Sample collection, DNA extraction and next generation sequencing
A subset of 2 year-old fish ranging from 10 to 25 (mean size = 182g) was randomly selected from each pen (Supplementary Table 1), and the fish were humanely euthanized and sacrificed to sample the gut contents. To obtain gut content samples for DNA extraction, the body cavity was cut open with a sterile scalpel and the hindgut of each offspring was collected. The gut samples were immediately stored in RNAlaterTM for transport to the research facility, where it was stored in the freezer at -20oC until DNA extraction.
We extracted DNA from gut content of the distal intestine using commercially available E.Z.N.A Stool DNA Kit (OMEGA Bio-tek) following the manufacturer’s protocol. Next generation sequencing library construction was completed in two steps as previously described (Heet al., 2017). Briefly, the universal primer set of 787F (V5F) (ATTAGATACCCNGGTAG) and 1046R (V6R) (CGACAGCCATGCANCACCT) was first used to PCR amplify the 16S rRNA encoding gene sequences containing the V5-V6 hypervariable regions. A short, Ion Torrent adaptor sequence was added to the 5’ end of the forward (acctgcctgccg) and reverse (acgccaccgagc) primers. The PCR product was visualized for amplification success on a 2% agarose gel, and PCR product purification was then carried out using Agencourt AMPure XP beads (Beckman Coulter Genomics GmbH, Mississauga, ON, Canada). A second short-cycle of PCR was conducted to ligate adaptor and the barcode sequences to the amplicon using purified PCR product from the first round PCR. The second round of PCR used: forward primer UniA (CCATCTCATCCCTGCGTGTCTCCGACTCAGXXXXXXXXXX GATacctgcctgccg), and reverse primer UniB (CCTCTCTATGGGCAGTCGGTGATacgccaccgagc), where the underlined sequence in UniA consisted of unique 10-12 bp barcode sequences necessary for the sample demultiplexing in sequence analysis and the lower-case sequence were the reverse compliment of the added sequence in the first primer set. Barcoded samples were combined based on PCR band intensity and a commercially available kit (GenCatchTM, Epoch Life Science, Inc., Sugar Land, TX., USA) was used to purify the PCR product from incomplete amplicons and primer dimers. The final library was sequenced with an Ion Torrent™ Personalized Genome Machine (Thermo Fisher Scientific, Inc., Mississauga, Canada).