Zymo-Seq Methyl Spike-in Control is comprised of six unique double-stranded synthetic amplicons (180-200 bp) with distinct sequences derived from the E. coli K12 genome, and each amplicon represents a different CpG methylation level ranging from 0% to 100%. This control serves as an in situ control for next generation sequencing library preparations, providing an unbiased way of calculating the efficiency of the bisulfite conversion reaction. It can also be used to validate bioinformatics pipeline calibration by demonstrating a strong correlation between observed and expected methylation levels of the amplicons.