Entering edit mode
6.1 years ago
L. A. Liggett
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120
I am building a probeset of oligos for targeted gDNA capture that will be used for sequencing. The idea is fundamentally similar to primer design, where I am building a panel consisting of targeting arms that bind specific loci in order to capture specified genomic regions.
I am using primer3-py for Tm, dimerization, and secondary structures, but the trouble is that I don't know how to measure the off-target binding potential of an oligo. Is there a good way to do this? Ideally the solution can be implemented programmatically.
Is there reason why you do not use any of the established tools like NimbleDesign from Roche?
The problem with such software is that they are often black boxes. You have no idea what they do. In my view, using them is not very scientific.
What do you mean by off-target potential ? The first thing to do is look for potential matches elsewhere in the target genome. This is a sequence alignment task easily automated.
Right, and I am looking for the best way of doing this that will output useful information like Gibb's free energy of binding association which can be helpful to understand the specificity of a particular probe.