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Old 07-07-2009, 09:16 AM   #13
Exeplex
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Location: Exeter, UK

Join Date: Jun 2009
Posts: 10
Default Multiplexing with custom adapters

Quote:
Originally Posted by PUGenomeLab View Post
Hello,
I'm new to this site, but I wanted get feedback on a couple of ideas that our lab is tossing around.
1) Illumina's multiplexing adapters contain ID tags on the end of the fragment. Does this lead to any significant loss of the tag or misreading of the tag during sequencing?
2) We wondered if anyone has ever moved the tag so that it would essentially be the first 3/4 base pairs of the read? This would cut down on the read length, but it would make tags that will not be lost and could bypass any additional cost that sequencing centers charge for multiplexing. The decoding of the tags would be done by our computational people, thus reducing the cost.

I'd appreciate feedback or suggestions. Thanks.
Hi,

Re. the second part of this post, various groups (e.g. Cronn et al., NAR 2008; Harismendy & Frazer, Biotechniques 2009) have used custom adapters incorporating the barcode so that it becomes part of the read at the 5' end. In a bid to get my own head around the various stages, I've adapted the excellent figure posted by greigite (in the Tech Summary: Illumina's Solexa Sequencing Technology thread) to include some details of the approach used by Cronn et al., as well as a generic strategy to follow through the PCR and sequencing steps (any errors, please let me know!) - hope this is useful.

Also, check out the other posts in this thread for great ideas re. barcode design and bioinformatic processing of sequences.

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