title: 16S rRNA amplicon sequencing of Pimephales promelas: skin microbiome
design: Frozen fish skin swab tubes were thawed, vortexed for 5 minutes, and centrifuged at 10,000 x g for 30 minutes to pellet any bacteria remaining in the preservation solution (sterile 1X PBS).  The solution supernatant was removed, and any pellet formation was resuspended and transferred directly into a PowerWater Bead Tube (Qiagen).  The swab tip was clipped aseptically and placed in the same PowerWater Bead Tube. The swabs and bacteria pellets were extracted using the Qiagen DNeasy PowerWater Kit according to the manufacturers instructions.  Frozen intestinal tracts were thawed, aseptically transferred into a PowerSoil Bead Tube, and then extracted with the Qiagen DNeasy PowerSoil Pro Kit.  DNA concentrations were measured with the Quantus fluorometer (Promega; Madison, Wisconsin, USA).   Genomic DNA was delivered frozen to the Michigan State University Research Support Technology Facility Genomics Core (MSU RTSF; East Lansing, MI) for 16S rRNA amplicon sequencing on the Illumina MiSeq sequencing platform (Illumina; San Diego, California, USA) for library preparation and sequencing.  The V4 hypervariable region of the bacterial 16S rRNA gene was amplified with dual-indexed, Illumina-compatible primers (515F: GTGCCAGCMGCCGCGGTAA /806R:  GGACTACHVGGGTWTCTAAT) following the protocol described by Kozich et al. 2013, except that the index sequences were revised to unique dual index pairs.  Following polymerase chain reaction (PCR), all amplified products were batch normalized using an Invitrogen (Carlsbad, California, USA) SequalPrep DNA Normalization plate, and the products recovered from this plate were pooled. Individual pools were concentrated and cleaned using a QIAquick Spin column (Qiagen) and AMPure XP magnetic beads (Beckman Coulter; Brea, California, USA).  The pools were then verified and quantified using a combination of the Qubit dsDNA HS (ThermoFisher Scientific; Waltham, Massachusetts, USA), Agilent 4200 TapeStation High Sensitivity DNA1000 (Agilent; Santa Clara, California, USA), Invitrogen Collibri Illumina Library Quantification qPCR assays (ThermoFisher Scientific; Waltham, Massachusetts, USA). Each pool was sequenced using an Illumina MiSeq v2 standard flow cell in a 2 x 250 base pair (bp) paired end format using a v2 500 cycle reagent cartridge. Custom sequencing and index primers complementary to the 515F/806R target-specific sequences were added to appropriate wells of the reagent cartridge.  Base calling was done by Illumina Real Time Analysis v1.18.54, and the output was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.20.0.