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
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
use std;

use rust_htslib::bam;
use rust_htslib::bam::record::Cigar;

use mosdepth_genome_coverage_estimators::*;
use bam_generator::*;
use coverage_takers::*;
use FlagFilter;
use ReadsMapped;

pub fn contig_coverage<R: NamedBamReader,
                       G: NamedBamReaderGenerator<R>,
                       T: CoverageTaker>(
    bam_readers: Vec<G>,
    coverage_taker: &mut T,
    coverage_estimators: &mut Vec<CoverageEstimator>,
    print_zero_coverage_contigs: bool,
    flag_filters: FlagFilter)
    -> Vec<ReadsMapped> {

    let mut reads_mapped_vector = vec!();
    for mut bam_generator in bam_readers {
        let mut bam_generated = bam_generator.start();

        let stoit_name = &(bam_generated.name().to_string());
        coverage_taker.start_stoit(stoit_name);
        let mut record: bam::record::Record = bam::record::Record::new();
        let mut last_tid: i32 = -2; // no such tid in a real BAM file
        let mut ups_and_downs: Vec<i32> = Vec::new();
        let header = bam_generated.header().clone();
        let target_names = header.target_names();

        let mut num_mapped_reads_total: u64 = 0;
        let mut num_mapped_reads_in_current_contig: u64 = 0;
        let mut total_indels_in_current_contig: u32 = 0;
        let mut total_edit_distance_in_current_contig: u32 = 0;

        let mut process_previous_contigs = |last_tid, tid,
        coverage_estimators: &mut Vec<CoverageEstimator>,
        ups_and_downs,
        num_mapped_reads_in_current_contig,
        total_edit_distance_in_current_contig,
        total_indels_in_current_contig,
        num_mapped_reads_total: &mut u64| {
            if last_tid != -2 {
                debug!("Found {} reads mapped to tid {}, with total edit \
                        distance {} and {} indels",
                       num_mapped_reads_in_current_contig, last_tid,
                       total_edit_distance_in_current_contig,
                       total_indels_in_current_contig);
                for estimator in coverage_estimators.iter_mut() {
                    estimator.add_contig(
                        &ups_and_downs,
                        num_mapped_reads_in_current_contig,
                        total_edit_distance_in_current_contig -
                            total_indels_in_current_contig)
                }
                let coverages: Vec<f32> = coverage_estimators.iter_mut()
                    .map(|estimator| estimator.calculate_coverage(0)).collect();
                let has_nonzero_coverage = coverages.iter().any(
                    |&coverage| coverage > 0.0);
                debug!("Found nonzero coverage?: {}", has_nonzero_coverage);
                if has_nonzero_coverage {
                    *num_mapped_reads_total += num_mapped_reads_in_current_contig;
                }
                if print_zero_coverage_contigs || has_nonzero_coverage {
                    coverage_taker.start_entry(
                        last_tid as usize,
                        std::str::from_utf8(target_names[last_tid as usize]).unwrap());
                    for (coverage, mut estimator) in coverages.iter().zip(coverage_estimators.iter_mut()) {
                        estimator.print_coverage(
                            &coverage,
                            coverage_taker);
                        estimator.setup();
                    }
                    coverage_taker.finish_entry();
                }
            }
            if print_zero_coverage_contigs {
                print_previous_zero_coverage_contigs(
                    match last_tid { -2 => -1, _ => last_tid},
                    tid, coverage_estimators, &target_names, coverage_taker,
                    &header);
            }
        };


        // for record in records
        while bam_generated.read(&mut record).is_ok() {
            debug!("Starting with a new read.. {:?}", record);
            if (!flag_filters.include_supplementary && record.is_supplementary()) ||
                (!flag_filters.include_secondary && record.is_secondary()) ||
                (!flag_filters.include_improper_pairs && !record.is_proper_pair()){
                    debug!("Skipping read based on flag filtering");
                    continue;
                }
            // if reference has changed, print the last record
            let tid = record.tid();
            if !record.is_unmapped() { // if mapped
                // if reference has changed, print the last record
                if tid != last_tid {
                    process_previous_contigs(
                        last_tid,
                        tid,
                        coverage_estimators,
                        ups_and_downs,
                        num_mapped_reads_in_current_contig,
                        total_edit_distance_in_current_contig,
                        total_indels_in_current_contig,
                        &mut num_mapped_reads_total);
                    ups_and_downs = vec![0; header.target_len(tid as u32).expect("Corrupt BAM file?") as usize];
                    debug!("Working on new reference {}",
                           std::str::from_utf8(target_names[tid as usize]).unwrap());
                    last_tid = tid;
                    num_mapped_reads_in_current_contig = 0;
                    total_edit_distance_in_current_contig = 0;
                    total_indels_in_current_contig = 0;
                }

                num_mapped_reads_in_current_contig += 1;

                // for each chunk of the cigar string
                debug!("read name {:?}", std::str::from_utf8(record.qname()).unwrap());
                let mut cursor: usize = record.pos() as usize;
                for cig in record.cigar().iter() {
                    debug!("Found cigar {:} from {}", cig, cursor);
                    match cig {
                        Cigar::Match(_) | Cigar::Diff(_) | Cigar::Equal(_) => {
                            // if M, X, or = increment start and decrement end index
                            debug!("Adding M, X, or = at {} and {}", cursor, cursor + cig.len() as usize);
                            ups_and_downs[cursor] += 1;
                            let final_pos = cursor + cig.len() as usize;
                            if final_pos < ups_and_downs.len() { // True unless the read hits the contig end.
                                ups_and_downs[final_pos] -= 1;
                            }
                            cursor += cig.len() as usize;
                        },
                        Cigar::Del(_) => {
                            cursor += cig.len() as usize;
                            total_indels_in_current_contig += cig.len() as u32;
                        },
                        Cigar::RefSkip(_) => {
                            // if D or N, move the cursor
                            cursor += cig.len() as usize;
                        },
                        Cigar::Ins(_) => {
                            total_indels_in_current_contig += cig.len() as u32;
                        },
                        Cigar::SoftClip(_) | Cigar::HardClip(_) | Cigar::Pad(_) => {}
                    }
                }

                // Determine the number of mismatching bases in this read by
                // looking at the NM tag.
                total_edit_distance_in_current_contig += match
                    record.aux("NM".as_bytes()) {
                        Some(aux) => {
                            aux.integer() as u32
                        },
                        None => {
                            panic!("Mapping record encountered that does not have an 'NM' \
                                    auxiliary tag in the SAM/BAM format. This is required \
                                    to work out some coverage statistics");
                        }
                    };

                debug!("At end of loop")
            }
        }

        process_previous_contigs(
            last_tid,
            target_names.len() as i32,
            coverage_estimators,
            ups_and_downs,
            num_mapped_reads_in_current_contig,
            total_edit_distance_in_current_contig,
            total_indels_in_current_contig,
            &mut num_mapped_reads_total);

        let reads_mapped = ReadsMapped {
            num_mapped_reads: num_mapped_reads_total,
            num_reads: bam_generated.num_detected_primary_alignments()
        };
        info!("In sample '{}', found {} reads mapped out of {} total ({:.*}%)",
              stoit_name, reads_mapped.num_mapped_reads,
              reads_mapped.num_reads, 2,
              (reads_mapped.num_mapped_reads * 100) as f64 / reads_mapped.num_reads as f64);
        reads_mapped_vector.push(reads_mapped);

        if bam_generated.num_detected_primary_alignments() == 0 {
            warn!("No primary alignments were observed for sample {} \
                   - perhaps something went wrong in the mapping?",
                  stoit_name);
        }

        bam_generated.finish();
    }
    return reads_mapped_vector;
}


fn print_previous_zero_coverage_contigs<T: CoverageTaker>(
    last_tid: i32,
    current_tid: i32,
    coverage_estimators: &Vec<CoverageEstimator>,
    target_names: &Vec<&[u8]>,
    coverage_taker: &mut T,
    header: &bam::HeaderView) {
    let mut my_tid = last_tid + 1;
    while my_tid < current_tid {
        debug!("printing zero coverage for tid {}", my_tid);
        coverage_taker.start_entry(
            my_tid as usize,
            std::str::from_utf8(target_names[my_tid as usize]).unwrap());
        for ref coverage_estimator in coverage_estimators.iter() {
            coverage_estimator.print_zero_coverage(
                coverage_taker, header.target_len(my_tid as u32).unwrap());
        }
        coverage_taker.finish_entry();
        my_tid += 1;
    };
}



#[cfg(test)]
mod tests {
    use super::*;
    use std::io::Cursor;
    use std::str;
    use mapping_parameters::*;

    fn test_with_stream<R: NamedBamReader,
                        G: NamedBamReaderGenerator<R>>(
        expected: &str,
        bam_readers: Vec<G>,
        coverage_estimators: &mut Vec<CoverageEstimator>,
        print_zero_coverage_contigs: bool,
        proper_pairs_only: bool) -> Vec<ReadsMapped> {
        let mut stream = Cursor::new(Vec::new());
        let flag_filters = FlagFilter {
            include_improper_pairs: !proper_pairs_only,
            include_secondary: false,
            include_supplementary: false,
        };
        let reads_mapped_vec;
        {
            let mut coverage_taker = CoverageTakerType::new_single_float_coverage_streaming_coverage_printer(
                &mut stream);
            reads_mapped_vec = contig_coverage(
                bam_readers,
                &mut coverage_taker,
                coverage_estimators,
                print_zero_coverage_contigs,
                flag_filters);
        }
        assert_eq!(expected, str::from_utf8(stream.get_ref()).unwrap());
        return reads_mapped_vec;
    }

    #[test]
    fn test_one_genome_two_contigs_first_covered_no_zeros(){
        test_with_stream(
            "7seqs.reads_for_seq1_and_seq2\tgenome2~seq1\t1.2\n7seqs.reads_for_seq1_and_seq2\tgenome5~seq2\t1.2\n",
            generate_named_bam_readers_from_bam_files(
                vec!["tests/data/7seqs.reads_for_seq1_and_seq2.bam"]),
            &mut vec!(CoverageEstimator::new_estimator_mean(0.0,0,false)),
            false,
            false);
    }

    #[test]
    fn test_one_genome_two_contigs_first_covered(){
        test_with_stream(
            "7seqs.reads_for_seq1_and_seq2\tgenome1~random_sequence_length_11000\t0\n7seqs.reads_for_seq1_and_seq2\tgenome1~random_sequence_length_11010\t0\n7seqs.reads_for_seq1_and_seq2\tgenome2~seq1\t1.2\n7seqs.reads_for_seq1_and_seq2\tgenome3~random_sequence_length_11001\t0\n7seqs.reads_for_seq1_and_seq2\tgenome4~random_sequence_length_11002\t0\n7seqs.reads_for_seq1_and_seq2\tgenome5~seq2\t1.2\n7seqs.reads_for_seq1_and_seq2\tgenome6~random_sequence_length_11003\t0\n",
            generate_named_bam_readers_from_bam_files(
                vec!["tests/data/7seqs.reads_for_seq1_and_seq2.bam"]),
            &mut vec!(CoverageEstimator::new_estimator_mean(0.0,0,false)),
            true,
            false);
    }

    #[test]
    fn test_proper_pairs_only(){
        test_with_stream(
            "",
            generate_named_bam_readers_from_bam_files(
                vec!["tests/data/1.bam"]),
            &mut vec!(CoverageEstimator::new_estimator_mean(0.0,0,false)),
            false,
            true);
    }

    #[test]
    fn test_one_contig_variance(){
        test_with_stream(
            &("2seqs.reads_for_seq1\tseq1\t0.9489489\n".to_owned()+
                "2seqs.reads_for_seq1\tseq2\t0\n"),
            generate_named_bam_readers_from_bam_files(
                vec!["tests/data/2seqs.reads_for_seq1.bam"]),
            &mut vec!(CoverageEstimator::new_estimator_variance(0.0,0)),
            true,
            false);
    }

    #[test]
    fn test_streaming_bam_file(){
        test_with_stream(
            "7seqs.fna/reads_for_seq1_and_seq2.1.fq.gz\tgenome2~seq1\t1.2\n\
             7seqs.fna/reads_for_seq1_and_seq2.1.fq.gz\tgenome5~seq2\t1.2\n",
            vec![
                generate_named_bam_readers_from_reads(
                    "tests/data/7seqs.fna",
                    "tests/data/reads_for_seq1_and_seq2.1.fq.gz",
                    Some("tests/data/reads_for_seq1_and_seq2.2.fq.gz"),
                    ReadFormat::Coupled,
                    4,
                    None,
                    false,
                    None,
                    true)],
            &mut vec!(CoverageEstimator::new_estimator_mean(0.0,0,false)),
            false,
            false);
    }

    #[test]
    fn test_multiple_coverage_methods(){
        test_with_stream(
            &("2seqs.reads_for_seq1\tseq1\t1.2\t0.9489489\n".to_owned()+
                "2seqs.reads_for_seq1\tseq2\t0\t0\n"),
            generate_named_bam_readers_from_bam_files(
                vec!["tests/data/2seqs.reads_for_seq1.bam"]),
            &mut vec!(
                CoverageEstimator::new_estimator_mean(0.0,0,false),
                CoverageEstimator::new_estimator_variance(0.0,0)
            ),
            true,
            false);
    }

    #[test]
    fn test_julian_error(){
        test_with_stream(
            "2seqs.reads_for_seq1.with_unmapped\tseq1\t1.497\n\
             2seqs.reads_for_seq1.with_unmapped\tseq2\t1.5\n",
            // has unmapped reads, which caused problems with --proper-pairs-only.
            generate_named_bam_readers_from_bam_files(
                vec!["tests/data/2seqs.reads_for_seq1.with_unmapped.bam"]),
            &mut vec!(CoverageEstimator::new_estimator_mean(0.0,0,true)),
            true,
            false);
    }

    #[test]
    fn test_trimmed_mean_bug(){
        test_with_stream(
            &("2seqs.reads_for_seq1\tseq1\t0\n".to_owned()+
                "2seqs.reads_for_seq1\tseq2\t0\n"),
            generate_named_bam_readers_from_bam_files(
                vec!["tests/data/2seqs.reads_for_seq1.bam"]),
            &mut vec!(
                CoverageEstimator::new_estimator_trimmed_mean(0.0,0.05,0.0,0)
            ),
            true,
            false);
    }

    #[test]
    fn test_multiple_outputs_one_zero_no_print_zeroes(){
        test_with_stream(
            "2seqs.reads_for_seq1\tseq1\t1.2\t0\n",
            generate_named_bam_readers_from_bam_files(
                vec!["tests/data/2seqs.reads_for_seq1.bam"]),
            &mut vec!(
                CoverageEstimator::new_estimator_mean(0.0,0,false),
                // covered fraction is 0.727, so go lower so trimmed mean is 0,
                // mean > 0.
                CoverageEstimator::new_estimator_trimmed_mean(0.0,0.05,0.0,0)
            ),
            false,
            false);
    }

    #[test]
    fn test_multiple_outputs_one_zero_no_print_zeroes_reverse_order(){
        test_with_stream(
            "2seqs.reads_for_seq1\tseq1\t0\t1.2\n",
            generate_named_bam_readers_from_bam_files(
                vec!["tests/data/2seqs.reads_for_seq1.bam"]),
            &mut vec!(
                // covered fraction is 0.727, so go lower so trimmed mean is 0,
                // mean > 0.
                CoverageEstimator::new_estimator_trimmed_mean(0.0,0.05,0.0,0),
                CoverageEstimator::new_estimator_mean(0.0,0,false),
            ),
            false,
            false);
    }

    #[test]
    fn test_contig_end_exclusion(){
        // From https://bitbucket.org/berkeleylab/metabat/issues/48/jgi_summarize_bam_contig_depths-coverage
        test_with_stream(
            &("7seqs.reads_for_seq1_and_seq2\tgenome2~seq1\t1.4117647\t1.3049262\n".to_owned()+
              "7seqs.reads_for_seq1_and_seq2\tgenome5~seq2\t1.2435294\t0.6862065\n"),
            generate_named_bam_readers_from_bam_files(
                vec!["tests/data/7seqs.reads_for_seq1_and_seq2.bam"]),
            &mut vec!(
                CoverageEstimator::new_estimator_mean(0.0,75,false),
                // covered fraction is 0.727, so go lower so trimmed mean is 0,
                // mean > 0.
                CoverageEstimator::new_estimator_variance(0.0,75)
            ),
            false,
            false);
    }

    #[test]
    fn test_one_read_of_pair_mapped(){
        // In the second read, tid is != -1, because the first in the pair is assigned somewhere.
        test_with_stream(
            &("1read_of_pair_mapped\t73.20100900_E1D.16_contig_9606\t0.011293635\n".to_owned()),
            generate_named_bam_readers_from_bam_files(
                vec!["tests/data/1read_of_pair_mapped.bam"]),
            &mut vec!(
                CoverageEstimator::new_estimator_mean(0.0,75,true),
            ),
            false,
            false);
    }

    #[test]
    fn test_variance_estimator_all_bases_covered(){
        // In the past this threw up a underflow error
        test_with_stream(
            &("k141_2005182	k141_2005182	5.107387\n".to_owned()),
            generate_named_bam_readers_from_bam_files(
                vec!["tests/data/k141_2005182.bam"]),
            &mut vec!(
                CoverageEstimator::new_estimator_variance(0.0,75),
            ),
            false,
            false);
    }

    #[test]
    fn test_reads_not_counting_when_sufficient_min_covered(){
        // In the past this threw up a underflow error
        let reads_mapped = test_with_stream(
            &("2seqs.reads_for_seq1_and_seq2	seq1	1.3049262\n\
               2seqs.reads_for_seq1_and_seq2	seq2	0.6862065\n".to_owned()),
            generate_named_bam_readers_from_bam_files(
                vec!["tests/data/2seqs.reads_for_seq1_and_seq2.bam"]),
            &mut vec!(
                CoverageEstimator::new_estimator_variance(0.0,75),
            ),
            false,
            false);
        assert_eq!(vec!(ReadsMapped{
            num_mapped_reads: 24,
            num_reads: 24
        }), reads_mapped);
    }


    #[test]
    fn test_reads_not_counting_when_insufficient_min_covered(){
        // In the past this threw up a underflow error
        let reads_mapped = test_with_stream(
            &("".to_owned()),
            generate_named_bam_readers_from_bam_files(
                vec!["tests/data/2seqs.reads_for_seq1_and_seq2.bam"]),
            &mut vec!(
                CoverageEstimator::new_estimator_variance(0.99,75),
            ),
            false,
            false);
        assert_eq!(vec!(ReadsMapped{
            num_mapped_reads: 0,
            num_reads: 24
        }), reads_mapped);
    }

    #[test]
    fn test_reads_per_base_estimator(){
        test_with_stream(
            "7seqs.fna/reads_for_seq1_and_seq2.1.fq.gz\tgenome1~random_sequence_length_11000\t0\n\
             7seqs.fna/reads_for_seq1_and_seq2.1.fq.gz\tgenome1~random_sequence_length_11010\t0\n\
             7seqs.fna/reads_for_seq1_and_seq2.1.fq.gz\tgenome2~seq1\t0.012\n\
             7seqs.fna/reads_for_seq1_and_seq2.1.fq.gz\tgenome3~random_sequence_length_11001\t0\n\
             7seqs.fna/reads_for_seq1_and_seq2.1.fq.gz\tgenome4~random_sequence_length_11002\t0\n\
             7seqs.fna/reads_for_seq1_and_seq2.1.fq.gz\tgenome5~seq2\t0.012\n\
             7seqs.fna/reads_for_seq1_and_seq2.1.fq.gz\tgenome6~random_sequence_length_11003\t0\n",
            vec![
                generate_named_bam_readers_from_reads(
                    "tests/data/7seqs.fna",
                    "tests/data/reads_for_seq1_and_seq2.1.fq.gz",
                    Some("tests/data/reads_for_seq1_and_seq2.2.fq.gz"),
                    ReadFormat::Coupled,
                    4,
                    None,
                    false,
                    None,
                    true)],
            &mut vec!(CoverageEstimator::new_estimator_reads_per_base()),
            true,
            false);
    }
}