Bacteria (16S ssu rRNA) in an Antarctic snow sample

Luigi Michaud1, Angelina Lo Giudice1, Mohamed Mysara2, Pieter Monsieurs3, Carmella Raffa1, Natalie Leys3, Stefano Almafitano4, Rob Van Houdt3, Maxime Sweetlove5

1 University of Messina, Messina, Italy; 2 Vrije Universiteit Brussel, Brussels, Belgium; 3 Belgian Nuclear Research Centre (SCK CEN), Mol, Belgium; 4 National Research Council (IRSA-CNR), Rome, Italy; 5 Royal Belgian Institute for Natural Sciences, Rue Vautier 29, Brussels

Corresponding author(s): Luigi MichaudAngelina Lo Giudice (alogiudice@unime.it)Mohamed MysaraPieter MonsieursCarmella RaffaNatalie LeysStefano AlmafitanoRob Van Houdt, Maxime Sweetlove (msweetlove@naturalsciences.be)

Received {date}; Revised {date}; Accepted {date}; Published {date}

Citation: Combination of authors, year of data paper publication (in parentheses), Title, Journal Name, Volume, Issue number (in parentheses), and doi of the data paper.

Resource Citation

Michaud L, Lo Giudice A, Mysara M, Monsieurs P, Raffa C, Leys N, Almafitano S, Van Houdt R (2019): Bacteria (16S ssu rRNA) in an Antarctic snow sample. v1.0. SCAR - Microbial Antarctic Resource System. Dataset/Metadata. http://ipt.biodiversity.aq/resource?r=bacteria_antarctic_snow&v=1.0

Abstract

Amplicon sequencing sample of Bacteria (16S ssu rRNA gene, v1-v3 region) from a snow sample taken from the "clean Area”, 2 km South from the Antarctic Research Base “Concordia” (75°06′S–123°20′E).

Keywords: Metadata

Project details

Project title: Bacteria (16S ssu rRNA) in an Antarctic snow sample
Personnel: Luigi Michaud, Angelina Lo Giudice
Funding: Support was given by the Italian “Programma Nazionale di Richerche in Antartide” (PNRA) and the MNA (Museo Nazionale dell'Antartide)

Data published through GBIF: http://ipt.biodiversity.aq/resource?r=bacteria_antarctic_snow

Taxonomic coverage

General taxonomic coverage description: Bacteria (16S ssu rRNA gene, v1-v3 region)
Taxonomic ranks
Domain: Bacteria
Common names: Bacteria

Spatial coverage

General spatial coverage: Antarctic snow surface sample collected in the “Clean Area” 2 km South from the Research Base “Concordia” (75°06′S–123°20′E) 
Coordinates: 75°0'0''S and 75°0'0''S Latitude; 123°0'0''W and 123°0'0''W Longitude 

Temporal coverage: 2010

Methods

Method step description: 
- . 	Collected samples were allowed to thaw at 4°C for 24–48 h in the laboratory, with 100 litres of packed snow per sample resulting in approximately 20 litres of snowmelt. 
- . 	15 L melted snow for DNA extraction was filtered through a 0.2-µm-pore-size Sterivex filter unit (Millipore). The filters were stored at −20°C in lysis buffer (50 mM tris, 40 mM EDTA, and 750 mM sucrose).
- . 	Genomic DNA was extracted in triplicate using the phenol-chloroform method according to Zhou et al., and precipitated by adding 0.7 volumes of 100% isopropanol followed by a wash with ice-cold 70% ethanol.  After air-drying, DNA was resuspended in 50 µl of deionizated sterile water. 
- . 	PCR of a bacterial 16S rRNA gene fragment (V1–V3 region, 507 bp) and subsequent tag-encoded pyrosequencing were performed at DNAVision (Charleroi, Belgium). The 16S rRNA genes were amplified using the two universal primers 8F (5′- AGAGTTTGATCCTGGCTCAG -3′) and 518R (5′- ATTACCGCGGCTGCTGG -3′). The forward primer contained the sequence of the Titanium A adaptor (5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3′) and a barcode sequence. For each sample, a PCR mix of 100 µl was prepared containing 1×PCR buffer, 2U of KAPA HiFi Hotstart polymerase blend and dNTPs (Kapabiosystems), 300 nM primers (Eurogentec, Liege, Belgium), and 60 ng gDNA. Thermal cycling consisted of initial denaturation at 95°C for 5 min, followed by 25 cycles of denaturation at 98°C for 20 s, annealing at 56°C for 40 s, and extension at 72°C for 20 s, with a final extension of 5 min at 72°C. 3 µl of PCR product were added to a new PCR mix (identical as first round of PCR) for the nested PCR of 15 cycles. Amplicons were visualized on 1% agarose gels using GelGreen Nucleic Acid gel stain in 1× TAE (Biotium) and were cleaned using the Wizard SV Gel and PCR Clean-up System (Promega) according to the manufacturer's instructions. Pyrosequencing was carried out using the forward primer on a 454 Life Sciences Genome Sequencer FLX instrument (Roche) following titanium chemistry.
Study extent description: Snow surface sample was collected in triplicate from a “Clean Area” 2 km from the Research Base “Concordia” (75°06′S–123°20′E) 
Sampling description: Sampling was performed by using polyethylene boxes pre-treated with 1M hydrogen chloride and hydrogen peroxide. Sterile gloves and suit, and an ethanol flame-sterilized shovel were used. 
Quality control description: Autoclave-sterilized Milli-Q water was treated in tandem with the snow samples as a negative-control field blank. Quantity and quality of extracted DNA was checked by nanodrop ND-1000 device and the Quant-iT PicoGreen dsDNA reagent and kit (Life Tech, Carlsbad, USA) following the manufacturer's instructions.

Metadata language: English
Date of metadata creation: 2019-01-04
Hierarchy level: Dataset

References
Michaud, L., Giudice, A. L., Mysara, M., Monsieurs, P., Raffa, C., Leys, N., ... & Van Houdt, R. (2014). Snow surface microbiome on the High Antarctic Plateau (DOME C). PloS one, 9(8), e104505.