Ice cave metagenome
SOM1, SOM2, SOM3, SOM4, and SOM5. Purified DNA was quantified, and 1 ng of input DNA was used for an initial PCR of 20 cycles with Q5 Hot Start High-Fidelity DNA Polymerase and 100 nM primers targeting the 16S region. A second PCR of 15 cycles was then carried out using the same polymerase and 400 nM primers from the Access Array Barcode Library for Illumina Sequencers. The resulting amplicons were validated and quantified with a Bioanalyzer, and an equimolar pool was purified using AMPure beads. This pool was titrated by quantitative PCR with the KAPA SYBR FAST qPCR kit for LightCycler 480, using a reference standard for accurate quantification. Amplicons were denatured and loaded onto a flow cell at a concentration of 10 pM, where cluster generation and sequencing were performed with a MiSeq Reagent Kit v3 in a 2  300 bp paired-end run on a MiSeq system, yielding approximately 100,000 reads per sample. Genomic data were analyzed using the BaseSpace platform.