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Commit fc115178 authored by BIOPZ-Gypas Foivos's avatar BIOPZ-Gypas Foivos
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Addition of scripts that determines the p-site offset based on different read lengths

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snakemake/process_data/Snakefile
+ 59
− 1
View file @ fc115178
configfile: "config.yaml"
#from snakemake.utils import listfiles
localrules: create_output_and_log_directories, remove_multimappers, read_length_histogram, count_reads, sfinish
localrules: create_output_and_log_directories, remove_multimappers, read_length_histogram, count_reads, determine_p_site_offset, finish
#################################################################################
### Finish rule
......@@ -9,6 +9,7 @@ localrules: create_output_and_log_directories, remove_multimappers, read_length_
rule finish:
input:
p_site_offsets = expand(os.path.join(config["output_dir"], "{sample}/p_site_offsets/alignment_offset.json"), 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"])
......@@ -227,6 +228,28 @@ rule remove_multimappers:
shell:
"(grep -P \"^@|\tNH:i:1\t\" {input.sam} > {output.sam}) &> {log}"
################################################################################
### SAM to BAM sort and index
################################################################################
rule sam2bam_sort_and_index:
input:
sam = os.path.join(config["output_dir"], "{sample}/transcripts.mapped.sam")
output:
bam = os.path.join(config["output_dir"], "{sample}/transcripts.mapped.unique.sorted.bam"),
bai = os.path.join(config["output_dir"], "{sample}/transcripts.mapped.unique.sorted.bam.bai")
params:
cluster_log = os.path.join(config["cluster_log"], "sam2bam_sort_and_index_{sample}.log")
log:
os.path.join(config["local_log"], "sam2bam_sort_and_index_{sample}.log")
threads: 1
singularity:
"docker://zavolab/samtools:1.8"
shell:
"(samtools view -bS {input.sam} \
| samtools sort - > {output.bam}; \
samtools index {output.bam};) &> {log}"
################################################################################
### Read length histogram
################################################################################
......@@ -258,6 +281,8 @@ rule count_reads:
transcript_id_gene_id_CDS = config["transcript_id_gene_id_CDS"]
output:
counts = os.path.join(config["output_dir"], "{sample}/counts.tsv")
log:
os.path.join(config["local_log"], "count_reads_{sample}.log")
threads: 1
singularity:
"docker://perl:5.24-slim"
......@@ -266,3 +291,36 @@ rule count_reads:
{input.sam} \
{input.transcript_id_gene_id_CDS} \
> {output.counts})"
################################################################################
### Determine P-site offset
################################################################################
rule determine_p_site_offset:
input:
bam = os.path.join(config["output_dir"], "{sample}/transcripts.mapped.unique.sorted.bam"),
transcript_id_gene_id_CDS = config["transcript_id_gene_id_CDS"]
output:
p_site_offsets = os.path.join(
config["output_dir"],
"{sample}/p_site_offsets/alignment_offset.json"
),
p_site_offset = os.path.join(config["output_dir"],
"{sample}/p_site_offsets")
params:
outdir = os.path.join(config["output_dir"], "{sample}/p_site_offsets"),
cluster_log = os.path.join(config["cluster_log"], "determine_p_site_offset_{sample}.log")
log:
os.path.join(config["local_log"], "determine_p_site_offset_{sample}.log")
threads: 1
singularity:
"docker://fgypas/python_pysam:3.6.5_0.15.1"
shell:
"(python scripts/determine_p_site_offsets.py \
--bam {input.bam} \
--cds_coordinates {input.transcript_id_gene_id_CDS} \
--outdir {params.outdir}) &> {log}"
################################################################################
### Filter reads based on selected read lengths
################################################################################
snakemake/process_data/cluster.json
+ 7
− 0
View file @ fc115178
......@@ -19,5 +19,12 @@
"time": "06:00:00",
"threads":"8",
"mem":"50G"
},
"sam2bam_sort_and_index":
{
"queue":"6hours",
"time": "06:00:00",
"threads":"1",
"mem":"10G"
}
}
snakemake/process_data/scripts/determine_p_site_offsets.py 0 → 100755
+ 211
− 0
View file @ fc115178
#!/usr/bin/env python
# _____________________________________________________________________________
# -----------------------------------------------------------------------------
# import needed (external) modules
# -----------------------------------------------------------------------------
from argparse import ArgumentParser, RawTextHelpFormatter
import itertools
import sys
import os
import pysam
import json
# _____________________________________________________________________________
# -----------------------------------------------------------------------------
# Main function
# -----------------------------------------------------------------------------
def main():
""" Main function """
__doc__ = "Determine p-site offset"
__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(
"--cds_coordinates",
dest="cds_coordinates",
help="CDS coordinates tsv table",
required=True,
metavar="FILE"
)
parser.add_argument(
"--filterReadsForPeriodicity",
dest="filterReadsForPeriodicity",
help="Filter reads for periodicity",
required=False,
default=False,
action='store_true'
)
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("Creating output directory: {} {}".format(options.outdir, os.linesep))
if not os.path.exists(options.outdir):
os.mkdir(options.outdir)
if options.verbose:
sys.stdout.write("Parsing {} {}".format(options.cds_coordinates, os.linesep))
sys.stdout.write("and parsing {} {}".format(options.bam, os.linesep))
# Open alignment file
bam = pysam.AlignmentFile(options.bam, "rb")
### Calculating P-site offset
possible_offsets = [10, 11, 12, 13, 14, 15, 16]
readsDict = {}
# loop over CDS coordinates
with open(options.cds_coordinates) as cds_coordinates:
for line in cds_coordinates:
sp_line = line.strip().split("\t")
transcript_id = sp_line[0]
cds_start = int(sp_line[2]) - 1
cds_end = int(sp_line[3])
for read in bam.fetch(transcript_id,
cds_start,
cds_end):
# keep only + strand
if read.is_reverse:
continue
read_len = len(str(read.seq))
# Create a dictionary to score the different offsets for the distinct read lenghts
if read_len not in readsDict.keys():
readsDict[read_len] = {}
for offset in possible_offsets:
readsDict[read_len][offset] = [0, 0, 0, 0, 0] #1st,2nd,3rd,UTR,CDS
readsDict[read_len]["reads"] = []
read_start = read.reference_start
for offset in possible_offsets:
psite_pos = (read_start + offset) #P-site
asite_pos = psite_pos + 3 #A-site
rel_pos_psite = psite_pos - cds_start
rel_pos_asite = asite_pos - cds_start - 3
if options.filterReadsForPeriodicity:
#There's NO peak at start codon
if rel_pos_asite < 0:
readsDict[read_len][offset][3] += 1
elif rel_pos_asite > (cds_end - cds_start):
readsDict[read_len][offset][3] += 1
else:
if rel_pos_asite > 60: #ignore first 20 codons
frame_rel_pos = rel_pos_asite % 3
readsDict[read_len][offset][frame_rel_pos] += 1
else:
#There's a peak at start codon
if rel_pos_psite < 0:
readsDict[read_len][offset][3] += 1
elif rel_pos_psite > 2:
readsDict[read_len][offset][4] += 1
elif rel_pos_psite in [0, 1, 2]: #anchor at start codon
readsDict[read_len][offset][rel_pos_psite] += 1
# Close alignment file
bam.close()
alignment_offset = {}
for key, value in readsDict.items():
best_offset = 0
best_offset_score = 0
for offset in possible_offsets:
raw_counts = readsDict[key][offset][0:3]
if sum(raw_counts) > 0:
if ((sum(raw_counts) > 200) \
and (raw_counts[0] > raw_counts[1]) \
and (raw_counts[0] > raw_counts[2])):
if ((raw_counts[0] > best_offset_score) \
and ((not options.filterReadsForPeriodicity) \
or (float(raw_counts[0])/sum(raw_counts) > 0.4))):
best_offset = offset
best_offset_score = raw_counts[0]
if best_offset != 0:
alignment_offset[key] = best_offset
# self.filtered_alignments += (readsDict[rl]["reads"])
sys.stderr.write("PEAK CALL:" + \
str(key) + \
": peak found at position " + \
str(best_offset) \
+ os.linesep)
else:
sys.stderr.write("PEAK CALL:" + \
str(key) + \
": peak not found" + \
os.linesep)
sys.stdout.write(str(alignment_offset) + os.linesep)
with open(os.path.join(options.outdir, "alignment_offset.json"), 'w') as file:
file.write(json.dumps(alignment_offset))
# _____________________________________________________________________________
# -----------------------------------------------------------------------------
# Call the Main function and catch Keyboard interrups
# -----------------------------------------------------------------------------
if __name__ == '__main__':
try:
main()
except KeyboardInterrupt:
sys.stderr.write("User interrupt!" + os.linesep)
sys.exit(0)
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