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TAXAassign.sh
executable file
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TAXAassign.sh
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#!/bin/bash
# ***************************************************************
# Name: TAXAassign.sh
# Purpose: Script to annotate sequences at different taxonomic levels using NCBI's taxonomy
#
# Dependencies:
# GNU Parallel
# Install: http://www.gnu.org/software/parallel/
# Blastn 2.28+ (The previous versions dont give Taxa IDs)
# Software: http://www.ncbi.nlm.nih.gov/books/NBK1763/
# For nt database, go to your local blastn db folder and use update_blastdb.pl:
# update_blastdb.pl --showall
# update_blastdb.pl nt
# Blastn output format used:
# blastn -db <path_to_nt> -query <fasta_file>
# -out <blast_file> -outfmt "6 qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore qcovs staxids" <other options>
# BioSQL
# Download BioSQL from http://biosql.org/DIST/biosql-1.0.1.tar.gz. Once the software is installed,
# setup a database and import the BioSQL schema. The following command line should create a new database
# on your own computer called bioseqdb, belonging to the root user account:
# mysqladmin -u root create bioseqdb
# We can then tell MySQL to load the BioSQL scheme we downloaded above. Change to the scripts subdirectory
# from the unzipped BioSQL download, then use the command:
# mysql -u root bioseqdb < biosqldb-mysql.sql
# To update the NCBI taxonomy, change to the scripts subdirectory from the unzipped BioSQL download, then use
# the command (output is also shown):
# ./load_ncbi_taxonomy.pl --dbname bioseqdb --driver mysql --dbuser root --download true
# Loading NCBI taxon database in taxdata:
# ... retrieving all taxon nodes in the database
# ... reading in taxon nodes from nodes.dmp
# ... insert / update / delete taxon nodes
# ... (committing nodes)
# ... rebuilding nested set left/right values
# ... reading in taxon names from names.dmp
# ... deleting old taxon names
# ... inserting new taxon names
# ... cleaning up
# Done.
#
# You can also use sqlite3 to store the database in case you don't want to go for MySQL server option.
# Last time I checked BioSQL didn't have any option to load database schema in sqlite directly or loading data with load_ncbi_taxonomy.pl script
# A work around is to dump your MySQL database to sqlite3 and place the database as db.sqlite in the database folder.
# You can then edit the parameters section in blast_concat_taxon.py and set use_MySQL=False. The section is as follows
#
# # Parameters #########################################
# use_MySQL=True
# #MySQL server settings for BioSQL
# MySQL_server='localhost'
# MySQL_user='root'
# MySQL_password=''
# MySQL_database='bioseqdb'
# #sqlite3 database
# sqlite3_database=os.getcwd()+"/../database/db.sqlite"
# #####################################################
#
# There are several conversion scripts out there to export data from MySQL to sqlite3 but not all of them work. The only thing that worked for me
# was ruby-gem. The commands are as follows:
#
# sudo gem install sequel
# sudo gem install sqlite3
# sudo gem install mysql
# sequel mysql://root@localhost/bioseqdb -C sqlite://db.sqlite
#
# Make sure that you have development version of both sqlite3 and MySQL installed.
#
# Version: 0.4
# Authors: Umer Zeeshan Ijaz (Umer.Ijaz@glasgow.ac.uk)
# http://userweb.eng.gla.ac.uk/umer.ijaz
# Last modified: 2013-07-18
# License: Copyright (c) 2013 Computational Microbial Genomics Group, University of Glasgow, UK
#
# 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/>.
# **************************************************************/
HELPDOC=$( cat <<EOF
Script to annotate sequences at different taxonomic levels using NCBI's taxonomy
Usage:
bash `basename $0` -f <fasta_file.fasta> [options]
Options:
-p Turn parallel processing on
-c Number of cores to use (Default: 10)
-r Number of reference matches (Default: 10)
-m Minimum percentage identity in blastn (Default: 97)
-q Minimum query coverage in blastn (Default: 97)
-a Threshold at different taxonomic levels (Default:"-m,-m,-m,-m,-m,-m" where -m is the minimum percentage identity argument)
The order is as follows: Phylum,Class,Order,Family,Genus,Species
For example, -a "60,70,80,95,95,97"
-t Consensus threshold (Default: 90)
EOF
)
set -o errexit
# = Parameters to set ============== #
LOGFILE="`pwd`/TAXAassign.log" # Where to save the log
BLASTN_DIR="/home/opt/ncbi-blast-2.2.28+/bin"; # Path where blastn is installed
BLASTDB_DIR="/home/opt/ncbi-blast-2.2.28+/db"; # Path where nt is installed
FASTA_FILE="" # This field should be empty
PARALLELIZE_FLAG=0
NUMBER_OF_CORES=10
NUMBER_OF_REFERENCE_MATCHES=10
MINIMUM_PERCENT_IDENT=97
MINIMUM_QUERY_COVERAGE=97
CONSENSUS_THRESHOLD=90
TAXONOMIC_LEVELS_THRESHOLD=""
# =/Parameters to set ============== #
CURRENT_DIR=`pwd`
# = Enable FP support ============== #
# By default, there is limited capability in bash to handle floating point
# operations. In this script bc is used to calculate the floating point operations.
# $float_scale parameter specifies the precision of the floating point.
# Reference: http://www.linuxjournal.com/content/floating-point-math-bash
float_scale=5
# Evaluate a floating point number expression.
function float_eval()
{
local stat=0
local result=0.0
if [[ $# -gt 0 ]]; then
result=$(echo "scale=$float_scale; $*" | bc -q 2>/dev/null)
stat=$?
if [[ $stat -eq 0 && -z "$result" ]]; then stat=1; fi
fi
echo $result
return $stat
}
# Evaluate a floating point number conditional expression.
function float_cond()
{
local cond=0
if [[ $# -gt 0 ]]; then
cond=$(echo "$*" | bc -q 2>/dev/null)
if [[ -z "$cond" ]]; then cond=0; fi
if [[ "$cond" != 0 && "$cond" != 1 ]]; then cond=0; fi
fi
local stat=$((cond == 0))
return $stat
}
function ceil () {
echo "define ceil (x) {if (x<0) {return x/1} \
else {if (scale(x)==0) {return x} \
else {return x/1 + 1 }}} ; ceil($1)" | bc;
}
# =/Enable FP support ============== #
# Create directories if they don't exist yet
function create_dirs() {
local dir
for dir in "$@"
do
if [ ! -d "$dir" ]; then
mkdir "$dir"
fi
done
}
# Check if files exist
function check_prog() {
local prog
for prog in "$@"
do
if which $prog >/dev/null; then
TAXAassign_print 'Using ' $prog
else
echo "$prog not in your path" >&2; exit 1;
fi
done
}
function skip_gen_file(){
if [ -f "$1" ]; then
echo "true"
else
echo "false"
fi
}
function skip_gen_dir(){
if [ -d "$1" ]; then
echo "true"
else
echo "false"
fi
}
function TAXAassign_print() {
echo [`date "+%Y-%m-%d %H:%M:%S"`] "$@" | tee -a $LOGFILE
}
# Parse options
while getopts ":phc:r:m:f:t:q:a:" opt; do
case $opt in
p)
PARALLELIZE_FLAG=1
;;
f)
FASTA_FILE=$OPTARG
;;
m)
MINIMUM_PERCENT_IDENT=$OPTARG
;;
c)
NUMBER_OF_CORES=$OPTARG
;;
r)
NUMBER_OF_REFERENCE_MATCHES=$OPTARG
;;
t)
CONSENSUS_THRESHOLD=$OPTARG
;;
q)
MINIMUM_QUERY_COVERAGE=$OPTARG
;;
a)
TAXONOMIC_LEVELS_THRESHOLD=$OPTARG
;;
h)
echo "$HELPDOC"
exit 0
;;
\?)
echo "$HELPDOC"
echo "Invalid option: -$OPTARG" >&2
exit 1
;;
esac
done
if [ -z $FASTA_FILE ]
then
echo "$HELPDOC"
exit 1
fi
if [ -z "$TAXONOMIC_LEVELS_THRESHOLD" ]; then
TAXONOMIC_LEVEL_THRESHOLD="$MINIMUM_PERCENT_IDENT,$MINIMUM_PERCENT_IDENT,$MINIMUM_PERCENT_IDENT,$MINIMUM_PERCENT_IDENT,$MINIMUM_PERCENT_IDENT,$MINIMUM_PERCENT_IDENT"
fi
OIFS=$IFS;
IFS=",";
TLTArray=($TAXONOMIC_LEVELS_THRESHOLD);
IFS=$OIFS;
if [ "${#TLTArray[@]}" != "6" ]; then
echo "$HELPDOC"
exit 1
fi
for i in "${TLTArray[@]}"
do
:
if ! [[ $i =~ ^-?[0-9]+$ ]]; then
echo "$HELPDOC"
exit 1
fi
done
if ! [[ $MINIMUM_PERCENT_IDENT =~ ^-?[0-9]+$ ]] || ! [[ $NUMBER_OF_CORES =~ ^-?[0-9]+$ ]] || ! [[ $NUMBER_OF_REFERENCE_MATCHES =~ ^-?[0-9]+$ ]] || ! [[ $CONSENSUS_THRESHOLD =~ ^-?[0-9]+$ ]] || ! [[ $MINIMUM_QUERY_COVERAGE =~ ^-?[0-9]+$ ]]; then
echo "$HELPDOC"
exit 1
fi
# Using /usr/bin/dirname to get the full path to this script location
# without being affected from where this script was invoked
export TAXAASSIGN_DIR=$(cd "$(dirname "$0")"; pwd)
TAXAassign_print "TAXAassign v0.4. Copyright (c) 2013 Computational Microbial Genomics Group, University of Glasgow, UK"
check_prog $BLASTN_DIR/blastn
check_prog $TAXAASSIGN_DIR/scripts/blast_concat_taxon.py
check_prog $TAXAASSIGN_DIR/scripts/blast_gen_assignments.pl
if [ $PARALLELIZE_FLAG -eq 1 ]
then
check_prog parallel
fi
fileName=`echo "$(basename $FASTA_FILE)" | cut -d'.' -f1`
# Run blastn
TAXAassign_print "Blast against NCBI's nt database with minimum percent ident of $MINIMUM_PERCENT_IDENT%, maximum of $NUMBER_OF_REFERENCE_MATCHES reference sequences, and evalue of 0.0001 in blastn."
# Format for blastn
blastOutFmt="\"6 qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore qcovs staxids\""
blastFileName=$fileName'_B'
if [ "$(skip_gen_file $blastFileName'.out')" == "true" ];then
TAXAassign_print $blastFileName'.out' already exists. Skipping this step.
elif [ $PARALLELIZE_FLAG -eq 1 ]; then
# Get the file size in KB
sizeFileBytes=$(du -b ${FASTA_FILE} | sed 's/\([0-9]*\)\(.*\)/\1/')
sizeChunks=$(ceil $(float_eval "$sizeFileBytes / ($NUMBER_OF_CORES * 1024)"))
sizeChunksString="${sizeChunks}k"
startTime=`date +%s`
cat $FASTA_FILE | parallel --block $sizeChunksString --recstart '>' --pipe $BLASTN_DIR/blastn -perc_identity $MINIMUM_PERCENT_IDENT -evalue 0.00001 -dust no -num_threads 1 -outfmt $blastOutFmt -max_target_seqs $NUMBER_OF_REFERENCE_MATCHES -db $BLASTDB_DIR'/nt' -query - > $blastFileName'.out'
TAXAassign_print "blastn using GNU parallel took $(expr `date +%s` - $startTime) seconds for $FASTA_FILE".
TAXAassign_print $blastFileName'.out' generated successfully!
else
startTime=`date +%s`
$BLASTN_DIR/blastn -db $BLASTDB_DIR'/nt' -query $FASTA_FILE -perc_identity $MINIMUM_PERCENT_IDENT -out $blastFileName'.out' -outfmt "6 qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore qcovs staxids" -max_target_seqs $NUMBER_OF_REFERENCE_MATCHES -evalue 0.00001 -dust no -num_threads 1
TAXAassign_print "blastn took $(expr `date +%s` - $startTime) seconds for $FASTA_FILE".
TAXAassign_print $blastFileName'.out' generated successfully!
fi
TAXAassign_print "Filter blastn hits with minimum query coverage of $MINIMUM_QUERY_COVERAGE%."
blastFilteredFileName=$fileName'_BF'
if [ "$(skip_gen_file $blastFilteredFileName'.out')" == "true" ];then
TAXAassign_print $blastFilteredFileName'.out' already exists. Skipping this step.
else
cat $blastFileName'.out' | awk -F"\t" -v pattern=$MINIMUM_QUERY_COVERAGE '$13>pattern{print $0}' > $blastFilteredFileName'.out'
TAXAassign_print $blastFilteredFileName'.out' generated successfully!
fi
blastFileNameWithTaxonomy=$fileName'_BFT'
TAXAassign_print "Annotate blastn hits with NCBI's taxonomy data."
if [ "$(skip_gen_file $blastFileNameWithTaxonomy'.out')" == "true" ];then
TAXAassign_print $blastFileNameWithTaxonomy'.out' already exists. Skipping this step.
elif [ $PARALLELIZE_FLAG -eq 1 ]; then
cat $blastFilteredFileName'.out' | parallel -j $NUMBER_OF_CORES python $TAXAASSIGN_DIR/scripts/blast_concat_taxon.py -b {} > $blastFileNameWithTaxonomy'.out'
TAXAassign_print $blastFileNameWithTaxonomy'.out' generated successfully!
else
python $TAXAASSIGN_DIR/scripts/blast_concat_taxon.py -b $blastFilteredFileName'.out' > $blastFileNameWithTaxonomy'.out'
TAXAassign_print $blastFileNameWithTaxonomy'.out' generated successfully!
fi
TAXAassign_print "Generate taxonomic assignment tables from blastn hits with consensus threshold of $CONSENSUS_THRESHOLD%."
if [[ "$(skip_gen_file $fileName'_ASSIGNMENTS.csv')" == "true" || "$(skip_gen_file $fileName'_PHYLUM.csv')" == "true" || "$(skip_gen_file $fileName'_CLASS.csv')" == "true" || "$(skip_gen_file $fileName'_ORDER.csv')" == "true" || "$(skip_gen_file $fileName'_FAMILY.csv')" == "true" || "$(skip_gen_file $fileName'_GENUS.csv')" == "true" || "$(skip_gen_file $fileName'_SPECIES.csv')" == "true" ]];then
TAXAassign_print Assignment files already exists. Skipping this step.
else
perl $TAXAASSIGN_DIR/scripts/blast_gen_assignments.pl -b $blastFileNameWithTaxonomy'.out' -c $CONSENSUS_THRESHOLD -a "$TAXONOMIC_LEVELS_THRESHOLD" | grep -v "^HASH(" > $fileName'_ASSIGNMENTS.csv'
totalReads=$(grep -c ">" $FASTA_FILE)
phylumLevelAssignments=$(cut -d, -f2 $fileName'_ASSIGNMENTS.csv' | grep -v "__Unclassified__" | wc -l)
classLevelAssignments=$(cut -d, -f3 $fileName'_ASSIGNMENTS.csv' | grep -v "__Unclassified__" | wc -l)
orderLevelAssignments=$(cut -d, -f4 $fileName'_ASSIGNMENTS.csv' | grep -v "__Unclassified__" | wc -l)
familyLevelAssignments=$(cut -d, -f5 $fileName'_ASSIGNMENTS.csv' | grep -v "__Unclassified__" | wc -l)
genusLevelAssignments=$(cut -d, -f6 $fileName'_ASSIGNMENTS.csv' | grep -v "__Unclassified__" | wc -l)
speciesLevelAssignments=$(cut -d, -f7 $fileName'_ASSIGNMENTS.csv' | grep -v "__Unclassified__" | wc -l)
cut -d, -f2 $fileName'_ASSIGNMENTS.csv' | grep -v "__Unclassified__" | sed 's/ /_____/g'| sort | uniq -c | awk '{gsub("_____"," ",$2);print $2","$1}' > $fileName'_PHYLUM.csv'
if [ "$totalReads" -ne "$phylumLevelAssignments" ]; then
echo "__Unclassified__,$(float_eval "$totalReads - $phylumLevelAssignments ")" >> $fileName'_PHYLUM.csv'
fi
cut -d, -f3 $fileName'_ASSIGNMENTS.csv' | grep -v "__Unclassified__" | sed 's/ /_____/g'| sort | uniq -c | awk '{gsub("_____"," ",$2);print $2","$1}' > $fileName'_CLASS.csv'
if [ "$totalReads" -ne "$classLevelAssignments" ]; then
echo "__Unclassified__,$(float_eval "$totalReads - $classLevelAssignments ")" >> $fileName'_CLASS.csv'
fi
cut -d, -f4 $fileName'_ASSIGNMENTS.csv' | grep -v "__Unclassified__" | sed 's/ /_____/g'| sort | uniq -c | awk '{gsub("_____"," ",$2);print $2","$1}' > $fileName'_ORDER.csv'
if [ "$totalReads" -ne "$orderLevelAssignments" ]; then
echo "__Unclassified__,$(float_eval "$totalReads - $orderLevelAssignments ")" >> $fileName'_ORDER.csv'
fi
cut -d, -f5 $fileName'_ASSIGNMENTS.csv' | grep -v "__Unclassified__" | sed 's/ /_____/g'| sort | uniq -c | awk '{gsub("_____"," ",$2);print $2","$1}' > $fileName'_FAMILY.csv'
if [ "$totalReads" -ne "$familyLevelAssignments" ]; then
echo "__Unclassified__,$(float_eval "$totalReads - $familyLevelAssignments ")" >> $fileName'_FAMILY.csv'
fi
cut -d, -f6 $fileName'_ASSIGNMENTS.csv' | grep -v "__Unclassified__" | sed 's/ /_____/g'| sort | uniq -c | awk '{gsub("_____"," ",$2);print $2","$1}' > $fileName'_GENUS.csv'
if [ "$totalReads" -ne "$genusLevelAssignments" ]; then
echo "__Unclassified__,$(float_eval "$totalReads - $genusLevelAssignments ")" >> $fileName'_GENUS.csv'
fi
cut -d, -f7 $fileName'_ASSIGNMENTS.csv' | grep -v "__Unclassified__" | sed 's/ /_____/g'| sort | uniq -c | awk '{gsub("_____"," ",$2);print $2","$1}' > $fileName'_SPECIES.csv'
if [ "$totalReads" -ne "$speciesLevelAssignments" ]; then
echo "__Unclassified__,$(float_eval "$totalReads - $speciesLevelAssignments ")" >> $fileName'_SPECIES.csv'
fi
TAXAassign_print $fileName'_ASSIGNMENTS.csv', $fileName'_PHYLUM.csv', $fileName'_CLASS.csv', $fileName'_ORDER.csv', $fileName'_FAMILY.csv', $fileName'_GENUS.csv', and $fileName'_SPECIES.csv' generated successfully!
TAXAassign_print "Sequences assigned at phylum level: $phylumLevelAssignments/$totalReads ($(float_eval "($phylumLevelAssignments / $totalReads) * 100")%)"
TAXAassign_print "Sequences assigned at class level: $classLevelAssignments/$totalReads ($(float_eval "($classLevelAssignments / $totalReads) * 100")%)"
TAXAassign_print "Sequences assigned at order level: $orderLevelAssignments/$totalReads ($(float_eval "($orderLevelAssignments / $totalReads) * 100")%)"
TAXAassign_print "Sequences assigned at family level: $familyLevelAssignments/$totalReads ($(float_eval "($familyLevelAssignments / $totalReads) * 100")%)"
TAXAassign_print "Sequences assigned at genus level: $genusLevelAssignments/$totalReads ($(float_eval "($genusLevelAssignments / $totalReads) * 100")%)"
TAXAassign_print "Sequences assigned at species level: $speciesLevelAssignments/$totalReads ($(float_eval "($speciesLevelAssignments / $totalReads) * 100")%)"
fi