16S rRNA sequencing of the hellbender skin microbiome: animal 7316 on day d187
Extracted DNA from all samples and controls was prepared for 16S rRNA amplicon sequencing following previously described methods (Becker, Richards-Zawacki, Gratwicke, & Belden, 2014; Caporaso et al., 2011). Amplicons from all samples and negative controls were quantified with Qubit dsDNA HS assay kit (Invitrogen, Carlsbad, CA, USA) and pooled in equimolar quantities by sample. The resulting library was size-selected with QIAquick Gel Extraction Kit and purified with QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA). The purified library was visualized with a Bioanalyzer High Sensitivity DNA chip (Agilent Technologies, Santa Clara, CA, USA) and quantified with the KAPA Illumina Library Quantification Kit (Roche, Pleasanton, CA, USA). The library was spiked with 15% PhiX and sequenced on an Illumina MiSeq with v2 chemistry and 2 x 250 bp paired-end reads (Caporaso et al., 2011). Raw sequencing reads were adapter trimmed, joined, demultiplexed, and assigned to samples with MacQIIME version 1.9.1 (Caporaso, Kuczynski, et al., 2010).
d187_7316