URLhttps://www.bco-dmo.org/dataset/626821
Download URLhttps://www.bco-dmo.org/dataset/626821/data/download
Media Type text/tab-separated-values
Created November 20, 2015
Modified August 19, 2016
State Final no updates expected
Brief Description

16S rRNA gene amplicon sequence accessions, Hydrate Ridge, offshore Oregon

Acquisition Description

DNA extraction and purification

DNA was extracted, in duplicate, from 8 to 20 g of “Mobio” IODP sediment samples previously frozen at -80C using a Powersoil total RNA Isolation Kit with the DNA Elution Accessory Kit (MO-BIO Laboratories, Solana Beach, CA, USA) following the manufacturer protocol but without beads. Approximately 2 grams of sediments were utilized for each extraction, and DNA pellets from each depth were pooled together. DNA concentrations were measured using a Qubit 2.0 fluorometer with dsDNA High Sensitivity reagents (Invitrogen, Grand Island, NY, USA). The DNA from the extractions that yielded robust 16S rRNA and metagenomic sequences ranged from 3.75-15 ng of DNA per gram of sediments.

16s rRNA gene PCR and amplicon sequencing

The V4 hypervariable region of the 16S rRNA gene was amplified from extracted DNA with barcoded Illumina-specific adapters (38) with technical replication. DNA template (2 ng) was mixed with 5 uL of 10x Taq Mutant reaction buffer, 0.4 uL of Klentaq LA Taq Polymerase (DNA Polymerase Technology, St. Louis, MO, USA), 2 uL of 10 mM dNTP mix (Sigma Aldrich, St. Louis, MO, USA), 2 uL of reverse and forward V4-specific primers F515 and R806 (39) and the remainder DNA-free water to 50 uL (Ambion, Grand Island, NY, USA). PCR was performed on a Bio-Rad C1000 Touch thermal cycler (Bio-Rad, Hercules, CA, USA) with the following conditions: initial denaturation at 94oC (5 min), followed by 35 cycles of denaturation at 94oC (40 s), primer annealing at 55oC (40 s) and primer extension at 68oC (30 s). The ~400 bp PCR size fragment was confirmed on a 1% agarose gel and replicated samples were pooled. A 50 uL fraction of each pooled sample was purified using a QIAquick PCR Purification Kit (Qiagen, Germantown, MD, USA).  Barcoded amplicons for each sample were pooled in equimolar concentrations and sequenced on an Illumina MiSeq (School of Biology, Georgia Institute of Technology), running MiSeq Control Software v.2.4.0.4, using MiSeq reagent kit v2 (500 cycles) with 5% PhiX genomic library control. 

Processing Description

BCO-DMO Processing:
– Added cruise_id, lat and lon columns

Instruments

Instance Description

 Bio-Rad C1000 Touch Thermocycler

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)

Instance Description

Illumina MiSeq

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.

Parameters

cruise_id [cruise_id]
Details
cruise_id
cruise identification
cruise designation; name
site [site]
Details
site
site identification
Sampling site identification.
lat [latitude]
Details
lat
latitude; north is positive

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

lon [longitude]
Details
lon
longitude; east is positive

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

BioSample [unknown]
Details
BioSample
NCBI BioSample id
association with a community-wide standard parameter is not yet defined
sample_gene [unknown]
Details
sample_gene
sequenced gene
association with a community-wide standard parameter is not yet defined
Tax_id [unknown]
Details
Tax_id
NCBI Taxonomy id
association with a community-wide standard parameter is not yet defined
NCBI_accession [unknown]
Details
NCBI_accession
NCBI accession number with link
association with a community-wide standard parameter is not yet defined

Dataset Maintainers

NameAffiliationContact
Jennifer B. GlassGeorgia Institute of Technology (Georgia Tech)
Nancy CopleyWoods Hole Oceanographic Institution (WHOI BCO-DMO)

BCO-DMO Project Info

Project Title Functional gene diversity and expression in methane-hydrate bearing deep subsurface sediments
Acronym Methane-Hydrate Sediment Omics
URLhttps://www.bco-dmo.org/project/626690
Created November 19, 2015
Modified November 19, 2015
Project Description

Methane is a critical component of the deep subsurface. In shallow marine sediments, anaerobic oxidation of methane (AOM) is coupled to sulfate reduction. However, relatively little is known about which microbial metabolisms are active in deeply buried sediment containing methane hydrates, particularly with regard to alternative electron acceptors that could fuel deep AOM. We propose to determine which microbial population(s) and functional genes are active in the deep biosphere beneath Hydrate Ridge offshore Oregon by sequencing metagenomes and metatranscriptomes from samples drilled on ODP Leg 204 and archived for future molecular analysis. We will analyze gene diversity and expression in six geochemically distinct zones from 2 to 139 meters below the seafloor with the goal of evaluating the relationship between geochemical conditions (i.e. sulfate, iron and manganese availability) and microbial metabolic activity.

Related References:

Tréhu, A.M, Bohrmann, G., Rack, F.R., Torres, M.E., et al., 2003. Proc. ODP, Init. Repts., 204: College Station, TX (Ocean Drilling Program).doi:10.2973/odp.proc.ir.204.2003

Shipboard Scientific Party, 2003. Site 1244. In Tréhu, A.M, Bohrmann, G., Rack, F.R., Torres, M.E., et al., Proc. ODP, Init. Repts., 204: College Station, TX (Ocean Drilling Program), 1–132. doi:10.2973/odp.proc.ir.204.103.2003

Tréhu, A.M., Bohrmann, G., Torres, M.E., and Colwell, F.S. (Eds.), 2006. Proc. ODP, Sci. Results, 204: College Station, TX (Ocean Drilling Program). doi:10.2973/odp.proc.sr.204.2006

This work was supported by a C-DEBI Research Grant.

Data Project Maintainers
NameAffiliationRole
Jennifer B. GlassGeorgia Institute of Technology (Georgia Tech)Principal Investigator
Cecilia Batmalle KretzGeorgia Institute of Technology (Georgia Tech)Co-Principal Investigator
James McManusUniversity of Akron (UAkron)Scientist
Menu