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.