degenerate primers

Dear all,

I am trying to process some 454 titanium data with flow order=A (TACG over and over again).

Number of Flows: 800
Format Code: 1
Flow Chars: TACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACG
Key Sequence: TCAG

I have problem with trim.flows .
trim.flows junks pretty much all the seqs, giving a primer error.
The forward primer is CCTACGGGNGGCWGCAG

My question is…
in my oligos.file i have to write out all the possible combinations of the degenerate primer?

Thanks in advance!

Eleni

Hi Eleni,

The degenerate primer should work. Can you post one of the sequences and it’s flowgram that you think should pass along with the contents of the oligos file? Note that if you are using barcodes and there are errors in the barcodes, then the primer will also fail.

Pat