URLhttps://www.bco-dmo.org/dataset/626150
Download URLhttps://www.bco-dmo.org/dataset/626150/data/download
Media Typetext/tab-separated-values
CreatedNovember 5, 2015
ModifiedSeptember 30, 2016
StateFinal no updates expected
Brief DescriptionSub-seafloor metatranscriptomes from anaerobic Peru Margin sediments collected on JOIDES Resolution, Leg 201.

Acquisition Description

Sample collection and storage:  Subsurface sediment samples from the continental shelf of Peru, Ocean Drilling Program (ODP) Site 1229D (77° 57.4590′ W, 10° 58.5721′ S), were obtained during ODP Leg 201 on 6 March 2002. Careful precautions were taken to avoid contamination during the sampling process. For Integrated Ocean Drilling Program (IODP) cores, contamination tests were performed using perfluorocarbon tracers and fluorescent microspheres. Sediment samples were immediately frozen at −80 degrees C after sampling and stored at −80 degrees C until used for mRNA extractions in this study (10-year storage time at −80 degrees C).

RNA extraction and purification:  Extraction of sub-seafloor RNA was performed according to the protocol described previously. In brief, RNA was extracted from 25 g of sediment using the FastRNA Pro Soil-Direct Kit (MP Biomedicals). It was necessary to scale up the volume of sediment that is typically extracted with the kit (~0.5 g) owing to the low biomass inherent to marine subsurface samples. All tubes, tips and disposables used were certified RNase free and all extraction procedures were performed in a laminar flow hood to reduce aerosol contamination by bacterial and fungal cells/spores. Five 15-ml Lysing Matrix E tubes (MP Biomedicals) were filled with 5 g sediment and 5 ml of Soil Lysis Solution (MP Biomedicals). Tubes were vortexed to suspend the sediment and Soil Lysis Solution was added to the tube leaving 1 ml of headspace. Tubes were then homogenized for 60 s on the FastPrep-24 homogenizer (MP Biomedicals) with a setting of 4.5. Contents were pooled into two 50-ml tubes and centrifuged for 30 min at 4000 r.p.m. (3220g) at room temperature (25  degrees C). Supernatants were combined in a new 50-ml tube and 1/10 volume of 2 M sodium acetate (pH 4.0) was added. An equal volume of phenol-chloroform (pH 6.5) was added and vortexed for 30 s, incubated for 5 min at room temperature, and spun at 4000 r.p.m. (3220g) for 20 min at 4 degrees C. The aqueous phase was transferred to a new 50-ml tube. Nucleic acids were precipitated by adding 2.5 and 1/10 volumes 100% ethanol and 3 M sodium acetate, respectively, and incubating overnight at −80 degrees C. The next day, tubes were spun at 4000 r.p.m. (3220g) for 60 min at 4 degrees C and the supernatant removed. Pellets were washed with 70% ethanol, spun for 15 min at 4 degrees C and air-dried. Dried pellets were resuspended with 0.25 ml RNase-free sterile water and combined into a new 1.5-ml tube. 1/10 volume of 2 M sodium acetate (pH 4.0) and an equal volume of phenol-chloroform (pH 6.5) were added, vortexed for 1 min and incubated for 5 min at room temperature. This was necessary to remove residual organic material (that is, humic acids) resulting from the rather large pellet/precipitate. After centrifuging at 14000 r.p.m. (20817g) for 10 min at 4 degrees C, the top phase was removed into a new 1.5-ml tube. 0.7 volumes of 100% isopropanol was added and incubated for 1 h at −20 degrees C (to precipitate nucleic acids). Tubes were then centrifuged for 20 min at 14000 r.p.m. (20,817g) at 4 degrees C and the supernatant removed. Pellets were washed with 70% ethanol and centrifuged at 14000 r.p.m. (20817g) for 5 min at 4 degrees C. After removing ethanol and air-drying, pellets were re-suspended in 0.2 ml of RNase free sterile water. DNA was removed using the Turbo DNA-free kit (Life Technologies), increasing the incubation time to 1 h to ensure rigorous DNA removal. After this step, samples were taken through the protocol supplied with the FastRNA Pro Soil-Direct kit to the end (starting at the RNA Matrix and RNA Slurry addition step), including the column purification step to remove residual humic acids (see FastRNA Pro Soil-Direct Kit manual). Extraction blanks were performed (adding sterile water instead of sample) to ensure that aerosolized contaminants did not enter sample and reagent tubes during the extraction process. Absence of DNA and RNA contamination was confirmed by no visible amplification of small subunit (SSU) ribosomal RNA (rRNA) and rRNA genes from extraction blanks after 35 cycles of PCR and RT–PCR.

After RNA extraction, the MEGA-Clear RNA Purification Kit (Life Technologies) was used to purify the RNA. This kit removes short RNA fragments (mostly produced during the extraction protocol) and residual inhibitors (that is, humics). We followed the protocol all the way through the optional precipitation/concentration step, re-suspending the RNA pellet in 10 ul of RNase-free sterile water. Before cDNA amplification, the removal of contaminating DNA in RNA extracts was confirmed by the absence of visible amplification of SSU rRNA genes after 35 cycles of PCR using the RNA extracts as template.

cDNA amplification and Illumina sequencing: Five microlitres of purified RNA was used as template for whole-cDNA amplification using the Ovation RNA-Seq v2 System (NuGEN technologies, http://www.nugeninc.com/nugen/index.cfm/products/cs/ngs/rna-seq-v2/). We followed the manufacturer’s instructions for cDNA amplification, and the resulting quantity of cDNA was checked on a Nanodrop (Thermo Scientific) and Fluorometer (Qubit 2.0, Life Technologies). Quality of the amplified cDNA was checked on a Bioanalyzer (Agilent Biotechnologies) before Illumina sequencing. Illumina library preparation and paired-end sequencing was performed at the University of Delaware Sequencing and Genotyping Center (Delaware Biotechnology Institute).

Related references:
For more sampling information see www-odp.tamu.edu/publications/prelim/201_prel/201toc.html
The manuscript is at http://www.nature.com/nature/journal/v499/n7457/full/nature12230.html

Processing Description

Quality control of the data set was performed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), with a quality score cutoff of 28. Approximately 1 billion paired-end reads that passed quality control were imported into CLC Genomics Workbench 5.0 (CLC Bio) and assembled using the paired-end Illumina assembler. Contigs were assembled over a range of k-mer sizes (20, 50, 60, 64) with a minimum contig size cutoff of 300 nucleotides. The k-mer size of 50 resulted in the highest number of contigs and these contigs were chosen for use in downstream analyses. To reduce the formation of chimaeric assemblies, we used a paired-end sequencing approach and performed assemblies without scaffolding. Reads were mapped onto the contigs using the read mapping option in CLC Genomics Workbench to retain information on relative abundance of contigs. Quality-filtered reads and raw reads are publicly available through the NCBI SRA at http://www.ncbi.nlm.nih.gov/sra?term=SRA058813

Instruments

Details
Instance Description (PCR)

Absence of DNA and RNA contamination was confirmed by no visible amplification of small subunit (SSU) ribosomal RNA (rRNA) and rRNA genes from extraction blanks after 35 cycles of PCR and RT–PCR.

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)

Details
Instance Description (Nanodrop)

We followed the manufacturer’s instructions for cDNA amplification, and the resulting quantity of cDNA was checked on a Nanodrop (Thermo Scientific) and Fluorometer (Qubit 2.0, Life Technologies).

An instrument used to measure the relative absorption of electromagnetic radiation of different wavelengths in the near infra-red, visible and ultraviolet wavebands by samples.

Fluorometer [Fluorometer]
Details
Instance Description (Fluorometer)

We followed the manufacturer’s instructions for cDNA amplification, and the resulting quantity of cDNA was checked on a Nanodrop (Thermo Scientific) and Fluorometer (Qubit 2.0, Life Technologies).

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.

Bioanalyzer [Bioanalyzer]
Details
Instance Description (Bioanalyzer)

Quality of the amplified cDNA was checked on a Bioanalyzer (Agilent Biotechnologies) before Illumina sequencing.

A Bioanalyzer is a laboratory instrument that provides the sizing and quantification of DNA, RNA, and proteins. One example is the Agilent Bioanalyzer 2100.

Parameters

accession_number [accession number]
Details
accession_number

NCBI accession number.
 

Database identifier assigned by repository and linked to GenBank or other repository.
SRA_number [accession number]
Details
SRA_number

NCBI SRA accession number.
 

Database identifier assigned by repository and linked to GenBank or other repository.
description [brief_desc]
Details
description

Brief description of the sequence.

brief description, open ended, specific to the data set in which it appears

Details
SRA_URL

Hyperlink to NCBI SRA accession.

Database identifier assigned by repository and linked to GenBank or other repository.
accession_URL [accession number]
Details
accession_URL

Hyperlink to NCBI accession.

Database identifier assigned by repository and linked to GenBank or other repository.

Dataset Maintainers

NameAffiliationContact
William D. OrsiWoods Hole Oceanographic Institution (WHOI)
Glenn D. ChristmanUniversity of Delaware
Virginia P. EdgcombWoods Hole Oceanographic Institution (WHOI)
Jennifer F. BiddleUniversity of Delaware
Virginia P. EdgcombWoods Hole Oceanographic Institution (WHOI)
William D. OrsiWoods Hole Oceanographic Institution (WHOI BCO-DMO)
Shannon RauchWoods Hole Oceanographic Institution (WHOI BCO-DMO)

BCO-DMO Project Info

Project TitleWorld-wide exploration of microbial eukaryote diversity and activity in the marine subsurface
AcronymMicrobial Euk Div Mar Subsurface
URLhttps://www.bco-dmo.org/project/626119
CreatedNovember 5, 2015
ModifiedNovember 10, 2015
Project Description

Project description obtained from C-DEBI:
Practically nothing is known about microbial eukaryotes (mEuks) in the marine subsurface. mEuks are pivotal members of microbial communities because they regenerate nutrients and modify or remineralize organic matter through grazing on prokaryotic and other eukaryotic prey. Thus, mEuks help determine metabolic potentials of microbial communities and influence elemental cycling. Only one study has addressed mEuk diversity in the marine subsurface (Edgcomb et al. 2010), which suggested Fungi dominate the eukaryotic subsurface community and are active in sediments 35 mbsf at the Peru Margin. Thus, some mEuks may be specifically adapted to the deep subsurface and may play significant roles in the utilization and regeneration of organic matter and nutrients in deep-sea sediments. 

One objective of this study will be to further investigate whether Fungi are consistently the dominant group of mEuks in the marine subsurface by examining mEuk diversity in a broad range of subsurface samples from ODP expeditions spanning the world’s oceans. Deep sequencing of SSU rRNA in these samples will provide a proxy for mEuk diversity and activity in the marine subsurface. A second objective will be to ‘ground truth’ an mRNA isolation protocol for mEuks in marine subsurface sediments. Once established, this protocol will enable the third objective, which is the creation of a eukaryotic metatranscriptome from ODP site 1229. This metatranscriptome will provide insights into the functional role of mEuks in the marine subsurface and perhaps new insights into microbial evolution.

This project was funded by a C-DEBI Postdoctoral Fellowship.

Project Maintainers
NameAffiliationRoleContact
William D. OrsiUniversity of MunichPrincipal Investigator
Glenn D. ChristmanUniversity of DelawareCo-Principal Investigator
Virginia P. EdgcombWoods Hole Oceanographic Institution (WHOI)Co-Principal Investigator
Jennifer F. BiddleUniversity of DelawareCo-Principal Investigator
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