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Old 11-30-2011, 11:20 PM   #1
Location: Singapore

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Default Custom SOLID Adapters for RNA-Seq

Has anyone here designed custom, SOLID-like adapters for preparing RNA-Seq libraries? Specifically, I'd like to use forward (F) and reverse (R), splint-like adapters (with poly-N overhangs) that include sequences used for the Illumina flow cell. Would this be as simple as just adding random-sequence overhangs to a sequence of my choosing?

As an example:



Also, do the SOLID adapters for RNA ligation consist of all DNA bases, or do they include some uracils (I've read posts with both)? And, besides the 5' PO4 on the R adapter, are there any other modifications required, or that aid, in the ligation?

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Old 12-01-2011, 04:21 AM   #2
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You should take a look at the patent disclosure filing for more information. (Someone, ECO, I think, posted a link to it at some point.)

Briefly, the enzyme being used is probably T4 RNA ligase2. If you check out the specificities of this enzyme, [1] you will see that this enzyme can seal nicks between 2 RNA strands, but to seal a nick between a DNA strand and an RNA strand, the RNA strand must donate the 3'-hydroxyl and the DNA strand must donate the 5'-phosphate.

"RNA" appears to be distinguished as such by the presence of a 2'-hydroxyl at the first few bases of the terminus adjacent to the nick.

Further, and more bizarre to me, T4 DNA ligase would also be able to seal 3'-OH RNA/5'-phosphate DNA nicks as long as the bottom (splinting) strand is DNA! (Trivia in this case -- one of your ligations would be RNA/RNA.)

[1] Bullard DR, Bowater RP (2006) Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4. Biochemical Journal 398: 135-144 is an excellent source for this information.

Last edited by pmiguel; 12-01-2011 at 04:24 AM.
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Old 12-01-2011, 06:19 PM   #3
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Thanks much, Phillip!

I found the patent info you referred too, it helps a lot. Looks like I am going to have to do a lot of optimization if I go the custom route.

Very interesting about the ligase specificities. I might end up using the SOLID kit for the enzymes, but use my own adapters.

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