Dataset: DHAB halocline sediment pyrotags - eukaryotes
Deployment: AT18-14

454 pyrotags for Eukaryote taxa in deep hypersaline anoxic basin (DHAB) halocline sediments

Principal Investigator:

Joan M. Bernhard (Woods Hole Oceanographic Institution, WHOI)

Virginia P. Edgcomb (Woods Hole Oceanographic Institution, WHOI)

BCO-DMO Data Manager:

Nancy Copley (Woods Hole Oceanographic Institution, WHOI BCO-DMO)

Shannon Rauch (Woods Hole Oceanographic Institution, WHOI BCO-DMO)

Project:

Investigations into the Physiological State of DHAB Metazoans (DHAB Metazoans)

Pickled Protists or Community Uniquely Adapted to Hypersalinity? (Pickled Protists)

Current State:

Final no updates expected

Version:

2015-05-22

Version Date:

2015-05-22

Data URL:

https://www.bco-dmo.org/dataset-deployment/553962/data

Expand/Collapse All

Description

Pyrotags of eukaryotes obtained from Urania, Discovery, and L’Atalante DHAB halocline sediments.

Small subunit ribosomal RNA V4 hyper variable region eukaryotic pyrotags prepared from total RNA extracted from sediments below haloclines of 3 deep hypersaline anoxic basins in the Eastern Mediterranean Sea and from nearby control sites (normal saline deep marine sediments in close proximity to deep hypersaline anoxic basins). Total RNA was transcribed to cDNA, and eukaryotic V4 pyrotags amplified from cDNA.

Related References:
Bernhard, J.M., CM Morrison, E Pape, DJ Beaudoin, MA Todaro, MG Pachiadaki, K Ar Kormas, & VP Edgcomb. 2015 (submitted). Metazoans of redoxcline seidments in Mediterranean deep-sea hypersaline anoxic basins.

Bernhard, J.M., Kormas, K.A., Pachiadaki, M.G., Rocke, E., Beaudoin, D.J., Morrison, C., Visscher, P.T., Cobban, A., Starczak, V.R., and Edgcomb, V.P. 2014. Benthic protists and fungi of Mediterranean deep hypersaline anoxic basin redoxcline sediments. Frontiers in Microbiology, doi: 10.3389/fmicb.2014.00605.

Methods & Sampling

Dataset acquisition description

ROV Jason was used to collect push cores 6.35cm diameter obtained from the Deep Submergence Lab at Woods Hole Oceanographic Institution (www.whoi.edu/groups/DSL/) and configured with a seal to prevent contamination during ascent from the halocline sediments of L’ Atalante, Urania and Discovery basins in the eastern Mediterranean Sea at a depth of about 3500-4000m. Sediments were profiled for oxygen and immediately frozen upon return to surface.

Data Processing Description

Dataset Processing Description

RNA from approximately 8 g of the top 2 cm from each frozen syringe subcore was extracted using an optimized protocol with the RNA Power Soil kit (MoBio, USA). Major modifications included introduction of 3 cycles of freeze-thaw (-80 degrees C, 5 min., 65 degrees C, 5 min.), bead beating with 2× 5 min. intervals on a horizontal vortexer, an overnight nucleic acid precipitation, and a one-hour centrifugation during the precipitation step. In addition, we introduced two DNAase treatments using TurboDNAase (Ambion, USA). Removal of DNA was confirmed by PCR using general eukaryotic primers. RNA was purified using the MEGAclear kit (Ambion, USA). Reverse transcription of the purified RNA samples was performed using the QuantiTect kit (Qiagen, USA). Tag-pyrosequencing of the eukaryotic small subunit rRNA (18S rRNA) gene was performed using PCR amplification of the V4 region of the 18S rRNA gene and the primer pair TAReuk454FWD1 (5′-CCAGCA(G/C)C(C/T)GCGGTAATTCC-3′) and TAReukREV3 (5′-ACTTTCGTTCTTGAT(C/T)(A/G)A-3′). Following PCR, all amplicon products (ca. 450 bp) from different samples were purified using the MinElute Reaction Cleanup kit (Qiagen, USA). Amplicon libraries were sequenced utilizing the Roche 454 FLX Titanium platform and reagents following manufacturer’s guidelines at the MR DNA (Molecular Research LP, Shallowater, TX, USA) sequencing facility.

Denoising of the flowgrams was performed using Acacia. Processing of the resulting sequences, i.e. trimming and quality control, was performed using QIIME. Sequences with 380 bp or more and no ambiguous base calls and no homopolymers of 6 bp or more were included in further analysis. All sequences were binned into Operational Taxonomic Units (OTUs) and were clustered (average neighbor algorithm) at 97% sequence similarity identity. Taxonomic assignments were made using BLAST within QIIME.

BCO-DMO Processing:
- original file: EdgcombDHABeukaryoteTags.xlsx
- replaced positions for Discovery and Urania with those in event log for pushcores
- added html links to GenBank BioProject
- added cruise_id, lat, lon
- added html links to the GenBank BioSample (22 May 2015)

More information about this dataset deployment

Funding

Award Number	Funding Source
OCE-0849578	NSF Division of Ocean Sciences
OCE-1061391	NSF Division of Ocean Sciences

Instruments

Automated DNA Sequencer

Supplied Name: Automated Sequencer

Supplied Description:

Roche 454 FLX Titanium platform

Instrument Type

Generic Name: Automated DNA Sequencer

Acronym: Automated Sequencer

Generic Description:

General term for a laboratory instrument used for deciphering the order of bases in a strand of DNA. Sanger sequencers detect fluorescence from different dyes that are used to identify the A, C, G, and T extension reactions. Contemporary or Pyrosequencer methods are based on detecting the activity of DNA polymerase (a DNA synthesizing enzyme) with another chemoluminescent enzyme. Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step.

ROV Jason

Supplied Description:

Sampled with push cores of 6.35cm diameter and configured with a seal to prevent contamination during ascent.

Instrument Type

Generic Name: ROV Jason

Generic Description:

The Remotely Operated Vehicle (ROV) Jason is operated by the Deep Submergence Laboratory (DSL) at Woods Hole Oceanographic Institution (WHOI). WHOI engineers and scientists designed and built the ROV Jason to give scientists access to the seafloor that didn't require them leaving the deck of the ship. Jason is a two-body ROV system. A 10-kilometer (6-mile) fiber-optic cable delivers electrical power and commands from the ship through Medea and down to Jason, which then returns data and live video imagery. Medea serves as a shock absorber, buffering Jason from the movements of the ship, while providing lighting and a bird’s eye view of the ROV during seafloor operations. During each dive (deployment of the ROV), Jason pilots and scientists work from a control room on the ship to monitor Jason’s instruments and video while maneuvering the vehicle and optionally performing a variety of sampling activities. Jason is equipped with sonar imagers, water samplers, video and still cameras, and lighting gear. Jason’s manipulator arms collect samples of rock, sediment, or marine life and place them in the vehicle’s basket or on "elevator" platforms that float heavier loads to the surface. More information is available from the operator site at URL.

Thermal Cycler

Supplied Description:

Instrument Type

Generic Name: Thermal Cycler

Generic Description:

A thermal cycler or "thermocycler" is a general term for a type of laboratory apparatus, commonly used for performing polymerase chain reaction (PCR), that is capable of repeatedly altering and maintaining specific temperatures for defined periods of time. 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. They can also be used to facilitate other temperature-sensitive reactions, including restriction enzyme digestion or rapid diagnostics.

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

Parameters

Supplied Name	Supplied description	Supplied Units	Standard Name
project_id	GenBank Project identification	unitless	no_bcodmo_term
seq_target	sequence target	unitless	no_bcodmo_term
BioSample_id	GenBank BioSample identification number and hyperlink.	unitless	sample
SRA	GenBank Sequence Read Archive identification	unitless	no_bcodmo_term
sample_location	sampling location	unitless	station
cruise_id	cruise identification	unitless	cruise_id
lat	latitude; north is positive	decimal degrees	lat
lon	longitude; east is positive	decimal degrees	lon

Database

Contribute Data

Dataset: DHAB halocline sediment pyrotags - eukaryotes
Deployment: AT18-14

Dataset acquisition description

Dataset Processing Description

Database

Contribute Data

Dataset: DHAB halocline sediment pyrotags - eukaryotesDeployment: AT18-14

Dataset acquisition description

Dataset Processing Description

Dataset: DHAB halocline sediment pyrotags - eukaryotes
Deployment: AT18-14