URLhttps://www.bco-dmo.org/dataset/654429
Download URLhttps://www.bco-dmo.org/dataset/654429/data/download
Media Type text/tab-separated-values
Created August 19, 2016
Modified September 30, 2016
State Final no updates expected
Brief Description

The eukaryotic (poly-A) metatranscriptome for Canterbury Basin

Acquisition Description

During IODP leg 317 expedition (JOIDES Resolution), a sediment core was drilled at Site U1352 in 344 m water depth. A total depth of 1927.5 mbsf was cored, spanning the Holocene to late Eocene periods. For this work, core sections were obtained from this site from 3.76 mbsf, 11.60 mbsf, 24.60 mbsf, 345.50 mbsf, and 403.10 mbsf.

RNA was extracted from 16 g of sediment from the Canterbury core samples using the RNA PowerSoil Total RNA Isolation Kit (MoBIO Laboratories, Carlsbad, CA) following a modified manufacturer’s protocol. Modifications included: ten cycles of homogenization for 1-minute intervals with 1 minute rest between intervals using a FastPrep benchtop homogenizer (MP Biomedicals, Santa Ana, CA) set to 4.0 m/s and increasing the incubation time to 45 min following the addition of SR4 buffer. Trace DNA was removed by treatment with Turbo DNA-free (Life Technologies, Grand Island, NY) for 60 minutes at 37 degrees C. A final RNA purification step was performed using the MEGAclear kit (Life Technologies, USA). In order to avoid contamination, all manipulations were carried out in a dedicated PCR hood (AirClean Systems, USA) for RNA work. An extraction blank was also carried through the entire procedure to control for kit contamination and served as “negative control”. Removal of carry-over DNA in RNA extracts was confirmed by the absence of visible amplification of the V4 hypervariable region of SSU rDNA after 35 cycles of PCR using the RNA extracts as template with key-tagged bacterial primers (Flores et al., 2011). PCR conditions were: 95 degrees C for 2 minutes followed by 35 cycles of 95 degrees C for 15 seconds, 53 degrees C for 45 seconds and 68 degrees C for 45 seconds with a final incubation of 68 degrees C for 3 minutes. Total RNA was used as template for cDNA amplification using the Ovation 3’-DGE System (NuGEN, San Carlos, CA) to obtain an enriched eukaryotic metatranscriptome through selection of polyA transcripts. Double-stranded cDNA was purified using the DNA Clean & Concentrator kit (Zymo Research) as described in the modified user guide (https://www.biolynx.ca/pdf/admin/nu_threedgeuserguide.pdf) provided by NuGEN. The quantity of amplified cDNA was evaluated using a fluorometer (Qubit 2.0, Life Technologies).

Processing Description

Raw read files were deposited in GenBankSRA under accession number SRP072032.

Instruments

The JOIDES Resolution's Advanced Piston Corer (APC) is used in soft ooze and sediments. The APC is a hydraulically actuated piston corer designed to recover relatively undisturbed samples from very soft to firm sediments.

More information is available from IODP (PDF).

General term for a laboratory apparatus commonly used for performing polymerase chain reaction (PCR). The device has a thermal block with holes where tubes with the PCR reaction mixtures can be inserted. The cycler then raises and lowers the temperature of the block in discrete, pre-programmed steps.

(adapted from http://serc.carleton.edu/microbelife/research_methods/genomics/pcr.html)

Qubit 2.0, Life Technologies [Fluorometer]
Details

A fluorometer or fluorimeter is a device used to measure parameters of fluorescence: its intensity and wavelength distribution of emission spectrum after excitation by a certain spectrum of light. The instrument is designed to measure the amount of stimulated electromagnetic radiation produced by pulses of electromagnetic radiation emitted into a water sample or in situ.

Parameters

cruise_id [cruise_id]
Details
cruise_id
Cruise identifier
cruise designation; name
location [site]
Details
location
Sampling location
Sampling site identification.
lat [latitude]
Details
lat
Latitude of sampling location

latitude, in decimal degrees, North is positive, negative denotes South; Reported in some datasets as degrees, minutes

lon [longitude]
Details
lon
Longitude of sampling location

longitude, in decimal degrees, East is positive, negative denotes West; Reported in some datsets as degrees, minutes

core_depth [meters below seafloor]
Details
core_depth

Depth of the sediment core

Meters below seafloor (mbsf); convention used for depths below the seabed in geology, oceanography, petrology and ocean drilling; often used in reporting measurements made from sediment cores.

sample_depth [meters below seafloor]
Details
sample_depth

Depth from which the samples were taken

Meters below seafloor (mbsf); convention used for depths below the seabed in geology, oceanography, petrology and ocean drilling; often used in reporting measurements made from sediment cores.

accession_num [accession number]
Details
accession_num
GenBank SRA accession number
Database identifier assigned by repository and linked to GenBank or other repository.
accession_link [accession number]
Details
accession_link
Hyperlink to GenBank SRA for the accession number
Database identifier assigned by repository and linked to GenBank or other repository.

Dataset Maintainers

NameAffiliationContact
Virginia P. EdgcombWoods Hole Oceanographic Institution (WHOI)
Maria G. PachiadakiWoods Hole Oceanographic Institution (WHOI)
Gaëtan BurgaudUniversite de Brest
Shannon RauchWoods Hole Oceanographic Institution (WHOI BCO-DMO)

BCO-DMO Project Info

Project Title Exploring of the Ecological Role(s) of Marine Fungi in the Deep Subseafloor
Acronym Fungal and prokaryotic activity in subseafloor
URLhttps://www.bco-dmo.org/project/653744
Created August 10, 2016
Modified August 10, 2016
Project Description

The deep sedimentary biosphere, extending hundreds of meters below the seafloor harbors unexpected diversity of Bacteria, Archaea and microbial eukaryotes. Far less is known about microbial eukaryotes in subsurface habitats, albeit several studies have indicated that fungi dominate microbial eukaryotic communities and fungal molecular signatures (of both yeasts and filamentous forms) have been detected in samples as deep as 1740mbsf. Here we compare and contrast fungal ribosomal RNA gene signatures and whole community metatranscriptomes present in sediment core samples from 6 and 95mbsf from Peru Margin site 1229A and from samples from 12 and 345 mbsf from Canterbury Basin site U1352. The metatranscriptome analyses reveal higher relative expression of amino acid and peptide transporters in the less nutrient rich Canterbury Basin sediments compared to the nutrient rich Peru Margin, and higher expression of motility genes in the Peru Margin samples. Higher expression of genes associated with metals transporters and antibiotic resistance and production was detected in Canterbury Basin sediments. A poly-A focused metatranscriptome produced for the Canterbury Basin sample from 345 mbsf provides further evidence for active fungal communities in the subsurface in the form of fungal-associated transcripts for metabolic and cellular processes, cell and membrane functions, and catalytic activities. Fungal communities at comparable depths at the two geographically separated locations appear dominated by distinct taxa. Differences in taxonomic composition and expression of genes associated with particular metabolic activities may be a function of sediment organic content as well as oceanic province. Microscopic analysis of Canterbury Basin sediment samples from 4 and 403 mbsf produced visualizations of septate fungal filaments, branching fungi, conidiogenesis and spores. These images provide another important line of evidence supporting the occurrence and activity of fungi in the deep subseafloor biosphere.

This project was funded by C-DEBI sub-award #49538097.

Data Project Maintainers
NameAffiliationRole
Virginia P. EdgcombWoods Hole Oceanographic Institution (WHOI)Principal Investigator
Maria G. PachiadakiWoods Hole Oceanographic Institution (WHOI)Co-Principal Investigator
Gaëtan BurgaudUniversite de BrestInternational Collaborator
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