DNA extraction, RAD-seq and Reduced-Representation Bisulfite
Sequencing
We used QIAGEN DNeasy blood and tissue kits to extract DNA from blood
samples, following the provided instructions for nucleated blood
samples. DNA was quantified using a NanoDrop ND8000 spectrophotometer
and a Qubit 2.0 fluorometer with the DNA HS assay kit (Life
Technologies). DNA quality was examined on agarose gels. We then
performed RAD-sequencing and RRBS-sequencing using standard protocols.
For RAD-sequencing (restriction-site-associated DNA sequencing, Baird et
al., 2008), the library preparation was done by the Montpellier GenomiX
(MGX) platform (CNRS, Montpellier), using the enzyme SbfI. Each
individual was identified using a unique six nucleotides tag,
individuals were randomly multiplexed in equimolar proportions by
libraries of 37 individuals. Each library was sequenced on a lane of an
Illumina HiSeq 2500. Paired-end sequencing was used to produce 150 bp
reads. This generated an average of 4.9M reads per individual. The DNA
of the 60 individuals were processed twice to test for reliability of
the genotyping process. The RRBS-sequencing started with DNA digestion
using MspI restriction enzymes, which cuts CCGG sites and targets
regions that are CG rich, permitting to have a high proportion sequences
in promoter regions. Individuals were randomly multiplexed in equimolar
proportions by libraries of 10 individuals. Bisulfite treatment
converted unmethylated cytosines into uracil, then converted to thymine
after PCR amplification. Each library was then sequenced on a lane of an
Illumina HiSeq 2500. Paired-end sequencing was used to produce 50 bp
reads. This generated an average of 19.3M reads per individual.