Gecko mouse library validation

I have a colleague who has just amplified the mouse Gecko library (2 lentivirus system) as per the Joung and Addgene protocols and sequenced on the Miseq to validate coverage. The results have come back as less than ideal.

Skew ratio: 13.5%

% matching guides: 64.2%

% undetected guides: 1.5%

What is the most likely reason for this? Does this imply that there is a problem with the original library amplification in bacteria or could it just be a PCR issue? Either way what are the implications for proceeding with a library which is less than perfect like this?


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Fuchsia Kelpiealmost 4 years ago

For the GeCKO mouse library, we have seen that the skew tends to be higher and the percentage of matching guides lower than ideal, because the library itself is less than ideal. It does not mean that you did anything wrong during the library amplification. In the past, since the numbers are still quite close to the ideal numbers, people have proceeded with their libraries. If the screen does appear to be noisy at 500x coverage, you may want to increase your coverage.

Yellow Valkyrieover 3 years ago

Thanks for getting back to me about this. He will be proceeding with the screen using this library and using 1000X coverage due to a low selection pressure so hopefully this will be good enough.

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