Bacteria (16S) in growth laminae of a large conical mats from Lake Untersee, East Antarctic
Citation
Koo H, Mojib N, Hakim J, Hawes I, Tanabe Y, Andersen D, Bej A, Sweetlove M (2019). Bacteria (16S) in growth laminae of a large conical mats from Lake Untersee, East Antarctic. Version 1.1. SCAR - Microbial Antarctic Resource System. Metadata dataset https://doi.org/10.15468/surft2 accessed via GBIF.org on 2024-09-01.Description
Amplicon sequencing dataset (454 pyrosequencing) of Bacterial communities (based on the 16S ssu rRNA gene; using Bacteria and Cyanobacteria-specific primers), of three different strata in a laminated microbial mat sample from Lake Untersee (east Antarctica)Sampling Description
Study Extent
The samples were taken from a single core of a conical mat from Lake Untersee, which was collected by scientific divers utilizing SCUBA by accessing the lake ecosystem through a hole made on the ∼3.5 m surface ice.Sampling
The core was collected by gently inserting a 50 mm diameter sterile polycarbonate core tube through the top of a conical mat structure, then sealing it with rubber stoppers and returning it to the surface. The core was stored in Antarctic Logistics Centre International (ALCI), Cape Town, South Africa facility first at -20°C in a walk-in freezer and then transported to UAB in dry ice and kept in -20°C freezer until used. The top three laminae (herein U1, top lamina; U2, middle lamina; and U3, inner lamina) were separated from the core and selected individually for sequencing. Each lamina was carefully separated using sterile forceps and rinsed with distilled water to avoid carryover of microorganisms between laminae before DNA extraction.Method steps
- Five samples from different segments of each lamina of the large conical mat were sliced with a sterile scalpel, transferred into separate microcentrifuge tubes consisting of beads (MO BIO Laboratories Inc., Carlsbad, CA, United States), and homogenized in a high velocity bead beater (BIO101/Savant FastPrep FP120 – Qiagen, Inc., Valencia, CA, United States). DNA was then purified using the PowerBiofilm®DNA isolation Kit (MO BIO Laboratories). The large conical mats used in this study are unique, found only in Lake Untersee. Therefore, to minimize sample collection while obtaining sufficient DNA to investigate the microbial composition of the mat laminae, we used five spatially representative samples from each lamina, and pooled the purified DNA into single samples for amplicon sequencing. The concentration and the quality of the pooled DNA from each mat lamina was determined by using a Lambda II spectrophotometer (Perkin Elmer, Norwalk, Conn.) followed by agarose gel electrophoresis (1% wt/vol agarose in 1X Tris-Acetate-EDTA (TAE) buffer, pH 7.8) to confirm that each sample consists of mostly high molecular weight DNA (>2 kbp). The DNA was then dried in a Savant Speedvac Evaporator SVC 100H and stored at 4°C until used for sequencing.
- Bacterial tag-encoded amplicon pyrosequencing was performed using the primers 341F (5′–CCTACGGGAGGCAGCAG–3′) and 907R (5′–CCGTCAATTCMTTTGAGTTT–3′) targeting the V3–V5 region of the 16S rRNA, combined with group-specific primers for cyanobacterial: CYA106F (5′- CGGACGGGTGAGTAACGCGTGA-3′) and CYA781R (5′-GACTACWGGGGTATCTAATCCCWTT-3′). First, a sequencing library was established by using a one-step PCR method with a total of 30 cycles of template DNA amplification with the HotStarTaq Plus Master Mix kit (Qiagen, Inc., Valencia, CA, United States), and amplicons originating and extending from the aforementioned bacteria- and cyanobacteria-specific primers. Then, tag-encoded FLX amplicon pyrosequencing was performed on a Roche 454 FLX instrument with Titanium reagents following the Titanium procedures and RTL protocols at the Research and Testing Laboratory (Lubbock, TX, United States).
Taxonomic Coverages
Bacteria (16S ssu rRNA gene v3-v5)
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Bacteriacommon name: Bacteria rank: domain
Cyanobacteria (16S ssu rRNA gene, group specific primer)
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Cyanobacteriacommon name: Cyanobacteria rank: domain
Geographic Coverages
Lake Untersee, East Antarctica
Bibliographic Citations
- Koo, H., Mojib, N., Hakim, J. A., Hawes, I., Tanabe, Y., Andersen, D. T., & Bej, A. K. (2017). Microbial communities and their predicted metabolic functions in growth laminae of a unique large conical mat from Lake Untersee, East Antarctica. Frontiers in microbiology, 8, 1347. -
Contacts
Hyunmin Koooriginator
University of Alabama at Birmingham
Birmingham
US
Nazia Mojib
originator
University of Alabama at Birmingham
Birmingham
US
Joseph Hakim
originator
University of Alabama at Birmingham
Birmingham
US
Ian Hawes
originator
University of Canterbury
Christchurch
NZ
Yukiko Tanabe
originator
National Institute of Polar Research
Tachikawa
JP
Dale Andersen
originator
University of Alabama at Birmingham
Birmingham
US
Asim Bej
originator
University of Alabama at Birmingham
Birmingham
US
Maxime Sweetlove
metadata author
position: Research assistent
Royal Belgian Institute for Natural Sciences
Rue Vautier 29
Brussels
1000
BE
email: msweetlove@naturalsciences.be
Hyunmin Koo
administrative point of contact
University of Alabama at Birmingham
Birmingham
US