The penalty calculation is partly written in italic, which means that this part is substituted for another formula in case thermodynamic model is used. According to manual the formula for primer pair penalty goes like this:
\<CITATION FROM MANUAL
These are allways added (+) to the penalty:
(if the thermodynamic approach is used then the part in italic
is substituted with text below this calculation):
PRIMER_PAIR_WT_DIFF_TM*
\<difference in Tm between the left and the right primer>
+ PRIMER_PAIR_WT_COMPL_ANY * PRIMER_PAIR_4_COMPL_ANY
+ PRIMER_PAIR_WT_COMPL_END * PRIMER_PAIR_4_COMPL_END
+ PRIMER_PAIR_WT_LIBRARY_MISPRIMING * PRIMER_PAIR_4_LIBRARY_MISPRIMING
+ PRIMER_PAIR_WT_TEMPLATE_MISPRIMING * PRIMER_PAIR_4_TEMPLATE_MISPRIMING
If the thermodynamic approach is used then the part of italic in
the above calculation is replaced by this:
.............
If ((min(PRIMER_LEFT_4_TM,PRIMER_RIGHT_4_TM) - 5) ≤ PRIMER_PAIR_4_TEMPLATE_MISPRIMING_TH) then is added (+):
+ PRIMER_PAIR_WT_TEMPLATE_MISPRIMING_TH
(PRIMER_PAIR_4_TEMPLATE_MISPRIMING_TH - (min(PRIMER_LEFT_4_TM,PRIMER_RIGHT_4_TM) - 5 - 1))
else if ((min(PRIMER_LEFT_4_TM,PRIMER_RIGHT_4_TM) - 5) > PRIMER_PAIR_4_TEMPLATE_MISPRIMING_TH) then is added (+):
+ PRIMER_PAIR_WT_TEMPLATE_MISPRIMING_TH
(1/(min(PRIMER_LEFT_4_TM,PRIMER_RIGHT_4_TM) - 5 + 1 - PRIMER_PAIR_4_TEMPLATE_MISPRIMING_TH));
END OF CITATION>
The term for primer pair mispriming (PRIMER_PAIR_4_TEMPLATE_MISPRIMING) is not written in italic so if thermodynamic model is used, the penalty is calculateded twice - for classic model and for thermodynamic. I was not able to find any reasonable explanation for this that's why i opened the issue. I believe this is just a mistake in manual but I would appreciate if you check.
Best regards, Tarasov Kirill
The penalty calculation is partly written in italic, which means that this part is substituted for another formula in case thermodynamic model is used. According to manual the formula for primer pair penalty goes like this:
\<CITATION FROM MANUAL
These are allways added (+) to the penalty: (if the thermodynamic approach is used then the part in italic is substituted with text below this calculation):
PRIMER_PAIR_WT_DIFF_TM* \<difference in Tm between the left and the right primer> + PRIMER_PAIR_WT_COMPL_ANY * PRIMER_PAIR_4_COMPL_ANY + PRIMER_PAIR_WT_COMPL_END * PRIMER_PAIR_4_COMPL_END + PRIMER_PAIR_WT_LIBRARY_MISPRIMING * PRIMER_PAIR_4_LIBRARY_MISPRIMING + PRIMER_PAIR_WT_TEMPLATE_MISPRIMING * PRIMER_PAIR_4_TEMPLATE_MISPRIMING
If the thermodynamic approach is used then the part of italic in the above calculation is replaced by this:
............. If ((min(PRIMER_LEFT_4_TM,PRIMER_RIGHT_4_TM) - 5) ≤ PRIMER_PAIR_4_TEMPLATE_MISPRIMING_TH) then is added (+): + PRIMER_PAIR_WT_TEMPLATE_MISPRIMING_TH (PRIMER_PAIR_4_TEMPLATE_MISPRIMING_TH - (min(PRIMER_LEFT_4_TM,PRIMER_RIGHT_4_TM) - 5 - 1)) else if ((min(PRIMER_LEFT_4_TM,PRIMER_RIGHT_4_TM) - 5) > PRIMER_PAIR_4_TEMPLATE_MISPRIMING_TH) then is added (+): + PRIMER_PAIR_WT_TEMPLATE_MISPRIMING_TH (1/(min(PRIMER_LEFT_4_TM,PRIMER_RIGHT_4_TM) - 5 + 1 - PRIMER_PAIR_4_TEMPLATE_MISPRIMING_TH));
END OF CITATION>
The term for primer pair mispriming (PRIMER_PAIR_4_TEMPLATE_MISPRIMING) is not written in italic so if thermodynamic model is used, the penalty is calculateded twice - for classic model and for thermodynamic. I was not able to find any reasonable explanation for this that's why i opened the issue. I believe this is just a mistake in manual but I would appreciate if you check. Best regards, Tarasov Kirill