Materials and Methods
DNA submitted to DArT was extracted from 60 L. conditor tissue samples collected by staff during routine trapping events at Arid Recovery Reserve, South Australia, between 1999 and 2003. DNA extraction was completed following the methods described by Barclay et al. (2006) and samples were subsequently stored at -20°C prior to sequencing by DArT. Following library preparation and sequencing by DArT using their proprietary workflow, we obtained the raw Illumina data in FASTQ format. We used the Paleomix v1.2.14 pipeline to process these data: AdapterRemoval2 v2.3.1 was used to trim residual adapter sequences (using default parameters) and filter reads shorter than 30 bp, after which all remaining reads were mapped against the repeat-masked house mouse genome assembly (GRCm38) using BWA v0.7.17 mem algorithm. We then used the idxstats command in SAMtools v1.10 to extract the number of reads mapping to each scaffold of the reference assembly.
We visualised read-dosage for each sample by using RStudio v1.3.1073 to plot the number of reads mapping to the autosomes and X chromosome (as a proportion of the total reads per sample) versus scaffold length. To determine the sex of the Greater Stick-Nest Rat samples we used Gower et al.’s (2019) python script sexassign(https://github.com/grahamgower/sexassign). This python script uses a likelihood ratio test to assign samples to either male or female on the basis of the observed ratio of reads mapping to the X chromosome versus the autosomes. However, because an underlying assumption of the calculations made by sexassign is that the X chromosome in homogametic females should receive the same read-dosage as an autosome of the same length (i.e. read dosage of ~1X versus ~0.5X in heterogametic males), we first multiplied the number of reads mapping to the X chromosome for all samples (regardless of putative sex) by a factor of 2.12 (the expected read-dosage for the X chromosome, 0.065, divided by the observed mean read-dosage for the X chromosome in putatively female samples, 0.0308, see Results).