Freshwater environment of eukaryotes 18S
Total environmental DNA was extracted using column based, DNeasy PowerSoil Pro Kit (47016) (Qiagen, Germany) and purified using VAHTS DNA Clean Beads (N411-03, Vazyme, China). Afterwards, the hypervariable V4 region of eukaryotic 18S rRNA genes (~400 bp) was amplified with universal primer pair of 547F (5- CCAGCASCYGCGGTAATTCC-3) and V4R (5- ACTTTCGTTCTTGATYRA-3) (Stoeck et al. 2010). PCR products from each sample were purified and pooled in equal amounts, followed by pair-end 2 x 250 bp sequencing using the Illumina NovaSeq platform at Shanghai Personal Biotechnology Co., Ltd. Quality check and chimeras removal of raw amplicon sequence variants (ASVs) were conducted using DADA2 (Callahan et al. 2016). Filtered ASV sequences were further classified with SILVA database (Pruesse et al. 2007) using QIIME2 (Bolyen et al. 2019). ASVs annotated as multi-cellular metazoan and parasites (mostly fungi and some ameoba) that were not the focus of this study were excluded from further analysis.