Design PCR Primers - Outputs

The results are in the form of a tab-delimited text file listing the top 10 primer sets that meet all of the input constraints specified by the user. The most critical columns are the primers (forward_primers and reverse_primers) and their predicted cross-amplifications of non-target groups (mismatches_set). The titles and contents of the output columns are as follows:

1. identifier:

   Matches the target group set by the user.

2. start_forward, start_reverse:

   Approximate starting position of the forward/reverse primer in a consensus sequence representing the target group. The consensus sequence position does not include gap positions.

3. product_size:

   Approximate number of base pairs expected in the PCR product as determined by the distance between the start of the forward and reverse primers.

4. start_aligned_forward, start_aligned_reverse:

   Approximate positions in the alignment where the forward/reverse primer sequences are located, which includes gap positions.

5. permutations_forward, permutations_reverse:

   Number of forward or reverse primer permutations required to reach the desired coverage of the target group.

6. score_forward, score_reverse, score_set:

   Measures of the specificity of the forward, reverse, or set of primers to the target group. A score of zero is the highest specificity, while a more negative score is less specific. The forward and reverse primer's scores are calculated as the negative sum of all predicted non-target amplification efficiencies. The set score is calculated as the negative sum of geometric means of forward and reverse primer's amplification efficiency for each group.

7. forward_primer.x, reverse_primer.x:

   The sequence of each forward and reverse primer, where “x” ranges from 1 to the total number of permutations. “NA” indicates that the additional primer permutation was not necessary in order to achieve the desired coverage of the target group.

8. forward_efficiency.x, reverse_efficiency.x:

   The respective efficiency of each forward and reverse primer, which is required to be at least 80% at the specified annealing temperature.

9. forward_coverage.x, reverse_coverage.x:

   Fraction of the target group that perfectly matches each respective forward and reverse primer permutation.

10. mismatches_forward, mismatches_reverse, mismatches_set:

    Lists the predicted specificity of the forward and reverse primers, and any potential cross-amplifications are shown in the column mismatches_set. Each column lists any predicted efficiencies greater than 0.1% with non-target groups, as well as the aligned primer/template binding. Mismatches_set lists the product of forward and reverse efficiencies matching the same non-target group, but only if non-target groups are present in both the mismatches_forward and mismatches_reverse lists.

11. forward_MM.x, reverse_MM.x:

    If the option to induce a mismatch (MM) at the 6^th position from the primer's 3'-end was selected, then these columns provide the type of primer/template mismatch to the target group.

12. forward_efficiency_MM.x, reverse_efficiency_MM.x:

    Again, if the option to induce a mismatch was selected then these columns list the mismatch amplification efficiency. Note that this is the initial efficiency with which the target will amplify in the first PCR cycles, but the generated amplicons will replicate with perfect match efficiency in subsequent cycles (i.e., forward_efficiency.x).