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 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376
|
#!/usr/bin/python3
import argparse
import sys
from deeptools import parserCommon, bamHandler, utilities
from deeptools.mapReduce import mapReduce
from deeptools.utilities import smartLabels
from importlib.metadata import version
def parseArguments():
parser = argparse.ArgumentParser(
formatter_class=argparse.RawDescriptionHelpFormatter,
description="""
This tool estimates the number of reads that would be filtered given a set of
settings and prints this to the terminal. Further, it tracks the number of singleton reads. The following metrics will always be tracked regardless of what you specify (the order output also matches this):
* Total reads (including unmapped)
* Mapped reads
* Reads in blacklisted regions (--blackListFileName)
The following metrics are estimated according to the --binSize and --distanceBetweenBins parameters
* Estimated mapped reads filtered (the total number of mapped reads filtered for any reason)
* Alignments with a below threshold MAPQ (--minMappingQuality)
* Alignments with at least one missing flag (--samFlagInclude)
* Alignments with undesirable flags (--samFlagExclude)
* Duplicates determined by deepTools (--ignoreDuplicates)
* Duplicates marked externally (e.g., by picard)
* Singletons (paired-end reads with only one mate aligning)
* Wrong strand (due to --filterRNAstrand)
The sum of these may be more than the total number of reads. Note that alignments are sampled from bins of size --binSize spaced --distanceBetweenBins apart.
""",
usage='estimateReadFiltering -b sample1.bam sample2.bam\n'
'help: estimateReadFiltering -h / estimateReadFiltering --help'
)
required = parser.add_argument_group('Required arguments')
required.add_argument('--bamfiles', '-b',
metavar='FILE1 FILE2',
help='List of indexed bam files separated by spaces.',
nargs='+',
required=True)
general = parser.add_argument_group('General arguments')
general.add_argument('--outFile', '-o',
type=parserCommon.writableFile,
help='The file to write results to. By default, results are printed to the console')
general.add_argument('--sampleLabels',
help='Labels for the samples. The '
'default is to use the file name of the '
'sample. The sample labels should be separated '
'by spaces and quoted if a label itself'
'contains a space E.g. --sampleLabels label-1 "label 2" ',
nargs='+')
general.add_argument('--smartLabels',
action='store_true',
help='Instead of manually specifying labels for the input '
'BAM files, this causes deepTools to use the '
'file name after removing the path and extension.')
general.add_argument('--binSize', '-bs',
metavar='INT',
help='Length in bases of the window used to sample the genome. (Default: %(default)s)',
default=1000000,
type=int)
general.add_argument('--distanceBetweenBins', '-n',
metavar='INT',
help='To reduce the computation time, not every possible genomic '
'bin is sampled. This option allows you to set the distance '
'between bins actually sampled from. Larger numbers are sufficient '
'for high coverage samples, while smaller values are useful for '
'lower coverage samples. Note that if you specify a value that '
'results in too few (<1000) reads sampled, the value will be '
'decreased. (Default: %(default)s)',
default=10000,
type=int)
general.add_argument('--numberOfProcessors', '-p',
help='Number of processors to use. Type "max/2" to '
'use half the maximum number of processors or "max" '
'to use all available processors. (Default: %(default)s)',
metavar="INT",
type=parserCommon.numberOfProcessors,
default=1,
required=False)
general.add_argument('--verbose', '-v',
help='Set to see processing messages.',
action='store_true')
general.add_argument('--version', action='version',
version='%(prog)s {}'.format(version('deeptools')))
filtering = parser.add_argument_group('Optional arguments')
filtering.add_argument('--filterRNAstrand',
help='Selects RNA-seq reads (single-end or paired-end) in '
'the given strand. (Default: %(default)s)',
choices=['forward', 'reverse'],
default=None)
filtering.add_argument('--ignoreDuplicates',
help='If set, reads that have the same orientation '
'and start position will be considered only '
'once. If reads are paired, the mate\'s position '
'also has to coincide to ignore a read.',
action='store_true')
filtering.add_argument('--minMappingQuality',
metavar='INT',
help='If set, only reads that have a mapping '
'quality score of at least this are '
'considered.',
type=int)
filtering.add_argument('--samFlagInclude',
help='Include reads based on the SAM flag. For example, '
'to get only reads that are the first mate, use a flag of 64. '
'This is useful to count properly paired reads only once, '
'as otherwise the second mate will be also considered for the '
'coverage. (Default: %(default)s)',
metavar='INT',
default=None,
type=int,
required=False)
filtering.add_argument('--samFlagExclude',
help='Exclude reads based on the SAM flag. For example, '
'to get only reads that map to the forward strand, use '
'--samFlagExclude 16, where 16 is the SAM flag for reads '
'that map to the reverse strand. (Default: %(default)s)',
metavar='INT',
default=None,
type=int,
required=False)
filtering.add_argument('--blackListFileName', '-bl',
help="A BED or GTF file containing regions that should be excluded from all analyses. Currently this works by rejecting genomic chunks that happen to overlap an entry. Consequently, for BAM files, if a read partially overlaps a blacklisted region or a fragment spans over it, then the read/fragment might still be considered. Please note that you should adjust the effective genome size, if relevant.",
metavar="BED file",
nargs="+",
required=False)
return parser
def getFiltered_worker(arglist):
chrom, start, end, args = arglist
# Fix the bounds
if end - start > args.binSize and end - start > args.distanceBetweenBins:
end -= args.distanceBetweenBins
if end <= start:
end = start + 1
o = []
for fname in args.bamfiles:
fh = bamHandler.openBam(fname)
chromUse = utilities.mungeChromosome(chrom, fh.references)
prev_pos = set()
lpos = None
minMapq = 0
samFlagInclude = 0
samFlagExclude = 0
internalDupes = 0
externalDupes = 0
singletons = 0
filterRNAstrand = 0
nFiltered = 0
total = 0 # This is only used to estimate the percentage affected
for read in fh.fetch(chromUse, start, end):
filtered = 0
if read.pos < start:
# ensure that we never double count (in case distanceBetweenBins == 0)
continue
if read.flag & 4:
# Ignore unmapped reads, they were counted already
continue
if args.minMappingQuality and read.mapq < args.minMappingQuality:
filtered = 1
minMapq += 1
if args.samFlagInclude and read.flag & args.samFlagInclude != args.samFlagInclude:
filtered = 1
samFlagInclude += 1
if args.samFlagExclude and read.flag & args.samFlagExclude != 0:
filtered = 1
samFlagExclude += 1
if args.ignoreDuplicates:
# Assuming more or less concordant reads, use the fragment bounds, otherwise the start positions
if read.tlen >= 0:
s = read.pos
e = s + read.tlen
else:
s = read.pnext
e = s - read.tlen
if read.reference_id != read.next_reference_id:
e = read.pnext
if lpos is not None and lpos == read.reference_start \
and (s, e, read.next_reference_id, read.is_reverse) in prev_pos:
filtered = 1
internalDupes += 1
if lpos != read.reference_start:
prev_pos.clear()
lpos = read.reference_start
prev_pos.add((s, e, read.next_reference_id, read.is_reverse))
if read.is_duplicate:
filtered = 1
externalDupes += 1
if read.is_paired and read.mate_is_unmapped:
filtered = 1
singletons += 1
# filterRNAstrand
if args.filterRNAstrand:
if read.is_paired:
if args.filterRNAstrand == 'forward':
if read.flag & 144 == 128 or read.flag & 96 == 64:
pass
else:
filtered = 1
filterRNAstrand += 1
elif args.filterRNAstrand == 'reverse':
if read.flag & 144 == 144 or read.flag & 96 == 96:
pass
else:
filtered = 1
filterRNAstrand += 1
else:
if args.filterRNAstrand == 'forward':
if read.flag & 16 == 16:
pass
else:
filtered = 1
filterRNAstrand += 1
elif args.filterRNAstrand == 'reverse':
if read.flag & 16 == 0:
pass
else:
filtered = 1
filterRNAstrand += 1
total += 1
nFiltered += filtered
fh.close()
# Append a tuple to the output
tup = (total, nFiltered, minMapq, samFlagInclude, samFlagExclude, internalDupes, externalDupes, singletons, filterRNAstrand)
o.append(tup)
return o
def main(args=None):
args = parseArguments().parse_args(args)
if not args.sampleLabels and args.smartLabels:
args.sampleLabels = smartLabels(args.bamfiles)
if args.sampleLabels and len(args.sampleLabels) != len(args.bamfiles):
sys.stderr.write("\nError: --sampleLabels specified but it doesn't match the number of BAM files!\n")
sys.exit(1)
if args.outFile is None:
of = sys.stdout
else:
of = open(args.outFile, "w")
bhs = [bamHandler.openBam(x, returnStats=True, nThreads=args.numberOfProcessors) for x in args.bamfiles]
mapped = [x[1] for x in bhs]
unmappedList = [x[2] for x in bhs]
bhs = [x[0] for x in bhs]
# Get the reads in blacklisted regions
if args.blackListFileName:
blacklisted = []
for bh in bhs:
blacklisted.append(utilities.bam_blacklisted_reads(bh, None, args.blackListFileName, args.numberOfProcessors))
else:
blacklisted = [0] * len(bhs)
# Get the total and mapped reads
total = [x + y for x, y in list(zip(mapped, unmappedList))]
chrom_sizes = list(zip(bhs[0].references, bhs[0].lengths))
for x in bhs:
x.close()
# Get the remaining metrics
res = mapReduce([args],
getFiltered_worker,
chrom_sizes,
genomeChunkLength=args.binSize + args.distanceBetweenBins,
blackListFileName=args.blackListFileName,
numberOfProcessors=args.numberOfProcessors,
verbose=args.verbose)
totals = [0] * len(args.bamfiles)
nFiltered = [0] * len(args.bamfiles)
MAPQs = [0] * len(args.bamfiles)
flagIncludes = [0] * len(args.bamfiles)
flagExcludes = [0] * len(args.bamfiles)
internalDupes = [0] * len(args.bamfiles)
externalDupes = [0] * len(args.bamfiles)
singletons = [0] * len(args.bamfiles)
rnaStrand = [0] * len(args.bamfiles)
for x in res:
for idx, r in enumerate(x):
totals[idx] += r[0]
nFiltered[idx] += r[1]
MAPQs[idx] += r[2]
flagIncludes[idx] += r[3]
flagExcludes[idx] += r[4]
internalDupes[idx] += r[5]
externalDupes[idx] += r[6]
singletons[idx] += r[7]
rnaStrand[idx] += r[8]
# Print some output
of.write("Sample\tTotal Reads\tMapped Reads\tAlignments in blacklisted regions\tEstimated mapped reads filtered\tBelow MAPQ\tMissing Flags\tExcluded Flags\tInternally-determined Duplicates\tMarked Duplicates\tSingletons\tWrong strand\n")
for idx, _ in enumerate(args.bamfiles):
if args.sampleLabels:
of.write(args.sampleLabels[idx])
else:
of.write(args.bamfiles[idx])
of.write("\t{}\t{}\t{}".format(total[idx], mapped[idx], blacklisted[idx]))
# nFiltered
metric = 0.0
if totals[idx] > 0:
metric = blacklisted[idx] + float(nFiltered[idx]) / float(totals[idx]) * mapped[idx]
of.write("\t{}".format(min(round(metric, 1), mapped[idx])))
# MAPQ
metric = 0.0
if totals[idx] > 0:
metric = float(MAPQs[idx]) / float(totals[idx]) * mapped[idx]
of.write("\t{}".format(min(round(metric, 1), mapped[idx])))
# samFlagInclude
metric = 0.0
if totals[idx] > 0:
metric = float(flagIncludes[idx]) / float(totals[idx]) * mapped[idx]
of.write("\t{}".format(min(round(metric, 1), mapped[idx])))
# samFlagExclude
metric = 0.0
if totals[idx] > 0:
metric = float(flagExcludes[idx]) / float(totals[idx]) * mapped[idx]
of.write("\t{}".format(min(round(metric, 1), mapped[idx])))
# Internally determined duplicates
metric = 0.0
if totals[idx] > 0:
metric = float(internalDupes[idx]) / float(totals[idx]) * mapped[idx]
of.write("\t{}".format(min(round(metric, 1), mapped[idx])))
# Externally marked duplicates
metric = 0.0
if totals[idx] > 0:
metric = float(externalDupes[idx]) / float(totals[idx]) * mapped[idx]
of.write("\t{}".format(min(round(metric, 1), mapped[idx])))
# Singletons
metric = 0.0
if totals[idx] > 0:
metric = float(singletons[idx]) / float(totals[idx]) * mapped[idx]
of.write("\t{}".format(min(round(metric, 1), mapped[idx])))
# filterRNAstrand
metric = 0.0
if totals[idx] > 0:
metric = float(rnaStrand[idx]) / float(totals[idx]) * mapped[idx]
of.write("\t{}".format(min(round(metric, 1), mapped[idx])))
of.write("\n")
if args.outFile is not None:
of.close()
return 0
|