I am using direct sequencing (with no cloning, not possible) and not sure if it is necessary that I sequence with both the forward and the reverse for each sample. In theory, if I just used the forward, this should be enough? I could then align it to my reference genome, correct and extract final sequence.
I would suggest to always sequence both directions, as you are going to quantify the methylation (I assume that's the point, to determine the percentage of methylation?)
As you probably know, sequencing peak heights are dependant on the base before (i.e. a T after a G is usually lower than that G, while with 2 T's after eachother, the second T is usually higher than the first T. Because of this, the quantification gets a little bit more reliable if you are able to sequence both directions.
Hi Alice,
I agree with Arnoud that if you MUST (and really MUST) quantify from a chromatogram you should at least try to sequence on both ends, however, just out of curiosity: for what reason is cloning impossible? in line of principle, if you can use two primers to sequence on both ends, you could also use them to PCR amplify and clone.
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