Laboratory protocols for nuclear genomic markers
We extracted genomic DNA from fresh tissue samples using DNeasy tissue
extraction kit (Qiagen, CA, USA). To generate the genomic data we used
double-digest restriction site-associated DNA sequencing (ddRADseq)
following the protocol of Peterson et al. (2012) with modifications
described in Thrasher et al. (2018). Briefly, for each sample we
isolated ~500 ng of DNA, at a standardized concentration
of 20 ng/µl, and digested it with the restriction enzymes SbfI High
Fidelity (8 base bp recognition site; 5’-CCTGCAGG-3’) and MspI (4 base
bp recognition site; 5’-CCGG-3’) (New England BioLabs, MA, USA). The
digested DNA was ligated to P1 and P2 adapters on both ends (sequences
are available in Peterson et al. 2012). We size-selected groups of 20
uniquely barcoded samples (index groups), retaining fragments between
400 and 700 bp using Blue Pippin (Sage Science, MA, USA). To incorporate
the full Illumina TruSeq primer sequences and unique indexing primers
into each library, we performed low cycle number PCR with Phusion
High-Fidelity DNA Polymerase (New England BioLabs), with the following
thermocycling profile: 98°C for 30 sec followed by 11 cycles at 98°C for
5 sec, 60°C for 25 sec, and 72°C for 10 sec with a final extension at
72°C for 5 min. We visualized the product of this amplification on a 1%
agarose gel and performed a final 0.73 AMPure cleanup to eliminate DNA
fragments smaller than 200 bp. Finally, sequencing was performed on an
Illumina HiSeq 2500 lane at the Cornell University Biotechnology
Resource Center as part of a larger sequencing batch, obtaining
single-end 150-bp sequences, with an average mean sequencing depth of
151 reads per locus per individual (range: 104-220).