16S amplicon of zebrafish larvae
To identify the 16S rRNA gene diversity in our facility and fish collected from 4 other zebrafish facilities, fish were reared as described above. GF fish were sterilised as above, and uninfected germ-free and conventional fish were collected at 6 dpf and 11 dpf. Infection was carried out as above with F. columnare ALG for 3h by bath immersion, followed by transfer to clean water. Infected conventional fish were collected at 6 dpf 6h after infection with F. columnare and at 11 dpf, the same as uninfected fish. GF infected larvae were only collected at 6 dpf 6h post infection, as at 11 dpf all larvae had succumbed to infection. Triplicate pools of 10 larvae were euthanized, washed and lysed as above. Total DNA was extracted with the Mobio PowerLyzer Ultraclean kit as described above and quantified with a NanoDrop ND-1000 Spectrophotometer and sent to IMGM Laboratories GmbH (Germany) for Illumina sequencing. Primers Bakt_341F (5-CCTACGGGNGGCWGCAG-3) and Bakt_805R (5-GACTACHVGGGTATCTAATCC-3), amplifying variable regions 3 and 4 of the 16S gene were used for amplification (33). Each amplicon was purified with solid phase reversible immobilization (SPRI) paramagnetic bead-based technology (AMPure XP beads, Beckman Coulter) with a Bead:DNA ratio of 0.7:1 (v/v) following manufacturers instructions. Amplicons were normalized with the Sequal-Prep Kit (Life Technologies), so each sample contained approximately 1 ng/l DNA. Samples, positive and negative controls were generated in one library. The High Sensitivity DNA LabChip Kit (was used on the 2100 Bioanalyzer system (both Agilent Technologies) to check the quality of the purified amplicon library. For cluster generation and sequencing, MiSeq reagents kit 500 cycles Nano v2 (Illumina Inc.) was used. Before sequencing, cluster generation by two-dimensional bridge amplification was performed, followed by bidirectional sequencing, producing 2 x 250 bp paired-end (PE) reads.