Laboratory methods
All laboratory work was performed at the Center for Conservation
Genomics (CCG), Smithsonian Conservation Biology Institute, Washington,
DC. We extracted DNA from liver and ear punch samples using a DNeasy
Blood and Tissue Kit (Qiagen, Valencia CA) following the manufacturer’s
protocol. We amplified whole mitogenomes in two fragments using long
range PCR, fragmented the PCR products to an average length of 500 base
pairs (bps) using a Qsonica Q800R sonicator (QSonica, Newtown, CT, USA),
and prepared single-indexed DNA libraries for sequencing using a Kapa
LTP Library Preparation kit (Kapa Biosystems, Wilmington, MA) following
Hawkins et al. (2016). We pooled libraries equimolarly and sequenced on
an Illumina MiSeq with 2 × 100 base pair (bp) reads (Illumina, Inc., San
Diego, CA).
We used in-solution DNA hybridization to enrich genomic DNA for UCEs
following Hawkins et al. (2016). We sheared DNA extracts and constructed
indexed libraries as above. We quantified libraries using a Qubit
fluorometer (Life Technologies) with a 1× dsDNA HS assay kit and
multiplexed 4-8 samples equimolarly prior to enrichment. We used a
NimbleGen SeqCap EZ kit (Roche, Basel, Switzerland) containing 54,689
unique 60-bp DNA probes representing 5,561 vertebrate UCE loci with an
average of 4× tiling per base per locus to enrich multiplexed libraries
following the manufacturer’s protocol. Post-enrichment libraries were
amplified with 12–14 cycles of PCR using Kapa HiFi HotStart DNA
polymerase (Kapa Biosystems, Wilmington, MA) following the
manufacturer’s protocol.
Following visualization on a Bioanalyzer 2100 (Agilent Technologies,
Santa Clara, CA) with High Sensitivity DNA kits, enriched libraries were
quantified via qPCR using the Kapa Biosystems Illumina Library
Quantification Kit (Kapa Biosystems, Wilmington, MA). Samples were
pooled equimolarly and sequenced with 2 × 150 bp reads on Illumina
HiSeq2000 (Semel Institute of Neurosciences, UCLA, & University of
Copenhagen, Denmark) and MiSeq platforms (CCG).