-
Notifications
You must be signed in to change notification settings - Fork 6
/
fastq-multx.cpp
1247 lines (1098 loc) · 43 KB
/
fastq-multx.cpp
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
/*
Copyright (c) 2011 Expression Analysis / Erik Aronesty
Permission is hereby granted, free of charge, to any person obtaining a copy
of this software and associated documentation files (the "Software"), to deal
in the Software without restriction, including without limitation the rights
to use, copy, modify, merge, publish, distribute, sublicense, and/or sell
copies of the Software, and to permit persons to whom the Software is
furnished to do so, subject to the following conditions:
The above copyright notice and this permission notice shall be included in
all copies or substantial portions of the Software.
THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR
IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY,
FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE
AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER
LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM,
OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN
THE SOFTWARE.
*/
/*
See "void usage" below for usage.
*/
#include "fastq-lib.h"
#define MAX_BARCODE_NUM 6000
#define MAX_GROUP_NUM 500
// factor to divide max by
#define THFIXFACTOR 20
#define endstr(e) (e=='e'?"end":e=='b'?"start":"n/a")
const char * VERSION = "1.4";
#define SVNREV 3
// barcode
struct bc {
line id;
line seq;
char *out[6]; // one output per input
FILE *fout[6];
bool gzout[6];
long long int cnt; // count found
bool shifted; // count found in 1-shifted position
char * dual; // is this a dual-indexed barcode? if so, this points to the second index.
int dual_n; // length of dual
};
// group of barcodes
struct group {
char *id;
int tcnt; // number of codes past thresh
int i; // my index
};
// barcode group
struct bcg {
struct bc b; // barcode
line group; // group (fluidigm, truseq, etc)
int bcnt[6]; // matched begin of file n
int ecnt[6]; // matched end of file n
int bscnt[6]; // matched begin of file n, shifted by 1
int escnt[6]; // matched end of file n, shifted by 1
int dbcnt[6]; // dual matched begin of file n
int decnt[6]; // dual matched end of file n
struct group *gptr;
};
struct group* getgroup(char *s);
void usage(FILE *f);
static int debug=0;
// it's times like this when i think a class might be handy, but nah, not worth it
typedef struct bnode {
char *seq;
int cnt;
} bnode;
struct group grs[MAX_GROUP_NUM];
static int grcnt=0;
struct bc bc[MAX_BARCODE_NUM+1];
static int bcnt=0;
static int pickmax=0;
static int pickmax2=0;
static void *picktab=NULL;
void pickbest(const void *nodep, const VISIT which, const int depth);
int bnodecomp(const void *a, const void *b) {return strcmp(((bnode*)a)->seq,((bnode*)b)->seq);};
static float pickmaxpct=0.10;
void getbcfromheader(struct fq *fqin, struct fq *bc, char **s2=NULL, int *ns2=NULL);
void getbcfromheader(char *s, int *ns, char **q=NULL, char **s2=NULL, int *ns2=NULL);
int ignore;
size_t ignore_st;
int main (int argc, char **argv) {
char c;
bool trim = true;
int mismatch = 1;
int mismatchdual = -1;
int mismatchtotal = 1;
bool indepmm = false;
int distance = 2;
int poor_distance = 0; // count of skipped reads on distance only
int quality = 0;
char end = '\0';
char dend = '\0';
bool dual = false;
char *in[6];
const char *out[6];
int f_n=0;
int f_oarg=0;
const char* guide=NULL; // use an indexed-read
const char* list=NULL; // use a barcode master list
char verify='\0';
bool noexec = false;
const char *group = NULL;
bool usefile1 = false;
int phred = 33;
double threshfactor = 1;
int bcinheader = 0;
int i;
bool omode = false;
char *bfil = NULL;
while ((c = getopt (argc, argv, "-DzxnHhbeov:m:M:B:g:L:l:G:q:d:t:")) != -1) {
switch (c) t:{
case '\1':
if (omode) {
if (f_oarg<5)
out[f_oarg++] = optarg;
else {
usage(stderr); return 1;
}
} else if (!bfil && !guide && !list)
bfil = optarg;
else if (f_n<5) {
in[f_n++] = optarg;
} else {
usage(stderr); return 1;
}
break;
case 'o': omode=true; break;
case 'v':
if (strlen(optarg)>1) {
fprintf(stderr, "Option -v requires a single character argument");
exit(1);
}
verify = *optarg; break;
case 'b': end = 'b'; break;
case 'h': usage(stdout); exit(0); break;
case 'H': bcinheader = 1; usefile1=1; break;
case 'e': end = 'e'; break;
case 'G': group = optarg; break;
case 'g':
guide = optarg;
in[f_n++] = optarg;
out[f_oarg++] = "n/a";
break;
case 'l': list = optarg; usefile1=0; break;
case 'L': list = optarg; usefile1=1; break;
case 'B': bfil = optarg; list = NULL; break;
case 'x': trim = false; break;
case 'n': noexec = true; break;
case 't': threshfactor = atof(optarg); break;
case 'm': mismatch = atoi(optarg); break;
case 'M': mismatchdual = atoi(optarg); break;
case 'd': distance = atoi(optarg); break;
case 'q': quality = atoi(optarg); break;
case 'D': ++debug; break;
case '?':
if (strchr("vmBglG", optopt))
fprintf (stderr, "Option -%c requires an argument.\n", optopt);
else if (isprint(optopt))
fprintf (stderr, "Unknown option `-%c'.\n", optopt);
else
fprintf (stderr,
"Unknown option character `\\x%x'.\n",
optopt);
usage(stderr);
return 1;
}
}
mismatchtotal = mismatch;
if (mismatchdual >= 0) {
mismatchtotal += mismatchdual;
indepmm = true;
}
if (group && !list) {
fprintf(stderr, "Error: -G only works with -l\n");
return 1;
}
if ((list && guide) || (list && bfil) || (guide && bfil)) {
fprintf(stderr, "Error: Only one of -B -g or -l\n");
return 1;
}
if (f_n != f_oarg) {
fprintf(stderr, "Error: number of input files (%d) must match number of output files following '-o'.\n", f_n);
return 1;
}
if (argc < 3 || !f_n || (!bfil && !guide && !list)) {
usage(stderr);
return 1;
}
quality+=phred;
FILE *fin[6];
bool gzin[6]; meminit(gzin);
for (i = 0; i < f_n; ++i) {
fin[i]=gzopen(in[i],"r",&gzin[i]);
}
// set all to null, zero
meminit(bc);
// 3 ways to get barcodes
if (list) {
// use a list of barcode groups... determine the best set, then use the determined set
struct bcg *bcg = (struct bcg *) malloc(sizeof(*bcg) * MAX_GROUP_NUM * MAX_BARCODE_NUM);
if (!bcg) {
fprintf(stderr, "Out of memory\n");
return 1;
}
memset(bcg, 0, sizeof(*bcg) * MAX_GROUP_NUM * MAX_BARCODE_NUM);
int bgcnt=0;
int b;
FILE *lin = fopen(list, "r");
if (!lin) {
fprintf(stderr, "Error opening file '%s': %s\n",list, strerror(errno));
return 1;
}
// read barcode groups
int ok;
while (bgcnt < (MAX_GROUP_NUM * MAX_BARCODE_NUM) && (ok = read_line(lin, bcg[bgcnt].b.id))) {
if (ok <= 0) break;
if (bcg[bgcnt].b.id.s[0]=='#') continue;
bcg[bgcnt].b.id.s=strtok(bcg[bgcnt].b.id.s, "\t\n\r ");
bcg[bgcnt].b.seq.s=strtok(NULL, "\t\n\r ");
char *g=strtok(NULL, "\n\r");
if (!g) {
if (bgcnt==0){
fprintf(stderr,"Barcode guide list needs to be ID<whitespace>SEQUENCE<whitespace>GROUP");
return 1;
} else {
continue;
}
}
if (group) {
if (strcasecmp(group, g)) {
continue;
}
}
if (!strcmp(bcg[bgcnt].b.seq.s,"seq")) continue;
// dual indexed indicated by a dash in the sequence...
if (bcg[bgcnt].b.dual=strchr(bcg[bgcnt].b.seq.s,'-')) {
*bcg[bgcnt].b.dual = '\0';
++bcg[bgcnt].b.dual;
bcg[bgcnt].b.dual_n = strlen(bcg[bgcnt].b.dual);
}
// group pointer for this group
bcg[bgcnt].gptr = getgroup(g);
bcg[bgcnt].b.id.n=strlen(bcg[bgcnt].b.id.s);
bcg[bgcnt].b.seq.n=strlen(bcg[bgcnt].b.seq.s);
if (debug) fprintf(stderr, "BCG: %d bc:%s n:%d\n", bgcnt, bcg[bgcnt].b.seq.s, bcg[bgcnt].b.seq.n);
++bgcnt;
}
if (!bgcnt) {
fprintf(stderr,"No barcodes %s from guide list %s.\n", group ? "matched" : "read", list);
return 1;
}
int sampcnt = 200000;
struct stat st;
int fsum[f_n], fmax[f_n]; int bestcnt=0, besti=-1, bestdual=0;
int dfsum[f_n], dfmax[f_n]; int dbestcnt=0, dbesti=-1;
meminit(fsum); meminit(fmax); meminit(dfsum); meminit(dfmax);
// subsample to determine group to use
for (i=0;i<(usefile1?1:f_n);++i) {
char *s = NULL; size_t na = 0; int nr = 0, ns = 0;
char *q = NULL; size_t nq = 0;
double tots=0, totsq=0;
char *s2 = NULL; int ns2=0;
char *ignore_s=NULL;
stat(in[i], &st);
while ((ns=getline(&s, &na, fin[i])) > 0) {
if (*s != '@') {
fprintf(stderr,"Invalid fastq file: %s.\n", in[i]);
exit(1);
}
if (bcinheader) {
// read in 3 more lines (seq, comment, qual) and ignore them
ignore=getline(&ignore_s, &ignore_st, fin[i]);
ignore=getline(&ignore_s, &ignore_st, fin[i]);
ignore=getline(&ignore_s, &ignore_st, fin[i]);
getbcfromheader(s, &ns, &q, &s2, &ns2);
nq=ns;
} else {
// read in 3 more lines (seq, comment, qual)
if ((ns=getline(&s, &na, fin[i])) <=0)
break;
ignore=getline(&q, &ignore_st, fin[i]);
nq=getline(&q, &ignore_st, fin[i]);
s[--ns]='\0'; q[--nq]='\0';
}
// skip if quality is below average
if (st.st_size > (sampcnt * 500) && poorqual(i, ns, s, q))
continue;
for (b=0;b<bgcnt;++b) {
// matches front of read?
if (!strncasecmp(s, bcg[b].b.seq.s, bcg[b].b.seq.n)) {
++bcg[b].bcnt[i];
} else if (!strncasecmp(s+1, bcg[b].b.seq.s, bcg[b].b.seq.n)) {
// shifted read?
++bcg[b].bscnt[i];
}
if (ns >= bcg[b].b.seq.n && !strcasecmp(s+ns-bcg[b].b.seq.n, bcg[b].b.seq.s)) {
++bcg[b].ecnt[i];
} else if (ns > bcg[b].b.seq.n && !strncasecmp(s+ns-bcg[b].b.seq.n-1, bcg[b].b.seq.s, bcg[b].b.seq.n)) {
++bcg[b].escnt[i];
}
if (bcg[b].b.dual) {
const char * t=s;
int nt=ns;
if (bcinheader) { // barcode in header? use stuff after '+' sign
t=s2;
nt=ns2;
}
if (!strncasecmp(t, bcg[b].b.dual, bcg[b].b.dual_n)) {
++bcg[b].dbcnt[i];
}
if (ns >= bcg[b].b.dual_n && !strcasecmp(t+nt-bcg[b].b.dual_n, bcg[b].b.dual)) {
++bcg[b].decnt[i];
}
}
}
++nr;
// got enough reads?
if (nr >= sampcnt)
break;
}
for (b=0;b<bgcnt;++b) {
// highest count
int hcnt = (int) (max(bcg[b].bcnt[i],bcg[b].ecnt[i]) * log(bcg[b].b.seq.n));
fsum[i]+=hcnt;
if (hcnt > fmax[i])
fmax[i]=hcnt;
if (fsum[i] > bestcnt) {
if (debug > 1)
fprintf(stderr,"file %d(%s), bcg: %s, file-sum: %d, bestsum: %d\n", i, in[i], bcg[b].gptr->id, fsum[i], bestcnt);
bestcnt=fsum[i];
besti=i;
bestdual=(bcg[b].b.dual!=NULL);
}
if (debug > 1)
fprintf(stderr,"dual %d(%s), bcg: %s, file-sum: %d, bestsum: %d\n", i, in[i], bcg[b].gptr->id, dfsum[i], dbestcnt);
if (bcg[b].b.dual) {
// highest count
int dcnt = (int) (max(bcg[b].dbcnt[i],bcg[b].decnt[i]) * log(bcg[b].b.dual_n));
dfsum[i]+=dcnt;
if (dcnt > dfmax[i])
dfmax[i]=dcnt;
if (dfsum[i] > dbestcnt) {
if (debug > 1)
fprintf(stderr,"dual %d(%s), bcg: %s, file-sum: %d, bestsum: %d\n", i, in[i], bcg[b].gptr->id, dfsum[i], dbestcnt);
dbestcnt=dfsum[i];
dbesti=i;
}
}
}
if (debug > 0) fprintf(stderr,"file-best %d sum:%d, max:%d\n", besti, fsum[besti], fmax[besti]);
if (debug > 0 && bestdual) fprintf(stderr,"dual file-best %d sum:%d, max:%d\n", dbesti, dfsum[dbesti], dfmax[dbesti]);
}
// chosen file is "besti"
i=usefile1?0:besti;
int gmax=0, gindex=-1, scnt = 0, ecnt=0, dscnt = 0, decnt = 0;
int thresh = (int) (pickmaxpct*fmax[i]);
if (debug > 0) fprintf(stderr,"besti: %d thresh: %d, dual: %d\n", besti, thresh, bestdual);
for (b=0;b<bgcnt;++b) {
int hcnt = (int) (max(bcg[b].bcnt[i],bcg[b].ecnt[i]) * log(bcg[b].b.seq.n));
if (debug > 1) fprintf(stderr,"cnt: %s %s hc:%d bc:%d ec: %d\n", bcg[b].b.id.s, bcg[b].b.seq.s, hcnt, bcg[b].bcnt[i], bcg[b].ecnt[i]);
if (hcnt >= thresh) {
// increase group count
bcg[b].gptr->tcnt += hcnt;
if (bcg[b].gptr->tcnt > gmax) {
gindex=bcg[b].gptr->i;
gmax=bcg[b].gptr->tcnt;
}
}
}
if (gindex == -1) {
fprintf(stderr, "Unable to determine barcode group\n");
exit(1);
}
//printf("gmax: %d, gindex %d, %s, thresh: %d\n", gmax, gindex, grs[gindex].id, thresh);
for (b=0;b<bgcnt;++b) {
if (bcg[b].gptr->i == gindex) {
if (bcg[b].bcnt[i] > bcg[b].ecnt[i]) {
scnt+=bcg[b].dbcnt[i];
} else if (bcg[b].bcnt[i] < bcg[b].ecnt[i]) {
ecnt+=bcg[b].decnt[i];
}
if (bcg[b].dbcnt[dbesti] > bcg[b].decnt[dbesti]) {
dscnt+=bcg[b].dbcnt[dbesti];
} else if (bcg[b].dbcnt[dbesti] < bcg[b].decnt[dbesti]) {
decnt+=bcg[b].decnt[dbesti];
}
}
};
end = scnt >= ecnt ? 'b' : 'e';
if (debug) fprintf(stderr,"scnt: %d, ecnt, %d, end: %c\n", scnt, ecnt, end);
thresh/=threshfactor;
if (bestdual)
thresh/=5;
// since this is a known good set, use a very low threshold, just to catch them all
fprintf(stderr, "Using Barcode Group: %s on File: %s (%s), Threshold %2.2f%%\n",
grs[gindex].id, in[i], endstr(end), 100.0 * (float) ((float)thresh/THFIXFACTOR)/sampcnt);
if (bestdual) {
dend = dscnt >= decnt ? 'b' : 'e';
fprintf(stderr, "Dual index on File: %s (%s)\n", in[dbesti], endstr(dend));
dual=true;
for (b=0;b<bgcnt;++b) {
// trim down a bit, but later should trim down to "both-match"
if (bcg[b].gptr->i == gindex) {
if (bcg[b].decnt[dbesti] < bcg[b].ecnt[i])
bcg[b].ecnt[i] = bcg[b].decnt[dbesti];
if (bcg[b].dbcnt[dbesti] < bcg[b].bcnt[i])
bcg[b].bcnt[i] = bcg[b].dbcnt[dbesti];
}
}
}
for (b=0;b<bgcnt;++b) {
if (bcg[b].gptr->i == gindex) {
int cnt = (end == 'e' ? (bcg[b].ecnt[i]+bcg[b].escnt[i]) : ( bcg[b].bcnt[i] + bcg[b].bscnt[i] ));
if (cnt > thresh/THFIXFACTOR) {
// count exceeds threshold... use it
bc[bcnt]=bcg[b].b;
if ((end == 'e' && (bcg[b].escnt[i] < 1.2*bcg[b].ecnt[i])) ||
(end == 'b' && (bcg[b].bscnt[i] < 1.2*bcg[b].bcnt[i]))
) {
if (!dual)
fprintf(stderr, "Using Barcode %s (%s)\n", bcg[b].b.id.s, bcg[b].b.seq.s);
if (debug) fprintf(stderr, "Debug Barcode %s (%s-%s) ... ecnt:%d, escnt:%d,bcnt:%d, bscnt:%d\n", bcg[b].b.id.s, bcg[b].b.seq.s, bcg[b].b.dual, bcg[b].ecnt[i], bcg[b].escnt[i], bcg[b].bcnt[i], bcg[b].bscnt[i]);
} else {
bc[bcnt].shifted=1;
if (!dual)
fprintf(stderr, "Using Barcode %s (%s) shifted\n", bcg[b].b.id.s, bcg[b].b.seq.s);
if (debug) printf("Debug Barcode %s (%s-%s) shifted ... ecnt:%d, escnt:%d,bcnt:%d, bscnt:%d\n", bcg[b].b.id.s, bcg[b].b.seq.s, bcg[b].b.dual, bcg[b].ecnt[i], bcg[b].escnt[i], bcg[b].bcnt[i], bcg[b].bscnt[i]);
}
++bcnt;
}
}
}
if (i != 0) {
// in[0] needs to be the guide file
FILE *f = fin[0];
char *n = in[0];
const char *o = out[0];
bool gzi = gzin[0];
fin[0]=fin[i];
in[0]=in[i];
out[0]=out[i];
gzin[0]=gzin[i];
fin[i]=f;
in[i]=n;
out[i]=o;
gzin[i]=gzi;
// swap file in to position 1 if dual
if (dual && dbesti != 1) {
FILE *f = fin[1];
char *n = in[1];
const char *o = out[1];
bool gzi = gzin[1];
fin[1]=fin[dbesti];
in[1]=in[dbesti];
out[1]=out[dbesti];
gzin[1]=gzin[dbesti];
fin[dbesti]=f;
in[dbesti]=n;
out[dbesti]=o;
gzin[dbesti]=gzi;
}
}
if (bcg) free(bcg);
} else if (guide) {
// use the first file as a "guide file" ... and select a set of codes from that
FILE *gin = fin[0];
int blen = 0;
int sampcnt = 100000;
struct stat st;
stat(guide, &st);
char *s = NULL; size_t na = 0; int nr = 0, ns = 0;
char *q = NULL; size_t nq = 0;
// small sample to get lengths
double tots=0, totsq=0;
while ((ns=getline(&s, &na, gin)) > 0) {
if (*s != '@') {
fprintf(stderr,"Invalid fastq file: %s.\n", in[0]);
exit(1);
}
if (bcinheader) {
ignore=getline(&q, &ignore_st, fin[i]);
ignore=getline(&q, &ignore_st, fin[i]);
ignore=getline(&q, &ignore_st, fin[i]);
/// no dual barcode detection allowed
getbcfromheader(s, &ns);
printf("bc is %s\n", s);
} else {
if ((ns=getline(&s, &na, gin)) <=0)
break;
ignore=getline(&q, &ignore_st, gin);
ignore=getline(&q, &ignore_st, gin);
}
--ns;
tots+=ns;
totsq+=ns*ns;
++nr;
if (nr >= 200) break;
}
double dev = stdev(nr, tots, totsq);
// short, and nonvarying (by much, depends on the tech used)
if (dev < .25 && roundl(tots/nr) < 12) {
// most probably a barcode-only read
blen = (int) round(tots/nr);
end = 'b';
} else if (round(tots/nr) < 12) {
fprintf(stderr, "File %s looks to be barcode-only, but it's length deviation is too high (%.4g)\n", in[0], dev);
return 1;
} else {
fprintf(stderr, "File %s isn't a barcode-only file, try using -l instead\n", in[0]);
return 1;
}
fprintf(stderr, "Barcode length used: %d (%s)\n", blen, endstr(end));
// load a table of possble codes
pickmax=0;
picktab=NULL;
bnode * ent = NULL;
while ((ns=getline(&s, &na, gin)) > 0) {
if (*s != '@') {
fprintf(stderr,"Invalid fastq file: %s.\n", in[i]);
exit(1);
}
if (bcinheader) {
ignore=getline(&q, &ignore_st, fin[i]);
ignore=getline(&q, &ignore_st, fin[i]);
ignore=getline(&q, &ignore_st, fin[i]);
getbcfromheader(s, &ns, &q);
printf("bc is %s\n", s);
} else {
if ((ns=getline(&s, &na, gin)) <=0)
break;
ignore=getline(&q, &ignore_st, gin);
if (getline(&q, &ignore_st, gin) != ns)
break;
s[--ns]='\0'; q[ns]='\0';
}
if (st.st_size > (sampcnt * 500) && poorqual(i, ns, s, q))
continue;
++nr;
char *p;
if (end == 'b') {
p=s;
} else {
p=s+nr-blen;
}
p[blen]='\0';
if (!ent) { // make a new ent
ent = (bnode *) malloc(sizeof(*ent));
ent->seq=(char*)malloc(blen+1);;
}
if (strchr(p, 'N')||strchr(p, 'n'))
continue;
ent->cnt=0;
strcpy(ent->seq, p);
bnode *fent = * (bnode**) tsearch(ent, &picktab, bnodecomp);
if (fent == ent) // used the ent, added to tree
ent = NULL; // need a new one
++fent->cnt;
if (fent->cnt > pickmax)
pickmax=fent->cnt;
else if (fent->cnt > pickmax2)
pickmax2=fent->cnt;
if (nr > sampcnt)
break;
}
pickmax=max(1,(int)(pickmaxpct*pickmax2));
fprintf(stderr, "Threshold used: %d\n", pickmax);
twalk(picktab, pickbest);
} else {
// user specifies a list of barcodes, indexed read is f[0] and f[1] if dual
FILE *bin = fopen(bfil, "r");
if (!bin) {
fprintf(stderr, "Error opening file '%s': %s\n",bfil, strerror(errno));
return 1;
}
bcnt = 0;
int ok;
while (bcnt < MAX_BARCODE_NUM && (ok = read_line(bin, bc[bcnt].id))) {
if (ok <= 0) break;
if (bc[bcnt].id.s[0]=='#') continue;
bc[bcnt].id.s=strtok(bc[bcnt].id.s, "\t\n\r ");
bc[bcnt].seq.s=strtok(NULL, "\t\n\r ");
if (!bc[bcnt].seq.s) {
fprintf(stderr, "Barcode file '%s' required format is 'ID SEQ'\n",bfil);
return 1;
}
if (bc[bcnt].dual=strchr(bc[bcnt].seq.s,'-')) {
*bc[bcnt].dual = '\0';
++bc[bcnt].dual;
bc[bcnt].dual_n = strlen(bc[bcnt].dual);
dual=true;
}
bc[bcnt].id.n=strlen(bc[bcnt].id.s);
bc[bcnt].seq.n=strlen(bc[bcnt].seq.s);
if (debug) fprintf(stderr, "BC: %d bc:%s n:%d\n", bcnt, bc[bcnt].seq.s, bc[bcnt].seq.n);
++bcnt;
}
fprintf(stderr, "Using Barcode File: %s\n", bfil);
}
if (noexec) {
int b;
for (b=0;b<bcnt;++b) {
fprintf(stdout, "%s %s\n", bc[b].id.s, bc[b].seq.s);
}
exit(0);
}
// for whatever reason, the end is not supplied... easy enough to determine accurately
// or it's dual... which means we need to resample stuff
if (bcinheader && !end) {
end = 'b';
}
if (end == '\0' || dual) {
for (i=0;i<f_n;++i) {
if (!gzin[i])
fseek(fin[i],0,0);
else {
pclose(fin[i]);
fin[i]=gzopen(in[i],"r",&gzin[i]);
}
}
int sampcnt = dual ? 200000 : 10000;
struct stat st;
stat(in[0], &st);
char *s = NULL; size_t na = 0; int nr = 0, ns = 0;
char *q = NULL; size_t nq = 0;
int ne=0, nb=0, dne=0, dnb=0, tcount=0, read_ok=0;
int *recount = dual ? ((int *) malloc(sizeof(int)*bcnt)) : NULL;
if (dual) memset(recount, 0, sizeof(int)*bcnt);
struct fq fq[2]; meminit(fq);
while (read_ok=read_fq(fin[0], nr, &fq[0])) {
fq[0].id.s[--fq[0].id.n]='\0';
if (dual)
read_fq(fin[1], nr, &fq[1]);
++nr;
if (st.st_size > (sampcnt * 500) && poorqual(0, fq[0].seq.n, fq[0].seq.s, fq[0].qual.s))
continue;
if (dual)
if (st.st_size > (sampcnt * 500) && poorqual(1, fq[1].seq.n, fq[1].seq.s, fq[1].qual.s))
continue;
for (i=0;i<bcnt;++i) {
int dok = 0;
if (debug > 1) fprintf(stderr, "check %s vs %s: %s vs %s", fq[0].id.s, bc[i].id.s, fq[0].seq.s, bc[i].seq.s);
if (!strncmp(fq[0].seq.s, bc[i].seq.s, bc[i].seq.n)) {
++nb;
++dok;
} else if (!strncmp(fq[0].seq.s+fq[0].seq.n-bc[i].seq.n, bc[i].seq.s, bc[i].seq.n)) {
++ne;
++dok;
}
if (dual) {
if (debug > 1) fprintf(stderr, ", dual: %s vs %s, ", fq[1].seq.s, bc[i].dual);
if (!strncmp(fq[1].seq.s, bc[i].dual, bc[i].dual_n)) {
++dnb;
++dok;
} else if (!strncmp(fq[1].seq.s+fq[1].seq.n-bc[i].dual_n, bc[i].dual, bc[i].dual_n)) {
++dne;
++dok;
}
}
if (debug > 1) fprintf(stderr, ", dok:%d, i:%d\n", dok, i);
if (dok == 2) {
++recount[i];
++tcount;
break;
}
}
if (nr >= sampcnt)
break;
}
end = (ne > nb) ? 'e' : 'b';
fprintf(stderr, "End used: %s\n", endstr(end));
if (dual && list) {
// trim down possibilities to reduce number of open files, and small stub files
dend = (dne > dnb) ? 'e' : 'b';
fprintf(stderr, "Dual-end used: %s\n", endstr(dend));
int ocnt = bcnt;
// this should allow up to a 300 plex
int thresh = max(1,tcount/1000);
thresh /= threshfactor;
bcnt=0;
if (debug)
fprintf(stderr, "dual resample threshold: %d out of %d\n", thresh, tcount);
for (i=0;i<ocnt;++i) {
if (recount[i] >= thresh) {
fprintf(stderr, "Using Barcode %s (%s-%s)\n", bc[i].id.s, bc[i].seq.s, bc[i].dual);
if (debug)
fprintf(stderr, "%d >= %d\n", recount[i], thresh);
bc[bcnt].seq=bc[i].seq;
bc[bcnt].id=bc[i].id;
bc[bcnt].dual=bc[i].dual;
bc[bcnt].dual_n=bc[i].dual_n;
++bcnt;
} else {
if (debug)
fprintf(stderr, "skipping barcode %s (%s-%s), %d < %d\n", bc[i].id.s, bc[i].seq.s, bc[i].dual, recount[i], thresh);
}
}
}
}
if (bcnt == 0) {
fprintf(stderr, "No barcodes defined, quitting.\n");
exit(1);
}
// one beyond barcode count is unmatched
bc[bcnt].id.s=(char *)"unmatched";
// TODO: output barcode read ...but only for unmatched?
int b;
for (b=0;b<=bcnt;++b) {
for (i=0;i<f_n;++i) {
if (!strcasecmp(out[i],"n/a") || !strcasecmp(out[i],"/dev/null")) {
bc[b].out[i] = NULL;
bc[b].fout[i] = NULL;
continue;
}
const char *p=strchr(out[i],'%');
if (!p) fail("Each output file name must contain a '%%' sign, which is replaced by the barcode id\n");
bc[b].out[i]=(char *) malloc(strlen(out[i])+strlen(bc[b].id.s)+100);
strncpy(bc[b].out[i], out[i], p-out[i]);
bc[b].out[i][p-out[i]]='\0';
strcat(bc[b].out[i], bc[b].id.s);
strcat(bc[b].out[i], p+1);
if (!(bc[b].fout[i]=gzopen(bc[b].out[i], "w", &bc[b].gzout[i]))) {
fprintf(stderr, "Error opening output file '%s': %s\n",bc[b].out[i], strerror(errno));
return 1;
}
}
}
// seek back to beginning of fastq
for (i=0;i<f_n;++i) {
if (!gzin[i])
fseek(fin[i],0,0);
else {
pclose(fin[i]);
fin[i]=gzopen(in[i],"r",&gzin[i]);
}
}
// don't trim if you're not outputting the read
struct fq fq[8];
meminit(fq);
long long int nrec=0;
int read_ok;
// ACTUAL DEMUX HAPPENS HERE
// read in 1 record from EACH file supplied
while (read_ok=read_fq(fin[0], nrec, &fq[0])) {
for (i=1;i<f_n;++i) {
int mate_ok=read_fq(fin[i], nrec, &fq[i]);
if (read_ok != mate_ok) {
fprintf(stderr, "# of rows in mate file '%s' doesn't match primary file, quitting!\n", in[i]);
return 1;
}
if (verify) {
// verify 1 in 100
if (0 == (nrec % 100)) {
char *p=strchr(fq[i].id.s,verify);
if (!p) {
fprintf(stderr, "File %s is missing id verification char %c at line %lld", in[i], verify, nrec*4+1);
return 1;
}
int l = p-fq[i].id.s;
if (strncmp(fq[0].id.s, fq[i].id.s, l)) {
fprintf(stderr, "File %s, id doesn't match file %s at line %lld", in[0], in[i], nrec*4+1);
return 1;
}
}
}
}
++nrec;
if (read_ok < 0) continue;
int i, best=-1, bestmm=mismatchtotal+distance+1, bestd=mismatchtotal+distance+1, next_best=mismatchtotal+distance*2+1;
if (bcinheader) {
for (i=f_n-1;i>=0;--i) {
fq[i+(dual?2:1)]=fq[i];
}
meminit(fq[0]);
if (dual) {
meminit(fq[1]);
getbcfromheader(&fq[2], &fq[0], &fq[1].seq.s, &fq[1].seq.n);
} else {
getbcfromheader(&fq[2], &fq[0]);
}
}
if (debug) {
if (!bcinheader) fq[0].id.s[fq[0].id.n-1] = '\0';
fprintf(stderr, "id: %s, seq: %s %d", fq[0].id.s, fq[0].seq.s, fq[0].seq.n);
if (dual) fprintf(stderr, ", sdual: %s %d", fq[1].seq.s, fq[1].seq.n);
if (!bcinheader) fq[0].id.s[fq[0].id.n] = '\n';
if (debug > 1) printf("\n");
}
if (quality > 0) {
// low quality base = 'N'
for (i=0;i<fq[0].seq.n;++i) {
if (fq[0].qual.s[i]<quality) {
fq[0].seq.s[i]='N';
}
}
}
// for each barcode
for (i =0; i < bcnt; ++i) {
int d;
int dd = 0;
if (end == 'e') {
if (bc[i].shifted) {
if (fq[0].seq.n > bc[i].seq.n) {
d=hd(fq[0].seq.s+fq[0].seq.n-bc[i].seq.n-1, bc[i].seq.s, bc[i].seq.n);
} else {
d=bc[i].seq.n;
}
} else {
if (fq[0].seq.n >= bc[i].seq.n) {
d=hd(fq[0].seq.s+fq[0].seq.n-bc[i].seq.n, bc[i].seq.s, bc[i].seq.n);
} else {
d=bc[i].seq.n;
}
}
if (dual) {
// distance is added in for duals
if (indepmm) {
if (fq[1].seq.n >= bc[i].dual_n) {
dd+=hd(fq[1].seq.s+fq[1].seq.n-bc[i].dual_n, bc[i].dual, bc[i].dual_n);
} else {
dd+=bc[i].dual_n;
}
} else {
if (fq[1].seq.n >= bc[i].dual_n) {
d+=hd(fq[1].seq.s+fq[1].seq.n-bc[i].dual_n, bc[i].dual, bc[i].dual_n);
} else {
d+=bc[i].dual_n;
}
}
}
} else {
if (bc[i].shifted)
d=hd(fq[0].seq.s+1,bc[i].seq.s, bc[i].seq.n);
else
d=hd(fq[0].seq.s,bc[i].seq.s, bc[i].seq.n);
// distance is added in for duals
if (dual) {
if (indepmm)
dd+=hd(fq[1].seq.s,bc[i].dual, bc[i].dual_n);
else
d+=hd(fq[1].seq.s,bc[i].dual, bc[i].dual_n);
}
//if (debug > 1) {
// fprintf(stderr, "index: %d dist: %d bc:%s n:%d", i, d, bc[i].seq.s, bc[i].seq.n);
// if (dual) fprintf(stderr, ", idual: %s %d", bc[i].dual, bc[i].dual_n);
// fprintf(stderr, "\n");
//}
}
// simple...
if ((d + dd) < bestd) {
next_best=bestd;
bestd=d+dd;
if (debug > 1) fprintf(stderr,"next_dist: %d, best_seq: %s:%d\n", next_best, bc[i].seq.s, bestd);
}
// if exact match
if ((d+dd)==0) {
if (debug) fprintf(stderr, ", found bc: %d bc:%s n:%d, bestd: %d, next_best: %d", i, bc[i].seq.s, bc[i].seq.n, bestd, next_best);
best=i;
break;
} else if ((d+dd) <= mismatchtotal && (!indepmm || (d <= mismatch && dd <= mismatchdual))) {
// if ok match
if ((d+dd) == bestmm) {
best=-1; // more than 1 match... bad
} else if ((d+dd) < bestmm) {
bestmm=d+dd; // best match...ok
best=i;
}
}
}
if ((best >= 0) && distance && (next_best-bestd) < distance) {
if (debug) fprintf(stderr, "%d<%d, skipping", next_best-bestd, distance);
// match is ok, but distance is poor
++poor_distance;
best=-1;
}
bool trimmed = false;
// only trim if you're outputting the sequence
if (trim && best >= 0 && bc[best].fout[0]) {