I am planning a MiSeq experiment in which I will be doing a PCR mutagenesis of a sequence, with the intention of obtaining just a single mutation in the ~100bp sequence. To do this, I will test 9 different PCR conditions + negative control. The entire sequenced region would be 130-140bp.
I am having a bit of a hard time thinking about indexing my samples. I am not entirely sure that doing a pair-end run would be very useful. From what I've seen, the majority of primer kits only provide 8 forward primers, and I think I'd need a unique forward and reverse primer in order for the alignment to work. Or, I can do single-sided, which I think would be good enough in my case, but how does it work from a sample preparation point of view - do I just use 10 P7 primers and a single P5 primer for all my samples?
I am fairly new to NGS and don't know how any of these choices affects data analysis in the later stage.
I am having a bit of a hard time thinking about indexing my samples. I am not entirely sure that doing a pair-end run would be very useful. From what I've seen, the majority of primer kits only provide 8 forward primers, and I think I'd need a unique forward and reverse primer in order for the alignment to work. Or, I can do single-sided, which I think would be good enough in my case, but how does it work from a sample preparation point of view - do I just use 10 P7 primers and a single P5 primer for all my samples?
I am fairly new to NGS and don't know how any of these choices affects data analysis in the later stage.
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