Program/script to count how many sequences have mismatches on each position of my primers, and also count how many mismatches each sequence has with the corresponding primers

Question

[gastonviarengo]

Hello everyone. I'm working with primer analysis and design, and so far I've performing it in Excel but: a) I don't like Excel, b) I do it manually, c) I don't like Excel, d) it gets tedious when working with thousands of sequences and tens of primers. My analysis consists of establishing how many sequences have mismatches on each position of my primers, and also count how many mismatches each sequence has with the corresponding primers. This last analysis I've been able to (partially) solve it by using ncbi-blast+ tool. However, I will find very helpful a program/script where you input your sequences and primers fasta files and retrieve the number of mismatches at every primer position. Does anyone know something that could help me achieve all this? If it also has an output tab-separated file as the one attached that would be perfect for easy visualization of results. Suggestion/orientations are also grateful! I just want to know if there's something to help me out, otherwise I'll try writing a script to automatize my analysis. Thank you!

Answer 1

[Kevin Blighe]

Kudos to you on your question title.

To help, you could follow a Standard Operating Procedure (SOP) that I wrote a few years ago with my colleague, Karen: Designing a single set of primers and probe for a genomic region of interest

Basically, there are two main programs that you can use:

• Primer3
• Primer Express

There are other tools mentioned in the SOP.

By following the SOP, I do not ever recall designing primers that failed. Some regions of the genome will always be difficult to target, though, due to sequence similarity, GC content, etc.

Kevin

Answer 2

[Mensur Dlakic]

There are some answers to a similar question in this thread. You would probably need to adapt the solutions that are shown there, but it might save you some time.

I suggest shortening your question title. It has two questions in that can be condensed and still remain informative.

[Kevin Blighe]

Thanks Mensur