perfectly matched guides in a custom library

you mentioned that an ideal sgRNA library should have >70% perfectly matching guides, <0.5% undetected guides, and a skew ratio of less than 10.

How did you reach these numbers?

I made 4 costume libraries . Undetected guides in all are less than 5 (0.2). The skew ratio is less than 10 (4.3-5.6). But the percentage of matched guides is 68%, 68%, 72% and 82 % for library 1 to 4 respectively.

Do you think I should remake the library 1 and 2 ?

I have no idea what to do at this step. I highly appreciate your opinion .


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Fuchsia Kelpiealmost 2 years ago

The recommended numbers were based on our past experience with generating several sgRNA libraries (ie GeCKO and SAM) - please see my earlier response as to why we don't expect to see 100% perfectly matching guides. It looks like your custom libraries are close enough to the recommended guidelines (68% vs 70%) that it will be fine to proceed.

Mint Baloralmost 2 years ago

Many many thanks for your reply!

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