PRC primers for NGS after screening

Im working with the Gecko v2 (1-vector system) library and followed the Joung et al. (2017) Nature Protocols paper very closely.

Now i'm in the screening section and organizing next PCR for NGS step.

Then, i have questions.

1. If i am followed the protocol, i'll need 10 different forward primers. Why cannot i use single forward primer??

I'm hesitating to use all of them because it's too expensive to prepare 10 primers ( w/ 100 bases ).

2. Instead of 1 step PCR, can i try with 2 step PCR (done in 2014, science) because it's cheaper than using w/ 10 primers.


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Fuchsia Kelpieover 4 years ago

To answer your questions:

1. Having 10 different forward primers staggers your target sequence such that you will not have sequencing cycles with all of the same base at the beginning of the read, which will crash your run. Usually people will spike in PhiX control to diversify the library, but this reduces the number of useful reads that you get per run, so it's better to stagger with PCR primers.

2. With 2 step PCR, you will be using twice as much PCR mastermix, and it's a lot of work to set up 2 step PCR for the number of reactions you need to maintain screening coverage.

For a genome-scale screen, over all having 10 different long forward primers will actually save you more money and effort than doing 2 step PCR or a single long forward primer.

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