Powersoil pro extraction kit

DNA was extracted from mussel gut tissue using a PowerSoil Pro extraction kit (Qiagen, Germantown, MD, USA) as described previously [30 (link)]. For seston and sediment microbiome analysis, filters or 30 mg of sediment were added directly into the bead beating tube of the extraction kit, before performing the same procedure.
Dual-indexed barcoded primers were used to amplify the V4 region of the 16S rRNA gene of the extracted DNA following established techniques [6 (link), 30 (link)] using primers from [31 ]. The amplified 16S rRNA gene fragments were combined and spiked with 20% PhiX before being sequenced on an Illumina MiSeq at the University of Mississippi Medical Centre Molecular and Genomics Core facility. Sequence data were obtained as FASTQ files, which were further processed using Mothur and DADA2. Sequencing quality was assessed using fastqc reports and was summarized using ‘fastqcr’ R-package [32 ] and provided in S3 Table in S2 File.

All five pools of cecal droppings originating from the second production cycle were used for DNA extraction (n = 178) using the PowerSoil Pro extraction kit (Qiagen, Hilden, Germany). DNA yield was measured by use of nanodrop spectrophotometry (ThermoFisher Scientific, Waltham, MA, USA) and Quantus fluorometry (Promega Corporation, Fitchburg, WI, USA). Samples were diluted according to their total DNA yield, as measured by the Quantus double-stranded DNA assay. If >100 ng/µL: 1:5 dilution, if <100 ng/µL: 1:2 dilution, if <10 ng/µL: no dilution. Subsequently, amplicon sequencing of the bacterial V3–V4 region of the 16S rRNA gene was performed as described by the Illumina protocol and the primers of Klindworth et al. [53 (link)] on an Illumina MiSeq sequencer with 2 × 300 bp reads (Admera Health, South Plainfield, NJ, USA). The sequencing data have been deposited with links to the BioProject accession number PRJNA643676 in the NCBI SRA database.