2. ISA-Tab format

Important

As a pre-requisite to reading this specification, please make sure you have read and understood the ISA Abstract Model that the ISA-Tab format is based on.

For detail on ISA framework terminology, please read the ISA Abstract Model specification.

This document describes the ISA Abstract Model reference implementation specified in the ISA-Tab format. ISA-Tab files are tab separated value (tsv) files, with specific labeled column structures specified below.

Below we provide the schemas and the content rules for valid ISA-Tab documents. Full examples of ISA content as ISA-Tab can be found in the ISA datasets repository, here https://git.io/vD1vC We recommend that you study these examples to better understand the structure of ISA-Tab documents.

2.1. Format

ISA-Tab uses three types of file to capture the experimental metadata:
  • Investigation file
  • Study file
  • Assay file (with associated data files)

The Investigation file contains all the information needed to understand the overall goals and means used in an experiment; experimental steps (or sequences of events) are described in the Study and in the Assay file(s). For each Investigation file there may be one or more Studies defined with a corresponding Study file; for each Study there may be one or more Assays defined with corresponding Assay files.

Files SHOULD be encoded using UTF-8.

Column delimiters SHOULD be the Unicode Horizontal Tab character (Unicode U+0009).

In order to facilitate identification of ISA-Tab component files, specific naming patterns SHOULD follow:

  • i_*.txt for identifying the Investigation file, e.g. i_investigation.txt
  • s_*.txt for identifying Study file(s), e.g. s_gene_survey.txt
  • a_*.txt for identifying Assay file(s), e.g. a_transcription.txt

All labels are case-sensitive:

  • In the Investigation file, section headers MUST be completely written in upper case (e.g. STUDY), field headers MUST have the first letter of each word in upper case (e.g. Study Identifier); with the exception of the referencing label (REF).
  • In the Study and Assay files, column headers MUST also have the first letter of each word in upper case, with the exception of the referencing label (REF).

Dates SHOULD be supplied in the ISO8601 format.

All values of cells MAY be enveloped with the Unicode Quotation Mark, Unicode U+0022 (the " character).

For maximal portability file names should only contain only ASCII characters not excluded already (that is A-Za-z0-9._!#$%&+,;=@^(){}'[] - we exclude space as many utilities do not accept spaces in file paths): non-English alphabetic characters cannot be guaranteed to be supported in all locales. It would be good practice to avoid the shell metacharacters (){}'[]$.".

2.2. Investigation File

The Investigation file fulfils four needs:

  1. to declare key entities, such as factors, protocols, which may be referenced in the other files
  2. to track provenance of the terminologies (controlled vocabularies or ontologies) there are used, where applicable
  3. to relate Assay files to Studies
  4. to relate each Study file to an Investigation (this only becomes necessary when two or more Study files need to be grouped).

An Investigation file is structured as a table with vertical headings along the first column, and corresponding values in the subsequent columns. The following section headings MUST appear in the Investigation file (in order), and the study block (headings from STUDY to STUDY CONTACTS) can be repeated, one block per study associated with the investigation.

  • ONTOLOGY SOURCE REFERENCE
  • INVESTIGATION
  • INVESTIGATION PUBLICATIONS
  • INVESTIGATION CONTACTS
  • STUDY
  • STUDY DESIGN DESCRIPTORS
  • STUDY PUBLICATIONS
  • STUDY FACTORS
  • STUDY ASSAYS
  • STUDY PROTOCOLS
  • STUDY CONTACTS

In the following sections, examples of each section block are given beside the specification of each section.

For a full example of a complete Investigation File, please see https://git.io/vD1va.

Attention

Rows in which the first character in the first column is Unicode U+0023 (the # character) MUST be interpreted as comments, where reference implementation parsers SHOULD ignore those lines entirely.

Rows where the label Comment[<comment name>] appear can also appear within any of the section blocks. Where these appear, the comment name must be unique within the context of a single block (e.g. you cannot have multiple occurences of Comment[external DB REF] within STUDY ASSAYS. Also, the value cells MUST match the number of values indicated by the rest of the section in context.

2.2.1. Ontology Source Reference section

The Ontology Source section of the Investigation file is used to declare Ontology Sources used elsewhere in the ISA-Tab files within the context of an Investigation.

Where a row labelled with Term Source REF suffixed in the Investigation file, the value of the cell SHOULD match one of the Term Source Name value declared in this section.

Where a column labelled with Term Source REF in a Study file or Assay file associated with the Investigation, the value of the cell SHOULD match one of the Term Source Name value declared in this section.

This section implements a list of Ontology Source from the ISA Abstract Model.

This section MUST contain zero or more values.

ONTOLOGY SOURCE REFERENCE

This section MUST contain the following labels, with the specified datatypes for values supported:

Label Datatype Description
Term Source Name String The name of the source of a term; i.e. the source controlled vocabulary or ontology. These names will be used in all corresponding Term Source REF fields that occur elsewhere.
Term Source File String (file name or URI) A file name or a URI of an official resource.
Term Source Version String The version number of the Term Source to support terms tracking.
Term Source Description String Use for disambiguating resources when homologous prefixes have been used.

For example, the ONTOLOGY SOURCE REFERENCE section of an ISA-Tab i_*.txt file may look as follows:

ONTOLOGY SOURCE REFERENCE
Term Source Name	"CHEBI"	"EFO"	"OBI"	"NCBITAXON"	"PATO"
Term Source File	"http://data.bioontology.org/ontologies/CHEBI"	"http://data.bioontology.org/ontologies/EFO"	"http://data.bioontology.org/ontologies/OBI"	"http://data.bioontology.org/ontologies/NCBITAXON"	"http://data.bioontology.org/ontologies/PATO"
Term Source Version	"78"	"111"	"21"	"2"	"160"
Term Source Description	"Chemical Entities of Biological Interest Ontology"	"Experimental Factor Ontology"	"Ontology for Biomedical Investigations"	"National Center for Biotechnology Information (NCBI) Organismal Classification"	"Phenotypic Quality Ontology"

2.2.2. Investigation section

This section is organized in several subsections, described in detail below. The Investigation section provides a flexible mechanism for grouping two or more Study files where required. When only one Study is created, the values in this section SHOULD be left empty and the relevant metadata values recorded in the Study section only.

These sections implement an Investigation from the ISA Abstract Model.

INVESTIGATION

This section MUST contain zero or one values.

This section MUST contain the following labels, with the specified datatypes for values supported:

Label Datatype Description
Investigation Identifier String A identifier or an accession number provided by a repository. This SHOULD be locally unique.
Investigation Title String A concise name given to the investigation.
Investigation Description String A textual description of the investigation.
Investigation Submission Date String formatted as ISO8601 date YYYY-MM-DD The date on which the investigation was reported to the repository.
Investigation Public Release Date String formatted as ISO8601 date YYYY-MM-DD The date on which the investigation was released publicly.

For example, the INVESTIGATION section of an ISA-Tab i_*.txt file may look as follows:

INVESTIGATION
Investigation Identifier	"BII-I-1"
Investigation Title	"Growth control of the eukaryote cell: a systems biology study in yeast"
Investigation Description	"Background Cell growth underlies many key cellular and developmental processes, yet a limited number of studies have been carried out on cell-growth regulation. Comprehensive studies at the transcriptional, proteomic and metabolic levels under defined controlled conditions are currently lacking. Results Metabolic control analysis is being exploited in a systems biology study of the eukaryotic cell. Using chemostat culture, we have measured the impact of changes in flux (growth rate) on the transcriptome, proteome, endometabolome and exometabolome of the yeast Saccharomyces cerevisiae. Each functional genomic level shows clear growth-rate-associated trends and discriminates between carbon-sufficient and carbon-limited conditions. Genes consistently and significantly upregulated with increasing growth rate are frequently essential and encode evolutionarily conserved proteins of known function that participate in many protein-protein interactions. In contrast, more unknown, and fewer essential, genes are downregulated with increasing growth rate; their protein products rarely interact with one another. A large proportion of yeast genes under positive growth-rate control share orthologs with other eukaryotes, including humans. Significantly, transcription of genes encoding components of the TOR complex (a major controller of eukaryotic cell growth) is not subject to growth-rate regulation. Moreover, integrative studies reveal the extent and importance of post-transcriptional control, patterns of control of metabolic fluxes at the level of enzyme synthesis, and the relevance of specific enzymatic reactions in the control of metabolic fluxes during cell growth. Conclusion This work constitutes a first comprehensive systems biology study on growth-rate control in the eukaryotic cell. The results have direct implications for advanced studies on cell growth, in vivo regulation of metabolic fluxes for comprehensive metabolic engineering, and for the design of genome-scale systems biology models of the eukaryotic cell."
Investigation Submission Date	"2007-04-30"
Investigation Public Release Date	"2009-03-10"

INVESTIGATION PUBLICATIONS

This section MUST contain zero or more values.

This section MUST contain the following labels, with the specified datatypes for values supported:

Label Datatype Description
Investigation PubMed ID String formatted as valid PubMed ID The PubMed IDs of the described publication(s) associated with this investigation.
Investigation Publication DOI String formatted as valid DOI A Digital Object Identifier (DOI) for that publication (where available).
Investigation Publication Author List String The list of authors associated with that publication.
Investigation Publication Title String The title of publication associated with the investigation.
Investigation Publication Status String, or Ontology Annotation by providing accompanying Term Accession Number and Term Source REF A term describing the status of that publication (i.e. submitted, in preparation, published).
Investigation Publication Status Term Accession Number String or URI The accession number from the Term Source associated with the selected term.
Investigation Publication Status Term Source REF String Identifies the controlled vocabulary or ontology that this term comes from. The Source REF has to match one the Term Source Name declared in the in the Ontology Source Reference section.

For example, the INVESTIGATION PUBLICATIONS section of an ISA-Tab i_*.txt file may look as follows:

INVESTIGATION PUBLICATIONS
Investigation PubMed ID	"17439666"
Investigation Publication DOI	"doi:10.1186/jbiol54"
Investigation Publication Author List	"Castrillo JI, Zeef LA, Hoyle DC, Zhang N, Hayes A, Gardner DC, Cornell MJ, Petty J, Hakes L, Wardleworth L, Rash B, Brown M, Dunn WB, Broadhurst D, O'Donoghue K, Hester SS, Dunkley TP, Hart SR, Swainston N, Li P, Gaskell SJ, Paton NW, Lilley KS, Kell DB, Oliver SG."
Investigation Publication Title	"Growth control of the eukaryote cell: a systems biology study in yeast."
Investigation Publication Status	"indexed in Pubmed"
Investigation Publication Status Term Accession Number	""
Investigation Publication Status Term Source REF	""

INVESTIGATION CONTACTS

This section MUST contain zero or more values.

This section MUST contain the following labels, with the specified datatypes for values supported:

Label Datatype Description
Investigation Person Last Name String The last name of a person associated with the investigation.
Investigation Person First Name String Investigation Person Name
Investigation Person Mid Initials String The middle initials of a person associated with the investigation.
Investigation Person Email String formatted as email The email address of a person associated with the investigation.
Investigation Person Phone String The telephone number of a person associated with the investigation.
Investigation Person Fax String The fax number of a person associated with the investigation.
Investigation Person Address String The address of a person associated with the investigation.
Investigation Person Affiliation String The organization affiliation for a person associated with the investigation.
Investigation Person Roles String or Ontology Annotation if accompanied by Term Accession Numbers and Term Source REFs Term to classify the role(s) performed by this person in the context of the investigation, which means that the roles reported here need not correspond to roles held withing their affiliated organization. Multiple annotations or values attached to one person can be provided by using a semicolon (“;”) Unicode (U0003+B) as a separator (e.g.: submitter;funder;sponsor) .The term can be free text or from, for example, a controlled vocabulary or an ontology. If the latter source is used the Term Accession Number and Term Source REF fields below are required.
Investigation Person Roles Term Accession Number String The accession number from the Term Source associated with the selected term.
Investigation Person Roles Term Source REF String Identifies the controlled vocabulary or ontology that this term comes from. The Source REF has to match one of the Term Source Names declared in the Ontology Source Reference section.

For example, the INVESTIGATION CONTACTS section of an ISA-Tab i_*.txt file may look as follows:

INVESTIGATION CONTACTS
Investigation Person Last Name	"Stephen"	"Castrillo"	"Zeef"
Investigation Person First Name	"Oliver"	"Juan"	"Leo"
Investigation Person Mid Initials	"G"	"I"	"A"
Investigation Person Email	""	""	""
Investigation Person Phone	""	""	""
Investigation Person Fax	""	""	""
Investigation Person Address	"Oxford Road, Manchester M13 9PT, UK"	"Oxford Road, Manchester M13 9PT, UK"	"Oxford Road, Manchester M13 9PT, UK"
Investigation Person Affiliation	"Faculty of Life Sciences, Michael Smith Building, University of Manchester"	"Faculty of Life Sciences, Michael Smith Building, University of Manchester"	"Faculty of Life Sciences, Michael Smith Building, University of Manchester"
Investigation Person Roles	"corresponding author"	"author"	"author"
Investigation Person Roles Term Accession Number	""	""	""
Investigation Person Roles Term Source REF	""	""	""

2.2.3. Study section

This section is organized in several subsections, described in detail below. This section also represents a repeatable block, which is replicated according to the number of Studies to report (i.e. two Studies, two Study blocks are represented in the Investigation file). The subsections in the block are arranged vertically; the intent being to enhance readability and presentation, and possibly to help with parsing. These subsections MUST remain within this repeatable block, although their order MAY vary; the fields MUST remain within their subsection.

These sections implement the metadata for a Study from the ISA Abstract Model and a list of Assay (i.e. Study and Assay without graphs; graphs are implemented in ISA-Tab as table files).

STUDY

This section MUST contain zero or one values.

This section MUST contain the following labels, with the specified datatypes for values supported:

Label Datatype Description
Study Identifier String A unique identifier, either a temporary identifier supplied by users or one generated by a repository or other database. For example, it could be an identifier complying with the LSID specification.
Study Title String A concise phrase used to encapsulate the purpose and goal of the study.
Study Description String A textual description of the study, with components such as objective or goals.
Study Submission Date String formatted as ISO8601 date The date on which the study is submitted to an archive.
Study Public Release Date String formatted as ISO8601 date The date on which the study SHOULD be released publicly.
Study File Name String formatted as file name or URI A field to specify the name of the Study Table file corresponding the definition of that Study. There can be only one file per cell.

For example, the STUDY section of an ISA-Tab i_*.txt file may look as follows:

Study Identifier	"BII-S-3"
Study Title	"Metagenomes and Metatranscriptomes of phytoplankton blooms from an ocean acidification mesocosm experiment"
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 Submission Date	"2008-08-15"
Study Public Release Date	"2008-08-15"
Study File Name	"s_BII-S-3.txt"

STUDY DESIGN DESCRIPTORS

This section MUST contain zero or more values.

This section MUST contain the following labels, with the specified datatypes for values supported:

Label Datatype Description
Study Design Type String A term allowing the classification of the study based on the overall experimental design, e.g cross-over design or parallel group design. The term can be free text or from, for example, a controlled vocabulary or an ontology. If the latter source is used the Term Accession Number and Term Source REF fields below are required.
Study Design Type Term Accession Number String The accession number from the Term Source associated with the selected term.
Study Design Type Term Source REF String Identifies the controlled vocabulary or ontology that this term comes from. The Study Design Term Source REF has to match one the Term Source Name declared in the Ontology Source Reference section.

For example, the STUDY DESIGN DESCRIPTORS section of an ISA-Tab i_*.txt file may look as follows:

STUDY DESIGN DESCRIPTORS
Study Design Type	"time series design"
Study Design Type Term Accession Number	"http://purl.obolibrary.org/obo/OBI_0500020"
Study Design Type Term Source REF	"OBI"

STUDY PUBLICATIONS

This section MUST contain zero or more values.

This section MUST contain the following labels, with the specified datatypes for values supported:

Label Datatype Description
Study PubMed ID String formatted as valid PubMed ID The PubMed IDs of the described publication(s) associated with this study.
Study Publication DOI String formatted as valid DOI A Digital Object Identifier (DOI) for that publication (where available).
Study Publication Author List String The list of authors associated with that publication.
Study Publication Title String The title of publication associated with the investigation.
Study Publication Status String, or Ontology Annotation by providing accompanying Term Accession Number and Term Source REF A term describing the status of that publication (i.e. submitted, in preparation, published).
Study Publication Status Term Accession Number String or URI The accession number from the Term Source associated with the selected term.
Study Publication Status Term Source REF String Identifies the controlled vocabulary or ontology that this term comes from. The Source REF has to match one the Term Source Name declared in the in the Ontology Source Reference section.

For example, the STUDY PUBLICATIONS section of an ISA-Tab i_*.txt file may look as follows:

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"
Study Publication Status Term Accession Number	""	""
Study Publication Status Term Source REF	""	""

STUDY FACTORS

This section MUST contain zero or more values.

This section MUST contain the following labels, with the specified datatypes for values supported:

Label Datatype Description
Study Factor Name String The name of one factor used in the Study and/or Assay files. A factor corresponds to an independent variable manipulated by the experimentalist with the intention to affect biological systems in a way that can be measured by an assay. The value of a factor is given in the Study or Assay file, accordingly. If both Study and Assay have a Factor Value, these must be different.
Study Factor Type String A term allowing the classification of this factor into categories. The term can be free text or from, for example, a controlled vocabulary or an ontology. If the latter source is used the Term Accession Number and Term Source REF fields below are required.
Study Factor Type Term Accession Number String The accession number from the Term Source associated with the selected term.
Study Factor Type Term Source REF String Identifies the controlled vocabulary or ontology that this term comes from. The Source REF has to match one of the Term Source Name declared in the Ontology Source Reference section.

For example, the STUDY FACTORS section of an ISA-Tab i_*.txt file may look as follows:

STUDY FACTORS
Study Factor Name	"dose"	"compound"	"collection time"
Study Factor Type	"dose"	"chemical substance"	"time"
Study Factor Type Term Accession Number	"http://www.ebi.ac.uk/efo/EFO_0000428"	"http://purl.obolibrary.org/obo/CHEBI_59999"	"http://purl.obolibrary.org/obo/PATO_0000165"
Study Factor Type Term Source REF	"EFO"	"CHEBI"	"PATO"

STUDY ASSAYS

This section MUST contain zero or more values.

This section MUST contain the following labels, with the specified datatypes for values supported:

Label Datatype Description
Study Assay Measurement Type String A term to qualify the endpoint, or what is being measured (e.g. gene expression profiling or protein identification). The term can be free text or from, for example, a controlled vocabulary or an ontology. If the latter source is used the Term Accession Number and Term Source REF fields below are required.
Study Assay Measurement Type Term Accession Number String The accession number from the Term Source associated with the selected term.
Study Assay Measurement Type Term Source REF String The Source REF has to match one of the Term Source Name declared in the Ontology Source Reference section.
Study Assay Technology Type String Term to identify the technology used to perform the measurement, e.g. DNA microarray, mass spectrometry. The term can be free text or from, for example, a controlled vocabulary or an ontology. If the latter source is used the Term Accession Number and Term Source REF fields below are required.
Study Assay Technology Type Term Accession Number String The accession number from the Term Source associated with the selected term.
Study Assay Technology Type Term Source REF String Identifies the controlled vocabulary or ontology that this term comes from. The Source REF has to match one of the Term Source Names declared in the Ontology Source Reference section.
Study Assay Technology Platform String Manufacturer and platform name, e.g. Bruker AVANCE
Study Assay File Name String A field to specify the name of the Assay Table file corresponding the definition of that assay. There can be only one file per cell.

For example, the STUDY ASSAYS section of an ISA-Tab i_*.txt file may look as follows:

STUDY ASSAYS
Study Assay File Name	"a_gilbert-assay-Gx.txt"	"a_gilbert-assay-Tx.txt"
Study Assay Measurement Type	"metagenome sequencing"	"transcription profiling"
Study Assay Measurement Type Term Accession Number	""	""
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 GS FLX"	"454 GS FLX"

STUDY PROTOCOLS

This section MUST contain zero or more values.

This section MUST contain the following labels, with the specified datatypes for values supported:

Label Datatype Description
Study Protocol Name String The name of the protocols used within the ISA-Tab document. The names are used as identifiers within the ISA-Tab document and will be referenced in the Study and Assay files in the Protocol REF columns. Names can be either local identifiers, unique within the ISA Archive which contains them, or fully qualified external accession numbers.
Study Protocol Type String Term to classify the protocol. The term can be free text or from, for example, a controlled vocabulary or an ontology. If the latter source is used the Term Accession Number and Term Source REF fields below are required.
Study Protocol Type Term Accession Number String The accession number from the Term Source associated with the selected term.
Study Protocol Type Term Source REF String Identifies the controlled vocabulary or ontology that this term comes from. The Source REF has to match one of the Term Source Name declared in the Ontology Source Reference section.
Study Protocol Description String A free-text description of the protocol.
Study Protocol URI String Pointer to protocol resources external to the ISA-Tab that can be accessed by their Uniform Resource Identifier (URI).
Study Protocol Version String An identifier for the version to ensure protocol tracking.
Study Protocol Parameters Name String A semicolon-delimited (“;”) list of parameter names, used as an identifier within the ISA-Tab document. These names are used in the Study and Assay files (in the “Parameter Value []” column heading) to list the values used for each protocol parameter. Refer to section Multiple values fields in the Investigation File on how to encode multiple values in one field and match term sources
Study Protocol Parameters Term Accession Number String The accession number from the Term Source associated with the selected term.
Study Protocol Parameters Term Source REF String Identifies the controlled vocabulary or ontology that this term comes from. The Source REF has to match one of the Term Source Name declared in the Ontology Source Reference section.
Study Protocol Components Name String A semicolon-delimited (“;”) list of a protocol’s components; e.g. instrument names, software names, and reagents names. Refer to section Multiple values fields in the Investigation File on how to encode multiple components in one field and match term sources.
Study Protocol Components Type String Term to classify the protocol components listed for example, instrument, software, detector or reagent. The term can be free text or from, for example, a controlled vocabulary or an ontology. If the latter source is used the Term Accession Number and Term Source REF fields below are required.
Study Protocol Components Type Term Accession Number String The accession number from the Source associated to the selected terms.
Study Protocol Components Type Term Source REF String Identifies the controlled vocabulary or ontology that this term comes from. The Source REF has to match a Term Source Name previously declared in the ontology section

For example, the STUDY PROTOCOLS section of an ISA-Tab i_*.txt file may look as follows:

STUDY PROTOCOLS
Study Protocol Name	"environmental material 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	"sample collection"	"nucleic acid extraction"	"nucleic acid extraction"	"nucleic acid extraction"	"reverse transcription"	"library construction"	"nucleic acid sequencing"	"data transformation"
Study Protocol Type Term Accession Number	""	""	""	""	""	""	""	""
Study Protocol Type Term Source REF	""	""	""	""	""	""	""	""
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 11000 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 MinElute 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."	""
Study Protocol URI	""	""	""	""	""	""	""	""
Study Protocol Version	""	""	""	""	""	""	""	""
Study Protocol Parameters Name	"filter pore size"	""	""	""	""	"library strategy;library layout;library selection"	"sequencing instrument"	""
Study Protocol Parameters Name Term Accession Number	""	""	""	""	""	";;"	""	""
Study Protocol Parameters Name Term Source REF	""	""	""	""	""	";;"	""	""
Study Protocol Components Name	""	""	""	""	""	""	""	""
Study Protocol Components Type	""	""	""	""	""	""	""	""
Study Protocol Components Type Term Accession Number	""	""	""	""	""	""	""	""
Study Protocol Components Type Term Source REF	""	""	""	""	""	""	""	""

STUDY CONTACTS

This section MUST contain zero or more values.

This section MUST contain the following labels, with the specified datatypes for values supported:

abel Datatype Description
Study Person Last Name String The last name of a person associated with the study.
Study Person First Name String Study Person Name
Study Person Mid Initials String The middle initials of a person associated with the study.
Study Person Email String formatted as email The email address of a person associated with the study.
Study Person Phone String The telephone number of a person associated with the study.
IStudy Person Fax String The fax number of a person associated with the study.
Study Person Address String The address of a person associated with the study.
Study Person Affiliation String The organization affiliation for a person associated with the study.
Study Person Roles String or Ontology Annotation if accompanied by Term Accession Numbers and Term Source REFs Term to classify the role(s) performed by this person in the context of the study, which means that the roles reported here need not correspond to roles held withing their affiliated organization. Multiple annotations or values attached to one person can be provided by using a semicolon (“;”) Unicode (U0003+B) as a separator (e.g.: submitter;funder;sponsor) .The term can be free text or from, for example, a controlled vocabulary or an ontology. If the latter source is used the Term Accession Number and Term Source REF fields below are required.
Study Person Roles Term Accession Number String The accession number from the Term Source associated with the selected term.
Study Person Roles Term Source REF String Identifies the controlled vocabulary or ontology that this term comes from. The Source REF has to match one of the Term Source Names declared in the Ontology Source Reference section.

For example, the STUDY CONTACTS section of an ISA-Tab i_*.txt file may look as follows:

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;SRA Inform On Status;SRA Inform On Error"	"principal investigator role"	"principal investigator role"	"principal investigator role"	"principal investigator role"	"principal investigator role"	"principal investigator role"
Study Person Roles Term Accession Number	";;"	""	""	""	""	""	""
Study Person Roles Term Source REF	";;"	""	""	""	""	""	""

2.3. Study and Assay files

Study and Assay Table files are structure with fields organized on a per-row basis. The first row MUST be used for column headers. Generally, objects such as Materials and Processes are indicated with <entity> Name, for example Sample Name to indicate a sample, or Assay Name to indicate a named instance of a process that has been applied. Object properties MUST follow this column, where materials MAY have Characteristics and Processes have MAY have Parameter Values. Both Characteristics and Parameter Values MUST be of type string, numeric, or an Ontology Annotation. <entity> File MAY be used to indicate a data file node.

Attention

Comments are also allowed in Study and Assay files, in a similar fashion to how they are used in the Investigation file. Columns headed with Comment[<comment name>] MAY appear after any named node in the Study and Assay files (e.g. if Comment[ORCID ID] appears after the Source Name column, we know that the comment regarding ORCID ID applies to the relevant Source node based on the row.

Specific types of nodes are specified in the Assay Table file section below.

2.3.1. Ontology Annotations

Where a value is an Ontology Annotation in a table file, Term Accession Number and Term Source REF fields MUST follow the column cell in which the value is entered. For example, a characteristic type Organism with a value of Homo sapiens can be qualified with an Ontology Annotation of a term from NCBI Taxonomy as follows:

Characteristics[Organism] Term Source REF Term Accession Number
Homo sapiens NCBITaxon http://…/NCBITAXON/9606

An Ontology Annotation MAY be applied to any appropriate Characteristics or Parameter Value.

This implements Ontology Annotation from the ISA Abstract Model.

2.3.2. Unit

Where a value is numeric, a Unit MAY be used to qualify the quantity. In this case, following the column in which a Unit is used, a Unit heading MUST be present, and MAY be further annotated as an Ontology Annotation.

For example, to qualify the value 300 with a Unit Kelvin qualified as an Ontology Annotation from the Units Ontology declared in the Ontology Sources with UO:

Parameter Value[Temperature] Unit Term Source REF Term Accession Number
300 Kelvin UO http://…/obo/UO_0000012

2.3.3. Processes

A Process MUST be indicated with the column heading Protocol REF. The value of Protocol REF cells MUST reference a Protocol declared in the investigation file.

2.3.4. Characteristics

Characteristics are used as an attribute column following Source Name, Sample Name. This column contains terms describing each material according to the characteristics category indicated in the column header in the pattern Characteristics [<category term>]. For example, a column header Characteristics [organ part] would contain terms describing an organ part. Characteristics SHOULD be used as an attribute column following Source Name, or Sample Name. The value MUST be free text, numeric, or an Ontology Annotation.

For example, a characteristic type Organism with a value of Homo sapiens can be qualified with an Ontology Annotation of a term from NCBI Taxonomy as follows:

Characteristics[organ part] Term Source REF Term Accession Number
Liver MeSH D008099

2.3.5. Factor Value

A factor is an independent variable manipulated by an experimentalist with the intention to affect biological systems in a way that can be measured by an assay. This field holds the actual data for the Factor Value named between the square brackets (as declared in the Investigation file) so MUST match; for example, Factor Value [compound]. The value MUST be free text, numeric, or an Ontology Annotation.

Factor Value[Gender] Term Source REF Term Accession Number
Male MeSH D008297

2.3.6. Study Table file

The Study file contains contextualizing information for one or more assays, for example; the subjects studied; their source(s); the sampling methodology; their characteristics; and any treatments or manipulations performed to prepare the specimens.

For a full example of a complete Study Table file, please see https://git.io/vD1vi

Study Table files SHOULD have file names corresponding to the pattern s_*.txt, e.g. s_Study01.txt

In Study files, there are two types of Material nodes implemented: Source and Sample.

These are linked with a Process node, incidcated with a value under a column headed Protocol REF that MUST be of a Protocol type that is of a type sample collection declared in the Investigation file.

A Source MUST be indicated with the column heading Source Name.

The protocol referenced MUST be of protocol type sample collection.

A Sample MUST be indicated with the column heading Sample Name.

For example, a simple source to sample may be represented as:

Source Name Protocol REF Sample Name
source1 sample collection sample1

Where a graph splits or pools, we use the Name column to represent the same nodes.

For example, if we split a source into two samples, we might represent this as:

Source Name Protocol REF Sample Name
source1 sample collection sample1
source1 sample collection sample2

If we pool two sources into a single sample, we might represent this as:

Source Name Protocol REF Sample Name
source1 sample collection sample1
source2 sample collection sample1

Node properties, such as Characteristics (for Material nodes), Parameter Value (for Process nodes) and additional Name columns for special cases of Process node to disambiguate Protocol REF entries of MUST follow the named node of context.

For example,

"Source Name"	"Characteristics[organism]"	"Term Source REF"	"Term Accession Number"	"Characteristics[strain]"	"Term Source REF"	"Term Accession Number"	"Characteristics[genotype]"	"Term Source REF"	"Term Accession Number"	"Characteristics[mating type]"	"Term Source REF"	"Term Accession Number"	"Protocol REF"	"Sample Name"
"Saccharomyces cerevisiae FY1679 "	"Saccharomyces cerevisiae (Baker's yeast)"	"NEWT"	""	"FY1679"	""	""	"KanMx4 MATa/MATalpha ura3-52/ura3-52 leu2-1/+trp1-63/+his3-D200/+ hoD KanMx4/hoD"	""	""	"mating_type_alpha"	""	""	"growth"	"NZ_0hrs_Grow_1"
"Saccharomyces cerevisiae FY1679 "	"Saccharomyces cerevisiae (Baker's yeast)"	"NEWT"	""	"FY1679"	""	""	"KanMx4 MATa/MATalpha ura3-52/ura3-52 leu2-1/+trp1-63/+his3-D200/+ hoD KanMx4/hoD"	""	""	"mating_type_alpha"	""	""	"growth"	"NZ_0hrs_Grow_2"

The Study Table file implements the Study graphs from the ISA Abstract Model.

2.3.7. Assay Table file

The Assay file represents a portion of the experimental graph (i.e., one part of the overall structure of the workflow); each Assay file must contain assays of the same type, defined by the type of measurement (e.g. gene expression) and the technology employed (e.g. DNA microarray). Assay-related information includes protocols, additional information relating to the execution of those protocols and references to data files (whether raw or derived).

For a full example of a complete Assay Table file, please see https://git.io/vD1vy.

Assay Table files SHOULD have file names corresponding to the pattern a_*.txt, e.g. a_Assay01.txt

A Sample MUST be provided as the first node in the experimental graph, indicated with the column heading Sample Name.

Protocol REF columns MUST be used to indicate Process nodes, with values referencing protocols declared in the Investigation file. The Protocol REF column MAY be qualified with Parameter Value [<parameter term>], ``Performer and Date. The Parameter Value [<parameter term>] field allows reporting the values taken by the parameter when applying in a protocol. Note that the term between [ ] must map to one (and only one) of parameters defined in the investigation file. Values can be qualitative or quantitative. The Performer field reports the name of the operator who carried out the protocol. This allows account to be taken of operator effects and can be part of a quality control data tracking. Date is the date on which a protocol is performed. This allows account to be taken of day effects and can be part of a quality control data tracking. Dates should be reported in ISO8601 format.

Extract Name MUST be used as an identifier for a Extract Material node within an Assay file. This column contains user-defined names for each portion of extracted material. Extracts MAY be qualified with Characteristics, Material Type and Description.

Labeled Extract Name MUST be used as an identifier for a Labeled Extract Material node within an Assay file. Labeled Extracts MAY be qualified with Label, Characteristics, Material Type, Description.

Assay Name MUST be used as an identifier for user-defined names for each assay.

Image File, Raw Data File or Derived Data File column heading MUST correspond to a relevant Data node to provide names or URIs of file locations. For submission or transfer, files MAY be packed with ISA-Tab files.

Data Transformation Name MUST be used as an identifier for a user-defined name for each data transformation Process applied.

Normalization Name MUST be used as an identifier for a user-defined name for each normalization Process applied.

Splitting and pooling is allowed as per the examples given in Study Table file.

For example,

"Sample Name"	"Protocol REF"	"Protocol REF"	"Extract Name"	"Material Type"	"Term Source REF"	"Term Accession Number"	"Protocol REF"	"Parameter Value[library strategy]"	"Parameter Value[library selection]"	"Parameter Value[library layout]"	"Protocol REF"	"Parameter Value[sequencing instrument]"	"Assay Name"	"Raw Data File"	"Comment[TraceDB]"
"GSM255770"	"nucleic acid extraction - standard procedure 2"	"genomic DNA extraction - standard procedure 4"	"GSM255770.e1"	"deoxyribonucleic acid"	"CHEBI"	"http://purl.obolibrary.org/obo/CHEBI_16991"	"library construction"	"WGS"	"RANDOM"	"SINGLE"	"pyrosequencing - standard procedure 6"	"454 GS FLX"	"assay1"	"EWOEPZA01.sff"	"ftp://ftp.ncbi.nih.gov/pub/TraceDB/ShortRead/SRA000266/EWOEPZA01.sff"
"GSM255771"	"nucleic acid extraction - standard procedure 2"	"genomic DNA extraction - standard procedure 4"	"GSM255771.e1"	"deoxyribonucleic acid"	"CHEBI"	"http://purl.obolibrary.org/obo/CHEBI_16991"	"library construction"	"WGS"	"RANDOM"	"SINGLE"	"pyrosequencing - standard procedure 6"	"454 GS FLX"	"assay2"	"EWOEPZA02.sff"	"ftp://ftp.ncbi.nih.gov/pub/TraceDB/ShortRead/SRA000266/EWOEPZA02.sff"
"GSM255772"	"nucleic acid extraction - standard procedure 2"	"genomic DNA extraction - standard procedure 4"	"GSM255772.e1"	"deoxyribonucleic acid"	"CHEBI"	"http://purl.obolibrary.org/obo/CHEBI_16991"	"library construction"	"WGS"	"RANDOM"	"SINGLE"	"pyrosequencing - standard procedure 6"	"454 GS FLX"	"assay3.1"	"EXHS9OF01.sff"	"ftp://ftp.ncbi.nih.gov/pub/TraceDB/ShortRead/SRA000266/EXHS9OF01.sff"
"GSM255772"	"nucleic acid extraction - standard procedure 2"	"genomic DNA extraction - standard procedure 4"	"GSM255772.e1"	"deoxyribonucleic acid"	"CHEBI"	"http://purl.obolibrary.org/obo/CHEBI_16991"	"library construction"	"WGS"	"RANDOM"	"SINGLE"	"pyrosequencing - standard procedure 6"	"454 GS FLX"	"assay3.2"	"EX398L102.sff"	"ftp://ftp.ncbi.nih.gov/pub/TraceDB/ShortRead/SRA000266/EX398L102.sff"
"GSM255773"	"nucleic acid extraction - standard procedure 2"	"genomic DNA extraction - standard procedure 4"	"GSM255773.e1"	"deoxyribonucleic acid"	"CHEBI"	"http://purl.obolibrary.org/obo/CHEBI_16991"	"library construction"	"WGS"	"RANDOM"	"SINGLE"	"pyrosequencing - standard procedure 6"	"454 GS FLX"	"assay4.1"	"EXHS9OF02.sff"	"ftp://ftp.ncbi.nih.gov/pub/TraceDB/ShortRead/SRA000266/EXHS9OF02.sff"
"GSM255773"	"nucleic acid extraction - standard procedure 2"	"genomic DNA extraction - standard procedure 4"	"GSM255773.e1"	"deoxyribonucleic acid"	"CHEBI"	"http://purl.obolibrary.org/obo/CHEBI_16991"	"library construction"	"WGS"	"RANDOM"	"SINGLE"	"pyrosequencing - standard procedure 6"	"454 GS FLX"	"assay4.2"	"EX398L101.sff"	"ftp://ftp.ncbi.nih.gov/pub/TraceDB/ShortRead/SRA000266/EX398L101.sff"

The Assay Table file implements the Assay graphs from the ISA Abstract Model.

2.3.8. Special cases

Assay with technology type: DNA microarray hybridization

If an Assay being described has a technology type of DNA microarray hybridization, the following additional nodes MAY apply.

Hybridization Assay Name (in place of Assay Name):
 Used as an identifier within the Assay file. This column contains an user-defined name for each hybridization. Qualifying headers for Hybridization Assay Name item include Array Design REF or Array Design File.
Scan Name:Used as an identifier within the Assay file. This column contains a user-defined name for each Scan event.
Array Data File (in place of Raw Data File):
 Column to provide name (or URI) of raw array data files.
Derived Array Data File (in place of Derived Data File):
 Column to provide name (or URI) of data files resulting from data transformation or processing.
Array Data Matrix File:
 Column to provide name (or URI) of raw data matrix files.
Derived Array Data Matrix File:
 Column to provide name (or URI) of processed data matrix files, resulting from data transformation or processing. Where data from multiple hybridizations is stored in a single file, the data should be mapped to the appropriate hybridization (or scan, or normalization) via the Data Matrix format itself
Array Design File:
 Column to provide name of file containing the array design, used for a particular hybridization. For submission or transfer, ADF files can be packaged with ISA-TAB files into an ISArchive, see section 2.4.
Array Design REF:
 This column is used to reference the identifier (or accession number) of an existing array design.

Assay file with technology type: Gel electrophoresis

If an Assay being described has a technology type of Gel electrophoresis, the following additional nodes MAY apply.

Gel Electrophoresis Assay Name (in place of Assay Name):
 Used as an identifier within the Assay file. This column contains user-defined names for each electrophoresis gel assay. For 2-dimensional gels, the following qualifying headers can be used instead:
First Dimension:
 The term can be free text or from, for example, a controlled vocabulary or an ontology. If the latter source is used the Term Accession Number and Term Source REF fields are required.
Second Dimension:
 The term can be free text or from, for example, a controlled vocabulary or an ontology. If the latter source is used the Term Accession Number and Term Source REF fields are required.
Scan Name:Used as an identifier within the Assay file. This column contains user-defined names for each Scan event.
Spot Picking File:
 Column to provide name (or URI) of files file holding protein spot coordinates and metadata for use by spot picking instruments.

Assay file with technology type: Mass Spectrometry (MS)

If an Assay being described has a technology type of Mass Spectrometry, the following additional nodes MAY apply.

MS Assay Name (in place of Assay Name):
 Used as an identifier within the Assay file. This column contains user-defined names for each MS Assay.
Raw Spectral Data File (in place of Raw Data File):
 Column to provide name (or URI) of ‘raw’ spectral data files.
Derived Spectral Data File (in place of Derived Data File):
 Column to provide name (or URI) of derived spectral data files, resulting from data transformation or processing.

When Mass Spectrometry is used in proteomics the following data files are required, according to PSI specifications and Pride submission requirements (6, 10):

Peptide Assignment File:
 Column to provide name (or URI) of file(s) containing peptide assignments.
Protein Assignment File:
 Column to provide name (or URI) of file(s) containing protein assignments.
Post Translational Modification Assignment File:
 Column to provide name (or URI) of file(s) containing posited post-translational modifications.

Capturing data resulting from the use of mass spectrometry in metabol/nomics requires a settled definition for a Metabolite Assignment File (inter alia); such a file is currently under development in collaboration with the Metabolomics Standards Initiative (MSI).

2.3.9. Data Files

ISA-Tab focuses on structuring experimental metadata; raw and derived data files are considered as external files. The Assay file can refer to one or more of these external data files. For guidelines on how to format these data files, users should refer to the relevant standards group or reference repository.

For submission or transfer, ISA-Tab files and associated data files MAY be packaged into an ISArchive, a zip file containing all the files together.