You can’t explicitly calculate Mutation rate as it’s impossible to observe all mutations. You can compare multiple sequence and extrapolate the substitution rate, but this is always lower than the real mutation rate.

(If this doesn’t make intuitive sense, consider the example where the transitions A -> T -> A happen between sampling times. You would never know this mutation (or rather, 2 mutations) had occurred because it spontaneously reverted, so your number would be off by 2 immediately).

You may also be interested in calculating the dN/dS ratio, which is a surrogate for measuring evolutionary pressure on a sequence.

We will need more information about what data you have to be able to help you much further though.

To calculate mutation rate, you could measure the differences between samples of different generations. Generally people find homologous sequences between species or different phenotypes over which to measure mutation rate. Or use the divergence of related species. You could compare between species separated by whatever you want to find the difference between (e.g., geographic barrier between two species).