Problems amplifying Ox-BS & BS template from TrueMethyl

[cbg301]

Howdy,

I recently ran an experiment with 2 samples through CEGX's oxidative bisulfite kit using the Illumina LT Truseq prep and have run into some issues. Was hoping to find some insight, suggestions or support here.

After creating my NGS Illumina Truseq LT adapted libraries, I split each of the 2 of them into 3 aliquots per sample (total of 6 libraries in the end; 2 oxbs, 2 BS & 2 NGS adapted). After successfully completing their detailed, elaborate and well thought out protocol I went to amplify my libraries. Unfortunately, only the NGS adapted libraries would amplify. I used the Qubit ssDNA kit to quantify the OXBS & BS libraries and have recovered plenty of template.

Through weeks of speaking with technical support, scratching my head & discussing with colleagues I have still made no ground. It then dawned on me earlier this week that the sequences will have definitely changed on the adapters & thus the primers that I were using would no longer amplify my template. The protocol calls for you to use the Illumina PCR primer cocktail with the supplied (by CEGX) uracil tolerant polymerase.

The point im making is that wouldn't the sequence the PCR primers would recognize have changed throughout the oxidation & bisulfite reactions whereas my primers would not have and thus the two would be incompatible?

If anyone has any insight/suggestions/support it would be much appreciated!

After speaking with Illumina & discovering that they will not disclose the 'proprietary PCR primer cocktail' sequence the only thing I can come up with to get the primers I need is to process a large portion of the primer cocktail through the TrueMethyl protocol.


.....Unless I am just overlooking something really simple here

Thanks,
Chris

[AnneH]

Hi there,

I'm not completely sure, but doesn't use Truseq methylated adapters? So they shouldn't be affected by the BS treatment and the amplification should still work, no?
I ran into a similar problem though, using NEBNext methylated adapters and qiagen epitect BS kit. After the subsequent PCR-enrichment step I got a really nice library for non-BS-treated DNA, but nothing for the BS treated. I also used a Uracil tolerant polymerase (and for DNA not containing Uracil it seems to work fine at least).

Right now I'm at a dead end as well and would be happy for any tips

[two_st]

My advice would be:

1) Check that the adapters are methylated. If they are not, then they will not be complementary to the PCR primers post conversion, so amplification will fail. If you are working in a lab that also runs MeDIP - make sure the adapters are correct, a simple oversight could mean you've made libraries with the non-methylated adapters (you would need to do this for MeDIP) by mistake.

2) The DNA polymerase in the PCR master mix provided in the TruSeq library construction kit is not U-tolerant. Using this master mix to amplify BS-converted DNA, amplification will fail.

3) You can use the ILMN PCR primer mix in combination with the CEGX PCR mix - this should work. But using the ILMN PCR primer mix and PCR master mix will fail (for reason 2).

4) Try lengthening the extension time. Uracil only "stalls" certain polymerases - doesn't completely inhibit them, so if your products are long and your extension time is short, you could be generating incomplete length fragments and not getting exponential amplification.

There should be no need to process the PCR primer mix through BS in order to generate a BS-converted PCR primer! There is no guarantee that their primers are methylated AND the fact of converting them will cause fragmentation which in turn could affect the specificity of resulting PCR.

Good luck!

[lyndaben]

I am about to try the TrueMethyl kit for the first time (for RRBS). Did you find a library prep protocol that worked for you? I don't think Illumina sell methylated adapters. I did find the NEBNext methylated adapters and primer index kit and thought that may work but I'm not sure which library prep kit to use

[pbest]

Hi Lyndaben,

my answer might be a bit late. However I've some experience using the TrueMethyl-Kit from CEGX.
I've successfully used the TM-Kit with Illuminas TrueSeq DNA Adapters and Primer-Cocktail. You have to make sure to use the correct kit. Illumina stated on their website:
"Are TruSeq DNA adapters methylated?
TruSeq DNA PCR-Free and TruSeq Nano DNA LT library prep kit adapters are methylated. However, TruSeq HT library prep kit adapters are not methylated and are therefore not suitable for bisulfite sequencing applications."

So the TruSeq Nano DNA LT-Kit should work fine.

http://support.illumina.com/sequencing/sequencing_kits/truseq_dna_lt_sample_prep_kit/contents_storage.html

Additionally I'm currently testing Nextflex Bisulfite Barcodes from Bioo-Scientific.

http://www.biooscientific.com/Next-Gen-Sequencing/Methylated-Illumina-Adapters/NEXTflex-Bisulfite-Seq-Barcodes

I hope this information helps for your experiments.

[lyndaben]

We used the NEBNext Methylated adapters and we got what I thought were pretty reasonable libraries. We do however have large peaks at 186-188bp in some of the libraries. Are these adapter dimers? And are they going to be a problem? According to BCM Core, our libraries are "failures"