DNA amplification and library
preparation
At the molecular laboratory we first discarded the alcohol from the
tubes and let the samples dry on large petri dishes with a sterile
filter paper inside a fume hood for 3 h. Once dry, we incubated the
samples in a lysis buffer (2 mL buffer ASL (Qiagen) + 250 µL proteinase
K) for 14 h at 56 °C with a gentle rotation. Subsequently, we recovered
the lysate and rinsed the arthropods with distilled water and 90%
ethanol, and we stored them in 80% ethanol. We used 225 µL of the
lysate to perform DNA extraction on a KingFisher Duo robot (Thermo
Fisher Scientific) with the Cell & Tissue kit (Thermo Fisher
Scientific), with an elution volume of 100 µL. We included lysis (empty
vial with just lysis buffer incubated alongside the samples) and
extraction (just for the robot part) blanks.
As metabarcoding markers, we amplified two fragments of the
mitochondrial cytochrome oxidase I (COI) and the 16S rRNA genes with the
primers COIBF2-COIBR1 (Elbrecht & Leese, 2017) and Chiar16SF-Chiar16SR
(Marquina et al., 2019b) respectively. Forward and reverse primers for
both markers had attached an 8 bp tag at the 5’ end for sample
multiplexing (Binladen et al., 2007) (see Table S4 for the multiplexing
information). The PCR reactions consisted of one Illustra Hot Start Taq
Mix RTG bead (GE Healthcare Life Sciences), 1 µL of each primer (10 nM),
2 µL DNA template and 21 µL of biology-grade water, for a final volume
of 25 µL; the temperature protocol was as follows: initial DNA
denaturation and Taq activation at 95 °C for 5 min, 40 cycles of
denaturation at 95 °C for 30 s, annealing at 48/50 °C (COI/16S) for 45 s
and extension at 68 °C for 45 s, and a final phase of extension at 72 °C
for 10 min. We ran all PCRs in duplicated, and a blank was included in
the batches of both markers. We confirmed PCR amplification in an
agarose gel, pooled the replicates together, and measured DNA
concentration using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific)
with the broad-range reagents. We discarded lysis, extraction and PCR
blanks after not producing a visible band in the gels and returning
undetectable values of DNA concentration. We then pooled the tagged PCR
products equimolarly, and cleaned-up primers and primer dimers using
MinElute columns (Qiagen). We prepared libraries with the TrueSeq
PCR-free kit (Illumina). We visualized ligation products on a 2%
agarose gel, cut the fragments of the desired length out of the gel, and
purified them using the QIAquick gel extraction kit (Qiagen). We pooled
the libraries to equimolar concentrations and sequenced them on a single
Illumina MiSeq lane using 2 x 300 bp paired-end v3 chemistry run at
SciLifeLab (National Genomics Infrastructure, Stockholm).