I am trying to understand the levels of methylation using LINE1 gene (surrogate marker for global methylation) in mice. My aim is to see the levels of methylation between control and test, hence i am doing bisulphite conversion followed by pyrosequencing of LINE1 gene.
Here, i am confused about sequence coverage.
LINE1 gene is 1.4kb, can anyone suggest me how much sequence i have to cover during sequencing so as to see overall methylation level?
Coverage recommendations for methylation analysis by whole-genome bisulfite sequencing, can be seen here: https://www.researchgate.net/deref/https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fm%2Fpubmed%2F25362363%2F
Hi Riddih,
If I understand you well, you will be designing primers to amplify a specific L1 insertion in the genome, and then use pyroseq to assess the level of methylation.
Sequencing coverage should not be a problem since you will be comparing signals between two experimental set ups and this is not a whole genome sequencing experiment.
Now, I believe you might be asking about the total amount of L1 sequence to cover in your assay. Most DNA methylation at L1s occurs over the 5'UTR (which is also the promoter and contains most CpG sites). You should be able to see changes in methylation by probing less than 1kb - I would say that the first 500bp should suffice, but it can vary between insertions (and there are thousand of them in the mouse genome).
I hope this helps.
Cheers,
Antoine.