2
Copyright (c) 1996,1997,1998,1999,2000,2001,2004,2006
3
Whitehead Institute for Biomedical Research, Steve Rozen
4
(http://jura.wi.mit.edu/rozen), and Helen Skaletsky
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Redistribution and use in source and binary forms, with or without
8
modification, are permitted provided that the following conditions are
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* Redistributions of source code must retain the above copyright
12
notice, this list of conditions and the following disclaimer.
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* Redistributions in binary form must reproduce the above
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copyright notice, this list of conditions and the following disclaimer
15
in the documentation and/or other materials provided with the
17
* Neither the names of the copyright holders nor contributors may
18
be used to endorse or promote products derived from this software
19
without specific prior written permission.
21
THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS
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"AS IS" AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT
23
LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR
24
A PARTICULAR PURPOSE ARE DISCLAIMED. IN NO EVENT SHALL THE COPYRIGHT
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OWNERS OR CONTRIBUTORS BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL,
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SPECIAL, EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT
27
LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE,
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DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER CAUSED AND ON ANY
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THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT
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(INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE
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OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE.
37
#include "primer3_release.h"
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#define MACRO_VALUE_AS_STRING(A) MACRO_STRING(A)
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static int check_intervals(const char *, const int,
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interval_array_t, const int, seq_args *);
43
static char dna_to_upper(char *, int);
44
static char *strstr_nocase(char *, char *);
47
/* Default parameter values. */
55
#define MAX_DIFF_TM 100.0
56
#define DEFAULT_OPT_GC_PERCENT PR_UNDEFINED_INT_OPT
59
#define SALT_CONC 50.0
61
#define NUM_NS_ACCEPTED 0
65
#define PAIR_COMPL_ANY 800
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#define PAIR_COMPL_END 300
68
#define EXPLAIN_FLAG 0
70
#define LIBERAL_BASE 0
71
#define PICK_INTERNAL_OLIGO 0
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#define INTERNAL_OLIGO_OPT_SIZE 20
74
#define INTERNAL_OLIGO_MIN_SIZE 18
75
#define INTERNAL_OLIGO_MAX_SIZE 27
76
#define INTERNAL_OLIGO_OPT_TM 60.0
77
#define INTERNAL_OLIGO_MIN_TM 57.0
78
#define INTERNAL_OLIGO_MAX_TM 63.0
79
#define INTERNAL_OLIGO_MIN_GC 20.0
80
#define INTERNAL_OLIGO_MAX_GC 80.0
81
#define INTERNAL_OLIGO_SALT_CONC 50.0
82
#define INTERNAL_OLIGO_DNA_CONC 50.0
83
#define INTERNAL_OLIGO_NUM_NS 0
84
#define INTERNAL_OLIGO_MAX_POLY_X 5
85
#define INTERNAL_OLIGO_SELF_ANY 1200
86
#define INTERNAL_OLIGO_SELF_END 1200
87
#define INTERNAL_OLIGO_REPEAT_SIMILARITY 1200
88
#define REPEAT_SIMILARITY 1200
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#define PAIR_REPEAT_SIMILARITY 2400
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#define FIRST_BASE_INDEX 0
93
#define QUALITY_RANGE_MIN 0
94
#define QUALITY_RANGE_MAX 100
95
#define DEFAULT_MAX_END_STABILITY 100.0
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#define PRIMER_PRODUCT_OPT_SIZE PR_UNDEFINED_INT_OPT
97
#define PRIMER_PRODUCT_OPT_TM PR_UNDEFINED_DBL_OPT
98
#define MAX_TEMPLATE_MISPRIMING PR_UNDEFINED_ALIGN_OPT
99
#define PAIR_MAX_TEMPLATE_MISPRIMING PR_UNDEFINED_ALIGN_OPT
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#define IO_MAX_TEMPLATE_MISHYB PR_UNDEFINED_ALIGN_OPT
102
#define LIB_AMBIGUITY_CODES_CONSENSUS 1
103
/* For backward compatibility. It turns out that
104
this _not_ what one normally wants, since many
105
libraries contain strings of N, which then match
106
every oligo (very bad).
109
/* Weights for objective functions for oligos and pairs. */
110
#define PRIMER_WT_TM_GT 1
111
#define PRIMER_WT_TM_LT 1
112
#define PRIMER_WT_SIZE_LT 1
113
#define PRIMER_WT_SIZE_GT 1
114
#define PRIMER_WT_GC_PERCENT_LT 0
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#define PRIMER_WT_GC_PERCENT_GT 0
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#define PRIMER_WT_COMPL_ANY 0
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#define PRIMER_WT_COMPL_END 0
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#define PRIMER_WT_NUM_NS 0
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#define PRIMER_WT_REP_SIM 0
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#define PRIMER_WT_SEQ_QUAL 0
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#define PRIMER_WT_END_QUAL 0
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#define PRIMER_WT_POS_PENALTY 1
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#define PRIMER_WT_END_STABILITY 0
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#define IO_WT_TM_GT 1
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#define IO_WT_TM_LT 1
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#define IO_WT_SIZE_LT 1
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#define IO_WT_SIZE_GT 1
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#define IO_WT_GC_PERCENT_LT 0
130
#define IO_WT_GC_PERCENT_GT 0
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#define IO_WT_COMPL_ANY 0
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#define IO_WT_COMPL_END 0
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#define IO_WT_NUM_NS 0
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#define IO_WT_REP_SIM 0
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#define IO_WT_SEQ_QUAL 0
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#define IO_WT_END_QUAL 0
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#define PAIR_WT_PRIMER_PENALTY 1
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#define PAIR_WT_IO_PENALTY 0
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#define PAIR_WT_DIFF_TM 0
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#define PAIR_WT_COMPL_ANY 0
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#define PAIR_WT_COMPL_END 0
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#define PAIR_WT_REP_SIM 0
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#define PAIR_WT_PRODUCT_TM_LT 0
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#define PAIR_WT_PRODUCT_TM_GT 0
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#define PAIR_WT_PRODUCT_SIZE_LT 0
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#define PAIR_WT_PRODUCT_SIZE_GT 0
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pr_set_default_global_args(a)
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memset(a, 0, sizeof(*a));
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a->primer_opt_size = OPT_SIZE;
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a->primer_min_size = MIN_SIZE;
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a->primer_max_size = MAX_SIZE;
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a->max_diff_tm = MAX_DIFF_TM;
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a->opt_gc_content = DEFAULT_OPT_GC_PERCENT;
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a->salt_conc = SALT_CONC;
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a->dna_conc = DNA_CONC;
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a->num_ns_accepted = NUM_NS_ACCEPTED;
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a->self_any = SELF_ANY;
168
a->self_end = SELF_END;
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a->pair_compl_any = PAIR_COMPL_ANY;
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a->pair_compl_end = PAIR_COMPL_END;
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a->file_flag = FILE_FLAG;
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a->explain_flag = EXPLAIN_FLAG;
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a->gc_clamp = GC_CLAMP;
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a->max_poly_x = MAX_POLY_X;
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a->liberal_base = LIBERAL_BASE;
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a->primer_task = PRIMER_TASK;
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a->first_base_index = FIRST_BASE_INDEX;
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a->num_return = NUM_RETURN;
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a->num_intervals = 1;
182
a->repeat_compl = REPEAT_SIMILARITY;
183
a->pair_repeat_compl = PAIR_REPEAT_SIMILARITY;
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a->min_quality = MIN_QUALITY;
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a->min_end_quality = MIN_QUALITY;
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a->quality_range_min = QUALITY_RANGE_MIN;
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a->quality_range_max = QUALITY_RANGE_MAX;
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a->outside_penalty = PR_DEFAULT_OUTSIDE_PENALTY;
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a->inside_penalty = PR_DEFAULT_INSIDE_PENALTY;
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a->max_end_stability = DEFAULT_MAX_END_STABILITY;
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a->product_max_tm = PR_DEFAULT_PRODUCT_MAX_TM;
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a->product_min_tm = PR_DEFAULT_PRODUCT_MIN_TM;
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a->product_opt_tm = PRIMER_PRODUCT_OPT_TM;
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a->product_opt_size = PRIMER_PRODUCT_OPT_SIZE;
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a->max_template_mispriming
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= MAX_TEMPLATE_MISPRIMING;
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a->pair_max_template_mispriming
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= PAIR_MAX_TEMPLATE_MISPRIMING;
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a->io_primer_opt_size = INTERNAL_OLIGO_OPT_SIZE;
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a->io_primer_min_size = INTERNAL_OLIGO_MIN_SIZE;
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a->io_primer_max_size = INTERNAL_OLIGO_MAX_SIZE;
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a->io_opt_tm = INTERNAL_OLIGO_OPT_TM;
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a->io_min_tm = INTERNAL_OLIGO_MIN_TM;
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a->io_max_tm = INTERNAL_OLIGO_MAX_TM;
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a->io_min_gc = INTERNAL_OLIGO_MIN_GC;
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a->io_opt_gc_content = DEFAULT_OPT_GC_PERCENT;
208
a->io_max_gc = INTERNAL_OLIGO_MAX_GC;
209
a->io_max_poly_x = INTERNAL_OLIGO_MAX_POLY_X;
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a->io_salt_conc = INTERNAL_OLIGO_SALT_CONC;
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a->io_dna_conc = INTERNAL_OLIGO_DNA_CONC;
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a->io_num_ns_accepted = INTERNAL_OLIGO_NUM_NS;
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a->io_self_any = INTERNAL_OLIGO_SELF_ANY;
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a->io_self_end = INTERNAL_OLIGO_SELF_END;
215
a->io_repeat_compl = INTERNAL_OLIGO_REPEAT_SIMILARITY;
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a->io_min_quality = MIN_QUALITY;
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a->io_min_end_quality = MIN_QUALITY;
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a->io_max_template_mishyb
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= IO_MAX_TEMPLATE_MISHYB;
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a->primer_weights.temp_gt = PRIMER_WT_TM_GT;
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a->primer_weights.temp_lt = PRIMER_WT_TM_LT;
223
a->primer_weights.length_gt = PRIMER_WT_SIZE_GT;
224
a->primer_weights.length_lt = PRIMER_WT_SIZE_LT;
225
a->primer_weights.gc_content_gt = PRIMER_WT_GC_PERCENT_GT;
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a->primer_weights.gc_content_lt = PRIMER_WT_GC_PERCENT_LT;
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a->primer_weights.compl_any = PRIMER_WT_COMPL_ANY;
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a->primer_weights.compl_end = PRIMER_WT_COMPL_END;
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a->primer_weights.num_ns = PRIMER_WT_NUM_NS;
230
a->primer_weights.repeat_sim = PRIMER_WT_REP_SIM;
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a->primer_weights.seq_quality = PRIMER_WT_SEQ_QUAL;
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a->primer_weights.end_quality = PRIMER_WT_END_QUAL;
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a->primer_weights.pos_penalty = PRIMER_WT_POS_PENALTY;
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a->primer_weights.end_stability = PRIMER_WT_END_STABILITY;
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a->io_weights.temp_gt = IO_WT_TM_GT;
237
a->io_weights.temp_lt = IO_WT_TM_LT;
238
a->io_weights.length_gt = IO_WT_SIZE_GT;
239
a->io_weights.length_lt = IO_WT_SIZE_LT;
240
a->io_weights.gc_content_gt = IO_WT_GC_PERCENT_GT;
241
a->io_weights.gc_content_lt = IO_WT_GC_PERCENT_LT;
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a->io_weights.compl_any = IO_WT_COMPL_ANY;
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a->io_weights.compl_end = IO_WT_COMPL_END;
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a->io_weights.num_ns = IO_WT_NUM_NS;
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a->io_weights.repeat_sim = IO_WT_REP_SIM;
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a->io_weights.seq_quality = IO_WT_SEQ_QUAL;
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a->io_weights.end_quality = IO_WT_END_QUAL;
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a->pr_pair_weights.primer_quality = PAIR_WT_PRIMER_PENALTY;
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a->pr_pair_weights.io_quality = PAIR_WT_IO_PENALTY;
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a->pr_pair_weights.diff_tm = PAIR_WT_DIFF_TM;
252
a->pr_pair_weights.compl_any = PAIR_WT_COMPL_ANY;
253
a->pr_pair_weights.compl_end = PAIR_WT_COMPL_END;
254
a->pr_pair_weights.repeat_sim = PAIR_WT_REP_SIM;
255
a->pr_pair_weights.product_tm_lt = PAIR_WT_PRODUCT_TM_LT;
256
a->pr_pair_weights.product_tm_gt = PAIR_WT_PRODUCT_TM_GT;
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a->pr_pair_weights.product_size_lt = PAIR_WT_PRODUCT_SIZE_LT;
258
a->pr_pair_weights.product_size_gt = PAIR_WT_PRODUCT_SIZE_GT;
260
/* a->short_match = 0; not used */
262
a->lib_ambiguity_codes_consensus = LIB_AMBIGUITY_CODES_CONSENSUS;
266
* Return 1 on error, 0 on success. Set sa->trimmed_seq and possibly modify
267
* sa->tar. Upcase and check all bases in sa->trimmed_seq.
270
_pr_data_control(pa, sa)
274
static char s1[MAX_PRIMER_LENGTH+1];
276
int seq_len = strlen(sa->sequence);
277
char offending_char = '\0';
279
if (pa->io_max_template_mishyb >= 0)
280
pr_append_new_chunk(&pa->glob_err,
281
"PRIMER_INTERNAL_OLIGO_MAX_TEMPLATE_MISHYB is not supported");
283
if (pa->primer_min_size < 1)
284
pr_append_new_chunk(&pa->glob_err, "PRIMER_MIN_SIZE must be >= 1");
286
if (pa->primer_max_size > MAX_PRIMER_LENGTH) {
287
pr_append_new_chunk(&pa->glob_err,
288
"PRIMER_MAX_SIZE exceeds built-in maximum of ");
289
pr_append(&pa->glob_err, MACRO_VALUE_AS_STRING(MAX_PRIMER_LENGTH));
293
if (pa->primer_opt_size > pa->primer_max_size) {
294
pr_append_new_chunk(&pa->glob_err,
295
"PRIMER_{OPT,DEFAULT}_SIZE > PRIMER_MAX_SIZE");
299
if (pa->primer_opt_size < pa->primer_min_size) {
300
pr_append_new_chunk(&pa->glob_err,
301
"PRIMER_{OPT,DEFAULT}_SIZE < PRIMER_MIN_SIZE");
305
if (pa->io_primer_max_size > MAX_PRIMER_LENGTH) {
306
pr_append_new_chunk(&pa->glob_err,
307
"PRIMER_INTERNAL_OLIGO_MAX_SIZE exceeds built-in maximum");
311
if (pa->io_primer_opt_size > pa->io_primer_max_size) {
312
pr_append_new_chunk(&pa->glob_err,
313
"PRIMER_INTERNAL_OLIGO_{OPT,DEFAULT}_SIZE > MAX_SIZE");
317
if (pa->io_primer_opt_size < pa->io_primer_min_size) {
318
pr_append_new_chunk(&pa->glob_err,
319
"PRIMER_INTERNAL_OLIGO_{OPT,DEFAULT}_SIZE < MIN_SIZE");
323
if (pa->gc_clamp > pa->primer_min_size) {
324
pr_append_new_chunk(&pa->glob_err,
325
"PRIMER_GC_CLAMP > PRIMER_MIN_SIZE");
329
if (NULL == sa->sequence_name && pa->file_flag) {
330
pr_append_new_chunk(&sa->error,
331
"Need PRIMER_SEQUENCE_ID if PRIMER_FILE_FLAG != 0");
335
if (0 == pa->num_intervals) {
336
pr_append_new_chunk(&pa->glob_err,
337
"Empty value for PRIMER_PRODUCT_SIZE_RANGE");
340
for (i = 0; i < pa->num_intervals; i++) {
341
if (pa->pr_min[i] > pa->pr_max[i] || pa->pr_min[i] < 0) {
342
pr_append_new_chunk(&pa->glob_err,
343
"Illegal element in PRIMER_PRODUCT_SIZE_RANGE");
349
for(i=0;i<pa->num_intervals;i++)
350
if(pa->pr_min[i]<pr_min) pr_min=pa->pr_min[i];
352
if (pa->primer_max_size > pr_min) {
353
pr_append_new_chunk(&pa->glob_err,
354
"PRIMER_MAX_SIZE > min PRIMER_PRODUCT_SIZE_RANGE");
358
if ((pick_pcr_primers_and_hyb_probe == pa->primer_task
359
|| pick_hyb_probe_only == pa->primer_task)
360
&& pa->io_primer_max_size > pr_min) {
361
pr_append_new_chunk(&pa->glob_err,
362
"PRIMER_INTERNAL_OLIGO_MAX_SIZE > min PRIMER_PRODUCT_SIZE_RANGE");
366
if (pa->num_return < 1) {
367
pr_append_new_chunk(&pa->glob_err,
368
"PRIMER_NUM_RETURN < 1");
372
if (sa->incl_l >= INT_MAX) {
373
pr_append_new_chunk(&sa->error, "Value for INCLUDED_REGION too large");
377
if (sa->incl_s < 0 || sa->incl_l < 0
378
|| sa->incl_s + sa->incl_l > seq_len) {
379
pr_append_new_chunk(&sa->error, "Illegal value for INCLUDED_REGION");
383
if (sa->incl_l < pr_min && pa->primer_task != pick_hyb_probe_only
384
&& pa->primer_task != pick_left_only
385
&& pa->primer_task != pick_right_only) {
386
pr_append_new_chunk(&sa->error,
387
"INCLUDED_REGION length < min PRIMER_PRODUCT_SIZE_RANGE");
391
if (pa->max_end_stability < 0) {
392
pr_append_new_chunk(&sa->error,
393
"PRIMER_MAX_END_STABILITY must be non-negative");
397
if (!PR_START_CODON_POS_IS_NULL(sa)) {
398
if (!PR_POSITION_PENALTY_IS_NULL(pa)) {
399
pr_append_new_chunk(&sa->error,
400
"Cannot accept both PRIMER_START_CODON_POSITION and non-default ");
401
pr_append(&sa->error,
402
"arguments for PRIMER_INSIDE_PENALTY or PRIMER_OUTSIDE_PENALTY");
404
if (sa->start_codon_pos > (sa->incl_s + sa->incl_l - 3))
405
pr_append_new_chunk(&sa->error,
406
"Start codon position not contained in INCLUDED_REGION");
408
if (sa->start_codon_pos >= 0
409
&& ((sa->sequence[sa->start_codon_pos] != 'A'
410
&& sa->sequence[sa->start_codon_pos] != 'a')
411
|| (sa->sequence[sa->start_codon_pos + 1] != 'T'
412
&& sa->sequence[sa->start_codon_pos + 1] != 't')
413
|| (sa->sequence[sa->start_codon_pos + 2] != 'G'
414
&& sa->sequence[sa->start_codon_pos + 2] != 'g')))
415
pr_append_new_chunk(&sa->error,
416
"No start codon at PRIMER_START_CODON_POSITION");
420
sa->trimmed_seq = pr_safe_malloc(sa->incl_l + 1);
421
_pr_substr(sa->sequence, sa->incl_s, sa->incl_l, sa->trimmed_seq);
423
sa->upcased_seq = pr_safe_malloc(strlen(sa->sequence) + 1);
424
strcpy(sa->upcased_seq, sa->sequence);
425
if ((offending_char = dna_to_upper(sa->upcased_seq, 1))) {
426
offending_char = '\0';
428
/* TODO add warning or error (depending on liberal base)
431
sa->upcased_seq_r = pr_safe_malloc(strlen(sa->sequence) + 1);
432
_pr_reverse_complement(sa->upcased_seq, sa->upcased_seq_r);
434
if (check_intervals("TARGET", sa->num_targets, sa->tar, seq_len, sa)
436
sa->start_codon_pos -= sa->incl_s;
438
if (check_intervals("EXCLUDED_REGION", sa->num_excl, sa->excl,
442
if (check_intervals("PRIMER_INTERNAL_OLIGO_EXCLUDED_REGION",
443
sa->num_internal_excl, sa->excl_internal,
447
if (NULL != sa->quality){
448
if(pa->min_quality != 0 && pa->min_quality < pa->quality_range_min) {
449
pr_append_new_chunk(&pa->glob_err,
450
"PRIMER_MIN_QUALITY < PRIMER_QUALITY_RANGE_MIN");
453
if(pa->min_quality != 0 && pa->min_quality > pa->quality_range_max) {
454
pr_append_new_chunk(&pa->glob_err,
455
"PRIMER_MIN_QUALITY > PRIMER_QUALITY_RANGE_MAX");
458
if(pa->io_min_quality != 0 && pa->io_min_quality <pa->quality_range_min) {
459
pr_append_new_chunk(&pa->glob_err,
460
"PRIMER_INTERNAL_OLIGO_MIN_QUALITY < PRIMER_QUALITY_RANGE_MIN");
463
if(pa->io_min_quality != 0 && pa->io_min_quality > pa->quality_range_max) {
464
pr_append_new_chunk(&pa->glob_err,
465
"PRIMER_INTERNAL_OLIGO_MIN_QUALITY > PRIMER_QUALITY_RANGE_MAX");
468
for(i=0; i< seq_len; i++) {
469
if(sa->quality[i] < pa->quality_range_min ||
470
sa->quality[i] > pa->quality_range_max) {
471
pr_append_new_chunk(&sa->error,
472
"Sequence quality score out of range");
477
else if (pa->primer_weights.seq_quality || pa->io_weights.seq_quality) {
478
pr_append_new_chunk(&sa->error,
479
"Sequence quality is part of objective function but sequence quality is not defined");
483
if ((offending_char = dna_to_upper(sa->trimmed_seq, 0))) {
484
if (pa->liberal_base) {
485
pr_append_new_chunk(&sa->warning,
486
"Unrecognized base in input sequence");
489
pr_append_new_chunk(&sa->error, "Unrecognized base in input sequence");
494
if(pa->opt_tm < pa->min_tm || pa->opt_tm > pa->max_tm) {
495
pr_append_new_chunk(&pa->glob_err,
496
"Optimum primer Tm lower than minimum or higher than maximum");
499
if(pa->io_opt_tm < pa->io_min_tm || pa->io_opt_tm > pa->io_max_tm) {
500
pr_append_new_chunk(&pa->glob_err,
501
"Illegal values for PRIMER_INTERNAL_OLIGO_TM");
504
if(pa->min_gc>pa->max_gc||pa->min_gc>100||pa->max_gc<0){
505
pr_append_new_chunk(&pa->glob_err,
506
"Illegal value for PRIMER_MAX_GC and PRIMER_MIN_GC");
509
if(pa->io_min_gc>pa->io_max_gc||pa->io_min_gc>100||pa->io_max_gc<0){
510
pr_append_new_chunk(&pa->glob_err,
511
"Illegal value for PRIMER_INTERNAL_OLIGO_GC");
514
if(pa->num_ns_accepted<0){
515
pr_append_new_chunk(&pa->glob_err,
516
"Illegal value for PRIMER_NUM_NS_ACCEPTED");
519
if(pa->io_num_ns_accepted<0){
520
pr_append_new_chunk(&pa->glob_err,
521
"Illegal value for PRIMER_INTERNAL_OLIGO_NUM_NS");
524
if(pa->self_any<0||pa->self_end<0
525
||pa->pair_compl_any<0||pa->pair_compl_end<0){
526
pr_append_new_chunk(&pa->glob_err,
527
"Illegal value for primer complementarity restrictions");
530
if(pa->io_self_any<0||pa->io_self_end<0){
531
pr_append_new_chunk(&pa->glob_err,
532
"Illegal value for internal oligo complementarity restrictions");
535
if(pa->salt_conc<=0||pa->dna_conc<=0){
536
pr_append_new_chunk(&pa->glob_err,
537
"Illegal value for primer salt or dna concentration");
540
if(pa->io_salt_conc<=0||pa->io_dna_conc<=0){
541
pr_append_new_chunk(&pa->glob_err,
542
"Illegal value for internal oligo salt or dna concentration");
545
if (!_PR_DEFAULT_POSITION_PENALTIES(pa) && sa->num_targets > 1) {
546
pr_append_new_chunk(&sa->error,
547
"Non-default inside penalty or outside penalty ");
548
pr_append(&sa->error,
549
"is valid only when number of targets <= 1");
551
if (!_PR_DEFAULT_POSITION_PENALTIES(pa) && 0 == sa->num_targets) {
552
pr_append_new_chunk(&sa->warning,
553
"Non-default inside penalty or outside penalty ");
554
pr_append(&sa->warning,
555
"has no effect when number of targets is 0");
557
if (pa->primer_task != pick_pcr_primers_and_hyb_probe
558
&& pa->primer_task != pick_hyb_probe_only
559
&& sa->internal_input) {
560
pr_append_new_chunk(&sa->error,
561
"Not specified to pick internal oligos");
562
pr_append(&sa->error,
563
" but a specific internal oligo is provided");
565
if (sa->internal_input) {
566
if (strlen(sa->internal_input) > pa->io_primer_max_size)
567
pr_append_new_chunk(&sa->error, "Specified internal oligo too long");
569
if (strlen(sa->internal_input) < pa->io_primer_min_size)
570
pr_append_new_chunk(&sa->error, "Specified internal oligo too short");
572
if (!strstr_nocase(sa->sequence, sa->internal_input))
573
pr_append_new_chunk(&sa->error,
574
"Specified internal oligo not in sequence");
575
else if (!strstr_nocase(sa->trimmed_seq, sa->internal_input))
576
pr_append_new_chunk(&sa->error,
577
"Specified internal oligo not in Included Region");
579
if (sa->left_input) {
580
if (strlen(sa->left_input) > pa->primer_max_size)
581
pr_append_new_chunk(&sa->error, "Specified left primer too long");
582
if (strlen(sa->left_input) < pa->primer_min_size)
583
pr_append_new_chunk(&sa->error, "Specified left primer too short");
584
if (!strstr_nocase(sa->sequence, sa->left_input))
585
pr_append_new_chunk(&sa->error,
586
"Specified left primer not in sequence");
587
else if (!strstr_nocase(sa->trimmed_seq, sa->left_input))
588
pr_append_new_chunk(&sa->error,
589
"Specified left primer not in Included Region");
591
if (sa->right_input) {
592
if (strlen(sa->right_input) < pa->primer_min_size)
593
pr_append_new_chunk(&sa->error, "Specified right primer too short");
594
if (strlen(sa->right_input) > pa->primer_max_size) {
595
pr_append_new_chunk(&sa->error, "Specified right primer too long");
596
} else { /* We do not want to overflow s1. */
597
_pr_reverse_complement(sa->right_input,s1);
598
if (!strstr_nocase(sa->sequence, s1))
599
pr_append_new_chunk(&sa->error,
600
"Specified right primer not in sequence");
601
else if (!strstr_nocase(sa->trimmed_seq, s1))
602
pr_append_new_chunk(&sa->error,
603
"Specified right primer not in Included Region");
607
if ((pa->pr_pair_weights.product_tm_lt ||
608
pa->pr_pair_weights.product_tm_gt)
609
&& pa->product_opt_tm == PR_UNDEFINED_DBL_OPT) {
610
pr_append_new_chunk(&pa->glob_err,
611
"Product temperature is part of objective function while optimum temperature is not defined");
615
if((pa->pr_pair_weights.product_size_lt ||
616
pa->pr_pair_weights.product_size_gt)
617
&& pa->product_opt_size == PR_UNDEFINED_INT_OPT){
618
pr_append_new_chunk(&pa->glob_err,
619
"Product size is part of objective function while optimum size is not defined");
623
if ((pa->primer_weights.gc_content_lt ||
624
pa->primer_weights.gc_content_gt)
625
&& pa->opt_gc_content == DEFAULT_OPT_GC_PERCENT) {
626
pr_append_new_chunk(&pa->glob_err,
627
"Primer GC content is part of objective function while optimum gc_content is not defined");
631
if ((pa->io_weights.gc_content_lt ||
632
pa->io_weights.gc_content_gt)
633
&& pa->io_opt_gc_content == DEFAULT_OPT_GC_PERCENT) {
634
pr_append_new_chunk(&pa->glob_err,
635
"Hyb probe GC content is part of objective function while optimum gc_content is not defined");
639
if ((pa->primer_task != pick_pcr_primers_and_hyb_probe
640
&& pa->primer_task != pick_hyb_probe_only ) &&
641
(pa->pr_pair_weights.io_quality)) {
642
pr_append_new_chunk(&pa->glob_err,
643
"Internal oligo quality is part of objective function while internal oligo choice is not required");
647
if (pa->primer_weights.repeat_sim && (!pa->repeat_lib.seq_num)) {
648
pr_append_new_chunk(&pa->glob_err,
649
"Mispriming score is part of objective function, but mispriming library is not defined");
653
if(pa->io_weights.repeat_sim && (!pa->io_mishyb_library.seq_num)){
654
pr_append_new_chunk(&pa->glob_err,
655
"Internal oligo mispriming score is part of objective function while mishyb library is not defined");
659
if(pa->pr_pair_weights.repeat_sim && (!pa->repeat_lib.seq_num)){
660
pr_append_new_chunk(&pa->glob_err,
661
"Mispriming score is part of objective function, but mispriming library is not defined");
665
if(pa->pr_pair_weights.io_quality
666
&& pa->primer_task != pick_pcr_primers_and_hyb_probe ) {
667
pr_append_new_chunk(&pa->glob_err,
668
"Internal oligo quality is part of objective function while internal oligo choice is not required");
672
return (NULL == sa->error.data && NULL == pa->glob_err.data) ? 0 : 1;
676
* Check intervals, and add to sa->error. Update the start of each interval to
677
* be relative to the start of the included region.
680
check_intervals(tag_name, num_intervals, intervals, seq_len, sa)
681
const char *tag_name;
682
const int num_intervals;
683
interval_array_t intervals;
688
int outside_warning_issued = 0;
689
for (i=0; i < num_intervals; i++) {
690
if (intervals[i][0] + intervals[i][1] > seq_len) {
691
pr_append_new_chunk(&sa->error, tag_name);
692
pr_append(&sa->error, " beyond end of sequence");
695
/* Cause the interval start to be relative to the included region. */
696
intervals[i][0] -= sa->incl_s;
697
/* Check that intervals are within the included region. */
698
if (intervals[i][0] < 0
699
|| intervals[i][0] + intervals[i][1] > sa->incl_l) {
700
if (!outside_warning_issued) {
701
pr_append_new_chunk(&sa->warning, tag_name);
702
pr_append(&sa->warning,
703
" outside of INCLUDED_REGION");
704
outside_warning_issued = 1;
707
if (intervals[i][1] < 0) {
708
pr_append_new_chunk(&sa->error, "Negative ");
709
pr_append(&sa->error, tag_name);
710
pr_append(&sa->error, " length");
717
static char * strstr_nocase(s1, s2)
723
if(s1 == NULL || s2 == NULL) return NULL;
724
n1 = strlen(s1); n2 = strlen(s2);
725
if(n1 < n2) return NULL;
727
tmp = pr_safe_malloc(n1 + 1);
731
while(q != '\0' && q != '\n'){
734
if(strcmp_nocase(p, s2)){
735
*(p + n2) = q; p++; continue;
737
else {free(tmp); return p;}
739
free(tmp); return NULL;
742
/* Upcase a DNA string, s, in place. If amibiguity_code_ok is false the
743
string can consist of acgtnACGTN. If it is true then the IUB/IUPAC
744
ambiguity codes are are allowed. Return the first unrecognized letter if
745
any is seen (and turn it to 'N' in s). Otherwise return '\0'. */
747
dna_to_upper(s, ambiguity_code_ok)
749
int ambiguity_code_ok;
752
int unrecognized_base = '\0';
755
case 'a': case 'A': *p='A'; break;
756
case 'c': case 'C': *p='C'; break;
757
case 'g': case 'G': *p='G'; break;
758
case 't': case 'T': *p='T'; break;
759
case 'n': case 'N': *p='N'; break;
761
if (ambiguity_code_ok) {
763
case 'r': case 'R': *p = 'R'; break;
764
case 'y': case 'Y': *p = 'Y'; break;
765
case 'm': case 'M': *p = 'M'; break;
766
case 'w': case 'W': *p = 'W'; break;
767
case 's': case 'S': *p = 'S'; break;
768
case 'k': case 'K': *p = 'K'; break;
769
case 'd': case 'D': *p = 'D'; break;
770
case 'h': case 'H': *p = 'H'; break;
771
case 'v': case 'V': *p = 'V'; break;
772
case 'b': case 'B': *p = 'B'; break;
775
if (!unrecognized_base) unrecognized_base = *p;
782
return unrecognized_base;
786
_pr_need_template_mispriming(pa)
787
const primer_args *pa;
790
pa->max_template_mispriming >= 0
791
|| pa->primer_weights.template_mispriming > 0.0
792
|| _pr_need_pair_template_mispriming(pa);
796
_pr_need_pair_template_mispriming(pa)
797
const primer_args *pa;
800
pa->pair_max_template_mispriming >= 0
801
|| pa->pr_pair_weights.template_mispriming > 0.0;
804
/* Takes substring of seq starting from n with length m and puts it to s. */
806
_pr_substr(const char *seq, int n, int m, char *s)
809
for(i=n;i<n+m;i++)s[i-n]=seq[i];
813
/* Reverse and complement the sequence seq and put the result in s. */
815
_pr_reverse_complement(const char *seq, char *s)
820
while (*p != '\0') p++;
825
case 'A': *q='T'; break;
826
case 'C': *q='G'; break;
827
case 'G': *q='C'; break;
828
case 'T': *q='A'; break;
829
case 'U': *q='A'; break;
831
case 'B': *q='V'; break;
832
case 'D': *q='H'; break;
833
case 'H': *q='D'; break;
834
case 'V': *q='B'; break;
835
case 'R': *q='Y'; break;
836
case 'Y': *q='R'; break;
837
case 'K': *q='M'; break;
838
case 'M': *q='K'; break;
839
case 'S': *q='S'; break;
840
case 'W': *q='W'; break;
842
case 'N': *q='N'; break;
844
case 'a': *q='t'; break;
845
case 'c': *q='g'; break;
846
case 'g': *q='c'; break;
847
case 't': *q='a'; break;
848
case 'u': *q='a'; break;
850
case 'b': *q='v'; break;
851
case 'd': *q='h'; break;
852
case 'h': *q='d'; break;
853
case 'v': *q='b'; break;
854
case 'r': *q='y'; break;
855
case 'y': *q='r'; break;
856
case 'k': *q='m'; break;
857
case 'm': *q='k'; break;
858
case 's': *q='s'; break;
859
case 'w': *q='w'; break;
861
case 'n': *q='n'; break;
added
removed
src/primer3.c
