library prep with sticky ends

[shawpa]

Whenever I attempt a protocol that uses sticky ends for adapter ligation I can never get it to work properly. Basically I am looking into methylation assays and have tried MSCC, RRBS, and a couple of others preps that use sticky ends. I can make blunt ended libraries with my eyes closed but no matter what I titrate my adapters to for sticky ended ligation all I get are adapter dimers. I always order my adapter oligos (IDT) with a 5'phoshate at the end. We have always used NEB's T4 DNA ligase and incubated at 16degrees for various lengths of time. I am wondering if anyone has any experience with making sticky ended libraries and if they know any "tricks" that may not be in the published protocols. At this point I am going to try using invitrogen ligase to see if that makes a difference but I am not hopeful.

[pmiguel]

It is not clear what you mean by "sticky ends". That is how long are they, from what (restriction enzyme) do they derive, etc. Nor did you mention the platform for which you are constructing these libraries.

But if your adapters have compatible and ligatable ends then you will mainly get adapter dimers because most ligations add adapters at much higher molar concentrations than inserts.

I am mystified by your focus on the ligase here. The ligase is apparently doing exactly what it should because you do see the adapter dimers. Why would changing ligases help?

Generally a great deal of effort is put into either preventing adapters from ligating to each other, or sifting the small number of adapter-insert molecules away from the large number of adapter-adapter molecules after ligation. For example, you could use 5' non-phosphorylated primers. But you would need to do a nick translation on the resultant products to repair the nicked duplex product that would result. Or you could blunt your "sticky ends", A-tail them, and then use your normal T-tail adapters. Or, you could do a size selection to rid your ligation of the majority of the adapter dimers using an agarose gel or AmPure.

Note that if you are running these libraries on Illumina instruments your stick ends will likely lead to another problem unless you employ some work-around to avoid it.

--
Phillip

[ophurtado]

Hi Phillip,
I just read your post and I got some questions. I am planning to use sticky end restriction enzymes for GBS and I want to blunt end my digested products. I am planning to use T4 DNA polymerase for blunting followed by heat inactivation. After this, I am planning to ligate my barcodes (which are A tailed-one base phosporilated), but I was wondering if I need to do any treatment to my blunt products prior to ligation of barcodes?. I am planning to do the ligation of barcodes using T4 ligase suplemmented with ATP.