Arctic-boreal bryophytes from sedimentary ancient DNA metabarcoding

Description

Methods (English)

DNA samples from the sediment core were subsampled under cool and clean conditions and ancient DNA work was conducted in the paleogenetic laboratories at Alfred Wegener Institute, Potsdam, Germany. DNA was extracted in batches, each of the batches included nine samples (3-10 g sample–1) and one extraction control, using the PowerMax® Soil DNA Isolation Kit (Mo Bio Laboratories, Inc., USA). All subsequent steps were performed according to the instructions of the manufacturer Qiagen, except for the final elution volume which varied between 1.6 and 2.0 mL. PCR reactions were conducted using g and h primer targeting the vascular plant trnL P6 loop of the chloroplast genome, each modified with unique 8 bp tags differing by at least five base pairs to distinguish samples post-sequencing. After pooling the PCR samples from each sediment core into a PCR Pool, sequencing for each pool was performed by Fasteris Gensupport SA sequencing service (Switzerland) with the paired-end sequencing on a HiSeq or NextSeq Illumina platform. Raw sequences were analysed using OBITools software (version 3.0.0) against a specific plant database (SibAla_2023). The customised database SibAla_2023 has been compiled using different R packages with the following steps. Taxa selection from a given region (55–90°N, 50–150°E and 40–90°N, 150°E–140°W) and taxa occurrences (>10) using the Global Biodiversity Information Facility (GBIF, 71) resulting in 233 families, 1059 genera, and 4849 species. GBIF occurrences were aligned with available trnL P6 loop sequence types from public databases. The SibAla_2023 database compared with given occurrences in GBIF amounts to 95.7% (family-level), 89.4% (genus-level), and 70.1%(species-level) of taxonomic coverage. Finally, the SibAla_2023 database includes a total of 4939 entries, comprising 3398 species, 947 genera, and 223 families that collapse into 2371 unique P6 loop sequence types.