raw fastqs of Eastern Spruce Budworm: QC 2014, 555 individuals
DNA extraction was performed on 6 to 33 individuals per site (Larroque et al. 2019), using a Qiagen DNA Blood and Tissue kit. Male samples had wings removed before extraction, and female samples had wings and abdomen removed before extraction. DNA concentration and purity was assessed using spectrophotometry in a Nanodrop 1000 (Techno Scientific), retaining only samples with 260/280nm absorbance ratios > 1.6 and 260/230nm ratios > 0.95. Due to comparatively lower DNA quality, DNA extraction from 277 L2s in New Brunswick and 11 in Maine was performed using a modified NucleoSpin tissue XS kit (Macherey-Nagel) protocol. Samples were pre-lysed with 80 L buffer B3 and 8 L proteinase K solution, vortexed and spun in a mini centrifuge. Incubation was done at 56 C overnight. Lyse was completed by adding 80 L buffer B3, vortexing briefly and incubating 5 min RT before adding 80 L 96100% EtOH, vortexing and spinning down. DNA binding was done by pipetting the supernatant into a column (2 ml collection tube+NucleoSpin tissue XS column). Samples were then centrifuged 1 min and washed twice by adding 50 L Buffer B5 to the column and centrifuging 1 min the first time, 2 min the second time. DNA was then eluted with 1520 L buffer BE (incubation RT 5 min before centrifugate 1 min to elute). All centrifugations were done at 11,000g. All DNA samples were prepared for genotype-by-sequencing (GBS, Elshire et al. 2011) following the protocol described in Brunet et al. (2017). GBS libraries (96 plex) were prepared using the PstI-MspI enzyme combination by the Institut of Biologie Intgrative et des Systmes (IBIS) at Universit Laval (Quebec City, Canada), following the protocol described in Poland et al. (2012). After final amplification of the pooled fragments, 600ng of each amplified library was normalized in order to remove repetitive fractions by treatment with duplex-specific nuclease (Zhulidov et al. 2004), followed by an additional PCR step using a selective reverse primer extending a single base (C) inserted past the 3 restriction site, selectively amplifying 0.25 of total fragments, increasing read depth (Sonah et al. 2013). Single-end sequencing (100bp reads) was performed using a Illumina HiSeq2000 sequencer at the McGill University-Gnome Qubec Innovation Centre, Montreal, Canada.