forked from nf-core/clipseq
-
Notifications
You must be signed in to change notification settings - Fork 0
/
Copy pathmain.nf
1534 lines (1212 loc) · 50.8 KB
/
main.nf
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
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
829
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859
860
861
862
863
864
865
866
867
868
869
870
871
872
873
874
875
876
877
878
879
880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
916
917
918
919
920
921
922
923
924
925
926
927
928
929
930
931
932
933
934
935
936
937
938
939
940
941
942
943
944
945
946
947
948
949
950
951
952
953
954
955
956
957
958
959
960
961
962
963
964
965
966
967
968
969
970
971
972
973
974
975
976
977
978
979
980
981
982
983
984
985
986
987
988
989
990
991
992
993
994
995
996
997
998
999
1000
#!/usr/bin/env nextflow
/*
========================================================================================
nf-core/clipseq
========================================================================================
nf-core/clipseq Analysis Pipeline.
#### Homepage / Documentation
https://github.com/nf-core/clipseq
----------------------------------------------------------------------------------------
*/
import java.util.zip.GZIPInputStream
def helpMessage() {
log.info nfcoreHeader()
log.info"""
Usage:
The typical command for running the pipeline is as follows:
nextflow run nf-core/clipseq --input metadata.csv -profile docker
Mandatory arguments:
--input [file] Comma-separated file with details of samples and reads
-profile [str] Configuration profile to use. Can use multiple (comma separated)
Available: conda, docker, singularity, test, awsbatch, <institute> and more
Options:
--genome [str] Name of iGenomes reference
--smrna_org [str] Organism for small RNA reference. Available: human, mouse, rat, zebrafish, fruitfly, yeast
References: If not specified in the configuration file or you wish to overwrite any of the references
--fasta [file] Path to genome fasta reference
--gtf [file] Path to genome annotation gtf reference
--star_index [folder] Path to genome STAR index
--smrna_fasta [file] Path to small RNA fasta reference
Adapter trimming:
--adapter [str] Adapter to trim from reads (default: AGATCGGAAGAGC)
Deduplication:
--deduplicate [bool] Deduplicate using UMIs (default: true)
--umi_separator [str] UMI separator character in read header/name (default: :)
Peak calling:
--peakcaller [str] Peak caller. Can use multiple (comma separated), or specify 'all'. Available: icount, paraclu, pureclip, paraclu
--segment [file] Path to iCount segment file
--half_window [int] iCount half-window size (default: 3)
--merge_window [int] iCount merge-window size (default: 3)
--min_value [int] Paraclu minimum cluster count/value (default: 10)
--min_density_increase [int] Paraclu minimum density increase (default: 2)
--max_cluster_length [int] Paraclu maximum cluster length (default: 2)
--bc [int] PureCLIP flag to set parameters according to binding characteristics of protein (default: 0)
--dm [str] PureCLIP merge distnace (default: 8)
--bin_size_both [int] Piranha bin size (default: 3)
--cluster_dist [int] Piranha cluster distance (default: 3)
Other options:
--outdir [file] The output directory where the results will be saved
--publish_dir_mode [str] Mode for publishing results in the output directory. Available: symlink, rellink, link, copy, copyNoFollow, move (Default: copy)
--email [email] Set this parameter to your e-mail address to get a summary e-mail with details of the run sent to you when the workflow exits
--email_on_fail [email] Same as --email, except only send mail if the workflow is not successful
--max_multiqc_email_size [str] Threshold size for MultiQC report to be attached in notification email. If file generated by pipeline exceeds the threshold, it will not be attached (Default: 25MB)
--save_index [bool] If the STAR index is generated by the pipeline use this parameter to save it to your results folder. These can then be used for future pipeline runs, reducing processing times (Default: false)
-name [str] Name for the pipeline run. If not specified, Nextflow will automatically generate a random mnemonic
AWSBatch options:
--awsqueue [str] The AWSBatch JobQueue that needs to be set when running on AWSBatch
--awsregion [str] The AWS Region for your AWS Batch job to run on
--awscli [str] Path to the AWS CLI tool
""".stripIndent()
}
// --iv [str] PureCLIP genomic chromosomes to learn HMM parameters, (default: 'chr1;chr2;chr3')
// Show help message
if (params.help) {
helpMessage()
exit 0
}
/*
================================================================================
SET UP CONFIGURATION VARIABLES
================================================================================
*/
// Check if genome exists in the config file
if (params.genomes && params.genome && !params.genomes.containsKey(params.genome)) {
exit 1, "The provided genome '${params.genome}' is not available in the iGenomes file. Currently the available genomes are ${params.genomes.keySet().join(", ")}"
}
// Option for user supplied fasta and gtf and pipeline supplied smRNA
def smrna_list = ['human', 'mouse', 'rat', 'zebrafish', 'fruitfly', 'yeast']
if (!params.genome && params.smrna_org) {
if (params.smrna_org in smrna_list) {
params.smrna_fasta = params.smrna[ params.smrna_org ].smrna_fasta
} else {
log.warn "There is no smRNA available for species '${params.smrna_org}'; pre-mapping will be skipped. Currently available options are: human, mouse, rat. Alternative you can supply your own smRNA fasta using --smrna_fasta"
}
} else {
params.smrna_fasta = params.genome ? params.smrna[ params.genome ].smrna_fasta ?: false : false
}
// Auto-load genome files from genome config
params.fasta = params.genome ? params.genomes[ params.genome ].fasta ?: false : false
params.gtf = params.genome ? params.genomes[ params.genome ].gtf ?: false : false
params.star_index = params.genome ? params.genomes[ params.genome ].star ?: false : false
// Check input path parameters to see if they exist
checkPathParamList = [
params.input,
params.fasta,
params.gtf,
params.star_index,
params.smrna_fasta,
params.fai
]
for (param in checkPathParamList) { if (param) { file(param, checkIfExists: true) } }
if(!params.smrna_fasta) {
if(params.genome) {
log.warn "There is no available smRNA fasta file associated with the provided genome '${params.genome}'; pre-mapping will be skipped. A smRNA fasta file can be specified on the command line with --smrna_fasta"
} else {
log.warn "There is no smRNA fasta file suppled for genome specified; pre-mapping will be skipped. A smRNA fasta file can be specified on the command line with --smrna_fasta or --smrna_org"
}
}
// Set up peak caller logic
def paraclu_check = false
def icount_check = false
def pureclip_check = false
def piranha_check = false
if (params.peakcaller){
def peak_list = params.peakcaller.split(',').collect()
peak_list.each {
if ( it == 'all') {
paraclu_check = true
icount_check = true
pureclip_check = true
piranha_check = true
} else if ( it == 'paraclu' && !paraclu_check ) {
paraclu_check = true
} else if ( it == 'icount' && !icount_check ) {
icount_check = true
} else if ( it == 'pureclip' && !pureclip_check ) {
pureclip_check = true
} else if ( it == 'piranha' && !piranha_check ) {
piranha_check = true
} else {
exit 1, "Invalid peak caller option: ${it}. Valid options: 'icount', 'paraclu', 'pureclip', 'piranha'"
}
}
}
// cannot run icount wihtout gtf file
if (!params.gtf && icount_check) {
icount_check = false
log.warn "iCount can only be run with a gtf annotation file - iCount will be skipped"
}
def gtf_check = false
String gtf_file_str = ""
String gtf_col_3 = ""
if (params.gtf && icount_check) {
if (hasExtension(params.gtf, 'gz')) {
gtf_file_str = "${workflow.workDir}/tmp_gtf.txt"
decompressGzipFile(params.gtf, gtf_file_str)
} else {
gtf_file_str = params.gtf
}
File gtf_file = new File(gtf_file_str)
boolean compatibility = check_gtf_by_line( gtf_file, 30 )
if (hasExtension(params.gtf, 'gz')) {
boolean fileSuccessfullyDeleted = new File("${workflow.workDir}/tmp_gtf.txt").delete()
}
if (compatibility) {
gtf_check = true
}
if (!gtf_check) {
icount_check = false
log.warn "The supplied gtf file is not compatible with iCount. Peakcalling with iCount will be skipped"
}
}
// Has the run name been specified by the user?
// this has the bonus effect of catching both -name and --name
custom_runName = params.name
if (!(workflow.runName ==~ /[a-z]+_[a-z]+/)) {
custom_runName = workflow.runName
}
// Check AWS batch settings
if (workflow.profile.contains('awsbatch')) {
// AWSBatch sanity checking
if (!params.awsqueue || !params.awsregion) exit 1, "Specify correct --awsqueue and --awsregion parameters on AWSBatch!"
// Check outdir paths to be S3 buckets if running on AWSBatch
// related: https://github.com/nextflow-io/nextflow/issues/813
if (!params.outdir.startsWith('s3:')) exit 1, "Outdir not on S3 - specify S3 Bucket to run on AWSBatch!"
// Prevent trace files to be stored on S3 since S3 does not support rolling files.
if (params.tracedir.startsWith('s3:')) exit 1, "Specify a local tracedir or run without trace! S3 cannot be used for tracefiles."
}
// Stage config files
ch_multiqc_config = file("$projectDir/assets/multiqc_config.yaml", checkIfExists: true)
ch_multiqc_custom_config = params.multiqc_config ? Channel.fromPath(params.multiqc_config, checkIfExists: true) : Channel.empty()
ch_output_docs = file("$projectDir/docs/output.md", checkIfExists: true)
ch_output_docs_images = file("$projectDir/docs/images/", checkIfExists: true)
/*
================================================================================
SET-UP INPUTS
================================================================================
*/
params.adapter = "AGATCGGAAGAGC"
params.umi_separator = ":"
params.deduplicate = true
if (params.smrna_fasta) ch_smrna_fasta = Channel.value(params.smrna_fasta)
if (params.star_index) ch_star_index = Channel.value(params.star_index)
if (params.gtf) ch_check_gtf = Channel.value(params.gtf)
// fai channels
if (params.fai) ch_fai_crosslinks = Channel.value(params.fai)
if (params.fai) ch_fai_icount = Channel.value(params.fai)
if (params.fai) ch_fai_icount_motif = Channel.value(params.fai)
if (params.fai) ch_fai_paraclu_motif = Channel.value(params.fai)
if (params.fai) ch_fai_size = Channel.value(params.fai)
// MultiQC empty channels from peakcaller checks
if (!paraclu_check) ch_paraclu_qc = Channel.empty()
if (!icount_check) ch_icount_qc = Channel.empty()
if (!piranha_check) ch_piranha_qc = Channel.empty()
if (!pureclip_check) ch_pureclip_qc = Channel.empty()
if (params.input) {
Channel
.fromPath(params.input, checkIfExists: true)
.splitCsv(header:true)
.map{ row -> [ row.sample_id, file(row.data1, checkIfExists: true) ] }
.into{ ch_fastq; ch_fastq_fastqc_pretrim }
} else {
exit 1, "Samples comma-separated input file not specified"
}
/*
================================================================================
HEADER LOG
================================================================================
*/
// Header log info
log.info nfcoreHeader()
def summary = [:]
if (workflow.revision) summary['Pipeline Release'] = workflow.revision
summary['Run Name'] = custom_runName ?: workflow.runName
summary['Input'] = params.input
if (params.fasta) summary['Fasta ref'] = params.fasta
if (params.gtf) summary['GTF ref'] = params.gtf
if (params.star_index) summary['STAR index'] = params.star_index
if (params.save_index) summary['Save STAR index?'] = params.save_index
if (params.smrna_org) summary['SmallRNA organism ref'] = params.smrna_org
if (params.smrna_fasta) summary['SmalRNA ref'] = [params.smrna_fasta]
if (params.deduplicate) summary['Deduplicate'] = params.deduplicate
if (params.deduplicate && params.umi_separator) summary['UMI separator'] = params.umi_separator
if (params.peakcaller) summary['Peak caller'] = params.peakcaller
if (params.segment) summary['iCount segment'] = params.segment
if (icount_check) summary['Half window'] = params.half_window
if (icount_check) summary['Merge window'] = params.merge_window
if (paraclu_check) summary['Min value'] = params.min_value
if (paraclu_check) summary['Max density increase'] = params.min_density_increase
if (paraclu_check) summary['Max cluster length'] = params.max_cluster_length
if (pureclip_check) summary['Protein binding parameter'] = params.bc
if (pureclip_check) summary['Crosslink merge distance'] = params.dm
if (piranha_check) summary['Bin size'] = params.bin_size_both
if (piranha_check) summary['Cluster distance'] = params.cluster_dist
summary['Max Resources'] = "$params.max_memory memory, $params.max_cpus cpus, $params.max_time time per job"
if (workflow.containerEngine) summary['Container'] = "$workflow.containerEngine - $workflow.container"
summary['Output dir'] = params.outdir
summary['Launch dir'] = workflow.launchDir
summary['Working dir'] = workflow.workDir
summary['Script dir'] = workflow.projectDir
summary['User'] = workflow.userName
if (workflow.profile.contains('awsbatch')) {
summary['AWS Region'] = params.awsregion
summary['AWS Queue'] = params.awsqueue
summary['AWS CLI'] = params.awscli
}
summary['Config Profile'] = workflow.profile
if (params.config_profile_description) summary['Config Profile Description'] = params.config_profile_description
if (params.config_profile_contact) summary['Config Profile Contact'] = params.config_profile_contact
if (params.config_profile_url) summary['Config Profile URL'] = params.config_profile_url
summary['Config Files'] = workflow.configFiles.join(', ')
if (params.email || params.email_on_fail) {
summary['E-mail Address'] = params.email
summary['E-mail on failure'] = params.email_on_fail
summary['MultiQC maxsize'] = params.max_multiqc_email_size
}
log.info summary.collect { k,v -> "${k.padRight(18)}: $v" }.join("\n")
log.info "-\033[2m--------------------------------------------------\033[0m-"
// Check the hostnames against configured profiles
checkHostname()
Channel.from(summary.collect{ [it.key, it.value] })
.map { k,v -> "<dt>$k</dt><dd><samp>${v ?: '<span style=\"color:#999999;\">N/A</a>'}</samp></dd>" }
.reduce { a, b -> return [a, b].join("\n ") }
.map { x -> """
id: 'nf-core-clipseq-summary'
description: " - this information is collected when the pipeline is started."
section_name: 'nf-core/clipseq Workflow Summary'
section_href: 'https://github.com/nf-core/clipseq'
plot_type: 'html'
data: |
<dl class=\"dl-horizontal\">
$x
</dl>
""".stripIndent() }
.set { ch_workflow_summary }
/*
* Parse software version numbers
*/
process get_software_versions {
publishDir "${params.outdir}/pipeline_info", mode: params.publish_dir_mode,
saveAs: { filename ->
if (filename.indexOf(".csv") > 0) filename
else null
}
output:
file 'software_versions_mqc.yaml' into ch_software_versions_yaml
file "software_versions.csv"
script:
"""
echo $workflow.manifest.version > v_pipeline.txt
echo $workflow.nextflow.version > v_nextflow.txt
fastqc --version > v_fastqc.txt
multiqc --version > v_multiqc.txt
cutadapt --version > v_cutadapt.txt
bowtie2 --version > v_bowtie2.txt
STAR --version > v_star.txt
samtools --version > v_samtools.txt
umi_tools --version > v_umi_tools.txt
bedtools --version > v_bedtools.txt
preseq 2> v_preseq.txt
# subread-align -v 2> v_subread.txt
bam2fq.py --version > v_rseqc.txt
iCount --version > v_icount.txt
pureclip --version > v_pureclip.txt
Piranha -about 2> v_piranha.txt
echo "9" > v_paraclu.txt # Paraclu does not output a version
meme -version > v_meme.txt
echo \$(R --version 2>&1) > v_R.txt
python --version > v_python.txt
pygmentize -V > v_pygments.txt
pigz --version > v_pigz.txt
perl -v > v_perl.txt
scrape_software_versions.py &> software_versions_mqc.yaml
"""
}
/*
================================================================================
PREPROCESSING
================================================================================
*/
/*
* Generating premapping index
*/
if (params.smrna_fasta) {
process generate_premap_index {
tag "$smrna_fasta"
label 'process_low'
input:
path(smrna_fasta) from ch_smrna_fasta
output:
path("${smrna_fasta.simpleName}.*.bt2") into ch_bt2_index
script:
"""
bowtie2-build --threads $task.cpus $smrna_fasta ${smrna_fasta.simpleName}
"""
}
}
/*
* Decompression
*/
// Recognise if fasta and/or gtf are compressed and decompress if so for STAR index generation
if (params.fasta) {
if (hasExtension(params.fasta, 'gz')) {
ch_fasta_gz = Channel
.fromPath(params.fasta, checkIfExists: true)
.ifEmpty { exit 1, "Genome reference fasta not found: ${params.fasta}" }
} else {
Channel
.fromPath(params.fasta, checkIfExists: true)
.ifEmpty { exit 1, "Genome reference fasta not found: ${params.fasta}" }
.into { ch_fasta; ch_fasta_fai; ch_fasta_dreme_icount; ch_fasta_dreme_paraclu; ch_fasta_pureclip; ch_fasta_dreme_pureclip; ch_fasta_dreme_piranha }
}
}
if (params.fasta) {
if (hasExtension(params.fasta, 'gz')) {
process decompress_fasta {
tag "$fasta_gz"
label 'process_low'
input:
path(fasta_gz) from ch_fasta_gz
output:
path("*.fa") into (ch_fasta, ch_fasta_fai, ch_fasta_dreme_icount, ch_fasta_dreme_paraclu, ch_fasta_pureclip, ch_fasta_dreme_pureclip, ch_fasta_dreme_piranha)
script:
"""
pigz -d -c $fasta_gz > ${fasta_gz.baseName}
"""
}
}
}
/*
* Generating fai index
*/
if (!params.fai) {
process generate_fai {
tag "$fasta"
label 'process_low'
input:
path(fasta) from ch_fasta_fai
output:
path("*.fai") into (ch_fai_crosslinks, ch_fai_icount, ch_fai_icount_motif, ch_fai_paraclu_motif, ch_fai_pureclip_motif, ch_fai_piranha_motif, ch_fai_size)
script:
command = "samtools faidx $fasta"
"""
${command}
"""
}
}
/*
* Generating STAR index
*/
if (!params.star_index) {
if (params.gtf) {
if (hasExtension(params.gtf, 'gz')) {
ch_gtf_gz_star = Channel
.fromPath(params.gtf, checkIfExists: true)
.ifEmpty { exit 1, "Genome reference gtf not found: ${params.gtf}" }
} else {
ch_gtf_star = Channel
.fromPath(params.gtf, checkIfExists: true)
.ifEmpty { exit 1, "Genome reference gtf not found: ${params.gtf}" }
}
}
// Calculate genomeSAindexNbases for building star index
process check_genome_size {
tag "$name"
label 'process_low'
input:
path(fai) from ch_fai_size
output:
path("genome_size.txt") into ch_genome_size
script:
"""
awk '{total = total + \$2}END{if ((log(total)/log(2))/2 - 1 > 14) {printf "%.0f", 14} else {printf "%.0f", (log(total)/log(2))/2 - 1}}' $fai > genome_size.txt
"""
}
// transform genome size to calculate genomeSAindexNbases to generate STAR index
ch_genomeSAindexNbases = ch_genome_size
.map { it -> it.getText("UTF-8") as int }
// .map { it -> (it / 2) - 1 }
// .map { it -> Math.round(Math.log(it) / Math.log(2)) }
// .map { it -> Math.min( 14, it ).shortValue() }
// .map { it -> it.toString() }
if (params.gtf) {
if (hasExtension(params.gtf, 'gz')) {
process decompress_gtf {
tag "$gtf_gz"
label 'process_low'
input:
path(gtf_gz) from ch_gtf_gz_star
output:
path("*.gtf") into ch_gtf_star
script:
"""
pigz -d -c $gtf_gz > ${gtf_gz.baseName}
"""
}
}
}
if (params.gtf) {
process generate_star_index {
tag "$fasta"
label 'process_high'
publishDir path: { params.save_index ? "${params.outdir}/STAR_index" : params.outdir },
saveAs: { params.save_index ? it : null }, mode: params.publish_dir_mode
input:
path(fasta) from ch_fasta
path(gtf) from ch_gtf_star
val(sa_ind_base) from ch_genomeSAindexNbases
output:
path("STAR_${fasta.baseName}") into ch_star_index
script:
"""
mkdir STAR_${fasta.baseName}
STAR --runMode genomeGenerate --runThreadN ${task.cpus} \
--genomeDir STAR_${fasta.baseName} \
--genomeFastaFiles $fasta \
--genomeSAindexNbases $sa_ind_base \
--sjdbGTFfile $gtf
"""
}
} else if (!params.gtf){
process generate_star_index_no_gtf {
tag "$fasta"
label 'process_high'
publishDir path: { params.save_index ? "${params.outdir}/STAR_index" : params.outdir },
saveAs: { params.save_index ? it : null }, mode: params.publish_dir_mode
input:
path(fasta) from ch_fasta
val(sa_ind_base) from ch_genomeSAindexNbases
output:
path("STAR_${fasta.baseName}") into ch_star_index
script:
"""
mkdir STAR_${fasta.baseName}
STAR --runMode genomeGenerate --runThreadN ${task.cpus} \
--genomeDir STAR_${fasta.baseName} \
--genomeFastaFiles $fasta \
--genomeSAindexNbases $sa_ind_base \
"""
}
}
}
/*
* Generating iCount segment file
*/
// iCount GTF input autodetects gz
if (params.peakcaller && icount_check) {
if(!params.segment) {
ch_gtf_icount = Channel
.fromPath(params.gtf, checkIfExists: true)
.ifEmpty { exit 1, "Genome reference gtf not found: ${params.gtf}" }
process icount_segment {
tag "$gtf"
publishDir "${params.outdir}/icount", mode: params.publish_dir_mode
input:
path(gtf) from ch_gtf_icount
path(fai) from ch_fai_icount
output:
path("icount_${gtf}") into ch_segment
script:
"""
iCount segment $gtf icount_${gtf} $fai
"""
}
} else {
ch_segment = Channel.value(params.segment)
}
}
/*
================================================================================
CLIP PIPELINE
================================================================================
*/
/*
* STEP 1 - Pre-trimming FastQC
*/
process fastqc {
tag "$name"
label 'process_medium'
publishDir "${params.outdir}/fastqc", mode: params.publish_dir_mode,
saveAs: { filename ->
filename.indexOf(".zip") > 0 ? "zips/$filename" : "$filename"
}
input:
tuple val(name), path(reads) from ch_fastq_fastqc_pretrim
output:
file "*fastqc.{zip,html}" into ch_fastqc_pretrim_mqc
// tuple val(name), path(reads) into ch_fastqc_pretrim_mqc
script:
read_ext = reads.getName().split('\\.', 2)[1]
read_name = reads.getName().split('\\.', 2)[0]
new_reads = "${name}_reads_fastqc.${read_ext}"
new_reads_simple = "${name}_reads_fastqc"
"""
cp ${reads} ${new_reads}
fastqc --quiet --threads $task.cpus ${new_reads}
mv ${new_reads_simple}*.html ${name}_reads_fastqc.html
mv ${new_reads_simple}*.zip ${name}_reads_fastqc.zip
"""
}
/*
* STEP 2 - Read trimming
*/
process cutadapt {
tag "$name"
label 'process_high'
publishDir "${params.outdir}/cutadapt", mode: params.publish_dir_mode
input:
tuple val(name), path(reads) from ch_fastq
output:
tuple val(name), path("${name}.trimmed.fastq.gz") into ch_trimmed
path "*.log" into ch_cutadapt_mqc
script:
"""
ln -s $reads ${name}.fastq.gz
cutadapt -j $task.cpus -a ${params.adapter} -m 12 -o ${name}.trimmed.fastq.gz ${name}.fastq.gz > ${name}_cutadapt.log
"""
}
/*
* STEP 3 - Post-trimming FastQC
*/
/*
* STEP 4 - Premapping
*/
if (params.smrna_fasta) {
process premap {
tag "$name"
label 'process_high'
publishDir "${params.outdir}/premap", mode: params.publish_dir_mode
input:
tuple val(name), path(reads) from ch_trimmed
path(index) from ch_bt2_index.collect()
output:
tuple val(name), path("${name}.unmapped.fastq.gz") into ch_unmapped
tuple val(name), path("${name}.premapped.bam"), path("${name}.premapped.bam.bai")
path "*.log" into ch_premap_mqc, ch_premap_qc
script:
"""
bowtie2 -p $task.cpus -x ${index[0].simpleName} --un-gz ${name}.unmapped.fastq.gz -U $reads 2> ${name}.premap.log | \
samtools sort -@ $task.cpus /dev/stdin > ${name}.premapped.bam && \
samtools index -@ $task.cpus ${name}.premapped.bam
"""
}
} else {
ch_unmapped = ch_trimmed
ch_premap_mqc = Channel.empty()
}
/*
* STEP 5 - Aligning
*/
process align {
tag "$name"
label 'process_high'
publishDir "${params.outdir}/mapped", mode: params.publish_dir_mode
input:
tuple val(name), path(reads) from ch_unmapped
path(index) from ch_star_index.collect()
output:
tuple val(name), path("${name}.Aligned.sortedByCoord.out.bam"), path("${name}.Aligned.sortedByCoord.out.bam.bai") into ch_aligned, ch_aligned_preseq
path "*.Log.final.out" into ch_align_mqc, ch_align_qc
script:
clip_args = "--outFilterMultimapNmax 1 \
--outFilterMultimapScoreRange 1 \
--outSAMattributes All \
--alignSJoverhangMin 8 \
--alignSJDBoverhangMin 1 \
--outFilterType BySJout \
--alignIntronMin 20 \
--alignIntronMax 1000000 \
--outFilterScoreMin 10 \
--alignEndsType Extend5pOfRead1 \
--twopassMode Basic \
--outSAMtype BAM Unsorted"
"""
STAR --runThreadN $task.cpus --runMode alignReads --genomeDir $index \
--readFilesIn $reads --readFilesCommand gunzip -c \
--outFileNamePrefix ${name}. $clip_args
samtools sort -@ $task.cpus -o ${name}.Aligned.sortedByCoord.out.bam ${name}.Aligned.out.bam
samtools index -@ $task.cpus ${name}.Aligned.sortedByCoord.out.bam
"""
}
/*
* STEP 5 - Aligning QC
*/
process preseq {
tag "$name"
label 'process_low'
publishDir "${params.outdir}/preseq", mode: params.publish_dir_mode
input:
tuple val(name), path(bam), path(bai) from ch_aligned_preseq
output:
path '*.ccurve.txt' into ch_preseq_mqc
path '*.log'
script:
"""
preseq lc_extrap \
-output ${name}.ccurve.txt \
-verbose \
-bam \
-seed 42 \
$bam
cp .command.err ${name}.command.log
"""
}
/*
* STEP 6 - Deduplicate
*/
if (params.deduplicate) {
process dedup {
tag "$name"
label 'process_high'
publishDir "${params.outdir}/dedup", mode: params.publish_dir_mode
input:
tuple val(name), path(bam), path(bai) from ch_aligned
output:
tuple val(name), path("${name}.dedup.bam"), path("${name}.dedup.bam.bai") into ch_dedup, ch_dedup_pureclip, ch_dedup_rseqc
path "*.log" into ch_dedup_mqc, ch_dedup_qc
script:
"""
umi_tools dedup --umi-separator="$params.umi_separator" -I $bam -S ${name}.dedup.bam --output-stats=${name} --log=${name}.log
samtools index -@ $task.cpus ${name}.dedup.bam
"""
}
} else {
ch_dedup = ch_aligned
ch_dedup_mqc = Channel.empty()
ch_dedup_qc = Channel.empty()
ch_dedup_rseqc = ch_aligned
}
/*
* STEP 6a - RSeQC
*/
if (params.gtf) {
ch_gtf_rseqc = Channel
.fromPath(params.gtf, checkIfExists: true)
.ifEmpty { exit 1, "Genome reference gtf not found: ${params.gtf}" }
process rseqc {
tag "$name"
label 'process_low'
publishDir "${params.outdir}/rseqc", mode: params.publish_dir_mode
input:
tuple val(name), path(bam), path(bai) from ch_dedup_rseqc
path(gtf) from ch_gtf_rseqc.collect()
output:
path '*.read_distribution.txt' into ch_rseqc_mqc
script:
"""
gtf2bed $gtf > gene.bed
read_distribution.py \
-i $bam \
-r gene.bed \
> ${name}.read_distribution.txt
"""
}
} else {
ch_rseqc_mqc = Channel.empty()
}
/*
* STEP 6 - Identify crosslinks
*/
process get_crosslinks {
tag "$name"
label 'process_medium'
publishDir "${params.outdir}/xlinks", mode: params.publish_dir_mode
input:
tuple val(name), path(bam), path(bai) from ch_dedup
path(fai) from ch_fai_crosslinks.collect()
output:
tuple val(name), path("${name}.xl.bed.gz") into ch_xlinks_icount, ch_xlinks_paraclu, ch_xlinks_piranha
tuple val(name), path("${name}.xl.bedgraph.gz") into ch_xlinks_bedgraphs
path "*.xl.bed.gz" into ch_xlinks_qc
script:
"""
bedtools bamtobed -i $bam > dedup.bed
bedtools shift -m 1 -p -1 -i dedup.bed -g $fai > shifted.bed
bedtools genomecov -dz -strand + -5 -i shifted.bed -g $fai | awk '{OFS="\t"}{print \$1, \$2, \$2+1, ".", \$3, "+"}' > pos.bed
bedtools genomecov -dz -strand - -5 -i shifted.bed -g $fai | awk '{OFS="\t"}{print \$1, \$2, \$2+1, ".", \$3, "-"}' > neg.bed
cat pos.bed neg.bed | sort -k1,1 -k2,2n | pigz > ${name}.xl.bed.gz
zcat ${name}.xl.bed.gz | awk '{OFS = "\t"}{if (\$6 == "+") {print \$1, \$2, \$3, \$5} else {print \$1, \$2, \$3, -\$5}}' | pigz > ${name}.xl.bedgraph.gz
"""
}
/*
* STEP 7a - Peak-call (iCount)
*/
if (params.peakcaller && icount_check) {
process icount_peak_call {
tag "$name"
label 'process_low'
publishDir "${params.outdir}/icount", mode: params.publish_dir_mode
input:
tuple val(name), path(xlinks) from ch_xlinks_icount
path(segment) from ch_segment.collect()
output:
tuple val(name), path("${name}.${half_window}nt.sigxl.bed.gz") into ch_sigxls_icount
tuple val(name), path("${name}.${half_window}nt_${merge_window}nt.peaks.bed.gz") into ch_peaks_icount
path "*.peaks.bed.gz" into ch_icount_qc
script:
half_window = params.half_window
merge_window = params.merge_window
"""
iCount peaks $segment $xlinks ${name}.${half_window}nt.sigxl.bed.gz --half_window ${half_window} --fdr 0.05
pigz -d -c ${name}.${half_window}nt.sigxl.bed.gz | \
bedtools sort | \
bedtools merge -s -d ${merge_window} -c 4,5,6 -o distinct,sum,distinct | \
pigz > ${name}.${half_window}nt_${merge_window}nt.peaks.bed.gz
"""
}
process icount_motif_dreme {
tag "$name"
label 'process_low'
publishDir "${params.outdir}/icount_motif", mode: params.publish_dir_mode
input:
tuple val(name), path(peaks) from ch_peaks_icount
path(fasta) from ch_fasta_dreme_icount.collect()
path(fai) from ch_fai_icount_motif.collect()
output:
tuple val(name), path("${name}_dreme/*") into ch_motif_dreme_icount
script:
"""
pigz -d -c $peaks | awk '{OFS="\t"}{if(\$6 == "+") print \$1, \$2, \$2+1, \$4, \$5, \$6; else print \$1, \$3-1, \$3, \$4, \$5, \$6}' | \
bedtools slop -s -l 20 -r 20 -i /dev/stdin -g $fai > resized_peaks.bed
bedtools getfasta -fi $fasta -bed resized_peaks.bed -fo resized_peaks.fasta
dreme -norc -o ${name}_dreme -p resized_peaks.fasta
"""
}
}
/*
* STEP 7b - Peak-call (paraclu)
*/
if (params.peakcaller && paraclu_check) {
process paraclu_peak_call {
tag "$name"