The chemical identities of DNA bases in cells are not just A, T, C and G. A number of modifications add epigenetic complexity to DNA molecules. These modifications regulate and are regulated by various cellular activities, and they bear importance in areas ranging from cell biology to disease research. High-throughput sequencing technology has been widely employed for reading either genetic or epigenetic base identities, but often in separate experiments. To combine the two into a single assay, Joanna Holbrook from Cambridge Epigenetix, Shankar Balasubramanian from the University of Cambridge, and their colleagues developed five- and six-letter sequencing.
The main innovation of this elegant method lies in using a two-base coding strategy for the four canonical bases plus 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). In the five-letter sequencing workflow, after hairpin addition and splitting, another copy of the DNA strand is synthesized in which modified cytosine is protected through oxidation and glycosylation while unmodified cytosine is deaminated to uracil. Following PCR and sequencing of the resulting constructs, the bases and status of cytosine modification can be efficiently decoded. “Our technology combined with Illumina sequencing is more accurate at identifying specific bases in the genetic sequence than Illumina sequencing alone,” says Holbrook; “it took a significant amount of time to do the comparisons, as we had to run multiple other workflows to compare against our single workflow.” The team further expanded the technology to six-letter sequencing, which allows 5mC and 5hmC to be distinguished. Much effort was spent to make sure there is no drop-off in performance of the usual properties of sequencing technologies — for instance, uniformity of read coverage and/or recovery of material through the process. Holbrook notes “we optimized the original protocol until we could show it was competitive in all these aspects.”
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