Mitochondrial DNA amplification and high-throughput sequencing
We amplified mitochondrial (mt)-genomes of 95 randomly selected European brown hares equally distributed across all sampling sites (n= 1 to 5 per site, n=36 sites). Randomisation was performed using Research Randomizer 4.0 (http://www.randomizer.org/). In an initial step, we performed two independent long range PCRs using Lepus europaeus -specific primers covering the mt-genome range (KY211031) 19-9,464 (mtF1_lepus_S_Roos19 5’-AAA GCA AAG CAC TGA AAA TGC T and mtF1_lepus_AS_Roos19 5’-CCA AAA CTA ACT GAT TGG AAG T) and 8,500-484 (mtF2_lepus_S_Roos19 5’-ATT AGT CCA ACA ACA GCC CTA and mtF2_lepus_AS_Roos19 5’-CTT AGC TAT CGT GAG TTC GAA). Primers were designed based on available mt-genome data in GenBank using Geneious Prime 2021.2.2 software. PCR reactions were adjusted to 50 µl and included the following components: 10 µl 5x PrimeSTAR GXL buffer (Takara Bio Europe SAS/Clontech Labs, Saint-Germain-en-Laye, France), 1 µl PrimeSTAR GXL DNA Polymerase (Takara Bio Europe SAS, Saint-Germain-en-Laye, France), 4 µl dNTP mixture (2.5 mM each), 28.5 µl RNase free water, 2 µl of each 10 µM primer and ~250 ng DNA (1-3.5 µl depending on DNA concentration). Cycling conditions were as follows: 35 cycles at 98°C for 10 sec, 59°C for 15 sec and 68°C for 12 min. Subsequently DNA amplicons were purified using SPRISelect magnetic beads (Beckman Coulter, Inc., Krefeld, Germany) followed by the determination of DNA concentration utilizing the Qubit 4.0 fluorometer (Thermofisher Scientific, Darmstadt, Germany). Next, we pooled 250 ng of each PCR product and adjusted the final volume to 26 µl that were subsequently used for next-generation library preparation. Libraries were prepared using the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs, Frankfurt am Main, Germany) according to the manufacturer’s instructions in combination with the NEBNext Multiplex Oligos for Illumina (NEBE6440, 96 Unique Dual Index Primers plate) to ensure that all samples can be sequenced at once. DNA was enzymatically fragmented to an average size of 300-700bps. Following the manufacturer’s guidance and prior to sequencing, library quantification was performed with the NEBNext Library Quant Kit for Illumina (New England Biolabs) run on a StepOnePlus RealTime PCR System (Thermo Fisher Scientific, Darmstadt, Germany). In a final step, we normalized the samples to a concentration of 10 nM each using the previously generated quantification data. Samples were then pooled and sent to the NGS Integrative Genomics Core Unit (NIG, University Medical Center, Goettingen, Germany) for Illumina MiSeq 250 bp paired-end sequencing.