Accurate simultaneous sequencing of genetic and epigenetic bases in DNA

We present a single-base-resolution sequencing methodology that will simultaneously sequence complete genetics and complete epigenetics in a single workflow. The approach is non-destructive to DNA and provides a digital readout of bases, which we exemplify by simultaneous sequencing of G, C, T, A, and 5mC/5hmC (5-letter sequencing) or 5mC and 5hmC (6-Letter sequencing). We demonstrate sequencing of human genomic DNA and also cell-free DNA taken from a blood sample of a cancer patient. The approach is accurate, requires low DNA input and has a simple workflow and analysis pipeline. We envisage it will be versatile across many applications in life sciences. Overall design: 5-letter and 6-letter sequencing technologies unambiguously resolve the 4 genetic bases with the 5-th letter being 5mC or 5hmC (also referred to as modC) or the 5-th and 6-th letter being 5mC and 5hmC, respectively. This experiment evaluates the performance of these technologies and compares 5-letter sequencing with Whole-genome Bisulfite Sequencing and Enzymatic Methyl Sequencing.