TypOn: the microbial typing ontology

Domain description

Several typing methods have been used in outbreak detection and epidemiological surveillance ranging from phenotypic methods to fragment based methods and sequence based methods [5, 7].

Multilocus Sequence Typing (MLST) [8, 9] is a widely adopted methodology to type several diferent species of microorganisms. This method is based on determining the sequence of internal fragments of multiple (typically seven) loci encoding proteins with housekeeping functions. By a locus we understand a specific location in the chromosome, where different sequences occupying a given locus define distinct alleles of that locus. Each set of loci defining an MLST scheme is proposed by a group of researchers that usually also provide evidence supporting its discriminatory power and suitability for its intended purpose. When applying the methodology, the set of alleles identified at the loci considered define a sequence type (ST), a key identifier with this methodology. The loci chosen are usually different for each species, although some species may share some or even all loci in their MLST schemas. The number of loci can be also variable and can be greater or smaller than the seven loci more commonly adopted. MLST’s large appeal for the community was the reproducibility and portability of results, which allowed the deployment of databases for several bacterial species [10–12]. The strain nomenclature developed by MLST facilitated the global tracking and immediate comparison of microbial strains in clinical and research settings.

Another sequence-based method that derived its success from a common nomenclature and the ease of strain classification, was spa typing for Staphylococcus aureus[13], an important pathogen that is one of the major causes of nosocomial infections [14]. This method is based on repeat sequences present at a single locus, the spa gene. These repeats are short sequences of DNA (in the case of spa about thirty nucleotides) that, although sharing consensus characteristics, can be variable in their sequence. Their number is also variable and, in the case of spa typing, this is expressed by a string of numbers representing the identity and number of repeats present at each spa allele. An updated list of identified repeats and spa types can be found at http://spaserver.ridom.de/.

Multilocus Variable Number of Tandem Repeats Analysis (MLVA) [15] is a method that is based on the number of repeat patterns present on several defined locus that are, similarly to MLST, defined in a schema. Several schemas are also available in multiple websites such as http://www.mlva.eu/, http://www.mlva.net/ or http://www.miru-vntrplus.org/.

With the advent of Next Generation Sequencing (NGS) technologies, and the ability to produce a draft genome sequence of a microbial strain in a couple of days instead of weeks or months, researchers can use this information to classify the strain according to new or previously available sequence-based typing methods. Furthermore, novel typing methods are being developed that are able to probe tens, hundreds or even thousands of different loci across the genome [16], effectively extending the MLST concepts. An example of such a method is ribosomal MLST [17]. Other whole genome methodologies probe the genome for Single Nucleotide Polymorphisms (SNPs) when compared to reference genomes [18, 19] in order to discriminate strains.

The main goal of typing methods is the characterization of individuals existing in a given sample. The sample under study can be recovered from sick or healthy subjects or directly from the environment. The first process is the isolation of the microorganisms to be characterized from the sample collected. Each individual, or in the case of bacteria, each colony isolated from the microbial population then becomes an “isolate”, referring to the process of isolating it from a sample that contains many microorganisms potentially representing distinct species. In particular each isolate can then be identified at subspecies, species, or genus level, identifying it as belonging to a given taxonomic unit, i.e., taxon. Moreover, since most microorganisms reproduce asexually the subsequent propagation of this isolate in the laboratory as an axenic culture, would be expected to generate a clonal population.

Each isolate can be associated with typing information and ancillary details. The isolate can have the nucleotide sequence of its genetic material determined and multiple typing characteristics defined through different typing methods, such as MLST, spa typing or MLVA [15]. There are several categories of typing methods. For instance, although MLST and spa typing are both genotypic methods, MLST is a multi-locus typing method while spa typing is a single-locus typing method.

For the specific case of multi-locus typing methods, several schemas have been defined as indicated above. Each schema is characterized by the (possibly ordered) set of loci selected for a given taxon, usually defined at the species level. Each schema is then administrated by one or a group of microbiologist, or by an institution, and each isolate’s ancillary data and respective typing information are deposited in public repositories and validated by a curator. Different schemas can be defined for the same taxon and an ontology will facilitate understanding the relationships between different schemas. Ancillary data include information about the place where the microorganism was isolated, information about environment or host, and other possible contextual details. Later on, we will discuss how this information can be added and annotated in the context of TypOn.

We also note that data ownership is a particular delicate issue in the surveillance of communicable diseases and, as we will discuss later, the approach proposed in this paper allows a straightforward implementation of the agreed upon policies. The sensitive nature of the information and the ethical issues associated can be also safeguarded through the application of suitable access control levels based on the ownership structure embedded in the ontology.

Ontology modeling

The main concepts and properties defined in TypOn are depicted in Figures 1 and 2. The ontology, which is an extended version of a previous prototype ontology [6], is available at http://purl.phyloviz.net/ontology/typon. In this current version, we added new concepts and refined existing ones, based on comments made by domain experts, including microbiologists and industry partners. The aim of TypOn is to represent knowledge about any of the currently used sequence-based microbial typing methods. TypOn can be reused as well as expanded, whenever new requirements and new technologies demand it. For backwards compatibility, older versions can still be used by explicitly stating the TypOn version. For instance, version 20140606 may be accessed and referenced as http://purl.phyloviz.net/ontology/20140606/typon. This ontology has been developed in the context of the Patho-NGen-Trace project http://patho-ngen-trace.eu/project/ as a way to standardize microbial data exchange between online databases and current software using those databases.

Ongoing developments, new versions, as well as use cases and examples, can be found in the project repository https://bitbucket.org/phyloviz/typon, and documented in the project wiki https://bitbucket.org/phyloviz/typon/wiki. The base URL for TypOn, http://purl.phyloviz.net/ontology/typon, redirects transparently to the last stable version of the ontology, in RDF/XML format, located in the master branch in the development repository.

Although we will discuss examples of bacterial typing, we believe that TypOn is equally applicable to typing methods used to characterize any microorganism. Naturally, this implies that other loci, in addition to those used in the examples, will have to be entered into the ontology and new schemas will have to be defined. The ontology offers a flexible framework with which the existing and future sequence typing methods can be described and the examples are meant to illustrate its application as well as its flexibility. As described above, the first process in microbial typing is the recovery of the microorganisms to be characterized from the sample collected and, thus, Isolate is a main concept in TypOn and it is characterized by several properties.

Figure 1 shows an overview of the Isolate class and its related concepts and properties.

Each Isolate when identified at subspecies, species, or genus level, belongs to a certain Taxon, a relation that we express through the property isFromTaxon. The Taxon concept is reused from the Uniprot Core Ontology [27] for classifying life forms. Moreover, we define that each Isolate is an Organism, a concept that is reused from the Ontology for Biomedical Investigations (OBI) [28]. We also know the Place from where each Isolate was recovered, which we describe through the property isolatedAt. As with the Taxon concept, the Place concept is reused from another ontology, in this case the DBpedia ontology [29]. One can also define the environment material or system where a given Isolate was collected. These concepts are already found in the environment ontology (EnvO) [22] and, hence, we reused them and we add the properties isolatedOnMaterial and isolatedOnSystem, relating these concepts with the concept Isolate. Each Isolate, can have Sequence information, i.e. the nucleotide sequence of its genetic material, and Typing Information. The property hasTypingInformation commonly has cardinality higher than one for each Isolate, since several typing methods, such as MLST or MLVA, can be applied to the same Isolate. Later we will present an example of an isolate with both MLST and spa typing information.

In this context, it is important to note that TypingInformation is the root of a class hierarchy (see Figure 2). This hierarchy can be extended by including new, and already known, typing techniques, such as phenotypic information related to antibiotic susceptibility. In particular, we are able to distinguish different categories of typing methods. Let us consider MLST and spa Typing. Both are Genotypic methods, but the first is a Multilocus method while the latter is a Single Locus method.

Let us consider the concepts Locus, Allele, Schema and MLST, as depicted in Figure 3. In MLST we can have several typing schemas administrated by some Agent, a concept reused from FOAF ontology [25], for instance the database curator, and composed by a set of Schema Parts. Such schemas are represented through the class Schema, which has associated the property hasSchemaPart. The Schema Part concept allows us to identify a particular Locus and provide the index order for that locus in the underlying schema through object property hasLocus and data property index, respectively. In the case of MLST, each locus identifies a region within the coding sequence of an housekeeping gene. Thus, the object property hasLocus associated to the Schema Part concept has cardinality of exactly one.

As depicted in Figure 1 and indicated above, each Isolate may have been characterized by more than one typing method. In the case of MLST, this kind of typing information can be subject to different schemas, resulting in different sequence types, which are characterized by the alleles found at each locus. Therefore, in our ontology, we associate to each MLST both a schema and a set of observed alleles through properties hasSchema and hasIdentifiedAllele, respectively. The property hasSchema has cardinality of exactly one and, hence, MLST and MLVA instances must be related with one and only one given Schema. Notice that STAllele is an Allele (see Figure 1) defined in a MLST Schema, disjoint from Spa Allele and VNTAllele. So, we only associate to MLST typing information the concept of STAllele. This is necessary given the different ways in which the distinct alleles are defined in these typing methods.

The entity Spa Allele is used in the context of spa typing. Each spa typing information has a matching spa Allele, which corresponds to a specific sequence of repeats found as a result of the amplification of the locus of the spa gene of Staphylococcus aureus. In Figure 2 we can observe that a given Spa Allele has repeat profile parts, i.e., an entity that stores the index order of a given Spa Repeat in a given Spa Allele. Notice that each Spa Repeat may occur more than once in a given Spa Allele and that different Spa Alleles can have distinct number of repeats.

Notice also the difference between properties hasIdentifiedAllele and hasDefinedAllele relating respectively MLST and Locus concepts with the Allele concept. One could imagine that alleles associated to a given MLST instance could be obtained through the defined Schema, since property hasDefinedAllele allows to relate loci and alleles. But this is not the case. A Locus may have associated many alleles, with each of them belonging to many MLST instances, and hence we cannot identify the allele belonging to a particular MLST instance. That can be accomplished through property hasIdentifiedAllele which relates each MLST instance with its identified alleles. Although we did not add those kind of assertions in our ontology, we can still use this information to assert that the identified alleles for a given MLST instance are sufficient against a given Schema. We could even infer for which schemas a given MLST instance provides enough information.

It is also important to note that, by knowing only the Locus, it is possible to identify the Taxon that it belongs to, using the isOfTaxon property (see Figure 1).

Additional information for each class, such as sample collection date and other id, are described through data properties. For instance, the class Allele has data properties such as id and date entered. The class Isolate has data properties such as sample collection date and date entered.

Whenever possible, we reuse concepts from and establish relations with other ontologies as indicated in Table 1. An Agent is a concept imported from the FOAF ontology [25] and it can be a person, a group of persons or even an institution. In TypOn it allows the description of a person or a group of persons who have curated the information about the isolate, who have submitted that information to the database and who own the isolate. These relations are described by the object properties curated by, sent by and owned by, respectively. Reusing the Agent concept is extremely useful because it will allow, for instance, the use of TypOn together with the Web Access Control ontology [30] for defining access control levels in microbial typing databases, an important issue as mentioned above. Several applications in defining access control have been widely discussed and are well known to the research community [31, 32].

All TypOn concepts were derived from the Basic Formal Ontology (BFO) [20], the Ontology for Biomedical Investigations (OBI) [23] and the Sequence Ontology (SO) [21] to ensure upper-level interoperability with other ontologies. In order to avoid huge imports in TypOn we have used OntoFox [33] to query and import only relevant concepts in top level ontologies. These are then imported as, and are available at, https://bitbucket.org/phyloviz/typon/raw/master/imported.owl.

TypOn was also submitted to the BioPortal (https://bioportal.bioontology.org/), hosted by the National Center of Biomedical Ontologies (NCBO), being also available at http://bioportal.bioontology.org/ontologies/TYPON.

Concepts such as Schema and Typing Information are qualities (BFO _0000019), i.e., a categorical property. As discussed, we have classified an Isolate as an organism (OBI _0100026). More details in Figure 4.

Figure 5 depicts the Typon concepts that are related to the Sequence Ontology. Notice that we define Locus as a region (SO:0000001), since it is a named region on a genome.

Another example is the Spa Repeat concept which is a repeat unit (SO:0000657). Both Locus and Spa Repeat are also generically dependent continuant (BFO _0000031) since region (SO:0000001) and repeat unit (SO:0000657) concepts are subclasses of BFO _0000031 as defined in the sequence ontology (SO). Thus these concepts are related to both SO and BFO (see Figures 4 and 5).

Use case

In this section, we illustrate how we can represent typing information annotated with the TypOn ontology. Our example makes use of data regarding the characterization of a Staphylococcus aureus isolate for the purpose of this case. We will use the Turtle language [34] in the description of our isolate.

We can represent the isolate named Sa66296 as follows:

This is an instance of typon:Isolate labelled as “Sa66296”. rdfs:label is an instance of rdf:Property that may be used to provide a human-readable version for the name of a resource. We further specify that it has two pieces of typing information mlst105 and spa _t002 (instances of typon:MLST and typon:spaTyping, respectively). Thus, these pieces of typing information are other individuals annotated with our ontology. We can keep track of the dates when the isolate was collected and when the isolate was entered into the system using properties sampleCollectionDate and dateEntered, respectively. We also describe the origin of the isolate, with the individual labelled Lisbon which has type dbpedia-owl:Place. Note that this isolate was recovered in Lisbon, Portugal, represented as a resource in DBpedia. Figure 6 depicts the isolate information.

The individual labelled mlst 105 represents the sequence based typing method with a schema defined by the sequence of seven housekeeping loci, represented as follows:

Note that the individual labelled schema1 identifies the seven housekeeping loci, using individuals of type typon:SchemaPart for keeping track of the index of each locus:

Let us now describe the individual that represent the arcc locus, the first locus in the considered schema as described by the individual labelled schema_part_1:

Note that the identified alleles of the loci can be directly obtained from the individual labelled mlst105. Furthermore all possible defined alleles can be obtained from the respective loci. It is also possible to obtain the locus that is associated to each allele, namely by property typon:isOfLocus. Figure 7 summarizes the representation of the MLST typing information concerning the isolate in our example. The complete example is available at https://bitbucket.org/phyloviz/typon/raw/master/test/Sa66296.ttl.