Melting Temperature Problems When Calling Working Python Program Through BASH/(***)

[AnushaC]

Hi,

I have a working python program that also calculates melting temperature of the primer that I am calling through bash.

I am getting errors like while I am running bash script but not while I am running python script

fmt=str(mt.Tm_Wallace(forpri))
File "/usr/local/lib/python2.7/site-packages/Bio/SeqUtils/MeltingTemp.py", line 630, in Tm_Wallace
'allowed when strict=True')
ValueError: ambiguous bases B, D, H, K, M, N, R, V, Y not allowed when strict=True

this is my part of the code that runs this in python
import sys
from Bio.SeqUtils import MeltingTemp as mt
from Bio.Seq import Seq

fmt=str(mt.Tm_Wallace(forpri))
rmt=str(mt.Tm_Wallace(revpri))

which is essentially forward and reverse melting temperature

I am also getting confused with the results of melting temperature because most of the online tools giving very different results than what bio python would do.
what is the right way to calculate melting temperature through program

[GenoMax]

Looks like you have IUPAC ambiguity codes in your sequence which the python script does not like.

[MarkusP]

1. If you have ambiguity codes in your sequence (B, D, etc.) you have to tell Biopython's Tm methods to accept these with "strict=False", e.g.

fmt=str(mt.Tm_Wallace(forpri, strict=False))

When you do so, some intermediate values will be used for ambiguous bases.

2. Tm_Wallace gives the melting temperature according to the "Wallace rule", which represents a very simple formula:
Tm = 4°C * (G+C) + 2°C * (A+T))
For a (much) more precise melting temperature calculation you have to use Tm_NN, which calculates the melting temperature with nearest neighbor thermodynamics. This is what many other programs (Primer3, Melting, etc) use. Unfortunately, there are different thermodynamics tables and parameter which are used by different programs. When you tell me, which program/web tool you are using, I can try to give you the correct parameters for Tm_NN.
In the meantime, you can simply use the default values with:
mt.Tm_NN(forpri)