ONTOLOGY SOURCE REFERENCE								
Term Source Name	UO	OBI	CHEBI	PATO	NCBITax		ENVO	
Term Source File								
Term Source Version	v 1.26	beta	v 1.26	v 1.26	v 1.26		v 1.26	
Term Source Description	Unit Ontology	Ontology of Biomedical Investigation	Chemical Entities of Biological Interest	Phenotypic qualities (properties)	NCBI Taxonomy Database		Environment Ontology	
INVESTIGATION								
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INVESTIGATION PUBLICATIONS								
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INVESTIGATION CONTACTS								
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STUDY								
Study Identifier	BII-S-3							
Study Title	Metagenomes and Metatranscriptomes of phytoplankton blooms from an ocean acidification mesocosm experiment							
Study Submission Date	15/08/2008							
Study Public Release Date	15/08/2008							
Study Description	"Sequencing the metatranscriptome can provide information about the response of organisms to varying environmental conditions. We present a methodology for obtaining random whole-community mRNA from a complex microbial assemblage using Pyrosequencing. The metatranscriptome had, with minimum contamination by ribosomal RNA, significant coverage of abundant transcripts, and included significantly more potentially novel proteins than in the metagenome. This experiment is part of a much larger experiment. We have produced 4 454 metatranscriptomic datasets and 6 454 metagenomic datasets. These were derived from 4 samples."							
Study File Name	s_BII-S-3.txt							
STUDY DESIGN DESCRIPTORS								
Study Design Type	time series design							
Study Design Type Term Accession Number	500020							
Study Design Type Term Source REF	OBI							
STUDY PUBLICATIONS								
Study PubMed ID	18725995	18783384						
Study Publication DOI	10.1371/journal.pone.0003042	10.1111/j.1462-2920.2008.01745.x						
Study Publication Author list	"Gilbert JA, Field D, Huang Y, Edwards R, Li W, Gilna P, Joint I."	"Gilbert JA, Thomas S, Cooley NA, Kulakova A, Field D, Booth T, McGrath JW, Quinn JP, Joint I."						
Study Publication Title	Detection of large numbers of novel sequences in the metatranscriptomes of complex marine microbial communities.	Potential for phosphonoacetate utilization by marine bacteria in temperate coastal waters.						
Study Publication Status	indexed in PubMed	indexed in PubMed						
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STUDY FACTORS								
Study Factor Name	dose	compound	collection_date					
Study Factor Type	dose	chemical compound	time					
Study Factor Type Term Accession Number		37577						
Study Factor Type Term Source REF		CHEBI						
STUDY ASSAYS								
Study Assay Measurement Type	metagenome sequencing	transcription profiling						
Study Assay Measurement Type Term Accession Number		424						
Study Assay Measurement Type Term Source REF	OBI	OBI						
Study Assay Technology Type	nucleotide sequencing	nucleotide sequencing						
Study Assay Technology Type Term Accession Number								
Study Assay Technology Type Term Source REF	OBI	OBI						
Study Assay Technology Platform	454 Genome Sequencer FLX	454 Genome Sequencer FS						
Study Assay File Name	a_gilbert-assay-Gx.txt	a_gilbert-assay-Tx.txt						
STUDY PROTOCOLS								
Study Protocol Name	sample collection - standard procedure 1	nucleic acid extraction - standard procedure 2	mRNA extraction - standard procedure 3	genomic DNA extraction - standard procedure 4	reverse transcription - standard procedure 5	library construction	pyrosequencing - standard procedure 6	sequence analysis - standard procedure 7
Study Protocol Type	environmental material collection	nucleic acid extraction	RNA extraction	DNA extraction	reverse transcription	library construction	nucleic acid sequencing	nucleic acid sequencing
Study Protocol Type Term Accession Number	600012	666667	666666	257			626	626
Study Protocol Type Term Source REF	OBI	OBI	OBI	OBI			OBI	OBI
Study Protocol Description	"Waters samples were prefiltered through a 1.6 um GF/A glass fibre filter to reduce Eukaryotic contamination. Filtrate was then collected on a 0.2 um Sterivex (millipore) filter which was frozen in liquid nitrogen until nucelic acid extraction. CO2 bubbled through 11,000 L mesocosm to simulate ocean acidification predicted conditions. Then phosphate and nitrate were added to induce a phytoplankton bloom."	Total nucleic acid extraction was done as quickly as possible using the method of Neufeld et al. 2007.	"RNA MinElute + substrative Hybridization + MEGAclear For transcriptomics, total RNA was separated from the columns using the RNA MinEluteTM clean-up kit (Qiagen) and checked for integrity of rRNA using an Agilent bioanalyser (RNA nano6000 chip). High integrity rRNA is essential for subtractive hybridization. Samples were treated with Turbo DNA-free enzyme (Ambion) to remove contaminating DNA. The rRNA was removed from mRNA by subtractive hybridization (Microbe Express Kit, Ambion), and absence of rRNA and DNA contamination was confirmed using the Agilent bioanalyser. The mRNA was further purified with the MEGAclearTM kit (Ambion). Reverse transcription of mRNA was performed using the SuperScript III enzyme (Invitrogen) with random hexamer primers (Promega). The cDNA was treated with RiboShredderTM RNase Blend (Epicentre) to remove trace RNA contaminants. To improve the yield of cDNA, samples were subjected to random amplification using the GenomiPhi V2 method (GE Healthcare). GenomiPhi technology produces branched DNA molecules that are recalcitrant to the pyrosequencing methodology. Therefore amplified samples were treated with S1 nuclease using the method of Zhang et al.2006."		superscript+random hexamer primer		"1. Sample Input and Fragmentation: The Genome Sequencer FLX System supports the sequencing of samples from a wide variety of starting materials including genomic DNA, PCR products, BACs, and cDNA. Samples such as genomic DNA and BACs are fractionated into small, 300- to 800-base pair fragments. For smaller samples, such as small non-coding RNA or PCR amplicons, fragmentation is not required. Instead, short PCR products amplified using Genome Sequencer fusion primers can be used for immobilization onto DNA capture beads as shown below under Emulsion Making, 2. Library Preparation: Using a series of standard molecular biology techniques, short adaptors (A and B), specific for both the 3' and 5' ends,  are added to each fragment. The adaptors are used for purification, amplification, and sequencing steps. Single-stranded fragments with A and B adaptors compose the sample library used for subsequent workflow steps. 3. Emulsion Making: The single-stranded DNA library is immobilized onto specifically designed DNA Capture Beads. Each bead carries a unique single-stranded DNA library fragment. The bead-bound library is emulsified with amplification reagents in a water-in-oil mixture resulting in microreactors containing just one bead with one unique sample-library fragment.4. emPCR (Emulsion PCR) Clonal Amplification: Each unique sample-library fragment is amplified within its own microreactor, excluding competing or contaminating sequences. Amplification of the entire fragment collection is done in parallel; for each fragment, this results in several million identical copies per bead. Subsequently, the emulsion mixture is broken while the amplified fragments remain bound to their specific beads. 5.Sequencing by Synthesis:The DNA-carrying Capture Beads are loaded onto the PicoTiterPlate device for sequencing. The diameter of the PicoTiterPlate wells allows for only one bead per well. After the PicoTiterPlate has been loaded into the Genome Sequencer FLX Instrument, individual nucleotides are flowed in a fixed order across the open wells and DNA Capture Beads. Addition of one (or more) nucleotide(s) complementary to the template strand results in a chemiluminescent signal recorded by the CCD camera of the Genome Sequencer FLX Instrument."""""	"SEED-MG-RAST: The combination of signal intensity and positional information generated across the PicoTiterPlate device allows the software to determine the sequence of more than 400,000 individual reads per 7.5-hour instrument run simultaneously. For sequencing-data analysis, three different bioinformatics tools are available supporting the following applications: de novo assembly up to 120 megabases; resequencing up to 3 gigabases; and amplicon variant detection by comparison with a known reference sequence."
Study Protocol URI								
Study Protocol Version								
Study Protocol Parameters Name	filter pore size						sequencing instrument	
Study Protocol Parameters Name Term Accession Number								
Study Protocol Parameters Name Term Source REF								
Study Protocol Components Name							454 GS-FLX	
Study Protocol Components Type							DNA sequencer	
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STUDY CONTACTS								
Study Person Last Name	Gilbert	Field	Huang	Edwards	Li		Gilna	Joint
Study Person First Name	Jack	Dawn	Ying	Rob	Weizhong		Paul	Ian
Study Person Mid Initials	A							
Study Person Email	jagi@pml.ac.uk							
Study Person Phone								
Study Person Fax								
Study Person Address	"Prospect Place, Plymouth, United Kingdom"	"CEH Oxford, Oxford, United Kingdom"	"San Diego State University, San Diego, California, United States of America"	"Argonne National Laboratory, Argonne, Illinois, United States of America"	"San Diego State University, San Diego, California, United States of America"		"San Diego State University, San Diego, California, United States of America"	"Prospect Place, Plymouth, United Kingdom"
Study Person Affiliation	Plymouth Marine Laboratory	NERC Centre for Ecology and Hydrology	California Institute for Telecommunications and Information Technology	"Department of Computer Science, Mathematics and Computer Science Division,"	California Institute for Telecommunications and Information Technology		California Institute for Telecommunications and Information Technology	Plymouth Marine Laboratory
Study Person Roles	principal investigator role	principal investigator role	principal investigator role	principal investigator role	principal investigator role		principal investigator role	principal investigator role
Study Person Roles Term Accession Number	103	103	103	103	103		103	103
Study Person Roles Term Source REF	OBI	OBI	OBI	OBI	OBI		OBI	OBI
