Hi all. Apologies if my question is basic or my terminology isn't 100% right as I'm fairly new to the world of NGS.
Basically, I will have a library of, let's say 100,000 clones, each differing by a 100bp sequence. This 100bp section of the library will be randomly mutagenized to have a single random mutation (obviously this won't be possible and many clones will remain WT, and many will have more than one mutation).
What I'll then need to do is run this on MiSeq, and analyse it in a way that I know both the coverage of the new mutated library, as well as what percentage of the new library is mutated to the extent that I want (ie. a single mutation). I am not sure what tool I can use to align a fastq to 100,000 references, not to mention any analysis down the line.
Sounds like a classical mutation calling workflow, just use a single reference with your insert and perform mutation calling for each sample. You won't know the reference until you sequenced them anyway, right?
I will actually know the reference - the 100,000 inserts are cloned oligos with specific sequences.
Not sure I understand the other part though - as far as I understand, mutation calling works for a single reference but in my case, there will be a 100,000 short reference sequences. The whole sequencing will be done with just one sample - the full library PCRed with a single set of MiSeq adapters.