Comparative Analysis of Single Base Resolution Whole Genome Bisulfite Sequencing Methods for Low-Input Samples

Whole genome bisulfite sequencing is the most accurate way to measure genome-wide DNA methylation levels at single-base resolution. However, it has been thought difficult to apply to low-input samples, such as sorted cells or rare samples. Here we systematically evaluated the performances of four whole genome bisulfite sequencing methods/kits, two bisulfite conversion kits, and four PCR enzymes using low amount of Arabidopsis thaliana genomic DNA sample from a single tube. We prepared and sequenced total 27 libraries from serially diluted genomic DNA, and compared 8 libraries against 1 control library in detail. We showed combination of MethylC-seq method, MethylCode bisulfite conversion kit and KAPA HiFi Uracil+ PCR enzyme performed best in all metrics with the least bias, although MethylC-seq is the most laborious method. On the other hand, EpiGnome kit and EpiTect bisulfite conversion kit caused sequence-specific biases. We also showed that MethylC-seq enabled accurate methylomes sequencing from at least 1 ng Arabidopsis genomic DNA and that MethylC-seq is the most versatile methods for this rage of input amount­­.