MiSeq Illumina of Spring seawater at 100 m depth
The DNA of euphotic zone and trap samples was extracted using a QIAGEN DNeasy Blood and Tissue Kit and following the protocol described in De Martini et al., 2018. The 16S rRNA gene amplicon libraries were prepared and sequenced using an Illumina MiSeq platform (reagent kit v3; 2x300bp paired-end; Illumina Inc.) by the Dalhousie University Integrated Microbiome Resource (IMR). The universal V4-V5 primer set: 515F-Y (5-GTGYCAGCMGCCGCGGTAA-3) (Parada et al., 2016) and 926-R (5-CCGYCAATTYMTTTRAGTTT-3) (Parada et al., 2016; Quince et al., 2011) were employed. The demultiplexed reads received from IMR were analyzed using QIIME2 (v2021.4) (Boylen et al., 2019), including diversity measures. After importing the FASTQ files into QIIME2, a median Q-score above 30 was chosen to avoid error rates larger than 0.1%. Primers were then trimmed using the q2-plugin cutadapt. Amplicon Sequence Variants (ASVs) from trimmed reads were prepared with q2-plugin DADA2 (Callahan et al., 2016), were we tested different sequence base positions as truncation parameters to ensure sufficient read recovery during the denoising step. The best non-chimeric recovery was obtained at ca. 70% for most samples using position 243 (forward) and 166 (reverse) with a 12-base minimum overlap. ASVs were classified using the SILVA (v 138.1) database (Quast et al., 2013) and the PR2 (v 14.4) database (Guillou et al., 2013), which includes PhytoREF (Decelle et al., 2015) as of April 2021, for the photoautotrophs.