Comparative genomic analysis of hepadnaviruses using GLUE

This is Hepadnavirus-GLUE, a GLUE project supporting comparative genomic and evolutionary analysis of hepadnaviruses (family Hepadnaviridae).

Hepadnaviruses ( family Hepadnaviridae) are reverse-transcribing DNA viruses that infect vertebrates. The type species - hepatitis B virus (HBV) - is estimated to infect ~300 million people worldwide, causing substantial morbidity and mortality. Recent studies have revealed that hepadnaviruses infect a diverse range of vertebrate species, ranging from fish to mammals. They are associated with disease in many of these species.

Species known to be afflicted by hepadnaviruses

Some of the vertebrate species known to be infected by hepadnaviruses: left to right: woolly monkeys, shrews, domestic cats, ducks.

Since the emergence of the SARS-COV2 pandemic, many have become familiar with the use of virus genome data to track the spread and evolution of pathogenic viruses - e.g. via tools such as NextStrain. However, it is less widely appreciated that the same kinds of data sets and comparative genomic approaches can also be used to explore the structural and functional basis of virus adaptations.

The GLUE software framework provides an extensible platform for implementing computational genomic analysis of viruses in an efficient, standardised and reproducible way. GLUE projects can not only incorporate all of the data items typically used in comparative genomic analysis (e.g. sequences, alignments, genome feature annotations) but can also represent the complex semantic links between these data items via a relational database. This 'poises' sequences and associated data for application in computational analysis, minimising the requirement for labour-intensive pre-processing of datasets.

GLUE projects are equally suited for carrying out exploratory work (e.g. using virus genome data to investigate structural and functional properties of viruses) as they are for implementing operational procedures (e.g. producing standardised reports in a public or animal health setting).

Hosting of GLUE projects in an online version control system (e.g. GitHub) provides a mechanism for their stable, collaborative development, as shown below.

GitHub illustration

What is a GLUE project?

GLUE is an open, integrated software toolkit that provides functionality for storage and interpretation of sequence data.

GLUE supports the development of “projects” containing the data items required for comparative genomic analysis (e.g. sequences, multiple sequence alignments, genome feature annotations, and other sequence-associated data).

GLUE web

Projects are loaded into the GLUE "engine", creating a relational database that represents the semantic relationships between data items. This provides a robust foundation for the implementation of systematic comparative analyses and the development of sequence-based resources.

Projects are loaded into the GLUE "engine", creating a relational database that represents the semantic relationships between data items. This provides a robust foundation for the implementation of systematic comparative analyses and the development of sequence-based resources. The database schema can be extended to accommodate the idiosyncrasies of different projects. GLUE provides a scripting layer (based on JavaScript) for developing custom analysis tools.

What does building the Hepadnavirus-GLUE project offer?

Hepadnavirus-GLUE offers a number of advantages for performing comparative sequence analysis of hepadnaviruses:

  1. Reproducibility. For many reasons, bioinformatics analyses are notoriously difficult to reproduce. The GLUE framework supports the implementation of fully reproducible comparative genomics through the introduction of data standards and the use of a relational database to capture the semantic links between data items.

  2. Reusable data objects and analysis logic. For many - if not most - comparative genomic analyses, data preparation is nine tenths of the battle. The GLUE framework has been designed to ensure that work spent preparing high-value data items such as multiple sequence alignments need only be performed once. Hosting of GLUE projects in an online version control system such as GitHub allows for collaborative management of important data items and community testing of hypotheses.

  3. Validation. Building GLUE projects entails mapping the semantic links between data items (e.g. sequences, tabular data, multiple sequence alignments). This process provides an opportunity for cross-validation, and thereby enforces a high level of data integrity.

  4. Standardisation of the genomic co-ordinate space. GLUE projects allow all sequences to utilise the coordinate space of a chosen reference sequence. Contingencies associated with insertions and deletions (indels) are handled in a systematic way.

  5. Predefined, fully annotated reference sequences: This project includes fully-annotated reference sequences for major lineages within the Hepadnaviridae family.

  6. Alignment trees: GLUE allows linking of alignments constructed at distinct taxonomic levels via an "alignment tree" data structure. In the alignment tree, each alignment is constrained to a standard reference sequence, thus all multiple sequence alignments are linked to one another via a standardised coordinate system.

There are a wide variety of ways in which the Hepadnavirus-GLUE resource can be used:

Installing Hepadnaviridae-GLUE

On computers with GLUE installed, the Hepadnavirus-GLUE project can be instantiated by navigating to the project folder, initiating GLUE, and issuing the following command in the GLUE shell:

  Mode path: /
  GLUE> run file buildCoreProject.glue

This will build the Hepadnavirus-GLUE core project by executing the commands in this file.

The Hepadnavirus-GLUE project can be further extended to incorporate EVE sequences by executing the commands in this file, as follows.

  Mode path: /
  GLUE> run file buildPaleoProject.glue

The paleovirus extension of incorporates a set of endogenous viral elements (EVEs) recovered from the genomes of metazoan species. Building the paleovirus extension allows automated alignment and phylogeny reconstruction for individual eHBV lineages in the project, based on the classifications in these files. Individual eHBV sequences have been classified into sets considered likely to have arisen from the same germline colonisation event. Loci have been named using a systematic approach (see here for details).

Related Publications

Lytras S, Arriagada G, and RJ Gifford (2020)
Ancient evolution of hepadnaviral paleoviruses and their impact on host genomes.
Virus Evolution [view]

Singer JB, Thomson EC, McLauchlan J, Hughes J, and RJ Gifford (2018)
GLUE: A flexible software system for virus sequence data.
BMC Bioinformatics [view]

Zhu H, Dennis T, Hughes J, and RJ Gifford (2018)
Database-integrated genome screening (DIGS): exploring genomes heuristically using sequence similarity search tools and a relational database. [preprint]

Gifford RJ, Blomberg B, Coffin JM, Fan H, Heidmann T, Mayer J, Stoye J, Tristem M, and WE Johnson (2018)
Nomenclature for endogenous retrovirus (ERV) loci.
Retrovirology [view]

Katzourakis A. and RJ. Gifford (2010)
Endogenous viral elements in animal genomes.
PLoS Genetics [view]


Robert J. Gifford ( (Project Leader)

Spyros Lytras (


This project is licensed under the GNU Affero General Public License v. 3.0.