Data Collection
We used epiRADseq (Schield et al. 2016) to screen variation in DNA methylation among house sparrows on the Ion Torrent PGM platform (Thermo Fisher Scientific, Waltham, MA). epiRADseq is a ddRADseq protocol, developed for species without well-annotated genomes. This method uses a DNA methylation sensitive restriction enzyme, HpaII , which fails to cut when its CCGG restriction site is modified by DNA methylation at the internal CG. The enzyme thus generates a variable fragment library among individuals based on the DNA methylation state of the HpaII restriction site. If the site is methylated, no fragments are generated to be sequenced. Thus, variation in DNA methylation is assayed as read count variation among individuals, which estimates the differences in DNA methylation of the screened CCGG sites. epiRADseq generates data in which zero read count result for an individual is meaningful, and therefore, we did not use cutoffs for differences in methylation.
We followed a genotype-by-sequencing (GBS) protocol developed for the Ion Torrent platform (Mascher et al. 2013), substituting the DNA methylation sensitive restriction enzyme HpaII for MspI (New England Biolabs, Ipswich, MA) to construct the epiRADseq library. After restriction digestion, we ligated Ion Torrent IonXpress barcoded adaptors and y-adapters. We ran emulsion polymerase chain reactions (PCR) following manufacturer protocols of the Ion PGM-Hi-Q-View OT2-200 kit on the Ion Express OneTouch2 platform. We sequenced resultant fragments following manufacturer protocols of the Ion PGM-Hi-Q-View Sequencing 200 Kit using an Ion 316v2 BC Chips.
The epiRADseq technique is a vast improvement on MS-AFLP (Schrey et al. 2013), yet it maintains many of the same limitations (i.e., anonymous CCGG sites, analysis focused on variable sites among individuals) and benefits (not requiring a reference genome, using standard RNA-seq analysis methods, and being economical) of MS-AFLP. We believe that epiRADseq is best used to ask questions about variation in DNA methylation among experimental units, rather than to address specific questions about the functional role of DNA methylation at the molecular level. Importantly, epiRADseq is not comparable to bisulfite- or enzymatic-methyl sequencing-like approaches. As such we have intentionally maintained a separation of our analysis to that typically expected of these techniques to avoid confusion or overinterpretation of our results.