title: 18_sediment_analysis
design: Tufts of seal hair, trimmed adjacent to the skin, and the gut content of brittle stars were homogenized in bead-beating tubes at maximum speed for five minutes in 25:24:1 phenol-chloroform-isoamyl (Sigma-Aldrich).  DNA was subsequently purified with the DNeasy PowerMax Soil Extraction kit (Qiagen), according to manufacturers instruction.  DNA from surface sediment was extracted using the DNeasy PowerMax Soil Exraction kit, according to manufacturers protocols. Extracted DNA was amplified with the TAReuk454FWD1 5'-CCAGCASCYGCGGTAATTCC-3' and TAReukREV3 5'-ACTTTCGTTCTTGATYRA-3' primer set (Stoeck et al. 2010) to target the V4 region of the 18S rRNA locus.  Polymerase chain reaction was conducted using Platinum Taq (ThermoFisher) with 40 cycles of 94C melting (30 seconds), 53C annealing (45 seconds), and 72C extension (1 minutes), followed by 72C for 5 minutes.  To supplement gut content analysis with the 18S V4 region, brittle star gut content and DNA from the surrounding sediment were amplified with the LR0R 5'- ACCCGCTGAACTTAAGC - 3' and LR21 5' - ACTTCAAGCGTTTCCCTTT -3' primer set (Cubeta et al. 1991; Hopple and Vilgalys, 1999) that amplifies the first several hundred bases of the 28S rRNA gene region.  This supplementary study was conducted to explore the phylogenetic position of anticipated fungal sequences. Secondary PCR and Illumina sequencing was conducted by the University of Alaska Core Lab and Michigan State Universitys RTSF Genomics Core for seal hair and gut content-sediment samples, respectively.  The amplicon pool was loaded onto an Illumina MiSeq v2 Standard flow cell and sequencing was performed in a 2x250bp paired-end format.