Dear all,
This might be a stupid question, but it's better asking than wasting a whole lane of HiSeq (and my PI hating me for the rest of my degree ).
I have different regions I'd like to amplify using PCR with primers that already have ligated Illumina PE adaptors to them, as in the following scheme:
Forward primer: [P1adaptor][forward.primer.sequence]
Reverse primer: [P2adaptor][reverse.primer.sequence]
So the PCR product would look something like:
[P1adaptor][forward.primer.sequence]XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX[reverse.primer.sequence][P2adaptor]
My question is, would it matter if I swap the order of adaptors so that instead of forward primer having PE1 adaptor it has PE2??
Like this:
Reverse primer: [P1adaptor][reverse.primer.sequence]
Forward primer: [P2adaptor][forward.primer.sequence]
I think it shouldn't alter anything, just basically the order in which the primer sequence is reported (reverse primer now being reported in the 1st PE reads rather than the 2nd reads).
Thanks!!!
This might be a stupid question, but it's better asking than wasting a whole lane of HiSeq (and my PI hating me for the rest of my degree ).
I have different regions I'd like to amplify using PCR with primers that already have ligated Illumina PE adaptors to them, as in the following scheme:
Forward primer: [P1adaptor][forward.primer.sequence]
Reverse primer: [P2adaptor][reverse.primer.sequence]
So the PCR product would look something like:
[P1adaptor][forward.primer.sequence]XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX[reverse.primer.sequence][P2adaptor]
My question is, would it matter if I swap the order of adaptors so that instead of forward primer having PE1 adaptor it has PE2??
Like this:
Reverse primer: [P1adaptor][reverse.primer.sequence]
Forward primer: [P2adaptor][forward.primer.sequence]
I think it shouldn't alter anything, just basically the order in which the primer sequence is reported (reverse primer now being reported in the 1st PE reads rather than the 2nd reads).
Thanks!!!
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