RRBS-Seq adapters

[jae_tan]

Hi guys,

I have the TruSeq DNA sample prep kit. From what I understand, this kit contains methylated adapters and I was thinking of using the adapters for RRBS-Seq. I know the current RRBS-Seq protocol from Gu et al., Nature protocols, 2011 used adapters of ~30bp while the TruSeq ones are at ~60bp.

Is it ok to still use the TruSeq adapters and their reagents instead of synthesizing new ones?

Any help is appreciated!

[lterhune]

We have tried using RRBS with TruSeq adapters and have had some success, but it is inconsistent. Sometimes it works, sometimes it doesn't. Using methylated PE adapters has been much better for us-- we get a better yield, and it consistently works. We've tried TruSeq adapters with a variety of applications other than the intended protocol (ChIP-seq, RRBS, WGBS) and tend to get inconsistent results and usually return to PE adapters. TruSeq adapters tend to anneal together easily, which can cause problems with the library. We're still learning more about them as we go, but I'd personally stick with the methylated PE adapters, unless you have some time and reagents to spare for optimization. Also, if you are interested in using small input DNA quantities for RRBS, this paper looks promising, although we haven't tried their protocol yet:

http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1002781

[jae_tan]

Hi Iterhune,
did you guys try to run the TruSeq adapter-ligated DNA on the gel before or after PCR? whehn you said inconsistent results with TruSeq adapters, do you mean the pcr amplification was not as good as the PE adapters? do you tend to see a lot of adapter dimers on the gel?

[C.R.]

I am not sure if you still can buy the methylated PE Adapters from Illumina but they worked good in our hands. We successfully used the TruSeq Adapters for RRBS, but you have to adjust the Gel conditions and use a SYBR gold prestained gel.

[jae_tan]

hi C.R,

did you see adapter dimers on your gel? I was thinking of cutting back on the adapter amount to reduce adapter dimers. Do you also use the TruSeq primers as well?

[C.R.]

well, I did some RRBS sample prep. using the TruSeq Adapter. These libraries have been sequenced and it looks OK. We needed to use custom made primers very soon, since these were limited in the original kit. It seems to work when using the SYBR gold and eluting the purified sample after Ligation in the Illumina resuspension buffer. The separation of the DNA in the gel is much better under these conditions.
But still adapter dimers quite often appeared to be a problem, whereas with the old PE adapters it used to work more robust. Thats why we switched back to the old protocol. We will put each sample in a separate lane for sequencing in the end anyway.
I never tried using lower amounts of adapter. Running the gel after a first PCR (which is performed after bisulfite conversion of course) is also an option. There is a nice RRBS protocol following this approach published: http://www.nature.com/ng/journal/v43...ll/ng.864.html

[jae_tan]

Hi C.R.,

after bisulfite conversion, I PCR using Pfu Turbo Cx Hotstart polymerase (15 cycles) then ran a gel but the DNA smear was hardly visible. Do you/anyone out there think it's ok to re-amplify the size-selected DNA using Pfx for another 10 cycles?

im not sure if this is due to the damage bisulfite treatment had done to the DNA...

thanks..