Fig. 4
Targeted oxBS accurately differentiates 5mC from 5hmC in post-mortem brain. a A 100 bp spike-in digestion control sequence contains a 5hmC mark at the TCGA Taq α1 restriction enzyme digestion site located at position 42 (h C). An efficient oxidation reaction will convert this 5hmC to thymine after bisulfite conversion, which will prevent the enzyme from cutting (T in the oxBS arm). In the bisulfite-alone reaction, the 5hmC will remain a cytosine after the bisulfite reaction and will be cut by the enzyme (C in the BS arm). As per manufacturer’s guidelines, digestion control reactions were run on a 1.5% agarose gel with (+) and without (-) the restriction enzyme and an additional cutting control was included, also with (+) and without (-) the restriction enzyme. b The targeted bisulfite sequencing approach presented in this manuscript involves consecutive PCR reactions to add the universal adaptors and indices, therefore, we expected to see an increase the size of the amplicons. Gel electrophoresis was performed on Agilent’s Tape Station. Each lane represents a different subject. In the red box, the 3rd and 4th bands represent amplicons with the universal adaptors, while the 1st and 2nd bands include the universal adaptors and the indices. c During the target amplification, primers were also designed to interrogate spiked-in sequencing controls that contain a variety of 5hmC, 5mC, 5fC, and C bases (provided by manufacturer). Conversion rates of cytosine to thymine in the oxBS (left) and BS (right) reactions were as expected, with 5hmC being converted to thymine after oxidation and bisulfite conversion. d Representative CpGs showing varying levels of 5mC and 5hmC between 0 and 100% were selected to design a standard curve showing the range of detection of 5hmC and 5mC in the loci we interrogated. e The correlation of %5mC and %5hmC across all 30 CpGs shows an inverse relationship between the two modifications