DOI: 10.5061/dryad.80gb5mkzz
title: Tammar & parma wallaby GBS data from NZ

description: While conducting a landscape genomics study of invasive tammar wallabies
 (Notamacropus eugenii) in Aotearoa New Zealand we discovered that parma
 wallabies (N. parma) are also present in the North Island. This population
 has gone undetected for at least 30 years (and potentially for over a
 century), hidden amongst the morphologically similar tammar wallabies. The
 fact that an invasive wallaby species could remain undetected for so long,
 highlights the need for greater monitoring efforts for invasive species
 including genomic species identification.
description: <strong>Laboratory and bioinformatic
 methods</strong><br>We used a DNeasy blood and tissue
 extraction kit on a QiaCube to extract the DNA from c. 0.5 cm2 of tissue,
 with an overnight digest using proteinase K according to the
 manufacturer’s protocols (QIAGEN, Hilden, Germany). DNA was eluted into
 200 μL of Buffer AE and then stored at − 20 °C. The quality and quantity
 of the DNA were evaluated using a denovix DS-11 nucleic acid
 spectrophotometer, examining the 260/230 and 260/280 ratios to determine
 if there was any contamination. Any sample that did not meet the criteria
 for purity (260/280 = 1.7–2.1, 260/230 = 1.9–2.2) were removed.
 All DNA extractions were then diluted to a uniform 50 ng/μL (with
 a concentration step using a SpeedVac for samples that had low
 concentration), with 1 μg of DNA sent for each sample for GBS sequencing.
 This GBS was performed at GenomNZ Animal Genomics Group (AgResearch, New
 Zealand). Procedures followed Dodds et al. (2015) after Elshire et al.
 (2011), with the following modifications. Briefly, genomic DNA was
 digested with PstI and MspI restriction enzymes (NEB R140L and R0106L, New
 England Biolabs, Ipswich, United States). We chose enzymes based on
 bioanalyser traces (2100 Bioanalyser, Agilent Technologies, Santa Clara,
 United States) showing an even digestion pattern with no evidence of
 repeat sequences through the region of interest. Following ligation to
 barcoded adapters, the uniquely barcoded individuals were pooled into two
 multiplexed libraries of 94 samples. Libraries post-pooling were run
 through PCR in multiples of four and pooled again before column clean-up,
 then each library was further purified and size selected (193–500 bp)
 using a Pippin (SAGE Science, Beverly, United States; 2% agarose, dye-free
 with internal standards CDF2050, Marker L CDF2010). We then sequenced each
 library on an Illumina HiSeq2500 using single-end reads, with 101 cycles
 in high-output mode (v4 chemistry). Quality checks and
 adapter removal followed Dodds et al. (2015). Raw fastq files were quality
 checked using FastQC v. 0.10.1
 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Barcodes and
 adapters were removed using cutadapt (Martin 2011), then a random 15,000
 reads were checked for contamination using BLAST + against the NT database
 (https://blast.ncbi.nlm.nih.gov ), with the following settings: blastn
 -query—-task blastn—num_threads 2 -db nt -evalue 1.0e-10 -dust ‘20 64 1’
 -max_target_seqs 1 -outfmt ‘7 qseqid sseqid pident evalue staxids
 sscinames scomnames sskingdoms stitle’. We then produced a catalogue of
 SNP loci (single nucleotide polymorphisms, or single base variants)
 following Dodds et al. (2015) and the general guidelines of (Benestan et
 al. 2016). After trimming adapters with cutadapt
 (Martin 2011), reads were mapped and SNPs detected via a modified
 reference-based pipeline in bcftools (Danecek et al. 2011) using the
 recently completed tammar wallaby genome, which was assembled from a male
 obtained by lead author Dr Veale from the Waimangu thermal area in the Bay
 of Plenty, New Zealand (mMacEug1.pri: GCA_028372415.1). All following data
 filtering steps were then conducted using vcftools (Danecek et al. 2011).
 Filtering parameters for loci were: Minor allele count = 3, Maximum
 average depth = 45, Minimum depth = 5, Maximum missingness = 0.3. To
 retain an individual, a minimum average depth of 2 was required (measured
 before other filtering). <strong>GBS
 analyses</strong><br>We performed a Principal Coordinate
 Analyses (PCoA) within KGD (Dodds et al. 2015) on the genomic relatedness
 between individuals. Based on the KGD analyses there were clearly two
 highly divergent groups (which corresponded to the two species, see
 results), though two individuals had slight intermediate values between
 these groups. To further investigate this, we converted the VCF file to a
 Geste file to enable us to calculate the number of fixed differences
 between the two species (excluding the two potential hybrids). Once the
 alleles that were fixed differences were identified between the two
 species were identified we then counted the proportion of each of these
 categories in the two potentially hybridised individuals (as per
 Etherington et al., 202219). To map the distribution of samples we used
 the R package ggmap (Kahle & Wickham 2013).
description: # Tammar & Parma Wallaby GBS data from NZ
 [https://doi.org/10.5061/dryad.80gb5mkzz](https://doi.org/10.5061/dryad.80gb5mkzz) ## Description of the data and file structure Metadata for each sample including ID, Species, Date, Sex, Latitude, and Longitude, as well as the first 4 Principal Coordinates can be found in the .csv file.   The VCF file contains the filtered SNP data for all samples. ## Code/software No specific software is required, the csv can be read in any text editor or database software.  The vcf can be read in any text editor and can be analysed with many packages such as vcftools, bcftools, plink etc.