Base resolution methylome profiling: considerations in platform selection, data preprocessing and analysis

Abstract

Bisulfite treatment-based methylation microarray (mainly Illumina 450K Infinium array) and next-generation sequencing (reduced representation bisulfite sequencing, Agilent SureSelect Human Methyl-Seq, NimbleGen SeqCap Epi CpGiant or whole-genome bisulfite sequencing) are commonly used for base resolution DNA methylome research. Although multiple tools and methods have been developed and used for the data preprocessing and analysis, confusions remains for these platforms including how and whether the 450k array should be normalized; which platform should be used to better fit researchers' needs; and which statistical models would be more appropriate for differential methylation analysis. This review presents the commonly used platforms and compares the pros and cons of each in methylome profiling. We then discuss approaches to study design, data normalization, bias correction and model selection for differentially methylated individual CpGs and regions.

Keywords: DNA methylation; bisulfite sequencing; differential methylation; methylation 450K array; normalization; reduced representation bisulfite sequencing; study design.

Figure 1.. Reduced representation bisulfite sequencing mechanism and flow diagram.

(A) Original DNA with CCGG motif at both ends. In human cytosine methylation occurs at CpG site (marked with m) and non-CpG cytosine is generally not methylated. The arrows point to the MSP1 cut sites, which is methylation independent. (B) After MSP1 digestion, DNA fragments are generated with sticky ends. Fragments in right sizes (generally 40–250 bps) for sequencing are selected. (C) The end repair adds CG (in blue, generally not methylated) from media that are not part of human sequence and needs to be removed in the analysis step. (D) The bisulfite treatment converts unmethylated cytosine to uracil but the methylated cytosine remains as cytosine. (E) The PCR amplification step converts/interprets uracil (U) as thymine (T). The amplification is based upon the original top and bottom strands, which are no longer complementary and generate their respective offspring sequences. For single end sequencing, only OT and OB sequences are used, however, for pair end sequencing all four strands are generated. Analysis need to group them correctly. OB: Original bottom strand; OBC: Original bottom strand complementary; OT: Original top strand; OTC: Original top strand complementary.