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run_spotrna2.sh
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run_spotrna2.sh
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#!/bin/bash
start=`date +%s`
input="$(cd "$(dirname "$1")"; pwd)/$(basename "$1")"
input_dir=$(dirname $input)
seq_id=$(basename $(basename $input) | cut -d. -f1)
program_dir=$(dirname $(readlink -f $0))
path_blastn=$program_dir/ncbi-blast-*+/bin # set path to the folder contains executable binary files of Blast package
path_blastn_database=$program_dir/nt_database/nt # set path to the formatted NCBI's database file without extension
path_infernal=$program_dir/infernal-*-linux-intel-gcc/binaries # set path to the folder contains executable binary files Infernal package
path_infernal_database=$program_dir/nt_database/nt # set path to the NCBI's database database file
mkdir -p $input_dir/${seq_id}_features && mkdir -p $input_dir/${seq_id}_outputs
echo ">"$seq_id > $input_dir/${seq_id}_features/$seq_id.fasta
awk -i inplace '/^>/ {printf("\n%s\n",$0);next; } { printf("%s",$0);} END {printf("\n");}' $input
tail -n1 $input >> $input_dir/${seq_id}_features/$seq_id.fasta
feature_dir=$input_dir/${seq_id}_features
output_dir=$input_dir/${seq_id}_outputs
#exit 1
if [ ! -f $path_blastn_database ]; then
echo ""
echo "========================================================================================"
echo " Looks like nt database doesn't exists in the path $path_blastn_database. "
echo " If you want to download the database now, please make sure you have enough "
echo " space in mounted directory and internet connection have enough bandwidth as "
echo " file is of size 270 GBs after unzip. It may take forever to download if "
echo " internet is slow! "
echo "========================================================================================"
echo ""
echo -n "Type 'y' for download or any other key to exit: "
read userinput
if [[ $(echo $userinput | tr '[A-Z]' '[a-z]') == 'y' ]]; then
echo ""
echo "=============================================================================================="
echo " Downloading NCBI's database form ftp://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/nt.gz link. "
echo " May take few hours to download. "
echo "=============================================================================================="
echo ""
wget -c "ftp://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/nt.gz" -O $program_dir/nt_database/nt.gz
if [[ $? -eq 0 ]]; then
echo ""
echo "======================================================================="
echo " nt database is completed successfully. "
echo "======================================================================="
echo ""
else
echo ""
echo "======================================================================="
echo " Error! Unable to download database sucessfully. "
echo " Check wget command or internet connection. "
echo "======================================================================="
echo ""
exit 1
fi
echo ""
echo "======================================================================"
echo " Unziping the downloaded nt database. "
echo " May take few hours as size of unzipped file is around 270 GBs. "
echo "======================================================================"
echo ""
############ unzip the nt data base file ############
gunzip $program_dir/nt_database/nt.gz
if [[ $? -eq 0 ]]; then
echo ""
echo "======================================================================="
echo " nt database unzip completed successfully. "
echo "======================================================================="
echo ""
else
echo ""
echo "======================================================================="
echo " Error! unable to unzip database sucessfully. "
echo " Please check if gunzip program exists! "
echo "======================================================================="
echo ""
exit 1
fi
else
echo ""
echo "==========================================================="
echo " Exiting the program because nt database is missing! "
echo "==========================================================="
echo ""
exit 1
fi
fi
###### check if aligned homologous sequences file already exists ############
if [ -f $feature_dir/$seq_id.a2m ]; then
echo ""
echo "======================================================================"
echo " MSA file $feature_dir/$seq_id.a2m from Infernal Pipeline already "
echo " exists for query sequence $feature_dir/$seq_id.fasta. "
echo " "
echo " Delete existing $feature_dir/$seq_id.a2m if want to generate new "
echo " alignment file "
echo "======================================================================"
echo ""
else
#### check if formatted nt database exists or not #####
if [[ ! -f "$path_blastn_database.nal" ]]; then
echo ""
echo "====================================================================="
echo " Nucleotide database file $path_database/nt need to formated "
echo " formated to use with 'makeblastdb' program in BLAST-N program. "
echo ""
echo " Formatting may take 2-3 hours as size of file is around 270 GBs. "
echo "====================================================================="
echo ""
$path_blastn/makeblastdb -in $path_database/nt -dbtype nucl
if [[ $? -eq 0 ]]; then
echo ""
echo "======================================================="
echo " nt database formatted successfully. "
echo "======================================================="
echo ""
else
echo ""
echo "=================================================================="
echo " Error occured while formatting the nt database. "
echo ""
echo " Check for '$path_blastn/makeblastdb' program in BLAST package "
echo "=================================================================="
echo ""
exit 1
fi
fi
#################### check if blastn alignment file ready exists ######################
if [ -f $feature_dir/$seq_id.bla ]; then
echo ""
echo "======================================================================="
echo " MSA-1 file $feature_dir/$seq_id.bla from Infernal Pipeline already "
echo " exists for query sequence $feature_dir/$seq_id.fasta. "
echo " "
echo " Delete existing $feature_dir/$seq_id.a2m if want to generate new "
echo " alignment file. "
echo "======================================================================="
echo ""
else
echo ""
echo "==========================================================================================================================="
echo " Running BLASTN for first round of homologous sequence search for query sequence $feature_dir/$seq_id.fasta. "
echo " May take 5 mins to few hours depending on sequence length and no. of homologous sequences in database. "
echo "==========================================================================================================================="
echo ""
$path_blastn/blastn -db $path_blastn_database -query $feature_dir/$seq_id.fasta -out $feature_dir/$seq_id.bla -evalue 0.001 -num_descriptions 1 -num_threads 8 -line_length 1000 -num_alignments 50000
fi
if [ $? -eq 0 ]; then
echo ""
echo "==========================================================="
echo " First round of MSA-1 search completed successfully. "
echo "==========================================================="
echo ""
else
echo ""
echo "=================================================================="
echo " Error occured while formatting the nt database. "
echo ""
echo " Check for '$path_blastn/makeblastdb' program in BLAST package "
echo "=================================================================="
echo ""
exit 1
fi
######## reformat the output ################
echo ""
echo "========================================================================================"
echo " Converting $feature_dir/$seq_id.bla from BLASTN to $feature_dir/$seq_id.sto. "
echo "========================================================================================"
echo ""
$program_dir/utils/parse_blastn_local.pl $feature_dir/$seq_id.bla $feature_dir/$seq_id.fasta $feature_dir/$seq_id.aln
$program_dir/utils/reformat.pl fas sto $feature_dir/$seq_id.aln $feature_dir/$seq_id.sto
if [ $? -eq 0 ]; then
echo ""
echo "=========================================="
echo " Converison completed successfully. "
echo "=========================================="
echo ""
else
echo ""
echo "============================================================================================="
echo " Error occured while Converting $feature_dir/$seq_id.bla to $feature_dir/$seq_id.sto "
echo " "
echo " Check for $program_dir/utils/parse_blastn_local.pl and $program_dir/utils/reformat.pl file."
echo "============================================================================================="
echo ""
exit 1
fi
######## predict secondary structure from SPOT-RNA ################
echo ""
echo "==============================================================================================================================="
echo " Predicting Consensus Secondary Structure (CSS) of query sequence $feature_dir/$seq_id.fasta using SPOT-RNA predictor. "
echo "==============================================================================================================================="
echo ""
source $program_dir/venv/bin/activate || conda activate venv
cd $program_dir/SPOT-RNA
python3 SPOT-RNA.py --inputs $feature_dir/$seq_id.fasta --outputs $feature_dir
cd -
export PERL5LIB=$program_dir/utils/FreeKnot
perl $program_dir/utils/FreeKnot/remove_pseudoknot.pl -i bpseq -s bp $feature_dir/$seq_id.bpseq > $feature_dir/$seq_id.bpseq.unknotted
python3 $program_dir/utils/bpseq2dbn.py --inputs $feature_dir --outputs $feature_dir --rna_id $seq_id
tail -n +3 $feature_dir/$seq_id.dbn > $feature_dir/$seq_id.db
deactivate || conda deactivate
################ reformat ss with according to gaps in reference sequence of .sto file from blastn ################
for i in `awk '{print $2}' $feature_dir/$seq_id.sto | head -n5 | tail -n1 | grep -b -o - | sed 's/..$//'`; do sed -i "s/./&-/$i" $feature_dir/$seq_id.db; done
######### add reformated ss from last step to .sto file of blastn ##############
head -n -1 $feature_dir/$seq_id.sto > $feature_dir/temp.sto
echo "#=GC SS_cons "`cat $feature_dir/$seq_id.db` > $feature_dir/temp.txt
cat $feature_dir/temp.sto $feature_dir/temp.txt > $feature_dir/$seq_id.sto
echo "//" >> $feature_dir/$seq_id.sto
if [ $? -eq 0 ]; then
echo ""
echo "=================================================================="
echo " Consensus Secondary Structure (CSS) generated successfully. "
echo "=================================================================="
echo ""
else
echo ""
echo "=============================================================================="
echo " Error occured while generating structure from SPOT-RNA. "
echo " "
echo " Please raise issue at 'https://github.com/jaswindersingh2/SPOT-RNA2/issues'."
echo "=============================================================================="
echo ""
exit 1
fi
######## run infernal ################
echo ""
echo "=============================================================================================================="
echo " Building Covariance Model from BLASTN alignment (with SS from SPOT-RNA) from $feature_dir/$seq_id.sto file. "
echo "=============================================================================================================="
echo ""
$path_infernal/cmbuild --hand -F $feature_dir/$seq_id.cm $feature_dir/$seq_id.sto
if [ $? -eq 0 ]; then
echo ""
echo "============================================================================"
echo " Covariance Model (CM) built successfully from $feature_dir/$seq_id.sto. "
echo "============================================================================"
echo ""
else
echo ""
echo "==============================================================================================="
echo " Error occured while building Covariance Model (CM) from $path_infernal/cmbuild. "
echo " "
echo " Please check for $path_infernal/cmbuild program. "
echo "==============================================================================================="
echo ""
exit 1
fi
echo ""
echo "===================================================================="
echo " Calibrating the Covariance Model $feature_dir/$seq_id.cm. "
echo "===================================================================="
echo ""
$path_infernal/cmcalibrate $feature_dir/$seq_id.cm
if [ $? -eq 0 ]; then
echo ""
echo "==========================================================="
echo " CM calibrated $feature_dir/$seq_id.cm successfully. "
echo "==========================================================="
echo ""
else
echo ""
echo "==============================================================="
echo " Error occured while calibrating $feature_dir/$seq_id.cm. "
echo " "
echo " Please check for $path_infernal/cmcalibrate program. "
echo "==============================================================="
echo ""
exit 1
fi
echo ""
echo "======================================================================================================================"
echo " Second round of homologous sequences search using the calibrated covariance model $feature_dir/$seq_id.cm. "
echo " May take 15 mins to few hours for this step. "
echo "======================================================================================================================"
echo ""
$path_infernal/cmsearch -o $feature_dir/$seq_id.out -A $feature_dir/$seq_id.msa --cpu 24 --incE 10.0 $feature_dir/$seq_id.cm $path_infernal_database
if [ $? -eq 0 ]; then
echo ""
echo "==========================================================="
echo " Second round of MSA-2 search completed successfully. "
echo "==========================================================="
echo ""
else
echo ""
echo "===================================================================================="
echo " Error occured during the second round search using CM $feature_dir/$seq_id.cm. "
echo " "
echo " Please check for $path_infernal/cmsearch program. "
echo "===================================================================================="
echo ""
exit 1
fi
######### reformat the alignment without gaps and dashes ###############
echo ""
echo "======================================================================="
echo " Reformatting the output alignment $feature_dir/$seq_id.msa "
echo " for PSSM and DCA features by removing the gaps and dashes. "
echo "======================================================================="
echo ""
##### check if .msa is not empty #########
if [[ -s $feature_dir/$seq_id.msa ]]
then
$path_infernal/esl-reformat --replace acgturyswkmbdhvn:................ a2m $feature_dir/$seq_id.msa > $feature_dir/temp.a2m
else
cat $feature_dir/$seq_id.fasta > $feature_dir/temp.a2m
cat $feature_dir/$seq_id.fasta >> $feature_dir/temp.a2m
sed -i '$ s/.$/./' $feature_dir/temp.a2m
fi
# $path_infernal/esl-reformat --replace acgturyswkmbdhvn:................ a2m $feature_dir/$seq_id.msa > $feature_dir/temp.a2m
if [ $? -eq 0 ]; then
echo ""
echo "==========================================================="
echo " Reformatted the $feature_dir/$seq_id.msa successfully. "
echo "==========================================================="
echo ""
else
echo ""
echo "========================================================================================"
echo " Error occured during the refomatting the alignment file $feature_dir/$seq_id.msa. "
echo " "
echo " Please check for $path_infernal/esl-reformat program. "
echo "========================================================================================"
echo ""
exit 1
fi
######### remove duplicates sequences from the alignment ###############
echo ""
echo "======================================================================="
echo " Removing duplicates from the alignment. "
echo "======================================================================="
echo ""
$program_dir/utils/seqkit rmdup -s $feature_dir/temp.a2m > $feature_dir/$seq_id.a2m
if [ $? -eq 0 ]; then
echo ""
echo "==============================================="
echo " Duplicate sequences removed successfully. "
echo "==============================================="
echo ""
else
echo ""
echo "========================================================================================"
echo " Error occured during the removel of duplicates from MSA-2. "
echo " "
echo " Please check for $program_dir/utils/seqkit program. "
echo "========================================================================================"
echo ""
exit 1
fi
############# multiline fasta to single line fasta file #############
awk '/^>/ {printf("\n%s\n",$0);next; } { printf("%s",$0);} END {printf("\n");}' < $feature_dir/$seq_id.a2m | sed '/^$/d' > $feature_dir/temp.a2m
############# add query sequence at the top of MSA file #############
cat $feature_dir/$seq_id.fasta $feature_dir/temp.a2m > $feature_dir/$seq_id.a2m
fi
############# check if pssm file already exists otherwise generate from alignment file #############
if [ -f $feature_dir/$seq_id.pssm ]; then
echo ""
echo "=============================================================================================================================================="
echo " PSSM feature file $feature_dir/$seq_id.pssm already exists for query sequence $feature_dir/$seq_id.fasta. "
echo "=============================================================================================================================================="
echo ""
else
echo ""
echo "======================================================================================"
echo " Extracting PSSM features from the alignment $feature_dir/$seq_id.a2m. "
echo "======================================================================================"
echo ""
$program_dir/utils/getpssm.pl $feature_dir/$seq_id.fasta $feature_dir/$seq_id.a2m $feature_dir/$seq_id.pssm
if [ $? -eq 0 ]; then
echo ""
echo "==============================================================="
echo " PSSM extracted successfully from $feature_dir/$seq_id.a2m. "
echo "==============================================================="
echo ""
else
echo ""
echo "========================================================================="
echo " Error occured while extracting PSSM from $feature_dir/$seq_id.a2m. "
echo " "
echo " Please check for $program_dir/utils/getpssm.pl program. "
echo "========================================================================="
echo ""
exit 1
fi
fi
######### run linearpartition RNA secondary structure base-pair probability predictor ###############
echo ""
echo "============================================================================"
echo " Running LinearPartition-V for base-pair probabilty features. "
echo "============================================================================"
echo ""
tail -n +2 $feature_dir/$seq_id.fasta | $program_dir/LinearPartition/linearpartition -V -r $feature_dir/$seq_id.prob
if [ $? -eq 0 ]; then
echo ""
echo "===================================================================="
echo " Base-pair probabilty successfully obtained from LinearPartition. "
echo "===================================================================="
echo ""
else
echo ""
echo "============================================================================="
echo " Error occured while running LinearPartition. "
echo " "
echo " Please check for $program_dir/LinearPartition/linearpartition program. "
echo "============================================================================="
echo ""
exit 1
fi
############# check if dca file already exists otherwise generate from alignment file #############
if [ -f $feature_dir/$seq_id.dca ]; then
echo ""
echo "==============================================================="
echo " GRELMLIN feature file $feature_dir/$seq_id.dca already "
echo " exists for query sequence $feature_dir/$seq_id.fasta. "
echo " "
echo " Delete the existing file if want to generate new dca file. "
echo "==============================================================="
echo ""
else
echo ""
echo "============================================================================"
echo " Running GREMLIN for DCA features. "
echo "============================================================================"
echo ""
$program_dir/GREMLIN_CPP/gremlin_cpp -alphabet rna -i $feature_dir/$seq_id.a2m -o $feature_dir/$seq_id.dca > $feature_dir/$seq_id.log_gremlin
if [ $? -eq 0 ]; then
echo ""
echo "===================================================="
echo " DCA features successfully obtained from GREMLIN. "
echo "===================================================="
echo ""
else
echo ""
echo "============================================================================="
echo " Error occured while running GREMLIN. "
echo " "
echo " Please check for $program_dir/GREMLIN_CPP/gremlin_cpp program. "
echo "============================================================================="
echo ""
exit 1
fi
fi
echo ""
echo "============================================================================"
echo " Running SPOT-RNA2 for RNA secondary structure prediction. "
echo "============================================================================"
echo ""
source $program_dir/venv/bin/activate || conda activate venv
python3 $program_dir/utils/SPOT-RNA2.py --inputs $feature_dir/$seq_id.fasta --outputs $output_dir --motifs True
deactivate || conda deactivate
end=`date +%s`
runtime=$((end-start))
echo -e "\ncomputation time = "$runtime" seconds"