Bar-code short amplicon for Myseq or HiSeq

[groad]

Hi,

I need to sequence ~50 short amplicon (100-150bp each) from 4 samples. I want to bar-code each sample so I could sequence all of them in one lane. I have a thought about simplified way of bar coding and not sure whether it would work. Could I add the 6 base barcode at the 5' of my forward primers? In that case, I would be able to know which sample the sequence is from just by looking at the reading from the first 6 bases? Does anyone know why illumina design the third run of sequencing just to get the sequence of the barcodes? Thanks.

Jim

[pmiguel]

What you describe is a common method, although not really supported by Illumina. I call these "in-line" barcodes. So the down side is that Illumina sequencers really want well-balanced and diverse sequence in the first 4 or 5 bases of the first read to do cluster calling, etc. Barcodes rarely meet these criteria because generally you will only deploy a few of them. Also, the first few bases are generally of high quality, so it seems a shame to waste them on barcodes. Hence the idea of obtaining the barcodes in a separate read.

--
Phillip

[groad]

Hi Phillip,

Thanks a lot for clarifying it for me. This is very helpful!

Cheers,
Jim