Download URLhttps://www.bco-dmo.org/dataset/661056/data/download
Media Type text/tab-separated-values
Created October 6, 2016
Modified October 11, 2016
State Final no updates expected
Brief Description

Accession numbers for clone and Illumina amplicon sequence libraries from Dorado Outcrop basalts

Acquisition Description

Acquisition methods are described in the following publication:
Lee, M.D., Walworth, N.G., Sylvan, J.B., Edwards, K.J., and Orcutt, B.N. 2015. Microbial Communities on Seafloor Basalts at Dorado Outcrop Reflect Level of Alteration and Highlight Global Lithic Clades. Front. Microbiol. doi:10.3389/fmicb.2015.01470

In summary (excerpted from above):
Over the span of December 7–23, 2013, 12 seafloor rock samples (11 basalts and 1 lithified carbonate, hereafter R1–R12) were collected from across Dorado Outcrop while aboard R/V Atlantis during cruise AT26-09 following previously developed protocols. Using the ROV Jason II, samples were collected and those for DNA analysis were placed in either sterile whirl-pak bags or centrifuge tubes and frozen at −80 degrees C. In addition to the basalts, two bottom water samples were collected using a Niskin bottle mounted to an elevator, and 1.25 L were filtered onto 0.2 um pore size polycarbonate Nucleopore filters that were then frozen at −80 degrees C.

DNA Extraction and Sequencing of the 16S rRNA Gene
Frozen rock pieces were crushed in a flame-sterilized impact mortar into sand-sized grains which were then transferred to sterile plastic centrifuge tubes and stored at −80 degrees C until DNA extractions were performed. DNA extractions were carried out with the FastDNA Spin Kit for Soil (MP Biomedicals, Santa Ana, CA, USA) following the manufacturer’s specifications. About 0.5 g of crushed material were placed directly into the lysis tubes of the kit, as were the bottom water filters. Protocol blanks were performed with each extraction (no samples or DNA added to lysis tubes) to track the potential for contamination. DNA concentrations were quantified with the Qubit HS dsDNA Assay kit with a Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) according to manufacturer protocols.

DNA extracts from the 12 rock samples (plus one technical replicate from the same sample), one green-colored, potential biofilm sample from R11, two bottom water samples, and four protocol blanks for a total of 20 samples were sent for DNA sequencing by a commercial vendor (Molecular Research LP; MR DNA; Shallowater, TX, USA). Illumina MiSeq paired-end (2 × 300 base pair) tag sequencing was carried out using the Earth Microbiome Project universal primers 515f and 806r, which flank the V4 region of the 16S rRNA gene. Library preparation and sequencing was carried out at the facility. In brief, the 515f/806r PCR primers with 8-base barcodes on the forward primer were used in a PCR reaction with the HotStarTaq Plus Master Mix Kit (QIAGEN:USA, Valencia, CA, USA). Based on their DNA concentrations and molecular weight, multiple samples were pooled together in equal proportions, purified with Ampure XP beads, and then used to prepare the library by following the Illumina TruSeq DNA library preparation protocol.

Sequence Data Analysis
Tag data curation and processing were carried out using mothur v.1.34.4 (Schloss et al., 2009) following the mothur Illumina MiSeq Standard Operating Procedure (Kozich et al., 2013). Refer to Lee et al (2015) for more information including treatment of extraction blanks and OTU filtering.

Clone Library Processing
For the clone library, near full-length contigs were assembled using Geneious v6.1.8 (Kearse et al., 2012). Sequences were oriented and trimmed manually and then screened for chimeras using the online program Decipher version 1.14.4 (Wright et al., 2012). The resulting sequences were used in phylogenetic tree construction and submitted to BLAST (Altschul et al., 1990) to search for nearest cultured neighbors as well as environmental samples.

Accession Numbers
The clone sequences recovered from this project are publicly available through NCBI′s GenBank, accession numbers KT748562–KT748628, and the raw tag data are available through NCBI′s Sequence Read Archive under project accession number SRP063681. Additionally, a fasta-formatted file containing the representative OTU sequences identified is available as a Datasheet 2 in Supplementary Material to Lee et al. (2015).

Processing Description

BCO-DMO Processing:
– BCO-DMO created file of accession numbers and links using information provided in the dataset metadata form.


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)


data_type [datatype]
Type of sequence
sampling method - instrument type, e.g. MOCNESS-10, CTD, etc.
repository [brief_desc]
Name of repository

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

description [brief_desc]
Description of the sequences

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

accession_number [accession number]
Accession number
Database identifier assigned by repository and linked to GenBank or other repository.
accession_link [accession number]
Hyperlink to accession in the repository
Database identifier assigned by repository and linked to GenBank or other repository.

Dataset Maintainers

Beth N. OrcuttBigelow Laboratory for Ocean Sciences
Shannon RauchBigelow Laboratory for Ocean Sciences

BCO-DMO Project Info

Project Title The Dorado Outcrop low-temperature ridge flank environment
Acronym Dorado ridge flank environment
Created September 30, 2016
Modified October 6, 2016
Project Description

Project description obtained from C-DEBI
With support from this project, I participated in two research cruises to the Dorado Outcrop in 2013 and 2014, leading efforts to collect samples of fluids, rocks, and sediment for microbiological characterization. In 2013, we were successful in discovering low-temperature seeps on the outcrop, although sampling issues prevented the collection of pristine fluid samples for detailed microbiological characterization. High-quality fluids were collected during the 2014 cruise, and several C-DEBI student and postdoc collaborators that participated in the 2014 cruise are currently analyzing them. We collected seafloor basalts from around the outcrop, both near and far to active venting. A student is currently analyzing 16S rRNA gene sequence libraries from these samples; preliminary analysis indicates some microbial community overlap with seafloor basalts from other deep-sea environments, but also some unique groups that may reflect the age of the Dorado Outcrop environment compared to other studies. We collected push cores and gravity cores during both cruises to document the diffusion of potentially altered basement fluids into sediment, and were successful in documenting the presence of oxygen in these cores, confirming our hypotheses about fluid-rock reactions in basement surrounding Dorado Outcrop. Several C-DEBI supported students and postdocs are further characterizing the sediment biogeochemistry and microbial community activity, structure and function. Both cruises were used as opportunities to engage the general public in deep-sea science through news articles, microblogging, and a feature on Al Jazeera America.

Related Publications:
Lee, M.D., Walworth, N.G., Sylvan, J.B., Edwards,K.J., Orcutt, B.N. 2015. Microbial Communities on Seafloor Basalts at Dorado Outcrop Reflect Level of Alteration and Highlight Global Lithic Clades. Frontiers in Microbiology 6:1470. C-DEBI Contribution 286. 

Orcutt, B. N. et al. 2013. Oxygen consumption rates in subseafloor basaltic crust derived from a reaction transport model. Nat. Commun. 4:2539. C-DEBI Contribution 166.

Note: This project was funded by a C-DEBI Research Grant.

Data Project Maintainers
Beth N. OrcuttBigelow Laboratory for Ocean SciencesPrincipal Investigator