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Old 01-12-2011, 10:23 AM   #8
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Location: Redwood City, CA

Join Date: Oct 2010
Posts: 4

Hi amango,

I should clarify that we did not use our 'Homebrew' RNA-Seq protocol on the multiplexing study I mentioned in my earlier post. For multiplexing we used Illumina's now obsolete Multiplexing Sample Preparation Oligonucleotide Kit. The protocol can be found on Illumina's website.

From your sequences it looks as if you are trying to have the barcode incorporated into the read itself. Just be aware that Illumina does not recommend this strategy (although that does not mean it can't be done).

To answer your question, since you are just adding a barcode to the end of your adapters, I would imagine you should be able to use the standard Illumina primers.

As for the problems you are having, some questions for you:

-Are you annealing your adapter oligos together (for example, with heating, slow-cooling) before you use them in ligation?
-Have you estimated the concentration of annealed adapter to use in ligation relative to your input template?

FYI, Illumina's new TruSeq RNA-Seq kits have multiplexing capability built in and are much less expensive than their earlier RNA-Seq Kits on a per sample basis (about $66/sample by my figuring). You might want to give that a try if you are still hitting a wall with your homebrew.

Good Luck,

JMorlan is offline   Reply With Quote