#!/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" trnadetection="yes" rrnadetection="yes" idprefix="no" organism="no" country="no" specimen="no" project="no" resetorganism="yes" types="chloro" partial=0 minlength=0 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 ' 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 CDS' echo echo ' -D | --no-cds-pass1' echo ' Do not annotate core CDS' echo echo ' -E | --no-cds-pass2' echo ' Do not annotate rps12 CDS' 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 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:NICDETR -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-trna,no-rrna -- "$@") 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 ;; -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";; -T|--no-trna) trnadetection="no";; -R|--no-rrna) rrnadetection="no";; (--) shift; break;; (-*) echo "$0: error - unrecognized option $1" 1>&2; exit 1;; (*) break;; esac shift done loginfo "NCBI taxid provided......: $taxid" if [[ "$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 "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=$(seqlength "toannotate.fasta") if (( sl >= minlength )) ; then seqid=$($AwkCmd '(NR==1) {print substr($1,2,1000)}' toannotate.fasta) case "$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 if [[ "$trnadetection" == "yes" ]] ; then loginfo "Annotating the tRNA..." ${PROG_DIR}/detectors/trna/bin/go_trna.sh "${RESULTS}.norm.fasta" >> "${RESULTS}.annot" loginfo "Done." fi if [[ "$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 \ ${PROG_DIR}/detectors/cds/bin/go_cds.sh "${RESULTS}.norm.fasta" >> "${RESULTS}.annot" loginfo "Done." fi if (( partial == 0 )) ; then topology="circular" defline="plastid, complete genome" else topology="linear" defline="plastid, partial sequence" fi ;; nucrdna) loginfo "Annotating a plant rDNA cistron..." if [[ "$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 \ | awk '{printf("%s;",$0)}' \ | awk '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 exit 1 ;; *) 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 sequence_number 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." if [[ "$idprefix" != "no" ]] ; then loginfo "Adding locus tags..." cat "${RESULTS}.sorted.annot" \ | $AwkCmd -v idprefix="$idprefix" ' BEGIN {n=1} /^FT +\/locus_tag=""/ { sub(/locus_tag=""/,"locus_tag=\""idprefix"_"n"\"",$0); n++; } { print $0 } ' loginfo "Locus tags done." else loginfo "Clearing locus tags done." egrep -v '^FT +\/locus_tag=""' \ "${RESULTS}.sorted.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."