The ISA-TAB-Nano specification makes use of the ISA-TAB Investigation, Study, and Assay files to record and share the assay metadata. In addition to sharing the assay metadata, we also want to share the endpoint measurements and description (chemical composition, characteristics) of both nanomaterials and small molecules (e.g., drugs, imaging agents). To accommodate this additional information, we have made extensions to ISA-TAB. In particular, the ISA-TAB Study file is suited for recording the source and characteristics of biological specimens and for specifying any treatments performed on them. However, the Study file is not designed to represent information about the chemical composition and characteristics of material samples
(nanomaterials, drugs, imaging agents, and other small molecules). To represent this information, we have introduced a fourth ISA-TAB-Nano file called the Material file. We use the Material file to describe material samples
while using the ISA-TAB Study file to describe biospecimens
. We have modified the definitions of some of the field names in the ISA-TAB Investigation, Study, and Assay files and added new fields names to expand the scope of information that needs to be shared using the ISA-TAB-Nano specification. Detailed information of the field names and definitions of each file format can be found in Supporting Information. For more detailed information about the structure and contents of the ISA-TAB files, the reader may refer to the ISA-TAB specification [25
]. The rules for naming the four file formats defined in the ISA-TAB-Nano specification are i_xxxx.txt for the Investigation file, s_xxxx.txt for the Study file, a_xxxx.txt for the Assay file, and m_xxxx.txt for the Material file, where ‘xxxx’ denotes a unique string.
The following sections describe the different components of the ISA-TAB-Nano files. Supporting Information provides additional materials to supplement the manuscript discussion, including a summary of extensions to the ISA-TAB format, the standard specification, and examples.
Types of information shared using ISA-TAB-Nano files
The types of information shared using ISA-TAB-Nano pertain to the material samples, biospecimens, assays, and protocols. As listed in Table , we use the ISA-TAB-Nano Material file to capture information about material samples and references to the external material data files (e.g., image files, structural data files); the Investigation file to enter reference information about each investigation, study, assay, protocol: the Study file to capture information about biospecimen source and characteristics, names and attributes of protocols used in the preparation of samples analyzed in an assay; and, the Assay file to capture information about measured values of endpoint variables and references to external files (raw/derived data files, image files) related to each analyzed biospecimen or material sample. In the following sections, we explain how to record and share the different types of information in each ISA-TAB-Nano file.
Types of information entered in the four ISA-TAB-Nano files
Sharing material sample description
In the ISA-TAB-Nano framework, all material samples are viewed as formulations having one or more chemical components (e.g., nanoparticles, small molecules, solvent medium, etc.). The ISA-TAB-Nano material file has been designed for recording descriptions of various types of formulations, ranging from a simple chemical compound to a complex multi-component nanoparticle formulation.
The layout of the ISA-TAB-Nano Material file is same as that of the Study and Assay files, wherein the field names are arranged as columns and material sample information is entered as rows. Columns in the ISA-TAB-Nano Material file provide information on the material and its components, the relationship between materials and components, and any associated data files, as mentioned in Table .
Using the ISA-TAB-Nano Material file to describe a material sample and its components
The Material file is used to describe a material sample and its structural and chemical components. There are different ways to identify the components, depending on how the sample is viewed at its various levels of molecular and structural granularities. Figure shows a process flowchart that can be used as a basis for identifying different components of the material sample. Once a component is identified, its description can be entered as a row in the Material file.
Figure 1 Flowchart depicting the ways to identify different components of a material sample, and to guide the creation of rows for each component in the ISA-TAB-Nano Material file. (A) The overall ISA-TAB-Nano file creation process which includes (B) the creation (more ...)
In Figure , we show an example of the ISA-TAB-Nano Material file that contains information about a hypothetical dendrimer sample (LAB-1) and its components. The first row (row 2) of the Material file is used to identify the material sample name (identifier, chemical name), type, synthesis type, design rationale, intended application, and nominal characteristics. Subsequent rows of the Material file are used to identify the different structural parts and chemical components of the sample including their name, type, nominal characteristics, and intended application. While row 2 represents the dendrimer sample LAB-1, rows 3, 4, and 5 represent the components (core molecule, branch, and terminal group) of the dendrimer sample. From the given information, one can understand that the dendrimer sample (LAB-1) is a 4.5 generation dendrimer that has a molecular weight of 27 kilodaltons (kDa). Ontology terms can be identified by the name of the source ontology (defined by Term Source REF labels) and their identification numbers (defined by Term Accession Number labels) used within the ontology. Thus, we use row 2 to describe the material sample as a whole, and subsequent rows to describe each material sample component. If a material sample that is described in a Material file is also a component of another material sample, then the components of the former sample need not be specified in the Material file of the latter sample. To illustrate this scenario, we consider another hypothetical sample called LAB-2 that contains both LAB-1 and an image contrast agent called Magnevist® (LAB-3). Figure shows the LAB-2 Material file, where row 2 represents the whole LAB-2 material sample and rows 3 and 4 represent its components (LAB-1 and LAB-3). Note that we did not have to enter information about each component of LAB-1 since that information is already present in the LAB-1 Material file (see Figure ). The LAB-2 Material file describes the material sample LAB-2 which consists of nanoparticles composed of carboxyl-terminated PAMAM dendrimer complexed with Magnevist®, a small molecule gadolinium-based image contrast agent. The dendrimer is conjugated to the Magnevist® and is intended to be used as a delivery vehicle for image contrast agents like Magnevist® in MRI applications.
Example of an ISA-TAB-Nano Material file with information about a dendrimer nanoparticle sample, identified as LAB-1.
Example of an ISA-TAB-Nano Material file with information about a dendrimer-magnevist nanoparticle complex sample, identified as LAB-2.
Using the ISA-TAB-Nano Material file to describe linkages in a material sample
The ISA-TAB-Nano Material file is used to describe the whole material sample and its components with respect to their name, type, characteristics, synthesis, design rationale, and intended application. In addition, the Material file can be used to identify the chemical components and represent how they are linked or connected to each other with respect to a nanoparticle. Any two chemical components in a typical multi-component nanoparticle formulation can be linked to each other by one of the three types of linkages: encapsulation, entrapment, or covalent linkage [28
]. We can describe linkages between the different chemical components of a nanoparticle sample as shown in Figure . In the figure, one can identify that there is a covalent linkage that exists between the dendrimer and the Magnevist®
of LAB-2 sample. The term “covalent linkage” is used here as a generic term. Normally, if the specific type of covalent linkage is known (e.g., amide linkage, disulfide linkage, etc.), then one should use the appropriate term identifying that linkage. The NPO [28
] provides several terms (their names and definitions) for describing different types of linkages that can be typically found in a nanoparticle sample. Additionally, we have recently developed a simple nomenclature system for describing the specific chemical linkages in nanoparticle formulations [30
]. It is important to ensure that the names of the two chemical components specified in the Material Constituent
field (Figure ) match their respective names given in the Material Name
An example showing the representation of linkages among components of a nanoparticle sample in the ISA-TAB-Nano Material file.
Using the ISA-TAB-Nano Material file to record information about external files that provide additional information about the material sample
The Material file is also used to record the name or URI of external files that contain additional information about the material sample. Examples include image files displaying the chemical structures of molecules, or documents (e.g., spreadsheet, Word, PDF, etc.) that contain detailed descriptions of the sample. The example given in Figure references one file called “magnevist.jpg” that contains an image of the molecular structure of Magnevist®.
In practice, one ISA-TAB-Nano Material file should be created for each material sample in a study. When using the format “m_xxxx.txt” to name the Material files, the string “xxxx” should exactly match the string used in the Material Source Name field of the sample’s Material file. For example, we would name the Material file of samples LAB-1 and LAB-2 as m_LAB-1.txt and m_LAB-2.txt, respectively.
Sharing assay information
Different types of assays are performed to measure the physicochemical, in vitro, and in vivo properties of material samples. We can identify the type of each assay based on the measured endpoint, measurand, and technique used. For example, if an assay is related to a size characterization study, the endpoint could be “size” and the technique could be “dynamic light scattering (DLS)”, or “scanning electron microscopy (SEM)”, or other relevant technique. The corresponding assay types are “size by DLS assay”, “size by SEM”, etc. Assays of the same type may be performed using different protocols, by different people, at different dates, and under different experimental conditions. To interpret the characterization results of a material sample meaningfully, we need to have information about the assay type; biospecimen information (if it is a bioassay); protocol descriptions; dates of synthesis and analysis of the material sample; the measured endpoint variables; as well as the protocol variables of the experiment. Based on the ISA-TAB specification, variables that are kept constant in an assay experiment are termed “parameters”, while variables that are changed for studying their effects on the measured endpoint values are termed “study factors”.
Representing physiochemical characterization data of material samples using ISA-TAB-Nano files
In a physicochemical characterization study, one measures the properties of a material sample and studies how these properties are affected by the study factors. Typically, one prepares several dilutions of same material sample and may even suspend each sample in different test media to achieve solution conditions suitable for performing a specific assay. Therefore, each analyzed sample may have its own identifier while its (source) material sample to be characterized has a different identifier (the Material Source Name).
Figure and Figure show an example of how to use the Study and Assay files, respectively, to represent the assay data (metadata, summary data) of each sample analyzed in a size characterization assay using DLS technique. In this example, the goal of the study was to determine the effect of varying temperature and solvent medium on the size of nanoparticles in the two material samples - LAB-1 and LAB-2.
An example showing the layout of the ISA-TAB-Nano Study file.
An example showing the layout of the ISA-TAB-Nano Assay file.
In the ISA-TAB Study file, a column Source Name is used to define the source of a biospecimen. ISA-TAB-Nano extends the ISA-TAB Study file by adding a column called Material Source Name REF to refer the source name of the material sample, given in the Material Source Name column of the sample’s Material file. Thus, as shown in Figure , the first column of the Study file (Material Source Name REF) represents the identifier of the material sample that is to be characterized. The second column contains the name of the protocol applied to prepare each individual biospecimen or material sample for the analysis. After the protocol column, we create columns to capture the attributes of the protocol including the name of the person who performed the protocol, the date when the protocol was applied, and the values of each parameter in the protocol. After these, we add two more columns to record the sample name (identifier) and its material type.
The first column of the Assay file is used to enter the biospecimen or material sample names under the header name Sample Name (see Figure ) – note that these names should match the names entered in the Sample Name column of the Study file. The names of the assay and protocol are entered in the next two columns. As in the Study file, we also create columns to capture the attributes of each protocol, including investigator, date, and parameter values. We also create columns for entering the values of each factor value and measurement value variables, followed by columns to record the URIs or names of associated data files, as shown in Figure .
The values for the parameters, factors, and measurements can be qualified by a unit of measurement and a statistical type (if applicable) using column headers Unit and Statistic, respectively, in both the Study as well as in the Assay files (see Figure and Figure ). Note that the Measurement Value [<measurement name>] and Statistic columns are newly added by the ISA-TAB-Nano specification. If terms from an ontology are used as valid values in any of the columns of the Study and Assay files, it is necessary to enter the term’s identifier and source ontology name in adjacent columns named as Term Accession Number and Term Source REF, respectively (not shown in the Figure and Figure ). One may choose to enter the factor values either in the Study file and/or in the Assay file, as appropriate to the actual experimental procedure(s). Furthermore, to enter any additional type of information in the Study or Assay file, one may create a column with header name, Comment [<string>], as specified by ISA-TAB.
The Investigation file is a reference for the different assays and protocols that have been conducted in a single study. The Investigation file is divided into several sections (listed in Table ), including Study, Study Assays, Study Factors, and Study Protocols. As summarized in Table , the Study section is used to record information about each study and its Study file (see Figure A). The Study Assays section is used to record information about each assay and its assay file, particularly, the assay and technology types, measurement variable names, and the assay file names (see example in Figure B). The Study Factors section is used to record the names of factor variables and the factor type, as shown in Figure C. The Study Protocols section is used to record information about each protocol, including the name, type, description, URI file references and version of each protocol; as well as names of parameter variables and components (instruments, reagents, and software) of each protocol (an example is given in Figure ). It should be noted the above information must be recorded in the Investigation file for reference to them in the individual Assay and Study files.
Types of information entered in the eleven sections of the ISA-TAB-Nano Investigation file
Examples showing how information is represented in the following sections of the Investigation file: (A) Study, (B) Study Assays, and (C) Study Factors.
An example showing how information is represented in the Study Protocols section of the Investigation file.
Note that the ontologies or controlled vocabularies referenced in any of the ISA-TAB-Nano files must be declared in the Ontology Reference section of the Investigation file (noted in Table ). The Investigation file can also contain information about publications and point of contact associated with an investigation or a specific study within the investigation (also noted in Table ). For more details on the Investigation file, we refer the reader to the ISA-TAB specification [25
] and ASTM ISA-TAB-Nano specification [31
Representing material sample in vitro and in vivo characterization data of material samples using ISA-TAB-Nano files
For in vitro and in vivo characterization studies, one is interested in measuring the effects of a material sample on a biological system, such as a cell line or animal model. In these studies, the material sample plays the role of a factor, and the sample analyzed is a biospecimen. The way we enter the in vitro/in vivo characterization data is similar to how we entered the physicochemical characterization data in the Investigation, Study, and Assay files. The only additional information to be entered is the description of the source and characteristics of the biospecimen in the Study file, as illustrated in Figure . Note that we use the column Characteristic [<name>] to enter the type of the biological source, as specified by ISA-TAB. The material type of the sample is entered as biospecimen in the Material Type column, and the material representing the nanoparticle or other non-biological material is entered in the Factor Value column. Particularly, the non-biological material entered in the Factor Value column should be the same as that entered in the Material Source Name column of the sample’s Material file.
Figure 9 A representative Study file for a hypothetical in vitro cytotoxicity characterization study, showing the description of a biological specimen and attributes of the sample preparation protocols. Note that the entered information is not from an actual study (more ...)
Integrated view of ISA-TAB-Nano files and external referenced documents
ISA-TAB-Nano files support the aggregation of all information pertaining to the description and characterization of material samples. In Figure , we show how the ISA-TAB-Nano files and the external files, such as protocol documents, raw data files, derived data files, image files, and material sample description files, can be assembled within the ISA-TAB-Nano framework. The names of all Study and Assay files are recorded in the Investigation file in columns corresponding to the fields Study File Name (Study section) and Study Assay File Name (Study Assays section), respectively. As a result, the Investigation, Study and Assay files are linked. The data in the Study and Assay files for each sample are integrated through the Sample Name fields found in both of the files. This is illustrated in Figure and Figure , where the sample names in Column A of the assay file (a_LABsizeDLS.xls) are the same as those found in Column P of the study file (s_LABsizeDLS.xls). Thus, the information of each biospecimen or material sample in the Study and Assay files can be linked to each other. If the sample that is analyzed is a material sample, then it is necessary to ensure that the value of Material Source Name REF entered in the Study file matches the string entered in the Material Source Name of its Material file (Figure ). Similarly, the same string must be used in the Factor Value column of the study file or assay file for material samples that are used as factors in an in vitro or an in vivo study. As shown in Figure , the ISA-TAB-Nano files can also be associated with any external files, as long as the URI or the names of these files are referenced in the appropriate fields in the ISA-TAB-Nano files. Specifically, the ISA-TAB-Nano files are associated with protocol documents by referencing the URIs or names of these documents in the Study Protocol URI field of the Investigation file. The external files related to the description of a material sample are associated via the Material Data File field of the material file. Finally, the Assay file associates the raw data, derived data and image files via the column fields, Raw Data File, Derived Data File, and Image File, respectively.
Links (black arrows) between ISA-TAB-Nano files and references (blue arrows) to documents (protocol documents, raw/derived data files, image files, and material description files).
Validation of ISA-TAB-Nano files
A key feature of ISA-TAB-Nano (and the parent ISA-TAB format) is that it can be readily created and edited with any spreadsheet software, without the need for specialized bioinformatics support. However, this also creates challenges because such software does not enforce validation rules for ISA-TAB-Nano files. One possible solution to this problem is to adapt the suite of open source tools provided by the ISA-TAB community [16
], such as: (1) ISACreator, which provides a graphical user interface for entering in data into ISA-TAB files; (2) ISAValidator, which assesses the validity of ISA-TAB files; (3) ISAConverter, which converts an ISA-TAB file to one of several other formats if so desired, (4) ISAConfigurator, which creates the XML files that define specialized versions of ISA-TAB, and (5) bioinvindex, which is a database for storing ISA-TAB files. These tools are part of our future plans to be tested for editing and validating ISA-TAB-Nano files.
XML-based validation is also of interest and would involve developing an ISA-TAB-Nano XML schema based on the ISA-TAB-Nano specification such that any ISA-TAB-Nano XML file converted from the ISA-TAB-Nano files can be formally validated against the XML schema. Having an XML-based validation service would also enable software systems to import and export validated ISA-TAB-Nano files to and from distributed nanotechnology resources.