metabarcoding of tardigrades from environmental sample of bryophyte (ALT-293)
The protocol used for sample processing followed that of Surmacz et al. (2024). Bryophyte sample was carefully fragmented by hand and soaked overnight in tap water in a 0.5-liter plastic beaker. The following day, the samples were sieved through a set of metal sieves with mesh sizes of 500 m, 250 m, and 36 m. DNA was extracted from the sediment collected in the finest sieve (0.25 g of sediment) using the DNeasy PowerSoil Pro Kit (Qiagen), following the manufacturer's protocol with a modification involving preincubation with proteinase K, as described in Surmacz et al. (2025). Extraction blank samples were included to monitor for potential contamination. The final DNA was eluted in 100 l of elution buffer and stored in a freezer. 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.
SUB15047094
ALT-293