Sodium Bisulfite Treatment is the gold standard for measuring the level of DNA methylation, it converts the unmethylated cytosines to uracils - which convert to thymines after PCR - but keeps methylated cytosines unchanged.
Here is my question: Suppose the DNA fragment (Methylated cytosines are in upper case: C, and unmethylated cytosines in lower case: c)
5' ACGATGc 3' (Top strand) 3' TGCTAcG 5' (Bottom strand)
After bisulfite treatment we will have:
5' ACGATGT 3' (Top strand) 3' TGCTATG 5' (Bottom strand)
And after PCR there each of top and bottom strands will be changed to a double-stranded DNA like below. The strands 1 and 2 are complementary, made from the Top Strand above, and strands 3 and 4 are made from the bottom strand
5' ACGATGT 3 (1, Top strand, forward) 3' TGCTATG 5' (3, Bottom strand, forward)
3' TGCTACA 5' (2, Top strand, reverse) 5' ACGATAC 3' (4, Bottom strand, reverse)
Now we have 4 different strands which align to the same genomic location (either forward or reverse strands). But the problem is that each of them makes a different measurement of DNA methylation. For instance in the sequence (1): ACGATGT the the last base is T meaning an unmethylated cytosines, which is correct. However in the strand (4) ACGATAC the last letter is C that means a methylated cytosine, which is a wrong assumption.
How to infer the correct methylation status of each base according to the 4 different reads?