Applications of sequence capture bisulphite sequencing for targeted analysis of DNA methylation patterns in complex plant genomes

DNA methylation may contribute to epigenetic variation among individuals of the same species and could provide additional information for predicting phenotype.  We are interested in documenting DNA methylation profiles of maize lines in order to assess the contributions of development, abiotic stress and genotype on DNA methylation variation.  There are a variety of approaches for studying whole-genome DNA methylation profiles but the highest quality information is obtained via whole-genome sequencing of bisulphite treated DNA.  This approach requires high >10X coverage of the genome and can be quite costly for organisms with large genomes such as maize (~2500 Mb).  The combination of sequence capture and bisulphite treatment provide the opportunity for targeted analysis of DNA methylation for a portion of the maize genome.  Sequence capture probes were designed to capture approximately 3 Mb of the maize genome.  These regions were selected to include regions of high methylation in different sequence contexts, low methylation or variable DNA methylation between inbreds B73 and Mo17.  Bisulphite sequence capture followed by multiplex sequencing on an Illumina Mi-Seq (~3-5M reads per sample) was performed on several maize inbreds and hybrids as well as several mutant lines.   The sequence capture resulted in >100 fold-enrichment for target sequences and resulted in at least 10X coverage for 50-65% of the targeted regions even with relatively minimal sequencing depth.  The obtained DNA methylation levels were in strong concordance with genome-wide DNA methylation analyses and successfully validate differential DNA methylation detected between inbred lines.  The analysis of hybrid genotypes provides evidence for capture of both methylated and umethylated alleles from the same sample.  This sequence capture bisulphite sequencing application provides an opportunity for low-cost high resolution analysis of DNA methylation for portions of complex genomes.