methylKit: a comprehensive R package for the analysis of genome-wide DNA methylation profiles

DNA methylation is a critical epigenetic modification that guides development, cellular differentiation and the manifestation of some cancers [1,2]. Specifically, cytosine methylation is a widespread modification in the genome, and it most often occurs in CpG dinucleotides, although non-CpG cytosines are also methylated in certain tissues such as embryonic stem cells [3]. DNA methylation is one of the many epigenetic control mechanisms associated with gene regulation. Specifically, cytosine methylation can directly hinder binding of transcription factors and methylated bases can also be bound by methyl-binding-domain proteins that recruit chromatin-remodeling factors [4,5]. In addition, aberrant DNA methylation patterns have been observed in many human malignancies and can also be used to define the severity of leukemia subtypes [6]. In malignant tissues, DNA is either hypo-methylated or hyper-methylated compared to the normal tissue. The location of hyper- and hypo-methylated sites gives distinct signatures within many diseases [7]. Often, hypomethylation is associated with gene activation and hypermethylation is associated with gene repression, although there are many exceptions to this trend [7]. DNA methylation is also involved in genomic imprinting, where the methylation state of a gene is inherited from the parents, but de novo methylation also can occur in the early stages of development [8,9].A common technique for measuring DNA methylation is bisulfite sequencing, which has the advantage of providing single-base, quantitative cytosine methylation levels. In this technique, DNA is treated with sodium bisulfite, which deaminates cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. Single-base resolution, %methylation levels are then calculated by counting the ratio of C/(C+T) at each base. There are multiple techniques that leverage high-throughput bisulfite sequencing such as: reduced representation bisulfite sequencing (RRBS)[10] an