TruSeq methylated adapters?

[epistatic]

Sorry for post, solved with quick phone call to Illumina.

For anyone else interested, the TruSeq adapters that come in the kits are methylated, so they can be used for bisulfite conversion prior to the PCR step. This is great because we can now take advantage of the multiplexing.

[lterhune]

Hi there, have you tried the bisulfite conversion after TruSeq and been successful? Because we just tried after our first TruSeq library-- which looked great on a gel-- and got nothing after conversion! We had been using the same Zymo kit for months with a homemade Illumina pair-end library protocol without problems. We called Illumina like you did and expected it to work. If it worked for you maybe it was a problem with our kit or library.

[atgc1]

The Truseq LT kit adapters are methylated but the HT kit adapters are NOT methylated

[SeqR&D]

I have perfomed BSC on TruSeq libraries several times and have had no problems other than losses/degradation. I recently tested BSC kits (Promega methyledge, Qiagen Epitect fast, and both the Zymo Gold/lightning kits (still waiting for my Epigentek kit)) on both a ~1kb fragment and gDNA. Overall the Promega kit had the least fragmentation and was similar in yield to the Zymo kits (the lightning kit was better than the gold kit for fragmentation). Qiagen had the worst recovery at about 60% (others were around 80%), but I did not use the carrier RNA supplied with the kit as i was converting 500ng (it was also the most time consuming purification).

Since I can't figure out how to post an image here...if you care to see my results, just give me your email address and I'll send it to you. I don't work for any of these companies.

[Lovro]

Hi.

I would like to bisulfite sequence (Illumina platform) selected loci on human genome.
I have ordered custom hybridization probes from IDT in order to enrich the human DNA. I was under impression that I also need methylated adapters/indexes in order to be able to bisulfite treat the multiplexed libraries.
Are you saying there is no need for the latter ?
Idealy i would like to multiplex 96 probands, however 48 would be acceptable.

Besides IDT which offers custom oligos, I have also found stock methylated adapters here:

http://www.biooscientific.com/Next-Gen-Sequencing/Illumina-Library-Prep-Kits/Methyl-Seq/NEXTflex-Bisulfite-Seq-Kit

Does anyone know of another company supplying methylated Illumina adapters/indexes?
Are they needed at all?

TNX for help!

[SeqR&D]

Hi

Yes, you definitely need methylated adapters...Assuming you are BSC the libraries before adding the adapters, which is likely no.

What is your custom hybridization probe plexity? How many probes? Unless you have some amazing process, anything below 20k probes will likely not yield much product after enrichment and BSC...you'll sequence more duplicates than not. Maybe you have a pooling plan to increase the amount of DNA going into BSC?

Hasn't anyone made this assay kit yet?

I don't know anywhere else to get specific methylated adapters.