Dataset: 16S rRNA gene from RNA
Deployment: Chikyu-331

16S rRNA gene (from RNA)

View Data: For data, See Dataset Metadata Page: https://www.bco-dmo.org/dataset/781114

Principal Investigator:

Christopher House (Pennsylvania State University, PSU)

Scientist:

Leah Brandt (Pennsylvania State University, PSU)

BCO-DMO Data Manager:

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

Project:

An In-Depth analysis of the subvent biosphere within Okinawa Backarc Basin (IODP 331, Iheya North Hydrothermal Field) sediments (Subvent_Biosphere_Sediments)

Version:

Deployment Synonyms:

IODP-331

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Description

Next-generation sequencing data across a hydrothermal gradient; Okinawa Backarc Basin IODP 331 – subvent biosphere. RNA extractions were attempted from sediments at Sites C0014 and C0017 to selectively investigate the active microbial populations at these sites. This study was conducted to supplement the gene-based and metagenomic studies. However, the results from sequenced reverse transcribe 16S rRNA demonstrate the sensitivity of extraction and amplification to external nucleic acids and attest to the challenges in working with low biomass environmental samples. Inconsistencies between primersets and replicates make conclusions about the results of this RNA study extremely tenuous.

Methods & Sampling

Dataset acquisition description

Sampling of cores was done in accordance with IODP protocols.

All C0014 samples were cored on 16 September 2010 via via HPCS (hydraulic piston coring system).
All C0015 samples were cored on 18 September 2010 via HPCS.
All C0017 samples were cored on 27 September 2010 via HPCS.

Core sections were kept frozen (-80°C) until DNA extractions. RNA was extracted using a phenol chloroform protocol. The RNA extract was DNAse treated using the Invitrogen™ Ambion™ Turbo DNA-free Kit and purified using the Qiagen® RNeasy Mini Kit. A cDNA library was made using the Qiagen ® QuantiTec Reverse Transcription Kit. Polymerase chain reactions used the KAPABiosystems® KAPA2G Robust HotStart ReadyMix PCR Kit for 32 cycles.

Sequencing of the product was performed on the Illumina® MiSeq 2500.

Data Processing Description

Dataset Processing Description

Data Processing: Demultiplexing was performed in Mothur (v.1.30.1).

Problem report: Upon analysis, the RNA dataset does not appear to reflect the same or similar information as the published 16S rRNA gene amplicon dataset. Rather, it indicates significant background noise from extraction and not an indigenous representation of the subsurface biosphere.

More information about this dataset deployment

Funding

Award Number	Funding Source
OCE-0939564	NSF Division of Ocean Sciences

Instruments

Advanced Piston Corer

Supplied Name: hydraulic piston coring system (HPCS)

Supplied Description:

Instrument Type

Generic Name: Advanced Piston Corer

Acronym: APC

Generic Description:

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).

Automated DNA Sequencer

Supplied Name: Illumina MiSeq 2500

Supplied Description:

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.

Parameters

Supplied Name	Supplied description	Supplied Units	Standard Name
Cruise_ID	Cruise ID number	unitless	cruise_id
Sample_Name	Sample name	unitless	sample
Latitude	Latitude; positive values = North	decimal degrees	lat
Longitude	Longitude; positive values = East	decimal degrees	lon
Water_depth	Water depth	meters	depth_w
Top_Depth	Top depth	meters below seafloor	depth_bsf
Bottom_Depth	Bottom depth	meters below seafloor	depth_bsf
Sediment_type	Sediment type	unitless	sample_descrip
Estimated_Temp	Estimated temperature; based on 3 degrees C/m	degrees Celsius	temp
Nucleic_Acid_amplified	Type of nucleic acid amplified	unitless	sample_descrip
Primer_Set_16S_rRNA	16S rRNA primer set	unitless	sample_descrip
Illumina_dataset	Illumina dataset ID	unitless	sample_descrip
Notes	Notes	unitless	comment

Database

Contribute Data

Dataset: 16S rRNA gene from RNA
Deployment: Chikyu-331

Dataset acquisition description

Dataset Processing Description

Database

Contribute Data

Dataset: 16S rRNA gene from RNADeployment: Chikyu-331

Dataset acquisition description

Dataset Processing Description

Dataset: 16S rRNA gene from RNA
Deployment: Chikyu-331