Illumina sequencing was applied to the nine replicates of glacial ice: ROJ 16S R1. Purified DNA was quantified and 1ng 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 and 18S 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.
bioproject_accession
PRJNA738680
collection_date
2015-02-01
depth
0,5 m
description
Illumina sequencing was applied to the nine replicates of glacial ice: ROJ 16S R1. Purified DNA was quantified and 1ng 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 and 18S 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.
elev
not applicable
env_broad_scale
not applicable
env_local_scale
not applicable
env_medium
not applicable
geo_loc_name
Antarctica
lat_lon
62.925 S 60.54444444 W
rel_to_oxygen
microaerophilic
samp_collect_device
drilling
sample_title
MAC
seq_methods
Illumina MiSeq
BioSampleModel
MIMARKS.survey
BioSampleModel
MIGS/MIMS/MIMARKS.soil