#!/bin/bash # # # # Annotate Organelle # # The org-annotate pipeline aims to annotate fasta files produced by assembling # genome skimming. It has been developped in the context of the PhyloAlps # (http://phyloalps.org) and of the PhyloNorway (http://phylonorway.no) projects. # # Today it is able to produce EMBL flat files suitable for submission to ENA/EBI # It provides annotation procedure for : # # - Plant chloroplast genomes. # - Nuclear rDNA Region. # # #======================================================================================== # # The template used for generating the EMBL files follows the recommendation presented # at ENA documentation website (at the date of 2021-11-04). # # https://ena-docs.readthedocs.io/en/latest/submit/fileprep/sequence-flatfile.html # #======================================================================================== # -- CAUTION -- Works as long as the script # is not called through a symlink THIS_DIR="$(dirname ${BASH_SOURCE[0]})" source "${THIS_DIR}/scripts/bash_init.sh" # # Management of options # taxid="no" normalization="yes" irdetection="yes" cdsdetection="yes" cdsdetection_pass1="yes" cdsdetection_pass2="yes" cdsdetection_pass3="yes" trnadetection="yes" rrnadetection="yes" idprefix="no" tagprefix="no" locusshift=1 organism="no" country="no" specimen="no" project="no" resetorganism="yes" types="chloro" partial=0 minlength=0 listfile="no" function usage { echo "Usage:" ; echo " $1 "'[-t|--ncbi-taxid ###] [-n|--no-normalization] \' echo ' [-i|--no-ir-detection] [-h|--help] \ ' echo ' [-o|--organism ] \ ' echo ' [-c|--chloroplast|-r|--nuclear-rdna|-m|--mitochondrion] ' echo echo "Options:" echo echo ' Defining the sequence category' echo ' -c | --chloroplast' echo ' Selects for the annotation of a chloroplast genome' echo ' This is the default mode' echo echo ' -r | --nuclear-rdna' echo ' Selects for the annotation of the rDNA nuclear cistron' echo echo ' -m | --mitochondrion' echo ' Selects for the annotation of an animal mitochondrion genome' echo echo ' Providing information about the sequence' echo ' -s | --specimen ###' echo ' Represents the specimen voucher identifier ' echo ' (e.g. for herbarium sample TROM_V_991090' echo ' will be added to the /specimen_voucher qualifier' echo echo ' -t | --ncbi-taxid ###' echo ' Represents the ncbi taxid associated to the sequence' echo echo ' -o | --organism ' echo ' Allows for specifiying the organism name in the embl generated file' echo ' Spaces have to be substituted by underscore ex : Abies_alba' echo echo ' -b | --country "[:][, ]"' echo ' Location (at least country) of collection' echo echo ' -f | --not-force-ncbi' echo ' Do not force the name of the organism to match the NCBI scientific name.' echo ' if the provided NCB taxid is publically defined' echo echo ' Information related to an ENA project' echo ' -P | --project ' echo ' The accession number of the related project in ENA' echo echo ' -i | --id-prefix ' echo ' prefix used to build the sequence identifier' echo ' the number of the contig is append to the prefix' echo ' to build the complete id. This id is used only if' echo ' an ENA project is specified.' echo echo ' -L | --locus-prefix ' echo ' Prefix used to build the locus tag of every annotated genes' echo ' generated locus tags follow the pattern : prefix_###,' echo ' where ### is a number following the order of gene in the embl file' echo ' starting at locus tag shift (default 1).' echo echo ' -S | --locus-shift <###>' echo ' Start number for building locus tags' echo echo ' --list-file ' echo ' The chomosome list file name to file for ENA submmission' echo echo ' Annotation of partial sequences' echo ' -p | --partial' echo ' Indicates that the genome sequence is partial and therefore in several contigs' echo echo ' -l | --min-length' echo ' Indicates for partial mode the minimum length of contig to annotate' echo echo ' Setting up the sequence annotation' echo ' -N | --no-normalization' echo ' Does not normalize the sequence befire annotation' echo echo ' -I | --no-ir-detection' echo ' Does not look for inverted repeats in the plastid genome' echo echo ' -C | --no-cds' echo ' Do not annotate CDSs' echo echo ' -D | --no-cds-pass1' echo ' Do not annotate core CDSs' echo echo ' -E | --no-cds-pass2' echo ' Do not annotate rps12 CDS' echo echo ' -F | --no-cds-pass3' echo ' Do not annotate shell and dust CDSs' echo echo ' -T | --no-trna' echo ' Do not look for transfert RNA' echo echo ' -R | --no-rrna' echo ' Do not look for ribosomal RNA' exit $2 } function ncbiscientificname { local efetch='https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi' local params='db=taxonomy&id='$1'&mode=text&report=xml' local url="${efetch}?${params}" curl $url \ | grep '' \ | sed 's///' \ | sed 's@@@' \ | sed 's@^ *@@' \ | sed 's@ *$@@' \ | head -1 } function split80 { local text=$1 local pattern local header if (( $# >= 2 )) ; then header=$2 else header="" fi if (( $# >= 3 )) ; then pattern=$3 else pattern=" " fi echo $text \ | $AwkCmd -v pattern="$pattern" -v header="$header" ' BEGIN { line = header } { n = split($0,parts,pattern) j = 1 for (i = 1; i <= n; i++) { if (length(line) + length(parts[i]) > 79) { print line line = header j = i } if (i > j) line = line pattern line = line parts[i] } $0 = line } END { print $0 } ' } function over_junction() { local genome_length=$1 $AwkCmd -v genome_length=$genome_length ' function split_location(location, p, status, newloc) { delete(state) delete(newloc) level = 0 state[level] = "" subloc = location newloc[level] = "" locpart = "" start = 0 pending = 0 while(length(subloc)>0) { first_letter = substr(subloc,1,1) switch (first_letter) { case "j" : print "entereing in join" > /dev/stderr subloc = substr(subloc,6) level++ state[level] = "join" break case "c" : print "entereing in complement" > /dev/stderr subloc = substr(subloc,12) level++ state[level] = "complement" break case "," : print "next exon" > /dev/stderr subloc = substr(subloc,2) newloc[level] = newloc[level] "," break case ")" : print "Ending "state[level] > /dev/stderr subloc = substr(subloc,2) level-- newloc[level] = newloc[level] state[level+1] "(" newloc[level+1] ")" if (pending == 1) { level-- newloc[level] = newloc[level] state[level+1] "(" newloc[level+1] ")" } break default : print "Matching location: " subloc > /dev/stderr p = match(subloc,/[0-9]+\.\.[0-9]+/) piece = substr(subloc,RSTART,RLENGTH) subloc= substr(subloc,RLENGTH+1) p = match(piece,/[0-9]+/) from = substr(piece,RSTART,RLENGTH) piece = substr(piece,RLENGTH+1) p = match(piece,/[0-9]+/) to = substr(piece,RSTART,RLENGTH) if ((genome_length+0 >= from+0) && (genome_length+0 <= to+0)) { status = "overlap" level++ state[level] = "join" pending = 1 piece = from ".." genome_length ",1.."(to - genome_length) } else { status = "out" } newloc[level] = newloc[level] piece print p, RSTART, from, to, status, piece > /dev/stderr break } } return newloc[0] } function cut_location(location) { pos = match(location,/,[^,]*$/) right = "" if (pos > 80) { while (pos > 80) { right = substr(location,pos+1) right location = substr(location,1,pos) pos = match(location,/,[^,]*,$/) } if (pos > 0) { right = substr(location,pos+1) right location = substr(location,1,pos) } } if (right !="") { right = "\nFT " right if (length(right) > 80) { right = cut_location(right) } location = location right } return location } /^FT [^ ]/ && (feature != "") { if (status != "remove") { if (status == "overlap") { print cut_location(fttype location) print "FT /note=\"" fttype " crosses the IR boundary\"" } else { print cut_location(fttype location) } print feature } feature = "" fttype = "" position= "" } (feature != "") { feature = feature "\n" $0 } # # Begining of a feature # /^FT [^ ]/ { fttype=$2 plocation=$3 fttype=substr($0,1,21) } # # Following of a location # /^FT +[^ \/]/ && (plocation != "") { plocation = plocation $2 } # # End of the location # And begining of the feature description # /^FT +\// && (plocation != "") { feature = $0 location = plocation split(location,parts,/\.\./) delete p pmin=1000000000 pmax=0 for (i in parts) { pos = match(parts[i],/[0-9]+/) if (pos+0 > 0) { j++ p[i] = substr(parts[i], RSTART , RLENGTH) if (p[i]+0 > pmax+0) { pmax = p[i]} if (p[i]+0 < pmin+0) { pmin = p[i]} } } status = "ok" if (pmin+0 > genome_length+0) { status = "remove" } else { if (pmax+0 > genome_length+0) { status = "overlap" location = split_location(location) } } plocation="" } END { if (status != "remove") { if (status == "overlap") { print fttype location print "FT /note=\"CDS crosses the IR boundary\"" } else { print fttype location } print feature } } ' $2 } function fastaIterator() { $AwkCmd '/^>/ {if (seq) printf("%s\f",seq); seq=""} \ {if (seq) seq=seq"\n"; seq=seq $1} \ END {print seq}' "$1" } # options may be followed by one colon to indicate they have a required argument if ! options=$(getopt -o s:t:o:b:P:i:fcrmhpl:NICDEFTRL:S: -l specimen:,ncbi-taxid:,organism:,country:,project:,id-prefix:,not-force-ncbi,chloroplast,nuclear-rdna,mitochondrion,partial,min-length:,help,no-normalization,no-ir-detection,no-cds,no-cds-pass1,no-cds-pass2,no-cds-pass3,no-trna,no-rrna,locus-prefix:,locus-shift:,list-file: -- "$@") then # something went wrong, getopt will put out an error message for us usage $0 1 fi eval set -- "$options" while [ $# -gt 0 ] do case $1 in -c|--chloroplast) types="chloro" ;; -r|--nuclear-rdna) types="nucrdna" ;; -m|--mitochondrion) types="mito" ;; -t|--ncbi-taxid) taxid="$2" ; shift ;; -s|--specimen) specimen="$2" ; shift ;; -b|--country) country="$2" ; shift ;; -o|--organism) organism="$2" ; shift ;; -P|--project) project="$2" ; shift ;; -i|--id-prefix) idprefix="$2" ; shift ;; -L|--locus-prefix) tagprefix="$2" ; shift ;; -S|--locus-shift) locusshift="$2" ; shift ;; -f|--not-force-ncbi) resetorganism="no" ;; -p|--partial) partial="1" ;; -l|--min-length) minlength="$2" ; shift ;; -h|--help) usage $0 0;; -N|--no-normalization) normalization="no" ;; -I|--no-ir-detection) irdetection="no" ;; -C|--no-cds) cdsdetection="no";; -D|--no-cds-pass1) cdsdetection_pass1="no";; -E|--no-cds-pass2) cdsdetection_pass2="no";; -F|--no-cds-pass3) cdsdetection_pass3="no";; -T|--no-trna) trnadetection="no";; -R|--no-rrna) rrnadetection="no";; --list-file) listfile="$2" ; shift;; (--) shift; break;; (-*) echo "$0: error - unrecognized option $1" 1>&2; exit 1;; (*) break;; esac shift done loginfo "Locus tag prefix provided: $tagprefix" loginfo "Locus tag numbered from..: $locusshift" loginfo "NCBI taxid provided......: $taxid" if [[ "$listfile" != "no" ]] ; then loginfo "contig info saved in.....: $listfile" fi if [[ "$taxid" != "no" ]] ; then scientificname=$(ncbiscientificname $taxid) loginfo "NCBI scientific name.....: $scientificname" if [[ -z "$scientificname" ]] ; then loginfo " Unknown taxid." else loginfo "Organism name from taxid.: $resetorganism" if [[ "$resetorganism" == "yes" ]] ; then organism=$(echo $scientificname | tr ' ' '_') fi fi fi loginfo "Annotating mode..........: $types" loginfo "Sequence normalization...: $normalization" loginfo "IR detection mode........: $irdetection" loginfo "CDS detection mode.......: $cdsdetection" loginfo " pass 1........: $cdsdetection_pass1" loginfo " pass 2........: $cdsdetection_pass2" loginfo " pass 3........: $cdsdetection_pass3" loginfo "tRNA detection mode......: $trnadetection" loginfo "rRNA detection mode......: $rrnadetection" loginfo "Organism.................: $organism" loginfo "Country..................: $country" loginfo "Partial mode.............: $partial" loginfo "Minimum length...........: $minlength" ############################# pushTmpDir ORG.organnot if [[ ! "$1" =~ ^/ ]]; then QUERY="${CALL_DIR}/$1" else QUERY="$1" fi RESULTS=$(basename ${QUERY/.*/}) LOG="${CALL_DIR}/${RESULTS}.log" rm -f ${LOG} openLogFile ${LOG} IFS=$'\f' sequence_number=0 for sequence in $(fastaIterator "${QUERY}") ; do let sequence_number=sequence_number+1 unset IFS if [[ ! -z "${sequence}" ]] ; then echo "${sequence}" > toannotate.fasta sl=$(seqlength "toannotate.fasta") if (( sl >= minlength )) ; then loginfo "Annotated genome length: $sl bp" seqid=$($AwkCmd '(NR==1) {print substr($1,2,1000)}' toannotate.fasta) # seqid=$(tr "." "_" <<< ${seqid}) case "$types" in chloro) loginfo "Annotating a plant chloroplast genome..." if [[ "$irdetection" == "yes" ]] && (( partial == 0 )) ; then if [[ "$normalization" == "yes" ]] ; then loginfo "Normalizing the structure of the Chloroplast sequence..." loginfo " LSC + IRB + SSC + IRA" ${PROG_DIR}/detectors/normalize/bin/go_normalize.sh toannotate.fasta > "${RESULTS}.norm.fasta" loginfo "Done." else loginfo "No normalization the structure of the Chloroplast sequence..." cat toannotate.fasta > "${RESULTS}.norm.fasta" fi loginfo "Annotating the Inverted repeats and Single copies (LSC and SSC)..." ${PROG_DIR}/detectors/ir/bin/go_ir.sh "${RESULTS}.norm.fasta" > "${RESULTS}.annot" loginfo "Done." else loginfo "No normalization of the structure of the Chloroplast sequence..." cat toannotate.fasta > "${RESULTS}.norm.fasta" rm -f "${RESULTS}.annot" touch "${RESULTS}.annot" fi # # We are annotating a circular sequence # # 2kb of the beginnig of the sequence is added to its end # to allow for overlaping feature # add_circular_extension=0 if (( partial == 0 )) ; then loginfo "Extends the sequence to allows for features overlaping juction" cp "${RESULTS}.norm.fasta" "${RESULTS}.norm.orig.fasta" cp "${RESULTS}.norm.fasta" "${RESULTS}.norm.frgs.fasta" cutseq "${RESULTS}.norm.orig.fasta" 1 2000 >> "${RESULTS}.norm.frgs.fasta" joinfasta "${RESULTS}.norm.frgs.fasta" > "${RESULTS}.norm.fasta" add_circular_extension=1 fi if [[ "$trnadetection" == "yes" ]] ; then loginfo "Annotating the tRNA..." ${PROG_DIR}/detectors/trna/bin/go_trna.sh "${RESULTS}.norm.fasta" "11" >> "${RESULTS}.annot" loginfo "Done." fi if [[ "$rrnadetection" == "yes" ]] ; then loginfo "Annotating the rRNA genes..." ${PROG_DIR}/detectors/rrna/bin/go_rrna.sh "${RESULTS}.norm.fasta" >> "${RESULTS}.annot" loginfo "Done." fi if [[ "$cdsdetection" == "yes" ]] ; then loginfo "Annotating the CDS..." cdsdetection_pass1=$cdsdetection_pass1 \ cdsdetection_pass2=$cdsdetection_pass2 \ cdsdetection_pass3=$cdsdetection_pass3 \ ${PROG_DIR}/detectors/cds/bin/go_cds.sh "${RESULTS}.norm.fasta" "$sl" >> "${RESULTS}.annot" loginfo "Done." fi if (( add_circular_extension == 1 )) ; then mv "${RESULTS}.norm.orig.fasta" "${RESULTS}.norm.fasta" cp "${RESULTS}.annot" "${CALL_DIR}/${RESULTS}.annot.circular" over_junction $sl "${RESULTS}.annot" > "${RESULTS}.circular.annot" mv "${RESULTS}.circular.annot" "${RESULTS}.annot" add_circular_extension=0 fi if (( partial == 0 )) ; then topology="circular" defline="plastid, complete genome" else topology="linear" defline="plastid, partial sequence" fi if [[ "$listfile" != "no" ]] ; then if (( partial == 0 )) ; then echo "${seqid} CHL Circular-Chromosome Chloroplast" >> ${CALL_DIR}/$listfile else echo "${seqid} CHL" >> ${CALL_DIR}/$listfile fi fi notAnnoted "${RESULTS}.annot" "${RESULTS}.norm.fasta" 100 > ${CALL_DIR}/not_annotated.fasta ;; nucrdna) loginfo "Annotating a plant rDNA cistron..." if [[ "$normalization" == "yes" ]] ; then loginfo "Normalizing the structure of the cistron sequence..." ${PROG_DIR}/detectors/normalizerdna/bin/go_normalizerdna.sh toannotate.fasta > "${RESULTS}.norm.fasta" loginfo "Done." else loginfo "No normalization of the structure of the cistron sequence..." cat toannotate.fasta > "${RESULTS}.norm.fasta" fi loginfo "Annotating the rRNA genes..." ${PROG_DIR}/detectors/nucrrna/bin/go_nucrrna.sh "${RESULTS}.norm.fasta" > "${RESULTS}.annot" loginfo "Done." topology="linear" defline=$(cat "${RESULTS}.annot" \ | grep "/gene=" \ | sed -E 's@^FT */gene="([^"]*)".*$@\1@' \ | sort -u \ | $AwkCmd '{printf("%s;",$0)}' \ | $AwkCmd 'BEGIN {i=1} /18S rRNA;/ {gene[i]="18S rRNA"; i++} /ITS1;/ {gene[i]="ITS1"; i++} /5.8S rRNA;/ {gene[i]="5.8S rRNA"; i++} /ITS2;/ {gene[i]="ITS2"; i++} /28S rRNA;/ {gene[i]="28S rRNA"; ii++} END { for (i=1;i <= length(gene); i++) { separator ="" if (i < (length(gene)-1)) separator=", " if (i == (length(gene)-1)) separator=" and " printf("%s gene%s",gene[i],separator) } }') # defline="18S rRNA gene, ITS1, 5.8S rRNA gene, ITS2 and 28S rRNA gene" ;; mito) loginfo "Annotating an animal mitochondrial genome..." # logerror "Not yet implemented" if (( partial == 0 )) ; then topology="circular" defline="mitochondrion, complete genome" else topology="linear" defline="mitochondrion, partial sequence" fi loginfo "No normalization of the structure of the Mitochondrial sequence..." cat toannotate.fasta > "${RESULTS}.norm.fasta" rm -f "${RESULTS}.annot" touch "${RESULTS}.annot" # # We are annotating a circular sequence # # 2kb of the beginnig of the sequence is added to its end # to allow for overlaping feature # add_circular_extension=0 if (( partial == 0 )) ; then loginfo "Extends the sequence to allows for features overlaping juction" cp "${RESULTS}.norm.fasta" "${RESULTS}.norm.orig.fasta" cp "${RESULTS}.norm.fasta" "${RESULTS}.norm.frgs.fasta" cutseq "${RESULTS}.norm.orig.fasta" 1 2000 >> "${RESULTS}.norm.frgs.fasta" joinfasta "${RESULTS}.norm.frgs.fasta" > "${RESULTS}.norm.fasta" add_circular_extension=1 fi if [[ "$trnadetection" == "yes" ]] ; then loginfo "Annotating the tRNA..." ${PROG_DIR}/detectors/trna/bin/go_trna.sh "${RESULTS}.norm.fasta" "vert" >> "${RESULTS}.annot" loginfo "Done." fi if [[ "$rrnadetection" == "yes" ]] ; then loginfo "Annotating the rRNA genes..." ${PROG_DIR}/detectors/rrna/bin/go_rrna.sh "${RESULTS}.norm.fasta" >> "${RESULTS}.annot" loginfo "Done." fi if (( add_circular_extension == 1 )) ; then mv "${RESULTS}.norm.orig.fasta" "${RESULTS}.norm.fasta" cp "${RESULTS}.annot" "${CALL_DIR}/${RESULTS}.annot.circular" over_junction $sl "${RESULTS}.annot" > "${RESULTS}.circular.annot" mv "${RESULTS}.circular.annot" "${RESULTS}.annot" add_circular_extension=0 fi if (( partial == 0 )) ; then topology="circular" defline="mitochondrion, complete genome" else topology="linear" defline="mitochondrion, partial sequence" fi ;; *) usage $0 1;; esac if [[ "${organism}" == "no" ]]; then organism="{organism}" else organism="$(echo ${organism} | tr '_' ' ')" fi if [[ "$specimen" != "no" ]] ; then defline="${defline}, voucher ${specimen}" fi sl=$(seqlength "${RESULTS}.norm.fasta") loginfo "Printing minimal header..." echo "ID XXX; XXX; ${topology}; genomic DNA; XXX; XXX; ${sl} BP." echo "XX" echo "AC XXX;" echo "XX" if [[ "${project}" != "no" ]] ; then if [[ "$idprefix" != "no" ]] ; then seqid="${idprefix}${sequence_number}" fi echo "AC * _${seqid};" echo "XX" echo "PR Project:${project};" echo "XX" echo "DE XXX" else split80 "${organism} ${defline}." "DE " fi echo "XX" loginfo "Done." loginfo "Printing annotations header..." echo "FH Key Location/Qualifiers" loginfo "Done." loginfo "Printing the source feature" echo "FT source 1..${sl}" if [[ "${organism}" != "no" ]] ; then echo "FT /organism=\"${organism}\"" fi case "${types}" in chloro) echo "FT /organelle=\"plastid:chloroplast\"" ;; mito) echo "FT /organelle=\"mitochondrion\"" ;; *) loginfo "Nuclear sequence" ;; esac echo "FT /mol_type=\"genomic DNA\"" if [[ "${specimen}" != "no" ]] ; then echo "FT /specimen_voucher=\"${specimen}\"" fi if [[ "${taxid}" != "no" ]] ; then echo "FT /db_xref=\"taxon:${taxid}\"" fi if [[ "${country}" != "no" ]] ; then echo "FT /country=\"${country}\"" fi loginfo "Done." loginfo "Ordering annotations..." $AwkCmd '(entry && /^.....(misc|repeat|rRNA|tRNA|CDS|source)/) { \ print pos,entry } \ /^.....(misc|repeat|rRNA|tRNA|CDS|source)/ { \ match($3,"[0-9][0-9]*"); \ pos=substr($3,RSTART,RLENGTH)*1000 + 1; \ entry=$0; \ next} \ { entry=entry "@" $0} \ END {print pos,entry}' "${RESULTS}.annot" | \ sort -nk1 |\ $AwkCmd '{ \ match($0,"^[0-9]* ");\ line=substr($0,RLENGTH+1);\ gsub("@","\n",line); \ print line}' > "${RESULTS}.sorted.annot" loginfo "Done." loginfo "Unifying gene names" $AwkCmd ' (FNR==NR) && /^FT \/gene="/ { gene = substr($0,29,100) gene = substr(gene,0,length(gene)-1) occurrence[gene]++ } (FNR==1) && (FNR!=NR) { for(gene in occurrence){ if (occurrence[gene]==1) { delete occurrence[gene] } else { occurrence[gene] = 1 } } } (FNR!=NR) && /^FT \/gene="/ { gene = substr($0,29,100) gene = substr(gene,0,length(gene)-1) n = occurrence[gene] if (n > 0) { $0="FT /gene=\""gene"_"n"\"" occurrence[gene]++ } } (FNR!=NR) { print $0 } ' "${RESULTS}.sorted.annot" "${RESULTS}.sorted.annot" \ > "${RESULTS}.uniq_gene.annot" loginfo "Done." if [[ "$tagprefix" != "no" ]] ; then loginfo "Adding locus tags from number: $locusshift..." cat "${RESULTS}.uniq_gene.annot" \ | $AwkCmd -v tagprefix="$tagprefix" \ -v locusshift="$locusshift" ' /^FT +\/locus_tag=""/ { sub(/locus_tag=""/,"locus_tag=\""tagprefix"_"locusshift"\"",$0); locusshift++; } { print $0 } ' loginfo "Locus tags done." else loginfo "Clearing locus tags done." egrep -v '^FT +\/locus_tag=""' \ "${RESULTS}.uniq_gene.annot" loginfo "Clearing of tags done." fi loginfo "Closing annotations table..." echo "XX" loginfo "Done." loginfo "Computing statistics on nucleotide usage..." $AwkCmd '! /^>/ { \ seq=toupper($0); \ gsub(" ","",seq); \ lseq=length(seq); \ for (i=0; i < lseq; i++) { \ freq[substr(seq,i,1)]++}\ } \ END { \ other=0; \ for (i in freq) { \ if (i!="A" && i!="C" && i!="G" && i!="T") {\ other+=freq[i] \ } \ }; \ print "SQ Sequence "\ (freq["A"]+freq["C"]+freq["G"]+freq["T"]+other) \ " BP; "\ freq["A"]" A; "\ freq["C"]" C; "\ freq["G"]" G; "\ freq["T"]" T; "\ other" other;" \ }' "${RESULTS}.norm.fasta" loginfo "Done." loginfo "Reformating sequences..." lines=$(wc -l "${RESULTS}.norm.fasta" | $AwkCmd '{print $1}') loginfo "Sequence length $(seqlength ${RESULTS}.norm.fasta)" loginfo "lines $lines" formatfasta "${RESULTS}.norm.fasta" | \ $AwkCmd -v lines=$lines ' \ ! /^>/ { \ seq=tolower($0); \ gsub(" ","",seq); \ printf(" ") ;\ for (i=0; i < 6; i++) { \ f=substr(seq,i * 10 + 1, 10); \ pos+=length(f); \ f = f substr(" ",1,10-length(f)); \ printf("%s ",f) \ }; \ printf(" %6d\n",pos) \ }' loginfo "Done." loginfo "Closing sequence part..." echo "//" loginfo "Done." fi # End of the minimum length condition fi # End of not empty sequence condition IFS=$'\f' done # End of the loop over the sequences popTmpDir loginfo "Annotation done."