Insights into the microbiota of Asian seabass (Lates calcarifer) with tenacibaculosis symptoms and description of sp. nov. Tenacibaculum singaporense
Healthy fishes were equally distributed into two tanks which underwent different treatments; one that was exposed to an experimental infection and another that was kept as healthy control.  Deceased fish with tenacibaculosis symptoms from a local aquaculture farm described earlier were used as inoculum. After inoculation, fish was left for a few days until the tenacibaculosis symptoms appeared. Once the symptoms occurred, fish was sacrificed humanely by dipping into ice for up to 30 seconds and spiking immediately. All fish were sacrificed 75 weeks after birth. Fish gut, kidney, brain and infected skin parts were collected from 10 fish per treatment by sterile scalpels, individually placed in sterile vials and flash frozen by liquid nitrogen. The samples were stored at -20?C until processed. Experimental infection and dissection in the laboratory was performed according to the approved IACUC protocol TLL(F)-14004.Samples were transferred to sterile bead beading vials containing 0.7g zirconium beads, and 200l of 20% SDS, 282l Buffer A, 268l Buffer PM, 550l phenol/chloroform/isoamylalcohol (25:24:1, pH 8) were added. The pelleted cells were subject to phenol-chloroform-based extraction by Rius et al. that combined mechanical and chemical lysis of cells. The samples were quantified and diluted to 40 ng/l per sample and stored in -20 C for further use.The extracted DNA was processed according to the Earth Microbiome Project (http://www.earthmicrobiome.org/protocols-and-standards/16s/) as described in Thompson et al except for two points. Firstly, primer pair 27f and 338r spanning hypervariable region V1-3 was used instead as the designed primer pair (515FB and 806RB) also amplified host fish DNA. Secondly, a dual-indexing strategy was used as described by Fadrosh et al to reduce the number of barcoded primers required. PCR was conducted with QIAGEN Taq MasterMix (CAT NO 201445, QIAGEN, Germany) in triplicates (plus one negative control per sample) under the condition: 94 C for 3 mins; 35 cycles of 94 C for 45 secs, 50 C for 60 C, 72 C for 90 secs; 72 C for 10 mins; 4 C hold. The amplicons were quantified with Quant IT Picogreen (CAT NO P7589, Thermo Fisher Scientific, USA), pooled together in equimolar concentrations and sequenced at Singapore Centre for Environmental Life Sciences Engineering (SCELSE) at Nanyang Technological University using Illumina MiSeq paired-end chemistry (251x251bp). The output raw sequences were analysed with MOTHUR v1.39.1 according to the standard MiSeq protocol. The sequence pairs were merged, de-multiplexed and quality-filtered accordingly.