I am screening a library of 10-15k variants through cell-based assays. After the selection, I perform the NGS sample prep using NEB ultra II kits. When I try to validate any potential hits from the data, they all turn out to be false positives.
One of the things that I think might be going wrong is that I have a lower-fold coverage of DNA copies in my input DNA compared to the total library diversity. The recommended fold-coverage is 500-1000x i.e. for a library of 10k, I will need to process DNA from 10^3 x 500 = 500k cells, which comes to about 10-15 µg of DNA. The NEB kits have a limit of 1 µg per reaction. It becomes unaffordable when I have multiple samples and replicates. So I am looking for a way to scale up these reactions with alternative kits or methods. Any ideas?
Thanks,
One of the things that I think might be going wrong is that I have a lower-fold coverage of DNA copies in my input DNA compared to the total library diversity. The recommended fold-coverage is 500-1000x i.e. for a library of 10k, I will need to process DNA from 10^3 x 500 = 500k cells, which comes to about 10-15 µg of DNA. The NEB kits have a limit of 1 µg per reaction. It becomes unaffordable when I have multiple samples and replicates. So I am looking for a way to scale up these reactions with alternative kits or methods. Any ideas?
Thanks,
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