Dataset: sea_fan_sequences
Deployment: Coral_Dive_HM

Accession numbers for Gorgonia ventalina (sea fan) sequencing data

Principal Investigator:

Drew Harvell (Cornell University, Cornell)

Co-Principal Investigator:

Laura Mydlarz (University of Texas at Arlington, UT Arlington)

Contact:

Dr Colleen Burge (Cornell University, Cornell)

BCO-DMO Data Manager:

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

Project:

Influence of Temperature and Acidification on the Dynamics of Coral Co-Infection and Resistance (Climate_CoralDisease)

Current State:

Final no updates expected

Version:

13 Sept 2012

Version Date:

2012-09-13

Data URL:

https://osprey.bco-dmo.org/dataset-deployment/455436/data

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Description

Accession numbers to NCBI's Gene Expression Omnibus (GEO) are provided for the Gorgonia ventalina Illumina sequences collected from reefs off the coast of Puerto Rico.

References:
Burge CA, Mouchka, ME, Harvell, CD & S Roberts. (In review) Immune response of the Caribbean sea fan, Gorgonia ventalina exposed to an Aplanochytrium parasite as revealed by transcriptome sequencing.

Methods & Sampling

Dataset acquisition description

Sea fan collection and husbandry
Twelve G. ventalina individuals were collected at Laurel Patch Reef, La Parguera, Puerto Rico in May 2010. Sea fans were cut into two, 6x9 cm pieces, and suspended in situ to heal on the reef for three days. Sea fans were then collected from the reef and moved into static 38 L aquariums at the University of Puerto Rico, Isla Magueyes Laboratories in La Parguera, PR. Fans were distributed equally, in a clonally replicated design, between six aquariums (n=4 sea fans per aquarium with sea fan specimens 1-4, 5-8, 9-12 held in individual aquariums in duplicate). Sea fans were acclimated for two days and water was changed twice daily.

Experimental Inoculations
One piece of each sea fan (n=12) was injected three times with a solution of a 5 day culture of an Aplanochytrium previously isolated from a sea fan. Complementary clonal pieces (n=12) were injected with QPX media only to serve as a control. After 24 hours, an area including each of the three injection points was collected from each sea fan and flash frozen in liquid nitrogen and subsequently stored at -80 C. Sea fan samples were shipped on dry ice overnight to Cornell University and stored at -80 C until sample preparation.

Sample preparation
Each individual sea fan sample was ground in liquid nitrogen using a mortar and pestle, and the resulting powder was placed in a 2.0 mL microcentrifuge tube. Total RNA was extracted using a modified Trizol/Qiagen RNeasy protocol, whereby following the ethanol precipitation step of the Trizol manufacturer's instructions (Invitrogen, The Life Technologies CorporationTM, Grand Island, NY ), the aqueous solution was added to an RNeasy column, and the Qiagen RNeasy manufacturer's instructions were subsequently followed (Qiagen, Valencia, California). DNA was removed from extracted RNA using the Turbo DNA-free treatment according to the manufacturer's instructions (Ambion Inc, The Life Technologies CorporationTM, Grand Island, NY). Removal of DNA was confirmed by using RNA (1 µl) as template in a PCR reaction targeting 18s ribosomal DNA as previously described. RNA concentrations were quantified using the NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE).

Sequencing and Analysis
For each treatment, samples were pooled (n=12) using 900 ng of total RNA from each sea fan. RNA quality was assessed using an Agilent BioAnalyzer 2100 at the Cornell University Life Sciences Core Laboratory Center (CLC). Library preparation was done at the Cornell Microarray facility using the mRNA-Seq 8-Sample Prep Kit (Illumina, San Diego, California) followed by sequencing preparation using the standard cluster generation kit and 36 cycle Illumnia sequencing kit (Illumina, San Diego, California) at the Cornell University CLC. Each library was sequenced in its own individual lane where 86 bp reads were captured using an Illumina/Solexa Genome Analyzer at the Cornell University CLC. The data have been deposited in NCBI's Gene Expression Omnibus.

Data Processing Description

Dataset Processing Description

Data Processing:
Initially, all sequences were trimmed based on quality scores of 0.05 and the number of ambiguous nucleotides (>2 on ends). Sequences smaller than 20 bp were also removed. De novo assembly was carried out using CLC Genomics Workbench v4.0 (CLC Bio) with the following parameters: similarity=0.90, length fraction=0.8, insertion cost=3, deletion cost=3, mismatch cost=2 and minimum size = 400.

Consensus sequences (or contigs) were compared to the UniProtKB/Swiss-Prot database. Comparisons were made using the BLASTx algorithm with a maximum of 1E-6 e-value threshold.

RNA-Seq analysis was performed to determine differential gene expression patterns between Aplanochytrium exposed and control libraries. CLC Genomics Workbench v4.0 (CLC Bio) was used to map the reads to the assembled transcriptome and to obtain raw counts of sequencing reads using following parameters: unspecific match limit = 5, maximum number of mismatches = 2, minimum number of reads = 10. Statistical comparisons of count data between the control and Aplanochytrium exposed libraries was carried out using DESeq. Genes were considered differentially expressed in a given library when the adjusted p-value was less than or equal to 0.05.

To identify enriched biological themes and GO terms, the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 was used. Specifically, corresponding Swiss-Prot accession numbers for differentially expressed genes were used as the gene list and corresponding Swiss-Prot accession numbers for the assembled transcriptome was used as the background. Biological Process terms were considered significantly enriched when the p-value was less than 0.05.

More information about this dataset deployment

Funding

Award Number	Funding Source
OCE-0849776	NSF Division of Ocean Sciences

Instruments

Automated DNA Sequencer

Supplied Description:

Each library was sequenced using an Illumina/Solexa Genome Analyzer at the Cornell University Core Laboratory Center.

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
treatment	Exposure type: Aplanochytrium exposed = sea fan injected with a solution of Aplanochytrium culture. Control = sea fan injected with QPX media as a control.	text	no_bcodmo_term
accession_number	Assigned accession number and link to NCBI GEO.	unitless	accession_number
site	Name of the reef where G. ventalina was collected.	text	site
lat	Latitude of the collection site. North = positive.	decimal degrees	lat
lon	Longitude of the collection site. West = negative.	decimal degrees	lon

Database

Contribute Data

Dataset: sea_fan_sequencesDeployment: Coral_Dive_HM

Dataset acquisition description

Dataset Processing Description

Dataset: sea_fan_sequences
Deployment: Coral_Dive_HM