methylnick

If you have infinium data already, I would look up minfi also consider peak-based correction for type I and type II chemistry.

it is possible to get technical artefact due to inefficient removal of protein by your extraction method, if you are using a kit then generally it should be okay, but if your protienase K has gone off and you don't know it, well that is a problem.

nick

Anne sorry for the long absence and late reply. for a positive control for your enrichment sequencing, without knowing your organism, you could try a centromeric or repeat element sequence? This maybe messy as you would also get signal in your input fractions because of the relative copy number. Another, if your organism is female, sequences on the X chromosome may have a similar pattern as imprinted control regions if there is X-inactivation?

As for direct sequencing, I have put the forward and revers primer phenomenon down to how the polymerase behaves differently when incorporating particular nucleotides, in the forward direction you are mainly incorporating TAG nucleotides, but in the reverse it's TAC. As for the spike in C's it's because the majority of your amplicon is TAG and as these are depleted during the sequencing reaction, there is an abundance of C's which give rise to the potential that more extensions are terminated at C leading to a higher signal.

most liekly a primer design problem, I would try to redesign them again, maybe using MethylPrimer Express which is freely available.

have you tried spiking the reaction with more enzyme after incubation? i.e.: like having two rounds of digestion?