Benthic microbial communities (Bacteria, 16S) of coastal terrestrial and ice shelf Antarctic meltwater ponds

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Archer S, McDonald I, Herbold C, Lee C, Cary C (2019): Benthic microbial communities (Bacteria, 16S) of coastal terrestrial and ice shelf Antarctic meltwater ponds. v1.0. SCAR - Microbial Antarctic Resource System. Dataset/Metadata. http://ipt.biodiversity.aq/resource?r=benthic_microbial_communities_16s_antarctic_meltwater_ponds&v=1.0

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The publisher and rights holder of this work is SCAR - Microbial Antarctic Resource System. This work is licensed under a Creative Commons Attribution (CC-BY) 4.0 License.

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此資源已向GBIF註冊，並指定以下之GBIF UUID: 994a6181-41fa-42ce-ab39-6efee8d43881。  SCAR - Microbial Antarctic Resource System 發佈此資源，並經由Scientific Committee on Antarctic Research同意向GBIF註冊成為資料發佈者。

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地理涵蓋範圍

Antarctica: Pond P70E, Huey and Legin on Bratina Island, and ponds Finch, Canary and Morepork in Miers Valley

分類群涵蓋範圍

Bacteria 16S ssu rRNA gene

計畫資料

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The personnel involved in the project:

取樣方法

Samples were aseptically collected using a disposable push-corer developed from a 50 mL syringe (BD, Singapore). The corer (with the plunger removed) was inserted 4–6 cm into the sediment, the plunger reinserted and core removed carefully to retain the sediment structure. After excess sediment was removed, each core was sub-sectioned into four one-centimeter samples, placed in sterile 15 oz whirlpack (Nasco, WI, USA), then frozen for transportation to the laboratory.

方法步驟描述:

1. DNA was extracted from 0.5 g ± 0.1 g of individual sediment sections using a modified bead-beating method (Coyne et al., 2001). Briefly, sediment was added to 0.5 g each of 0.1 mm and 2.5 mm silica-zirconia beads. To each sample 270 μL of phosphate buffer (100 mM NaH2PO4) and 270 μL of SDS lysis buffer (100 mM NaCl, 500 mM Tris pH 8.0, 10% SDS) were added and samples were horizontally shaken on a Vortex Genie 2 (MO BIO Laboratories Inc, Carlsbad, CA, USA) for 15 min. Samples were centrifuged at 12,500 rpm for 30 s and 180 μL of cetyltrimethylammonium bromide-polyvinylpyrrolidone (CTAB) extraction buffer (100 mM Tis-HCl, 1.4 M NaCl, 20 mM EDTA, 2% CTAB, 1% polyvinylpyrrlidone and 0.4% β-mecaptoethanol) was added. Samples were vortexed for 10 s prior to incubation at 60°C and 300 rpm for 30 min on a rocking bed. Samples were centrifuged at 12,500 rpm for 30 s and then 350 μL of chloroform/isoamyl alcohol (24:1) was added. Samples were again vortexed for 10 s and centrifuged for 5 min at 12,500 rpm. The aqueous phase was transferred to a new eppendorf tube then 500 μL of chloroform/isoamyl alcohol (24:1) was added. Samples were vortexed and left on a rocking bed HulaMixer (Invitrogen, Carlsbad, CA, USA) for 20 min. Samples were centrifuged for 5 min at 13,500 rpm, the aqueous phase was removed and 10 M ammonium acetate was added to the samples to achieve a final concentration of 2.5 M. The samples were vortexed and centrifuged for 5 min at 13,500 rpm. The aqueous layer was removed to a new tube and 0.54 volumes of isopropanol was added and mixed. Samples were left overnight at −20°C then centrifuged for 20 min at 13,500 rpm. The supernatant was removed, the pellet washed with 1 mL of 70% AR grade ethanol and centrifuged for 1 min at 13,500 rpm. Ethanol was removed and DNA was re-suspended in 30 μL of sterile TE then quantified using the Qubit 2.0 Florometer (Invitrogen). The four individual sectioned samples from each core were diluted to 10 ng/μL, then 10 μL of each was pooled and frozen at −20°C until use.
2. The V5-V6 hypervariable region of the 16S rRNA gene was utilized to identify variation in bacterial community diversity and structure. 30 μL PCR reactions were run in triplicate for each sample using un-adapted primers Tx9F (5′-GGATTAGAWACCCBGGTAGTC-3′) and 1391R (5′-GACGGGCRGTGWGTRCA-3′). The triplicates were pooled then gel extracted on a 2% TAE agarose gel stained with “SYBR Safe” and DNA was retrieved using the UltraClean 15 (MoBio, Inc, Carlsbad, USA) DNA Purification Kit as per manufacturers instructions. A second round of triplicate PCR was run as above but with only 10 cycles and using 25 ng of the purified DNA from the previous step per reaction (milli-Q H2O volume adjusted accordingly). The primers used were adapted for one-way reads as per manufacturers instructions, including unique MID identifiers for each sample [BacX-Tx9F (5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG-MID-GGATTAGAWACCCBGGTAGTC-3′) and BacB-1391R (5′-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-GACGGGCRGTGWGTRCA-3′)]. A second gel extraction was performed as above. Samples went through a final cleanup step using the Agencourt AMPure XP system (Beckman Coulter Genomics, Danvers, MA, USA) as per the manufacturers instructions.
3. The diluted amplicons were mixed together in the desired proportions to create the 1 × 109 amplicon pool. Sequencing was performed using the GS Junior Titanium emPCR Kit (Lib-L), the GS Junior Titanium Sequencing Kit, PicoTiterPlate Kit and GS Junior System according to the manufacturers instructions (Roche 454 Life Sciences, Branford, CT, USA).

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