Recommended Skew Ratio

I have expanded the gecko library and submitted for NGS to check for the representation.

I currently have a skew ratio of 10.9 and 10.7 for libraries A and B, respectively.

Would you recommend re-expanding the libraries given the skew ratio is >10 or do you think it is close enough to proceed?

TitleLibrary ALibrary B
NGS Reads302148365736988
Percent of Reads with sgRNA86%87%
Percent of reads with perfect sgRNA match79%79%
Number of unique sgRNA6395056869
Percent of detected sgRNAs100%100%
Percent of undetected sgRNAs0.01%0.07%
Skew ratio10.9200910.70123

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Fuchsia Kelpiealmost 3 years ago

This really depends on your screen. Is this the mouse or human library? If this is the mouse GeCKO library, then this skew is expected. If this is the human, then you may be able to reduce the skew with a reamplification.

As for whether you should reamplify your library: if you are performing a very noisy, difficult, or costly screen, then I would recommend reamplifying your library. If not, then I would recommend screening at 600 cells/sgRNA coverage instead of 500, and proceed.

Navy Nagaalmost 3 years ago

Excellent; thank you for your response!

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