18S rRNA amplicon sequences from the Bronx River Estuary, New York City
DNA sequences were amplified by primers Euk7F (Medlin et al. 1988) and Euk570R (Weekers et al. 1994) (Euk7F: AACCTGGTTGATCCTGCCAGT + an identifier barcode; Euk570R: GCTATTGGAGCTGGAATTAC) using the Qiagen HotStarTaq Plus Master Mix Kit (Qiagen, USA) under standard conditions. After agarose gel checks, PCR products were purified with calibrated Ampure XP beads (Agencourt Bioscience, USA). DADA2 was used as a QIIME2 plugin to denoise and quality filter the sequences, assign amplicon sequence variants (ASVs), and generate a feature table of ASVs (features) and metadata (Callahan et al. 2016). Primers and low-quality base calls were trimmed at the ends of each single-end sequence during the DADA2 step. The decontam program was then run on the feature table to remove potential contaminants  (Davis et al. 2018).  To taxonomically classify the 18S reads, the q2-Nave Bayesian classifier as implemented in QIIME2 using the SILVA 138 reference database (Quast et al. 2013) trained on the entirety of the 18S rRNA gene was employed (Bokulich et al. 2018, Karst et al. 2018).  Once taxonomic classification was complete, we exported the ASV feature table, taxonomy, and sample metadata for analysis in R.