16S rRNA gene amplification and sequencing

Environmental DNA was extracted from all 54 sediment samples and the 16S rRNA gene was amplified using primers 515F and 806R targeting the V4 region of the 16S rRNA gene, and thus biased to the amplification of bacterial DNA (Caporaso et al., 2011; D'Amore et al., 2016) and sequenced using an Illumina MiSeq platform. The read counts before and after adapter trimming and quality control are summarized (Table S1). Further analysis used only R1 and R2 reads and the samples H18-3b and L18-6c were excluded from the dataset due to low pair reads [ < 100 base pairs (bp)]. Following adaptor sequence removal and quality trimming, the remaining 52 samples contained between 149,199 and 1,107,840 trimmed reads (Table S1). Amplicon generation targeting the 16S rRNA gene was performed for each of the 54 environmental DNA samples, and amplified by 10 cycles of PCR using the Kapa enzyme (see Supplementary Material for detailed methodology). DNA concentrations were recorded using a Qubit fluorometer (ThermoFisher) and scanned on the Fragment Analyser (Advanced Analytical). This allowed pooling of samples based on a size selection of 350–650 base pairs. Sequencing was carried out on an Illumina MiSeq at 2 × 250 base pair (bp) paired-end sequencing with v2 chemistry. Fragmented PhiX phage was added to the sequence library in order to increase the sequence complexity. Sequences are published in the European Nucleotide Archive (ENA) under the study accession number PRJEB13670 and sample accession numbers ERS1124371-ERS1124422).