive'.\n"; + print "Completed quartets within in this archive will be removed from the job queue.\n\n"; + + # Because it takes longer to append to a tarball than append to a text file, the tarball most + # likely has fewer quartet entries in it, we must therefore account for this ensuring that + # the tarball and csv have the same quartet entries + + # See which quartets in the tarball are complete + chomp(my @complete_quartets_tarball = `tar tf '$init_dir/$project_name/$bucky_archive'`); + + # Add quartets to a hash for easier lookup + my %complete_quartets_tarball; + foreach my $complete_quartet (@complete_quartets_tarball) { + $complete_quartets_tarball{$complete_quartet}++; + } + + # Load csv into memory + #open(my $quartet_output_file, "<", $quartet_output); + open(my $quartet_output_file, "<", "$init_dir/$project_name/$quartet_output"); + (my @quartet_info = <$quartet_output_file>); + close($quartet_output_file); + + # Remove header line + my $header = shift(@quartet_info); + + # Rewrite csv to include only quartets also contained in tarball + #open($quartet_output_file, ">", $quartet_output); + open($quartet_output_file, ">", "$init_dir/$project_name/$quartet_output"); + print {$quartet_output_file} $header; + foreach my $quartet (@quartet_info) { + my ($taxon1, $taxon2, $taxon3, $taxon4) = split(",", $quartet); + my $quartet_name = "$taxon1--$taxon2--$taxon3--$taxon4"; + my $quartet_name_tarball = $quartet_name.".tar.gz"; + + if (exists($complete_quartets_tarball{$quartet_name_tarball})) { + $complete_quartets{$quartet_name}++; + print {$quartet_output_file} $quartet; + } + } + close($quartet_output_file); +} + +my @taxa; +my @genes; +if ($input_is_mbsum) { + + # Unarchive input genes + chomp(@genes = `tar xvf '$init_dir/$project_name/$archive' -C $mb_sum_dir 2>&1`); + @genes = map { s/x //; $_ } @genes if ($os_name eq "darwin"); + + die "No genes found in '$archive'.\n" if (!@genes); + + # Move into MrBayes output directory + chdir($mb_sum_dir); + + # Remove subdirectories that may have been a part of the tarball + my @dirs; + my @gene_roots; + foreach my $gene (@genes) { + (my $gene_root = $gene) =~ s/.*\///; + next if ($gene eq $gene_root); + + # Move mbsum files so they are no longer in subdirectories + if (!-d $gene) { + system("mv '$gene' '$gene_root'"); + push(@gene_roots, $gene_root); + } + else { + push(@dirs, $gene); + #splice(@genes, $index, 1); + } + } + @genes = @gene_roots if (@gene_roots); + + # Remove any subdirectories that may have been in the input + foreach my $dir (@dirs) { + remove_tree($dir); + } + + # Parse taxa present in each gene, determine which are shared across all genes + my %taxa; + foreach my $gene (@genes) { + my @taxa = @{parse_mbsum_taxa($gene)}; + + # Count taxa present + foreach my $taxon (@taxa) { + $taxa{$taxon}++; + } + } + + # Add taxa present in all genes to analysis + foreach my $taxon (keys %taxa) { + if ($taxa{$taxon} == scalar(@genes)) { + push(@taxa, $taxon); + } + } + + # Archive genes + system("tar", "czf", $mbsum_archive, @genes); +} +else { + + # Unarchive input genes + chomp(@genes = `tar xvf '$init_dir/$archive' -C '$mb_out_dir' 2>&1`); + @genes = map { s/x //; $_ } @genes if ($os_name eq "darwin"); + + # Move into MrBayes output directory + chdir($mb_out_dir); + + # Check that each gene has a log file + my %taxa; + foreach my $gene (@genes) { + + # Unzip a single gene + chomp(my @mb_files = `tar xvf '$gene' 2>&1`); + @mb_files = map { s/x //; $_ } @mb_files if ($os_name eq "darwin"); + + # Locate the log file output by MrBayes + my $log_file_name; + foreach my $file (@mb_files) { + if ($file =~ /\.log$/) { + $log_file_name = $file; + last; + } + } + die "Could not locate log file for '$gene'.\n" if (!defined($log_file_name)); + + # Parse log file for run information + my $mb_log = parse_mb_log($log_file_name); + + # Check for taxa present + my @taxa = @{$mb_log->{TAXA}}; + foreach my $taxon (@taxa) { + $taxa{$taxon}++; + } + + # Clean up + unlink(@mb_files); + } + + # Add taxa present in all genes to analysis + foreach my $taxon (keys %taxa) { + if ($taxa{$taxon} == scalar(@genes)) { + push(@taxa, $taxon); + } + } +} + +# Create list of possible quartets +my @quartets = combine(\@taxa, 4); + +my $original_size = scalar(@quartets); + +# Remove completed quartets +if (%complete_quartets) { + foreach my $index (reverse(0 .. $#quartets)) { + my $quartet = $quartets[$index]; + $quartet = join("--", @{$quartet}); + if (exists($complete_quartets{$quartet})) { + splice(@quartets, $index, 1); + } + } +} + +print "Found ".scalar(@taxa)." taxa shared across all genes in this archive, ".scalar(@quartets). + " of $original_size possible quartets will be run using output from ".scalar(@genes)." total genes.\n"; + +# Go back to working directory +chdir(".."); + +# Determine whether or not we need to run mbsum on the specified input +my $should_summarize = 1; +if (-e $mbsum_archive && $input_is_dir && !$input_is_mbsum) { + chomp(my @sums = `tar tf '$mbsum_archive'`) || die "Something appears to be wrong with '$mbsum_archive'.\n"; + + # Check that each gene has actually been summarized, if not redo the summaries + if (scalar(@sums) != scalar(@genes)) { + unlink($mbsum_archive); + } + else { + $should_summarize = 0; + } +} + +$should_summarize = 0 if ($input_is_mbsum); + +# Summarize MrBayes output if needed +if ($should_summarize) { + # Run mbsum on each gene + print "Summarizing MrBayes output for ".scalar(@genes)." genes.\n"; + + my @pids; + foreach my $gene (@genes) { + + # Wait until a CPU is available + until(okay_to_run(\@pids)) {}; + + my $pid; + until (defined($pid)) { $pid = fork(); usleep(30000); } + + # The child fork + if ($pid == 0) { + #setpgrp(); + run_mbsum($gene); + exit(0); + } + else { + push(@pids, $pid); + } + } + + # Wait for all summaries to finish + foreach my $pid (@pids) { + waitpid($pid, 0); + } + undef(@pids); + + # Remove directory storing mb output + remove_tree($mb_out_dir); + + # Archive and zip mb summaries + chdir($mb_sum_dir); + #system("tar", "czf", $mbsum_archive, glob("$archive_root_no_ext*.sum")); + system("tar", "czf", $mbsum_archive, glob("*.sum")); + system("cp", $mbsum_archive, ".."); + chdir(".."); +} + +die "\nAll quartets have already been completed.\n\n" if (!@quartets); + +# Returns the external IP address of this computer +chomp(my $server_ip = `dig +short myip.opendns.com \@resolver1.opendns.com 2>&1`); +if ($server_ip !~ /(?:[0-9]{1,3}\.){3}[0-9]{1,3}/) { + print "Could not determine external IP address, only local clients will be created.\n"; + $server_ip = "127.0.0.1"; +} + +# Initialize a server +my $sock = IO::Socket::INET->new( + LocalPort => $port, + Blocking => 0, + Reuse => 1, + Listen => SOMAXCONN, + Proto => 'tcp') +or die "Could not create server socket: $!.\n"; +$sock->autoflush(1); + +print "Job server successfully created.\n"; + +# Should probably do this earlier +# Determine server hostname and add to machines if none were specified by the user +chomp(my $server_hostname = `hostname`); +if (scalar(@machines) == 0) { + push(@machines, $server_hostname); + $machines{$server_hostname} = "127.0.0.1"; +} +elsif (scalar(@machines) == 1) { + # Check if the user input only the local machine in the config + if ($machines{$machines[0]} eq $server_ip) { + $machines{$machines[0]} = "127.0.0.1"; + } +} + +my @pids; +foreach my $machine (@machines) { + + # Fork and create a client on the given machine + my $pid = fork(); + if ($pid == 0) { + close(STDIN); + close(STDOUT); + close(STDERR); + + (my $script_name = $script_path) =~ s/.*\///; + + # Send over/copy files depending on where analyses will be run + if ($machines{$machine} ne "127.0.0.1" && $machines{$machine} ne $server_ip) { + + # Send MrBayes summaries to remote machines + if ($input_is_mbsum) { + system("scp", "-q", "$mb_sum_dir/$mbsum_archive", $machine.":/tmp"); + } + else { + system("scp", "-q", $mbsum_archive, $machine.":/tmp"); + } + + # Send this script to the machine + system("scp", "-q", $script_path, $machine.":/tmp"); + + # Send BUCKy executable to the machine + system("scp", "-q", $bucky, $machine.":/tmp"); + + # Execute this perl script in client mode on the given machine + # -tt forces pseudo-terminal allocation and lets us stop remote processes + exec("ssh", "-tt", $machine, "perl", "/tmp/$script_name", $mbsum_archive, "--server-ip=$server_ip:$port"); + } + else { + # Send this script to the machine + system("cp", $script_path, "/tmp"); + + # Send BUCKy executable to the machine + system("cp", $bucky, "/tmp"); + + # Execute this perl script in client mode + exec("perl", "/tmp/$script_name", "$init_dir/$project_name/$mb_sum_dir/$mbsum_archive", "--server-ip=127.0.0.1:$port"); + } + + exit(0); + } + else { + push(@pids, $pid); + } +} + +# Move into mbsum directory +chdir($mb_sum_dir); + +my $select = IO::Select->new($sock); + +# Stores which job is next in queue +my $job_number = 0; + +# Number of open connections to a client +my $total_connections; + +# Number of complete jobs (necessary?) +my $complete_count = 0; + +# Number of connections server has closed +my $closed_connections = 0; + +# Minimum number of connections server should expect +my $starting_connections = scalar(@machines); + +my $time = time(); +my $num_digits = get_num_digits({'NUMBER' => scalar(@quartets)}); + +# Begin the server's job distribution +my %complete_queue; +my $complete_queue_max_size = 100; +while ((!defined($total_connections) || $closed_connections != $total_connections) || $total_connections < $starting_connections) { + # Contains handles to clients which have sent information to the server + my @clients = $select->can_read(0); + + # Free up CPU by sleeping for 10 ms + usleep(10000); + + # Reap any children that we can + foreach my $pid (@pids) { + waitpid($pid, WNOHANG); + } + + # Handle each ready client individually + CLIENT: foreach my $client (@clients) { + + if (scalar(keys %complete_queue) > $complete_queue_max_size) { + my $cwd = abs_path("."); + chdir($mb_sum_dir); + dump_quartets(\%complete_queue); + chdir($cwd); + undef(%complete_queue); + } + + # Client requesting new connection + if ($client == $sock) { + $total_connections++; + $select->add($sock->accept()); + } + else { + + # Get client's message + my $response = <$client>; + next if (not defined($response)); # a response should never actually be undefined + + # Client wants to send us a file + if ($response =~ /SEND_FILE: (.*)/) { + my $file_name = $1; + receive_file({'FILE_PATH' => $file_name, 'FILE_HANDLE' => $client}); + } + + # Client has finished a job + if ($response =~ /DONE '(.*)' '(.*)' \|\|/) { + $complete_count++; + printf(" Analyses complete: %".$num_digits."d/%d.\r", $complete_count, scalar(@quartets)); + + my $completed_quartet = $1; + my $quartet_statistics = $2; + + #push(@unlink, glob("$completed_quartet*")); + push(@unlink, glob(abs_path(".")."/$completed_quartet*")); + $complete_queue{$completed_quartet} = $quartet_statistics; + } + + # Client wants a new job + if ($response =~ /NEW: (.*)/) { + my $client_ip = $1; + + # Check if jobs remain in the queue + if ($job_number < scalar(@quartets)) { + printf("\n Analyses complete: %".$num_digits."d/%d.\r", 0, scalar(@quartets)) if ($job_number == 0); + + my $quartet = join("--", @{$quartets[$job_number]}); + + # Tell local clients to move into mbsum directory + if ($client_ip eq $server_ip) { + print {$client} "CHDIR: ".abs_path(".")."\n"; + } + + # Invocation changes if we want to use a prior of infinity + if ($alpha =~ /(^inf(inity)?)/i) { + print {$client} "NEW: '$quartet' '--use-independence-prior -n $ngen'\n"; + } + else { + print {$client} "NEW: '$quartet' '-a $alpha -n $ngen'\n"; + } + $job_number++; + } + else { + # Client has asked for a job, but there are none remaining + print {$client} "HANGUP\n"; + $select->remove($client); + $client->close(); + $closed_connections++; + next CLIENT; + } + } + } + } +} + +# Dump remaining quartets +dump_quartets(\%complete_queue); + +# Wait until all children have completed +foreach my $pid (@pids) { + waitpid($pid, 0); +} + +print "\n All connections closed.\n"; +print "Total execution time: ", sec2human(time() - $time), ".\n\n"; + +rmdir("$initial_directory/$project_name/$mb_sum_dir"); + +sub client { + my ($opt_name, $address) = @_; + + my ($server_ip, $port) = split(":", $address); + + chdir("/tmp"); + my $bucky = "/tmp/bucky"; + + my $pgrp = $$; + setpgrp(); + + # Determine this host's IP + chomp(my $ip = `dig +short myip.opendns.com \@resolver1.opendns.com`); + + # Set IP to localhost if we don't have internet + if ($ip !~ /(?:[0-9]{1,3}\.){3}[0-9]{1,3}/) { + $ip = "127.0.0.1"; + } + + # Determine file name of mbsum archive the client should use + my @ARGV = split(/\s+/, $invocation); + shift(@ARGV); shift(@ARGV); # remove "perl" and "bucky.pl" + my $mbsum_archive = shift(@ARGV); + + # Extract files from mbsum archive + my @sums; + if ($mbsum_archive =~ /\//) { + chomp(@sums = `tar tf '$mbsum_archive'`); + } + else { + chomp(@sums = `tar xvf '$mbsum_archive' 2>&1`); + @sums = map { s/x //; $_ } @sums if ($os_name eq "darwin"); + } + + # Spawn more clients + my @pids; + # A slightly modification in order to limit forks to 10 + # my $total_forks = get_free_cpus(); + my $total_forks = 10; + if ($total_forks > 1) { + foreach my $fork (1 .. $total_forks - 1) { + my $pid = fork(); + if ($pid == 0) { + last; + } + else { + push(@pids, $pid); + } + } + } + + # The name of the quartet we are working on + my $quartet; + + # Stores names of unneeded files + my @unlink; + + # Change signal handling so killing the server kills these processes and cleans up + $SIG{HUP} = sub { unlink($0, $bucky); unlink(@sums); unlink($mbsum_archive); kill -15, $$; }; + $SIG{TERM} = sub { unlink(glob("$quartet*")) if (defined($quartet)); exit(0); }; + + # Connect to the server + my $sock = new IO::Socket::INET( + PeerAddr => $server_ip.":".$port, + Proto => 'tcp') + or exit(0); + $sock->autoflush(1); + + print {$sock} "NEW: $ip\n"; + while (chomp(my $response = <$sock>)) { + + if ($response =~ /CHDIR: (.*)/) { + chdir($1); + } + elsif ($response =~ /NEW: '(.*)' '(.*)'/) { + $quartet = $1; + my $bucky_settings = $2; + + # If client is local this needs to be defined now + #chomp(@sums = `tar tf $mbsum_archive`) if (!@sums); + + # Create prune tree file contents required for BUCKy + my $count = 0; + my $prune_tree_output = "translate\n"; + foreach my $member (split("--", $quartet)) { + $count++; + $prune_tree_output .= " $count $member"; + if ($count == 4) { + $prune_tree_output .= ";\n"; + } + else { + $prune_tree_output .= ",\n"; + } + } + + # Write prune tree file + my $prune_file_path = "$quartet-prune.txt"; + + push(@unlink, $prune_file_path); + + open(my $prune_file, ">", $prune_file_path); + print {$prune_file} $prune_tree_output; + close($prune_file); + + push(@unlink, "$quartet.input", "$quartet.out", "$quartet.cluster", "$quartet.concordance", "$quartet.gene"); + + # Run BUCKy on specified quartet + system($bucky, split(" ", $bucky_settings), "-cf", 0, "-o", $quartet, "-p", $prune_file_path, @sums); + unlink($prune_file_path); + + # Zip and tarball the results + my @results = glob($quartet."*"); + my $quartet_archive_name = "$quartet.tar.gz"; + + # Open concordance file and parse out the three possible resolutions + my $num_genes = get_used_genes("$quartet.out"); + my $split_info = parse_concordance_output("$quartet.concordance", $num_genes); + + # Archive and compress results + system("tar", "czf", $quartet_archive_name, @results); + + # Send the results back to the server if this is a remote client + if ($server_ip ne "127.0.0.1" && $server_ip ne $ip) { + send_file({'FILE_PATH' => $quartet_archive_name, 'FILE_HANDLE' => $sock}); + unlink($quartet_archive_name); + } + + unlink(@unlink); + undef(@unlink); + + print {$sock} "DONE '$quartet_archive_name' '$split_info' || NEW: $ip\n"; + } + elsif ($response eq "HANGUP") { + last; + } + } + + # Have initial client wait for all others to finish and clean up + if ($$ == $pgrp) { + foreach my $pid (@pids) { + waitpid($pid, 0); + } + unlink($0, $bucky); + unlink(@sums, $mbsum_archive); + #if ($server_ip ne $ip) { + # unlink(@sums, $mbsum_archive); + #} + #else { + # unlink(@sums); + #} + } + + exit(0); +} + +sub dump_quartets { + my $quartets = shift; + + my $pid; + until (defined($pid)) { $pid = fork(); usleep(30000); } + if ($pid == 0) { + + my @completed_quartets = keys %{$quartets}; + my @quartet_statistics = values %{$quartets}; + + $SIG{TERM} = sub { close(STDIN); close(STDOUT); close(STDERR); unlink(@unlink); exit(0); }; + + # Check if this is the first to complete, if so we must create CF output file + if (!-e "../$quartet_output") { + open(my $quartet_output_file, ">", "../$quartet_output"); + print {$quartet_output_file} "taxon1,taxon2,taxon3,taxon4,CF12_34,CF12_34_lo,CF12_34_hi,CF13_24,CF13_24_lo,CF13_24_hi,CF14_23,CF14_23_lo,CF14_23_hi,ngenes\n"; + foreach my $quartet (@quartet_statistics) { + print {$quartet_output_file} $quartet,"\n"; + } + close($quartet_output); + } + else { + # Obtain a file lock on archive so another process doesn't simultaneously try to add to it + open(my $quartet_output_file, ">>", "../$quartet_output"); + flock($quartet_output_file, LOCK_EX) || die "Could not lock '$quartet_output': $!.\n"; + seek($quartet_output_file, 0, SEEK_END) || die "Could not seek '$quartet_output': $!.\n"; + + # Add completed gene + #print {$quartet_output_file} @quartet_statistics,"\n"; + foreach my $quartet (@quartet_statistics) { + print {$quartet_output_file} $quartet,"\n"; + } + + # Release lock + flock($quartet_output_file, LOCK_UN) || die "Could not unlock '$quartet_output': $!.\n"; + close($quartet_output_file); + } + + # Check if this is the first to complete, if so we must create BUCKy tarball + # if (!-e "../$bucky_archive") { + # system("tar", "cf", "../$bucky_archive", @completed_quartets); + # unlink(@completed_quartets); + # } + # else { + # my @unlink; + # foreach my $completed_quartet (@completed_quartets) { + # (my $quartet = $completed_quartet) =~ s/\.tar\.gz//; + # push(@unlink, glob("$quartet*")); + # } + # #$SIG{TERM} = sub { close(STDIN); close(STDOUT); close(STDERR); unlink(glob("$quartet*")); exit(0); }; + # #$SIG{TERM} = sub { close(STDIN); close(STDOUT); close(STDERR); unlink(@unlink); exit(0); }; + + # # Obtain a file lock on archive so another process doesn't simultaneously try to add to it + # open(my $bucky_archive_file, "<", "../$bucky_archive"); + # flock($bucky_archive_file, LOCK_EX) || die "Could not lock '$bucky_archive': $!.\n"; + + # # Add completed gene + # system("tar", "rf", "../$bucky_archive", @completed_quartets); + # unlink(@completed_quartets); + + # # Release lock + # flock($bucky_archive_file, LOCK_UN) || die "Could not unlock '$bucky_archive': $!.\n"; + # close($bucky_archive_file); + # } + + unlink(@unlink); + undef(@unlink); + + exit(0); + } + else { + push(@pids, $pid); + } + +# unlink(@unlink); +# undef(@unlink); + + return; +} + +sub get_used_genes { + my $file_name = shift; + + # Number of genes actually used by BUCKy + my $num_genes; + + # Open up the BUCKy output file + open(my $bucky_out, "<", $file_name); + while (my $line = <$bucky_out>) { + if ($line =~ /Read (\d+) genes with a total of/) { + $num_genes = $1; + last; + } + } + close($bucky_out); + + die "Error determining number of genes used by BUCKy ($file_name).\n" if (!defined($num_genes)); + + return $num_genes; +} + +sub parse_concordance_output { + my ($file_name, $ngenes) = @_; + + my @taxa; + my %splits; + + # Open up the specified output file + open(my $concordance_file, "<", $file_name); + + my $split; + my $in_translate; + my $in_all_splits; + while (my $line = <$concordance_file>) { + + # Parse the translate table + if ($in_translate) { + if ($line =~ /\d+ (.*)([,;])/) { + my $taxon = $1; + my $line_end = $2; + push(@taxa, $taxon); + + $in_translate = 0 if ($line_end eq ';'); + } + } + + # Parse the split information + if ($in_all_splits) { + + # Set the split we are parsing information from + if ($line =~ /^(\{\S+\})/) { + my $current_split = $1; + if ($current_split eq "{1,4|2,3}") { + $split = "14|23"; + } + elsif ($current_split eq "{1,3|2,4}") { + $split = "13|24"; + } + elsif ($current_split eq "{1,2|3,4}") { + $split = "12|34"; + } + } + + # Parse mean number of loci for split + if ($line =~ /=\s+(\S+) \(number of loci\)/) { + $splits{$split}->{"CF"} = $1 / $ngenes; + } + + # Parse 95% confidence interval + if ($line =~ /95% CI for CF = \((\d+),(\d+)\)/) { + #$splits{$split}->{"95%_CI"} = "(".($1 / $ngenes).",".($2 / $ngenes).")"; + $splits{$split}->{"95%_CI_LO"} = ($1 / $ngenes); + $splits{$split}->{"95%_CI_HI"} = ($2 / $ngenes); + } + } + + $in_translate++ if ($line =~ /^translate/); + $in_all_splits++ if ($line =~ /^All Splits:/); + } + + # Concat taxa names together + my $return = join(",", @taxa); + $return .= ","; + + # Concat split proportions with their 95% CI to return + if (exists($splits{"12|34"})) { + #$return .= $splits{"12|34"}->{"CF"}.$splits{"12|34"}->{"95%_CI"}."\t"; + $return .= $splits{"12|34"}->{"CF"}.",".$splits{"12|34"}->{"95%_CI_LO"}.",".$splits{"12|34"}->{"95%_CI_HI"}.","; + } + else { + #$return .= "0(0,0)\t"; + $return .= "0,0,0,"; + } + + if (exists($splits{"13|24"})) { + #$return .= $splits{"13|24"}->{"CF"}.$splits{"13|24"}->{"95%_CI"}."\t"; + $return .= $splits{"13|24"}->{"CF"}.",".$splits{"13|24"}->{"95%_CI_LO"}.",".$splits{"13|24"}->{"95%_CI_HI"}.","; + } + else { + #$return .= "0(0,0)\t"; + $return .= "0,0,0,"; + } + + if (exists($splits{"14|23"})) { + #$return .= $splits{"14|23"}->{"CF"}.$splits{"14|23"}->{"95%_CI"}; + $return .= $splits{"14|23"}->{"CF"}.",".$splits{"14|23"}->{"95%_CI_LO"}.",".$splits{"14|23"}->{"95%_CI_HI"}; + } + else { + #$return .= "0(0,0)"; + $return .= "0,0,0"; + } + + # Append number of genes used + $return .= ",$ngenes"; + + return $return; +} + +sub parse_mb_log { + my $log_file_name = shift; + + # Open the specified mb log file and parse useful information from it + + my @taxa; + my $ngen; + my $nruns; + my $burnin; + my $burninfrac; + my $samplefreq; + open(my $log_file, "<", $log_file_name); + while (my $line = <$log_file>) { + if ($line =~ /Taxon\s+\d+ -> (\S+)/) { + push(@taxa, $1); + } + elsif ($line =~ /Setting number of runs to (\d+)/) { + $nruns = $1; + } + elsif ($line =~ /Setting burnin fraction to (\S+)/) { + $burninfrac = $1; + } + elsif ($line =~ /Setting chain burn-in to (\d+)/) { + $burnin = $1; + } + elsif ($line =~ /Setting sample frequency to (\d+)/) { + $samplefreq = $1; + } + elsif ($line =~ /Setting number of generations to (\d+)/) { + $ngen = $1; + } + } + close($log_file); + + if (defined($burnin)) { + #return {'SAMPLEFREQ' => $samplefreq, 'NRUNS' => $nruns, 'BURNIN' => $burnin }; + return {'SAMPLEFREQ' => $samplefreq, 'NRUNS' => $nruns, 'BURNIN' => $burnin, 'TAXA' => \@taxa}; + } + else { + return {'NGEN' => $ngen, 'NRUNS' => $nruns, 'BURNINFRAC' => $burninfrac, + 'SAMPLEFREQ' => $samplefreq, 'TAXA' => \@taxa}; + } +} + +sub parse_mbsum_taxa { + my $mbsum_file_name = shift; + + # Open the specified mbsum file and parse its taxa list + + my @taxa; + my $in_translate_block = 0; + open(my $mbsum_file, "<", $mbsum_file_name); + while (my $line = <$mbsum_file>) { + $in_translate_block++ if ($line =~ /translate/); + + if ($in_translate_block == 1 && $line =~ /\d+\s+([^,;]+)/) { + push(@taxa, $1); + } + } + close($mbsum_file); + + # Check if there were multiple translate blocks in the file which is indicative of an error with the creation of the file + die "Something is amiss with '$mbsum_file_name', multiple translate blocks ($in_translate_block) were detected when there should only be one.\n" if ($in_translate_block > 1); + + # Check that we actually parsed something, otherwise input is improperly formatted/not mbsum output + die "No taxa parsed for file '$mbsum_file_name', does '$archive' actually contain mbsum output?.\n" if (!@taxa); + + return \@taxa; +} + +sub run_mbsum { + my $tarball = shift; + + # Unzip specified tarball + chomp(my @mb_files = `tar xvf '$mb_out_dir$tarball' -C '$mb_out_dir' 2>&1`); + @mb_files = map { s/x //; $_ } @mb_files if ($os_name eq "darwin"); + + # Determine name for this partition's log file + my $log_file_name; + foreach my $file (@mb_files) { + if ($file =~ /\.log$/) { + $log_file_name = $file; + last; + } + } + die "Could not locate log file for '$tarball'.\n" if (!defined($log_file_name)); + + # Parse log file + my $mb = parse_mb_log("$mb_out_dir$log_file_name"); + + (my $gene_name = $tarball) =~ s/\.nex\.tar\.gz//; + + # Determine number of trees mbsum should remove from each file + + my $trim; + if ($mb->{BURNIN}) { + $trim = $mb->{BURNIN} + 1; + } + else { + $trim = ((($mb->{NGEN} / $mb->{SAMPLEFREQ}) * $mb->{NRUNS} * $mb->{BURNINFRAC}) / $mb->{NRUNS}) + 1; + } + + # Summarize gene's tree files + system("$mbsum '$mb_out_dir$gene_name.'*.t -n $trim -o '$mb_sum_dir$gene_name.sum' >/dev/null 2>&1"); + + # Clean up extracted files + chdir($mb_out_dir); + unlink(@mb_files); +} + +sub okay_to_run { + my $pids = shift; + + # Free up a CPU by sleeping for 10 ms + usleep(10000); + + my $current_forks = scalar(@{$pids}); + foreach my $index (reverse(0 .. $current_forks - 1)) { + next if ($index < 0); + + my $pid = @{$pids}[$index]; + my $wait = waitpid($pid, WNOHANG); + + # Successfully reaped child + if ($wait > 0) { + $current_forks--; + #splice(@pids, $index, 1); + splice(@{$pids}, $index, 1); + } + } + + return ($current_forks < $max_forks); +} + +sub hashsum { + my $settings = shift; + + my $file_path = $settings->{'FILE_PATH'}; + + open(my $file, "<", $file_path) or die "Couldn't open file '$file_path': $!.\n"; + my $md5 = Digest::MD5->new; + my $md5sum = $md5->addfile(*$file)->hexdigest; + close($file); + + return $md5sum; +} + +sub send_file { + my $settings = shift; + + my $file_path = $settings->{'FILE_PATH'}; + my $file_handle = $settings->{'FILE_HANDLE'}; + + my $hash = hashsum({'FILE_PATH' => $file_path}); + print {$file_handle} "SEND_FILE: $file_path\n"; + + open(my $file, "<", $file_path) or die "Couldn't open file '$file_path': $!.\n"; + while (<$file>) { + print {$file_handle} $_; + } + close($file); + + print {$file_handle} " END_FILE: $hash\n"; + + # Stall until we know status of file transfer + while (defined(my $response = <$file_handle>)) { + chomp($response); + + last if ($response eq "TRANSFER_SUCCESS"); + die "Unsuccessful file transfer, checksums did not match.\n" if ($response eq "TRANSFER_FAILURE"); + } +} + +sub receive_file { + my $settings = shift; + + my $file_path = $settings->{'FILE_PATH'}; + my $file_handle = $settings->{'FILE_HANDLE'}; + + my $check_hash; + open(my $file, ">", $file_path); + while (<$file_handle>) { + if ($_ =~ /(.*) END_FILE: (\S+)/) { + print {$file} $1; + $check_hash = $2; + last; + } + else { + print {$file} $_; + } + } + close($file); + + # Use md5 hashsum to make sure transfer worked + my $hash = hashsum({'FILE_PATH' => $file_path}); + if ($hash ne $check_hash) { + die "Unsuccessful file transfer, checksums do not match.\n'$hash' - '$check_hash'\n"; # hopefully this never pops up + print {$file_handle} "TRANSFER_FAILURE\n" + } + + else { + print {$file_handle} "TRANSFER_SUCCESS\n"; + } +} + +sub INT_handler { + #dump_quartets(\%complete_queue); + + unlink(@unlink); + + # Kill ssh process(es) spawned by this script + foreach my $pid (@pids) { + #kill(-9, $pid); + #kill(15, $pid); + kill(1, $pid); + } + + # Move into gene directory + chdir("$initial_directory/$project_name"); + + rmdir($mb_out_dir); + + # Try to delete directory once per second for five seconds, if it can't be deleted print an error message + # I've found this method is necessary for analyses performed on AFS drives + my $count = 0; + until (!-e $mb_sum_dir || $count == 5) { + $count++; + + remove_tree($mb_sum_dir, {error => \my $err}); + sleep(1); + } + #logger("Could not clean all files in './$gene_dir/'.") if ($count == 5); + print "Could not clean all files in './$mb_sum_dir/'.\n" if ($count == 5); + + exit(0); +} + +sub clean_up { + my $settings = shift; + + my $remove_dirs = $settings->{'DIRS'}; + my $current_dir = getcwd(); + +# chdir($alignment_root); +# unlink(glob($gene_dir."$alignment_name*")); +# #unlink($server_check_file) if (defined($server_check_file)); +# +# if ($remove_dirs) { +# rmdir($gene_dir); +# } + chdir($current_dir); +} + +sub get_num_digits { + my $settings = shift; + + my $number = $settings->{'NUMBER'}; + + my $digits = 1; + while (floor($number / 10) != 0) { + $number = floor($number / 10); + $digits++; + } + + return $digits; +} + +sub sec2human { + my $secs = shift; + + # Constants + my $secs_in_min = 60; + my $secs_in_hour = 60 * 60; + my $secs_in_day = 24 * 60 * 60; + + $secs = int($secs); + + return "0 seconds" if (!$secs); + + # Calculate units of time + my $days = int($secs / $secs_in_day); + my $hours = ($secs / $secs_in_hour) % 24; + my $mins = ($secs / $secs_in_min) % 60; + $secs = $secs % 60; + + # Format return nicely + my $time; + if ($days) { + $time .= ($days != 1) ? "$days days, " : "$days day, "; + } + if ($hours) { + $time .= ($hours != 1) ? "$hours hours, " : "$hours hour, "; + } + if ($mins) { + $time .= ($mins != 1) ? "$mins minutes, " : "$mins minute, "; + } + if ($secs) { + $time .= ($secs != 1) ? "$secs seconds " : "$secs second "; + } + else { + # Remove comma + chop($time); + } + chop($time); + + return $time; +} + +sub get_free_cpus { + + my $os_name = $^O; + + # Returns a two-member array containing CPU usage observed by top, + # top is run twice as its first output is usually inaccurate + my @percent_free_cpu; + if ($os_name eq "darwin") { + # Mac OS + chomp(@percent_free_cpu = `top -i 1 -l 2 | grep "CPU usage"`); + } + else { + # Linux + chomp(@percent_free_cpu = `top -b -n2 -d0.05 | grep "Cpu(s)"`); + } + + my $percent_free_cpu = pop(@percent_free_cpu); + + if ($os_name eq "darwin") { + # Mac OS + $percent_free_cpu =~ s/.*?(\d+\.\d+)%\s+id.*/$1/; + } + else { + # linux + $percent_free_cpu =~ s/.*?(\d+\.\d)\s*%?ni,\s*(\d+\.\d)\s*%?id.*/$1 + $2/; # also includes %nice as free + $percent_free_cpu = eval($percent_free_cpu); + } + + my $total_cpus; + if ($os_name eq "darwin") { + # Mac OS + $total_cpus = `sysctl -n hw.ncpu`; + } + else { + # linux + $total_cpus = `grep --count 'cpu' /proc/stat` - 1; + } + + my $free_cpus = ceil($total_cpus * $percent_free_cpu / 100); + + if ($free_cpus == 0 || $free_cpus !~ /^\d+$/) { + $free_cpus = 1; # assume that at least one cpu can be used + } + + return $free_cpus; +} + +sub run_cmd { + my $command = shift; + + my $return = system($command); + + if ($return) { + logger("'$command' died with error: '$return'.\n"); + #kill(2, $parent_pid); + exit(0); + } +} + +sub check_path_for_exec { + my $exec = shift; + + my $path = $ENV{PATH}.":."; # include current directory as well + my @path_dirs = split(":", $path); + + my $exec_path; + foreach my $dir (@path_dirs) { + $dir .= "/" if ($dir !~ /\/$/); + $exec_path = abs_path($dir.$exec) if (-e $dir.$exec && -x $dir.$exec && !-d $dir.$exec); + } + + die "Could not find the following executable: '$exec'. This script requires this program in your path.\n" if (!defined($exec_path)); + return $exec_path; +} + +# I grabbed this from StackOverflow so that's why its style is different #DontFixWhatIsntBroken: +# https://stackoverflow.com/questions/10299961/in-perl-how-can-i-generate-all-possible-combinations-of-a-list +sub combine { + my ($list, $n) = @_; + die "Insufficient list members" if ($n > @$list); + + return map [$_], @$list if ($n <= 1); + + my @comb; + + for (my $i = 0; $i+$n <= @$list; ++$i) { + my $val = $list->[$i]; + my @rest = @$list[$i + 1 .. $#$list]; + push(@comb, [$val, @$_]) for combine(\@rest, $n - 1); + } + + return @comb; +} + +sub check_bucky_version { + my $bucky = shift; + + print "\nChecking for BUCKy version >= 1.4.4...\n"; + + # Run BUCKy with --version and extract version info + chomp(my @version_info = grep { /BUCKy version/ } `$bucky --version`); + my $version_info = shift(@version_info); + + die " Could not determine BUCKy version.\n" if (!defined($version_info)); + + # Get the actual version number + my $version; + if ($version_info =~ /BUCKy\s+version\s+([^\s|,]+)/) { + $version = $1; + } + die " Could not determine BUCKy version.\n" if (!defined($version_info)); + + # Version testing + #my @versions = qw/2.1b 1.2.1000 1 0.9.8 2.3 1.4.5 1.4.3 1.4 1.500.2 1.4.4 1.4.4.1/; + #foreach my $version (@versions) { + + print " BUCKy version: $version.\n"; + + # Split version number based on period delimiters + my @version_parts = split(/\./, $version); + + # Future proofing if letters are ever used (we won't ever care about them) + @version_parts = map { s/[a-zA-Z]+//g; $_ } @version_parts; + die " Error determining BUCKy version.\n" if (!@version_parts); + + # Check that version is >= 1.4.4 + if (defined($version_parts[0]) && int($version_parts[0]) > 1) { + print " BUCKy version check passed.\n"; + return; + } + elsif ((defined($version_parts[0]) && int($version_parts[0]) == 1) && (defined($version_parts[1]) && int($version_parts[1]) > 4)) { + print " BUCKy version check passed.\n"; + return; + } + elsif (((defined($version_parts[0]) && int($version_parts[0]) == 1) && (defined($version_parts[1]) && int($version_parts[1]) == 4)) + && defined($version_parts[2]) && int($version_parts[2]) >= 4) { + print " BUCKy version check passed.\n"; + return; + } + else { + die " BUCKy version check failed, update to version >= 1.4.4.\n"; + } + + #print "\n"; + #} +} + +sub usage { + return "Usage: bucky.pl [MRBAYES TARBALL]\n"; +} + +sub help { +print < +EOF +exit(0); +} diff --git a/99.scripts_v2/utils/classify_frameshifts.py b/99.scripts_v2/utils/classify_frameshifts.py new file mode 100644 index 0000000..39bf9c5 --- /dev/null +++ b/99.scripts_v2/utils/classify_frameshifts.py @@ -0,0 +1,269 @@ +#!/usr/bin/env python3 + +import argparse +import csv +from collections import Counter +from pathlib import Path + + +LOCUS_HEADER = [ + "OG", + "sequence_count", + "independent_event_count", + "max_single_sequence_unique_events", + "carrier_pattern", + "severe", +] + + +def read_fasta(fasta_path: Path) -> dict[str, str]: + sequences: dict[str, list[str]] = {} + sequence_id: str | None = None + + with fasta_path.open() as fasta: + for line_number, raw_line in enumerate(fasta, start=1): + line = raw_line.strip() + if not line: + continue + if line.startswith(">"): + sequence_id = line[1:].split()[0] + if not sequence_id: + raise ValueError( + f"{fasta_path}:{line_number}: empty FASTA identifier" + ) + if sequence_id in sequences: + raise ValueError( + f"{fasta_path}:{line_number}: duplicate FASTA identifier " + f"{sequence_id!r}" + ) + sequences[sequence_id] = [] + elif sequence_id is None: + raise ValueError( + f"{fasta_path}:{line_number}: sequence before first FASTA header" + ) + else: + sequences[sequence_id].append(line) + + if not sequences: + raise ValueError(f"{fasta_path}: no FASTA sequences found") + + joined = {name: "".join(parts) for name, parts in sequences.items()} + lengths = {len(sequence) for sequence in joined.values()} + if len(lengths) != 1: + raise ValueError(f"{fasta_path}: aligned sequences have different lengths") + return joined + + +def event_mode(carrier_count: int, sequence_count: int) -> str: + if carrier_count == 1: + return "single_species" + if carrier_count == sequence_count - 1: + return "all_but_one" + if carrier_count == sequence_count: + return "all_taxa" + return "branch_shared" + + +def locus_pattern( + event_modes: list[str], + event_count: int, + max_unique_events: int, +) -> str: + modes = set(event_modes) + if max_unique_events >= 2: + return "single_species_scattered_multiple_events" + if event_count == 1 and modes == {"single_species"}: + return "single_species_single_event" + if modes == {"all_but_one"}: + return "all_but_one" + if modes == {"branch_shared"}: + return "branch_shared" + if modes == {"all_taxa"}: + return "all_taxa" + if not modes: + return "no_frameshift_detected" + return "mixed" + + +def merge_overlapping_triplets( + triplets: list[tuple[tuple[int, int, int], str]], +) -> list[tuple[tuple[int, ...], list[str]]]: + components: list[tuple[set[int], set[str]]] = [] + + for columns, carrier in triplets: + column_set = set(columns) + matching = [ + index + for index, (component_columns, _) in enumerate(components) + if component_columns & column_set + ] + + if not matching: + components.append((column_set, {carrier})) + continue + + merged_columns = set(column_set) + merged_carriers = {carrier} + for index in reversed(matching): + component_columns, component_carriers = components.pop(index) + merged_columns.update(component_columns) + merged_carriers.update(component_carriers) + components.append((merged_columns, merged_carriers)) + + return [ + (tuple(sorted(columns)), sorted(carriers)) + for columns, carriers in components + ] + + +def classify_fasta(fasta_path: Path) -> tuple[list[str], list[list[str]]]: + sequences = read_fasta(fasta_path) + sequence_ids = list(sequences) + sequence_count = len(sequence_ids) + unique_events_by_sequence: Counter[str] = Counter() + event_rows: list[list[str]] = [] + modes: list[str] = [] + complete_triplets: list[tuple[tuple[int, int, int], str]] = [] + events: list[tuple[tuple[int, ...], list[str]]] = [] + + for sequence_id, sequence in sequences.items(): + marker_columns = [ + column_index + 1 + for column_index, character in enumerate(sequence) + if character == "!" + ] + for index in range(0, len(marker_columns), 3): + event_columns = tuple(marker_columns[index : index + 3]) + if len(event_columns) == 3: + complete_triplets.append((event_columns, sequence_id)) + else: + # A trailing 1-2 markers still form one unique event, but cannot + # be classified as shared because they are not a complete triplet. + events.append((event_columns, [sequence_id])) + + events.extend(merge_overlapping_triplets(complete_triplets)) + events.sort(key=lambda event: (event[0], event[1])) + + for event_columns, carriers in events: + carriers = [ + sequence_id for sequence_id in sequence_ids if sequence_id in carriers + ] + mode = event_mode(len(carriers), sequence_count) + modes.append(mode) + if len(carriers) == 1: + unique_events_by_sequence[carriers[0]] += 1 + event_rows.append( + [ + fasta_path.stem, + ",".join(str(column) for column in event_columns), + str(len(carriers)), + ",".join(carriers), + mode, + ] + ) + + event_count = len(event_rows) + max_unique_events = max(unique_events_by_sequence.values(), default=0) + severe = event_count >= 3 or max_unique_events >= 2 + locus_row = [ + fasta_path.stem, + str(sequence_count), + str(event_count), + str(max_unique_events), + locus_pattern(modes, event_count, max_unique_events), + "yes" if severe else "no", + ] + return locus_row, event_rows + + +def fasta_paths_from_list(list_path: Path) -> list[Path]: + paths: list[Path] = [] + seen: set[Path] = set() + with list_path.open() as fasta_list: + for line_number, raw_line in enumerate(fasta_list, start=1): + value = raw_line.strip() + if not value: + continue + path = Path(value) + if not path.is_absolute(): + path = list_path.parent / path + path = path.resolve() + if not path.is_file(): + raise FileNotFoundError( + f"{list_path}:{line_number}: FASTA file not found: {path}" + ) + if path not in seen: + paths.append(path) + seen.add(path) + return paths + + +def write_results( + output_dir: Path, + locus_rows: list[list[str]], + event_rows: list[list[str]], +) -> None: + output_dir.mkdir(parents=True, exist_ok=True) + + with (output_dir / "frameshift_events.tsv").open("w", newline="") as output: + writer = csv.writer(output, delimiter="\t", lineterminator="\n") + writer.writerow(["OG", "alignment_column", "carrier_count", "carriers", "mode"]) + writer.writerows(event_rows) + + with (output_dir / "frameshift_locus_classification.tsv").open( + "w", newline="" + ) as output: + writer = csv.writer(output, delimiter="\t", lineterminator="\n") + writer.writerow(LOCUS_HEADER) + writer.writerows(locus_rows) + + severe_rows = [row for row in locus_rows if row[-1] == "yes"] + with (output_dir / "frameshift_severe_subset.tsv").open( + "w", newline="" + ) as output: + writer = csv.writer(output, delimiter="\t", lineterminator="\n") + writer.writerow(LOCUS_HEADER) + writer.writerows(severe_rows) + + pattern_counts = Counter(row[4] for row in locus_rows) + with (output_dir / "frameshift_classification_summary.tsv").open( + "w", newline="" + ) as output: + writer = csv.writer(output, delimiter="\t", lineterminator="\n") + writer.writerow(["metric", "count"]) + writer.writerow(["total_FS_loci", len(locus_rows)]) + writer.writerow(["severe_loci", len(severe_rows)]) + for pattern, count in sorted(pattern_counts.items()): + writer.writerow([pattern, count]) + + +def main() -> None: + parser = argparse.ArgumentParser( + description="Classify frameshift loci by event count and carrier pattern." + ) + parser.add_argument( + "--fasta-list", + required=True, + type=Path, + help="File containing one aligned FASTA path per line.", + ) + parser.add_argument( + "--output-dir", + required=True, + type=Path, + help="Directory for classification tables.", + ) + args = parser.parse_args() + + fasta_paths = fasta_paths_from_list(args.fasta_list.resolve()) + locus_rows: list[list[str]] = [] + event_rows: list[list[str]] = [] + for fasta_path in fasta_paths: + locus_row, rows = classify_fasta(fasta_path) + locus_rows.append(locus_row) + event_rows.extend(rows) + write_results(args.output_dir, locus_rows, event_rows) + + +if __name__ == "__main__": + main() diff --git a/99.scripts_v2/utils/get_og_seqs.py b/99.scripts_v2/utils/get_og_seqs.py new file mode 100644 index 0000000..49c7a68 --- /dev/null +++ b/99.scripts_v2/utils/get_og_seqs.py @@ -0,0 +1,148 @@ +#! /usr/bin/env python3 +# -*- coding: utf-8 -*- + +""" +Extract Single Copy OG sequences from a comprehensive FASTA file based on OG definitions from results of orthofinder. +""" + +import sys +from Bio import SeqIO +from Bio.SeqRecord import SeqRecord +from Bio.Seq import Seq +from pathlib import Path +import argparse + + +def get_og_fastas(og_dir: Path, ext: str) -> dict[str, list[str]]: + """ + Get a dictionary of OGs and their corresponding sequence IDs from FASTA files in a directory. + + Args: + og_dir (Path): Path to the directory containing OG FASTA files. + ext (str): Extension of the FASTA files. Default is ".fa". + + Returns: + dict: Dictionary with OG names as keys and lists of sequence IDs as values. + """ + og_dict: dict[str, list[str]] = {} + try: + for fasta_file in og_dir.glob(f"*{ext}"): + og_name = fasta_file.stem + seq_ids: list[str] = [] + for record in SeqIO.parse(fasta_file, "fasta"): + seq_ids.append(record.id) + og_dict[og_name] = seq_ids + except Exception as e: + print(f"Error processing OG FASTA files in {og_dir}: {e}") + sys.exit(1) + return og_dict + + +def parse_all_fasta(fasta_file: Path) -> dict[str, Seq]: + """ + Parse a FASTA file and return a dictionary of sequence IDs and their corresponding Seq objects. + + Args: + fasta_file (Path): Path to the FASTA file. + Returns: + dict: Dictionary with sequence IDs as keys and Seq objects as values. + """ + seq_dict: dict[str, Seq] = {} + try: + for record in SeqIO.parse(fasta_file, "fasta"): + seq_dict[record.id] = record.seq + except Exception as e: + print(f"Error parsing FASTA file {fasta_file}: {e}") + sys.exit(1) + return seq_dict + + +def output_og_seqs( + all_seq_dict: dict[str, Seq], og_dict: dict[str, list[str]], output_dir: Path +): + """ + Output sequences for each OG into separate FASTA files, remove gene id and only keep taxon name. + + Args: + all_seq_dict (dict): Dictionary of all sequences with sequence IDs as keys. + og_dict (dict): Dictionary of OGs with OG names as keys and lists of sequence IDs as values. + output_dir (Path): Path to the output directory. + """ + for og_name, seq_ids in og_dict.items(): + og_seqs: list[SeqRecord] = [] + for seq_id in seq_ids: + if seq_id in all_seq_dict: + seq_record = SeqRecord( + all_seq_dict[seq_id], id=seq_id.split("@")[0], description="" + ) + og_seqs.append(seq_record) + else: + print(f"Warning: Sequence ID {seq_id} not found in all sequences.") + output_fasta = output_dir / f"{og_name}.fa" + try: + SeqIO.write(og_seqs, output_fasta, "fasta") + except Exception as e: + print(f"Error writing to FASTA file {output_fasta}: {e}") + sys.exit(1) + + +def write_og_list(og_dict: dict[str, list[str]]): + """ + Write the OG list to a text file. + + Args: + og_dict (dict): Dictionary of OGs with OG names as keys and lists of sequence IDs as values. + """ + try: + with open("og_list.tsv", "w") as f: + for og_name, seq_ids in og_dict.items(): + line = f"{og_name}\t" + "\t".join(seq_ids) + "\n" + f.write(line) + print("OG list written to og_list.tsv") + except Exception as e: + print(f"Error writing OG list to file: {e}") + sys.exit(1) + + +def main(og_dir_path: str, all_fasta_path: str, output_dir_path: str, ext: str = ".fa"): + og_dir = Path(og_dir_path) + all_fasta = Path(all_fasta_path) + output_dir = Path(output_dir_path) + output_dir.mkdir(parents=True, exist_ok=True) + + og_dict = get_og_fastas(og_dir, ext) + all_seqs = parse_all_fasta(all_fasta) + output_og_seqs(all_seqs, og_dict, output_dir) + write_og_list(og_dict) + + +if __name__ == "__main__": + parser = argparse.ArgumentParser( + description="Extract OG sequences from a comprehensive FASTA file based on OG definitions." + ) + parser.add_argument( + "-d", + "--og_dir", + required=True, + help="Directory containing OG FASTA files.", + ) + parser.add_argument( + "-a", + "--all_fasta", + required=True, + help="FASTA file containing all sequences.", + ) + parser.add_argument( + "-o", + "--output_dir", + required=True, + help="Output directory for OG FASTA files.", + ) + parser.add_argument( + "-e", + "--ext", + default=".fa", + help="Extension of OG FASTA files (default: .fa).", + ) + args = parser.parse_args() + main(args.og_dir, args.all_fasta, args.output_dir, args.ext) diff --git a/99.scripts_v2/utils/mbblock.txt b/99.scripts_v2/utils/mbblock.txt new file mode 100644 index 0000000..e32a24d --- /dev/null +++ b/99.scripts_v2/utils/mbblock.txt @@ -0,0 +1,12 @@ +begin mrbayes; +set nowarnings=yes; +set usebeagle=yes; +set autoclose=yes; +set seed=12345; +set swapseed=12345; +lset nst=6 rates=gamma; +mcmcp ngen=2000000 burninfrac=.25 samplefreq=1000 printfreq=10000 checkpoint=no +diagnfreq=10000 nruns=3 nchains=3 temp=0.40 swapfreq=10 stoprule=no; +mcmc; +sumt; +end; \ No newline at end of file diff --git a/99.scripts_v2/utils/mrbayes.sh b/99.scripts_v2/utils/mrbayes.sh new file mode 100644 index 0000000..c6f6f7a --- /dev/null +++ b/99.scripts_v2/utils/mrbayes.sh @@ -0,0 +1,20 @@ +#! /bin/bash +set -e + +dirname=$(dirname "$0") +if [ "$#" -ne 2 ]; then + echo "Usage: $0 " + exit 1 +fi + +in_fasta=$(readlink -f "$1") +out_nex=$2 + +# Convert fasta to nexus +out_dir=$(dirname "$out_nex") +mkdir -p "$out_dir" +seqmagick convert --output-format nexus --alphabet dna --input-format fasta "$in_fasta" "$out_nex" +# append MrBayes block to nexus file +cat "$dirname/mbblock.txt" >>"$out_nex" +# Run MrBayes +mb "$out_nex" >"${out_dir}"/mrbayes.log 2>&1 diff --git a/99.scripts_v2/utils/raxml.sh b/99.scripts_v2/utils/raxml.sh new file mode 100755 index 0000000..a12fd30 --- /dev/null +++ b/99.scripts_v2/utils/raxml.sh @@ -0,0 +1,20 @@ +#! /bin/bash +set -e +if [ "$#" -ne 4 ]; then + echo "Usage: $0 " + echo "Run modeltest-ng and raxml-ng on a given alignment" + exit 1 +fi + +aln=$(readlink -f "$1") +out=$2 +threads=$3 +outgroup=$4 +outdir=$(dirname "$out") +mkdir -p "$outdir" +# run modeltest-ng +modeltest-ng -p "$threads" -r 12345 --force -i "$aln" -d nt -t ml -o "$out".modeltest +# run raxml-ng +cmd=$(grep "raxml-ng" "$out".modeltest.out | tail -n 1 | sed 's/> //') +params="--blopt nr_safe --all --bs-trees 1000 --outgroup $outgroup --redo --threads $threads --seed 12345 --prefix $out" +bash -c "$cmd $params" >/dev/null diff --git a/99.scripts_v2/utils/snaq.jl b/99.scripts_v2/utils/snaq.jl new file mode 100644 index 0000000..c51f230 --- /dev/null +++ b/99.scripts_v2/utils/snaq.jl @@ -0,0 +1,195 @@ +#! /usr/bin/env julia +# Installing Dependencies +## using Pkg +## Pkg.add("Distributed") +## Pkg.add("DataFrames") +## Pkg.add("CSV") +## Pkg.add("SNaQ") +## Pkg.add("PhyloNetworks") +## Pkg.add("RCall") +## Pkg.add("PhyloPlots") +## Pkg.add("QuartetNetworkGoodnessFit") + +# Running SNaQ Analysis +using PhyloNetworks, SNaQ; +using Distributed; +addprocs(9); +@everywhere using PhyloNetworks, SNaQ; +nruns = 100; # number of runs for each hmax +astralfile = joinpath("..", "species_tree", "coal.tre"); +astraltree = readnewick(astralfile); + +### Reading RAxML gene trees and ASTRAL species tree +### running in raxml_snaq/ folder +# raxmltrees = joinpath("..", "..", "species_tree", "all.trees"); +# inputCF = readtrees2CF(raxmltrees); +# net0 = snaq!(astraltree, inputCF, hmax=0, filename="net0", seed=123, outgroup="ZJ", runs=nruns); +# net1 = snaq!(net0, inputCF, hmax=1, filename="net1", seed=123, outgroup="ZJ", runs=nruns); +# net2 = snaq!(net1, inputCF, hmax=2, filename="net2", seed=123, outgroup="ZJ", runs=nruns); +# net3 = snaq!(net2, inputCF, hmax=3, filename="net3", seed=123, outgroup="ZJ", runs=nruns); +# net4 = snaq!(net3, inputCF, hmax=4, filename="net4", seed=123, outgroup="ZJ", runs=nruns); + +### Alternatively, reading in the input files from Bucky +### running in input_snaq/ folder +inputCFfile = joinpath("..", "bucky", "bucky_1", "input.mb.CFs.csv"); +inputCF = readtableCF(inputCFfile); +net0 = snaq!(astraltree, inputCF, hmax=0, filename="net0", seed=123, outgroup="ZJ", runs=nruns); +net1 = snaq!(net0, inputCF, hmax=1, filename="net1", seed=123, outgroup="ZJ", runs=nruns); +net2 = snaq!(net1, inputCF, hmax=2, filename="net2", seed=123, outgroup="ZJ", runs=nruns); +net3 = snaq!(net2, inputCF, hmax=3, filename="net3", seed=123, outgroup="ZJ", runs=nruns); +net4 = snaq!(net3, inputCF, hmax=4, filename="net4", seed=12345, outgroup="ZJ", runs=nruns); + + +# Plotting the SNaQ results +using PhyloPlots, RCall; +using PhyloNetworks, SNaQ; +## Network scores vs. hmax +net0 = readsnaqnetwork("net0.out"); +net1 = readsnaqnetwork("net1.out"); +net2 = readsnaqnetwork("net2.out"); +net3 = readsnaqnetwork("net3.out"); +net4 = readsnaqnetwork("net4.out"); +scores = [loglik(net0), loglik(net1), loglik(net2), loglik(net3), loglik(net4)]; +hmax = collect(0:4); +R"pdf"("snaq_network_scores.pdf", width=12, height=8); +R"plot"(hmax, scores, type="b", ylab="network score", xlab="hmax", col="blue"); +R"dev.off"(); + +## Rerooting and rotating the networks for better visualization +rootatnode!(net1, "ZJ"); +rootatnode!(net2, "ZJ"); +rootatnode!(net3, "ZJ"); +rootatnode!(net4, "ZJ"); +### rotate!(net1, -2); +### rotate!(net2, -2); +### rotate!(net3, -2); +### rotate!(net4, -2); + +## Plotting the networks +R"pdf"("snaq_networks_fulltree.pdf", width=14, height=10); +R"layout(matrix(1:4, 2, 2, byrow=TRUE))"; # to get 4 plots into a single figure: 2 row, 2 columns +R"par"(mar=[0, 0, 1.5, 0]); # for smaller margins +xmin, xmax = PhyloPlots.PhyloPlots.edgenode_coordinates(net1, false, false)[13:14]; +xmax += (xmax - xmin) * 0.3; +plot(net1, showgamma=true, tipoffset=0.1, xlim=[xmin, xmax]); +R"mtext"(string("hmax=1, loglik=-", round(loglik(net1), digits=2)), font=2); +xmin, xmax = PhyloPlots.PhyloPlots.edgenode_coordinates(net2, false, false)[13:14]; +xmax += (xmax - xmin) * 0.3; +plot(net2, showgamma=true, tipoffset=0.1, xlim=[xmin, xmax]); +R"mtext"(string("hmax=2, loglik=-", round(loglik(net2), digits=2)), font=2); +xmin, xmax = PhyloPlots.PhyloPlots.edgenode_coordinates(net3, false, false)[13:14]; +xmax += (xmax - xmin) * 0.3; +plot(net3, showgamma=true, tipoffset=0.1, xlim=[xmin, xmax]); +R"mtext"(string("hmax=3, loglik=-", round(loglik(net3), digits=2)), font=2); +xmin, xmax = PhyloPlots.PhyloPlots.edgenode_coordinates(net4, false, false)[13:14]; +xmax += (xmax - xmin) * 0.3; +plot(net4, showgamma=true, tipoffset=0.1, xlim=[xmin, xmax]); +R"mtext"(string("hmax=4, loglik=-", round(loglik(net4), digits=2)), font=2); +R"dev.off"(); + +R"pdf"("snaq_networks_majortree.pdf", width=14, height=10); +R"layout(matrix(1:4, 2, 2, byrow=TRUE))"; # to get 4 plots into a single figure: 2 row, 2 columns +R"par"(mar=[0, 0, 1.5, 0]); # for smaller margins +xmin, xmax = PhyloPlots.PhyloPlots.edgenode_coordinates(net1, false, false)[13:14]; +xmax += (xmax - xmin) * 0.3; +plot(net1, style=:majortree, showgamma=true, tipoffset=0.1, xlim=[xmin, xmax]); +R"mtext"(string("hmax=1, loglik=-", round(loglik(net1), digits=2)), font=2); +xmin, xmax = PhyloPlots.PhyloPlots.edgenode_coordinates(net2, false, false)[13:14]; +xmax += (xmax - xmin) * 0.3; +plot(net2, style=:majortree, showgamma=true, tipoffset=0.1, xlim=[xmin, xmax]); +R"mtext"(string("hmax=2, loglik=-", round(loglik(net2), digits=2)), font=2); +xmin, xmax = PhyloPlots.PhyloPlots.edgenode_coordinates(net3, false, false)[13:14]; +xmax += (xmax - xmin) * 0.3; +plot(net3, style=:majortree, showgamma=true, tipoffset=0.1, xlim=[xmin, xmax]); +R"mtext"(string("hmax=3, loglik=-", round(loglik(net3), digits=2)), font=2); +xmin, xmax = PhyloPlots.PhyloPlots.edgenode_coordinates(net4, false, false)[13:14]; +xmax += (xmax - xmin) * 0.3; +plot(net4, style=:majortree, showgamma=true, tipoffset=0.1, xlim=[xmin, xmax]); +R"mtext"(string("hmax=4, loglik=-", round(loglik(net4), digits=2)), font=2); +R"dev.off"(); + +## expected vs. observed quartet concordance factors +using CSV, DataFrames, Distributions, Random, RCall; +using PhyloNetworks, SNaQ; +using QuartetNetworkGoodnessFit; +using Statistics, Printf; + +inputCFfile = joinpath("..", "bucky", "bucky_1", "input.mb.CFs.csv"); +inputCF = readtableCF(inputCFfile); +Random.seed!(123); +jitter = 0.005; +hmax_list = 0:3; + +R"pdf"("expCFs_obsvsfitted.pdf", width=6, height=6); +R"par(mfrow=c(2,2), mar=c(4,4,3,1))" +gof_results = DataFrame(h=Int[], pvalue_normal=Float64[], pvalue_empirical=Float64[], z_uncorrected=Float64[], sigma=Float64[], nsim=Int[]) + +for h in hmax_list + net = readsnaqnetwork("net$(h).out") + topologymaxQpseudolik!(net, inputCF) + + df_long = fittedquartetCF(inputCF, :long) + df_jittered = deepcopy(df_long) + df_jittered.obsCF .+= rand(Uniform(-jitter/2, jitter/2), nrow(df_long)) + df_jittered.expCF .+= rand(Uniform(-jitter/2, jitter/2), nrow(df_long)) + + res = quarnetGoFtest!(net, inputCF, false; seed=123, nsim=1000) + z_obs = res[2] + z_sim = sort!(res[6]) + p_emp = mean(z_sim .>= z_obs) # 经验 p 值：模拟 z 超过观测 z 的比例 + + # 存表时两种 p 值都留，加上模拟分布的特征备查 + push!(gof_results, (h=h, + pvalue_normal=res[1], + pvalue_empirical=p_emp, + z_uncorrected=z_obs, + sigma=res[3], + nsim=1000)) + + + ptxt = p_emp == 0 ? "p < 0.001 (emp.)" : @sprintf("p = %.3f (emp.)", p_emp) + ttl = "h = $h, $ptxt" + R"plot"(df_jittered.obsCF, df_jittered.expCF, + xlim=[0, 1.05], ylim=[0, 1.05], + xlab="quartet CF observed in gene trees", + ylab="quartet CF expected from network", + main=ttl, + pch=16, col="#008080", cex=0.5) + R"abline"(0, 1, col="#008080", lwd=0.5) +end + +R"dev.off"(); + +CSV.write("gof_results.csv", gof_results) +println(gof_results) + + +net_final = readsnaqnetwork("net0.out"); +topologymaxQpseudolik!(net_final, inputCF); +df_long = fittedquartetCF(inputCF, :long); + +### Adding jitter to the points for better visualization +df_jittered = deepcopy(df_long); +df_jittered.obsCF .+= rand(Uniform(-jitter / 2, jitter / 2), nrow(df_long)); +df_jittered.expCF .+= rand(Uniform(-jitter / 2, jitter / 2), nrow(df_long)); +df_wide = fittedquartetCF(inputCF, :wide) +println(names(df_wide)) +df_wide.outlier_p = res1[4] +outliers = sort(df_wide[df_wide.outlier_p .< 0.01, :], :outlier_p) +CSV.write("outlier_quartets_h1.csv", outliers) + + +R"plot"(df_jittered.obsCF, df_jittered.expCF, xlab="quartet CF observed in gene trees", + ylab="quartet CF expected from network", pch=16, + col="#008080", + cex=0.7); +R"abline"(0, 1, col="#008080", lwd=0.5); +R"dev.off"(); + +# Goodness of fit of the SNaQ networks +using QuartetNetworkGoodnessFit; +res1 = quarnetGoFtest!(net_final, inputCF, true; seed=123, nsim=1000); +res1[[1, 2, 3]] # p-value, uncorrected z, σ +z_obs = res1[2] +z_sim = sort!(res1[6]) +p_emp = mean(z_sim .>= z_obs) diff --git a/99.scripts_v2/utils/snaq_raw.jl b/99.scripts_v2/utils/snaq_raw.jl new file mode 100644 index 0000000..71570b3 --- /dev/null +++ b/99.scripts_v2/utils/snaq_raw.jl @@ -0,0 +1,19 @@ +#! /usr/bin/env julia + + +# Running SNaQ Analysis +using PhyloNetworks, SNaQ; +using Distributed; +addprocs(9); +@everywhere using PhyloNetworks, SNaQ; +nruns = 100; # number of runs for each hmax +astralfile = joinpath("..", "species_tree", "coal.tre"); +astraltree = readnewick(astralfile); + +inputCFfile = joinpath("..", "bucky", "bucky_1", "input.mb.CFs.csv"); +inputCF = readtableCF(inputCFfile); +net0 = snaq!(astraltree, inputCF, hmax=0, filename="net0", seed=123, outgroup="ZJ", runs=nruns); +net1 = snaq!(net0, inputCF, hmax=1, filename="net1", seed=123, outgroup="ZJ", runs=nruns); +net2 = snaq!(net1, inputCF, hmax=2, filename="net2", seed=123, outgroup="ZJ", runs=nruns); +net3 = snaq!(net2, inputCF, hmax=3, filename="net3", seed=123, outgroup="ZJ", runs=nruns); +net4 = snaq!(net3, inputCF, hmax=4, filename="net4", seed=12345, outgroup="ZJ", runs=nruns); diff --git a/pixi.lock b/pixi.lock index ad2d6f1..2a30e0b 100644 --- a/pixi.lock +++ b/pixi.lock @@ -7,1010 +7,368 @@ platforms: - 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