Fig. 2

Targeted BS-Seq performs to the same standard as Illumina’s NEXTERA XT kit. Three genomic regions located in the human OPRK1 gene were amplified by quantitative PCR and then used for library preparation with either (i) the commercially available, gold standard NEXTERA XT kit (Illumina) or (ii) our targeted BS-Seq approach. a In targeted BS-Seq, successive rounds of PCR are performed to amplify BS-DNA and add universal and reusable primers (CS1 and CS2), followed by adapters to the MiSeq flow cell (P5 and P7 + index). Gel electrophoresis was performed on Agilent’s Tape Station and shows the expected increases in size of PCR products for 2 replicates after each round of PCR. b–d Both NEXTERA XT and targeted BS-Seq yielded very high coverages throughout the amplicons of interest, with the exception of 4 CpG sites in NEXTERA XT results (c, d). Note that while the difference in coverage between the 2 methods only reflects the amount of material loaded onto the sequencer, the targeted BS-Seq method allows for a more even distribution of coverage along the amplicons and lower variability in coverage across biological samples. Values represent mean ± S.E.M. e NEXTERA XT (y-axis) and targeted BS-Seq (x-axis) measured very similar DNA modification levels at all CpGs interogated (r ² = 0.9947, p < 0.0001), confirming that our methodology matches a gold standard, commercially available solution to call DNA modifications with high precision. f This correlation remains highly significant (r ² = 0.7476, p = 0.0012) even when looking at the 10 CG sites that showed intermediate methylation levels, corresponding to sites with more variable methylation states across individual cells