DNA extraction, PCR amplification, and Illumina sequencing
Bulk specimen subsamples were DNA extracted using the DNeasy Blood and
Tissue Kit (Qiagen) in a Kingfisher Flex robotic system (Thermo
Scientific). The bulk of Coleoptera was extracted non-destructively by
splitting specimens into parts or puncturing the body. The mesofauna
sample was homogenized in 1.5 ml vials with glass pestles. DNA extracts
were quantified using Nanodrop 1000 UV–Vis spectrophotometer (Thermo
Scientific), and the corresponding Coleoptera and mesofaunal subsample
pairs were combined at a ratio of 1:10 in the amount of DNA (in
accordance with the expected species diversity for these two fractions
(Arribas et al., 2021)). The bc3’ fragment, corresponding to the 3’ 418
bp of the COI barcode region was amplified, using tailed primers
corresponding to the Illumina P5 and P7 sequencing adapters for
subsequent library preparation. Three independent PCR reactions were
performed for each sample, and amplicons were pooled. All information
regarding primers, PCR reagents and conditions is provided in Table S2.
Amplicons were then cleaned using Ampure XP magnetic beads and used as
the template for limited-cycle secondary PCR amplification to add
dual-index barcodes and the Illumina sequencing adapters (Nextera XT
Index Kit; Illumina, San Diego, CA, USA). Metabarcoding libraries were
then sequenced on an Illumina MiSeq sequencer (2 x 300 bp paired-end
reads), dedicating approximately 1% of a flow cell to each library,
producing paired reads (R1 and R2) with a dual tag combination for each
sample.