We were recently doing some small scale Gibson-based NNK site saturation mutagenesis PCR reactions. In this scheme, we are independently transforming each position separately, so the number of transformants (ie. colonies) on a given plate should be directly related to the likelihood that all of the desired variants that we want to see are there at least once.

In fact, there are three parameters that factor into how good the variant coverage is at a given position. This is going to be 1) nucleotide biases in the creation of the NNK degenerate region of the primer, 2) the number of transformants, and 3) the fraction of the number of total transformants are actually variants, rather than undesired molecules such as carryover of the WT plasmid used for the template.

For any given experiment, you’re not going to know what the nucleotide bias is like until you actually Illumina sequence your library…. but at that point, you’ll already know the variant coverage of your library, so no need to estimate it anymore. On the other hand, if you know the nucleotide biases you observed for similar libraries, then you can do this estimation far before you get around to Illumina sequencing. Based on previous libraries, I have a pretty good idea of what the biases from machine-mixed NNK primers from IDT are like. For simplicity sake, I’m using 40% G, 20% C, 20% A, and 20%T as a rough estimate for the nucleotide bias I saw in the most biased NNK libraries.

The other two parameters are going to be very much experiment specific, and can be determined shortly after generating the library. The number of transformants can be determined by counting colonies from the transformation. And the amount of template contamination can be roughly determined by performing Sanger sequencing on a handful of colonies from those plates. Thus, I chose a few reasonable values for each: colony counts ranging from the very small (10 and 20) to quite large (400 and 1000), and template contamination percentages from almost impossibly low (0%) and much more likely (10 or 20%) all the way to possibly prohibitively high (50% and 75%). I then simulated the entire process, bootstrapped 20 times to get a sense of the average output, and made a plot showing what types of variant coverages you get depending on the combinations of those observed parameters. This is what the plot looks like:

So there you go. In a reasonable condition where you have, let’s say 10 or 20% template contamination, then you’d really be hoping to see at least 200 colonies, and hopefully around 400, where you can then really pat yourself on the back. If things went awry with the DPNI step, for example, and you were getting between a quarter to a half of colonies being template, then you’d minimally want 400 or so colonies and don’t feel too safe until you got a fair bit more than that. Though that’s only to make sure you at least have one copy of every variant at that position. If your library is half template, then chances are you’ll be running into a bunch of other problems down the line.