6 Primer evaluation (in silico)

Primers can be evaluated on any sequence alignment using the function evaluate_primer(). Unlike other in silico primer evaluation software (e.g. ecoPCR), PrimerMiner considers:

• Position of mismatches (give higher penalties for 3' mismatches, a scoring table can be provided in mm_position or the default table can be used with mm_position = "Position_v1" (recommended). See folder primer_scoring for examples).
• Type of mismatch (optional, provide scoring table as csv in 'mm_type', or use the default tables with mm_type = "Type_v1" (recommended))
• If two mismatches that are directly adjacent each other, their penalties scores are doubled (considering that 2 adjacent mismatches will lead to higher instability, adjacent = 2. Set adjacent = 1 to deactivate this option!). Penalty will increase up to 4 times with 3 adjacent mismatches!
• N and gaps (-) in the sequence will be considered as missing data by default and no primer penalty score will be calculated. To give penalty scores for gaps & N instead, set gaps_NA = F and N_NA = F (this will take scores from primer_scoring, as no information over mismatch type is available).

For mismatches / gaps of each sequence to the tested primer a penalty score is given, based on the specified parameters above. The scores for each position as well as the cumulative penalty score for each evaluated sequence are saved in a csv file (which are both already integrated in PrimerMiner and used by default). These csv files can however also be customised and be imported into R (see Sample_Data/primer_scoring for examples). All primers above a certain threshold (e.g. penalty of above 100) can be considered "not working" or the individual penalty scores can be used for a more detailed statistical analysis. Notice that primer evaluation is always done for a single primer, not a primer pair. To evaluate how well a primer pair will be working, evaluate the forward and reverse primer individually and add up the penalty scores form both primers.

How to use evaluate_primer()

Primer evaluation works on the basis of single primers, not primer pairs. This makes sense, as evaluating primer pairs can hide problems present in just one and makes it difficult to figure out why a primer set is not working well. However, primer pairs can be easily evaluated with PrimerMiner by combining the penalty scores from both primers!

PrimerMiner can evaluate any sequence alignment (wobble bases are supported) and requires information about the primer sequence, binding position and penalty scoring matrix (see folder primer_scoring, Position_v1.csv). Additionally penalty scores for different mismatch types can be adjusted in the Type_v1.csv file (see Stadhouders et al. 2010), and penalties can be increased for adjacent = 2 mismatches. Note that penalties for mismatch position and mismatch scores are fully adjustable (feel free to use your own tables, but make sure to include them together with the used settings in the supplements of your publication)!

An example command could look like this:

evaluate_primer("07_Plecoptera.fasta", "GCYCCHGAYATRGCHTTYCC", 243, 262, save="save_evaluation_table.csv", mm_position ="Position_v1", adjacent=2, mm_type="Type_v1")

Evaluation of the different parameters / penalties is processed in the following order:

1. Scoring of mismatches (mm_position)

2. Adjustment of mismatch types (mm_type)

3. Increasing of penalties for adjacent mismatches

4. Reduction of penalty if wobble bases are present in the target sequence, which only partially match the primer.

Evaluate primer pairs

After evaluating single primers with evaluate_primer() primer pairs can tested using both output tables and primer_threshold(table1, table2, threshold=100). Penalties from forward and reverse primers are summed and sequences above a given threshold are considered "failed". If NA values are present for one sequence, it is considered as missing data. For more information see ?primer_threshold

Primer versions

With the command primerversions("PRIMER_SEQUENCE") one can generate all possible primer versions contained in a degenerated primer sequence for further evaluation of e.g. Tm.