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DNA metabarcoding of the prey and microbiome of museum specimen Antarctic trematomid fishes

版本 1.0 由 SCAR - Microbial Antarctic Resource System 發佈於 Nov 14, 2019 SCAR - Microbial Antarctic Resource System

In this dataset, stomachs and hindguts were sampled from 225 Trematomus specimens from the Natural History Museum London. Fish specimen were collected between 20 and 100 years ago and fixed in either formaldehyde or ethanol. A 313 bp fragment of the cytochrome c oxidase subunit I (COI) was amplified and sequenced for prey item identification in the stomach and a 450 bp region of the 16S rRNA gene to investigate microbiome composition in the gut system.

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Sweetlove M (2019): DNA metabarcoding of the prey and microbiome of museum specimen Antarctic trematomid fishes. v1.0. SCAR - Microbial Antarctic Resource System. Dataset/Occurrence. https://ipt.biodiversity.aq/resource?r=historic_antarctic_fish_dataset_2019&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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關鍵字

Samplingevent

聯絡資訊

資源建立者:

Maxime Sweetlove
Data officer
Royal Belgian Institute of Natural Sciences Rue Vautier 29 1000 Brussels BE

可回覆此資源相關問題者:

Heindler Franz
University of Leuven Leuven BE

元數據填寫者:

Heindler Franz
University of Leuven Leuven BE
Hendrik Christiansen
University of Leuven Leuven BE
Bruno Frédérich
University of Liège Liège BE
Agnes Detaï
Muséum National d'Histoire Naturelle Paris FR
Gilles Lepont
University of Liège Liège BE
Gregory Maes
University of Leuven Leuven BE
Anton Van de Putte
Royal Belgian Institute of Natural Sciences 1000 Brussels BE
Filip Volkaert
University of Leuven Leuven BE

與此資源的相關者:

內容提供者
Heindler Franz
University of Leuven Leuven BE

地理涵蓋範圍

Various places in the Souther Ocean and the coastal waters of the Antarctic continent. Geographic coordinates not available for all the museum specimen.

界定座標範圍 緯度南界 經度西界 [-90, -180], 緯度北界 經度東界 [-54, 180]

分類群涵蓋範圍

Fish specimen of the genus Trematomus, with DNA samples of the stomach and gut for prey composition and micro biome.

Species  Trematomus eulepidotus,  Trematomus penellii,  Trematomus hansoni,  Trematomus eulepidotus,  Trematomus brachysoma,  Trematomus bernacchii,  Trematomus borchgrevinki,  Trematomus newnesi,  Trematomus loennbergii,  Trematomus scotti

Bacterial and Archaeal microbiome in the stomach and gut of the fish was profiled with the 16S ssu rRNA gene

Domain  Bacteria (Bacteria),  Archaea (Archaea)

Prey (eukaryotes) of the fish were investigated with the COI marker gene

Domain  Eukaryota (Eukaryotes)

時間涵蓋範圍

彙整期間 1899-2018

計畫資料

無相關描述

計畫名稱 SYNTHESYS-RECTO-VERSO
經費來源 This research received support from the SYNTHESYS Project (http://www.synthesys.info/), which is financed by European Community Research Infrastructure Action under the FP7 Capacities Program. It furthermore received funds through the Brilliant Marine Research Idea Philanthropy Award 2017 issued by the Vlaams Instituut voor de Zee (VLIZ), Belgium. Research was funded by the Refugia and Ecosystem Tolerance in the Southern Ocean project (RECTO; BR/154/A1/RECTO) as well as the Ecosystem Responses to global change—a multiscale approach in the Southern Ocean project (vERSO; BR/132/A1/vERSO) (http://rectoversoprojects.be), both funded by the Belgian Science Policy Office (BELSPO). This is contribution number 003 of the RECTO project and contribution number 029 of the vERSO project. HC was supported by a grant from the former Flemish agency for Innovation by Science and Technology (IWT), now managed through Flanders Innovation & Entrepreneurship (VLAIO, Grant No. 141328).

The personnel involved in the project:

作者
Heindler Franz

取樣方法

Fish were carefully dissected: stomachs were opened to remove stomach content and a small portion of the hindgut (1 cm) was removed. Stomach content and hindgut were stored separately in 70% ethanol.

研究範圍 Stomach and hindgut samples of 225 specimens of the genus Trematomus were obtained from the Natural History Museum, London. Sampling dates ranged from 1901 to 1988, sampling locations were in the Southern Ocean around the Antarctic continent. Contemporary samples were caught with hook and line in the vicinity of the Gerlache Strait, Antarctic Peninsula in the season of 2017–2018.
品質控管 During molecular laboratory work special care was taken to prevent (cross-) contamination of samples. Workbench wipes (workbench contamination), human saliva wipes (human contamination) and no-template extractions (blanks) were included as contamination controls for amplification and sequencing.

方法步驟描述:

  1. A large piece of stomach content (0.5 × 0.5 cm) or the entire piece of hindgut (1 cm) was placed into screwcap microtubes with 500 μl of Phosphate Buffered Saline (PBS) at pH 9. Tissue was fragmented thoroughly in each tube to ameliorate efficiency. Samples were heated to 100°C for 10 min, left to cool on ice for 5 min and then spun down with 20,000 × g for 5 min. PBS was carefully removed without taking any tissue and replaced by 500 μl of PBS at pH 7.2 and again heated to 100°C for 10 min. PBS was again carefully removed and further purification steps were conducted using the commercial Nucleospin® Tissue (Macherey-Nagel, Accession number: 740952) DNA extraction kit following the manufacturer's protocol.
  2. For prey identification a 313 bp region of the COI gene was amplified from the stomach content using the tailed primers NGSmlCOIint and NGSjgHCO2198. The V3 and V4 region (460 bp) of the 16S rRNA gene was amplified using the tailed primers 16s-IllumTS-F and 16s-IllumTS-R to assess the microbiome composition. The reaction mix for the amplicon PCR for COI contained 12.5 μl of MytaqTM 2x Mix (Bioline, Accession number: BIO-25041), 0.5 μl of each primer (20 μM), 10.5 μl of molecular grade water and 1 μl of DNA template with a PCR profile of 10 s of denaturation at 95°C, 30 s of annealing at 62°C and 60 s elongation at 72°C for 16 cycles with the annealing temperature dropping every cycle by 1°C, followed by 25 cycles with an annealing temperature at 46°C. The reaction mix for the amplicon PCR for 16S contained 12.5 μl of MytaqTM 2x Mix, 2.5 μl of each primer (1 μM), 2.5 μl of DNA template (5 ng ul−1) and 5 μl of molecular grade water with a PCR profile of 60 s of initial denaturation at 95°C followed by 25 cycles of 15 s denaturation at 95°C, 15 s of annealing at 55°C and 10 s elongation at 72°C, finishing with a final extension of 72°C for 300 s. PCR products were cleaned up using Agencourt AMPure XP beads (Beckman Coulter, Accession number: A63882) following the manufacturer's instructions with a bead to template ratio of 0.8 to 1. Thereafter followed an indexing PCR, which binds a unique primer barcode to each respective sample following Lange et al. (2014) with a PCR mix of 10 μl of MytaqTM 2x Mix, 0.5 μl of each forward and reverse indexing-primer (to form a unique identifiable primer combination for each sample; 20 μM) and 9 μl of DNA template with a PCR profile of an initial denaturation of 1 min at 95°C followed by 15 cycles of denaturation for 15 s at 95°C, 15 s of annealing at 51°C and 10 s of extension at 72°C finishing with a final extension of 5 min at 72°C. The PCR product was cleaned up again, then quantified using the commercial Quant-iTTM Picogreen® kit (Thermo Fisher) and pooled, if sufficient template (20 ng) was available. Sequencing took place on an Illumina MiSeq PE 3000 (Genomics Core, KU Leuven, Belgium).

收藏資料

蒐藏名稱 Antarctic Fish, London Natural History Museum

引用文獻

  1. Heindler F.M., Christiansen H., Frédérich B., et al. (2018) ‘Historical DNA Metabarcoding of the Prey and Microbiome of Trematomid Fishes Using Museum Samples’. Frontiers in Ecology and Evolution 6: 151, https://doi.org/10.3389/fevo.2018.00151. https://doi.org/10.3389/fevo.2018.00151.