MethMarker Tutorial - HTML Tutorial 1 - Step 3/3

Search for CpG sites as biomarkers

Click on "Design Methylation Assays..." to simulate COBRA, Bisulfite SNuPE, Bisulfite Pyrosequencing, MSP, MethyLight and/or MeDIP Assays. A window will appear in which you can adjust several options for the respective methods. For this tutorial, just select Cobra, SNuPE and Pyrosequencing to be used in this analysis and click "OK", accepting default parameters for each method

MethMarker searches the genomic target region for CpG sites that are accessible by each of the individual methods. For example, for Cobra it tries to find restriction sites of CpG cutting enzymes; for SNuPE and Pyrosequencing, it will find primers that fulfill the corresponding criteria. If you have chosen MSP, this method may take a while, since MethMarker has to check a lot of combinations of possible MSP primers, similarly for MethyLight. When finished, found CpG sites are displayed for each method and highlighted in blue. Restriction enzymes and primers needed for the CpG analysis assay are also shown at the right place.

Now you should have a close look at the data. MethMarker lists all experimentally accessible CpG sites. For the primers and enzymes, MethMarker will provide more information if you hold the mouse over them.

In most cases, you want to select primers and restriction enzymes you want to use. E.g. you might not want to use three (redundant) primers that all analyse the same CpG sites. Just deselect CpG sites, enzymes or primers that you don't want to take into account for biomarker development. In our case, we deselect all redundant primers in Pyrosequencing.

Note: Finding CpG sites in this step is independent of loaded samples. That's why you can use MethMarker simply for primer finding, without loading any samples. Only if you have loaded at least two samples and performed at least one CpG analysis method, you can proceed with the generation and evaluation of biomarkers.

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