Provided Primer fix, better sequencing stats

src/libprimer3.cc

@@ -1279,18 +1279,18 @@ choose_primers(const p3_global_settings *pa,
      scope, has not yet been initialized. */
   if (dpal_arg_to_use == NULL)
     dpal_arg_to_use = create_dpal_arg_holder();
-   if(thal_arg_to_use == NULL) {
-      thal_arg_to_use = create_thal_arg_holder(&pa->p_args);
-   } else {
-      destroy_thal_arg_holder(thal_arg_to_use);
-      thal_arg_to_use = create_thal_arg_holder(&pa->p_args);
-   }
-   if(thal_oligo_arg_to_use == NULL) {
-      thal_oligo_arg_to_use = create_thal_arg_holder(&pa->o_args);
-   } else {
-      destroy_thal_arg_holder(thal_oligo_arg_to_use);
-      thal_oligo_arg_to_use = create_thal_arg_holder(&pa->o_args);
-   } 
+  if(thal_arg_to_use == NULL) {
+    thal_arg_to_use = create_thal_arg_holder(&pa->p_args);
+  } else {
+    destroy_thal_arg_holder(thal_arg_to_use);
+    thal_arg_to_use = create_thal_arg_holder(&pa->p_args);
+  }
+  if(thal_oligo_arg_to_use == NULL) {
+    thal_oligo_arg_to_use = create_thal_arg_holder(&pa->o_args);
+  } else {
+    destroy_thal_arg_holder(thal_oligo_arg_to_use);
+    thal_oligo_arg_to_use = create_thal_arg_holder(&pa->o_args);
+  }
   if (pa->primer_task == pick_primer_list) {
     make_complete_primer_lists(retval, pa, sa,
                                dpal_arg_to_use,thal_arg_to_use,thal_oligo_arg_to_use);
@@ -2411,6 +2411,7 @@ pick_sequencing_primer_list(p3retval *retval,
           length = n - start;
         }
         /* Pick all good in the given range */
+        retval->fwd.expl.sequencing_location++;
         pick_only_best_primer(start, length, &retval->fwd,
                               pa, sa, dpal_arg_to_use, thal_arg_to_use, retval);
       }
@@ -2427,13 +2428,16 @@ pick_sequencing_primer_list(p3retval *retval,
           length = n - start;
         }
         /* Pick all good in the given range */
+        retval->rev.expl.sequencing_location++;
         pick_only_best_primer(start, length, &retval->rev,
                               pa, sa, dpal_arg_to_use, thal_arg_to_use, retval);
       }
     }
 
   } /* End of Target Loop */
 
+  retval->fwd.expl.ok = retval->fwd.num_elem;
+  retval->rev.expl.ok = retval->rev.num_elem;
   /* Print an error if not all primers will be printed */
   if (retval->fwd.num_elem > pa->num_return 
                   || retval->rev.num_elem > pa->num_return) {
@@ -6055,6 +6059,7 @@ p3_oligo_explain_string(const oligo_stats *stat)
   size_t bsize = 10000;
   size_t r;
 
+  IF_SP_AND_CHECK("sequencing locations %d, ", stat->sequencing_location)
   SP_AND_CHECK("considered %d", stat->considered)
   IF_SP_AND_CHECK(", would not amplify any of the ORF %d", stat->no_orf)
   IF_SP_AND_CHECK(", too many Ns %d", stat->ns)
@@ -7126,6 +7131,14 @@ _pr_data_control(const p3_global_settings *pa,
       else if (!strstr_nocase(sa->trimmed_seq, s1))
         pr_append_new_chunk(nonfatal_err,
                             "Specified right primer not in Included Region");
+      else if (sa->left_input) {
+        if (strcmp(sa->left_input, s1) == 0)
+          pr_append_new_chunk(nonfatal_err,
+                            "Specified left and right primer are reverse complementary");
+        if (strcmp(sa->left_input, sa->right_input) == 0)
+          pr_append_new_chunk(nonfatal_err,
+                            "Specified left and right primer are identical");
+      }
     }
   }
 

src/libprimer3.h

@@ -717,6 +717,7 @@ const int *
 interval_array_t2_get_pair(const interval_array_t2 *array, int i);
 
 typedef struct oligo_stats {
+  int sequencing_location; /* Number of locations for sequencing primers    */
   int considered;          /* Total number of tested oligos of given type   */
   int ns;                  /* Number of oligos rejected because of Ns       */
   int target;              /* Overlapping targets.                          */

src/release_notes.txt

@@ -13,14 +13,25 @@ based on the 3' portion of the oligo that binds to the template.
 
 Minor fixes:
 1. Handling of N in PRIMER_MAX_POLY_X was fixed. Now 'GGGNNN' violates
-MAX_POLY_X=5. It is now based on the worst possible case (all 3 Ns could
-be Gs).
+   MAX_POLY_X=5. It is now based on the worst possible case (all 3 Ns
+   could be Gs).
 
 2. ntthal allows multiple queries using the -i command line option.
-ntthal runs in an interactive mode, each line is an oligo, where pairs of
-consecutive lines are pairs of oligos to test (only for dimers).
+   ntthal runs in an interactive mode, each line is an oligo, where pairs
+   of consecutive lines are pairs of oligos to test (only for dimers).
 
-3. GitHub default branch was renamed to main.
+3. GitHub default branch was renamed to main. Please run these commands
+   on old repos:
+     git branch -m master main
+     git fetch origin
+     git branch -u origin/main main
+     git remote set-head origin -a
+
+4. Provided left and right primer may be not identical or the reverse
+   complement of each other.
+
+5. More informative primer statistics with
+   PRIMER_TASK=pick_sequencing_primers.
 
 
 release 2.5.0 2019-08-17  ===============================================

test/primer_new_tasks_formatted_output

@@ -2290,3 +2290,11 @@ considered 0, ok 0
 libprimer3 release 2.2.3
 
 
+PRIMER PICKING RESULTS FOR rev_comp_primers
+
+INPUT PROBLEM: Specified left and right primer are reverse complementary
+
+PRIMER PICKING RESULTS FOR identical_primers
+
+INPUT PROBLEM: Specified left and right primer are identical
+

test/primer_new_tasks_input

@@ -282,3 +282,13 @@ PRIMER_FIRST_BASE_INDEX=1
 PRIMER_MIN_SIZE=16
 SEQUENCE_PRIMER_PAIR_OK_REGION_LIST=1,20,49,17
 =
+SEQUENCE_ID=rev_comp_primers
+PRIMER_TASK=check_primers
+SEQUENCE_PRIMER=CACCCCCATCAAACTCTTACTCA
+SEQUENCE_PRIMER_REVCOMP=TGAGTAAGAGTTTGATGGGGGTG
+=
+SEQUENCE_ID=identical_primers
+PRIMER_TASK=check_primers
+SEQUENCE_PRIMER=CACCCCCATCAAACTCTTACTCA
+SEQUENCE_PRIMER_REVCOMP=CACCCCCATCAAACTCTTACTCA
+=

test/primer_new_tasks_output

@@ -1861,3 +1861,15 @@ PRIMER_LEFT_14_SELF_ANY=11.00
 PRIMER_LEFT_14_SELF_END=11.00
 PRIMER_LEFT_14_END_STABILITY=6.6000
 =
+SEQUENCE_ID=rev_comp_primers
+PRIMER_TASK=check_primers
+SEQUENCE_PRIMER=CACCCCCATCAAACTCTTACTCA
+SEQUENCE_PRIMER_REVCOMP=TGAGTAAGAGTTTGATGGGGGTG
+PRIMER_ERROR=Specified left and right primer are reverse complementary
+=
+SEQUENCE_ID=identical_primers
+PRIMER_TASK=check_primers
+SEQUENCE_PRIMER=CACCCCCATCAAACTCTTACTCA
+SEQUENCE_PRIMER_REVCOMP=CACCCCCATCAAACTCTTACTCA
+PRIMER_ERROR=Specified left and right primer are identical
+=

test/primer_sec_struct_thal_output

@@ -5544,7 +5544,7 @@ PRIMER_PICK_LEFT_PRIMER=1
 SEQUENCE_ID=pick_sequencing_primers_th_2
 SEQUENCE_TEMPLATE=GANAATGCAGTAATGAGTTGGCAAATTATGAATCGCAATCAAATTTTTTCTATGACCTGATAGAAACGCCAACTCACTGTATGAGTCTGTTGTCAAAGTCTAACGTTGTCAAAGTCTATTCCAAAACGCAAAGCACGCTCCCGATC
 PRIMER_WARNING=No left primer found in range 1 - 40
-PRIMER_LEFT_EXPLAIN=considered 56, too many Ns 5, low tm 51, ok 0
+PRIMER_LEFT_EXPLAIN=sequencing locations 1, considered 56, too many Ns 5, low tm 51, ok 0
 PRIMER_LEFT_NUM_RETURNED=0
 PRIMER_RIGHT_NUM_RETURNED=0
 PRIMER_INTERNAL_NUM_RETURNED=0
@@ -5555,8 +5555,8 @@ PRIMER_PICK_RIGHT_PRIMER=1
 SEQUENCE_ID=pick_sequencing_primers_th_3
 SEQUENCE_TEMPLATE=GANAATGCAGTAATGAGTTGGCAAATTATGAATCGCAATCAAATTTTTTCTATGACCTGATAGAAACGCCAACTCACTGTATGAGTCTGTTGTCAAAGTCTAACGTTGTCAAAGTCTATTCCAAAACGCAAAGCACGCTCCCGATC
 PRIMER_WARNING=No left primer found in range 1 - 40
-PRIMER_LEFT_EXPLAIN=considered 56, too many Ns 5, low tm 51, ok 0
-PRIMER_RIGHT_EXPLAIN=considered 63, low tm 33, high tm 13, ok 1
+PRIMER_LEFT_EXPLAIN=sequencing locations 1, considered 56, too many Ns 5, low tm 51, ok 0
+PRIMER_RIGHT_EXPLAIN=sequencing locations 1, considered 63, low tm 33, high tm 13, ok 1
 PRIMER_PAIR_EXPLAIN=considered 0, ok 0
 PRIMER_LEFT_NUM_RETURNED=0
 PRIMER_RIGHT_NUM_RETURNED=1

test/test_left_to_right_of_right_input

@@ -1,5 +1,5 @@
-P3_COMMENT=The SEQUENCE_PRIMER and SEQUENCE_PRIMER_REVCOMP are reverse compliments of each other, and each is found at two positions in the template. The causes there to be 4 pairs to be considered, including one in which the left primer it to the right of the right primer.
-SEQUENCE_TEMPLATE=AGGCTAGGCGAGCTGAAAAATCCTACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGGCTAGGCGAGCTGAAAAATCCTAC
+P3_COMMENT=The SEQUENCE_PRIMER and SEQUENCE_PRIMER_REVCOMP are found at two positions in the template. The causes there to be 2 pairs to be considered, including one in which the left primer it to the right of the right primer.
+SEQUENCE_TEMPLATE=AGGCTAGGCGAGCTGAAAAATCCTACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGGCTAGGCGAGCTGTTTTTTCCTACAGGCTAGGCGAGCTGAAAAATCCTAC
 PRIMER_MAX_TEMPLATE_MISPRIMING=12
 PRIMER_PICK_ANYWAY=1
 PRIMER_PRODUCT_SIZE_RANGE=30-100
@@ -16,7 +16,7 @@ PRIMER_NUM_RETURN=1
 PRIMER_MAX_SIZE=27
 PRIMER_TM_FORMULA=1
 SEQUENCE_PRIMER=AGGCTAGGCGAGCTGAAAAATCCTAC
-SEQUENCE_PRIMER_REVCOMP=GTAGGATTTTTCAGCTCGCCTAGCCT
+SEQUENCE_PRIMER_REVCOMP=GTAGGAAAAAACAGCTCGCCTAGCCT
 PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT=1
 PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT=1
 PRIMER_THERMODYNAMIC_PARAMETERS_PATH=./primer3_config/

test/test_left_to_right_of_right_output

@@ -1,5 +1,5 @@
-P3_COMMENT=The SEQUENCE_PRIMER and SEQUENCE_PRIMER_REVCOMP are reverse compliments of each other, and each is found at two positions in the template. The causes there to be 4 pairs to be considered, including one in which the left primer it to the right of the right primer.
-SEQUENCE_TEMPLATE=AGGCTAGGCGAGCTGAAAAATCCTACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGGCTAGGCGAGCTGAAAAATCCTAC
+P3_COMMENT=The SEQUENCE_PRIMER and SEQUENCE_PRIMER_REVCOMP are found at two positions in the template. The causes there to be 2 pairs to be considered, including one in which the left primer it to the right of the right primer.
+SEQUENCE_TEMPLATE=AGGCTAGGCGAGCTGAAAAATCCTACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGGCTAGGCGAGCTGTTTTTTCCTACAGGCTAGGCGAGCTGAAAAATCCTAC
 PRIMER_MAX_TEMPLATE_MISPRIMING=12
 PRIMER_PICK_ANYWAY=1
 PRIMER_PRODUCT_SIZE_RANGE=30-100
@@ -16,7 +16,7 @@ PRIMER_NUM_RETURN=1
 PRIMER_MAX_SIZE=27
 PRIMER_TM_FORMULA=1
 SEQUENCE_PRIMER=AGGCTAGGCGAGCTGAAAAATCCTAC
-SEQUENCE_PRIMER_REVCOMP=GTAGGATTTTTCAGCTCGCCTAGCCT
+SEQUENCE_PRIMER_REVCOMP=GTAGGAAAAAACAGCTCGCCTAGCCT
 PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT=1
 PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT=1
 PRIMER_THERMODYNAMIC_PARAMETERS_PATH=./primer3_config/
@@ -25,23 +25,23 @@ PRIMER_MAX_HAIRPIN_TH=47
 PRIMER_MAX_SELF_ANY_TH=47
 PRIMER_MAX_SELF_END_TH=47
 PRIMER_PAIR_MAX_COMPL_ANY_TH=30
-PRIMER_WARNING=More than one position in template for input oligo AGGCTAGGCGAGCTGAAAAATCCTAC; More than one position in template for input oligo GTAGGATTTTTCAGCTCGCCTAGCCT
-PRIMER_PAIR_EXPLAIN=considered 4, unacceptable product size 4, high any compl 3, high end compl 3, left primer to right of right primer 1, ok 3
+PRIMER_WARNING=More than one position in template for input oligo AGGCTAGGCGAGCTGAAAAATCCTAC
+PRIMER_PAIR_EXPLAIN=considered 2, unacceptable product size 2, high any compl 1, high end compl 1, left primer to right of right primer 1, ok 1
 PRIMER_LEFT_NUM_RETURNED=1
 PRIMER_RIGHT_NUM_RETURNED=1
 PRIMER_INTERNAL_NUM_RETURNED=0
 PRIMER_PAIR_NUM_RETURNED=1
-PRIMER_PAIR_0_PENALTY=14.595379
+PRIMER_PAIR_0_PENALTY=14.210545
 PRIMER_LEFT_0_PENALTY=7.297689
-PRIMER_RIGHT_0_PENALTY=7.297689
+PRIMER_RIGHT_0_PENALTY=6.912856
 PRIMER_LEFT_0_PROBLEMS= Hairpin stability too high;
-PRIMER_RIGHT_0_PROBLEMS= Hairpin stability too high;
+PRIMER_RIGHT_0_PROBLEMS= Hairpin stability too high; Contains too-long poly nucleotide tract;
 PRIMER_LEFT_0_SEQUENCE=AGGCTAGGCGAGCTGAAAAATCCTAC
-PRIMER_RIGHT_0_SEQUENCE=GTAGGATTTTTCAGCTCGCCTAGCCT
-PRIMER_LEFT_0=175,26
+PRIMER_RIGHT_0_SEQUENCE=GTAGGAAAAAACAGCTCGCCTAGCCT
+PRIMER_LEFT_0=1,26
 PRIMER_RIGHT_0=200,26
 PRIMER_LEFT_0_TM=58.702
-PRIMER_RIGHT_0_TM=58.702
+PRIMER_RIGHT_0_TM=59.087
 PRIMER_LEFT_0_GC_PERCENT=50.000
 PRIMER_RIGHT_0_GC_PERCENT=50.000
 PRIMER_LEFT_0_SELF_ANY_TH=15.46
@@ -52,7 +52,7 @@ PRIMER_LEFT_0_HAIRPIN_TH=47.75
 PRIMER_RIGHT_0_HAIRPIN_TH=50.05
 PRIMER_LEFT_0_END_STABILITY=3.1800
 PRIMER_RIGHT_0_END_STABILITY=4.5800
-PRIMER_PAIR_0_COMPL_ANY_TH=58.65
-PRIMER_PAIR_0_COMPL_END_TH=58.65
-PRIMER_PAIR_0_PRODUCT_SIZE=26
+PRIMER_PAIR_0_COMPL_ANY_TH=47.59
+PRIMER_PAIR_0_COMPL_END_TH=47.59
+PRIMER_PAIR_0_PRODUCT_SIZE=200
 =

test/th-w-other-tasks_output

@@ -10246,7 +10246,7 @@ PRIMER_PICK_LEFT_PRIMER=1
 SEQUENCE_ID=pick_sequencing_primers_th_2
 SEQUENCE_TEMPLATE=GANAATGCAGTAATGAGTTGGCAAATTATGAATCGCAATCAAATTTTTTCTATGACCTGATAGAAACGCCAACTCACTGTATGAGTCTGTTGTCAAAGTCTAACGTTGTCAAAGTCTATTCCAAAACGCAAAGCACGCTCCCGATC
 PRIMER_WARNING=No left primer found in range 1 - 40
-PRIMER_LEFT_EXPLAIN=considered 56, too many Ns 5, low tm 51, ok 0
+PRIMER_LEFT_EXPLAIN=sequencing locations 1, considered 56, too many Ns 5, low tm 51, ok 0
 PRIMER_LEFT_NUM_RETURNED=0
 PRIMER_RIGHT_NUM_RETURNED=0
 PRIMER_INTERNAL_NUM_RETURNED=0
@@ -10257,8 +10257,8 @@ PRIMER_PICK_RIGHT_PRIMER=1
 SEQUENCE_ID=pick_sequencing_primers_th_3
 SEQUENCE_TEMPLATE=GANAATGCAGTAATGAGTTGGCAAATTATGAATCGCAATCAAATTTTTTCTATGACCTGATAGAAACGCCAACTCACTGTATGAGTCTGTTGTCAAAGTCTAACGTTGTCAAAGTCTATTCCAAAACGCAAAGCACGCTCCCGATC
 PRIMER_WARNING=No left primer found in range 1 - 40
-PRIMER_LEFT_EXPLAIN=considered 56, too many Ns 5, low tm 51, ok 0
-PRIMER_RIGHT_EXPLAIN=considered 63, low tm 33, high tm 13, ok 1
+PRIMER_LEFT_EXPLAIN=sequencing locations 1, considered 56, too many Ns 5, low tm 51, ok 0
+PRIMER_RIGHT_EXPLAIN=sequencing locations 1, considered 63, low tm 33, high tm 13, ok 1
 PRIMER_PAIR_EXPLAIN=considered 0, ok 0
 PRIMER_LEFT_NUM_RETURNED=0
 PRIMER_RIGHT_NUM_RETURNED=1