Difference between nagative/positive screening in NGS terms

Hi everyone!

I'm trying to perform a drug screening and i know how to do it for positive selection (NGS before and after drug with a 500X coverage according to the last paper from 2017). But i'm also interested in the negative selection part of the screening and here is where i have doubts:

-I need to perform NGS in more time points (ex. before-during-after treatment) or just before-after is ok?
-The NGS parameters are the same?coverage, amount of gDNA to analyse?


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Fuchsia Kelpiealmost 5 years ago

To answer your questions: for negative selection screens, collecting 2 time points is sufficient, but if this is your first time setting up a particular screen it doesn't hurt to take multiple time points, especially if you are working with dividing cells. To collect multiple time points, you can freeze the cells as pellets at -80C and decide whether to harvest genomic DNA at other time points after you analyze the first two timepoints. Coverage for negative selection screens is also >500x, but if you can, I would recommend starting with 1,000x.

Purple Fenriralmost 5 years ago

Thnaks for the answer. I was planning to do a 500X coverage but i'll try to get more coverage (but the number of PCRs to perform is so high!).

This brings me to another question i have. I was going to follow the 2014 paper where they use Herculase II polimerase for the PCRs using 10 micrograms of gDNA in 100 microliters of PCR.

Following your paper:

-For a 1000x coverage i'll need to amplify 440 microg of gDNA i'll need 176 PCRs (2.5 microg in 50 microL) or 88 PCRs (5 microg in 100 microL)

Using the 2014 protocol and Herculase II:

-For a 1000x coverage i'll need to perform 44 PCRs (10 microg in 100 microL)

I know i have to test the performance of my gDNA with the polimerase but in your experience, which option is

Fuchsia Kelpiealmost 5 years ago

I believe the NEBNext HF MM is better for diverse sequences, so I would recommend going for the NEBNext MM as described in the 2017 Nat Protocol paper. I have noticed that using 100uL reactions with NEBNext MM is less efficient and produces primer dimers because the glycerol in the NEBNext MM settles over time, so I would recommend going with 50uL reactions.

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