2.2 DNA extraction, sexing and sequencing
High molecular weight DNA was extracted from flash frozen heart and
kidney using the Nanobind Tissue Big DNA Kit v1.0 11/19 (Circulomics). A
Qubit fluorometer was used to quantify DNA concentrations with the Qubit
dsDNA BR assay kit (Thermo Fisher Scientific). RNA was extracted from
heart, spleen, kidney, gonads, brain, and liver stored in RNA later
using the RNeasy Plus mini Kit (Qiagen) with RNAse-free DNAse (Qiagen)
digestion. RNA quality was assessed via Nanodrop (Thermo Fisher
Scientific). We extracted DNA for population genomics from blood and
feather samples using the Monarch® Genomic DNA Purification Kit (New
England BioLabs, Victoria, Australia). We quantified DNA concentrations
using a Qubit 3.0 fluorometer (yield range 10.3 – 209 ng
μl-1, Table S1) and standardised the concentration of
each sample to 10-30 ng µl-1 DNA for 20-25 μl and
determined the sex of individuals using a polymerase chain reaction
(PCR) protocol adapted from Fridolfsson and Ellegren (1999,
Supplementary file S1). We arranged the samples on a single 96 well
plate, containing five technical replicates of the samples with the
highest DNA concentrations, an additional 21 non-technical replicates
including all of the King Island samples, five extra samples from
mainland Tasmania and one negative control.
Double-digest restriction associated DNA (ddRAD) sequencing following
Peterson et al. (2012) was undertaken at the Australian Genome Research
Facility, Melbourne on an Illumina NovaSeq 6000 platform using 150bp
paired-end reads. Samples were first quantified using Quantifluor and
visualised on 1 % agarose e-gel to ensure all samples exceeded the
minimum input DNA quantity of 50 ng. Three establishment samples with at
least 250 ng DNA that were representative of the distribution of the
samples (2 Tasmanian scrubtits, 1 King Island scrubtit) were used to
determine the optimal combination of restriction enzymes, which wereEcoRI and HpyCH4IV . Further details on the library
preparation protocol are provided in Supplementary file S1.