-
Notifications
You must be signed in to change notification settings - Fork 2
/
agrvate
470 lines (411 loc) · 16.2 KB
/
agrvate
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
#!/bin/bash
#Vishnu Raghuram 2020-11-12
version="1.0.2"
USAGE=$(echo -e "\n\
AgrVATE: Agr Variant Assessment & Typing Engine \n\n\
VERSION: agrvate v$version \n\n\
USAGE: \
agrvate [options] -i filename.fasta \n\n\
FLAGS: \n\
-i | --input Input S. aureus genome in FASTA format\n\
-t | --typing-only Does agr typing only (skips agr operon extraction and frameshift detection)\n\
-m | --mummer Uses mummer instead of usearch (May not perform frameshift detection)\n\
-f | --force Force overwrite existing results directory\n\
-d | --databases Path to agrvate_databases (Not required if installed using Conda)\n\
-h | --help Print this help message and exit\n\
-v | --version Print version and exit\n\n\
SOURCE: https://github.com/VishnuRaghuram94/AgrVATE\n\n\
")
force=0
typing_only=0
mumm=0
script_dir=$( cd "$( dirname "${BASH_SOURCE[0]}" )" >/dev/null 2>&1 && pwd )
databases_path="$script_dir/agrvate_databases/"
extraction="pcr"
if [[ ! $1 ]]
then
echo -e "$USAGE"
exit
fi
while :; do
case $1 in
-h|-\?|--help) #Print usage and exit
echo -e "$USAGE"
shift
exit
;;
-i|--input) #Save input with specified path to $fasta_path
if [[ $2 && $2 != -* ]]
then
fasta_path=$2
bname=$(basename $fasta_path)
echo -e "Processing $fasta_path ..."
shift
else
1>&2 echo -e " -i requires an argument.\n$USAGE "
exit 1
fi
;;
-d|--databases)
if [[ $2 && $2 != -* ]]
then
databases_path=$2
shift
else
databases_path="$script_dir/agrvate_databases/"
shift
fi
;;
-f|--force)
force=$((force + 1))
shift
continue
;;
-t|--typing-only)
typing_only=$((typing_only + 1))
shift
continue
;;
-m|--mummer)
mumm=$((mumm + 1))
extraction="mummer"
shift
continue
;;
-v|--version)
echo -e "agrvate v$version"
shift
exit
;;
--)
shift
break
;;
-?*)
1>&2 echo -e " $1 is an invalid option. \n$USAGE "
shift
exit 1
;;
*)
shift
break
esac
shift
done
###########################
#### ERROR REPORT VARS ####
###########################
input_check="fail"
databases_check="fail"
outdir_check="fail"
agr_typing="fail"
usearch_check="fail"
snippy_check="fail"
#Error report file
echo -e "#input_name\tinput_check\tdatabases_check\toutdir_check\tagr_typing\toperon_check\tsnippy_check" > $bname-error-report.tab
###########################
#### INPUT VALIDATIONS ####
###########################
# validate input fasta
if [[ -f $fasta_path ]] #Check if input is a file
then
if [[ $(file $fasta_path | grep -c "compressed") -eq 0 ]] #checks if file is compressed
then
if [[ $(grep -q "^@" $fasta_path ; echo $?) -eq 1 && $(seqkit seq -t dna -n --quiet $fasta_path | wc -l) -ge 1 ]] # if file is NOT fastq and checks if seqkit can parse the file
then
if [[ $(grep -v ">" $fasta_path | grep -q -i "[^ATCGNWSMKRY]"; echo $?) -eq 1 ]] #check if seqence has characters other than ATGCN
then
fna_name="${bname%.*}"
input_check="pass"
else
1>&2 echo -e "Seqence has non-standard nucleic acid characters\n$USAGE"
#Error report file
echo -e "$bname\t$input_check\t$databases_check\t$outdir_check\t$agr_typing\t$usearch_check\t$snippy_check" >> $bname-error-report.tab
exit 1
fi
else
1>&2 echo -e "Input file not in FASTA format\n$USAGE"
#Error report file
echo -e "$bname\t$input_check\t$databases_check\t$outdir_check\t$agr_typing\t$usearch_check\t$snippy_check" >> $bname-error-report.tab
exit 1
fi
else
1>&2 echo -e "Compressed input not supported (for now)\n$USAGE"
#Error report file
echo -e "$bname\t$input_check\t$databases_check\t$outdir_check\t$agr_typing\t$usearch_check\t$snippy_check" >> $bname-error-report.tab
exit 1
fi
else
1>&2 echo -e "Invalid input\n$USAGE"
#Error report file
echo -e "$bname\t$input_check\t$databases_check\t$outdir_check\t$agr_typing\t$usearch_check\t$snippy_check" >> $bname-error-report.tab
exit 1
fi
#Databases folder checks
if [[ -e $databases_path ]]
then #Check if all necessary files are present
if [[ \
$(find $databases_path -name gp1234_all_motifs.fna | wc -l) -eq 1 && \
$(find $databases_path -name agr_operon_primers.fa | wc -l) -eq 1 && \
$(find $databases_path -name agrD_hmm.hmm* | wc -l) -eq 5 && \
$(find $databases_path/references -name gp[1234]-operon_ref.gbk | wc -l) -eq 4 ]]
then
echo -e "$databases_path is valid"
databases_check="pass"
else
1>&2 echo -e "$databases_path is not a valid databases directory.\nPlease download the agrvate_databases folder from https://github.com/VishnuRaghuram94/AgrVATE/"
#Error report file
echo -e "$bname\t$input_check\t$databases_check\t$outdir_check\t$agr_typing\t$usearch_check\t$snippy_check" >> $bname-error-report.tab
exit 1
fi
else
1>&2 echo $databases_path ' - provided database path does not exist'
#Error report file
echo -e "$bname\t$input_check\t$databases_check\t$outdir_check\t$agr_typing\t$usearch_check\t$snippy_check" >> $bname-error-report.tab
exit 1
fi
#check if results directory already exists
if [[ -d $fna_name-results ]]
then
if [[ $force > 0 ]]
then
echo "Results directory already exists, -f specified, overwriting..."
rm -rf $fna_name-results
mkdir $fna_name-results
outdir_check="pass"
else
1>&2 echo "Results directory already exists, cannot overwrite. Use option -f to force overwrite"
#Error report file
echo -e "$bname\t$input_check\t$databases_check\t$outdir_check\t$agr_typing\t$usearch_check\t$snippy_check" >> $bname-error-report.tab
exit 1
fi
else
mkdir $fna_name-results
outdir_check="pass"
fi
#create summary file
echo -e "#filename\tagr_group\tmatch_score\tcanonical_agrD\tmultiple_agr\tframeshifts" > $fna_name-results/$fna_name-summary.tab
################################
#### AGR TYPING KMER SEARCH ####
################################
#fna file searched against agr typing kmers using blastn and write output to agrgp_tab
blastn -query $databases_path/gp1234_all_motifs.fna -subject $fasta_path -outfmt "6 sseqid qseqid evalue pident sstart send" -max_hsps 1 -perc_identity 100 -qcov_hsp_perc 100 -out $fna_name-results/$fna_name-agr_gp.tab &>$fna_name-results/$fna_name-blastn_log.txt
#identify agr group from search_oligodb results
#check no. of agr group kmers matched.
if [[ -f $fna_name-results/$fna_name-agr_gp.tab ]]
then
agr_typing="pass"
if [[ $(cat $fna_name-results/$fna_name-agr_gp.tab | cut -f2 | sort | uniq | wc -l) -ge 2 ]]
then #multiple agr groups
echo -e "Fasta file has more than one agr type (multiple S. aureus sequences)\n Choosing highest confidence agr type"
#top agr gp match
agr_gp=$(cat $fna_name-results/$fna_name-agr_gp.tab | cut -f2 | sort | uniq -c | sort -k1 -nr | head -1 | sed 's/^\s\+//g' | cut -f2 -d " ")
#score for top agr gp match
agr_match=$(cat $fna_name-results/$fna_name-agr_gp.tab | cut -f2 | sort | uniq -c | sort -k1 -nr | head -1 | sed 's/^\s\+//g' | cut -f1 -d " ")
multiple="m"
canonical=1
#if top match is low confidence
if [[ $agr_match -le 4 ]]
then
echo -e "Multiple agr types found all with low confidence. Cannot proceed to frameshift detection"
canonical="u"
fs="u"
#summary file
echo -e "$fna_name\t$agr_gp\t$agr_match\t$canonical\t$multiple\t$fs" >> $fna_name-results/$fna_name-summary.tab
#Error report file
echo -e "$bname\t$input_check\t$databases_check\t$outdir_check\t$agr_typing\t$usearch_check\t$snippy_check" >> $bname-error-report.tab
exit
fi
else #single agr group
agr_match=$(cat $fna_name-results/$fna_name-agr_gp.tab | wc -l)
#Check match score. if 0 then unknown type. if less than 4 then low confidence type. In both cases hmm search for agrD will be done.
if [[ $agr_match -eq 0 ]]
then
echo -e "Unable to agr type"
multiple="u"
agr_gp="u"
agrD_nhmmer=1
elif [[ $agr_match -le 4 ]]
then
echo -e "Low confidence agr-typing"
multiple="s"
agrD_nhmmer=1
agr_gp=$(cat $fna_name-results/$fna_name-agr_gp.tab | cut -f2 | sort | uniq | cut -f1 -d"|" | uniq)
else
multiple="s"
agr_gp=$(cat $fna_name-results/$fna_name-agr_gp.tab | cut -f2 | sort | uniq | cut -f1 -d"|" | uniq)
canonical=1
echo -e "agr typing successful, $agr_gp"
fi
fi
else
1>&2 echo -e "Agr group typing kmer search failed"
#Error report file
echo -e "$bname\t$input_check\t$databases_check\t$outdir_check\t$agr_typing\t$usearch_check\t$snippy_check" >> $bname-error-report.tab
exit 1
fi
###########################
#### NOVEL agrD SEARCH ####
###########################
if [[ $agrD_nhmmer -eq 1 ]]
then
#use nhmmer to search for sequence matching canonical S. aureus agrD
nhmmer --noali --tblout $fna_name-results/$fna_name-hmm.tab -E 0.01 $databases_path/agrD_hmm.hmm $fasta_path > $fna_name-results/$fna_name-hmm-log.txt
#Check if hmmer hit ~120bp long. if yes - possible non-canonical agrD.
if [[ -z $(grep -v "#" $fna_name-results/$fna_name-hmm.tab | sed 's/\s\+/\t/g' | awk 'BEGIN{FS=OFS="\t"};{if($6-$5 > 120) print $0};') ]]
then
echo -e "Unable to find agrD"
canonical=u
else
echo -e "Possible non-canonical S. aureus agrD found"
canonical=0
fi
fi
#Check if --typing-only is enabled
if [[ $typing_only > 0 ]]
then
fs="u"
#summary file
echo -e "$fna_name\t$agr_gp\t$agr_match\t$canonical\t$multiple\t$fs" >> $fna_name-results/$fna_name-summary.tab
#Error report file
echo -e "$bname\t$input_check\t$databases_check\t$outdir_check\t$agr_typing\t$usearch_check\t$snippy_check" >> $bname-error-report.tab
exit
fi
#################################
##### EXTRACTING AGR OPERON #####
#################################
if [[ $mumm -eq 0 ]] #If option --mummer not enabled, do usearch
then
#Get operating system
if [[ $OSTYPE == *[lL]inux* ]]
then
usearch_bin=usearch11.0.667_i86linux32
elif [[ $OSTYPE == *[dD]arwin* ]]
then
usearch_bin=usearch11.0.667_i86osx32
else
1>&2 echo -e "Error running usearch, unable to determine usearch version. Use flag -m to use mummer instead"
fs="u"
#summary file
echo -e "$fna_name\t$agr_gp\t$agr_match\t$canonical\t$multiple\t$fs" >> $fna_name-results/$fna_name-summary.tab
#Error report file
echo -e "$bname\t$input_check\t$databases_check\t$outdir_check\t$agr_typing\t$usearch_check\t$snippy_check" >> $bname-error-report.tab
exit 1
fi
#Check if usearch11.0.667 binary is in PATH
path_to_usearch=$(which $usearch_bin)
if [[ -x $path_to_usearch ]]
then
#Check usearch exit status
if [[ $($usearch_bin &> /dev/null; echo $?) > 0 ]]
then
1>&2 echo -e "Error running usearch, please make sure usearch is installed correctly. Otherwise, use flag -m to use mummer instead"
fs="u"
#summary file
echo -e "$fna_name\t$agr_gp\t$agr_match\t$canonical\t$multiple\t$fs" >> $fna_name-results/$fna_name-summary.tab
#Error report file
echo -e "$bname\t$input_check\t$databases_check\t$outdir_check\t$agr_typing\t$usearch_check\t$snippy_check" >> $bname-error-report.tab
exit 1
else
echo -e "usearch found"
usearch_check="pass"
fi
else
1>&2 echo -e "$usearch_bin not in path, cannot perform frameshift detection\n please download usearch11.0.667 from https://www.drive5.com/usearch/download.html"
1>&2 echo -e "For example:\n\tcurl 'https://www.drive5.com/downloads/$usearch_bin.gz' --output $usearch_bin.gz\n\tgunzip $usearch_bin.gz\n\tchmod 755 $usearch_bin\n\tcp ./$usearch_bin $script_dir/"
1>&2 echo -e "Otherwise, use flag -m to use mummer"
fs="u"
#summary file
echo -e "$fna_name\t$agr_gp\t$agr_match\t$canonical\t$multiple\t$fs" >> $fna_name-results/$fna_name-summary.tab
#Error report file
echo -e "$bname\t$input_check\t$databases_check\t$outdir_check\t$agr_typing\t$usearch_check\t$snippy_check" >> $bname-error-report.tab
exit 0
fi
#In-silico PCR using predefined primers(agr_operon_primers.fa) to extract agr operon
$usearch_bin -search_pcr $fasta_path -db $databases_path/agr_operon_primers.fa -strand both -maxdiffs 8 -minamp 3000 -maxamp 5000 -ampout $fna_name-results/$fna_name-agr_operon.fna &>$fna_name-results/$fna_name-pcr-log.txt
else
#if --mummer is enabled, use mummer.
mkdir $fna_name-results/$fna_name-mummer/
dnadiff $databases_path/references/mummer_ref_operon.fna $fasta_path -p $fna_name-results/$fna_name-mummer/out &>$fna_name-results/$fna_name-mummer-log.txt
if [[ $(echo $?) == 0 ]]
then
usearch_check="pass"
echo "Mummer successful"
echo "Extracting agr operon from mummer output"
cut -f3,4,13 $fna_name-results/$fna_name-mummer/out.1coords | awk '{if($1<$2) print $3":"$1"-"$2; else print $3":"$2"-"$1;}' | xargs -I {} samtools faidx $fasta_path {} > $fna_name-results/$fna_name-agr_operon.fna
if [[ $(cat $fna_name-results/$fna_name-mummer/out.1coords | wc -l) -eq 1 ]]
then
echo "Mummer alignment is contiguous"
elif [[ $(cat $fna_name-results/$fna_name-mummer/out.1coords | wc -l) > 1 ]]
then
echo "Mummer alignment is not contiguous, extracted all fragments"
fi
else
1>&2 echo -e "Error running mummer, please make sure mummer is installed correctly"
fs="u"
#summary file
echo -e "$fna_name\t$agr_gp\t$agr_match\t$canonical\t$multiple\t$fs" >> $fna_name-results/$fna_name-summary.tab
#Error report file
echo -e "$bname\t$input_check\t$databases_check\t$outdir_check\t$agr_typing\t$usearch_check\t$snippy_check" >> $bname-error-report.tab
exit 1
fi
fi
#Check if agr operon file is empty
if [[ -s $fna_name-results/$fna_name-agr_operon.fna ]]
then
echo -e "agr operon extraction successful"
else
echo -e "Unable to find agr operon, check $fna_name-results/$fna_name-$extraction-log.txt"
fs="u"
#summary file
echo -e "$fna_name\t$agr_gp\t$agr_match\t$canonical\t$multiple\t$fs" >> $fna_name-results/$fna_name-summary.tab
#Error report file
echo -e "$bname\t$input_check\t$databases_check\t$outdir_check\t$agr_typing\t$usearch_check\t$snippy_check" >> $bname-error-report.tab
exit
fi
if [[ $agr_gp == "u" ]]
then
echo -e "Unable to determine agr group, cannot perform frameshift detection"
fs="u"
#summary file
echo -e "$fna_name\t$agr_gp\t$agr_match\t$canonical\t$multiple\t$fs" >> $fna_name-results/$fna_name-summary.tab
#Error report file
echo -e "$bname\t$input_check\t$databases_check\t$outdir_check\t$agr_typing\t$usearch_check\t$snippy_check" >> $bname-error-report.tab
exit
fi
################################
##### FRAMESHIFT DETECTION #####
################################
#Running snippy with a group specific reference to call variants in the agr operon
snippy --outdir $fna_name-results/$fna_name-snippy --ctgs $fna_name-results/$fna_name-agr_operon.fna --ref $databases_path/references/$agr_gp-operon_ref.gbk --minqual 1 --mincov 2 2> $fna_name-results/$fna_name-snippy-log.txt
#Check if snps file is empty
if [[ -s $fna_name-results/$fna_name-snippy/snps.tab ]]
then
echo -e "Snippy successful"
snippy_check="pass"
else
1>&2 echo -e "Snippy unsuccessful, check $fna_name-results/$fna_name-snippy-log.txt"
#Error report file
echo -e "$bname\t$input_check\t$databases_check\t$outdir_check\t$agr_typing\t$usearch_check\t$snippy_check" >> $bname-error-report.tab
fs="u"
#summary file
echo -e "$fna_name\t$agr_gp\t$agr_match\t$canonical\t$multiple\t$fs" >> $fna_name-results/$fna_name-summary.tab
exit 1
fi
#Filtering out frameshifts in coding regions from snippy data
echo -e "#filename\tposition\ttype\teffect\tgene" > $fna_name-results/$fna_name-agr_operon_frameshifts.tab
awk -v i="$fna_name" 'BEGIN{FS=OFS="\t"};{if($7=="CDS") print i,$2,$3,$11,$13}' $fna_name-results/$fna_name-snippy/snps.tab | sed 's/ /\t/g' | grep -E -v '[conservative|disruptive]_inframe_[insertion|deletion]|splice_region_variant&stop_retained_variant|intergenic_region|initiator_codon_variant|gene_fusion|missense_variant' | grep -E -v 'synonymous_variant' >> $fna_name-results/$fna_name-agr_operon_frameshifts.tab
#Subtract 1 from $fs to account for the header
fs=$(($(cat $fna_name-results/$fna_name-agr_operon_frameshifts.tab | wc -l)-1))
#summary file
echo -e "$fna_name\t$agr_gp\t$agr_match\t$canonical\t$multiple\t$fs" >> $fna_name-results/$fna_name-summary.tab
#Error report file
echo -e "$bname\t$input_check\t$databases_check\t$outdir_check\t$agr_typing\t$usearch_check\t$snippy_check" >> $bname-error-report.tab
#Check if frameshifts found
if [[ $fs > 0 ]]
then
echo -e "Frameshifts in agr operon found, check $fna_name-results/$fna_name-agr_operon_frameshifts.tab"
else
echo -e "No frameshifts found"
exit
fi