-
Notifications
You must be signed in to change notification settings - Fork 0
/
others_pipeline.sh
executable file
·679 lines (544 loc) · 47.8 KB
/
others_pipeline.sh
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
#/bin/sh -S
#########
#Please address any bugs to Cheng.
#Date 2018.9.13
#########
#echo $(readlink -f $1)
sample_metadata=$(readlink -f $1)
depth=$2
min_freq=$3
category_sum=${4//,/ }
reference_trained=$(readlink -f $5)
close_reference_trained=$(readlink -f $6)
manifest_file=$(readlink -f $7)
not_rda=${8//\;/ }
classifier_type=$9
echo "Check wheather your categories are the following:"
for i in $category_sum;do echo $i;done
echo "Check wheather the group of enviromental factors excluded from rda are the following:"
for i in $not_rda;do echo $i;done
declare -A tax_aa;
tax_aa=([k]=Kingdom [p]=Phylum [c]=Class [o]=Order [f]=Family [g]=Genus [s]=Species);
tax_levels["1"]="Kingdom"
tax_levels["2"]="Phylum"
tax_levels["3"]="Class"
tax_levels["4"]="Order"
tax_levels["5"]="Family"
tax_levels["6"]="Genus"
tax_levels["7"]="Species"
#tax_levels1["k"]="Kingdom"
#tax_levels1["p"]="Phylum"
#tax_levels1["c"]="Class"
#tax_levels1["o"]="Order"
#tax_levels1["f"]="Family"
#tax_levels1["g"]="Genus"
#tax_levels1["s"]="Species"
if [ -z "$9" ]; then
echo "##########
Please prepare the directory structure before starting the program like below:
raw/fastq_files ...
mapping_file
manifest_file
\n\n"
echo "Please provide following input parameters
1) Path of the mapping file. (Accept both .csv or txt format.)
2) Depth of the for subsampleing. (Suggested value: 4000)
3) Mininum frequence for OTU to be selected. (Suggested value: 1000)
4) The name of categories in the mapping file seprated by commas.
5) Path of the classifier for alignment.
6) Path of the reference sequences for close reference alignment.
7) Path of the manifest file.
8) Specify numeric variables excluded from rda seprated by commas,use 'none' if all numeric variables is expected
9) Specify the type of classifier, either silva or gg
Sample Usage:
bash ~/github/Bayegy/16S_pipeline.V9.sh ../data/sample-metadata.tsv auto 1000 Group ~/database_16S/GG/338-806/gg_13_8_99_338_806_classifier.qza ~/database_16S/GG/338-806/gg_13_5_97_338_806_ref_seqs.qza ../data/manifest.txt none gg
"
exit 0
else
echo "################
Running: sh $0 $1 $2 $3 $4 $5 $6 $7 $8 $9"
fi
check_file() {
echo "Checking file for $1 ..."
file_name=$1
if [ -f $file_name ]; then
echo "File $file_name exists"
else
echo "File $file_name does not exist"
exit
fi
}
function assign_taxa() {
loop_id=$1
if [ $loop_id == 1]; then
echo "Kingdom"
elif [ $loop_id == 2]; then
echo "Phylum"
elif [ $loop_id == 3]; then
echo "Class"
elif [ $loop_id == 4]; then
echo "Order"
elif [ $loop_id == 5]; then
echo "Family"
elif [ $loop_id == 6]; then
echo "Genus"
elif [ $loop_id == 7]; then
echo "Species"
fi
}
#for f in 1 2 3 4 5 6 7;
# do echo $f;
# tax=$(assign_taxa ${f});
# echo $tax;
#done;
##Activate Qiime2 Version
echo "##############################################################\n#Initiate directory name and set up the directory structure"
SCRIPTPATH="$( cd "$(dirname "$0")" ; pwd -P )"
source activate qiime2-2018.11
echo "##############################################################\n#paired end analysis using DADA2"
qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path $manifest_file --output-path demux.qza --input-format PairedEndFastqManifestPhred33
qiime demux summarize --i-data demux.qza --o-visualization demux.qzv
qiime dada2 denoise-paired --i-demultiplexed-seqs demux.qza --p-trunc-len-f 245 --p-trunc-len-r 245 --p-trim-left-f 26 --p-trim-left-r 26 --o-representative-sequences rep-seqs-dada2.qza --o-table table-dada2.qza --p-n-threads 0 --o-denoising-stats stats-dada2.qza --verbose
qiime metadata tabulate --m-input-file stats-dada2.qza --o-visualization stats-dada2.qzv
<<com1
echo "##############################################################\n#Single end analysis using DADA2"
qiime tools import --type 'SampleData[SequencesWithQuality]' --input-path $manifest_file --output-path demux.qza --input-format SingleEndFastqManifestPhred33
qiime demux summarize --i-data demux.qza --o-visualization demux.qzv
qiime dada2 denoise-single --i-demultiplexed-seqs demux.qza --p-trim-left 26 --p-trunc-len 240 --o-representative-sequences rep-seqs-dada2.qza --o-table table-dada2.qza --p-n-threads 0 --o-denoising-stats stats-dada2.qza --verbose
qiime metadata tabulate --m-input-file stats-dada2.qza --o-visualization stats-dada2.qzv
com1
<<comment1
echo "############################################################\nAlternative methods of read-joining using deblur"
qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path $manifest_file --output-path demux.qza --input-format PairedEndFastqManifestPhred33
#qiime cutadapt trim-paired --i-demultiplexed-sequences demux.qza --p-front-f CCAGCASCYGCGGTAATTCC --p-front-r ACTTTCGTTCTTGATYRA --p-overlap 10 --o-trimmed-sequences trimmed-demux.qza
#qiime tools import --type 'SampleData[JoinedSequencesWithQuality]' --input-path $manifest_file --output-path demux-joined.qza --input-format SingleEndFastqManifestPhred33
qiime vsearch join-pairs --p-maxdiffs 5 --p-minovlen 15 --p-truncqual 2 --i-demultiplexed-seqs demux.qza --o-joined-sequences demux-joined.qza
qiime demux summarize --i-data demux-joined.qza --o-visualization demux.qzv
qiime quality-filter q-score-joined --i-demux demux-joined.qza --o-filtered-sequences demux-joined-filtered.qza --o-filter-stats demux-joined-filter-stats.qza
qiime deblur denoise-16S --i-demultiplexed-seqs demux-joined-filtered.qza --p-trim-length 400 --p-sample-stats --o-representative-sequences rep-seqs-dada2.qza --o-table table-dada2.qza --o-stats stats-dada2.qza --p-jobs-to-start 16 --verbose
qiime deblur visualize-stats --i-deblur-stats stats-dada2.qza --o-visualization stats-dada2.qzv
comment1
mv rep-seqs-dada2.qza rep-seqs.withCandM.qza
mv table-dada2.qza table.withCandM.qza
echo "##############################################################\n#Filter out Choloroplast and Mitochondira"
check_file $reference_trained
qiime feature-classifier classify-sklearn --p-n-jobs 16 --i-classifier $reference_trained --i-reads rep-seqs.withCandM.qza --o-classification taxonomy.withCandM.qza
qiime metadata tabulate --m-input-file taxonomy.withCandM.qza --o-visualization taxonomy.withCandM.qzv
#Archaea,
qiime taxa filter-table --i-table table.withCandM.qza --i-taxonomy taxonomy.withCandM.qza --p-exclude mitochondria,chloroplast,Archaea,Unassigned --o-filtered-table table-no-mitochondria-no-chloroplast.qza
mv table-no-mitochondria-no-chloroplast.qza table.qza
qiime taxa filter-seqs --i-sequences rep-seqs.withCandM.qza --i-taxonomy taxonomy.withCandM.qza --p-exclude mitochondria,chloroplast,Archaea,Unassigned --o-filtered-sequences rep-seqs-no-mitochondria-no-chloroplast.qza
mv rep-seqs-no-mitochondria-no-chloroplast.qza rep-seqs.qza
echo "##############################################################\n#Classify the taxonomy"
qiime feature-classifier classify-sklearn --p-n-jobs 16 --i-classifier $reference_trained --i-reads rep-seqs.qza --o-classification taxonomy.qza
if [[ $classifier_type == 'silva' ]];
then python $SCRIPTPATH/format_silva_to_gg.py -i taxonomy.qza;
else python $SCRIPTPATH/format_silva_to_gg.py -i taxonomy.qza -c;
fi;
echo "##############################################################\n#Generate tree";
qiime alignment mafft --i-sequences rep-seqs.qza --o-alignment aligned-rep-seqs.qza
qiime alignment mask --i-alignment aligned-rep-seqs.qza --o-masked-alignment masked-aligned-rep-seqs.qza
qiime phylogeny fasttree --i-alignment masked-aligned-rep-seqs.qza --o-tree unrooted-tree.qza
qiime phylogeny midpoint-root --i-tree unrooted-tree.qza --o-rooted-tree rooted-tree.qza
qiime feature-table tabulate-seqs --i-data rep-seqs.qza --o-visualization rep-seqs.qzv
echo "##############################################################\n#Generate OTUStats";
for f in rep-seqs.qza table.qza taxonomy.qza ; do echo $f; qiime tools export --input-path $f --output-path exported; done
#for f in alpha-rarefaction.qzv table.qzv taxa-bar-plots.qzv; do echo $f; qiime tools export --input-path $f --output-path exported_qzv; done
qiime tools export --input-path rooted-tree.qza --output-path exported/
mv exported/tree.nwk exported/tree.rooted.nwk
qiime tools export --input-path unrooted-tree.qza --output-path exported/
mv exported/tree.nwk exported/tree.unrooted.nwk
biom add-metadata -i exported/feature-table.biom -o exported/feature-table.taxonomy.biom --observation-metadata-fp exported/taxonomy.tsv --observation-header OTUID,taxonomy,confidence
biom convert -i exported/feature-table.taxonomy.biom -o exported/feature-table.taxonomy.txt --to-tsv --header-key taxonomy
for f in $(find . -type f -name "*.qzv"); do echo $f; base=$(basename $f .qzv); dir=$(dirname $f); new=${dir}/${base}; qiime tools export --input-path $f --output-path ${new}.qzv.exported; done
source deactivate
source activate qm2
echo "##############################################################\n#Generate the figure for the percentage of annotated level"
perl ${SCRIPTPATH}/stat_otu_tab.unspecifiedadded.pl -unif min exported/feature-table.taxonomy.txt -prefix exported/Relative/otu_table --even exported/Relative/otu_table.even.txt -spestat exported/Relative/classified_stat_relative.xls
perl ${SCRIPTPATH}/bar_diagram.pl -table exported/Relative/classified_stat_relative.xls -style 1 -x_title "Sample Name" -y_title "Sequence Number Percent" -right -textup -rotate='-45' --y_mun 1,7 > exported/Relative/Classified_stat_relative.svg
for svg_file in exported/Relative/*svg; do echo $svg_file; n=$(basename "$svg_file" .svg); echo $n; rsvg-convert -h 3200 -b white $svg_file > exported/Relative/${n}.png; done
if [ -d "./Result_AmpliconSequencing" ];then
rm -r ./Result_AmpliconSequencing;
fi;
mkdir -p ./Result_AmpliconSequencing/1-OTUStats/3-RepresentiveSequence/;
cp -r demux.qzv* stats-dada2.qzv* ./Result_AmpliconSequencing/1-OTUStats/
cp -r ./rep-seqs.qzv* ./exported/*nwk ./exported/dna-sequences.fasta ./Result_AmpliconSequencing/1-OTUStats/3-RepresentiveSequence/
for f in $(find ./Result_AmpliconSequencing/1-OTUStats/ -type f -name "*qzv"); do echo $f; base=$(basename $f .qzv); dir=$(dirname $f); mv $f ${f}.exported; mv ${f}.exported ${dir}/${base}; done
for f in $(find ./Result_AmpliconSequencing/1-OTUStats/ -type f -name "index.html") ; do echo $f; base=$(basename $f .html); dir=$(dirname $f); new=${dir}/Summary_请点此文件查看.html; mv $f $new; done
mv ./Result_AmpliconSequencing/1-OTUStats/stats-dada2 ./Result_AmpliconSequencing/1-OTUStats/2-Stats-dada2
mv ./Result_AmpliconSequencing/1-OTUStats/demux/ ./Result_AmpliconSequencing/1-OTUStats/1-Stats-demux
cp -r exported/feature-table.taxonomy.txt exported/feature-table.taxonomy.biom exported/Relative/Classified_stat_relative.png ./Result_AmpliconSequencing/1-OTUStats/
cp -r exported/Relative/otu_table.even.txt ./Result_AmpliconSequencing/1-OTUStats/feature-table.taxonomy.even.txt
echo "##############################################################\n#Generate the results of each group"
for category_set in $category_sum;
do echo $category_set;
source deactivate;
source activate qiime2-2018.11;
#mkdir ${category_set}_Results;
python $SCRIPTPATH/split_source_by_group.py -i table.qza -t taxonomy.qza -r rep-seqs.qza -m $sample_metadata -c $category_set -o ${category_set}_Results;
cd ${category_set}_Results;
mapping_file=$(readlink -f './mapping_file.txt');
qiime metadata tabulate --m-input-file taxonomy.qza --o-visualization taxonomy.qzv;
echo "##############################################################\n#Generate tree";
qiime alignment mafft --i-sequences rep-seqs.qza --o-alignment aligned-rep-seqs.qza
qiime alignment mask --i-alignment aligned-rep-seqs.qza --o-masked-alignment masked-aligned-rep-seqs.qza
qiime phylogeny fasttree --i-alignment masked-aligned-rep-seqs.qza --o-tree unrooted-tree.qza
qiime phylogeny midpoint-root --i-tree unrooted-tree.qza --o-rooted-tree rooted-tree.qza
echo "##############################################################\n#Visulize of the table without Choloroplast and Mitochondira"
qiime feature-table summarize --i-table table.qza --o-visualization table.qzv --m-sample-metadata-file $mapping_file
qiime taxa barplot --i-table table.qza --i-taxonomy taxonomy.qza --m-metadata-file $mapping_file --o-visualization taxa-bar-plots.qzv
#########calculate the min sample depth
#for f in rep-seqs.qza table.qza taxonomy.qza ; do echo $f; qiime tools export --input-path $f --output-path exported; done
qiime tools export --input-path table.qzv --output-path exported_qzv
if [[ $depth == 'auto' ]];
then min_depth=$(echo \($(cut -f2 -d ',' exported_qzv/sample-frequency-detail.csv | sort -n | head -n1)/1000\)*1000 | bc);
else min_depth=$depth;
fi;
echo "##############################################################The selected sample depth is $min_depth"
echo "##############################################################\n#Core alpha and beta diversity analysis"
qiime diversity core-metrics-phylogenetic --i-phylogeny rooted-tree.qza --i-table table.qza --p-sampling-depth $min_depth --m-metadata-file $mapping_file --output-dir core-metrics-results
qiime diversity alpha-group-significance --i-alpha-diversity core-metrics-results/faith_pd_vector.qza --m-metadata-file $mapping_file --o-visualization core-metrics-results/faith-pd-group-significance.qzv
qiime diversity alpha-group-significance --i-alpha-diversity core-metrics-results/evenness_vector.qza --m-metadata-file $mapping_file --o-visualization core-metrics-results/evenness-group-significance.qzv
qiime diversity alpha-group-significance --i-alpha-diversity core-metrics-results/shannon_vector.qza --m-metadata-file $mapping_file --o-visualization core-metrics-results/shannon-group-significance.qzv
qiime diversity alpha-group-significance --i-alpha-diversity core-metrics-results/observed_otus_vector.qza --m-metadata-file $mapping_file --o-visualization core-metrics-results/observed_otus-group-significance.qzv
for category_1 in $category_set;
do echo $category_1;
qiime diversity beta-group-significance --i-distance-matrix core-metrics-results/unweighted_unifrac_distance_matrix.qza --m-metadata-file $mapping_file --p-method permanova --m-metadata-column $category_1 --o-visualization 'core-metrics-results/unweighted_unifrac-permanova-'$category_1'-significance.qzv' --p-pairwise;
#qiime diversity beta-group-significance --i-distance-matrix core-metrics-results/unweighted_unifrac_distance_matrix.qza --m-metadata-file $mapping_file --p-method permanova --m-metadata-column $category_2 --o-visualization 'core-metrics-results/unweighted_unifrac-permanova-'$category_2'-significance.qzv' --p-pairwise
qiime diversity beta-group-significance --i-distance-matrix core-metrics-results/weighted_unifrac_distance_matrix.qza --m-metadata-file $mapping_file --p-method permanova --m-metadata-column $category_1 --o-visualization 'core-metrics-results/weighted_unifrac-permanova-'$category_1'-significance.qzv' --p-pairwise;
#qiime diversity beta-group-significance --i-distance-matrix core-metrics-results/weighted_unifrac_distance_matrix.qza --m-metadata-file $mapping_file --p-method permanova --m-metadata-column $category_2 --o-visualization 'core-metrics-results/weighted_unifrac-permanova-'$category_2'-significance.qzv' --p-pairwise
qiime diversity beta-group-significance --i-distance-matrix core-metrics-results/bray_curtis_distance_matrix.qza --m-metadata-file $mapping_file --p-method permanova --m-metadata-column $category_1 --o-visualization 'core-metrics-results/bray_curtis-permanova-'$category_1'-significance.qzv' --p-pairwise;
#qiime diversity beta-group-significance --i-distance-matrix core-metrics-results/bray_curtis_distance_matrix.qza --m-metadata-file $mapping_file --p-method permanova --m-metadata-column $category_2 --o-visualization 'core-metrics-results/bray_curtis-permanova-'$category_2'-significance.qzv' --p-pairwise
qiime diversity beta-group-significance --i-distance-matrix core-metrics-results/unweighted_unifrac_distance_matrix.qza --m-metadata-file $mapping_file --p-method anosim --m-metadata-column $category_1 --o-visualization 'core-metrics-results/unweighted_unifrac-anosim-'$category_1'-significance.qzv' --p-pairwise;
#qiime diversity beta-group-significance --i-distance-matrix core-metrics-results/unweighted_unifrac_distance_matrix.qza --m-metadata-file $mapping_file --p-method anosim --m-metadata-column $category_2 --o-visualization 'core-metrics-results/unweighted_unifrac-anosim-'$category_2'-significance.qzv' --p-pairwise
qiime diversity beta-group-significance --i-distance-matrix core-metrics-results/weighted_unifrac_distance_matrix.qza --m-metadata-file $mapping_file --p-method anosim --m-metadata-column $category_1 --o-visualization 'core-metrics-results/weighted_unifrac-anosim-'$category_1'-significance.qzv' --p-pairwise;
#qiime diversity beta-group-significance --i-distance-matrix core-metrics-results/weighted_unifrac_distance_matrix.qza --m-metadata-file $mapping_file --p-method anosim --m-metadata-column $category_2 --o-visualization 'core-metrics-results/weighted_unifrac-anosim-'$category_2'-significance.qzv' --p-pairwise
qiime diversity beta-group-significance --i-distance-matrix core-metrics-results/bray_curtis_distance_matrix.qza --m-metadata-file $mapping_file --p-method anosim --m-metadata-column $category_1 --o-visualization 'core-metrics-results/bray_curtis-anosim-'$category_1'-significance.qzv' --p-pairwise;
done;
#qiime diversity beta-group-significance --i-distance-matrix core-metrics-results/bray_curtis_distance_matrix.qza --m-metadata-file $mapping_file --p-method anosim --m-metadata-column $category_2 --o-visualization 'core-metrics-results/bray_curtis-anosim-'$category_2'-significance.qzv' --p-pairwise
qiime diversity alpha-rarefaction --i-table table.qza --i-phylogeny rooted-tree.qza --p-max-depth $min_depth --m-metadata-file $mapping_file --o-visualization alpha-rarefaction.qzv --p-steps 50
##These following two commands work only for column with numeric values:
##qiime emperor plot --i-pcoa core-metrics-results/unweighted_unifrac_pcoa_results.qza --m-metadata-file $mapping_file --p-custom-axis $category_2 --o-visualization 'core-metrics-results/unweighted_unifrac-emperor-'$category_2'.qzv'
##qiime emperor plot --i-pcoa core-metrics-results/bray_curtis_pcoa_results.qza --m-metadata-file $mapping_file --p-custom-axis $category_2 --o-visualization 'core-metrics-results/bray-curtis-emperor-'$category_2'.qzv'
echo "##############################################################\n#alpha dviersity summary"
mkdir alpha
qiime diversity alpha --i-table table.qza --p-metric chao1 --output-dir alpha/chao1
qiime diversity alpha --i-table table.qza --p-metric shannon --output-dir alpha/shannon
qiime diversity alpha --i-table table.qza --p-metric observed_otus --output-dir alpha/observed_otus
qiime diversity alpha-phylogenetic --i-table table.qza --i-phylogeny rooted-tree.qza --p-metric faith_pd --output-dir alpha/faith_pd
qiime tools export --input-path alpha/chao1/alpha_diversity.qza --output-path alpha/chao1/
qiime tools export --input-path alpha/shannon/alpha_diversity.qza --output-path alpha/shannon/
qiime tools export --input-path alpha/observed_otus/alpha_diversity.qza --output-path alpha/observed_otus/
qiime tools export --input-path alpha/faith_pd/alpha_diversity.qza --output-path alpha/faith_pd/
paste alpha/observed_otus/alpha-diversity.tsv alpha/chao1/alpha-diversity.tsv alpha/shannon/alpha-diversity.tsv alpha/faith_pd/alpha-diversity.tsv | awk -F'\t' 'BEGIN{OFS="\t"}{print $1, $2, $4, $6, $8}' > alpha/alpha-summary.tsv
echo "##############################################################\n#Export necessary files for future analysis"
for f in rep-seqs.qza table.qza taxonomy.qza ; do echo $f; qiime tools export --input-path $f --output-path exported; done
#for f in alpha-rarefaction.qzv table.qzv taxa-bar-plots.qzv; do echo $f; qiime tools export --input-path $f --output-path exported_qzv; done
qiime tools export --input-path rooted-tree.qza --output-path exported/
mv exported/tree.nwk exported/tree.rooted.nwk
qiime tools export --input-path unrooted-tree.qza --output-path exported/
mv exported/tree.nwk exported/tree.unrooted.nwk
biom add-metadata -i exported/feature-table.biom -o exported/feature-table.taxonomy.biom --observation-metadata-fp exported/taxonomy.tsv --observation-header OTUID,taxonomy,confidence
biom convert -i exported/feature-table.taxonomy.biom -o exported/feature-table.taxonomy.txt --to-tsv --header-key taxonomy
biom convert -i exported/feature-table.taxonomy.biom -o exported/feature-table.txt --to-tsv
sed 's/taxonomy/Consensus Lineage/' < exported/feature-table.taxonomy.txt | sed 's/ConsensusLineage/Consensus Lineage/' > exported/feature-table.ConsensusLineage.txt
echo "##############################################################\n#Generate heatmaps for top OTUs with different levels with minimum frequence reads supported"
mkdir exported/collapsed
mkdir exported/${min_freq}
for n in 2 3 4 5 6 7;
do echo $n;
qiime taxa collapse --i-table table.qza --i-taxonomy taxonomy.qza --p-level $n --o-collapsed-table exported/collapsed/collapsed-${tax_levels[${n}]}.qza;
qiime feature-table summarize --i-table exported/collapsed/collapsed-${tax_levels[${n}]}.qza --o-visualization exported/collapsed/collapsed-${tax_levels[${n}]}.qzv '--m-sample-metadata-file' $mapping_file;
qiime feature-table filter-features --i-table exported/collapsed/collapsed-${tax_levels[${n}]}.qza --p-min-frequency $min_freq --o-filtered-table exported/${min_freq}/table-${tax_levels[${n}]}.${min_freq}.qza;
for category_1 in $category_set;
do echo $category_1;
Rscript ${SCRIPTPATH}/clean_na_of_inputs.R -m $mapping_file --group $category_1 -t exported/${min_freq}/table-${tax_levels[${n}]}.${min_freq}.qza -o media_files
qiime feature-table heatmap --i-table media_files/filtered_feature_table.qza --m-metadata-file media_files/cleaned_map.txt --m-metadata-column $category_1 --o-visualization exported/${min_freq}/${category_1}-table-${tax_levels[${n}]}.${min_freq}.qzv;
done;
done;
source deactivate
source activate qm2
echo "##############################################################\n#Generate the figure for the percentage of annotated level"
perl ${SCRIPTPATH}/stat_otu_tab.unspecifiedadded.pl -unif min exported/feature-table.taxonomy.txt -prefix exported/Relative/otu_table --even exported/Relative/otu_table.even.txt -spestat exported/Relative/classified_stat_relative.xls
perl ${SCRIPTPATH}/bar_diagram.pl -table exported/Relative/classified_stat_relative.xls -style 1 -x_title "Sample Name" -y_title "Sequence Number Percent" -right -textup -rotate='-45' --y_mun 1,7 > exported/Relative/Classified_stat_relative.svg
for key in ${!tax_aa[*]};do mv exported/Relative/otu_table.${key}.relative.mat exported/Relative/otu_table.${tax_aa[$key]}.relative.txt;done;
#mv exported/Relative/otu_table.p.relative.mat exported/Relative/otu_table.Phylum.relative.txt
#mv exported/Relative/otu_table.c.relative.mat exported/Relative/otu_table.Class.relative.txt
#mv exported/Relative/otu_table.o.relative.mat exported/Relative/otu_table.Order.relative.txt
#mv exported/Relative/otu_table.f.relative.mat exported/Relative/otu_table.Family.relative.txt
#mv exported/Relative/otu_table.g.relative.mat exported/Relative/otu_table.Genus.relative.txt
#mv exported/Relative/otu_table.s.relative.mat exported/Relative/otu_table.Species.relative.txt
# for n7 in "Phylum" "Class" "Order" "Family" "Genus" "Species";
# do echo $n7;
#echo "mv exported/Relative/otu_table.${n7}.relative.mat exported/Relative/otu_table.${tax_levels[${n7}]}.relative.txt"
#mv exported/Relative/otu_table.${n7}.relative.mat exported/Relative/otu_table.${tax_levels[${n7}]}.relative.txt
#echo ${tax_levels[$n7]}
#echo ${tax_levels[${n7}]}
# perl -lane '$,="\t";pop(@F);print(@F)' exported/Relative/otu_table.${n7}.relative.txt > exported/Relative/otu_table.${n7}.relative.lastcolumn.txt;
# perl ${SCRIPTPATH}/get_table_head2.pl exported/Relative/otu_table.${n7}.relative.lastcolumn.txt 20 -trantab > exported/Relative/otu_table.${n7}.relative.lastcolumn.trans;
#perl ${SCRIPTPATH}/bar_diagram.pl -table exported/Relative/otu_table.${n7}.relative.lastcolumn.trans -style 1 -x_title "Sample Name" -y_title "Sequence Number Percent" -right -textup -rotate='-45' --y_mun 1,1 > exported/Relative/otu_table.${n7}.relative.svg;
# perl ${SCRIPTPATH}/bar_diagram.pl -table exported/Relative/otu_table.${n7}.relative.lastcolumn.trans -style 1 -x_title "Sample Name" -y_title "Sequence Number Percent (%)" -right -textup -rotate='-45' --y_mun 0.2,5 --micro_scale --percentage > exported/Relative/otu_table.${n7}.relative.svg
# done;
for svg_file in exported/Relative/*svg; do echo $svg_file; n=$(basename "$svg_file" .svg); echo $n; rsvg-convert -h 3200 -b white $svg_file > exported/Relative/${n}.png; done
source deactivate
source activate qiime2-2018.11
echo "ANCOM analaysis for differential OTU"
mkdir exported/ANCOM
for n2 in 2 3 4 5 6 7;
do echo $n2;
for category_1 in $category_set;
do echo $category_1;
Rscript ${SCRIPTPATH}/clean_na_of_inputs.R -m $mapping_file --group $category_1 -t exported/collapsed/collapsed-${tax_levels[${n2}]}.qza -o media_files
qiime composition add-pseudocount --i-table media_files/filtered_feature_table.qza --o-composition-table exported/ANCOM/composition.${tax_levels[${n2}]}.qza;
qiime composition ancom --i-table exported/ANCOM/composition.${tax_levels[${n2}]}.qza --m-metadata-file media_files/cleaned_map.txt --m-metadata-column $category_1 --o-visualization exported/ANCOM/${category_1}.ANCOM.${tax_levels[${n2}]}.qzv;
done;
#qiime composition ancom --i-table exported/ANCOM/composition.${tax_levels[${n2}]}.qza --m-metadata-file $mapping_file --m-metadata-column $category_2 --o-visualization exported/ANCOM/SecondaryGroup/ANCOM.${tax_levels[${n2}]}.qzv;
done;
echo "##############################################################\n#Run for PICRUST analysis and STAMP visulization"
qiime vsearch cluster-features-closed-reference --i-sequences rep-seqs.qza --i-table table.qza --i-reference-sequences $close_reference_trained --p-perc-identity 0.97 --p-threads 0 --output-dir closedRef_forPICRUSt
qiime feature-table summarize --i-table closedRef_forPICRUSt/clustered_table.qza --o-visualization closedRef_forPICRUSt/clustered_table.qzv --m-sample-metadata-file $mapping_file
qiime feature-table tabulate-seqs --i-data closedRef_forPICRUSt/unmatched_sequences.qza --o-visualization closedRef_forPICRUSt/unmatched_sequences.qzv
qiime tools export --input-path closedRef_forPICRUSt/clustered_table.qza --output-path closedRef_forPICRUSt/
biom convert -i closedRef_forPICRUSt/feature-table.biom -o closedRef_forPICRUSt/feature-table.txt --to-tsv
<<COMMENT
source deactivate
source activate qm2
normalize_by_copy_number.py -i closedRef_forPICRUSt/feature-table.biom -o closedRef_forPICRUSt/feature-table.normalized.biom
predict_metagenomes.py -i closedRef_forPICRUSt/feature-table.normalized.biom -o closedRef_forPICRUSt/feature-table.metagenome.biom
categorize_by_function.py -i closedRef_forPICRUSt/feature-table.metagenome.biom -o closedRef_forPICRUSt/feature-table.metagenome.L1.txt -c KEGG_Pathways -l 1 -f
categorize_by_function.py -i closedRef_forPICRUSt/feature-table.metagenome.biom -o closedRef_forPICRUSt/feature-table.metagenome.L2.txt -c KEGG_Pathways -l 2 -f
categorize_by_function.py -i closedRef_forPICRUSt/feature-table.metagenome.biom -o closedRef_forPICRUSt/feature-table.metagenome.L3.txt -c KEGG_Pathways -l 3 -f
cd closedRef_forPICRUSt
for n3 in 1 2 3;
do echo $n3;
python ${SCRIPTPATH}/convert_percent.py -i feature-table.metagenome.L${n3}.txt;
#perl ${SCRIPTPATH}/get_table_head2.pl percent.feature-table.metagenome.L${n3}.txt 35 -trantab > percent.feature-table.metagenome.L${n3}.tab
#perl ${SCRIPTPATH}/top10_bar_diagram.pl -right -grid -rotate='-45' -x_title 'Sample Name' -y_title 'Relative Abundance' --y_mun 0.25,4 --height 350 -table percent.feature-table.metagenome.L${n3}.tab > percent.feature-table.metagenome.L${n3}.svg
Rscript ${SCRIPTPATH}/abundance_barplot.R -n 20 -m $mapping_file -c $category_set -i feature-table.metagenome.L${n3}.txt -o function-bar-plots-top20/ -p L${n3}_${category_set}_ordered_ -b F -s T;
Rscript ${SCRIPTPATH}/abundance_barplot.R -n 20 -m $mapping_file -c $category_set -i feature-table.metagenome.L${n3}.txt -o function-bar-plots-top20-group-mean/ -p ${category_set}_L${n3}_mean_ -b T -s T;
perl ${SCRIPTPATH}/cluster.pl -BC -Z -x percent.feature-table.metagenome.L${n3}.txt > level1.relative.tree
perl ${SCRIPTPATH}/draw_tree.pl -bun 0.25,4 -bline -type 4 level1.relative.tree percent.feature-table.metagenome.L${n3}.tab --flank_x 100 > tree.feature-table.metagenome.L${n3}.svg
#${SCRIPTPATH}/PCA.R.pl ${PWD}/percent.feature-table.metagenome.L${n3}.txt 0.2 ${PWD}/PCA_L${n3}
cp $mapping_file ./sample-metadata.PCA.txt
perl -p -i.bak -e 's/#//' ./sample-metadata.PCA.txt
tail -n +2 feature-table.metagenome.L${n3}.txt | grep -v "disease"> feature-table.metagenome.L${n3}.PCA.txt
perl -p -i.bak -e 's/#OTU ID/KEGG_function/' feature-table.metagenome.L${n3}.PCA.txt
done;
for svg_file in *svg; do echo $svg_file; base=$(basename $svg_file .svg); rsvg-convert -h 3200 -b white $svg_file > ${base}.png; done
cd ..
echo "##############################################################\n#Make phylogenetic trees for ITOL"
mkdir phylogeny
qiime feature-table filter-features --i-table table.qza --p-min-frequency $min_freq --o-filtered-table phylogeny/table.${min_freq}.qza
qiime tools export --input-path phylogeny/table.${min_freq}.qza --output-path phylogeny
biom convert -i phylogeny/feature-table.biom -o phylogeny/feature-table.txt --to-tsv
cut -f1 phylogeny/feature-table.txt | tail -n +3 > phylogeny/feature-table.list
seqtk subseq exported/dna-sequences.fasta phylogeny/feature-table.list > phylogeny/dna-sequences.${min_freq}.fasta
qiime tools import --input-path phylogeny/dna-sequences.${min_freq}.fasta --output-path phylogeny/dna-sequences.${min_freq}.qza --type 'FeatureData[Sequence]'
qiime alignment mafft --i-sequences phylogeny/dna-sequences.${min_freq}.qza --o-alignment phylogeny/dna-sequences.${min_freq}.aligned.qza
qiime alignment mask --i-alignment phylogeny/dna-sequences.${min_freq}.aligned.qza --o-masked-alignment phylogeny/dna-sequences.${min_freq}.aligned.masked.qza
qiime phylogeny fasttree --i-alignment phylogeny/dna-sequences.${min_freq}.aligned.masked.qza --o-tree phylogeny/dna-sequences.${min_freq}.unrooted-tree.qza
qiime phylogeny midpoint-root --i-tree phylogeny/dna-sequences.${min_freq}.unrooted-tree.qza --o-rooted-tree phylogeny/dna-sequences.${min_freq}.rooted-tree.qza
qiime feature-classifier classify-sklearn --i-classifier $reference_trained --i-reads phylogeny/dna-sequences.${min_freq}.qza --o-classification phylogeny/taxonomy.${min_freq}.qza
biom add-metadata -i phylogeny/feature-table.biom -o phylogeny/feature-table.taxonomy.biom --observation-metadata-fp exported/taxonomy.tsv --observation-header OTUID,taxonomy,confidence
biom convert -i phylogeny/feature-table.taxonomy.biom -o phylogeny/feature-table.taxonomy.txt --to-tsv --header-key taxonomy
qiime tools export --input-path phylogeny/dna-sequences.${min_freq}.rooted-tree.qza --output-path phylogeny/
mv phylogeny/tree.nwk phylogeny/tree.rooted.nwk
perl ${SCRIPTPATH}/generate_file_Itol.pl phylogeny/feature-table.taxonomy.txt
COMMENT
source deactivate
source activate qiime2-2018.11
qiime tools export --input-path masked-aligned-rep-seqs.qza --output-path ./
qiime tools export --input-path rep-seqs.qza --output-path ./
qiime tools export --input-path rooted-tree.qza --output-path ./
echo "##############################################################\n#export all qzv files into clickable folders"
#for f in $(find . -type f -name "*.qzv"); do echo $f; qiime tools export $f --output-dir ${f}.exported; done
for f in $(find . -type f -name "*.qzv"); do echo $f; base=$(basename $f .qzv); dir=$(dirname $f); new=${dir}/${base}; qiime tools export --input-path $f --output-path ${new}.qzv.exported; done
echo "##############################################################\n#Run Qiime1 for differOTU analysis"
source deactivate
source activate qm1
mkdir exported/DiffAbundance
biom convert -i exported/Relative/otu_table.even.txt -o exported/DiffAbundance/otu_table.even.biom --to-hdf5 --table-type="OTU table" --process-obs-metadata taxonomy
summarize_taxa.py -i exported/DiffAbundance/otu_table.even.biom -a -o exported/DiffAbundance/tax
summarize_taxa.py -i exported/DiffAbundance/otu_table.even.biom -a -L 7 -o exported/DiffAbundance/tax
source ~/.bash_profile
min_observation=$(echo \(`wc -l $mapping_file | sed 's/ .*//g'`-1\)/4 | bc)
echo "###############min observation of otu in samples is $min_observation"
for n4 in 2 3 4 5 6 7;
do echo $n4;
#the biom file should include taxonomy information for group_significance.py script
cut -f1 exported/DiffAbundance/tax/otu_table.even_L${n4}.txt > exported/DiffAbundance/tax/otu_table.even_L${n4}.1stColumn.txt
perl -p -i.bak -e 's/#OTU ID/taxonomy/' exported/DiffAbundance/tax/otu_table.even_L${n4}.1stColumn.txt
paste exported/DiffAbundance/tax/otu_table.even_L${n4}.txt exported/DiffAbundance/tax/otu_table.even_L${n4}.1stColumn.txt > exported/DiffAbundance/tax/otu_table.even_${tax_levels[${n4}]}.taxonomy.txt
biom convert -i exported/DiffAbundance/tax/otu_table.even_${tax_levels[${n4}]}.taxonomy.txt -o exported/DiffAbundance/tax/otu_table.even_${tax_levels[${n4}]}.taxonomy.biom --to-hdf5 --table-type="OTU table" --process-obs-metadata taxonomy
filter_otus_from_otu_table.py -i exported/DiffAbundance/tax/otu_table.even_${tax_levels[${n4}]}.taxonomy.biom -s $min_observation -o filtered_otu_table.biom
for category_1 in $category_set;
do echo $category_1;
#Rscript ${SCRIPTPATH}/clean_na_of_inputs.R -m $mapping_file --group $category_1 -o media_files
group_significance.py -i filtered_otu_table.biom -m $mapping_file -c $category_1 -s kruskal_wallis -o exported/DiffAbundance/kruskal_wallis_${category_1}_DiffAbundance_${tax_levels[${n4}]}.txt --biom_samples_are_superset --print_non_overlap;
group_significance.py -i filtered_otu_table.biom -m $mapping_file -c $category_1 -s ANOVA -o exported/DiffAbundance/ANOVA_${category_1}_DiffAbundance_${tax_levels[${n4}]}.txt --biom_samples_are_superset --print_non_overlap;
# python ${SCRIPTPATH}/auto_DESeq.py -m $mapping_file -g $category_1 -l ${tax_levels[${n4}]};
done;
for category_1 in $category_set;
do echo $category_1;
#Rscript ${SCRIPTPATH}/clean_na_of_inputs.R -m $mapping_file --group $category_1 -o media_files
# group_significance.py -i filtered_otu_table.biom -m $mapping_file -c $category_1 -s kruskal_wallis -o exported/DiffAbundance/kruskal_wallis_${category_1}_DiffAbundance_${tax_levels[${n4}]}.txt --biom_samples_are_superset --print_non_overlap;
# group_significance.py -i filtered_otu_table.biom -m $mapping_file -c $category_1 -s ANOVA -o exported/DiffAbundance/ANOVA_${category_1}_DiffAbundance_${tax_levels[${n4}]}.txt --biom_samples_are_superset --print_non_overlap;
python ${SCRIPTPATH}/auto_DESeq.py -m $mapping_file -g $category_1 -l ${tax_levels[${n4}]};
done;
done;
echo "##############################################################\n#Run R script for additional R related figure generation"
source deactivate
source activate qm2
mkdir R_output
#Change format of meta-data file for Rscript of PLSDA analysis
#cp $mapping_file ./R_output/sample-metadata.txt
#tail -n +2 exported/feature-table.txt > ./R_output/feature-table.PLSDA.txt
#perl -p -i.bak -e 's/#OTU ID//' ./R_output/feature-table.PLSDA.txt
#sort ./R_output/sample-metadata.txt > ./R_output/sample-metadata.PLSDA.txt
#Change format of meta-data file for Rscript of alpha diversity analysis
#cp $mapping_file ./alpha/sample-metadata_alphadiversity.txt
#perl -p -i.bak -e 's/#SampleID//' ./alpha/sample-metadata_alphadiversity.txt
for category_1 in $category_set;
do echo $category_1;
Rscript ${SCRIPTPATH}/clean_na_of_inputs.R -m $mapping_file --group $category_1 -o media_files
map=$(readlink -f ./media_files/cleaned_map.txt)
#otu=$(readlink -f ./media_files/cleaned_feature_table.txt)
Rscript ${SCRIPTPATH}/RRelatedOutput.R $map $category_1;
Rscript ${SCRIPTPATH}/alphaboxplotwitSig.R -m $map -c $category_1 -i ./alpha/alpha-summary.tsv -o ./alpha/;
done;
Rscript ${SCRIPTPATH}/beta_heatmap.R -i exported/feature-table.ConsensusLineage.txt -m $mapping_file -t exported/tree.rooted.nwk -r exported/dna-sequences.fasta -o R_output -c $category_set -p 'unclustered_';
Rscript ${SCRIPTPATH}/beta_heatmap.R -i exported/feature-table.ConsensusLineage.txt -m $mapping_file -t exported/tree.rooted.nwk -r exported/dna-sequences.fasta -o R_output;
perl ${SCRIPTPATH}/table_data_svg.pl --colors cyan-orange R_output/bray_curtis_matrix.txt R_output/weighted_unifrac_matrix.txt R_output/unweighted_unifrac_matrix.txt --symbol 'Beta Diversity' > R_output/BetaDiversity_heatmap.svg
rsvg-convert -h 3200 -b white R_output/BetaDiversity_heatmap.svg > R_output/BetaDiversity_heatmap.png
#python2 ${SCRIPTPATH}/biom_to_stamp.py -m KEGG_Pathways closedRef_forPICRUSt/feature-table.metagenome.biom > closedRef_forPICRUSt/feature-table.metagenome.KEGG_Pathways.STAMP.txt
echo "##############################################################\n#Generate the absolute directory for enviromental factors relational analysis"
# cd exported/
perl ${SCRIPTPATH}/stat_otu_tab.unspecifiedadded.pl -unif min exported/feature-table.taxonomy.txt --prefix exported/Absolute/otu_table -nomat -abs -spestat exported/Absolute/classified_stat.xls
# cd exported/Absolute/
for key in ${!tax_aa[*]};do mv exported/Absolute/otu_table.${key}.absolute.mat exported/Absolute/otu_table.${tax_aa[$key]}.absolute.txt;done;
#mv otu_table.k.absolute.mat otu_table.Kingdom.absolute.txt
#mv otu_table.p.absolute.mat otu_table.Pylumn.absolute.txt
#mv otu_table.c.absolute.mat otu_table.Class.absolute.txt
#mv otu_table.o.absolute.mat otu_table.Order.absolute.txt
#mv otu_table.f.absolute.mat otu_table.Family.absolute.txt
#mv otu_table.g.absolute.mat otu_table.Genus.absolute.txt
#mv otu_table.s.absolute.mat otu_table.Species.absolute.txt
# mkdir RDA
# for n6 in "Phylum" "Class" "Order" "Family" "Genus" "Species";
# do echo $n6;
# mkdir RDA/${n6}
# cp otu_table.${n6}.absolute.txt RDA/${n6}
# cd RDA/${n6}
# for category_1 in $category_set;do echo $category_1;python ${SCRIPTPATH}/RDA.py -i otu_table.${n6}.absolute.txt -m $mapping_file -g $category_1 -o ./ -n 25 -e $not_rda;done;
# cd ../../
# done;
# cd ../../
source deactivate
source activate qm2
perl ${SCRIPTPATH}/stat_otu_tab.pl -unif min exported/feature-table.taxonomy.txt -prefix otu_table_forlefse/otu_table
for key in ${!tax_aa[*]};do mv otu_table_forlefse/otu_table.${key}.relative.mat otu_table_forlefse/otu_table.${tax_aa[$key]}.relative.txt;done;
test=${not_rda// */}
if [ ! $test == "all" ];then
echo "##############################################################\nCorrelation heatmap analysis"
for nrda in $not_rda;
do echo $nrda;
prefix=${nrda//,/_}_excluded_;
prefix=${prefix//none_excluded_/};
for n7 in "Phylum" "Class" "Order" "Family" "Genus" "Species";
do echo $n7;
Rscript ${SCRIPTPATH}/cor_heatmap.R -i otu_table_forlefse/otu_table.${n7}.relative.txt -o 2-CorrelationHeatmap/${n7}/ -n 25 -m $mapping_file -e $nrda -p "$prefix";
for category_1 in $category_set;do echo $category_1;python ${SCRIPTPATH}/RDA.py -i exported/Relative/otu_table.${n7}.relative.txt -m $mapping_file -g $category_1 -o exported/Absolute/RDA/${n7} -n 25 -e $nrda -p "$prefix";done;
done;
done;
fi;
source deactivate
source activate qm2
echo "##############################################################\network and abundance heatmap"
for n7 in "Phylum" "Class" "Order" "Family" "Genus" "Species";
do echo $n7;
Rscript ${SCRIPTPATH}/network.R -c 0.5 -i otu_table_forlefse/otu_table.${n7}.relative.txt -o 3-NetworkAnalysis/${n7}/;
#Rscript ${SCRIPTPATH}/abundance_heatmap.R -n 20 -i exported/Relative/otu_table.${n7}.relative.txt -o Heatmap_top20/${n7}/;
Rscript ${SCRIPTPATH}/abundance_heatmap.R -m $mapping_file -c $category_set -n 20 -i exported/Absolute/otu_table.${n7}.absolute.txt -o Heatmap_top20/${n7}/ -l T;
Rscript ${SCRIPTPATH}/abundance_heatmap.R -m $mapping_file -c $category_set -n 20 -i exported/Absolute/otu_table.${n7}.absolute.txt -o Heatmap_top20/${n7}/ -b T -l T -p 'group_mean_';
done;
echo "###############################################################\nAdditional plot"
mkdir 4-VennAndFlower
for category_1 in $category_set;
do echo $category_1;
Rscript ${SCRIPTPATH}/venn_and_flower_plot.R -i ./exported/feature-table.taxonomy.txt -m $mapping_file -c $category_1 -o ./4-VennAndFlower;
python ${SCRIPTPATH}/phylotree_and_heatmap.py -i ./exported/feature-table.taxonomy.txt -m $mapping_file -g $category_1 -r aligned-dna-sequences.fasta -o AdditionalPhylogeny/ -n 30
done;
echo "##############################################################\n#Barplot and RDA according to group mean"
for category_1 in $category_set;
do echo $category_1;
for n7 in "Phylum" "Class" "Order" "Family" "Genus" "Species";
do echo $n7;
Rscript ${SCRIPTPATH}/abundance_barplot.R -n 20 -m $mapping_file -c $category_1 -i exported/Relative/otu_table.${n7}.relative.txt -o taxa-bar-plots-top20-group-ordered/ -p ${n7}_${category_1}_ordered_ -b F;
Rscript ${SCRIPTPATH}/abundance_barplot.R -n 20 -m $mapping_file -c $category_1 -i exported/Relative/otu_table.${n7}.relative.txt -o Barplot-of-Group-Mean/ -p ${category_1}_${n7}_mean_ -b T;
done;
done;
<<COMMENT
for n6 in "Phylum" "Class" "Order" "Family" "Genus" "Species";
do echo $n6;
Rscript ${SCRIPTPATH}/collapse_table_with_group_mean.R -i exported/Relative/otu_table.${n6}.relative.txt -m $mapping_file -c Group4 -o media_files -s F
for category_1 in Group3;do echo $category_1;python ${SCRIPTPATH}/RDA.py -i media_files/abundance_table_collapsed_with_group_mean.txt -m media_files/map_collapsed_with_group_mean.txt -g $category_1 -o exported/Absolute/RDA/${n6} -n 25 -e $not_rda;done;
done;
COMMENT
##########alpha rarefacation
Rscript ${SCRIPTPATH}/alphararefaction.R -i alpha-rarefaction.qzv.exported -o alpha-rarefaction-ggplot2
Rscript ${SCRIPTPATH}/alphararefaction.R -i alpha-rarefaction.qzv.exported -o alpha-rarefaction-ggplot2 -m $mapping_file -c $category_set -p "group_mean_"
echo "##############################################################\n#Run LEFSE for Group"
#perl ${SCRIPTPATH}/stat_otu_tab.pl -unif min exported/feature-table.taxonomy.txt -prefix otu_table_forlefse/otu_table
#for key in ${!tax_aa[*]};do mv otu_table_forlefse/otu_table.${key}.relative.mat otu_table_forlefse/otu_table.${tax_aa[$key]}.relative.txt;done;
source deactivate
source deactivate
source activate lefse
cd exported/Relative
mkdir Lefse/
for n7 in "Phylum" "Class" "Order" "Family" "Genus" "Species";
do echo $n7;
mkdir Lefse/${n7}
cp ../../otu_table_forlefse/otu_table.${n7}.relative.txt Lefse/${n7}
cd Lefse/${n7}
for category_1 in $category_set;
do echo $category_1;
Rscript ${SCRIPTPATH}/write_data_for_lefse.R otu_table.${n7}.relative.txt $mapping_file $category_1 ${category_1}_${n7}_lefse.txt F;
base="${category_1}_${n7}_lefse_LDA2"; format_input.py ${category_1}_${n7}_lefse.txt ${base}.lefseinput.txt -c 2 -u 1 -o 1000000; run_lefse.py ${base}.lefseinput.txt ${base}.LDA.txt -l 2;
# plot_res.py --left_space 0.3 --dpi 300 ${base}.LDA.txt ${base}.png; plot_cladogram.py ${base}.LDA.txt --dpi 300 ${base}.cladogram.png --format png --right_space_prop 0.45 --label_font_size 10;
plot_res.py --max_feature_len 200 --orientation h --format pdf --left_space 0.3 --dpi 300 ${base}.LDA.txt ${base}.pdf; plot_cladogram.py ${base}.LDA.txt --dpi 300 ${base}.cladogram.pdf --clade_sep 1.8 --format pdf --right_space_prop 0.45 --label_font_size 10;
base="${category_1}_${n7}_lefse_LDA4"; format_input.py ${category_1}_${n7}_lefse.txt ${base}.lefseinput.txt -c 2 -u 1 -o 1000000; run_lefse.py ${base}.lefseinput.txt ${base}.LDA.txt -l 4;
# plot_res.py --left_space 0.3 --dpi 300 ${base}.LDA.txt ${base}.png; plot_cladogram.py ${base}.LDA.txt --dpi 300 ${base}.cladogram.png --format png --right_space_prop 0.45 --label_font_size 10;
plot_res.py --max_feature_len 200 --orientation h --format pdf --left_space 0.3 --dpi 300 ${base}.LDA.txt ${base}.pdf; plot_cladogram.py ${base}.LDA.txt --dpi 300 ${base}.cladogram.pdf --clade_sep 1.8 --format pdf --right_space_prop 0.45 --label_font_size 10;
done;
cd ../../
done;
cd ../../
# source deactivate
# source activate lefse
# mkdir -p LEfSe/Genus/
# for category_1 in $category_set;
# do echo $category_1;
# Rscript ${SCRIPTPATH}/write_data_for_lefse.R exported/Absolute/otu_table.Genus.absolute.txt $mapping_file $category_1 LEfSe/Genus/${category_1}_table_for_lefse.txt F;
# base="${category_1}_Genus_LEfSe_LDA2"; format_input.py LEfSe/Genus/${category_1}_table_for_lefse.txt LEfSe/Genus/${base}.lefseinput.txt -c 2 -u 1 -o 1000000; run_lefse.py LEfSe/Genus/${base}.lefseinput.txt LEfSe/Genus/${base}.LDA.txt -l 2; plot_res.py --left_space 0.3 --dpi 300 LEfSe/Genus/${base}.LDA.txt LEfSe/Genus/${base}.png; plot_cladogram.py LEfSe/Genus/${base}.LDA.txt --dpi 300 LEfSe/Genus/${base}.cladogram.png --format png --right_space_prop 0.45 --label_font_size 10 --labeled_stop_lev 4;
# plot_res.py --max_feature_len 200 --orientation h --format pdf --left_space 0.3 --dpi 300 LEfSe/Genus/${base}.LDA.txt LEfSe/Genus/${base}.pdf; plot_cladogram.py LEfSe/Genus/${base}.LDA.txt --dpi 300 LEfSe/Genus/${base}.cladogram.pdf --clade_sep 1.8 --format pdf --right_space_prop 0.45 --label_font_size 10 --labeled_stop_lev 4;
# done;
# mkdir -p LEfSe/OTU/
# for category_1 in $category_set;
# do echo $category_1;
# Rscript ${SCRIPTPATH}/write_data_for_lefse.R exported/feature-table.taxonomy.txt $mapping_file $category_1 LEfSe/OTU/${category_1}_table_for_lefse.txt T;
#Rscript ${SCRIPTPATH}/write_data_for_lefse.R exported/Absolute/otu_table.Genus.absolute.txt $mapping_file $category_1 LEfSe/OTU/${category_1}_table_for_lefse.txt F;
# base="${category_1}_OTU_LEfSe_LDA4"; format_input.py LEfSe/OTU/${category_1}_table_for_lefse.txt LEfSe/OTU/${base}.lefseinput.txt -c 2 -u 1 -o 1000000; run_lefse.py LEfSe/OTU/${base}.lefseinput.txt LEfSe/OTU/${base}.LDA.txt -l 4; plot_res.py --left_space 0.3 --dpi 300 LEfSe/OTU/${base}.LDA.txt LEfSe/OTU/${base}.png; plot_cladogram.py LEfSe/OTU/${base}.LDA.txt --dpi 300 LEfSe/OTU/${base}.cladogram.png --format png --right_space_prop 0.45 --label_font_size 10;
# plot_res.py --max_feature_len 200 --orientation h --format pdf --left_space 0.3 --dpi 300 LEfSe/OTU/${base}.LDA.txt LEfSe/OTU/${base}.pdf; plot_cladogram.py LEfSe/OTU/${base}.LDA.txt --dpi 300 LEfSe/OTU/${base}.cladogram.pdf --clade_sep 1.8 --format pdf --right_space_prop 0.45 --label_font_size 10;
# done;
source deactivate
source activate qm2
category_report=($category_set)
echo "##############################################################\n#Organize the result files";
#cp -r ${SCRIPTPATH}/Result_AmpliconSequencing ./
sh ${SCRIPTPATH}/organize_dir_structure_others.sh $mapping_file $category_report ${SCRIPTPATH} $min_freq $prefix;
if [ -d "../Result_AmpliconSequencing/${category_report}_Result_AmpliconSequencing" ];then
rm -r ../Result_AmpliconSequencing/${category_report}_Result_AmpliconSequencing;
fi;
mv Result_AmpliconSequencing ../Result_AmpliconSequencing/${category_report}_Result_AmpliconSequencing
cd ../;
done;