High-throughput sequencing of cytosine methylation in plant DNA

Abstract

: Cytosine methylation is a significant and widespread regulatory factor in plant systems. Methods for the high-throughput sequencing of methylation have allowed a greatly improved characterisation of the methylome. Here we discuss currently available methods for generation and analysis of high-throughput sequencing of methylation data. We also discuss the results previously acquired through sequencing plant methylomes, and highlight remaining challenges in this field.

Figure 1

Alignment choices for bisulphite treated data. Biased and unbiased alignments of bisulphite treated data. Bisulphite treatment converts unmethylated cytosines to uracil, which are sequenced as thymine. In a biased alignment, sequenced thymines are treated as ambiguously cytosine/thymine (ambiguity code Y). In case (b), of a unmethylated read, this ambiguity allows the converted read to align to two separate locations on the reference genome (blue), while in case (c), which consists of the same nucleotide sequence but contains methylated cytosines, the read aligns to a single location. This results in a greater confidence in the alignment of the methylated read. In the case of an unbiased alignment, all cytosines on both the sequenced reads and the reference genome are converted to thymines, and the sequences in both (b) and (c) align to the same locations on the genome, with no additional confidence in the alignment of the methylated read. In case (a), the read aligns to a single location in both a biased and unbiased alignment; however, in the unbiased case less information is available to make this alignment.

Figure 2

Post-alignment data. An example of the data available for analysis of methylation following alignment of the sequenced reads to a reference genome. The data are taken from Lister et al[2] and show the first ten thousand bases of the positive strand of two samples of Arabidopsis in wild type (Col-0) and met1 knockout mutant. The number of times a cytosine is observed to be methylated is shown by the height of the coloured bars, with the colour indicating the context of methylation. The abundance of reported methylation is heavily dependent on the read coverage (black curve) at each base, which exhibits high variability.