RRBS 5ng multiplexing

[Lilip]

Hey everyone,

I succeed in RRBS protocol following Nature protocol, using low amount of DNA. http://www.ncbi.nlm.nih.gov/pubmed/?...tion+profiling

My problems started when I tryed Barcode adapters for multiplexing (from Truseq Kit). I followed the same protocol just changing the adapters and primers. 2 things happened:

- I got this huge band adapter size. I diluted the primers 3x and it keeps happening.
- I tryed a different approach ( from other protocol) where I bisulfite converted the DNA, amplified it (6 cycles), then went to the gel size selection step , and finally did more 12/14 cycles. This way, I am able to cut the adapters away. But following this I got almost nothing after final PCR amplification.
- Not to mention the smear is shorter when compared to smear from no barcode adapters, even the gel cut being exactly the same.

Has anyone succeed using 5ng or less to multiplexing?

[Lilip]

Ops I meant, I diluted the adapters 3X

[Lilip]

http://seqanswers.com/forums/showthread.php?t=23278

It seems that switching from Gu et al., Nature protocols, 2011 to TruSeq adapters could be a problem. So, how can I perfom Muliplexing for RRBS samples?

[C.R.]

I never used 5ng DNA but TrueSeq Adapters in generall are not a problem. OK, still we get more Adapter dimer. Next time I will use less Adapter. This is even more important when you use only 5ng. But it is really crucial that you carefully follow the general instructions for the TruSeq Library Prep when it comes to gel extraction. And run the gel very very slow! You may also consider to adjust the PCR parameters to those suggested for the TruSeq library prep. Finally have a look at this protocoll: http://genomebiology.com/2012/13/10/R92 Maybe we can just to it in a different way! This also looks good.

[Lilip]

Thxs CR.

Why should I run the gel very, very slow?

This link you sent me (gel free), the concentration of TruSeq (stock) adapters metioned on the text is right?

[C.R.]

I did not test the gel free protocol yet. I always had adapter dimers in the final library when the gel was running too fast and I never had this problem with the old PE adapters