A workflow to count genes, duplicates, singletons and TAG

workflow/families_and_TAGs/input.csv

@@ -0,0 +1,10 @@
+species,proteome,gff3,blastp
+Glycine max,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Glycine_max.Glycine_max_v2.1.pep.all.fa.gz,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Glycine_max.Glycine_max_v2.1.60.chr.gff3.gz,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Glycine_max_Blastp_longIsoforme
+Gossypium raimondii,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Gossypium_raimondii.Graimondii2_0_v6.pep.all.fa.gz,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Gossypium_raimondii.Graimondii2_0_v6.60.gff3.gz,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Gossypium_raimondii_Blastp_longIsoforme
+Malus domestica Golden,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Malus_domestica_golden.ASM211411v1.pep.all.fa.gz,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Malus_domestica_golden.ASM211411v1.60.gff3.gz,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Malus_domestica_golden_Blastp_longIsoforme2
+Musa acuminata,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Musa_acuminata.Musa_acuminata_v2.pep.all.fa.gz,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Musa_acuminata.Musa_acuminata_v2.60.gff3.gz,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Musa_acuminata_Blastp_longIsoforme
+Oryza sativa,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Oryza_sativa.IRGSP-1.0.pep.all.fa.gz,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Oryza_sativa.IRGSP-1.0.60.gff3.gz,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Oryza_sativa_Blastp_longIsoforme
+Prunus persica,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Prunus_persica.Prunus_persica_NCBIv2.pep.all.fa.gz,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Prunus_persica.Prunus_persica_NCBIv2.60.gff3.gz,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Prunus_persica_Blastp_longIsoforme
+Solanum tuberosum,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Solanum_tuberosum.SolTub_3.0.pep.all.fa.gz,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Solanum_tuberosum.SolTub_3.0.60.gff3.gz,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Solanum_tuberosum_Blastp_longIsoforme
+Vigna radiata,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Vigna_radiata.Vradiata_ver6.pep.all.fa.gz,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Vigna_radiata.Vradiata_ver6.60.gff3.gz,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Vigna_radiata_Blastp_longIsoforme
+Theobroma cacao,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Theobroma_cacao.Theobroma_cacao_20110822.pep.all.fa.gz,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Theobroma_cacao.Theobroma_cacao_20110822.60.gff3.gz,/media/data/sync/Documents/course/master/M2/S1/comparative_genomics/data/Theobroma_cacao_Blastp_longIsoforme

workflow/families_and_TAGs/modules/clustering.nf

@@ -36,7 +36,7 @@ process MCL {
 
     script:
     """
-    mcl "${abc}" --abc -o 'custering.mcl'
+    mcl "${abc}" --abc -o 'clustering.mcl'
     """
 }
 

workflow/families_and_TAGs/modules/filter_blastp.nf

@@ -1,6 +1,23 @@
 /** Filter blastp output based on coverage and identity percentage
 /**/
 
+process REMOVE_SUPERCONTIGS_CHLOROPLASTS_AND_MITOCHONDRIA {
+    input:
+    path proteome
+    path blastp
+    output:
+    path 'blastp_no_supercontigs_chloroplast_nor_mitochondria.tsv'
+    script:
+    """
+    awk -f "${baseDir}/scripts/black_gene_list.awk" "${proteome}" > 'blacklist.list'
+    if [ ! -s 'blacklist.list' ]; then
+        cp "${blastp}" 'blastp_no_supercontigs_chloroplast_nor_mitochondria.tsv'
+    else
+        awk -f "${baseDir}/scripts/filter_blastp_sequence_id_remove.awk" 'blacklist.list' "${blastp}" > 'blastp_no_supercontigs_chloroplast_nor_mitochondria.tsv'
+    fi
+    """
+}
+
 process REMOVE_LOOPS {
 
     input:
@@ -12,7 +29,7 @@ process REMOVE_LOOPS {
     script:
 
     """
-    awk 'BEGIN { FS=OFS="\t" }  \$1 != \$2 { print \$0 }'
+    awk 'BEGIN { FS=OFS="\t" }  \$1 != \$2 { print \$0 }' "${blastp}" > 'noloops.blastp.tsv'
     """
 }
 
@@ -27,11 +44,11 @@ process ADD_GENE_ID_AND_PROTEIN_LENGTHS {
 
     script:
     """
-    sort -k 1 "${blastp}" > blastp_s
+    LANG=en_EN sort -t \$'\t' -k 1 "${blastp}" > blastp_s
-    sort -k 1 "${protein_length}" > protein_length_s
+    LANG=en_EN sort -t \$'\t' -k 1 "${protein_length}" > protein_length_s
-    join -1 1 -2 1 -t \$'\t' blastp_s protein_length_s > join1.tsv
+    LANG=en_EN join -1 1 -2 1 -t \$'\t' blastp_s protein_length_s > join1.tsv
-    sort -k 2 join1.tsv > join1.tsv_s
+    LANG=en_EN sort -t \$'\t' -k 2 join1.tsv > join1.tsv_s
-    join -1 2 -2 1 -t \$'\t' join1.tsv_s protein_length_s > joined.blastp.tsv
+    LANG=en_EN join -1 2 -2 1 -t \$'\t' join1.tsv_s protein_length_s > joined.blastp.tsv
     """
 
 }
@@ -39,9 +56,9 @@ process ADD_GENE_ID_AND_PROTEIN_LENGTHS {
 process FILTER_COVERAGE_IDENTITY_BLASTP {
 
     input:
+    path blastp
     val min_coverage
     val min_identity
-    path blastp
 
     output:
     path 'filtered.blastp.tsv'
@@ -58,15 +75,17 @@ process FILTER_COVERAGE_IDENTITY_BLASTP {
 workflow FILTER_BLASTP {
     take:
     blastp
+    proteome
     protein_length
     min_coverage
     min_identity
 
     main:
-    REMOVE_LOOPS(blastp)
+    REMOVE_SUPERCONTIGS_CHLOROPLASTS_AND_MITOCHONDRIA(proteome, blastp)
-    ADD_GENE_ID_AND_PROTEIN_LENGTHS(REMOVE_LOOPS.out)
+    REMOVE_LOOPS(REMOVE_SUPERCONTIGS_CHLOROPLASTS_AND_MITOCHONDRIA.out)
-    FILTER_COVERAGE_IDENTITY_BLASTP(min_identity, min_coverage, ADD_GENE_ID_AND_PROTEIN_LENGTHS.out)
+    ADD_GENE_ID_AND_PROTEIN_LENGTHS(REMOVE_LOOPS.out, protein_length)
+    FILTER_COVERAGE_IDENTITY_BLASTP(ADD_GENE_ID_AND_PROTEIN_LENGTHS.out, min_identity, min_coverage)
 
     emit:
-    blastp=FILTER_COVERAGE_IDENTITY_BLASTP
+    blastp=FILTER_COVERAGE_IDENTITY_BLASTP.out
 }

workflow/families_and_TAGs/modules/filter_fasta.nf

@@ -1,16 +1,14 @@
-/** 1. Filter out proteome sequence belonging to 'supercontig' */
+/** 1. Filter out proteome sequence belonging to 'supercontig', Mt, or Pt */
 /** 2. Filter proteome to keep only the longest isoform */
 
-process FILTER_SUPERCONTIG {
+process REMOVE_SUPERCONTIGS_CHLOROPLASTS_AND_MITOCHONDRIA {
     input:
     path proteome
-
     output:
-    path 'proteome_nosupercontig.fasta'
+    path 'filtered_proteome.fasta'
-
     script:
     """
-    awk -f "${baseDir}/scripts/remove_supercontigs.awk" "${proteome}" > 'proteome_nosupercontig.fasta'
+    awk -f "${baseDir}/scripts/remove_supercontigs_chloroplasts_mitochondria_fasta.awk" "${proteome}" > 'filtered_proteome.fasta'
     """
 }
 
@@ -42,14 +40,16 @@ process PROTEOME_LONGEST {
     """
 }
 
+
+
 workflow FILTER_FASTA {
     take:
     proteome
 
     main:
-    FILTER_SUPERCONTIG(proteome)
+    REMOVE_SUPERCONTIGS_CHLOROPLASTS_AND_MITOCHONDRIA(proteome)
-    SEQUENCE_LENGTH(FILTER_SUPERCONTIG.out)
+    SEQUENCE_LENGTH(REMOVE_SUPERCONTIGS_CHLOROPLASTS_AND_MITOCHONDRIA.out)
-    PROTEOME_LONGEST(FILTER_SUPERCONTIG.out, SEQUENCE_LENGTH.out)
+    PROTEOME_LONGEST(REMOVE_SUPERCONTIGS_CHLOROPLASTS_AND_MITOCHONDRIA.out, SEQUENCE_LENGTH.out)
 
     emit:
     lengths = SEQUENCE_LENGTH.out

workflow/families_and_TAGs/modules/tags.nf

@@ -9,7 +9,7 @@ process TAG_FINDER {
 
     script:
     """
-    "./${baseDir}/../../rust/tagfinder/target/release/tagfinder" --positions "${positions}" --families "${families}" > 'tags.tsv'
+    "${baseDir}/../../rust/tagfinder/target/release/tagfinder" --positions "${positions}" --families "${families}" --definitions 0 > 'tags.tsv'
     """
 }
 
@@ -32,8 +32,8 @@ process GENE_POSITION_TABLE {
 workflow TAGs {
 
     take:
-    gff3
     families
+    gff3
 
     main:
     GENE_POSITION_TABLE(gff3)

workflow/families_and_TAGs/run_all.sh

@@ -0,0 +1,10 @@
+#!/usr/bin/env bash
+set -euo pipefail
+tail -n +2 input.csv | while IFS= read -r line; do
+    species="$(echo $line | cut -d ',' -f 1)"
+    proteome="$(echo $line | cut -d ',' -f 2)"
+    gff3="$(echo $line | cut -d ',' -f 3)"
+    blastp="$(echo $line | cut -d ',' -f 4)"
+    echo Launching for $species
+    nextflow run run_one.nf -resume -profile conda --species "$species" --proteome "$proteome" --gff3 "$gff3" --blastp "$blastp"
+done

workflow/families_and_TAGs/run_one.nf

@@ -0,0 +1,46 @@
+/** Run the workflow for all species in the csv table */
+
+include { GUNZIP as GUNZIP_1 } from "./modules/gunzip.nf"
+include { GUNZIP as GUNZIP_2 } from "./modules/gunzip.nf"
+include { FILTER_FASTA } from "./modules/filter_fasta.nf"
+include { FILTER_BLASTP } from "./modules/filter_blastp.nf"
+include { CLUSTERING } from "./modules/clustering.nf"
+include { TAGs } from "./modules/tags.nf"
+
+
+
+process CSV_ROW {
+    publishDir "results/", mode: 'copy'
+    input:
+    val(species)
+    path(proteome)
+    path(tags)
+    path(families)
+    output:
+    path "${species}.csv"
+    script:
+    """
+    nb_tag=\$(tail -n +2  "${tags}"| awk 'BEGIN { max = 0 } { if (\$3 != "-" && \$3 > max) max = \$3 } END { print max }')
+    nb_families=\$(tail -1 "${families}" | awk '{ print \$2 }')
+    nb_duplicates=\$(cat "${families}" | wc -l)
+    nb_genes=\$(awk '/^>/ { print \$4 }' "${proteome}" | sort | uniq | wc -l)
+    nb_singletons=\$((nb_genes - nb_duplicates + 1))
+    echo "${species},\${nb_genes},\${nb_duplicates},\${nb_singletons},\${nb_families},\${nb_tag}" > "${species}.csv"
+    """
+}
+
+
+
+workflow {
+    proteome = file(params.proteome)
+    gff3 = file(params.gff3)
+    blastp = file(params.blastp)
+
+    GUNZIP_1(proteome)
+    GUNZIP_2(gff3)
+    FILTER_FASTA(GUNZIP_1.out)
+    FILTER_BLASTP(blastp, GUNZIP_1.out, FILTER_FASTA.out.lengths, params.min_coverage, params.min_identity)
+    CLUSTERING(FILTER_BLASTP.out.blastp)
+    TAGs(CLUSTERING.out, GUNZIP_2.out)
+    CSV_ROW(params.species, GUNZIP_1.out, TAGs.out, CLUSTERING.out)
+}

workflow/families_and_TAGs/scripts/black_gene_list.awk

@@ -0,0 +1,12 @@
+#!/usr/bin/env -S awk -f
+
+/^>/ {
+    locus=$3
+    split(":", arr, locus)
+    chromosome=arr[3]
+    if (chromosome == "Mt" || chromosome == "Pt" || chromosome ~ /supercontig/) {
+        record_id=$1
+        gsub(">", "", record_id)
+        print record_id
+    }
+}

workflow/families_and_TAGs/scripts/filter_blastp_sequence_id.awk

@@ -9,13 +9,13 @@
 
 NR == FNR {
     sequence_id = $1
-    to_remove[sequence_id] = 1
+    kept[sequence_id] = 1
     next
 }
 
 {
     sequence_id = $1
-    if (!(sequence_id in to_remove)) {
+    if (sequence_id in kept || $2 in kept) {
         print $0
     }
 }

workflow/families_and_TAGs/scripts/filter_blastp_sequence_id_remove.awk

@@ -0,0 +1,25 @@
+#!/usr/bin/env -S awk -f
+# Filter BLASTp format 6 file
+# to keep only the records
+# with no ID in records.list
+#
+# Usage:
+# awk -f filter_blastp_sequence_id.awk \
+# records.list records.blastp.tsv
+
+BEGIN {
+    FS="\t"
+    OFS="\t"
+}
+
+NR==FNR {
+    sequence_id=$1
+    kept[sequence_id]=1
+    next
+}
+
+{
+    if (!($1 in kept) && !($2 in kept)) {
+        print $0
+    }
+}

workflow/families_and_TAGs/scripts/filter_records_fasta.awk

@@ -1,10 +1,10 @@
 #!/usr/bin/env -S awk -f
 # Filter FASTA file
-# to keep only the records
+# to keep or remove only the records
 # with ID in records.list
 #
 # Usage:
-# awk -f filter_record_fasta.awk \
+# awk -f (-v direction=keep|remove) filter_record_fasta.awk \
 # records.list records.fasta
 
 BEGIN {
@@ -22,9 +22,16 @@ NR == FNR {
     sequence_id = $1
     gsub(">", "", sequence_id)
     if (sequence_id in kept) {
+        if (direction == "keep") {
             print $0
             on_good_record = 1
         } 
+    } else {
+        if (direction == "remove") {
+            print $0
+            on_good_record = 1
+        }
+    }
 }
 
 /^[^>]/ && on_good_record {

workflow/families_and_TAGs/scripts/remove_supercontigs.awk

@@ -1,25 +0,0 @@
-#!/usr/bin/env -S awk -f
-# Filter out 'supercontigs' records
-# from an Ensembl proteome fasta file
-# Usage:
-# awk -f remove_supercontigs.awk \
-# "proteome.fasta" \
-# > "proteome_nosupercontigs.fasta"
-
-BEGIN {
-    on_good_sequence = 0
-}
-
-/^>/ {
-    on_good_sequence = 0
-    locus = $3
-    split(locus, locus_array, ":")
-    if (locus_array[1] != "supercontig") {
-        on_good_sequence = 1
-        print $0
-    }
-}
-
-/^[^>]/ && on_good_sequence {
-    print $0
-}

workflow/families_and_TAGs/scripts/remove_supercontigs_chloroplasts_mitochondria_fasta.awk

@@ -0,0 +1,21 @@
+#!/usr/bin/env -S awk -f
+
+BEGIN {
+    on_good_sequence = 0
+}
+
+/^>/ {
+    locus=$3
+    split(locus, arr, ":")
+    chromosome=arr[3]
+    if (chromosome == "Mt" || chromosome == "Pt" || chromosome ~ /supercontig/) {
+        on_good_sequence = 0
+    } else {
+        on_good_sequence = 1
+        print $0
+    }
+}
+
+/^[^>]/ && on_good_sequence == 1 {
+    print $0
+}