on barcodes and primers

Theory behind mothur
1

Hi,

I’m designing a new 454 run with quite some samples and hence I need a lot of barcodes.
Now, I was wondering which experiences you have with the Roche MIDs? I did a search of the first 4 ‘basic’ MIDs against the RDP-training set, and 1 barcode was retrieved completely, while the other 3 had a 9-out-of-10 bp match (last base not).
How does Mothur search for barcode and primer in a read? Is it required them to be adjacent or within X bases, or is the complete sequence screened? Or does the search stop when both barcode and primer were detected (at the beginning of the sequence)?
If the complete sequence is screened, could this lead to some complications, especially when some ambiguities are allowed in the barcode?

Would the MIDs proposed by Parameswaran et al. 2007 be a safer option?

I will use Berry et al’s 2011 2 PCR method. Anybody knows of other problems (biases) with any MID-set?

Thanks!

2

mothur looks for the barcodes at the beginning of the sequence. If the sequence doesn’t start with the barcode (and then the primer) it is tossed. Why bother coming up with your own barcodes? There is a list of ~192 at http://hmpdacc.org/doc/16S_Sequencing_SOP_4.2.2.pdf.