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).