diff --git a/ASSU.sh b/ASSU.sh
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+++ b/ASSU.sh
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+#!/bin/bash
+
+##############################################################################################################
+# #
+# ASSU: ASSembling SSU #
+# #
+ COPYRIGHT="Copyright (C) 2024 Institut Pasteur" #
+# #
+# This program is free software: you can redistribute it and/or modify it under the terms of the GNU #
+# General Public License as published by the Free Software Foundation, either version 3 of the License, or #
+# (at your option) any later version. #
+# #
+# This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even #
+# the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public #
+# License for more details. #
+# #
+# You should have received a copy of the GNU General Public License along with this program. If not, see #
+# <http://www.gnu.org/licenses/>. #
+# #
+# Contact: #
+# Alexis Criscuolo alexis.criscuolo@pasteur.fr #
+# Genome Informatics & Phylogenetics (GIPhy) giphy.pasteur.fr #
+# Centre de Ressources Biologiques de l'Institut Pasteur (CRBIP) research.pasteur.fr/en/b/VTq #
+# Institut Pasteur, Paris, FRANCE research.pasteur.fr #
+# #
+##############################################################################################################
+
+##############################################################################################################
+# #
+# ============ #
+# = VERSIONS = #
+# ============ #
+# #
+ VERSION=1.0; #
+# #
+##############################################################################################################
+
+##############################################################################################################
+# #
+# ============= #
+# = CONSTANTS = #
+# ============= #
+# #
+# -- PWD: directory containing the current script --------------------------------------------------------- #
+# #
+ PWD="$(cd "$(dirname "${BASH_SOURCE[0]}")" >/dev/null 2>&1 && pwd)";
+# #
+# -- extensions ------------------------------------------------------------------------------------------- #
+# #
+ declare -a FILE_EXTENSIONS=("fq" "fastq" "gz" "bz" "bz2" "dsrc" "dsrc2");
+# #
+# -- n/a -------------------------------------------------------------------------------------------------- #
+# #
+ NA="._N.o.N._.A.p.P.l.I.c.A.b.L.e_.";
+# #
+# -- headers ---------------------------------------------------------------------------------------------- #
+# #
+ H1="10 20 30 40 50 60 70 80 90 100";
+ H2=" | | | | | | | | | |";
+# #
+##############################################################################################################
+
+##############################################################################################################
+# #
+# ================ #
+# = REQUIREMENTS = #
+# ================ #
+# #
+# - SUMMARY - #
+# bwa_mem2/2.2.1 bzip2/1.0.6 dsrc/2.0.2 gawk/5.0.1 samtools/1.18
+# #
+# -- gawk ------------------------------------------------------------------------------------------------- #
+# #
+ GAWK_BIN=gawk;
+# #
+# -- bzip2 ------------------------------------------------------------------------------------------------ #
+# #
+ BZIP2_BIN=bzip2;
+# #
+# -- gunzip ----------------------------------------------------------------------------------------------- #
+# #
+ GUNZIP_BIN=gunzip;
+# #
+# -- zgrep ------------------------------------------------------------------------------------------------ #
+# #
+ ZGREP_BIN=zgrep;
+# #
+# -- pigz ------------------------------------------------------------------------------------------------- #
+# #
+ PIGZ_BIN=pigz;
+# #
+# -- DSRC ------------------------------------------------------------------------------------------------- #
+# #
+ DSRC_BIN=dsrc;
+# #
+# -- bwa-mem2 --------------------------------------------------------------------------------------------- #
+# #
+ BWAMEM2_BIN=bwa-mem2;
+# #
+# -- samtools --------------------------------------------------------------------------------------------- #
+# #
+ SAMTOOLS_BIN=samtools;
+# #
+##############################################################################################################
+
+##############################################################################################################
+# #
+# ================ #
+# = FITTINGS = #
+# ================ #
+# #
+# -- gawk ------------------------------------------------------------------------------------------------- #
+# #
+ BAWK="$GAWK_BIN";
+ CAWK="$GAWK_BIN -F,";
+ TAWK="$GAWK_BIN -F\\t";
+# #
+# -- bzip ------------------------------------------------------------------------------------------------- #
+# #
+ BUNZIP="$BZIP2_BIN --decompress";
+# #
+# -- DSRC ------------------------------------------------------------------------------------------------- #
+# #
+ DUNZIP="$DSRC_BIN d";
+# #
+# -- gunzip ----------------------------------------------------------------------------------------------- #
+# #
+ GUNZIP="$GUNZIP_BIN";
+# #
+# -- pigz ------------------------------------------------------------------------------------------------- #
+# #
+ PUNZIP="$PIGZ_BIN --decompress";
+# #
+# -- agrep ------------------------------------------------------------------------------------------------ #
+# #
+ ZGREP="$ZGREP_BIN";
+# #
+# -- bwa-mem2 --------------------------------------------------------------------------------------------- #
+# #
+ BWA_INDEX="$BWAMEM2_BIN index";
+ BWA_MEM="$BWAMEM2_BIN mem -v 0 -K 100000000 -Y -L 20";
+# #
+# -- samtools: exploring read alignments ------------------------------------------------------------------ #
+# #
+ SAMTOOLS_SORT="$SAMTOOLS_BIN sort --output-fmt BAM -l 1 -m 1073741824";
+ SAMTOOLS_CONSENSUS="$SAMTOOLS_BIN consensus --format FASTA --show-del no --show-ins yes";
+# #
+##############################################################################################################
+
+##############################################################################################################
+# #
+# ============ #
+# = DOC = #
+# ============ #
+# #
+mandoc() {
+ echo -e "\n\033[1m ASSU v$VERSION $COPYRIGHT\033[0m";
+ cat <<EOF
+
+ https://gitlab.pasteur.fr/GIPhy/ASSU
+
+ USAGE: ASSU [options] <infile> [<infile> ...]
+
+ OPTIONS:
+ -d <file> SSU databank file (default: SSUdb.gz in the same directory as ASSU.sh)
+ -p <string> restricts the SSU databank to the specified pattern (default: none)
+ -o <string> output SSU sequence file name (default: ssu.fasta)
+ -O <string> writes the selected reads into the specified file name (default: none)
+ -l <int> minimum sequence length (default: 1000)
+ -L <int> minimum read length (default: AUTO)
+ -Q <int> minimum base Phred quality value (default: 20)
+ -M <int> minimum mapping Phred quality value (default: 20)
+ -D <int> minimum coverage depth (default: 50)
+ -F <float> minimum proportion of the majority base to infer that base (default: 0.8)
+ -A <float> minimum ratio of the alternative base(s) to the majority one to add that
+ base(s) to the consensus (default: 0.2)
+ -N set N when multiple bases at a position (default: not set)
+ -w <dir> path to the tmp directory (default: \$TMPDIR, otherwise /tmp)
+ -t <int> thread numbers (default: 2)
+ -v verbose mode
+ -s prints the content of the SSU databank and exit
+ -c checks dependencies and exit
+ -h prints this help and exit
+
+ EXAMPLES:
+ ASSU -t 24 -o 16s.fasta fwd.fastq.gz rev.fastq.gz sgl.fastq.bz2
+ ASSU -d SSUdb.gz -O 16s.fastq -p Clostridium -L 75 -v *.fastq
+
+EOF
+}
+# #
+##############################################################################################################
+
+##############################################################################################################
+# #
+# ============= #
+# = FUNCTIONS = #
+# ============= #
+# #
+# -- echoxit() --------------------------------------------------------------------------------------------- #
+# >> prints in stderr the specified error message $1 and next exit 1 #
+# #
+echoxit() {
+ echo "$1" >&2 ; exit 1 ;
+}
+# #
+# -- chrono ----------------------------------------------------------------------------------------------- #
+# >> returns the elapsed time in well-formatted format #
+# #
+chrono() {
+ local s=$SECONDS; printf "[%02d:%02d]" $(( $s / 60 )) $(( $s % 60 )) ;
+}
+# #
+# -- dcontrol --------------------------------------------------------------------------------------------- #
+# >> controls required and optional dependancies #
+# #
+dcontrol() {
+ fail=false;
+ for binexe in bc fold grep $ZGREP_BIN sed $GAWK_BIN $BWAMEM2_BIN $SAMTOOLS_BIN
+ do
+ if [ ! $(command -v $binexe) ]; then echo "[ERROR] $binexe not found" >&2 ; fail=true; fi
+ done
+ if [ ! $(command -v $GUNZIP_BIN) ] && [ ! $(command -v $PIGZ_BIN) ]
+ then
+ echo "[ERROR] $GUNZIP_BIN or $PIGZ_BIN not found" >&2 ;
+ fail=true;
+ fi
+ for binexe in $BZIP2_BIN $DSRC_BIN
+ do
+ if [ ! $(command -v $binexe) ]; then echo "[WARNING] $binexe not found" ; fi
+ done
+ if $fail ; then exit 1 ; fi
+}
+# #
+# -- dcheck ----------------------------------------------------------------------------------------------- #
+# >> checks dependancies and exit #
+# #
+dcheck() {
+ echo "Checking required dependencies ..." ;
+ ## bc ##############################
+ echo -e -n "> \e[1mbc\e[0m >1.0 mandatory\t" ; binexe=bc;
+ echo -e -n "$binexe \t\t" ;
+ if [ ! $(command -v $binexe) ]; then echo -e "\e[31m[fail]\e[0m";
+ else echo -e -n "$(which $binexe)\t\t\e[32m[ok]\e[0m\t\t"; bc -v | head -1 ;
+ fi
+ ## fold ############################
+ echo -e -n "> \e[1mfold\e[0m >1.0 mandatory\t" ; binexe=fold;
+ echo -e -n "$binexe \t\t" ;
+ if [ ! $(command -v $binexe) ]; then echo -e "\e[31m[fail]\e[0m";
+ else echo -e -n "$(which $binexe)\t\t\e[32m[ok]\e[0m\t\t"; fold --version | head -1 ;
+ fi
+ ## grep ############################
+ echo -e -n "> \e[1mgrep\e[0m >1.0 mandatory\t" ; binexe=grep;
+ echo -e -n "$binexe \t\t" ;
+ if [ ! $(command -v $binexe) ]; then echo -e "\e[31m[fail]\e[0m";
+ else echo -e -n "$(which $binexe)\t\t\e[32m[ok]\e[0m\t\t"; grep -V | head -1 ;
+ fi
+ ## zgrep ###########################
+ echo -e -n "> \e[1zzgrep\e[0m >1.0 mandatory\t" ; binexe=$ZGREP_BIN;
+ echo -e -n "$binexe \t\t" ;
+ if [ ! $(command -v $binexe) ]; then echo -e "\e[31m[fail]\e[0m";
+ else echo -e -n "$(which $binexe)\t\t\e[32m[ok]\e[0m\t\t"; $ZGREP_BIN -V | head -1 ;
+ fi
+ ## sed #############################
+ echo -e -n "> \e[1msed\e[0m >1.0 mandatory\t" ; binexe=sed;
+ echo -e -n "$binexe \t\t" ;
+ if [ ! $(command -v $binexe) ]; then echo -e "\e[31m[fail]\e[0m";
+ else echo -e -n "$(which $binexe)\t\t\e[32m[ok]\e[0m\t\t"; sed --version | head -1 ;
+ fi
+ ## gawk >=4.0.0 ####################
+ echo -e -n "> \e[1mgawk\e[0m >4.0.0 mandatory\t" ; binexe=$GAWK_BIN;
+ echo -e -n "$binexe \t\t" ;
+ if [ ! $(command -v $binexe) ]; then echo -e "\e[31m[fail]\e[0m";
+ else echo -e -n "$(which $binexe)\t\t\e[32m[ok]\e[0m\t\t"; $GAWK_BIN -V | head -1 ;
+ fi
+ ## bzip2 ###########################
+ echo -e -n "> \e[1mbzip2\e[0m >1.0 optional\t" ; binexe=$BZIP2_BIN;
+ echo -e -n "$binexe \t\t" ;
+ if [ ! $(command -v $binexe) ]; then echo -e "\e[31m[fail]\e[0m";
+ else echo -e -n "$(which $binexe)\t\t\e[32m[ok]\e[0m\t\t"; $BZIP2_BIN --help 2>&1 | head -1 ;
+ fi
+ ## DSRC ############################
+ echo -e -n "> \e[1mDSRC\e[0m >=2.0 optional\t" ; binexe=$DSRC_BIN;
+ echo -e -n "$binexe \t\t" ;
+ if [ ! $(command -v $binexe) ]; then echo -e "\e[31m[fail]\e[0m";
+ else echo -e -n "$(which $binexe)\t\t\e[32m[ok]\e[0m\t\t"; $DSRC_BIN 2>&1 | sed -n 2p ;
+ fi
+ ## gunzip ##########################
+ echo -e -n "> \e[1mgunzip\e[0m >1.0 mandatory\t" ; binexe=$GUNZIP_BIN;
+ echo -e -n "$binexe \t\t" ;
+ if [ ! $(command -v $binexe) ]; then echo -e "\e[31m[fail]\e[0m";
+ else echo -e -n "$(which $binexe)\t\t\e[32m[ok]\e[0m\t\t"; $GUNZIP_BIN -V | head -1 ;
+ fi
+ ## pigz ############################
+ echo -e -n "> \e[1mpigz\e[0m >=2.4 optional\t" ; binexe=$PIGZ_BIN;
+ echo -e -n "$binexe \t\t" ;
+ if [ ! $(command -v $binexe) ]; then echo -e "\e[31m[fail]\e[0m";
+ else echo -e -n "$(which $binexe)\t\t\e[32m[ok]\e[0m\t\t"; $PIGZ_BIN -V ;
+ fi
+ ## bwa-mem2 >=2.2.1 ################
+ echo -e -n "> \e[1mBWA-mem2\e[0m >=2.2.1 mandatory\t" ; binexe=$BWAMEM2_BIN;
+ echo -e -n "$binexe\t\t" ;
+ if [ ! $(command -v $binexe) ]; then echo -e "\e[31m[fail]\e[0m";
+ else echo -e -n "$(which $binexe)\t\t\e[32m[ok]\e[0m\t\t"; $BWAMEM2_BIN version ;
+ fi
+ ## samtools >= 1.18 ################
+ echo -e -n "> \e[1mSamtools\e[0m >=1.18 mandatory\t" ; binexe=$SAMTOOLS_BIN;
+ echo -e -n "$binexe \t\t" ;
+ if [ ! $(command -v $binexe) ]; then echo -e "\e[31m[fail]\e[0m";
+ else echo -e -n "$(which $binexe)\t\t\e[32m[ok]\e[0m\t\t"; $SAMTOOLS_BIN --version | head -1;
+ fi
+ echo "[exit]" ;
+ exit ;
+}
+# -- dispdb ----------------------------------------------------------------------------------------------- #
+# >> displays the content of the specified SSU databank #
+# #
+dispdb() {
+# $ZGREP "^>" $1 |
+# tr -d '>' |
+# $BAWK ' {if($2=="A"){k="Archaea";++a}
+# else {k="Bacteria";++b}
+# if($3=="Candidatus")print k" | "$1" | "$3" "$4" "$5;
+# else print k" | "$1" | "$3" "$4}
+# END{print"# Archaea: "a;
+# print"# Bacteria: "b}' ;
+ $GUNZIP -c $1 2>/dev/null |
+ tr -d '>' | paste - - | tr '\t' ' ' |
+ $BAWK ' {if($2=="A"){k="Archaea";++a}
+ else {k="Bacteria";++b}
+ if($3=="Candidatus")print k" | "$1" | "length($NF)" bps | "$3" "$4" "$5;
+ else print k" | "$1" | "length($NF)" bps | "$3" "$4}
+ END{print"# Archaea: "a;
+ print"# Bacteria: "b}' ;
+}
+# #
+##############################################################################################################
+
+
+##############################################################################################################
+# #
+# READING OPTIONS #
+# #
+##############################################################################################################
+
+if [ $# -lt 1 ]; then mandoc ; exit 1 ; fi
+
+SSUDB=$PWD/SSUdb.gz; # SSU databank -d
+OUTFILE=ssu.fasta; # FASTA outfile -o
+OUTFQ="$NA"; # FASTQ outfile -O
+PATTERN="$NA"; # pattern -p
+SLGT=1000; # min seq lgt -l
+RLGT="00"; # min read lgt -L
+BQ=20; # min Phred -Q
+MQ=20; # min map Phread -M
+COV=50; # min cov depth -D
+FQ=0.8; # min freq -F
+FH=0.2; # min het -A
+AMB=true; # only N -N
+DISPDB=false; # display SSUdb -s
+TMP_DIR=${TMPDIR:-/tmp}; # tmp directory -w
+NTHREADS=2; # no. threads -t
+VERBOSE=false; # verbose mode -v
+
+while getopts d:p:o:O:l:L:Q:M:D:F:A:w:t:Nvcsh option
+do
+ case $option in
+ d) SSUDB="$OPTARG" ;;
+ p) PATTERN="$OPTARG" ;;
+ o) OUTFILE="$OPTARG" ;;
+ O) OUTFQ="$OPTARG" ;;
+ l) SLGT=$OPTARG ;;
+ L) RLGT=$OPTARG ;;
+ Q) BQ=$OPTARG ;;
+ M) MQ=$OPTARG ;;
+ D) COV=$OPTARG ;;
+ F) FQ="$OPTARG" ;;
+ A) FH="$OPTARG" ;;
+ N) AMB=false ;;
+ w) TMP_DIR="$OPTARG" ;;
+ t) NTHREADS=$OPTARG ;;
+ v) VERBOSE=true ;;
+ c) dcheck ;;
+ s) DISPDB=true ;;
+ h) mandoc ; exit 0 ;;
+ \?) mandoc ; exit 1 ;;
+ esac
+done
+
+shift "$(( $OPTIND - 1 ))"
+NFILE=$#; # no. input file(s)
+FQLIST="$@"; # specified input file(s)
+
+echo "# ASSU v$VERSION" ;
+echo "# $COPYRIGHT" ;
+echo "+ https://gitlab.pasteur.fr/GIPhy/ASSU" ;
+$VERBOSE && echo "> Syst: $MACHTYPE" ;
+$VERBOSE && echo "> Bash: $BASH_VERSION" ;
+
+[ ! -e $SSUDB ] && echoxit "[ERROR] databank not found (option -d): $SSUDB" ;
+[ -d $SSUDB ] && echoxit "[ERROR] not a file (option -d): $SSUDB" ;
+[ ! -s $SSUDB ] && echoxit "[ERROR] empty file (option -d): $SSUDB" ;
+[ ! -r $SSUDB ] && echoxit "[ERROR] no read permission (option -d): $SSUDB" ;
+if $DISPDB
+then
+ dispdb $SSUDB ;
+ exit ;
+fi
+[[ $SLGT =~ ^[0-9]+$ ]] || echoxit "[ERROR] incorrect value (option -l): $SLGT" ;
+[[ $RLGT =~ ^[0-9]+$ ]] || echoxit "[ERROR] incorrect value (option -L): $RLGT" ;
+[[ $BQ =~ ^[0-9]+$ ]] || echoxit "[ERROR] incorrect value (option -Q): $BQ" ;
+[[ $MQ =~ ^[0-9]+$ ]] || echoxit "[ERROR] incorrect value (option -M): $MQ" ;
+[[ $COV =~ ^[0-9]+$ ]] || echoxit "[ERROR] incorrect value (option -D): $COV" ;
+[[ $FQ =~ ^[0-9]+([.][0-9]+)?$ ]] || echoxit "[ERROR] incorrect value (option -F): $FQ" ;
+[ $(bc <<<"0<=$FQ&&$FQ<=1") -eq 0 ] && echoxit "[ERROR] incorrect value (option -F): $FQ" ;
+[[ $FH =~ ^[0-9]+([.][0-9]+)?$ ]] || echoxit "[ERROR] incorrect value (option -H): $FH" ;
+[ $(bc <<<"0<=$FH&&$FH<=1") -eq 0 ] && echoxit "[ERROR] incorrect value (option -H): $FH" ;
+[[ $NTHREADS =~ ^[0-9]+$ ]] || echoxit "[ERROR] incorrect value (option -t): $NTHREADS" ;
+ [ $NTHREADS -lt 1 ] && NTHREADS=1;
+
+
+##############################################################################################################
+# #
+# CHECKING DEPENDANCIES AND AVAILABLE ZIPPERS #
+# #
+##############################################################################################################
+BZ=false; [ -s "$(command -v $BZIP2_BIN)" ] && BZ=true;
+DZ=false; [ -s "$(command -v $DSRC_BIN)" ] && DZ=true;
+GZ=false; [ -s "$(command -v $GUNZIP_BIN)" ] && GZ=true;
+PZ=false; [ -s "$(command -v $PIGZ_BIN)" ] && PZ=true;
+
+dcontrol;
+
+
+##############################################################################################################
+# #
+# CHECKING INFILES #
+# #
+##############################################################################################################
+if [ $NFILE -eq 0 ]; then echoxit "[ERROR] no input file" ;
+elif [ $NFILE -eq 1 ]; then MESSAGE="checking input file" ;
+else MESSAGE="checking input files" ;
+fi
+echo -n "$(chrono) $MESSAGE ... " ;
+
+ouch=false;
+for f in $FQLIST
+do
+ if [ ! -e $f ]; then ! $ouch && echo ; echo "[ERROR] file not found: $f" >&2 ; ouch=true; continue; fi
+ if [ -d $f ]; then ! $ouch && echo ; echo "[ERROR] not a file: $f" >&2 ; ouch=true; continue; fi
+ if [ ! -s $f ]; then ! $ouch && echo ; echo "[ERROR] empty file: $f" >&2 ; ouch=true; continue; fi
+ if [ ! -r $f ]; then ! $ouch && echo ; echo "[ERROR] no read permission: $f" >&2 ; ouch=true; continue; fi
+ fext="${f##*.}";
+ valid=false;
+ for e in "${FILE_EXTENSIONS[@]}"
+ do
+ if [ "$e" == "$fext" ]; then valid=true; break; fi
+ done
+ if ! $valid ; then ! $ouch && echo ; echo "[ERROR] file extension not valid: $f" >&2 ; ouch=true; continue; fi
+ case $fext in
+ bz|bz2) if ! $BZ ; then ! $ouch && echo ; echo "[ERROR] bzip2 not available: $f" >&2 ; ouch=true; continue; fi ;;
+ dsrc|dsrc2) if ! $DZ ; then ! $ouch && echo ; echo "[ERROR] dsrc not available: $f" >&2 ; ouch=true; continue; fi ;;
+ esac
+done
+$ouch && exit 1 ;
+
+echo "[ok]" ;
+
+if $VERBOSE ; then for f in $FQLIST ; do echo "+ $f" ; done ; fi
+
+
+##############################################################################################################
+# #
+# CREATING TMP DIRECTORY, DEFINING TRAP AND TMP FILES, ESTIMATING RLGT #
+# #
+##############################################################################################################
+MESSAGE="creating tmp directory" ;
+echo -n "$(chrono) $MESSAGE ." ;
+
+[ "${TMP_DIR:0:1}" != "/" ] && TMP_DIR="$(pwd)/$TMP_DIR";
+if [ ! -e $TMP_DIR ]; then echo ; echo "[ERROR] tmp directory does not exist (option -w): $TMP_DIR" >&2 ; exit 1 ; fi
+if [ ! -d $TMP_DIR ]; then echo ; echo "[ERROR] not a directory (option -w): $TMP_DIR" >&2 ; exit 1 ; fi
+if [ ! -w $TMP_DIR ]; then echo ; echo "[ERROR] no write permission (option -w): $TMP_DIR" >&2 ; exit 1 ; fi
+
+echo -n "." ;
+
+TMP_DIR=$(mktemp -d -p $TMP_DIR ASSU.XXXXXXXXXX);
+finalize() { rm -r $TMP_DIR ; }
+trap 'finalize ; exit 1' SIGTERM SIGINT SIGQUIT SIGHUP TERM INT QUIT HUP ;
+
+SSU_IDX=$TMP_DIR/ssu;
+BAM=$TMP_DIR/aln.bam;
+SAM=$TMP_DIR/aln.sam;
+FQTMP=$TMP_DIR/htsr.fastq;
+REF_SEQ=$TMP_DIR/ref.fa;
+REF_IDX=$TMP_DIR/ref;
+TMP1=$TMP_DIR/tmp1.txt;
+TMP2=$TMP_DIR/tmp2.txt;
+
+echo -n "." ;
+
+if [ "$RLGT" == "00" ]
+then
+ [ $NTHREADS -gt 24 ] && DSRCopt="-t24 -s" || DSRCopt="-t$NTHREADS -s" ;
+ for f in $FQLIST
+ do
+ fext="${f##*.}";
+ case $fext in
+ fq|fastq) sed -n 2~4p $f | $BAWK '(FNR>10000){exit}{print length()}' ;;
+ gz) if $PZ ; then $PUNZIP -c -p $NTHREADS $f 2>/dev/null | sed -n 2~4p | $BAWK '(FNR>10000){exit}{print length()}' ;
+ else $GUNZIP -c $f 2>/dev/null | sed -n 2~4p | $BAWK '(FNR>10000){exit}{print length()}' ; fi ;;
+ bz|bz2) $BUNZIP -c $f 2>/dev/null | sed -n 2~4p | $BAWK '(FNR>10000){exit}{print length()}' ;;
+ dsrc|dsrc2) $DUNZIP $DSRCopt $f 2>/dev/null | sed -n 2~4p | $BAWK '(FNR>10000){exit}{print length()}' ;;
+ esac
+ done > $TMP1 ;
+
+ echo -n "." ;
+
+ sort -rn $TMP1 > $TMP2 ;
+ n=$(cat $TMP2 | wc -l)
+ n=$(( $n / 10 ));
+
+ if [ $n -eq 0 ]
+ then
+ echo "" ;
+ echo "> empty file(s)" ;
+ finalize ;
+ echo "$(chrono) exit" ;
+ exit ;
+ fi
+
+ RLGT=$(sed -n "$n"p $TMP2);
+ RLGT=$(( 90 * $RLGT / 100 )); # 90% of the 90th centile of the read lengths
+fi
+
+echo " [ok]" ;
+
+if $VERBOSE ; then echo "> TMP_DIR=$TMP_DIR" ; fi
+
+
+##############################################################################################################
+# #
+# SEARCHING SSU DATABANK #
+# #
+##############################################################################################################
+MESSAGE="examining SSU databank" ;
+echo -n "$(chrono) $MESSAGE ." ;
+
+## indexing SSU databank #######################################################
+if [ "$PATTERN" == "$NA" ]
+then
+ $BWA_INDEX -p $SSU_IDX $SSUDB &>/dev/null ;
+else
+ $ZGREP -A1 -F "$PATTERN" $SSUDB | grep -v -e "^--$" > $REF_SEQ ;
+
+ if [ ! -s $REF_SEQ ]
+ then
+ echo ".. [ok]" ;
+ echo "> no selected sequence with the specified pattern: $PATTERN" ;
+ finalize ;
+ exit ;
+ fi
+
+ $BWA_INDEX -p $SSU_IDX $REF_SEQ &>/dev/null ;
+ rm -f $REF_SEQ ;
+fi
+
+echo -n "." ;
+
+## aligning HTS reads ##########################################################
+touch $SAM ;
+[ $NTHREADS -gt 24 ] && DSRCopt="-t24" || DSRCopt="-t$NTHREADS" ;
+# BWAopt="-t $NTHREADS -k 21 -w 50 -D 1.0 -r 2.0 -T $(( $RLGT - 20 ))";
+BWAopt="-t $NTHREADS -k 21 -w 50";
+for fin in $FQLIST
+do
+ echo -n "." ;
+ fext="${fin##*.}";
+ case $fext in
+ fq|fastq|gz) f=$fin ;;
+ bz|bz2) $BUNZIP -c $fin > $FQTMP 2>/dev/null ; f=$FQTMP ;;
+ dsrc|dsrc2) $DUNZIP $DSRCopt $fin $FQTMP 2>/dev/null ; f=$FQTMP ;;
+ esac
+ echo -n "." ;
+ $BWA_MEM $BWAopt $SSU_IDX $f 2>/dev/null | $TAWK '/^@/||($2==4){next}{print}' >> $SAM ;
+done
+
+echo " [ok]" ;
+
+## getting ref #################################################################
+## maximizing the no. hits
+# accn=$($TAWK '{++c[$3]}END{for(a in c)if(c[a]>1)print c[a]" "a}' $SAM | sort -n | tail -1 | $BAWK '{print$2}');
+## maximizing the avg score among the two most hitted references (as longer sequences often wins when using the basic criterion above)
+accn=$($TAWK '{print$3"\t"$14}' $SAM |
+ sed 's/AS:i://g' |
+ $TAWK ' {m[$1]+=$2;++n[$1]}
+ END{for(a in m)if(n[a]>10)print n[a]" "(m[a]/n[a])" "a}' |
+ sort -rn | head -2 |
+ sort -rgk2 | head -1 | $BAWK '{print$3}');
+
+if [ -z "$accn" ]
+then
+ echo "> no SSU found" ;
+ finalize ;
+ echo "$(chrono) exit" ;
+ exit ;
+fi
+
+$ZGREP -m1 -A1 -F ">$accn" $SSUDB > $REF_SEQ ;
+
+line="$(head -1 $REF_SEQ)";
+case $($BAWK '{print$2}' <<<"$line") in
+A) kingdom="Archaea" ;;
+B) kingdom="Bacteria" ;;
+esac
+genus=$($BAWK '{print$3}' <<<"$line");
+if [ "$genus" == "Candidatus" ]
+then taxo="$($BAWK '{print$3" "$4" "$5}' <<<"$line")";
+else taxo="$($BAWK '{print$3" "$4}' <<<"$line")";
+fi
+line="$(tail -1 $REF_SEQ)";
+LREF=${#line};
+
+$VERBOSE && [ "$PATTERN" != "$NA" ] && echo "> selection pattern: $PATTERN" ;
+$VERBOSE && echo "> model: $kingdom | $taxo | $accn | $LREF bps" ;
+
+
+##############################################################################################################
+# #
+# BUILDING SSU SEQUENCE #
+# #
+##############################################################################################################
+MESSAGE="building SSU sequence" ;
+echo -n "$(chrono) $MESSAGE ." ;
+
+## getting HTS reads ###########################################################
+# $TAWK -v l=$RLGT '(length($10)>=l){print$10"\t"$11"\t"$12}' $SAM | sed 's/NM:i://g' | $TAWK '($3<10){print$1"\t"$2}' | # NOTE: <10 mismatches against the best hit
+$TAWK -v l=$RLGT '(length($10)>=l){print$10"\t"$11}' $SAM |
+ sort -u | $TAWK '{print"@SSU:"NR;
+ print$1;
+ print"+";
+ print$2}' > $FQTMP ;
+
+echo -n "." ;
+
+line=$(sed -n 2~4p $FQTMP | $TAWK '{b+=length()}END{print NR" "b}');
+NR=$($BAWK '{print$1}' <<<"$line"); # no. reads
+NB=$($BAWK '{print$2}' <<<"$line"); # no. bases
+DEPTH=$(( $NB / $LREF ));
+
+if [ $DEPTH -lt $COV ]
+then
+ echo ". [ok]" ;
+ echo "> low coverage depth: $DEPTH""""x" ;
+ finalize ;
+ echo "$(chrono) exit" ;
+ exit ;
+fi
+
+## indexing reference sequence #################################################
+$BWA_INDEX -p $REF_IDX $REF_SEQ &>/dev/null ;
+
+echo -n "." ;
+
+## mapping HTS reads ###########################################################
+t=$(( 2 * $NTHREADS / 3 ));
+[ $t -lt 1 ] && t=1;
+BWAopt="-t $t -D 0.1 -r 0.1 -k 7";
+t=$(( $NTHREADS - $t ));
+[ $t -lt 1 ] && t=1;
+STSopt="--threads $t -T $TMP_DIR" ;
+$BWA_MEM $BWAopt $REF_IDX $FQTMP 2>/dev/null |
+ $TAWK '/^@/||($2!=4)' |
+ $SAMTOOLS_SORT $STSopt -o $BAM &>/dev/null ;
+
+echo -n "." ;
+
+## building consensus ##########################################################
+# STCopt="--threads $NTHREADS --mode bayesian --ambig --min-MQ 2 --P-het 1.0e-100 --het-scale 1.0";
+STCopt="--threads $NTHREADS --mode simple --min-MQ $MQ --min-BQ $BQ --min-depth $COV --use-qual --call-fract $FQ --het-fract $FH";
+$AMB && STCopt="$STCopt --ambig";
+SEQ=$($SAMTOOLS_CONSENSUS $STCopt $BAM 2>/dev/null |
+ grep -v "^>" | tr -d '\n' |
+ sed -r 's/^[ACGT]{0,5}[acgtRrYySsWwKkMmBbDdHhVvNn]+//;s/[acgtRrYySsWwKkMmBbDdHhVvNn]+[ACGT]{0,5}$//;' |
+ sed -r 's/^[ACGT]{0,5}[acgtRrYySsWwKkMmBbDdHhVvNn]+//;s/[acgtRrYySsWwKkMmBbDdHhVvNn]+[ACGT]{0,5}$//;' |
+ sed -r 's/^[ACGT]{0,5}[acgtRrYySsWwKkMmBbDdHhVvNn]+//;s/[acgtRrYySsWwKkMmBbDdHhVvNn]+[ACGT]{0,5}$//;' |
+ sed -r 's/^[ACGT]{0,5}[acgtRrYySsWwKkMmBbDdHhVvNn]+//;s/[acgtRrYySsWwKkMmBbDdHhVvNn]+[ACGT]{0,5}$//;' |
+ sed -r 's/^[ACGT]{0,5}[acgtRrYySsWwKkMmBbDdHhVvNn]+//;s/[acgtRrYySsWwKkMmBbDdHhVvNn]+[ACGT]{0,5}$//;');
+
+echo " [ok]" ;
+
+
+##############################################################################################################
+# #
+# FINALIZING #
+# #
+##############################################################################################################
+l=${#SEQ};
+r=$(tr -cd 'ACGT' <<<"$SEQ" | wc -m);
+a=$(( $l - $r ))
+
+if $VERBOSE
+then
+ echo "> $NR selected reads ($NB bases; lgt > $(( $RLGT - 1 )))" ;
+ echo "> coverage depth: $DEPTH""""x" ;
+ if [ $a -eq 0 ]; then echo "> $l bps" ;
+ elif [ $a -eq 1 ]; then echo "> $l bps (ambiguous base: $a)" ;
+ else echo "> $l bps (ambiguous bases: $a)" ;
+ fi
+fi
+
+if $VERBOSE
+then
+ fold -w 100 <<<"$SEQ" |
+ $BAWK -v h1="$H1" -v h2="$H2" 'BEGIN{print" "h1; print" "h2;}
+ {printf("%5s %s\n", 1+100*(NR-1), $1); printf" "; w=split($1,c,""); i=0;
+ while(++i<=w) switch(c[i]){ case "A": case "C": case "G": case "T": printf" "; continue;
+ default: printf"*"; continue; }
+ print""; }' ;
+# for b in $(sed 's/./& /g' <<<$"$SEQ")
+# do
+# case $b in
+# A|C|G|T) echo -n "$b" ;;
+# *) echo -n -e "\e[31m$b\e[0m" ;;
+# esac
+# done
+# echo ;
+fi
+
+if [ $l -lt $SLGT ]
+then
+ echo "> sequence too short: $l bps" ;
+ finalize ;
+ echo "$(chrono) exit" ;
+ exit ;
+fi
+
+pa=$(( 100 * $a / $l ));
+if [ $pa -gt 5 ]
+then
+ echo "> too many ambiguous bases: $pa%" ;
+ finalize ;
+ echo "$(chrono) exit" ;
+ exit ;
+fi
+
+echo ">SSU_covr_$DEPTH""_lgt_$l""_amb_$a" > $OUTFILE ;
+echo "$SEQ" >> $OUTFILE ;
+if [ "$OUTFQ" == "$NA" ]
+then
+ echo "$(chrono) writing output file ... [ok]" ;
+ $VERBOSE && echo "+ FASTA: $OUTFILE" ;
+else
+ mv $FQTMP $OUTFQ ;
+ echo "$(chrono) writing output files ... [ok]" ;
+ $VERBOSE && echo "+ FASTA: $OUTFILE" ;
+ $VERBOSE && echo "+ FASTQ: $OUTFQ" ;
+fi
+
+finalize ;
+
+echo "$(chrono) exit" ;
+
+exit ;
+
diff --git a/COPYING b/COPYING
new file mode 100644
index 0000000000000000000000000000000000000000..be3f7b28e564e7dd05eaf59d64adba1a4065ac0e
--- /dev/null
+++ b/COPYING
@@ -0,0 +1,661 @@
+ GNU AFFERO GENERAL PUBLIC LICENSE
+ Version 3, 19 November 2007
+
+ Copyright (C) 2007 Free Software Foundation, Inc. <https://fsf.org/>
+ Everyone is permitted to copy and distribute verbatim copies
+ of this license document, but changing it is not allowed.
+
+ Preamble
+
+ The GNU Affero General Public License is a free, copyleft license for
+software and other kinds of works, specifically designed to ensure
+cooperation with the community in the case of network server software.
+
+ The licenses for most software and other practical works are designed
+to take away your freedom to share and change the works. By contrast,
+our General Public Licenses are intended to guarantee your freedom to
+share and change all versions of a program--to make sure it remains free
+software for all its users.
+
+ When we speak of free software, we are referring to freedom, not
+price. Our General Public Licenses are designed to make sure that you
+have the freedom to distribute copies of free software (and charge for
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+
+ Developers that use our General Public Licenses protect your rights
+with two steps: (1) assert copyright on the software, and (2) offer
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+and/or modify the software.
+
+ A secondary benefit of defending all users' freedom is that
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+software used on network servers, this result may fail to come about.
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+users of that server. Therefore, public use of a modified version, on
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+code of the modified version.
+
+ An older license, called the Affero General Public License and
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+ <one line to give the program's name and a brief idea of what it does.>
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+ (at your option) any later version.
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+ but WITHOUT ANY WARRANTY; without even the implied warranty of
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diff --git a/README.md b/README.md
index 79594b2d7d68ea5044cc125fa352638a1f4e196e..7a2699c084e1e6da59b1d0ec9b504d4b3e28074d 100644
--- a/README.md
+++ b/README.md
@@ -1,93 +1,527 @@
+[](https://www.gnu.org/licenses/)
+[](https://www.gnu.org/software/bash/)
+
+
# ASSU
+_ASSU_ (ASSemblig SSU) is a command line tool written in [Bash](https://www.gnu.org/software/bash/) to carry out the reference-guided assembly of small subunit (SSU) 16S ribosomal ribonucleic acid (rRNA) using short high-throughput sequencing (HTS) reads derived from whole genome sequencing of bacteria or archaea strains.
+
+This tool was developed to compensate the failure of several _de novo_ assembly programs to assemble a non-fragmented SSU segment when the sequenced genome contains different 16S rRNA copies with sequence variation, especially when using short HTS reads.
+
+
+
+## Dependencies
+
+You will need to install the required programs and tools listed in the following tables, or to verify that they are already installed with the required version.
+
+##### Mandatory dependencies
+
+<div align="center">
+| program | package | version | sources |
+|:----------------------------------------------------- |:-----------------------------------------------:| ------------:|:------------------------------------------------------------------------------------------------------------------------------------------------------- |
+| [_gawk_](https://www.gnu.org/software/gawk/) | - | > 4.0.0 | [ftp.gnu.org/gnu/gawk](http://ftp.gnu.org/gnu/gawk/) |
+| _gunzip_<br>_zgrep_ | [_gzip_](https://www.gnu.org/software/gzip/) | > 1.0 | [ftp.gnu.org/gnu/gzip](https://ftp.gnu.org/gnu/gzip/) |
+| [_bwa-mem2_](https://github.com/bwa-mem2/bwa-mem2) | - | ≥ 2.2.1 | [gitlab.pasteur.fr/GIPhy/contig_info](https://gitlab.pasteur.fr/GIPhy/contig_info) |
+| [_samtools_](http://www.htslib.org/) | - | ≥ 1.18 | [github.com/samtools/samtools](https://github.com/samtools/samtools)<br>[sourceforge.net/projects/samtools](https://sourceforge.net/projects/samtools) |
-## Getting started
+</div>
-To make it easy for you to get started with GitLab, here's a list of recommended next steps.
-Already a pro? Just edit this README.md and make it your own. Want to make it easy? [Use the template at the bottom](#editing-this-readme)!
+##### Optional dependencies
-## Add your files
+<div align="center">
-- [ ] [Create](https://docs.gitlab.com/ee/user/project/repository/web_editor.html#create-a-file) or [upload](https://docs.gitlab.com/ee/user/project/repository/web_editor.html#upload-a-file) files
-- [ ] [Add files using the command line](https://docs.gitlab.com/ee/gitlab-basics/add-file.html#add-a-file-using-the-command-line) or push an existing Git repository with the following command:
+| program | package | version | sources |
+|:----------------------------------------------------- |:-----------:| -----------:|:--------------------------------------------------------------------------------------- |
+| [_bzip2_](https://sourceware.org/bzip2/) | - | > 1.0.0 | [sourceware.org/bzip2/downloads.html](https://sourceware.org/bzip2/downloads.html) |
+| [_DSRC_](https://refresh-bio.github.io/software/) | - | ≥ 2.0 | [github.com/refresh-bio/DSRC](https://github.com/refresh-bio/DSRC) |
+| [_pigz_](https://zlib.net/pigz/) | - | ≥ 2.4 | [github.com/madler/pigz](https://github.com/madler/pigz) |
+</div>
+
+
+## Installation and execution
+
+**A.** Clone this repository with the following command line:
+
+```bash
+git clone https://gitlab.pasteur.fr/GIPhy/ASSU.git
```
-cd existing_repo
-git remote add origin https://gitlab.pasteur.fr/GIPhy/ASSU.git
-git branch -M main
-git push -uf origin main
+
+**B.** Go to the created directory and give the execute permission to the file `ASSU.sh`:
+
+```bash
+cd ASSU/
+chmod +x ASSU.sh
```
-## Integrate with your tools
+**C.** Check the dependencies (and their version) using the following command line:
-- [ ] [Set up project integrations](https://gitlab.pasteur.fr/GIPhy/ASSU/-/settings/integrations)
+```bash
+./ASSU.sh -c
+```
-## Collaborate with your team
+**D.** If at least one of the required program (see [Dependencies](#dependencies)) is not available on your `$PATH` variable (or if one compiled binary has a different default name), it should be manually specified.
+To specify the location of a specific binary, edit the file `ASSU.sh` and indicate the local path to the corresponding binary(ies) within the code block `REQUIREMENTS` (approximately lines 60-110).
+For each required program, the table below reports the corresponding variable assignment instruction to edit (if needed) within the code block `REQUIREMENTS`
-- [ ] [Invite team members and collaborators](https://docs.gitlab.com/ee/user/project/members/)
-- [ ] [Create a new merge request](https://docs.gitlab.com/ee/user/project/merge_requests/creating_merge_requests.html)
-- [ ] [Automatically close issues from merge requests](https://docs.gitlab.com/ee/user/project/issues/managing_issues.html#closing-issues-automatically)
-- [ ] [Enable merge request approvals](https://docs.gitlab.com/ee/user/project/merge_requests/approvals/)
-- [ ] [Set auto-merge](https://docs.gitlab.com/ee/user/project/merge_requests/merge_when_pipeline_succeeds.html)
+<div align="center">
+<sup>
-## Test and Deploy
+| program | variable assignment | | program | variable assignment |
+|:---------------- |:-------------------------------- | - |:---------------- |:-------------------------------- |
+| _bwa-mem2_ | `BWAMEM2_BIN=bwa-mem2;` | | _gunzip_ | `GUNZIP_BIN=gunzip;` |
+| _bzip2_ | `BZIP2_BIN=bzip2;` | | _pigz_ | `PIGZ_BIN=pigz;` |
+| _DSRC_ | `DSRC_BIN=dsrc;` | | _samtools_ | `SAMTOOLS_BIN=samtools;` |
+| _gawk_ | `GAWK_BIN=gawk;` | | _zgrep_ | `ZGREP_BIN=zgrep;` |
-Use the built-in continuous integration in GitLab.
+</sup>
+</div>
-- [ ] [Get started with GitLab CI/CD](https://docs.gitlab.com/ee/ci/quick_start/index.html)
-- [ ] [Analyze your code for known vulnerabilities with Static Application Security Testing (SAST)](https://docs.gitlab.com/ee/user/application_security/sast/)
-- [ ] [Deploy to Kubernetes, Amazon EC2, or Amazon ECS using Auto Deploy](https://docs.gitlab.com/ee/topics/autodevops/requirements.html)
-- [ ] [Use pull-based deployments for improved Kubernetes management](https://docs.gitlab.com/ee/user/clusters/agent/)
-- [ ] [Set up protected environments](https://docs.gitlab.com/ee/ci/environments/protected_environments.html)
-***
+**E.** Execute _ASSU_ with the following command line model:
-# Editing this README
+```bash
+./ASSU.sh [options] <infile> [<infile> ...]
+```
-When you're ready to make this README your own, just edit this file and use the handy template below (or feel free to structure it however you want - this is just a starting point!). Thanks to [makeareadme.com](https://www.makeareadme.com/) for this template.
+**F.** _ASSU_ also requires a databank of reference SSU sequences. By default, a version of this databank is provided as a file named `SSUdb.gz` (see details in `SSUdb.version.txt`). However, a more recent version can be quickly built using the provided script `makeSSUdb.sh` with the following command line:
-## Suggestions for a good README
+```bash
+./makeSSUdb.sh
+```
+
+After a few seconds, a new SSU databank file named `SSUdb.gz` will be automatically created from the [NCBI RefSeq Targeted Loci Project](https://www.ncbi.nlm.nih.gov/refseq/targetedloci/). Note that the previous command line will overwrite the provided version of `SSUdb.gz` when run in the same directory.
+
+
+## Usage
-Every project is different, so consider which of these sections apply to yours. The sections used in the template are suggestions for most open source projects. Also keep in mind that while a README can be too long and detailed, too long is better than too short. If you think your README is too long, consider utilizing another form of documentation rather than cutting out information.
+Run _ASSU_ without option to read the following documentation:
-## Name
-Choose a self-explaining name for your project.
+```
+ USAGE: ASSU [options] <infile> [<infile> ...]
+
+ OPTIONS:
+ -d <file> SSU databank file (default: SSUdb.gz in the same directory as ASSU.sh)
+ -p <string> restricts the SSU databank to the specified pattern (default: none)
+ -o <string> output SSU sequence file name (default: ssu.fasta)
+ -O <string> writes the selected reads into the specified file name (default: none)
+ -l <int> minimum sequence length (default: 1000)
+ -L <int> minimum read length (default: AUTO)
+ -Q <int> minimum base Phred quality value (default: 20)
+ -M <int> minimum mapping Phred quality value (default: 20)
+ -D <int> minimum coverage depth (default: 50)
+ -F <float> minimum proportion of the majority base to infer that base (default: 0.8)
+ -A <float> minimum ratio of the alternative base(s) to the majority one to add that
+ base(s) to the consensus (default: 0.2)
+ -N set N when multiple bases at a position (default: not set)
+ -w <dir> path to the tmp directory (default: $TMPDIR, otherwise /tmp)
+ -t <int> thread numbers (default: 2)
+ -v verbose mode
+ -s prints the content of the SSU databank and exit
+ -c checks dependencies and exit
+ -h prints this help and exit
+
+ EXAMPLES:
+ ASSU -t 24 -o 16s.fasta fwd.fastq.gz rev.fastq.gz sgl.fastq.bz2
+ ASSU -d SSUdb.gz -O 16s.fastq -p Clostridium -L 75 -v *.fastq
+```
-## Description
-Let people know what your project can do specifically. Provide context and add a link to any reference visitors might be unfamiliar with. A list of Features or a Background subsection can also be added here. If there are alternatives to your project, this is a good place to list differentiating factors.
-## Badges
-On some READMEs, you may see small images that convey metadata, such as whether or not all the tests are passing for the project. You can use Shields to add some to your README. Many services also have instructions for adding a badge.
+## Notes
-## Visuals
-Depending on what you are making, it can be a good idea to include screenshots or even a video (you'll frequently see GIFs rather than actual videos). Tools like ttygif can help, but check out Asciinema for a more sophisticated method.
+* In brief, _ASSU_ first quickly aligns the specified HTS reads against all the reference sequences available in the SSU databank using _bwa-mem2_. This first step enables to determine the most suited reference sequence (called model), as well as the subset of HTS reads that arise from SSU genome region(s). Next, every HTS read from the subset is accurately aligned against the model sequence, and the resulting alignments are processed by _samtools_ to build a final (consensus) sequence.
+
+* _ASSU_ requires at least one HTS read file. Input file(s) should be in FASTQ format and can be compressed using [_gzip_](https://www.gnu.org/software/gzip/), [_bzip2_](https://sourceware.org/bzip2/) or [_DSRC_](https://github.com/refresh-bio/DSRC) (Roguski and Deorowicz 2014). Note that input files compressed using [_bzip2_](https://sourceware.org/bzip2/) or [_DSRC_](https://github.com/refresh-bio/DSRC) require the associated decompression tool to be read (see [Dependencies](#dependencies)).
+
+* _ASSU_ is not working with long HTS reads, as _bwa-mem2_ is not developed to align HTS reads on significantly shorter reference sequences. The source code of _ASSU_ can be easily modified (on request) to deal with such a case, but long HTS reads generally lead to complete SSU segments via _de novo_ assembly.
+
+* By default, _ASSU_ expects that the SSU databank file `SSUdb.gz` is located in the same directory. However, an alternative SSU databank file (e.g. different version, different file name) can be specified using option `-d`. The content of the specified SSU databank can be summarized using option `-s`.
+
+* The running time of _ASSU_ is very dependent on the size of the input files, but faster running times can be obtained using multiple threads (option `-t`) and/or a temporary directory located on a hard drive with high speed (option `-w`). Faster running times can be also observed by restricting the reference SSU sequences using a specified pattern (e.g. sequence accession, genus name; option `-p`).
+
+* The assembled sequence is written in FASTA format into an output file (option `-o`; default name: `ssu.fasta`). Optionally, the selected HTS reads can be written in FASTQ format into a specified output file (option `-O`).
+
+* As the assembled SSU sequence is often the consensus of several copies with sequence variation within the sequenced genome (e.g. Větrovský and Baldrian 2013), it may contain ambiguous positions resulting from the consensus of different sequenced bases at those positions. In such cases, degenerated nucleotides are used to represent the consensus of different character states (see e.g. [Table 1](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2865858/table/T1/) in Johnson 2010), or lowercase characters when a deletion (i.e. gap) is involved in the consensus. Note that every degenerated nucleotide can be replaced by the character state `N` using option `-N`.
+
+* The (number of) ambiguous positions can be slightly modified by considering shorter HTS reads (option `-L`), putative sequencing errors (option `-Q`), weak alignments (option `-M`) or low coverage depth (option `-D`). The consensus definition can be modified by tuning the two options `-F` and `-A` (corresponding to the options `--call-fract` and `--het-fract` of [samtools consensus](http://www.htslib.org/doc/samtools-consensus.html), respectively).
+
+* No output file is written in several situations: <br>   • insufficient coverage depth (default: at least 50×; option `-D`), <br>   • too short assembled SSU sequence (default: at least 1,000 bps; option `-l`), <br>   • too many ambiguous positions (i.e. more than 5%).
+
+
+
+## Example
+
+In order to illustrate the usefulness of _ASSU_, the following example describes its usage for assembling the 16S rRNA (consensus) segment of _Escherichia coli_ [O113:H21](https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=1078021) strain FWSEC0011. Its genome assembly ([GCF_005171095.1](https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_005171095.1/)) consists of one chromosome ([NZ_CP031892.1](https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP031892.1/)) and one plasmid ([NZ_CP031893.1](https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP031893.1/)), built from short and long HTS reads ([SRS3815841](https://www.ncbi.nlm.nih.gov/sra/SRS3815841)).
+
+**Downloading input files**
+
+Paired-end sequencing of this genome was performed using Illumina Miseq, and the resulting pair of (compressed) FASTQ files (225 Mb and 249 Mb, respectively) can be downloaded using the following command lines:
+
+```bash
+wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR789/009/SRR7896249/SRR7896249_1.fastq.gz
+wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR789/009/SRR7896249/SRR7896249_2.fastq.gz
+```
+
+**Running _ASSU_**
+
+Use the following command line to run _ASSU_ on these two FASTQ files using 12 threads:
+
+```bash
+./ASSU.sh -t 12 -o FWSEC0011.ssu.fasta -v SRR7896249_*.fastq.gz
+```
+
+Note that the SSU databank used for this assembly is the version 2024-02-16 (20,404 sequences).
+As the verbose mode was set (option `-v`), this command line leads to the following output:
+
+```
+# ASSU v1.0
+# Copyright (C) 2024 Institut Pasteur
++ https://gitlab.pasteur.fr/GIPhy/ASSU
+> Syst: x86_64-redhat-linux-gnu
+> Bash: 4.4.20(1)-release
+[00:00] checking input files ... [ok]
++ SRR7896249_1.fastq.gz
++ SRR7896249_2.fastq.gz
+[00:00] creating tmp directory .... [ok]
+> TMP_DIR=/tmp/ASSU.f2PzH4mWpX
+[00:00] examining SSU databank ...... [ok]
+> model: Bacteria | Escherichia fergusonii | NR_074902.1 | 1542 bps
+[00:24] building SSU sequence .... [ok]
+> 3016 selected reads (903953 bases; lgt > 269)
+> coverage depth: 586x
+> 1543 bps (ambiguous bases: 14)
+ 10 20 30 40 50 60 70 80 90 100
+ | | | | | | | | | |
+ 1 AAATTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGRAARCAGCTTGCTGYTTYGCTGACG
+ * * * *
+ 101 AGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAG
+
+ 201 GGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTWGTWGGTGGGGTAACGGCTCACCWAGGCGACGATCCCTAGCTGGTCTGAGA
+ * * *
+ 301 GGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGC
+
+ 401 CGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAG
+
+ 501 CACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCA
+
+ 601 GATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCG
+
+ 701 TAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGG
+
+ 801 TAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCA
+
+ 901 AGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCA
+
+ 1001 CRGAASTTTYCAGAGATGaGAWTgGTGCCTTCGGGAACYGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCG
+ * * * * * * *
+ 1101 CAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGT
+
+ 1201 CATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTA
+
+ 1301 GTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACAC
+
+ 1401 CGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTA
+
+ 1501 ACAAGGTAACCGTAGGGGAACCTGCGGTTGGATCACCTCCTTA
+
+[00:27] writing output file ... [ok]
++ FASTA: FWSEC0011.ssu.fasta
+[00:27] exit
+```
+
+The SSU sequence [NR_074902.1](https://www.ncbi.nlm.nih.gov/nuccore/NR_074902.1/) was selected as a model to carry out the reference-guided assembly using 3,016 HTS reads, leading to an assembled (consensus) sequence of length 1,543 bps (coverage depth: 586×) written into the FASTA file _FWSEC0011.ssu.fasta_.
+The overall running time was < 30 seconds.
+
+The assembled SSU sequence contains 14 ambiguous bases, highlighted with a `*` in the above output. This suggests that the genome of _E. coli_ [O113:H21](https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=1078021) strain FWSEC0011 contains different 16S rRNA copies with sequence variations.
+
+In fact, its chromosome ([NZ_CP031892.1](https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP031892.1/)) contains seven 16S rRNA segments labeled with the following locus tags:
+<br> • [C8202_RS02200](https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP031892.1?from=445743&to=447284)
+<br> • [C8202_RS06240](https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP031892.1?from=1257192&to=1258733)
+<br> • [C8202_RS19645](https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP031892.1?from=3974035&to=3975576)
+<br> • [C8202_RS23325](https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP031892.1?from=4722664&to=4724205)
+<br> • [C8202_RS23525](https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP031892.1?from=4764044&to=4765585)
+<br> • [C8202_RS24135](https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP031892.1?from=4901279&to=4902820)
+<br> • [C8202_RS24625](https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP031892.1?from=4999827&to=5001368)
+
+
+Below is represented a multiple sequence alignment (MSA) of these seven 16S rRNA segments together with the assembled SSU sequence, showing that the 14 ambiguous bases (`*`) reflects as expected the variability between the different copies.
+
+<sup>
+
+```
+ 10 20 30 40 50 60 70 80 90 100
+ | | | | | | | | | |
+SSU AAATTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGRAARCAGCTTGCTGYTTYGCTGACG
+ |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||*||*||||||||||*||*|||||||
+RS02200 AAATTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGGAAACAGCTTGCTGTTTCGCTGACG
+RS06240 AAATTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGGAAACAGCTTGCTGTTTCGCTGACG
+RS19645 AAATTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGGAAGCAGCTTGCTGCTTCGCTGACG
+RS23325 AAATTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGAAAGCAGCTTGCTGCTTTGCTGACG
+RS23525 AAATTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGAAAGCAGCTTGCTGCTTTGCTGACG
+RS24135 AAATTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGGAAGCAGCTTGCTGCTTCGCTGACG
+RS24625 AAATTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGGAAGCAGCTTGCTGCTTTGCTGACG
+
+ 110 120 130 140 150 160 170 180 190 200
+ | | | | | | | | | |
+SSU AGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAG
+ ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
+RS02200 AGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAG
+RS06240 AGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAG
+RS19645 AGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAG
+RS23325 AGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAG
+RS23525 AGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAG
+RS24135 AGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAG
+RS24625 AGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAG
+
+ 210 220 230 240 250 260 270 280 290 300
+ | | | | | | | | | |
+SSU GGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTWGTWGGTGGGGTAACGGCTCACCWAGGCGACGATCCCTAGCTGGTCTGAGA
+ |||||||||||||||||||||||||||||||||||||||||||||||||*||*|||||||||||||||||||*|||||||||||||||||||||||||||
+RS02200 GGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGA
+RS06240 GGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGA
+RS19645 GGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGA
+RS23325 GGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTTGTTGGTGGGGTAACGGCTCACCAAGGCGACGATCCCTAGCTGGTCTGAGA
+RS23525 GGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTTGTTGGTGGGGTAACGGCTCACCAAGGCGACGATCCCTAGCTGGTCTGAGA
+RS24135 GGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTTGTTGGTGGGGTAACGGCTCACCAAGGCGACGATCCCTAGCTGGTCTGAGA
+RS24625 GGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTTGTTGGTGGGGTAACGGCTCACCAAGGCGACGATCCCTAGCTGGTCTGAGA
+
+ 310 320 330 340 350 360 370 380 390 400
+ | | | | | | | | | |
+SSU GGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGC
+ ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
+RS02200 GGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGC
+RS06240 GGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGC
+RS19645 GGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGC
+RS23325 GGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGC
+RS23525 GGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGC
+RS24135 GGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGC
+RS24625 GGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGC
+
+ 410 420 430 440 450 460 470 480 490 500
+ | | | | | | | | | |
+SSU CGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAG
+ ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
+RS02200 CGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAG
+RS06240 CGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAG
+RS19645 CGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAG
+RS23325 CGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAG
+RS23525 CGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAG
+RS24135 CGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAG
+RS24625 CGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTACTCATTGACGTTACCCGCAGAAGAAG
+
+ 510 520 530 540 550 560 570 580 590 600
+ | | | | | | | | | |
+SSU CACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCA
+ ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
+RS02200 CACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCA
+RS06240 CACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCA
+RS19645 CACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCA
+RS23325 CACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCA
+RS23525 CACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCA
+RS24135 CACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCA
+RS24625 CACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCA
+
+ 610 620 630 640 650 660 670 680 690 700
+ | | | | | | | | | |
+SSU GATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCG
+ ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
+RS02200 GATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCG
+RS06240 GATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCG
+RS19645 GATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCG
+RS23325 GATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCG
+RS23525 GATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCG
+RS24135 GATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCG
+RS24625 GATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCG
+
+ 710 720 730 740 750 760 770 780 790 800
+ | | | | | | | | | |
+SSU TAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGG
+ ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
+RS02200 TAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGG
+RS06240 TAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGG
+RS19645 TAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGG
+RS23325 TAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGG
+RS23525 TAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGG
+RS24135 TAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGG
+RS24625 TAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGG
+
+ 810 820 830 840 850 860 870 880 890 900
+ | | | | | | | | | |
+SSU TAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCA
+ ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
+RS02200 TAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCA
+RS06240 TAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCA
+RS19645 TAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCA
+RS23325 TAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCA
+RS23525 TAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCA
+RS24135 TAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCA
+RS24625 TAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCA
+
+ 900 920 930 940 950 960 970 980 990 1000
+ | | | | | | | | | |
+SSU AGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCA
+ ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
+RS02200 AGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCA
+RS06240 AGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCA
+RS19645 AGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCA
+RS23325 AGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCA
+RS23525 AGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCA
+RS24135 AGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCA
+RS24625 AGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCA
+
+ 1010 1020 1030 1040 1050 1060 1070 1080 1090 1100
+ | | | | | | | | | |
+SSU CRGAASTTTYCAGAGATGaGAWTgGTGCCTTCGGGAACYGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCG
+ |*|||*|||*||||||||*||*|*||||||||||||||*|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
+RS02200 CAGAACTTTCCAGAGATG-GATTGGTGCCTTCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCG
+RS06240 CAGAACTTTCCAGAGATG-GATTGGTGCCTTCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCG
+RS19645 CGGAAGTTTTCAGAGATGAGAAT-GTGCCTTCGGGAACCGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCG
+RS23325 CGGAAGTTTTCAGAGATGAGAAT-GTGCCTTCGGGAACCGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCG
+RS23525 CGGAAGTTTTCAGAGATGAGAAT-GTGCCTTCGGGAACCGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCG
+RS24135 CGGAAGTTTTCAGAGATGAGAAT-GTGCCTTCGGGAACCGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCG
+RS24625 CGGAAGTTTTCAGAGATGAGAAT-GTGCCTTCGGGAACCGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCG
+
+ 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200
+ | | | | | | | | | |
+SSU CAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGT
+ ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
+RS02200 CAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGT
+RS06240 CAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGT
+RS19645 CAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGT
+RS23325 CAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGT
+RS23525 CAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGT
+RS24135 CAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGT
+RS24625 CAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGT
+
+ 1210 1220 1230 1240 1250 1260 1270 1280 1290 1300
+ | | | | | | | | | |
+SSU CATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTA
+ ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
+RS02200 CATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTA
+RS06240 CATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTA
+RS19645 CATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTA
+RS23325 CATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTA
+RS23525 CATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTA
+RS24135 CATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTA
+RS24625 CATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTA
+
+ 1310 1320 1330 1340 1350 1360 1370 1380 1390 1400
+ | | | | | | | | | |
+SSU GTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACAC
+ ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
+RS02200 GTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACAC
+RS06240 GTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACAC
+RS19645 GTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACAC
+RS23325 GTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACAC
+RS23525 GTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACAC
+RS24135 GTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACAC
+RS24625 GTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACAC
+
+ 1410 1410 1430 1440 1450 1460 1470 1480 1490 1500
+ | | | | | | | | | |
+SSU CGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTA
+ ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
+RS02200 CGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTA
+RS06240 CGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTA
+RS19645 CGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTA
+RS23325 CGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTA
+RS23525 CGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTA
+RS24135 CGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTA
+RS24625 CGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTA
+
+ 1510 1520 1530 1540
+ | | | |
+SSU ACAAGGTAACCGTAGGGGAACCTGCGGTTGGATCACCTCCTTA
+ |||||||||||||||||||||||||||||||||||||||||||
+RS02200 ACAAGGTAACCGTAGGGGAACCTGCGGTTGGATCACCTCCTTA
+RS06240 ACAAGGTAACCGTAGGGGAACCTGCGGTTGGATCACCTCCTTA
+RS19645 ACAAGGTAACCGTAGGGGAACCTGCGGTTGGATCACCTCCTTA
+RS23325 ACAAGGTAACCGTAGGGGAACCTGCGGTTGGATCACCTCCTTA
+RS23525 ACAAGGTAACCGTAGGGGAACCTGCGGTTGGATCACCTCCTTA
+RS24135 ACAAGGTAACCGTAGGGGAACCTGCGGTTGGATCACCTCCTTA
+RS24625 ACAAGGTAACCGTAGGGGAACCTGCGGTTGGATCACCTCCTTA
+```
+</sup>
+
+
+As the HTS reads arise from an _E. coli_ genome, _ASSU_ can also be run by using option `-p` to specify this species as model (don't forget to use quotation marks when specifying a pattern with option `-p`):
+
+```bash
+./ASSU.sh -t 12 -p "Escherichia coli" -v SRR7896249_*.fastq.gz
+```
+
+This command line leads to the following output:
+
+```
+# ASSU v1.0
+# Copyright (C) 2024 Institut Pasteur
++ https://gitlab.pasteur.fr/GIPhy/ASSU
+> Syst: x86_64-redhat-linux-gnu
+> Bash: 4.4.20(1)-release
+[00:00] checking input files ... [ok]
++ SRR7896249_1.fastq.gz
++ SRR7896249_2.fastq.gz
+[00:00] creating tmp directory .... [ok]
+> TMP_DIR=/pasteur/appa/scratch/criscuol/ASSU.lc4DYy80KV
+[00:00] examining SSU databank ...... [ok]
+> selection pattern: Escherichia coli
+> model: Bacteria | Escherichia coli | NR_114042.1 | 1467 bps
+[00:15] building SSU sequence .... [ok]
+> 2868 selected reads (859565 bases; lgt > 269)
+> coverage depth: 585x
+> 1468 bps (ambiguous bases: 14)
+ 10 20 30 40 50 60 70 80 90 100
+ | | | | | | | | | |
+ 1 ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGRAARCAGCTTGCTGYTTYGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGG
+ * * * *
+ 101 GAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGG
+
+ 201 ATGTGCCCAGATGGGATTAGCTWGTWGGTGGGGTAACGGCTCACCWAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGA
+ * * *
+ 301 CACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTT
+
+ 401 GTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCG
+
+ 501 CGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGG
+
+ 601 GAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCG
+
+ 701 AAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACT
+
+ 801 TGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGG
+
+ 901 GGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACRGAASTTTYCAGAGATGaGAWTgGTG
+ * * * * * *
+ 1001 CCTTCGGGAACYGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTT
+ *
+ 1101 GCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTAC
+
+ 1201 ACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACT
+
+ 1301 CCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTG
+
+ 1401 CAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAG
+
+[00:18] writing output file ... [ok]
++ FASTA: ssu.fasta
+[00:18] exit
+```
+
+As expected, _ASSU_ assembles a similar 16S rRNA sequence using _E. coli_ as a model (e.g. same ambiguous positions).
+However, as the _E. coli_ model sequence ([NR_114042.1](https://www.ncbi.nlm.nih.gov/nuccore/NR_114042.1/); 1,467 bps) from the SSU databank is shorter than the _E. fergusonii_ one ([NR_074902.1](https://www.ncbi.nlm.nih.gov/nuccore/NR_074902.1/); 1,542 bps), the last assembled SSU sequence (1,468 bps) is also shorter than the previously assembled one (1,543 bps).
-## Installation
-Within a particular ecosystem, there may be a common way of installing things, such as using Yarn, NuGet, or Homebrew. However, consider the possibility that whoever is reading your README is a novice and would like more guidance. Listing specific steps helps remove ambiguity and gets people to using your project as quickly as possible. If it only runs in a specific context like a particular programming language version or operating system or has dependencies that have to be installed manually, also add a Requirements subsection.
-## Usage
-Use examples liberally, and show the expected output if you can. It's helpful to have inline the smallest example of usage that you can demonstrate, while providing links to more sophisticated examples if they are too long to reasonably include in the README.
-## Support
-Tell people where they can go to for help. It can be any combination of an issue tracker, a chat room, an email address, etc.
+## References
-## Roadmap
-If you have ideas for releases in the future, it is a good idea to list them in the README.
+Johnson AD (2010) _An extended IUPAC nomenclature code for polymorphic nucleic acids_. **Bioinformatics**, 26(10):1386-1389. [doi:10.1093/bioinformatics/btq098](https://doi.org/10.1093/bioinformatics/btq098).
-## Contributing
-State if you are open to contributions and what your requirements are for accepting them.
+Roguski L, Deorowicz S (2014) _DSRC 2: Industry-oriented compression of FASTQ files_. **Bioinformatics**, 30(15):2213-2215. [doi:10.1093/bioinformatics/btu208](https://doi.org/10.1093/bioinformatics/btu208).
-For people who want to make changes to your project, it's helpful to have some documentation on how to get started. Perhaps there is a script that they should run or some environment variables that they need to set. Make these steps explicit. These instructions could also be useful to your future self.
+Větrovský T, Baldrian P (2013) _The Variability of the 16S rRNA Gene in Bacterial Genomes and Its Consequences for Bacterial Community Analyses_. **PLoS One**, 8(2):e57923. [doi:10.1371/journal.pone.0057923](https://doi.org/10.1371/journal.pone.0057923).
-You can also document commands to lint the code or run tests. These steps help to ensure high code quality and reduce the likelihood that the changes inadvertently break something. Having instructions for running tests is especially helpful if it requires external setup, such as starting a Selenium server for testing in a browser.
-## Authors and acknowledgment
-Show your appreciation to those who have contributed to the project.
-## License
-For open source projects, say how it is licensed.
-## Project status
-If you have run out of energy or time for your project, put a note at the top of the README saying that development has slowed down or stopped completely. Someone may choose to fork your project or volunteer to step in as a maintainer or owner, allowing your project to keep going. You can also make an explicit request for maintainers.
diff --git a/SSUdb.gz b/SSUdb.gz
new file mode 100644
index 0000000000000000000000000000000000000000..a4fef7b44b49e701e1a26a1fab7dc7a472d7262b
Binary files /dev/null and b/SSUdb.gz differ
diff --git a/example/FWSEC0011.ssu.fasta b/example/FWSEC0011.ssu.fasta
new file mode 100644
index 0000000000000000000000000000000000000000..65f738a8cf6615806efa60f544ac4a946e983d69
--- /dev/null
+++ b/example/FWSEC0011.ssu.fasta
@@ -0,0 +1,2 @@
+>SSU_covr_257_lgt_1543_amb_14
+AAATTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGRAARCAGCTTGCTGYTTYGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTWGTWGGTGGGGTAACGGCTCACCWAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACRGAASTTTYCAGAGATGaGAWTgGTGCCTTCGGGAACYGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAACCGTAGGGGAACCTGCGGTTGGATCACCTCCTTA
diff --git a/makeSSUdb.sh b/makeSSUdb.sh
new file mode 100755
index 0000000000000000000000000000000000000000..70aeca9224efa9883a28aa8ff7c2b4e5dba58a4a
--- /dev/null
+++ b/makeSSUdb.sh
@@ -0,0 +1,168 @@
+#!/bin/bash
+
+##############################################################################################################
+# #
+# makeSSUdb: creating SSU databank from NCBI to be used by ASSU #
+# #
+ COPYRIGHT="Copyright (C) 2024 Institut Pasteur" #
+# #
+# This program is free software: you can redistribute it and/or modify it under the terms of the GNU #
+# General Public License as published by the Free Software Foundation, either version 3 of the License, or #
+# (at your option) any later version. #
+# #
+# This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even #
+# the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public #
+# License for more details. #
+# #
+# You should have received a copy of the GNU General Public License along with this program. If not, see #
+# <http://www.gnu.org/licenses/>. #
+# #
+# Contact: #
+# Alexis Criscuolo alexis.criscuolo@pasteur.fr #
+# Genome Informatics & Phylogenetics (GIPhy) giphy.pasteur.fr #
+# Centre de Ressources Biologiques de l'Institut Pasteur (CRBIP) research.pasteur.fr/en/b/VTq #
+# Institut Pasteur, Paris, FRANCE research.pasteur.fr #
+# #
+##############################################################################################################
+
+##############################################################################################################
+# #
+# ============ #
+# = VERSIONS = #
+# ============ #
+# #
+ VERSION=1.0; #
+# #
+##############################################################################################################
+
+##############################################################################################################
+# #
+# ================ #
+# = REQUIREMENTS = #
+# ================ #
+# #
+# -- gzip ------------------------------------------------------------------------------------------------- #
+# #
+ GZIP_BIN=gzip;
+ if [ ! $(command -v $GZIP_BIN) ]; then echo "no $GZIP_BIN detected" >&2 ; exit 1 ; fi
+ GZIP_STATIC_OPTIONS="--quiet";
+ GZIP="$GZIP_BIN $GZIP_STATIC_OPTIONS";
+ GUNZIP="$GZIP --decompress";
+# #
+# -- wget | curl ------------------------------------------------------------------------------------------ #
+# #
+ WGET_BIN=wget;
+ CURL_BIN=curl;
+ USE_WGET=true;
+ if [ $(command -v $WGET_BIN) ]
+ then
+ WGET_STATIC_OPTIONS="--quiet --retry-connrefused --no-check-certificate";
+ WGET="$WGET_BIN $WGET_STATIC_OPTIONS";
+ elif [ $(command -v $CURL_BIN) ]
+ then
+ USE_WGET=false;
+ CURL_STATIC_OPTIONS="--silent --location --continue-at -";
+ CURL="$CURL_BIN $CURL_STATIC_OPTIONS";
+ else
+ echo "neither $WGET_BIN nor $CURL_BIN detected" >&2 ;
+ exit 1 ;
+ fi
+# #
+##############################################################################################################
+
+echo "makeSSUdb v$VERSION $COPYRIGHT" ;
+
+##############################################################################################################
+# #
+# CREATING TMP DIR #
+# #
+##############################################################################################################
+TMP_DIR=$(mktemp -d -p $(pwd) XXXXXXXXXX);
+finalize() { rm -r $TMP_DIR ; }
+trap 'finalize;exit 1' SIGTERM SIGINT SIGQUIT SIGHUP TERM INT QUIT HUP ;
+
+##############################################################################################################
+# #
+# DOWNLOADING SEQUENCE FILES #
+# #
+##############################################################################################################
+FTPURL="https://ftp.ncbi.nlm.nih.gov/refseq/TargetedLoci";
+echo -n "downloading ." ;
+if $USE_WGET
+then while true ; do echo -n "." ; $WGET -O $TMP_DIR/arc.fna.gz $FTPURL/Archaea/archaea.16SrRNA.fna.gz && break ; done
+else while true ; do echo -n "." ; $CURL -o $TMP_DIR/arc.fna.gz $FTPURL/Archaea/archaea.16SrRNA.fna.gz && break ; done
+fi
+echo -n "." ;
+if $USE_WGET
+then while true ; do echo -n "." ; $WGET -O $TMP_DIR/bac.fna.gz $FTPURL/Bacteria/bacteria.16SrRNA.fna.gz && break ; done
+else while true ; do echo -n "." ; $CURL -o $TMP_DIR/bac.fna.gz $FTPURL/Bacteria/bacteria.16SrRNA.fna.gz && break ; done
+fi
+echo ". [ok]" ;
+
+##############################################################################################################
+# #
+# READING SEQUENCE FILES #
+# #
+##############################################################################################################
+echo -n "reading .." ;
+$GUNZIP -c $TMP_DIR/arc.fna.gz |
+ gawk '!/^>/ {s=s$0;next}
+ (s!=""){print s;s=""}
+ {print}
+ END {print s}' | # | starts with A == archaea
+ paste - - | tr '\t' ' ' | sed 's/>/A /g;s/16S ribosomal RNA, //g;s/ sequence//g' | tr -d []\' | sort -k3 > $TMP_DIR/arc.txt ;
+echo -n ".." ;
+rm -f $TMP_DIR/arc.fna.gz ;
+echo -n ".." ;
+$GUNZIP -c $TMP_DIR/bac.fna.gz |
+ gawk '!/^>/ {s=s$0;next}
+ (s!=""){print s;s=""}
+ {print}
+ END {print s}' | # | starts with B == bacteria
+ paste - - | tr '\t' ' ' | sed 's/>/B /g;s/16S ribosomal RNA, //g;s/ sequence//g' | tr -d []\' | sort -k3 > $TMP_DIR/bac.txt ;
+echo -n ".." ;
+rm -f $TMP_DIR/bac.fna.gz ;
+echo ". [ok]" ;
+
+##############################################################################################################
+# #
+# PROCESSING SEQUENCE FILES #
+# #
+##############################################################################################################
+echo -n "processing ..." ;
+for f in $TMP_DIR/arc.txt $TMP_DIR/bac.txt
+do
+ gawk 'function c(s) {return length(s)-gsub("N","N",s)}
+ (NR==1) {best=$0;gnsp=$3":"$4;max=c($NF);next}
+ ($3":"$4!=gnsp){print best;
+ best=$0;gnsp=$3":"$4;max=c($NF);next}
+ (max<c($NF)) {best=$0; max=c($NF)}
+ END {print best}' $f ;
+done | gawk '{printf">"$2" "$1;
+ i=2;while(++i < NF)printf" "$i;print""; print$NF}' > $TMP_DIR/ssu.db ;
+echo "... [ok]" ;
+
+##############################################################################################################
+# #
+# FINALIZING #
+# #
+##############################################################################################################
+echo -n "compressing .." ;
+n=$(grep -c "^>" $TMP_DIR/ssu.db);
+echo -n "." ;
+$GZIP -c $TMP_DIR/ssu.db > SSUdb.gz ;
+echo -n "." ;
+{ echo -n "SSUdb.gz " ;
+ date +"%Y-%m-%d %H:%M:%S" ;
+ echo "$n sequences" ;
+ echo "built using makeSSUdb v$VERSION" ; } > SSUdb.version.txt ;
+finalize ;
+echo ". [ok]" ;
+
+echo ">> SSU databank: SSUdb.gz" ;
+echo ">> SSUdb version: SSUdb.version.txt" ;
+echo ">> no. sequences: $n" ;
+
+echo "exit" ;
+
+exit ;