Whole Methylome Analysis by Ultra-Deep Sequencing Using Two-Base Encoding
Figure 1
Library construction to protect the adapter sequence from bisulfite conversion.
Genomic DNA (5 ug) was sheared by sonication and end-repaired to yield 5′-phosphorylated (5′P) blunt ends. Two double-stranded oligonucleotide adaptors, having only one preselected oligonucleotide protected by 5mC (5mC/black) against bisulfite conversion, were ligated to the DNA fragments. After nick-translation with 5mC-dNTP one adaptor consists of two fully 5mC-protected oligonucleotides, whereas the other adaptor still contains one oligonucleotide with unprotected regular Cs (Fig. 1A). Following a size selection to 175–225 bp on an agarose gel, equal amounts of DNA (240 ng) were used for bisulfite conversion in solution and in gel, respectively. During bisulfite conversion, the DNA is denatured and due to only three 5mC-protected adaptor strands, the fourth adaptor strand was bisulfite converted thus changing the sequence by altering C to U. During PCR amplification (scheme B) with regular four-base primers (A, G, C, and T) complement to the library adaptors, only one of the fragments was amplified. 5′P = 5′-phosphorylated blunt ends, 5mC = 5-methylcytosine.