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
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
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