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.