I´m not sure I quite understand how the oligos file should look like. I searched through the forum, but didn´t find what I was looking for.
So, I have both forward and reverse barcoded primers- cca 90 forward and 4 reverse and 2 linkers.
Do I somehow have to specify the combinations of barcodes and primers, or is it just enough to list them?
Can I trim both primers simultaneously or do I have to do forward first and then reverse?
Does it matter if I don´t know which was the forward sequencing primer, e.g. can I just name “forward” the actual forward primer (27f in this case)?
Is it necessary to specify linker sequences?
Thank you very much in advance for answering my questions.
THanks Pat for your quick answer, I actually read those before posting (sometimes I don´t express myself precise enough, sorry), and I also realized I could simply add linkers to the primer sequence in the meantime, which I also did (though i still don´t know if that is necessary; does bc have to “sit” directly on the primer?), but the part of it I don´t get is how to handle reverse primers and barcodes (reverse has bc 2,3,5,6 )? Run it twice, once for fw and once for rw (with silencing unnecessary parts)? Apart from linkers, my questions stay basically the same…
This is how my oligo for forward primer looks like:
Your strategy looks right. One small but important thing for processing the reverse sequences would be to relabel the reverse primer as forward and the forward as the reverse. It’s probably easiest to add the linkers/spacers to the primer sequences.
Thanks a lot for the answer.
Just to be sure I got it right: I should reverse the sequences and relabel the primers?
It´s my first 454 dataset and I don´t want to make some cardinal mistake in the first step already :).