Custom SOLID Adapters for RNA-Seq

nbogard · 12-01-2011, 12:20 AM

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:

F...
5' - ACTGACTGACTG
3' - TGACTGACTGACNNNNN

R...
5' - PO4 - TTGGAACCTTGGAA
3' - NNNNAACCTTGGAACCTT

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?

Thanks

pmiguel · 12-01-2011, 05:21 AM

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.)
--
Phillip

[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.

nbogard · 12-01-2011, 07:19 PM

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.

-Nick

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12-01-2011, 12:20 AM	#1
nbogard Member Location: Singapore Join Date: Apr 2011 Posts: 10	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: F... 5' - ACTGACTGACTG 3' - TGACTGACTGACNNNNN R... 5' - PO4 - TTGGAACCTTGGAA 3' - NNNNAACCTTGGAACCTT 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? Thanks

12-01-2011, 05:21 AM	#2
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	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.) -- Phillip [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 05:24 AM.

12-01-2011, 07:19 PM	#3
nbogard Member Location: Singapore Join Date: Apr 2011 Posts: 10	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. -Nick