The Zymo-Seq Trio WGBS Library Kit offers an optimized and reliable workflow for whole genome bisulfite sequencing (WGBS) library preparation from cell-free DNA (cfDNA), FFPE DNA, and gDNA. This all-inclusive kit features a straightforward procedure capable of preparing high-quality methyl-seq libraries from as little as 5 ng of cfDNA or fragmented DNA. The process is completed in three basic steps: bisulfite conversion, direct adapter ligation, and index PCR amplification. The initial bisulfite treatment is gentle on fragmented DNA, such as cfDNA, yet effective in converting any unmodified cytosines into uracil. Next, the innovative splinted adapters capture and directly ligate the Illumina-compatible adapters onto any size DNA fragment, allowing for sequencing of nicked, damaged, and short DNA fragments that may have otherwise been discarded with traditional library prep methods. The direct ligation of the adapters also eliminates the need for second-strand synthesis, end repair, and dA tailing steps, thus reducing bias by preserving the integrity of any native methylation present on the fragment termini. Finally, validated open-source bioinformatic tools and a step-by-step guide are available for customers to co-detect genetic information, enhancing downstream analysis power.