Ice cave metagenome DEV Genome sequencing and assembly

Illumina sequencing was applied to six samples of ice: SDEV-1, DEV-2, DEV-3, DEV-4, DEV-5, DEV-6. Purified DNA was quantified and 1 ng of input DNA was used in a first PCR of 20 cycles with Q5 Hot Start High-Fidelity DNA Polymerase in the presence of 100nM primers for 16S amplification. After the first PCR, a second PCR of 15 cycles was performed with Q5 Hot Start High-Fidelity DNA Polymerase in the presence of 400nM of primers of the Access Array Barcode Library for Illumina Sequencers. The finally obtained amplicons were validated and quantified by Bioanalyzer and an equimolecular pool was purified using AMPure beads and titrated by quantitative PCR using the Kapa-SYBR FAST qPCR kit for LightCycler 480 and a reference standard for quantification. The pool of amplicons were denatured prior to be seeded on a flowcell at a density of 10pM, where clusters were formed and sequenced using a MiSeq Reagent Kit v3, in a 2x300 pair-end sequencing run on a MiSeq sequencer to obtain 100000 reads per sample approximately. Genomic data were analyzed with Base Space platform.2019