title: COI metabarcoding of sieved bryophyte substrate; sample BM10
design: The protocol used for sample processing followed that of Surmacz et al. (2025). Bryophyte samples were fragmented by hand, soaked in water, sieved through a set of metal sieves with mesh sizes of 500 m, 250 m, and 36 m. Pool of ten samples (0.5 ml of sediment from the finest sieve of each sample) was used to form one bulk sample. DNA was extracted using the DNeasy PowerMax Soil Kit (Qiagen), following the manufacturer's protocol with a modification involving 30-minute preincubation with 30 ul of proteinase K. For metabarcoding, a fragment of the COI gene, referred to as COI_A, was used as the target.  Specific primers for this gene region, modified and tested by Surmacz et al. (2025). Library preparation was performed using a two-step PCR method. The first PCR reaction amplified the target region using region-specific primers with Illumina overhangs. This reaction involved an initial denaturation step of 5 minutes at 95C, followed by 30 cycles of 30 seconds of denaturation at 95C, 90 seconds of annealing at 55C, and 20 seconds of elongation at 72C, with a final elongation step of 10 minutes at 72C. The product of the first reaction was then used in a second, indexing PCR, which amplified the target DNA further. This step employed primer sets containing flow-cell binding domains and unique indices from the Nextera XT Index Kit (FC1311001/FC1311002) and followed the Illumina protocol (Illumina 2013). The resulting libraries were sequenced on an AVITI instrument (Element Biosciences, San Diego, CA) using 300-bp paired-end mode.