1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
|
#!/usr/bin/env python3
# encoding: utf-8
from __future__ import (absolute_import, division,
print_function, unicode_literals)
import os
import sys
import argparse
import subprocess
import shlex
import logging
import re
sys.path.insert(0, os.path.sep.join([os.path.dirname(os.path.realpath(__file__)),
"..", "..", "..", "PyLib"]))
import Pipeliner
logger = None
UTILDIR = os.path.sep.join([os.path.dirname(os.path.realpath(__file__)), "util"])
def main():
FORMAT = "%(asctime)-15s %(levelname)s %(module)s.%(name)s.%(funcName)s at %(lineno)d :\n\t%(message)s\n"
global logger
logger = logging.getLogger()
logging.basicConfig(filename='variant_calling.log', format=FORMAT, filemode='w', level=logging.DEBUG)
# add a new Handler to print all INFO and above messages to stdout
ch = logging.StreamHandler(sys.stdout)
ch.setLevel(logging.INFO)
logger.addHandler(ch)
parser = argparse.ArgumentParser(
description=str("This script requires you have the following dependencies:\n" +
"Samtools: \"samtools\" in your path\n" +
"Java: \"java\" in your path\n" +
"Picard-Tools: env var \"$PICARD_HOME\" with the path to Picard-Tools's bin\n" +
"STAR: \"STAR\" in your path\n" +
"GATK: env var \"$GATK_HOME\" with the path to GATK's bin\n"),
epilog="", formatter_class=argparse.RawTextHelpFormatter)
parser.add_argument('--st_fa', '--supertranscript_fasta', dest="st_fa", type=str, required=True,
help="Path to the SuperTranscripts fasta file.")
parser.add_argument('--st_gtf', '--supertranscript_gtf', dest="st_gtf", type=str, required=True,
help="Path to the SuperTranscript gtf file.")
group = parser.add_mutually_exclusive_group(required=True)
group.add_argument('-p', '--paired', dest="paired_reads", type=str, nargs=2,
help="Pair of paired ends read files.")
group.add_argument('-s', '--single', dest="single_reads", type=str,
help="Single reads file.")
group.add_argument("-S", "--samples_file", dest="samples_file", type=str,
help="Trinity samples file (fmt: condition_name replicate_name /path/to/reads_1.fq /path/to/reads_2.fq (tab-delimited, single sample per line))")
parser.add_argument('-o', '--output', dest="out_path", type=str, required=True,
help="Path to the folder where to generate the output.")
parser.add_argument('-l', '--sjdbOverhang', dest="sjdbOverhang", default=150, type=int,
help="Size of the reads (used for STAR --sjdbOverhang). default=150")
parser.add_argument('-t', '--threads', dest="nthreads", type=str, default="4",
help="Number of threads to use for tools that are multithreaded.")
parser.add_argument('-m', '--maxram', dest="maxram", type=str, default="50000000000",
help="Maximum amount of RAM allowed for STAR's genome generation step (only change if you get an error from STAR complaining about this value).")
parser.add_argument("--STAR_genomeGenerate_opts", type=str, default="",
help="options to pass through to STAR's genomeGenerate function")
args = parser.parse_args()
PICARD_HOME = os.getenv("PICARD_HOME")
if not PICARD_HOME:
exit("Error, missing path to Picard-Tools in $PICARD_HOME.")
GATK_HOME = os.getenv("GATK_HOME")
if not GATK_HOME:
exit("Error, missing path to GATK in $GATK.")
# get real paths before changing working directory in case they are relative paths
if args.paired_reads:
reads_paths = [os.path.realpath(f) for f in args.paired_reads]
elif args.single_reads:
reads_paths = [os.path.realpath(args.single_reads)]
elif args.samples_file:
left_fq_list = list()
right_fq_list = list()
with open(args.samples_file) as fh:
for line in fh:
line = line.rstrip()
if not re.match("\w", line):
continue
fields = line.split("\t")
left_fq = fields[2]
left_fq_list.append(os.path.realpath(left_fq))
if len(fields) > 3:
right_fq = fields[3]
right_fq_list.append(os.path.realpath(right_fq))
reads_paths = [",".join(left_fq_list)]
if right_fq_list:
reads_paths.append(",".join(right_fq_list))
else:
raise RuntimeError("no reads specified") # should never get here
st_fa_path = os.path.realpath(args.st_fa)
st_gtf_path = os.path.realpath(args.st_gtf)
# check if output directory exists, if not create
real_path = os.path.realpath(args.out_path)
if not os.path.isdir(real_path):
os.makedirs(real_path)
# move to output folder
os.chdir(real_path)
checkpoint_dir = os.path.abspath(os.path.basename(st_fa_path)) + ".gatk_chkpts"
pipeliner = Pipeliner.Pipeliner(checkpoint_dir)
# generate supertranscript index
logger.info("Generating SuperTranscript index.")
pipeliner.add_commands([Pipeliner.Command("samtools faidx {}".format(st_fa_path), "samtools_faidx_st.ok")])
pipeliner.run()
# generate supertranscript Picard dictionary
logger.info("Generating Picard dictionary.")
dict_file = re.sub("\.[^\.]+$", ".dict", st_fa_path)
if os.path.isfile(dict_file):
open(checkpoint_dir + "/picard_dict_st.ok", 'a').close()
else:
pipeliner.add_commands([Pipeliner.Command("java -jar " + PICARD_HOME + "/picard.jar" +
" CreateSequenceDictionary R=" + st_fa_path +
" O=" + dict_file +
" VALIDATION_STRINGENCY=LENIENT ",
"picard_dict_st.ok")])
pipeliner.run()
# generate genome folder for STAR's first pass
logger.info("Generating genome folder for STAR")
star_genome_generate_cmd = str("STAR --runThreadN " +
args.nthreads +
" --runMode genomeGenerate" +
" --genomeDir star_genome_idx " +
" --genomeFastaFiles {} ".format(st_fa_path) +
" --genomeSAindexNbases 8 " + # as per A. Dobin
" --sjdbGTFfile {} ".format(st_gtf_path) +
" --sjdbOverhang {} ".format(args.sjdbOverhang) +
" --limitGenomeGenerateRAM {}".format(args.maxram) +
" {} ".format(args.STAR_genomeGenerate_opts)
)
pipeliner.add_commands([
Pipeliner.Command("mkdir star_genome_idx", "mkdir_star_genome_idx.ok"),
Pipeliner.Command(star_genome_generate_cmd,
"star_genome_generate.ok")
])
pipeliner.run()
# run STAR's alignment
logger.info("Running STAR alignment.")
cmd = str("STAR --runThreadN " + args.nthreads
+ " --genomeDir star_genome_idx "
+ " --runMode alignReads "
+ " --twopassMode Basic "
+ " --alignSJDBoverhangMin 10 "
+ " --outSAMtype BAM SortedByCoordinate "
+ " --limitBAMsortRAM {} ".format(args.maxram)
+ " --readFilesIn " + " ".join(reads_paths) )
if re.search("\.gz$", reads_paths[0]):
cmd += " --readFilesCommand 'gunzip -c' "
pipeliner.add_commands([Pipeliner.Command(cmd, "star_aln.ok")])
pipeliner.run()
##
## GATK settings based on best practices:
## https://software.broadinstitute.org/gatk/documentation/article.php?id=3891
##
# clean and convert sam file with Picard-Tools
logger.info("Cleaning and Converting sam file with Picard-Tools.")
pipeliner.add_commands([
Pipeliner.Command("java -jar " + PICARD_HOME + "/picard.jar " +
" AddOrReplaceReadGroups " +
"I=Aligned.sortedByCoord.out.bam " +
"O=rg_added_sorted.bam " +
" VALIDATION_STRINGENCY=SILENT " +
"SO=coordinate RGID=id RGLB=library RGPL=platform RGPU=machine RGSM=sample",
"add_read_groups.ok"),
Pipeliner.Command("java -jar " + PICARD_HOME + "/picard.jar " +
" MarkDuplicates " +
"I=rg_added_sorted.bam O=dedupped.bam " +
"CREATE_INDEX=true VALIDATION_STRINGENCY=SILENT M=output.metrics",
"mark_dups.ok"),
Pipeliner.Command("java -jar " + PICARD_HOME + "/picard.jar ValidateSamFile " +
"I=dedupped.bam " +
"IGNORE_WARNINGS=true " +
"MAX_OUTPUT=100000 " +
"IGNORE=MATE_NOT_FOUND " +
"O=dedupped.bam.validation",
"bam_validate.ok",
ignore_error=True),
Pipeliner.Command(UTILDIR + "/clean_bam.pl dedupped.bam dedupped.bam.validation dedupped.valid.bam",
"make_valid_dedupped_bam.ok"),
Pipeliner.Command("java -jar " + GATK_HOME + "/GenomeAnalysisTK.jar " +
"-T SplitNCigarReads -R " + st_fa_path +
" -I dedupped.valid.bam -o splitNCigar.bam " +
" -rf ReassignOneMappingQuality -RMQF 255 -RMQT 60 -U ALLOW_N_CIGAR_READS --validation_strictness LENIENT",
"splitNCigarReads.ok")
])
pipeliner.run()
# do the actual variant calling
logger.info("Variant Calling using Haplotype Caller.")
pipeliner.add_commands([
Pipeliner.Command("java -jar " + GATK_HOME + "/GenomeAnalysisTK.jar " +
"-T HaplotypeCaller -R " + st_fa_path +
" -I ./splitNCigar.bam -dontUseSoftClippedBases -stand_call_conf 20.0 -o output.vcf",
"haplotypecaller.ok")
])
pipeliner.run()
# do some basic filtering
logger.info("Doing some basic filtering of vcf.")
pipeliner.add_commands([
Pipeliner.Command("java -jar " + GATK_HOME + "/GenomeAnalysisTK.jar " +
"-T VariantFiltration -R " + st_fa_path +
" -V output.vcf -window 35 -cluster 3 " +
"-filterName FS -filter \"FS > 30.0\" " +
"-filterName QD -filter \"QD < 2.0\" -o filtered_output.vcf",
"variant_filt.ok")
])
pipeliner.run()
logger.info("Done!")
if __name__ == "__main__":
main()
|
