Hi All,
I need an opinion about an experiment I am planning to do.
I am planning to do a genome wide CRISPR/Cas9 screening to identify genes responsible for drug resistance. I have purchased the Brunello lentiviral library and am planning on transducing cells with the virus particles. My question has to do with the screening part: according to the library preparation protocol from Broad Institute, the PCR is done with 96 different P7 primers in a 96-well format (protocol in link: https://media.addgene.org/cms/filer_public/61/16/611619f4-0926-4a07-b5c7-e286a8ecf7f5/broadgpp-sequencing-protocol.pdf) . I have come across other protocols, published both before and after my reference paper, which have used 8-10 P5 and 8-10 P7 primers (instead of the 96 P7 primers used here), as is outlined here, for example : https://star-protocols.cell.com/protocols/524.
Using 96 primers will increase the experiment cost, as these are 91-mer primers. Primers itself will cost $1700, whereas using 10 primers will cost $170.
Does anybody have any inputs as to what is to be kept in mind when I am deciding which option I should go for as far as coverage is concerned?
Thanks in advance!
Well, the primer cost is low compared to all the other associated costs such as salaries and time. I’d use the protocol that seems most likely to get you high quality results.
When doing the screens, each sample/time point/replicate has their own P7 primer. The Broad orders theirs in 96-well plates as they do LOTS of high throughput experiments so that they can multiplex and sequence them all in a large flow cell. In a typical screening experiment, you have at least a duplicate (triplicate is better) for each point you're interested in and each needs a unique P7 primer.
Therefore, if you think you will have 96 different samples to quantifiy, you will need 96 different P7 primers...if you think you will have 10 samples, then you will need 10 P7 primers. Each sample gets the same set of P5 primers which are staggered to allow for easier cluster identification on the flow cell.
One major and sort of important caveat is that many sequencing platforms do not like to mix amplicon sequencing with other sequencing experiments and you very likely will have to either get your own flow cell and/or lane. If many amplicon experiments are being sequenced simultaneously from different labs, you need to chose P7 indices that are unique; therefore, with a large of set of 96, you can increase the odds that you will have the choice of unique indices. However, this is usually not the case, but something to consider and will be determined by whichever facility does the sequencing.
Ultimately, the number of unique P7 primers is equal to the number of samples you wish to sequence.
Hope this helps.
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