EpiTYPER 1.0 DNA Methylation Analysis FAQ’s

How much DNA do I need for the experiment?

• While the bisulfite kit will work with as little as 500 pg, this will not give you representative or reproducible DNA methylation ratios in our experiment.  We recommend at least 500 ng of genomic DNA.  Remember that you cannot use WGA material for your methylation experiments.

How can I test the bisulfite reaction’s success before doing an EpiTYPER experiment?

• You can test your busulfite reaction by running the PCR reaction and if you don’t obtain the expected product (by agarose gel electrophoresis) then there is no point in continuing in our process.

Can I use a different bisulfite treatment kit?

• Yes, you may.  We have had the most success with the Zymo kit which we resell.  You may have good experience with a different technique.  There are two things to consider when using a different bisulfite kit: (1) Do I get PCR amplicons from the bisulfite treated DNA?  If not then your kit may not be as successful as ours.  (2) Will the different kit negatively affect the downstream processes?  If so then this can usually be remedied by inserting a DNA cleanup set somewhere after the bisulfite treatment.

How long does the whole process take?

• The bisulfite protocol takes about 6 hours to complete.  Once you have bisulfite treated material, then the Sequenom protocol takes about 9 hours to complete, most of which is “hands off” time.

What is the difference between a CpG unit and CpG site?

• A CpG site is a single CpG dinucleotide.  A CpG unit may contain one or more CpG sites.  If two CpG sites exist on the same CpG unit, there is no way to unambiguously differentiate between the two with our chemistry.  Our data analysis software will specify the same percentage of methylation for both CpG sites on the CpG unit.

Should all the peaks in my spectra be expected?  I have some peaks which are not assigned in the software.

• This is not uncommon.  Additional peaks in your spectra could come from a variety of sources.  The additional peaks could come from : (1) Additional amplicons from your PCR reaction indicating poor PCR primers.  (2) Genotypic variation in the sequence of interest.  If you have a sequence variation in your amplicon, different from the expected sequence, you would expect some peaks to appear and others to possibly disappear.  Our software is fairly forgiving regarding this.   (3) Adducts, such as salt, can be observed.

My CpG site of interest was not analyzed.  Why?

• There are several reasons this could be.  Is the CpG unit which your CpG site is on within the appropriate mass window?  Is your signal to noise level too low to get a percentage methylation within the appropriate uncertainty threshold?  These two problems are the most common.

How can I get my CpG site analyzed?

• If the CpG unit is not analyzable as it falls outside the analysis window, then you can run the C cleavage reaction and see if you can get that site analyzed.  If the problem was too low a signal to noise ratio, then you have to increase signal to noise by increasing your PCR reaction yield or checking your dispensing/mass spectrometer performance and taking the appropriate steps to improve them.

How do I design Epityper experiments?

• We have a new Epityper designer that you can access from mySequenom.com and all you need is your amplicon sequence of interest from the UCSC genome browser to paste into the designer.  We also have a new EpiPanel with 40 genes of interest and their associated primers.  Please visit www.sequenom.com to obtain more information on the EpiPanel.