# HG changeset patch
# User mingchen0919
# Date 1541794332 18000
# Node ID dad0996ce0b791069ae76ce0126a6a622faaaf7e
# Parent  6da16f4e1e3b482aa9c4a8d3fc98b831dc3f595c
planemo upload commit 0ab80de699747ead439fcaccfdd73f66066affc1-dirty

diff -r 6da16f4e1e3b -r dad0996ce0b7 findSSRs_altered.pl
--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/findSSRs_altered.pl	Fri Nov 09 15:12:12 2018 -0500
@@ -0,0 +1,1107 @@
+#!/usr/bin/env perl
+################################################################################
+# Author: Meg Staton & Stephen Ficklin
+#
+# DESCRIPTION
+# -----------
+# This script identifies simple sequence repeats (SSRs) and calls primers from
+# the sequences in a fastq formatted file.
+#
+# Dependencies:
+# ------------
+# Perl must have access to the packages:
+# Getopt::Long
+# Bio::SeqIO
+# Excel::Writer::XLSX
+# All are available from CPAN.
+#
+# Also path to the primer3 executable and primer3 config files must be specified
+# in the global variables section of the script.
+#
+# Usage:
+# -----
+# Usage: findSSRs.pl <arguments>
+#
+# The list of arguments includes:
+#
+# -f|--fasta_file <fasta_file>
+# Required.  The file of the sequences to be searched.
+#
+# -m|--masked_file <masked_fasta_file>
+# Required.  A soft-masked version of the fasta file (soft masked means low
+# complexity sequences are in lower case bases.)
+#
+# Output:
+# ------
+# <input-file-name>.ssr.fasta
+# A fasta file with sequences with a SSR. (Sequences with compound SSRs are included)
+#
+# <input-file-name>.ssr_stats.txt
+# A text file of statistics about the SSRs discovered.
+#
+# <input-file-name>.ssr_report.txt
+# A tab-delimited file with each SSR.  The columns are SSR ID,
+# motif, number of repeats, start position, end position.
+#
+# <input-file-name>.ssr_report.xlsx
+# A excel file with SSR results and stats
+#
+# <input-file-name>.di_primer_report.txt
+# <input-file-name>.tri_primer_report.txt
+# <input-file-name>.tetra_primer_report.txt
+# Tab-delimited files with sequences with a specified SSR motif length.  Columns are
+# SSR ID, motif, number of repeats, start position, end position, left primer,
+# right primer, left primer Tm, right primer Tm, amplicon size
+#
+# Details:
+# -------
+# By default the script finds:
+# 2 bp motifs repeated from 8 to 200 times,
+# 3 bp motifs repeated from 7 to 133 times,
+# 4 bp motifs repeated from 6 to 100 times,
+#
+# These parameters may be changed in the "GLOBAL PARAMETERS" part of
+# the script.
+#
+# Compound SSRs are defined as any SSRs that abut or are less than 15 bases
+# apart. These are essentially compound SSRs for the purposes of mapping
+# because it is unlikely that primers can be designed between the repeat 
+# segments.
+#
+
+
+use strict;
+
+#-------------------------------------------------------------------------------
+#  DEPENDENCIES
+#-------------------------------------------------------------------------------
+
+use Getopt::Long;
+use Bio::SeqIO;
+use Excel::Writer::XLSX;
+
+#-------------------------------------------------------------------------------
+# GLOBAL PARAMETERS
+#-------------------------------------------------------------------------------
+
+#--------------
+# REPEAT IDENTIFICATION PARAMETERS
+# Specify Motif Frequency
+# Motifs that occur less frequently than indicated below will be ignored.
+# A 0 indicates that this motif length will be ignored.
+
+our $MIN_REPS_2bp = 6;
+our $MIN_REPS_3bp = 6;
+our $MIN_REPS_4bp = 4;
+
+our $MAX_REPS_2bp = 20;
+our $MAX_REPS_3bp = 20;
+our $MAX_REPS_4bp = 20;
+
+#------------
+# PRIMER PARAMETERS
+
+# my $PRIMER3 = "/staton/software/primer3-2.3.6/src/primer3_core";
+# my $PRIMER3_CONFIG = "/staton/software/primer3-2.3.6/src/primer3_config/";
+
+my $PRIMER_OPT_SIZE="21";  # default 20
+my $PRIMER_MIN_SIZE="18";  # default 18
+my $PRIMER_MAX_SIZE="27";  # default 27
+
+my $PRIMER_NUM_NS_ACCEPTED = "0";  # default 0
+
+my $PRIMER_PRODUCT_SIZE_RANGE = "100-400";
+
+my $PRIMER_OPT_TM = "60.0";
+my $PRIMER_MIN_TM = "55.0";
+my $PRIMER_MAX_TM = "65.0";
+
+my $PRIMER_MIN_GC = "40";
+my $PRIMER_MAX_GC = "60";
+
+my $PRIMER_MAX_POLY_X = "3";
+my $PRIMER_GC_CLAMP = "2";
+
+my $PRIMER_LOWERCASE_MASKING = 1;
+
+#-------------------------------------------------------------------------------
+# GLOBAL HASHES
+#-------------------------------------------------------------------------------
+# This makes life much easier than passing a bunch of hash refs all over the place.
+#
+#-------------------------------------------------------------------------------
+# Data structures:
+
+## CONTIG_SSR_STARTS structure:
+## key: contig_name
+##  value: array of starts of SSRs in that contig
+my %CONTIG_SSR_STARTS = ();
+
+## SSR_STATS structure:
+## key: ssr_id
+##  value -> keys: MOTIF START END MOTIF_LENGTH NO_REPEATS
+my %SSR_STATS = ();
+my $SEQ_COUNT = 0;
+
+# Set up the Motif specifications, based on the chosen motif types:.
+my @MOTIF_SPECS;
+push(@MOTIF_SPECS,[2, $MIN_REPS_2bp, $MAX_REPS_2bp, 'dinucleotides']);
+push(@MOTIF_SPECS,[3, $MIN_REPS_3bp, $MAX_REPS_3bp, 'trinucleotides']);
+push(@MOTIF_SPECS,[4, $MIN_REPS_4bp, $MAX_REPS_4bp, 'tetranucleotides']);
+
+#-------------------------------------------------------------------------------
+# Generating statistics
+my $TIME = scalar localtime;                   # Get the current time
+my $SEQ_w_SSRS = 0;
+my $SSR_COUNT = 0;
+my $SSR_COUNT_COMPOUND = 0;
+my $SSR_COUNT_PRIMER = 0;
+
+my %MOTIFLEN = (2  => 0,
+                3  => 0,
+                4  => 0);
+
+my %MOTIFLEN_w_PRIMERS = (2  => 0,
+                          3  => 0,
+                          4  => 0);
+my %MOTIFS = ('|AT|TA|'                   => 0,
+              '|AG|GA|CT|TC|'             => 0,
+              '|AC|CA|TG|GT|'             => 0,
+              '|GC|CG|'                   => 0,
+
+              '|AAT|ATA|TAA|ATT|TTA|TAT|' => 0,
+              '|AAG|AGA|GAA|CTT|TTC|TCT|' => 0,
+              '|AAC|ACA|CAA|GTT|TTG|TGT|' => 0,
+
+              '|CCA|CAC|ACC|TGG|GTG|GGT|' => 0,
+              '|GGC|GCG|CGG|GCC|CCG|CGC|' => 0,
+              '|AGG|GAG|GGA|CCT|CTC|TCC|' => 0,
+
+              '|ATG|TGA|GAT|CAT|ATC|TCA|' => 0,
+              '|AGT|GTA|TAG|ACT|CTA|TAC|' => 0,
+              '|AGC|GCA|CAG|GCT|CTG|TGC|' => 0,
+              '|ACG|CGA|GAC|CGT|GTC|TCG|' => 0);
+
+
+
+#-------------------------------------------------------------------------------
+#  EXECUTE
+#-------------------------------------------------------------------------------
+main();
+#-------------------------------------------------------------------------------
+#  PUBLIC SUBROUTINES
+#-------------------------------------------------------------------------------
+# Function Name:  main()
+
+sub main{
+
+	##---------------------------------------------------------------
+	## Get input parameters
+    my $fasta_file;
+    my $masked_file;
+    my $project;
+
+    Getopt::Long::Configure ('bundling');
+    GetOptions('f|fasta_file=s'  => \$fasta_file,
+               'm|masked_file=s' => \$masked_file,
+               'p|project=s'     => \$project);
+
+    ## Check that all required parameters have been included
+    if(!$fasta_file){ print "A fasta file is required.\n"; _printUsage(); exit;}
+    if(!$masked_file){ print "A masked file is required.\n"; _printUsage(); exit;}
+
+    ## Check that fasta file exists
+    if(! -e $fasta_file) { print "Fasta file $fasta_file does not exist\n"; exit; }
+    if(! -e $masked_file) { print "Masked file $masked_file does not exist\n"; exit; }
+
+	##---------------------------------------------------------------
+	## Set up output files
+    my $p3_input         = "$fasta_file.p3in.txt";
+    my $p3_output        = "$fasta_file.p3out.txt";
+    my $ssr_out          = "$fasta_file.ssr_report.txt";
+    my $fasta_out        = "$fasta_file.ssr.fasta";
+    my $stats_out        = "$fasta_file.ssr_stats.txt";
+    my $di_primer_out    = "$fasta_file.di_primer_report.txt";
+    my $tri_primer_out   = "$fasta_file.tri_primer_report.txt";
+    my $tetra_primer_out = "$fasta_file.tetra_primer_report.txt";
+    my $ssr_xlsx         = "$fasta_file.ssr_report.xlsx";
+
+	##---------------------------------------------------------------
+    print "finding SSRs...\n";
+    process_file($fasta_file, $masked_file);
+	collapse_compound_ssrs();
+    flag_multiSSRs();
+    print "done.\n";
+
+	##---------------------------------------------------------------
+    print "running primer3...\n";
+	addToPrimer3InputFile ($p3_input);
+    print "primer3_core < $p3_input > $p3_output\n";
+    my $status = system("primer3_core < $p3_input > $p3_output");
+	print "$status\n";
+    parseP3_output($p3_output);
+    print "done.\n";
+
+	##---------------------------------------------------------------
+	## Producing output - Fasta files and flat files
+	print "printing output files...";
+	create_flat_files($ssr_out, $di_primer_out, $tri_primer_out, $tetra_primer_out);
+	create_fasta_file($fasta_out);
+
+	##---------------------------------------------------------------
+	## Producing output - statistics
+
+	calculate_stats();
+	print_stats($stats_out);
+
+	##---------------------------------------------------------------
+	## Producing output - Excel
+	create_excel_file($ssr_xlsx, $project);
+    print "done.\n";
+
+}
+
+###############################################################
+
+sub process_file{
+    my $fasta_file  = $_[0]; # file name
+    my $masked_file = $_[1]; # file name
+
+    my $seqio = Bio::SeqIO->new('-format' => 'fasta', -file => $fasta_file);
+    my $seqioM = Bio::SeqIO->new('-format' => 'fasta', -file => $masked_file);
+
+    # Get seq obj from io stream
+    while(my $seqobj = $seqio->next_seq){
+        my $seqobjM = $seqioM->next_seq;
+
+        $SEQ_COUNT++;
+
+        my $seqname = $seqobj->id;        # get actual sequence as a string
+        my $seqnameM = $seqobjM->id;        # get actual sequence as a string
+
+        my $seqstr = $seqobj->seq();        # get actual sequence as a string
+        my $seqstrM = $seqobjM->seq();        # get actual sequence as a string
+
+        if($seqname ne $seqnameM){
+            die "masked sequence $seqnameM not in same order as regular sequence $seqname\n";
+        }
+
+        process_seq($seqname, $seqstr, $seqstrM);
+    }
+
+}
+
+###############################################################
+
+sub process_seq{
+    my $contig_name = shift;
+    my $seq         = shift;
+    my $seq_masked  = shift;
+
+
+    my %seen;     # used to keep track of start positions we've already seen
+    my $index;    # used to iterate through the 2D motif specs array
+
+    ## LOOPB
+    # iterate through the motif specifications
+    for $index (0 .. (scalar @MOTIF_SPECS - 1)) {
+
+        # holds the motif size (1,2,3,4,5 or 6)
+        my $motifLength = $MOTIF_SPECS[$index][0];
+        # holds the minimum number of repeats
+        my $min_number_of_repeats = $MOTIF_SPECS[$index][1]-1;
+        # holds the maximum number of repeats
+        my $max_number_of_repeats = $MOTIF_SPECS[$index][2]-1;
+        # the regular expression for looking for SSRs
+        my $regex = "(([gatc]{$motifLength})\\2{$min_number_of_repeats,$max_number_of_repeats})";
+
+        # run through the sequence and check for this motif spec
+        while ($seq =~ /$regex/ig) {
+            # Get the ssr and motif that were found
+            my $ssr = $1;
+            my $motif = lc $2;
+			my $start_index = $-[0];
+			my $end_index = $+[0];
+
+			if(quality_check_ssr($contig_name, $ssr, $motif, $start_index, $end_index, $seq)){
+				process_ssr($contig_name, $ssr, $motif, $start_index, $end_index, $seq, $seq_masked);
+
+			}
+
+		}
+	}
+}
+
+
+
+###############################################################
+sub quality_check_ssr{
+	my $contig_name = shift;
+	my $ssr = shift;
+ 	my $motif = shift;
+ 	my $start_index = shift;
+ 	my $end_index = shift;
+	my $seq = shift;
+
+	##-------------------------------------
+	## CHECKS to see if this is a good ssr
+	my $flag_same_base = 0;
+	my $flag_already_seen = 0;
+
+	## Check #1
+	## ignore SSRs that are the same base repeated
+	if ($ssr !~ /^g+$/i &&
+	    $ssr !~ /^a+$/i &&
+	    $ssr !~ /^c+$/i &&
+	    $ssr !~ /^t+$/i ) {
+		$flag_same_base = 1;
+	}
+
+	# Check #2
+	# Make sure this isn't an already called SSR in disguise
+	# (a dinucleotide repeat posing as a tetranucleotide repeat, for instance)
+	if (!exists $SSR_STATS{$contig_name."_ssr".$start_index} &&
+	    !exists $SSR_STATS{$contig_name."_ssr".($start_index-1)} &&
+	    !exists $SSR_STATS{$contig_name."_ssr".($start_index-2)} &&
+	    !exists $SSR_STATS{$contig_name."_ssr".($start_index+1)} &&
+	    !exists $SSR_STATS{$contig_name."_ssr".($start_index+2)}
+	) {
+		$flag_already_seen = 1;
+	}
+
+	if($flag_same_base && $flag_already_seen){
+		return 1;
+	}
+	else{
+		return 0;
+	}
+
+}
+
+
+
+######################################################
+sub process_ssr{
+	my $contig_name = shift;
+	my $ssr = shift;
+ 	my $motif = shift;
+ 	my $start_index = shift;
+ 	my $end_index = shift;
+	my $seq = shift;
+	my $seq_masked = shift;
+
+	##-------------------------------------
+	## generate a few more stats and variables
+	my $motif_len = length $motif;
+	my $num_of_repeats = ($end_index-$start_index)/$motif_len;
+    my $ssr_id = $contig_name."_ssr".$start_index;
+
+	##-------------------------------------
+	## store in data structures
+
+    if(exists $CONTIG_SSR_STARTS{$contig_name}){
+        push @{ $CONTIG_SSR_STARTS{$contig_name} }, $start_index;
+    }
+    else{
+       $CONTIG_SSR_STARTS{$contig_name} = [$start_index];
+    }
+
+	$SSR_STATS{$ssr_id}{MOTIF}        = $motif;
+	$SSR_STATS{$ssr_id}{START}        = $start_index;
+	$SSR_STATS{$ssr_id}{END}          = $end_index;
+	$SSR_STATS{$ssr_id}{MOTIF_LENGTH} = $motif_len;
+	$SSR_STATS{$ssr_id}{NO_REPEATS}   = $num_of_repeats;
+	$SSR_STATS{$ssr_id}{SEQ}          = $seq;
+	$SSR_STATS{$ssr_id}{SEQM}         = $seq_masked;
+	$SSR_STATS{$ssr_id}{COMPOUND}     = 0; #assume its not until proven otherwise
+
+
+}
+
+#######################################################
+sub collapse_compound_ssrs{
+	foreach my $contig (keys %CONTIG_SSR_STARTS){
+		my @starts = @{ $CONTIG_SSR_STARTS{$contig}};
+		if(@starts > 1){
+			## this contig has multiple ssrs
+			my $previous_start = -1;
+			my $previous_end = -1;
+			my $previous_ssr_id = "";
+
+			foreach my $current_start (sort {$a <=> $b} @starts){
+				my $current_ssr_id = $contig."_ssr".$current_start;
+				my $current_end = $SSR_STATS{$current_ssr_id}{END};
+
+				if(too_close($previous_start, $previous_end, $current_start, $current_end)){
+						collapse($contig, $previous_ssr_id, $current_ssr_id);
+				}
+
+				$previous_start = $current_start;
+				$previous_end = $current_end;
+				$previous_ssr_id = $current_ssr_id;
+			}
+		}
+	}
+}
+
+################################################################
+sub too_close{
+	my $previous_start = shift;
+	my $previous_end = shift;
+	my $current_start = shift;
+	my $current_end  = shift;
+
+	# if start is a -1, then go ahead and return ok
+	if($previous_start == -1){
+		return 0;
+	}
+	# we want to know if they overlap, abut or are less than 15 bases apart
+	elsif($previous_end >= $current_start ||
+		($current_start - $previous_end) < 15){
+		#print "$previous_start - $previous_end, $current_start - $current_end\n";
+		return 1;
+	}
+	else{
+		return 0;
+	}
+}
+################################################################
+sub collapse{
+	my $contig = shift;
+	my $first_ssr_id = shift;
+	my $second_ssr_id = shift;
+	my $second_ssr_start = $SSR_STATS{$second_ssr_id}{START};
+
+	##fix SSR_STATS
+	$SSR_STATS{$first_ssr_id}{MOTIF}        = "COMPOUND";
+	$SSR_STATS{$first_ssr_id}{END}          = $SSR_STATS{$second_ssr_id}{END};
+	$SSR_STATS{$first_ssr_id}{MOTIF_LENGTH} = "COMPOUND";
+	$SSR_STATS{$first_ssr_id}{NO_REPEATS}   = "COMPOUND";
+	$SSR_STATS{$first_ssr_id}{COMPOUND}     = 1; #assume its not until proven otherwise
+
+	delete $SSR_STATS{$second_ssr_id}{MOTIF};
+	delete $SSR_STATS{$second_ssr_id}{START};
+	delete $SSR_STATS{$second_ssr_id}{END};
+	delete $SSR_STATS{$second_ssr_id}{MOTIF_LENGTH};
+	delete $SSR_STATS{$second_ssr_id}{NO_REPEATS};
+	delete $SSR_STATS{$second_ssr_id}{SEQ};
+	delete $SSR_STATS{$second_ssr_id}{SEQM};
+	delete $SSR_STATS{$second_ssr_id}{COMPOUND};
+	undef %{$SSR_STATS{$second_ssr_id}};
+	delete $SSR_STATS{$second_ssr_id};
+
+	print "\tdeleting $second_ssr_id, part of compound ssr\n";
+
+	if(exists $SSR_STATS{$second_ssr_id}){ print "\t still exists\n";}
+
+	##fix CONTIG_SSR_STARTS
+	# get rid of the start index for the second ssr (it is now part of the
+	# first ssr)
+
+	my $index = 0;
+	$index++ until $CONTIG_SSR_STARTS{$contig}[$index] == $second_ssr_start;
+	splice(@{$CONTIG_SSR_STARTS{$contig}}, $index, 1);
+
+}
+
+
+################################################################
+sub flag_multiSSRs{
+	# adds a MULTI flag to the data hash indicating if the
+	# ssr is the only one in the sequence or one of many
+
+	foreach my $contig (keys %CONTIG_SSR_STARTS){
+		my @starts = @{ $CONTIG_SSR_STARTS{$contig}};
+		if(@starts == 1){
+			## this contig has only one ssr
+			my $start_index = $starts[0];
+			my $ssr_id = $contig."_ssr".$start_index;
+			$SSR_STATS{$ssr_id}{MULTI} = 0;
+		}
+		else{
+			## this contig has multiple ssrs
+			foreach my $start_index (@starts){
+				my $ssr_id = $contig."_ssr".$start_index;
+				$SSR_STATS{$ssr_id}{MULTI} = 1;
+			}
+		}
+	}
+	close FASTA;
+
+}
+
+################################################################
+sub addToPrimer3InputFile{
+    my $p3_file = shift;
+
+	open OUT, ">$p3_file";
+
+	foreach my $ssr_id (keys %SSR_STATS){
+		#skip compound SSRS
+		if($SSR_STATS{$ssr_id}{COMPOUND} == 0){
+			my $ssrStart   = $SSR_STATS{$ssr_id}{START};
+			my $ssrEnd     = $SSR_STATS{$ssr_id}{END};
+			my $seq        = $SSR_STATS{$ssr_id}{SEQM};
+
+			# change from soft mask to hard mask
+			$seq =~ s/[actg]/N/g;
+
+			my $len         = $ssrEnd-$ssrStart;
+
+			printf OUT ("SEQUENCE_ID=$ssr_id\n");
+    		printf OUT ("SEQUENCE_TEMPLATE=$seq\n");
+    		printf OUT ("SEQUENCE_TARGET=$ssrStart,$len\n");
+    		printf OUT ("PRIMER_TASK=generic\n");
+    		printf OUT ("PRIMER_PICK_LEFT_PRIMER=1\n");
+    		printf OUT ("PRIMER_PICK_INTERNAL_OLIGO=0\n");
+    		printf OUT ("PRIMER_PICK_RIGHT_PRIMER=1\n");
+    		printf OUT ("PRIMER_OPT_SIZE=$PRIMER_OPT_SIZE\n");
+    		printf OUT ("PRIMER_MIN_SIZE=$PRIMER_MIN_SIZE\n");
+    		printf OUT ("PRIMER_MAX_SIZE=$PRIMER_MAX_SIZE\n");
+    		printf OUT ("PRIMER_NUM_NS_ACCEPTED=$PRIMER_NUM_NS_ACCEPTED\n");
+    		printf OUT ("PRIMER_PRODUCT_SIZE_RANGE=$PRIMER_PRODUCT_SIZE_RANGE\n");
+    		printf OUT ("PRIMER_OPT_TM=$PRIMER_OPT_TM\n");
+    		printf OUT ("PRIMER_MIN_TM=$PRIMER_MIN_TM\n");
+    		printf OUT ("PRIMER_MAX_TM=$PRIMER_MAX_TM\n");
+    		printf OUT ("PRIMER_MIN_GC=$PRIMER_MIN_GC\n");
+    		printf OUT ("PRIMER_MAX_GC=$PRIMER_MAX_GC\n");
+    		printf OUT ("PRIMER_MAX_POLY_X=$PRIMER_MAX_POLY_X\n");
+    		printf OUT ("PRIMER_GC_CLAMP=$PRIMER_GC_CLAMP\n");
+    		# printf OUT ("PRIMER_THERMODYNAMIC_PARAMETERS_PATH=$PRIMER3_CONFIG\n");
+    		printf OUT ("PRIMER_LOWERCASE_MASKING=$PRIMER_LOWERCASE_MASKING\n");
+    		printf OUT ("=\n");
+		}
+	}
+	close OUT;
+}
+################################################################
+sub parseP3_output{
+    my $p3_output = $_[0]; # file name
+
+	# We are going to keep track of a weird phenomenon only seen in one
+	# project - the generation of identical forward and reverse primers. The
+	# sequences from this project were overlapping paired ends that were joined.
+	# Apparently something went wrong and weird sequences were obtained, all of
+	# which yield the identical primers.
+	# This is not reported in the final stats, just as part of the standard output.
+    my $identical_primer_cnt = 0;
+
+    # The primers output file separates information about different sequences
+    # with an equal sign on a single line. So, we want to set the file line
+    # delimiter (for looping on the input file below) to a single equal sign
+    # followed by a line feed.  This way were guranteed to have all the primer
+    # information together per line
+    local $/ = "=\n";
+
+    open (P3O, $p3_output) || die "could not open $_\n";
+
+    # Read in all of the lines of the input file
+	my $primer_record;
+    while ($primer_record = <P3O>) {
+        my $start = "";
+        my $seq_id = "";
+        my $ssr_id = "";
+        my $forward = "";
+        my $reverse = "";
+        my $product_size = "";
+        my $left_tm = "";
+        my $right_tm = "";
+
+		if ($primer_record =~ /SEQUENCE_ID=(\S+)/) {
+		    $ssr_id = $1;
+		}
+		# get the primary primers only
+		if ($primer_record =~ /PRIMER_LEFT_0_SEQUENCE=(\S+)/) {
+		    $forward = $1;
+		}
+		if ($primer_record =~ /PRIMER_RIGHT_0_SEQUENCE=(\S+)/) {
+		    $reverse = $1;
+		}
+		if ($primer_record =~ /PRIMER_LEFT_0_TM=(\S+)/) {
+		    $left_tm = $1;
+		}
+		if ($primer_record =~ /PRIMER_RIGHT_0_TM=(\S+)/) {
+		    $right_tm = $1;
+		}
+		if ($primer_record =~ /PRIMER_PAIR_0_PRODUCT_SIZE=(\S+)/) {
+		    $product_size = $1;
+		}
+
+		if(length $forward > 1){
+			if($forward eq $reverse){
+				print "\tFLAG: identical primer problem with $ssr_id\n";
+				$identical_primer_cnt++;
+			}
+			else{
+				$SSR_STATS{$ssr_id}{FORWARD} = $forward;
+				$SSR_STATS{$ssr_id}{REVERSE} = $reverse;
+				$SSR_STATS{$ssr_id}{PRODUCT_SIZE} = $product_size;
+				$SSR_STATS{$ssr_id}{LEFT_TM} = $left_tm;
+				$SSR_STATS{$ssr_id}{RIGHT_TM} = $right_tm;
+
+			}
+		}
+	}
+
+    print "\ttotal identical primers: $identical_primer_cnt\n";
+}
+
+###################################################
+sub create_flat_files{
+	my $ssr_out = shift;
+	my $di_primer_out = shift;
+	my $tri_primer_out = shift;
+	my $tetra_primer_out = shift;
+
+	open OUTS, ">$ssr_out";
+	open OUT2, ">$di_primer_out";
+	open OUT3, ">$tri_primer_out";
+	open OUT4, ">$tetra_primer_out";
+
+	my $di_fh = *OUT2;
+	my $tri_fh = *OUT3;
+	my $tetra_fh = *OUT4;
+
+	##printer headers
+	print OUTS join("\t", "SSR ID",
+				"motif", "number of repeats", "start position", "end position");
+	print OUTS "\n";
+
+	print OUT2 join("\t", "SSR ID",
+				"motif", "number of repeats", "start position", 
+				"end position", "forward primer", "reverse primer",
+				"forward Tm", "reverse Tm","product size" );
+	print OUT2  "\n";
+
+	print OUT3 join("\t", "SSR ID",
+				"motif", "number of repeats", "start position", 
+				"end position", "forward primer", "reverse primer",
+				"forward Tm", "reverse Tm","product size" );
+	print OUT3  "\n";
+
+	print OUT4 join("\t", "SSR ID",
+				"motif", "number of repeats", "start position", 
+				"end position", "forward primer", "reverse primer",
+				"forward Tm", "reverse Tm","product size" );
+	print OUT4  "\n";
+
+	foreach my $ssr_id (keys %SSR_STATS){
+		## all ssrs including compound go in main ssr file
+		print OUTS join("\t",
+			$ssr_id,
+			$SSR_STATS{$ssr_id}{MOTIF},
+			$SSR_STATS{$ssr_id}{NO_REPEATS},
+			$SSR_STATS{$ssr_id}{START},
+			$SSR_STATS{$ssr_id}{END},
+		);
+		print OUTS "\n";
+
+		# for primer flat files, only print SSRs with 
+		# that have primers
+		if($SSR_STATS{$ssr_id}{COMPOUND} == 0 &&
+			$SSR_STATS{$ssr_id}{FORWARD} =~ /\S/
+		){
+			if($SSR_STATS{$ssr_id}{MOTIF_LENGTH} == 2){
+				_print_primer_flat_file_line($di_fh, $ssr_id);
+			}
+			elsif($SSR_STATS{$ssr_id}{MOTIF_LENGTH} == 3){
+				_print_primer_flat_file_line($tri_fh, $ssr_id);
+			}
+			elsif($SSR_STATS{$ssr_id}{MOTIF_LENGTH} == 4){
+				_print_primer_flat_file_line($tetra_fh, $ssr_id);
+			}
+		}
+	}
+	close OUTS;
+	close OUT2;
+	close OUT3;
+	close OUT4;
+
+}
+###################################################
+sub _print_primer_flat_file_line{
+	my $fh = shift;
+	my $ssr_id = shift;
+
+	print $fh join("\t", $ssr_id,
+		$SSR_STATS{$ssr_id}{MOTIF},
+		$SSR_STATS{$ssr_id}{NO_REPEATS},
+		$SSR_STATS{$ssr_id}{START},
+		$SSR_STATS{$ssr_id}{END},
+		$SSR_STATS{$ssr_id}{FORWARD},
+		$SSR_STATS{$ssr_id}{REVERSE},
+		$SSR_STATS{$ssr_id}{LEFT_TM},
+		$SSR_STATS{$ssr_id}{RIGHT_TM},
+		$SSR_STATS{$ssr_id}{PRODUCT_SIZE},
+	);
+	print $fh "\n";
+
+}
+
+###################################################
+sub create_fasta_file{
+	my $fasta_out = shift;
+	open FASTA, ">$fasta_out";
+
+	foreach my $contig (keys %CONTIG_SSR_STARTS){
+		my @starts = @{ $CONTIG_SSR_STARTS{$contig}};
+		my $seq = "";
+		print FASTA ">$contig (";
+		foreach my $start_index (sort {$a <=> $b} @starts){
+			my $ssr_id = $contig."_ssr".$start_index;
+
+			#if($SSR_STATS{$ssr_id}{START} >= 0){
+			$seq = $SSR_STATS{$ssr_id}{SEQ};
+			print FASTA "$SSR_STATS{$ssr_id}{START}-$SSR_STATS{$ssr_id}{END} ";
+			if($SSR_STATS{$ssr_id}{COMPOUND} == 1){ 
+				print FASTA "*Compound ";
+			}
+		}
+		#get the first ssr index just so we can get the sequence
+		print FASTA ")\n";
+		print FASTA "$seq\n";
+
+	}
+	close FASTA;
+
+}
+################################################################
+sub calculate_stats{
+
+	$SEQ_w_SSRS = keys %CONTIG_SSR_STARTS;
+
+	foreach my $ssr_id (keys %SSR_STATS){
+		$SSR_COUNT++;
+
+		if($SSR_STATS{$ssr_id}{COMPOUND} == 1){
+			$SSR_COUNT_COMPOUND++;
+		}
+		else{
+			my $motif_len = $SSR_STATS{$ssr_id}{MOTIF_LENGTH} ;
+			#print "motif length is $motif_len\n";
+			$MOTIFLEN{$motif_len}++;
+
+			my $motifUC = uc($SSR_STATS{$ssr_id}{MOTIF});
+			foreach my $group (keys %MOTIFS) {
+				if($group =~ /\|$motifUC\|/){
+					#print "Incrementing $group for $motifUC\n";
+					$MOTIFS{$group}++;
+				}
+			}
+
+			if($SSR_STATS{$ssr_id}{FORWARD} =~ /\S/){
+				$SSR_COUNT_PRIMER++;
+				$MOTIFLEN_w_PRIMERS{$motif_len}++;
+			}
+		}
+	}
+
+}
+sub print_stats{
+    my $stats_out = $_[0]; # file name
+
+    open (OUTS, ">".$stats_out) || die "ERROR cannot open $stats_out\n";
+
+    print OUTS 'SSR Summary Report\n';
+    print OUTS "Analysis of $SEQ_COUNT sequences\n";
+    print OUTS "$TIME\n";
+	print OUTS "\n";
+    print OUTS "Number of sequences with at least one SSR\t$SEQ_w_SSRS\n";
+    print OUTS "Number of SSRs identified\t$SSR_COUNT\n";
+	print OUTS "\n";
+    print OUTS "Number of compound SSRs*: $SSR_COUNT_COMPOUND\n";
+    print OUTS "Number of SSRs with primers**: $SSR_COUNT_PRIMER\n";
+	print OUTS "\n";
+	print OUTS "*Compound SSRs are defined as any SSRs next to each or separated by less than 15 bases\n";
+    print OUTS "**No primers are designed for compound SSRs\n";
+    print OUTS "\n";
+	print OUTS "Parameters used for identifying SSRS:\n";
+    print OUTS "Base Pairs in Motif\tMin # Reps\tMax # Reps\n";
+    print OUTS "--------------------------------------\n";
+    print OUTS "2 (Dinucleotides)\t$MIN_REPS_2bp\t$MAX_REPS_2bp\n";
+    print OUTS "3 (Trinucleotides)\t$MIN_REPS_3bp\t$MAX_REPS_3bp\n";
+    print OUTS "4 (Tetranucleotides)\t$MIN_REPS_4bp\t$MAX_REPS_4bp\n";
+    print OUTS "\n";
+	print OUTS "Chart of motif pattern frequence (compound SSRs excluded)\n";
+    print OUTS "Motif Patterns\tNumber of SSRs Found\n";
+    print OUTS "--------------------------------------\n";
+    my $group;
+    foreach $group (sort {length $a <=> length $b} keys %MOTIFS){
+        $group =~ s/^|//;
+        $group =~ s/|$//;
+        print OUTS "$group\t$MOTIFS{$group}\n";
+    }
+    print OUTS "\n";
+	print OUTS "Chart of motif pattern length frequence (compound SSRs excluded)\n";
+    print OUTS "Motif Pattern Length\tNumber of SSRs\n";
+    print OUTS "--------------------------------------\n";
+
+    foreach $group (sort keys %MOTIFLEN){
+        print OUTS "$group\t$MOTIFLEN{$group}\n";
+    }
+
+    print OUTS "\n";
+    print OUTS "SSRS with Primers \n";
+	print OUTS "Chart of motif pattern length frequence (compound SSRs excluded)\n";
+    print OUTS "Motif Pattern Length\tNumber of SSRs\n";
+    print OUTS "--------------------------------------\n";
+
+    foreach $group (sort keys %MOTIFLEN_w_PRIMERS){
+        print OUTS "$group\t$MOTIFLEN_w_PRIMERS{$group}\n";
+    }
+
+
+    close OUTS;
+
+}
+
+################################################################
+
+sub create_excel_file{
+	my $ssr_xlsx = shift;
+	my $project = shift;
+
+    # Create an excel workbook
+    my $workbook = Excel::Writer::XLSX->new("$ssr_xlsx");
+
+    # Setup the formats that will be necessary for the excel spreadsheet
+    my %header = (font         => 'Calibri',
+                size         => 12,
+                bold         => 1,
+                color        => 'black',
+                align        => 'left',
+                text_wrap    => 1);
+
+    my %text = (font         => 'Calibri',
+                size         => 12,
+                color        => 'black',
+                align        => 'left',
+                text_wrap    => 1);
+
+    #add the formats to the workbook
+    my $header_format = $workbook->add_format(%header);
+    my $text_format = $workbook->add_format(%text);
+
+	my $worksheet_stats = create_stats_worksheet($workbook, $header_format, $text_format, $project);
+
+	build_data_worksheets($workbook, $header_format, $text_format);
+
+	$worksheet_stats->activate();
+	$worksheet_stats->select();
+	$workbook->close();
+
+
+}
+
+sub create_stats_worksheet{
+    my $workbook      = shift;
+    my $header_format = shift;
+    my $text_format   = shift;
+    my $project       = shift;
+
+    my $worksheet = $workbook->add_worksheet("Summary");
+
+	## set all cells to text format
+	## only cells that need the header format will need to specify the format during write
+	## set column widths
+    $worksheet->set_column('A:A', 75, $text_format);
+    $worksheet->set_column('B:B', 30, $text_format);
+    $worksheet->set_column('C:C', 30, $text_format);
+
+    $worksheet->write('A1', "SSR Summary Report for $project", $header_format);
+    $worksheet->write('A2', "Analysis of $SEQ_COUNT sequences");
+    $worksheet->write('A3', "$TIME");
+
+    $worksheet->write('A5', "Number of sequences with at least one SSR");
+	$worksheet->write('B5', "$SEQ_w_SSRS");
+
+    $worksheet->write('A6', "Number of SSRs identified");
+	$worksheet->write('B6', "$SSR_COUNT");
+
+    $worksheet->write('A8', "Number of compound SSRs*:");
+	$worksheet->write('B8', "$SSR_COUNT_COMPOUND");
+
+    $worksheet->write('A9', "Number of SSRs with primers**");
+	$worksheet->write('B9', "$SSR_COUNT_PRIMER");
+
+    $worksheet->write('A11', "*Compound SSRs are defined as any SSRs next to each or separated by less than 15 bases\n");
+
+    $worksheet->write('A12', "**No primers are designed for compound SSRs\n");
+
+    $worksheet->write('A14', "Parameters used for identifying SSRS:\n", $header_format);
+
+    $worksheet->write('A15','Base Pairs in Motif', $header_format);
+    $worksheet->write('B15','Min # Reps', $header_format);
+    $worksheet->write('C15','Max # Reps', $header_format);
+
+    $worksheet->write('A16','2 (Dinucleotides)');
+    $worksheet->write('B16',"$MIN_REPS_2bp");
+    $worksheet->write('C16',"$MAX_REPS_2bp");
+
+    $worksheet->write('A17','3 (Trinucleotides)');
+    $worksheet->write('B17',"$MIN_REPS_3bp");
+    $worksheet->write('C17',"$MAX_REPS_3bp");
+
+    $worksheet->write('A18','4 (Tetranucleotides)');
+    $worksheet->write('B18',"$MIN_REPS_4bp");
+    $worksheet->write('C18',"$MAX_REPS_4bp");
+
+	##----------------------------------------------------------
+	##Chart of motif pattern frequence (compound SSRs excluded)
+
+    $worksheet->write('A20','Chart of motif pattern frequence (compound SSRs excluded)', $header_format);
+    $worksheet->write('A21','Motif Patterns', $header_format);
+    $worksheet->write('B21','Number of SSRs Found', $header_format);
+    my $group;
+    my $i = 21;
+    foreach $group (sort {length $a <=> length $b} keys %MOTIFS){
+        $group =~ s/^|//;
+        $group =~ s/|$//;
+        $i++;
+        $worksheet->write("A$i", $group);
+        $worksheet->write("B$i", $MOTIFS{$group});
+    }
+
+	##----------------------------------------------------------
+	## Chart of motif pattern length frequence (compound SSRs excluded)
+    $i++;
+    $i++;
+	$worksheet->write("A$i", "Chart of motif pattern length frequence (compound SSRs excluded)", $header_format);
+    $i++;
+    $worksheet->write("A$i",'Motif Pattern Length', $header_format);
+    $worksheet->write("B$i",'Number of SSRs Found', $header_format);
+    foreach $group (sort keys %MOTIFLEN){
+        $i++;
+        $worksheet->write("A$i", "$group bp");
+        $worksheet->write("B$i", $MOTIFLEN{$group});
+    }
+
+	##----------------------------------------------------------
+	## SSRs w primers:
+	## Chart of motif pattern length frequence (compound SSRs excluded)
+    $i++;
+    $i++;
+    $worksheet->write("A$i",'SSRs with Primers', $header_format);
+    $i++;
+    $worksheet->write("A$i",'Chart of motif pattern length frequence (compound SSRs excluded)', $header_format);
+    $i++;
+    $worksheet->write("A$i",'Motif Pattern Length', $header_format);
+        $worksheet->write("B$i",'Number of SSRs Found', $header_format);
+    foreach $group (sort keys %MOTIFLEN_w_PRIMERS){
+        $i++;
+        $worksheet->write("A$i", "$group bp");
+        $worksheet->write("B$i", $MOTIFLEN_w_PRIMERS{$group});
+    }
+
+	return $worksheet;
+}
+
+##############################################################
+sub build_data_worksheets{
+    my $workbook      = shift;
+    my $header_format = shift;
+    my $text_format   = shift;
+
+	my $di_worksheet  =  _initiate_worksheet($workbook, $header_format, $text_format, "Dinucleotides");
+	my $tri_worksheet  =  _initiate_worksheet($workbook, $header_format, $text_format, "Trinucleotides");
+	my $tetra_worksheet  =  _initiate_worksheet($workbook, $header_format, $text_format, "Tetranucleotides");
+
+	my $di_index = 3;
+	my $tri_index = 3;
+	my $tetra_index = 3;
+
+	foreach my $ssr_id (keys %SSR_STATS){
+		# for excel data files, only print SSRs 
+		# that have primers
+		if($SSR_STATS{$ssr_id}{COMPOUND} == 0 &&
+			$SSR_STATS{$ssr_id}{FORWARD} =~ /\S/
+		){
+			if($SSR_STATS{$ssr_id}{MOTIF_LENGTH} == 2){
+				_print_excel_file_line($di_worksheet, $di_index, $ssr_id);
+				$di_index++;
+			}
+			elsif($SSR_STATS{$ssr_id}{MOTIF_LENGTH} == 3){
+				_print_excel_file_line($tri_worksheet, $tri_index, $ssr_id);
+				$tri_index++;
+			}
+			elsif($SSR_STATS{$ssr_id}{MOTIF_LENGTH} == 4){
+				_print_excel_file_line($tetra_worksheet, $tetra_index, $ssr_id);
+				$tetra_index++;
+			}
+		}
+	}
+
+
+}
+
+##############################################################
+sub _initiate_worksheet{
+    my $workbook      = $_[0];
+    my $header_format = $_[1];
+    my $text_format   = $_[2];
+	my $name          = $_[3];
+
+    my $worksheet = $workbook->add_worksheet($name);
+    $worksheet->set_column('A:A', 60, $text_format);
+    $worksheet->set_column('B:E', 10, $text_format);
+    $worksheet->set_column('F:G', 30, $text_format);
+    $worksheet->set_column('H:J', 10, $text_format);
+
+    $worksheet->write('A1', "$name with primers", $header_format);
+    $worksheet->write('A2', 'SSR ID', $header_format);
+	$worksheet->write('B2', 'Motif', $header_format);
+    $worksheet->write('C2', '# Repeats', $header_format);
+    $worksheet->write('D2', 'Start', $header_format);
+    $worksheet->write('E2', 'End', $header_format);
+    $worksheet->write('F2', 'Forward Primer', $header_format);
+    $worksheet->write('G2', 'Reverse Primer', $header_format);
+    $worksheet->write('H2', 'Forward Tm', $header_format);
+    $worksheet->write('I2', 'Reverse Tm', $header_format);
+    $worksheet->write('J2', 'Fragment Size', $header_format);
+
+	return $worksheet;
+}
+
+
+################################################################
+sub _print_excel_file_line{
+	my $worksheet = shift;
+	my $index = shift;
+	my $ssr_id = shift;
+
+	$worksheet->write("A$index", $ssr_id);
+	$worksheet->write("B$index", $SSR_STATS{$ssr_id}{MOTIF});
+	$worksheet->write("C$index", $SSR_STATS{$ssr_id}{NO_REPEATS});
+	$worksheet->write("D$index", $SSR_STATS{$ssr_id}{START});
+	$worksheet->write("E$index", $SSR_STATS{$ssr_id}{END});
+	$worksheet->write("F$index", $SSR_STATS{$ssr_id}{FORWARD});
+	$worksheet->write("G$index", $SSR_STATS{$ssr_id}{REVERSE});
+	$worksheet->write("H$index", $SSR_STATS{$ssr_id}{LEFT_TM});
+	$worksheet->write("I$index", $SSR_STATS{$ssr_id}{RIGHT_TM});
+	$worksheet->write("J$index", $SSR_STATS{$ssr_id}{PRODUCT_SIZE});
+
+}
+
+###############################################################
+sub _printUsage {
+    print "Usage: $0.pl <arguments>";
+    print qq(
+    The list of arguments includes:
+
+    -f|--fasta_file <fasta_file>
+        Required.  The file of the sequences to be searched. 
+
+    -m|--masked_file <masked_fasta_file>
+        Required.  A soft-masked version of the fasta file (soft masked means low
+        complexity sequences are in lower case bases.)
+
+    -p|--project "project name"
+        Optional.  A project name for use in the Excel output.
+
+    );
+    print "\n";
+    return;
+}
+
+
+1;
diff -r 6da16f4e1e3b -r dad0996ce0b7 galaxy_outputs.sh
--- a/galaxy_outputs.sh	Fri Nov 09 12:23:25 2018 -0500
+++ b/galaxy_outputs.sh	Fri Nov 09 15:12:12 2018 -0500
@@ -11,6 +11,15 @@
 fi
 
 
-if [ -e `*.ssr.fasta` ]; then
-  cp `*.ssr.fasta` ${X_F}
-fi
\ No newline at end of file
+if [ -e "input.ssr.fasta" ]; then
+  cp "input.ssr.fasta" ${X_F}
+fi
+
+if [ -e "input.ssr_stats.txt" ]; then
+  cp "input.ssr_stats.txt" ${X_S}
+fi
+
+if [ -e "input.ssr_report.txt" ]; then
+  cp "input.ssr_report.txt" ${X_R}
+fi
+
diff -r 6da16f4e1e3b -r dad0996ce0b7 rmarkdown_report.Rmd
--- a/rmarkdown_report.Rmd	Fri Nov 09 12:23:25 2018 -0500
+++ b/rmarkdown_report.Rmd	Fri Nov 09 15:12:12 2018 -0500
@@ -1,5 +1,5 @@
 ---
-title: 'Aurora Tool Report'
+title: 'FindSSRs Report'
 output:
     html_document:
       highlight: pygments
@@ -55,16 +55,17 @@
 
 # Run `findSRRs_altered.pl`
 
-* `${REPORT_FILES_PATH}`: the output directory that stores all analysis outputs.
-* `${TOOL_INSTALL_DIR}`: the tool install directory where the `findSSRs_altered.pl` is located.
-* `$X_f`: the path to the fasta file.
-* `$X_m`: the path to the masked fasta file.
-
-```{bash}
-cd ${REPORT_FILES_PATH}
-perl ${TOOL_INSTALL_DIR}/findSSRs_altered.pl \
-  -f $X_f \
-  -m $X_m
+```{bash echo=FALSE}
+sh ${TOOL_INSTALL_DIR}/scripts_generator.sh
 ```
 
+```{r echo=FALSE,warning=FALSE,results='asis'}
+# display content of the job-script.sh file.
+cat('```bash\n')
+cat(readLines(paste0(Sys.getenv('REPORT_FILES_PATH'), '/findSSRs.sh')), sep = '\n')
+cat('\n```')
+```
 
+* `-f`: the path to the fasta file.
+* `-m`: the path to the masked fasta file.
+
diff -r 6da16f4e1e3b -r dad0996ce0b7 rmarkdown_report.xml
--- a/rmarkdown_report.xml	Fri Nov 09 12:23:25 2018 -0500
+++ b/rmarkdown_report.xml	Fri Nov 09 15:12:12 2018 -0500
@@ -35,7 +35,7 @@
   <inputs>
     <param type="data" name="fasta_file" argument="-f" label="Fasta file" help="The file of the sequences to be searched" optional="False" format="fasta,fa"/><param type="data" name="masked_file" argument="-m" label="Masked fasta file" help="A soft-masked version of the fasta file (soft masked means low complexity sequences are in lower case bases.)" optional="False" format="fasta,fa"/></inputs>
   <outputs>
-        <data format="html" name="report" label="${tool.name} report on ${on_string}"/><data name="ssr_fasta" format="fasta,fa" label="${tool.name} on ${on_string} (SSR fasta)" hidden="false"/><data name="ssr_stats" format="txt" label="${tool.name} on ${on_string} (SSR stats)" hidden="false"/><data name="ssr_report" format="txt" label="${tool.name} on ${on_string} (SSR report)" hidden="false"/></outputs>
+        <data format="html" name="report" label="${tool.name} report on ${on_string}"/><data name="ssr_fasta" format="fasta" label="${tool.name} on ${on_string} (SSR fasta)" hidden="false"/><data name="ssr_stats" format="txt" label="${tool.name} on ${on_string} (SSR stats)" hidden="false"/><data name="ssr_report" format="txt" label="${tool.name} on ${on_string} (SSR report)" hidden="false"/></outputs>
   <citations>
         <citation type="bibtex"><![CDATA[
             @article{allaire2016rmarkdown,
diff -r 6da16f4e1e3b -r dad0996ce0b7 scripts_generator.sh
--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/scripts_generator.sh	Fri Nov 09 15:12:12 2018 -0500
@@ -0,0 +1,16 @@
+# run SHELL_SCRIPT within tool outputs directory
+cd ${REPORT_FILES_PATH}
+
+cp $X_f input
+
+# generate findSSRs.sh script
+cat >findSSRs.sh <<EOF
+
+perl ${TOOL_INSTALL_DIR}/findSSRs_altered.pl \\
+  -f input \\
+  -m $X_m > log.txt 2>&1
+
+EOF
+
+# run dustmasker job
+sh findSSRs.sh
\ No newline at end of file