title: 16S amplicon sequencing of Deepwater clam
design: The DNA from all samples was subjected to a 16S amplicon sequencing following a previously published protocol (Pichler et al., 2018). Briefly, 5 ng of DNA was subject to PCR to amplify a 400 bp section of the V4 region of the bacterial 16S rRNA gene using 10 M of tagged, indexed primers forward: 5GTGCCAGCMGCCGCGGTAA3 and reverse: 5GGACTACHVGGGTWTCTAAT3. Indexed primers were designed allowing the samples to be multiplexed. The high fidelity NEBNext PCR mix was then used with cycling conditions of 99C for 30 secs followed by 30 cycles of 98C for 10 sec, 55C for 30 sec, 72C for 30 sec and a final step of 72C for 2 min. Each sample was subject to triplicate PCR reactions to reduce the error rate . The PCR product was cleaned up using AMPure XP beads at a ratio of 0.8:1. The cleaned-up PCR product was eluted in 10mM Tris pH 8.5. The quantity of tagged DNA was checked using the ProNex NGS Library Quantification Kit (Promega, UK).  The samples were diluted to 4 nm, pooled together and denatured using 0.2M NaOH. The pooled library was finally diluted to 12pM with HT1 buffer and run on an Illumina MiSeq using the 600 cycle V3 technology. To increase the cluster density, sequencing runs were spiked with the genomic DNA of lumpfish.