title: Atlantic salmon microbiota: Sample CK02
design: Studying the composition of microbial communities in skin mucus, and how the microbiota is structured in ulcers will enhance our understanding of ulcer etiology of Atlantic salmon. To achieve this we used seawater farmed Atlantic salmon and sampled the base and edge of ulcers at the end of winter (April) and at the end of summer (September), in addition to skin-mucus of healthy individuals. In order to assess microbiota associated to the host and to get a better insight into the environmental ecology we additionally sampled the seawater column and sediment layer underneath the farm facility in addition to the distal intestine of Atlantic salmon. Enzymatic lysis in combination with the UltraClean® Soil DNA Isolation Kit (MO BIO, Laboratories, Inc.) was usedfor the DNA extraction from water and sediment. Sampled skin-mucus and ulcer-tissue were first subjected to mechanical disruption before enzymatically digested and the DNA extraction was continued with the QIAamp® cador® Pathogen Kit. Intestinal samples were pretreated similarly by mechanical and enzymatic disruption before DNA extraction was conducted with the QIAamp DNA stool mini kit.  The DNA composition and diversity were characterized by PCR amplification of the V1–V3 region of the 16S region of rDNA using the universal primers 27F (AGAGTTTGATCCTGGCTCAG) and 534R (ATTACCGCGGCTGCTGG). The 16S rRNA genes were sequenced by paired end reads performed on an Illumina MiSeq. Forward and reverse fragments were paired using FLASH v. 1.2.11 allowing a minimum overlap of 60 bp, a maximum overlap of 250 bp and a mismatch density in the overlapping region of 20%. The reads were then quality filtered using mothur v. 1.33.3. All reads shorter than 310 bp, longer than 540 bp, or containing ambiguous bases or homopolymers of length >10 were discarded from the dataset, as well as all those reads with an average PHRED quality score <30. Read quality was further evaluated using a 6 bp long sliding window across the length of the sequence, which was discarded if the average PHRED quality score across the window fell below 20. The uchime algorithm as implemented in Qiime v. 1.8.0 was used for both de novo and reference based chimera detection against the GreenGenes database requiring a minimum divergence of 0.8% between putative parents and an abundance skew of 2. Sequences were considered chimeric and discarded whether they were identified by one or both of the de novo and reference-based analyses.