The Evidence and Conclusion Ontology (ECO): Supporting GO Annotations
The Evidence and Conclusion Ontology (ECO) is a community resource for describing the various types of evidence that are generated during the course of a scientific study and which are typically used to support assertions made by researchers. ECO describes multiple evidence types, including evidence resulting from experimental (i.e., wet lab) techniques, evidence arising from computational methods, statements made by authors (whether or not supported by evidence), and inferences drawn by researchers curating the literature. In addition to summarizing the evidence that supports a particular assertion, ECO also offers a means to document whether a computer or a human performed the process of making the annotation. Incorporating ECO into an annotation system makes it possible to leverage the structure of the ontology such that associated data can be grouped hierarchically, users can select data associated with particular evidence types, and quality control pipelines can be optimized. Today, over 30 resources, including the Gene Ontology, use the Evidence and Conclusion Ontology to represent both evidence and how annotations are made.
Key wordsAnnotation Biocuration Conclusion Confidence Evidence ECO Experiment Inference Literature curation Quality control
1 Describing Evidence in Scientific Investigations
1.1 Importance of Documenting Evidence
Recording evidence is essential because: (1) knowing what methodologies were used is central to the scientific method and can impact one’s evaluation of the data or results; (2) associating evidence with data maintained electronically allows for selective data queries and retrieval from even the largest of databases; and (3) a structured representation of evidence makes automated quality control possible, which is absolutely essential to managing the ever-increasing number and size of biological databases.
1.2 Multiple Types of Evidence and Ways of Associating Evidence with Assertions
Evidence can be associated with assertions in many ways. Manual curation is a common approach [3, 4], outlined in Fig. 1. However, text mining or other computational methods can also be used to extract biological assertions from the scientific literature [5, 6], and assertions can also be made directly via bioinformatic techniques , e.g. assigning of functional annotations as resulting from a functional genome annotation pipeline.
2 The Evidence and Conclusion Ontology (ECO)
2.1 The Argument for an Ontology of Evidence
Due to the diversity of ways that exist to describe the multitude of scientific research methodologies, a means of representing evidence in a descriptive but structured way is required in order to maximize utility. The most efficient way to achieve this is to use an ontology, a controlled vocabulary where each term is well-defined and linked to other terms via defined relationships [8, 9]. In an ontological framework, evidence descriptions are represented not as free text, but rather as networked ontology classes where each child term is more specific (granular) than its parent . High-level descriptions of types of evidence (such as “experimental evidence”) are contained in more basal classes closest to the root class evidence. Increasingly specific terms that are grouped under the more general classes describe particular sub-types of evidence (such as “chromatography evidence”). The most specific terms, the so-called “leaf nodes” that contain no child terms, represent the most granular types of evidence generated during the course of a scientific investigation (for example “thin layer chromatography evidence”). The Evidence and Conclusion Ontology (ECO) (http://evidenceontology.org) was created to enable the structured description of experimental, computational, and other evidence types to support the assertions captured by scientific databases .
2.2 A Brief History of ECO
As described throughout this book, the Gene Ontology (GO) uses terms organized into controlled vocabularies, and the relationships among these terms, to capture functional information about gene products. The need to systematically document evidence while curating annotations was recognized from the inception of the GO  and a set of “evidence codes” was created for this purpose . In time it was realized that a better-structured and more comprehensive way to represent evidence was required. Thus, the set of initially created GO codes, along with terms created by two model organism databases, FlyBase  and The Arabidopsis Information Resource , evolved into the first version of ECO, the “Evidence Code Ontology”. Since then, the use of ECO by other resources has continued to grow and the ontology has shifted its focus beyond GO in order to become a generalized ontology for the capture of evidence information. The official name of ECO is now the “Evidence and Conclusion Ontology”. ECO is presently being developed to define and broaden its scope, normalize its content, and enhance interoperability with related resources. The GO remains an active user and participant in developing ECO. It is anticipated that soon the three letter GO evidence codes to which so many are accustomed will be replaced by ECO term identifiers.
2.3 ECO Structure and Content
In addition to describing evidence, ECO can also describe the means by which assertions are made, i.e., by a human or a machine. ECO calls this the “assertion method” and defines it as “a means by which a statement is made about an entity” (Figs. 1c and 2b). For example, whether a curator makes an annotation after reading about an experimental result in a scientific paper or after manually evaluating pairwise sequence alignment results, ECO can express that a manual curation method was used (3,8). Conversely, if an algorithm was used to assign a predicted function to a protein, ECO can express that an automated computational method was used. Thus “assertion method” forms a second root class with two branches: “manual assertion” and “automatic assertion” (Fig. 3).
The current version of ECO comprises 630 terms that describe “evidence”, “assertion method”, or “evidence x assertion method” cross products. Ontology architecture of ECO was recently described in Chibucos et al. .
2.3.1 Extending ECO Beyond GO
Recent development efforts of ECO have emphasized meeting the needs of a larger research community; see for example [11, 16], while still capturing the needed information for GO annotation, such as by adding comments and synonyms to a term. Many high-level ECO term definitions were written with explicit GO usage notes contained therein because ECO originated during early efforts of the GO. However, in order to increase overall usability of ECO by resources other than the GO, such verbiage has been removed, while retaining the essence of the term’s meaning and applicability to GO. As ECO has been developed, more and more granular terms have been created to represent increasingly complex laboratory, computational, and even inferential techniques.
A discussion of ECO and GO would not be complete without mention of the GO evidence code IEA or “inferred from electronic annotation”. IEA is used to connote that an annotation was assigned through automated computational means, e.g., transferring annotations from one protein to another. Because IEA describes how an annotation was assigned, rather than the specific type of supporting evidence, this term belongs as a subclass of “assertion method”. As described above, “assertion method” has two child terms, “manual assertion” and “automatic assertion”, with the latter being equivalent to IEA. Now it is possible to more accurately model evidence and the annotation process using ECO.
Aside from rewording definitions and creating a second root class, the biggest conceptual modification of ECO is reflected by removal of the prefix “inferred from” from every term name (see the GO codes for a sense of how ECO terms were previously labeled). This was done because ECO considers not just inferences made during the curation process, per se, but other aspects of evidence documentation, such as what research methodologies were performed.
3 Fundamentals of Evidence-Based GO Annotation
Creating an association between a GO term and a gene product is the fundamental essence of the GO annotation process. Documenting the evidence for any given GO annotation is a critical component of this annotation process, and an annotation would be incomplete without the requisite evidence. In fact, evidence capture by the GO requires both a “GO evidence code” that describes in detail the type of work or analysis that was performed in support of the annotation, as well as a citation for the reference from which the evidence was derived. Curators go to great lengths to understand and properly apply the correct “evidence code” to a given annotation, and an online guide exists to explain the often-subtle distinctions between multiple related evidence types (http://geneontology.org/page/guide-go-evidence-codes) [4, 13].
3.1 ECO Terms Versus GO Codes
Although GO evidence codes are useful in themselves because they represent detailed descriptions of evidence types, they are maintained as a controlled vocabulary with a shallow hierarchical structure that lacks the advantages of a formal ontology like ECO. Further, the full set of terms within ECO provides the ability to capture more breadth and depth of evidence information than the GO evidence codes do. Additionally, as the field of biocuration evolves and the kinds of evidence being curated from the literature continue to grow both more detailed and nuanced, the number of two- and three-letter acronyms (e.g., IEA, IMP, EXP, and ISS) available for new terms will hit an upper limit (there are only 676 possibilities using all 26 two-letter combinations, as the first letter of the three-letter GO codes often stands for “inferred”). In fact, ECO developers have already received requests from different users to develop new, but unrelated, terms that had the same suggested three-letter acronyms. For all of these reasons, there are discussions underway about transitioning GO evidence storage to use ECO terms rather than GO evidence codes. Such a shift would combine the advantages of both systems and would still provide a mechanism for filtering evidence annotations by the previous codes if desired. If ECO terms were to be fully adopted by GO, the GAF format would change to require “ECO term” instead of “evidence code.” Since most GO evidence codes have a one-to-one mapping to ECO terms (while the remainder, i.e., IEA, IGC, ISS, map, in conjunction with various GO standard references [http://purl.obolibrary.org/obo/eco/gaf-eco-mapping.txt], to specific ECO terms), GO data depositors could use a straightforward replacement based on the mappings. Other resources outside of GO have modeled their annotation capture systems on the GAF format. For example, the Ontology of Microbial Phenotypes  uses a modified version of the GO GAF, but employs ECO terms instead of GO evidence codes. The full use of ECO terms by the GO would enhance the integration of data derived from such diverse sources.
4 Benefits of ECO and Applications for the GO
There are currently over 365 million annotations in the GO repository linked to an evidence term, and these can be queried and maintained better with the help of an ontology by leveraging its hierarchical structure. One of the most direct applications for using an ontology of evidence is selective data query, i.e., to query a database for records associated with a particular evidence type. For example, searching for “thin layer chromatography evidence” (at present a leaf term with no subclasses) would return only the records associated with that evidence type and no others. But grouping annotations is also possible with this approach. A query for “chromatography evidence” will return data associated not only with “chromatography evidence” but also its more specific subtypes including “thin layer chromatography evidence” and “high performance liquid chromatography evidence”.
To amplify the benefits of experimental knowledge that curators capture, the GO Consortium is using a phylogenetic tree-based approach to generate manually reviewed, homology-based annotations for a range of species . This phylogenetic annotation methodology necessitated a new set of evidence terms to capture the inference process (Fig. 4a). Currently over 150,000 annotations are associated with these new terms and the number continues to grow.
The GO curatorial process uses evidence to support computable rules about the kinds of information that must be associated with different evidence types. For example, one rule states that annotation of a protein based on alignment with another protein requires that the identity of the matching protein be captured, along with the evidence type “protein alignment evidence” (Fig. 4b). If such an evidence type were missing, this would flag the annotation for review.
The GO uses evidence as a quality control mechanism for annotation consistency. For example, expression pattern evidence is restricted to annotations for terms from the “biological process” ontology. Annotations to terms from either of the other two GO ontologies (“molecular function” or “cellular component”) would be flagged as suspect (Fig. 4c).
Evidence is used to prevent circular annotations based solely on computational predictions (Fig. 4d). Chains of evidence are computationally evaluated to ensure that inferential annotations are linked to experimental evidence. For example, annotations supported by “sequence alignment evidence” require the inclusion of a database identifier for the matching gene product that is itself linked to an annotation supported by experimental evidence.
Yet another application of ECO for the GO has been realized in the UniProt-Gene Ontology Annotation (UniProt-GOA) project. Arguably, UniProt is the most comprehensive and best-curated protein database available to the research community. ECO terms have replaced the original UniProtKB  evidence types and are available in UniProtKB XML . Novel ways of mapping and extending ontologies have been discussed with ECO and the GO Consortium to ensure appropriate development for UniProtKB annotation. The UniProt-GOA project provides >169 million manual and electronic evidence-based associations between GO terms and 26.5 million UniProtKB proteins covering >411,000 taxa . Of these, manual annotation provides 1.4 million annotations to ~260,000 proteins. Since 2010, UniProt-GOA has supplied GO annotations in a Gene Product Association Data (GPAD) file format, which allows inclusion of ECO terms. Because ECO terms are cross referenced to corresponding GO codes, even if evidence for annotations was supplied to UniProt as GO codes, the GPAD file will display the appropriate equivalent ECO term. Thus, UniProt annotations can be grouped by leveraging the structure of ECO.
Once the reader has gained a basic understanding of ECO and its connection to GO, we can perform the following simple exercise that displays a faceted query using ECO in AmiGO 2 (http://amigo2.geneontology.org/amigo).
5 The Future of ECO
What else can an ontology of evidence do? One aspect of active exploration for ECO is the evaluation of confidence or quality of evidence. Work has begun  to develop a mechanism to incorporate quality information into ECO or, as needed, to create a standalone system. It might one day be possible to use ECO to describe the quality of the evidence supporting an annotation in addition to the type of evidence that supports the annotation.
In summary, the Evidence and Conclusion Ontology can be used to support faceted queries of data, to establish computable rules about required types of evidence, as a quality control check for annotation consistency, and as a mechanism to prevent circular annotations rooted only in computational predictions. GO is already benefitting from these applications of ECO, and the future promises both additional new applications of ECO as well as advancements to current ones.
This material is based upon work supported by the National Science Foundation under Award Number 1458400 and National Institutes of Health/National Institute of General Medical Sciences under Grant Number 2R01 GM089636.
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