File: alignmentSieve.py

package info (click to toggle)
python-deeptools 3.5.6%2Bdfsg-1
  • links: PTS, VCS
  • area: main
  • in suites: sid, trixie
  • size: 34,456 kB
  • sloc: python: 14,503; xml: 4,212; sh: 33; makefile: 5
file content (440 lines) | stat: -rw-r--r-- 18,213 bytes parent folder | download
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
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
#!/usr/bin/python3
import argparse
import pysam
import os
import sys

from deeptools import parserCommon
from deeptools.bamHandler import openBam
from deeptools.mapReduce import mapReduce
from deeptools.utilities import getTLen, smartLabels, getTempFileName
from importlib.metadata import version


def parseArguments():
    parser = argparse.ArgumentParser(
        formatter_class=argparse.RawDescriptionHelpFormatter,
        description="This tool filters alignments in a BAM/CRAM file according the the specified parameters. It can optionally output to BEDPE format.",
        usage='alignmentSieve -b sample1.bam -o sample1.filtered.bam --minMappingQuality 10 --filterMetrics log.txt\n'
        'help: alignmentSieve -h / alignmentSieve --help')

    required = parser.add_argument_group('Required arguments')
    required.add_argument('--bam', '-b',
                          metavar='FILE1',
                          help='An indexed BAM file.',
                          required=True)

    required.add_argument('--outFile', '-o',
                          help='The file to write results to. These are the alignments or fragments that pass the filtering criteria.')

    general = parser.add_argument_group('General arguments')
    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('--filterMetrics',
                         metavar="FILE.log",
                         help="The number of entries in total and filtered are saved to this file")

    general.add_argument('--filteredOutReads',
                         metavar="filtered.bam",
                         help="If desired, all reads NOT passing the filtering criteria can be written to this file.")

    general.add_argument('--label', '-l',
                         metavar='sample1',
                         help='User defined label instead of the default label '
                         '(file name).')

    general.add_argument('--smartLabels',
                         action='store_true',
                         help='Instead of manually specifying a labels for the input '
                         'file, this causes deepTools to use the file name '
                         'after removing the path and extension.')

    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')))

    general.add_argument('--shift',
                         nargs='+',
                         type=int,
                         help='Shift the left and right end of a read (for BAM files) or a fragment (for BED files). A positive value shift an end to the right (on the + strand) and a negative value shifts a fragment to the left. Either 2 or 4 integers can be provided. For example, "2 -3" will shift the left-most fragment end two bases to the right and the right-most end 3 bases to the left. If 4 integers are provided, then the first and last two refer to fragments whose read 1 is on the left or right, respectively. Consequently, it is possible to take strand into consideration for strand-specific protocols. A fragment whose length falls below 1 due to shifting will not be written to the output. See the online documentation for graphical examples. Note that non-properly-paired reads will be filtered.')

    general.add_argument('--ATACshift',
                         action='store_true',
                         help='Shift the produced BAM file or BEDPE regions as commonly done for ATAC-seq. This is equivalent to --shift 4 -5 5 -4.')

    general.add_argument('--genomeChunkLength',
                         type=int,
                         default=int(1e6),
                         help='Size of the genome (in bps) to be processed per thread. (Default: %(default)s)')

    output = parser.add_argument_group('Output arguments')
    output.add_argument('--BED',
                        action='store_true',
                        help='Instead of producing BAM files, write output in BEDPE format (as defined by MACS2). Note that only reads/fragments passing filtering criterion are written in BEDPE format.')

    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.',
                           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.',
                           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)

    filtering.add_argument('--minFragmentLength',
                           help='The minimum fragment length needed for read/pair '
                           'inclusion. This option is primarily useful '
                           'in ATACseq experiments, for filtering mono- or '
                           'di-nucleosome fragments. (Default: %(default)s)',
                           metavar='INT',
                           default=0,
                           type=int,
                           required=False)

    filtering.add_argument('--maxFragmentLength',
                           help='The maximum fragment length needed for read/pair '
                           'inclusion. A value of 0 indicates no limit. (Default: %(default)s)',
                           metavar='INT',
                           default=0,
                           type=int,
                           required=False)

    return parser


def shiftRead(b, chromDict, args):
    if not b.is_proper_pair:
        return None
    tLen = getTLen(b, notAbs=True)
    start = b.pos
    end = start + b.query_alignment_end
    if b.is_reverse and not b.is_read2:
        end -= args.shift[2]
        deltaTLen = args.shift[3] - args.shift[2]
    elif b.is_reverse and b.is_read2:
        end += args.shift[1]
        deltaTLen = args.shift[1] - args.shift[0]
    elif not b.is_reverse and not b.is_read2:
        start += args.shift[0]
        deltaTLen = args.shift[1] - args.shift[0]
    else:
        start -= args.shift[3]
        deltaTLen = args.shift[3] - args.shift[2]

    # Sanity check
    if end - start < 1:
        if b.is_reverse:
            start = end - 1
        else:
            end = start + 1
    if start < 0:
        start = 0
    if end > chromDict[b.reference_name]:
        end = chromDict[b.reference_name]
    if end - start < 1:
        return None

    # create a new read
    b2 = pysam.AlignedSegment()
    b2.query_name = b.query_name
    b2.flag = b.flag
    b2.reference_id = b.reference_id
    b2.reference_start = start
    b2.mapping_quality = b.mapping_quality
    b2.cigar = ((0, end - start),)  # Returned cigar is only matches
    if tLen < 0:
        b2.template_length = tLen - deltaTLen
    else:
        b2.template_length = tLen + deltaTLen
    b2.next_reference_id = b.next_reference_id
    b2.next_reference_start = b.next_reference_start
    if b.is_proper_pair:
        if b2.is_read2 and b2.is_reverse:
            b2.next_reference_start += args.shift[0]
        elif not b2.is_read2 and b2.is_reverse:
            b2.next_reference_start -= args.shift[3]

    return b2


def filterWorker(arglist):
    chrom, start, end, args, chromDict = arglist
    fh = openBam(args.bam)
    mode = 'wb'
    oname = getTempFileName(suffix='.bam')
    if args.filteredOutReads:
        onameFiltered = getTempFileName(suffix='.bam')
    else:
        onameFiltered = None
    ofh = pysam.AlignmentFile(oname, mode=mode, template=fh)
    if onameFiltered:
        ofiltered = pysam.AlignmentFile(onameFiltered, mode=mode, template=fh)
    else:
        ofiltered = None

    prev_pos = set()
    lpos = None

    nFiltered = 0
    total = 0
    for read in fh.fetch(chrom, start, end):
        if read.pos < start:
            # ensure that we never double count (in case distanceBetweenBins == 0)
            continue

        total += 1
        if read.flag & 4:
            # Ignore unmapped reads, they were counted already
            nFiltered += 1
            if ofiltered:
                ofiltered.write(read)
            continue

        if args.minMappingQuality and read.mapq < args.minMappingQuality:
            nFiltered += 1
            if ofiltered:
                ofiltered.write(read)
            continue

        if args.samFlagInclude and read.flag & args.samFlagInclude != args.samFlagInclude:
            nFiltered += 1
            if ofiltered:
                ofiltered.write(read)
            continue
        if args.samFlagExclude and read.flag & args.samFlagExclude != 0:
            nFiltered += 1
            if ofiltered:
                ofiltered.write(read)
            continue

        tLen = getTLen(read)
        if args.minFragmentLength > 0 and tLen < args.minFragmentLength:
            nFiltered += 1
            if ofiltered:
                ofiltered.write(read)
            continue
        if args.maxFragmentLength > 0 and tLen > args.maxFragmentLength:
            nFiltered += 1
            if ofiltered:
                ofiltered.write(read)
            continue

        if args.ignoreDuplicates:
            # Assuming more or less concordant reads, use the fragment bounds, otherwise the start positions
            if tLen >= 0:
                s = read.pos
                e = s + tLen
            else:
                s = read.pnext
                e = s - 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:
                nFiltered += 1
                if ofiltered:
                    ofiltered.write(read)
                continue
            if lpos != read.reference_start:
                prev_pos.clear()
            lpos = read.reference_start
            prev_pos.add((s, e, read.next_reference_id, read.is_reverse))

        # filterRNAstrand
        if args.filterRNAstrand:
            if read.is_paired:
                if args.filterRNAstrand == 'forward':
                    if read.flag & 144 == 128 or read.flag & 96 == 64:
                        pass
                    else:
                        nFiltered += 1
                        if ofiltered:
                            ofiltered.write(read)
                        continue
                elif args.filterRNAstrand == 'reverse':
                    if read.flag & 144 == 144 or read.flag & 96 == 96:
                        pass
                    else:
                        nFiltered += 1
                        if ofiltered:
                            ofiltered.write(read)
                        continue
            else:
                if args.filterRNAstrand == 'forward':
                    if read.flag & 16 == 16:
                        pass
                    else:
                        nFiltered += 1
                        if ofiltered:
                            ofiltered.write(read)
                        continue
                elif args.filterRNAstrand == 'reverse':
                    if read.flag & 16 == 0:
                        pass
                    else:
                        nFiltered += 1
                        if ofiltered:
                            ofiltered.write(read)
                        continue

        if args.shift:
            read = shiftRead(read, chromDict, args)
            if not read:
                continue

        # Read survived filtering
        ofh.write(read)

    # The results from the workers will get sorted, so get the TID
    tid = fh.get_tid(chrom)

    ofh.close()
    if ofiltered:
        ofiltered.close()
    fh.close()
    return tid, start, total, nFiltered, oname, onameFiltered


def convertBED(oname, tmpFiles, chromDict):
    """
    Stores results in BEDPE format, which is:
    chromosome	frag_leftend	frag_rightend

    The fragment ends can be shifted
    """
    ofile = open(oname, "w")
    for tmpFile in tmpFiles:
        # Setting verbosity to avoid lack of index error/warning
        pysam.set_verbosity(0)
        fh = pysam.AlignmentFile(tmpFile)
        # Reset verbosity
        pysam.set_verbosity(3)
        for b in fh.fetch(until_eof=True):
            tLen = getTLen(b, notAbs=True)
            if tLen > 0:
                start = b.pos
                end = start + tLen
                if end > chromDict[b.reference_name]:
                    end = chromDict[b.reference_name]
                if end - start < 1:
                    continue
                ofile.write("{}\t{}\t{}\n".format(b.reference_name, start, end))
        fh.close()
        os.unlink(tmpFile)
    ofile.close()


def main(args=None):
    args = parseArguments().parse_args(args)
    if args.shift:
        if len(args.shift) not in [2, 4]:
            sys.exit("The --shift option can accept either 2 or 4 values only.")
        if len(args.shift) == 2:
            args.shift.extend([-args.shift[1], -args.shift[0]])
    elif args.ATACshift:
        args.shift = [4, -5, 5, -4]

    bam, mapped, unmapped, stats = openBam(args.bam, returnStats=True, nThreads=args.numberOfProcessors)
    total = mapped + unmapped
    chrom_sizes = [(x, y) for x, y in zip(bam.references, bam.lengths)]
    chromDict = {x: y for x, y in zip(bam.references, bam.lengths)}
    bam.close()

    # Filter, writing the results to a bunch of temporary files
    res = mapReduce([args, chromDict],
                    filterWorker,
                    chrom_sizes,
                    blackListFileName=args.blackListFileName,
                    numberOfProcessors=args.numberOfProcessors,
                    genomeChunkLength=args.genomeChunkLength,
                    verbose=args.verbose)

    res = sorted(res)  # The temp files are now in order for concatenation
    nFiltered = sum([x[3] for x in res])
    totalSeen = sum([x[2] for x in res])  # The * contig isn't queried

    tmpFiles = [x[4] for x in res]
    if not args.BED:
        arguments = ["-o", args.outFile]
        arguments.extend(tmpFiles)  # [..., *someList] isn't available in python 2.7
        pysam.samtools.cat(*arguments)
        for tmpFile in tmpFiles:
            os.unlink(tmpFile)
    else:
        convertBED(args.outFile, tmpFiles, chromDict)

    if args.filteredOutReads:
        tmpFiles = [x[5] for x in res]
        if not args.BED:
            arguments = ["-o", args.filteredOutReads]
            arguments.extend(tmpFiles)  # [..., *someList] isn't available in python 2.7
            pysam.samtools.cat(*arguments)
            for tmpFile in tmpFiles:
                os.unlink(tmpFile)
        else:
            convertBED(args.outFile, tmpFiles, chromDict, args)

    if args.filterMetrics:
        sampleName = args.bam
        if args.label:
            sampleName = args.label
        if args.smartLabels:
            sampleName = smartLabels([args.bam])[0]

        of = open(args.filterMetrics, "w")
        of.write("#bamFilterReads --filterMetrics\n")
        of.write("#File\tReads Remaining\tTotal Initial Reads\n")
        of.write("{}\t{}\t{}\n".format(sampleName, totalSeen - nFiltered, total))
        of.close()

    return 0