fumarolic microbial communities at Tramway Ridge, Mt. Erebus, Antarctica

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如何引用

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Herbold C, Lee C, McDonald I, Cary C (2019): fumarolic microbial communities at Tramway Ridge, Mt. Erebus, Antarctica. v1.2. SCAR - Microbial Antarctic Resource System. Dataset/Metadata. https://ipt.biodiversity.aq/resource?r=fumarolic_antarctic_microbial_communities_at_mount_erebus&v=1.2

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此資料的發布者及權利單位為 SCAR - Microbial Antarctic Resource System。 This work is licensed under a Creative Commons Attribution (CC-BY 4.0) License.

GBIF 註冊

此資源已向GBIF註冊，並指定以下之GBIF UUID: 53b30953-201f-4599-9377-1c41b5cba477。  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。

關鍵字

Metadata

聯絡資訊

地理涵蓋範圍

Tramway Ridge (ASPA 130), Mount Erebus, Antarctica

分類群涵蓋範圍

metagenomic data (amplicon and shotgun) of Bacteria

計畫資料

無相關描述

參與計畫的人員:

取樣方法

All suggested sterilization protocols for entering into this protected site were adhered to, following the ASPA 130 Management Plan ( http://www.scar.org/publications/bulletins/151/aspa130.html). Sites were chosen based on measuring a surface temperature of 65 °C with a stainless steel Checktemp1 temperature probe (Hanna Instruments, Rhode Island, USA), sterilized with 70% ethanol immediately before each use. Surface ‘crust’ was set aside before collecting samples. Samples were collected by aseptically removing the top 2 cm of sediment in an ~25 cm2 area. Sediment was placed into a fresh 50 ml Falcon tube. Sampling continued with the collection of a second (2–4 cm depth) and third (4–8 cm depth) layer following the same procedures. Temperature measurements were repeated for each layer sampled. All samples were immediately frozen, transported back to the University of Waikato frozen and maintained at −80 °C in the laboratory until analysed.

方法步驟描述:

1. DNA was extracted from samples using a modified CTAB (cetyltrimethylammonium bromide) bead-beating protocol. For shotgun sequencing, a portion of extracted genomic DNA was sequenced using standard protocols for the 454-Ti platform (Roche 454 Life Sciences, Branford, CT, USA) at the UCLA GenoSeq CORE.
2. PCR amplicons containing V5–V7 hypervariable regions of the 16S rRNA gene were generated from the same genomic DNA samples using primers Tx9 (5′-GGATTA GAWACCCBGGTAGTC-3′) and 1391R (5′-GACGGGCRGTGWGTRCA-3′). PCR was performed in triplicate on each sample and pooled to reduce stochastic variation. Three samples (site A 0–2 cm, site B 0–2 cm, site B 2–4 cm) were sequenced using the 454-GS-FLX platform by Taxon Biosciences (Tiburon, CA, USA) and three samples (site A 2–4 cm, site A 4–8 cm and site B 4–8 cm) were sequenced using the 454 Junior platform at the Waikato DNA Sequencing Facility (Hamilton, New Zealand). For the three samples sequenced using the 454-GS-FLX platform, each 30 μl reaction contained 2–10 ng of DNA extract, Pfx polymerase and platinum polymerase (0.5 U each; Invitrogen), 1 × Pfx PCR buffer with Pfx enhancer, 0.2 mM dNTPs, 1 mM MgCl2, 0.02 mg ml−1 BSA, 0.8 μM of forward and reverse primer and PCR-grade water. Thermal cycling conditions were 94 °C for 2 min; 24 cycles of 94 °C for 15 s, 55 °C for 30 s and 68 °C for 1 min; and 68 °C for 3 min. Amplicons were size-selected and purified using polyacrylamide gel electrophoresis before being prepared for pyrosequencing by Taxon Biosciences. For the three samples sequenced using the 454-junior platform, each 30 μl reaction contained 2–10 ng of DNA extract, PrimeStar polymerase (0.625 U; Takara), 1 × PCR buffer, 0.2 mM dNTPs, 0.4 μM of forward and reverse primer and PCR-grade water. Thermal cycling conditions were 94 °C for 3 min; 24 cycles of 94 °C for 20 s, 52 °C for 20 s and 72 °C for 45 s; and 72 °C for 3 min. Triplicate PCR reactions were pooled and gel-purified using the UltraCleanTM 15 DNA Purification Kit (MO BIO Laboratories Inc.), cleaned using the Agencourt AMPure XP Bead Cleanup kit (Beckman Coulter Inc.) and quantified (Quant-iTTM dsDNA HS Assay Kit, Invitrogen Ltd.). Cleaned amplicons were used as template (25 ng) in a second PCR reaction using fusion primers (forward: 5′-(454 Adapter A)-TCAG-MID-Tx9-3′; reverse: 5′-(454 Adapter B)-TCAG-1391R-3′). PCR conditions were exactly the same as the first round except only 10 cycles of PCR were performed. Triplicate PCR reactions were pooled and gel-purified using the UltraCleanTM 15 DNA Purification Kit, cleaned using the Agencourt AMPure XP Bead Cleanup kit and quantified (Quant-iTTM dsDNA HS Assay Kit) before being prepared for pyrosequencing using the 454 Junior platform by the University of Waikato sequencing facility.

引用文獻

1. Herbold, C. W., Lee, C. K., McDonald, I. R., & Cary, S. C. (2014). Evidence of global-scale aeolian dispersal and endemism in isolated geothermal microbial communities of Antarctica. Nature communications, 5, 3875.

額外的詮釋資料