added a new script for counting reads

snakemake/process_data/Snakefile

-configfile: "config.yaml"
+configfile: "config_hepb2.yaml"
 #from snakemake.utils import listfiles
 
 localrules: finish
@@ -11,7 +11,7 @@ rule finish:
 	input:
 		oligos_counts = expand(os.path.join(config["output_dir"], "{sample}", "oligos_counts.txt"), sample=config["sample"]),
 		pdf = expand(os.path.join(config["output_dir"], "{sample}/read_length/read_length_histogram.pdf"), sample=config["sample"]),
-		counts = expand(os.path.join(config["output_dir"], "{sample}/counts.tsv"), sample=config["sample"]),
+		counts = expand(os.path.join(config["output_dir"], "{sample}/counts/counts.tsv"), sample=config["sample"]),
 		bai = expand(os.path.join(config["output_dir"], "{sample}/transcripts.mapped.unique.a_site_profile.sorted.bam.bai"), sample=config["sample"]),
 		overrepresented_sequences_counts = expand(os.path.join(config["output_dir"], "{sample}/overepressented_sequences_other.txt"), sample=config["sample"])
 
@@ -317,20 +317,28 @@ rule read_length_histogram:
 
 rule count_reads:
 	input:
-		sam = os.path.join(config["output_dir"], "{sample}/transcripts.mapped.unique.sam"),
-		transcript_id_gene_id_CDS = config["transcript_id_gene_id_CDS"]
+		bam = os.path.join(config["output_dir"], "{sample}/transcripts.mapped.unique.sorted.bam"),
+		transcript_id_gene_id_CDS = config["transcript_id_gene_id_CDS"],
+                p_site_offsets = os.path.join(
+			config["output_dir"],
+			"{sample}/p_site_offsets/alignment_offset.json")
 	output:
-		counts = os.path.join(config["output_dir"], "{sample}/counts.tsv")
+		counts = os.path.join(config["output_dir"], "{sample}/counts/counts.tsv"),
+                count = os.path.join(config["output_dir"], "{sample}/counts")
+	params:
+		outdir = os.path.join(config["output_dir"], "{sample}/counts")
+
 	log:
 		os.path.join(config["local_log"], "count_reads_{sample}.log")
 	threads:	1
 	singularity:
-		"docker://perl:5.24-slim"
+		"docker://fgypas/python_pysam:3.6.5_0.15.1"
 	shell:
-		"(perl scripts/xp-count-reads-ribseq.pl \
-		{input.sam} \
-		{input.transcript_id_gene_id_CDS} \
-		> {output.counts})"
+		"(python scripts/count_reads.py \
+		--bam {input.bam} \
+		--tsv {input.transcript_id_gene_id_CDS} \
+                --json {input.p_site_offsets} \
+                --outdir {params.outdir})"
 
 ################################################################################
 ### Determine P-site offset

snakemake/process_data/scripts/count_reads.py

+#!/usr/bin/env python
+
+# _____________________________________________________________________________
+# -----------------------------------------------------------------------------
+# import needed (external) modules
+# -----------------------------------------------------------------------------
+
+from argparse import ArgumentParser, RawTextHelpFormatter
+import sys
+import os
+import pysam
+import json
+
+# _____________________________________________________________________________
+# -----------------------------------------------------------------------------
+# Main function
+# -----------------------------------------------------------------------------
+
+def main():
+    """ Main function """
+
+    __doc__ = "Count reads mapped to transcripts using BAM file and offsets from json file."
+    __version__ = "0.1"
+
+    parser = ArgumentParser(
+        description=__doc__,
+        formatter_class=RawTextHelpFormatter
+    )
+
+    parser.add_argument(
+        "--bam",
+        dest="bam",
+        help="Input bam file (sorted and indexed)",
+        required=True,
+        metavar="FILE"
+    )
+
+    parser.add_argument(
+        "--json",
+        dest="json",
+        help="Input json file",
+        required=True,
+        metavar="FILE"
+    )
+
+    parser.add_argument(
+        "--tsv",
+        dest="tsv",
+        help="Input tsv file",
+        required=True,
+        metavar="FILE"
+    )
+    parser.add_argument(
+        "--outdir",
+        dest="outdir",
+        help="Output directory",
+        required=True,
+        metavar="FILE"
+    )
+
+    parser.add_argument(
+        "--verbose",
+        action="store_true",
+        dest="verbose",
+        default=False,
+        required=False,
+        help="Be verbose"
+    )
+
+    parser.add_argument(
+        '--version',
+        action='version',
+        version=__version__
+    )
+
+    # _________________________________________________________________________
+    # -------------------------------------------------------------------------
+    # get the arguments
+    # -------------------------------------------------------------------------
+    try:
+        options = parser.parse_args()
+    except Exception:
+        parser.print_help()
+
+    if len(sys.argv) == 1:
+        parser.print_help()
+        sys.exit(1)
+
+    if options.verbose:
+        sys.stdout.write("and parsing {} {}".format(options.bam, os.linesep))
+
+    with open(options.json, 'r') as json_file:
+        data = json_file.read()
+
+    cds_start_ind = {}
+    cds_end_ind = {}
+
+    with open(options.tsv) as cds_coordinates:
+        for line in cds_coordinates:
+            sp_line = line.strip().split("\t")
+            transcript_id = sp_line[0]
+            cds_start_ind[transcript_id] = int(sp_line[2])
+            cds_end_ind[transcript_id] = int(sp_line[3])
+
+    count_discarded = 0
+    all_transcripts = {}
+    offset_obj = json.loads(data)
+    bam = pysam.AlignmentFile(options.bam, "rb")  
+    for read in bam.fetch():
+        if read.is_reverse:
+            continue
+        transcript = read.reference_name
+        if transcript not in all_transcripts:
+           all_transcripts[transcript] = [0, 0, 0] 
+        read_length = len(read.seq)
+        if str(read_length) in offset_obj:
+            read_start = read.reference_start
+            psite_pos = (read_start + offset_obj[str(read_length)])
+            if psite_pos >= cds_start_ind[transcript] and psite_pos <= cds_end_ind[transcript]:
+                all_transcripts[transcript][1] = all_transcripts[transcript][1] + 1
+            elif psite_pos < cds_start_ind[transcript]:
+                all_transcripts[transcript][0] = all_transcripts[transcript][0] + 1
+            else:
+                all_transcripts[transcript][2] = all_transcripts[transcript][2] + 1
+        else:
+            count_discarded = count_discarded + 1
+        
+    w = open(os.path.join(options.outdir, "counts.tsv"), 'w')
+    w.write("Transcript" + "\t" + "5' UTR" + "\t" + "CDS" + "\t" + "3' UTR" + "\t" + "CDS length" + "\n")
+    for trcpt_key in all_transcripts:
+        gene_length = cds_end_ind[trcpt_key]-cds_start_ind[trcpt_key]+1
+        w.write(trcpt_key + "\t" + str(all_transcripts[trcpt_key][0]) + "\t" + str(all_transcripts[trcpt_key][1]) + "\t" + str(all_transcripts[trcpt_key][2]) + "\t" + str(gene_length) + "\n")	
+    bam.close()
+    w.close() 
+
+if __name__ == '__main__':
+    try:
+        main()
+    except KeyboardInterrupt:
+        sys.stderr.write("User interrupt!" + os.linesep)
+        sys.exit(0)

snakemake/process_data/scripts/xp-count-reads-ribseq.pl

-use Data::Dumper;
-
-my %hash  = ();
-my %anno  = ();
-my $readsSum = 0;
-
-open(SAM,$ARGV[0]);
-
-#Annotation file included with beginning and end of every CDS
-my $annoFile = (defined($ARGV[1])) ? 1 : 0;
-
-if($annoFile){
-	open(AN,$ARGV[1]);
-	while( $line = <AN> ){
-		chomp $line;
-		my @F = split /\t/ , $line; #annotation line
-		my @a = ($F[1],$F[2]);
-		$anno{$F[0]} = \@a;
-	}
-} else {
-	print STDERR "Annotation file not specified, using len(5UTR)=200\n";
-}
-
-#print Dumper(%anno);
-
-#Parse SAM file and count reads
-while( $line = <SAM> ){
-
-	chomp $line;
-	next if $line =~ /^@/; #discard headers/comments
-
-	my @F = split /\t/ , $line; #sam line
-
-	my $gene_name = $F[2];
-        if($F[1] == 0) { #only consider hits in plus strand
-		if($annoFile){
-			if(($F[3] >= $anno{$gene_name}->[0]-15) & ($F[3] <= $anno{$gene_name}->[1]-15)) { #close to start and stop
-				$hash{$gene_name}++;
-			}
-		} {	
-			if($F[3] >= 186) { #consider that len(5UTR)==200
-				$hash{$gene_name}++;
-			}
-		}
-	}
-}
-
-#print Dumper(\%hash);
-print "gene\tcounts\n";
-for $key (keys %hash){
-	print $key,"\t",$hash{$key},"\n";
-}