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.