Bandage: interactive visualization of de novo genome assemblies

1Current de novo genome assemblers use graphs, most typically a de Bruijn graph. An ideal graph would contain one distinct path for each underlying sequence, but complexities such as repeated sequences usually prevent this. Instead, assembly graphs contain branching structures, where one node may lead into multiple others. The longest sequences in the graph that can be determined unambiguously are saved as contigs, which are often the final result of de novo assembly. However, an assembly graph contains more information because it also has connections between sequences. It can therefore be advantageous to work with assembly graphs instead of contigs.

2Bandage facilitates interaction with de Bruijn graphs made by de novo assemblers such as Velvet, SPAdes and Trinity. It displays the graph in a graphical user interface (GUI) using a simple, comprehensible representation. The program is interactive, allowing users to zoom, pan and manually move nodes to focus on areas of interest.

3Bandage is a GUI application written in C++ with Qt, giving it speed, memory efficiency and cross-platform portability. It runs on Linux, OS X and Windows. The Open Graph Drawing Framework library (http://www.ogdf.net/) is used to perform the graph layout using the fast multipole multilevel layout algorithm, which scales well for very large graphs.

4On a 3 GHz laptop, Bandage can load and display the de novo assembly graph for a bacterial genome (5 Mb) in a few seconds, using <100 MB of RAM. A large graph with hundreds of thousands of nodes, such a metagenome assembly (100 Mb or more), may take minutes and multiple gigabytes of RAM to display in its entirety, but the visualization can be limited to smaller regions of the graph to improve performance and reduce memory requirements.

5A full feature list and program screenshots are available in the Supplementary Material.

6Assemblies of whole genomes can be difficult to complete if repeated sequences occur in chromosomes or plasmids. Repeated sequences cause distinctive structures in the assembly graph, limiting contig length. Bandage’s visualization of the assembly graph makes it easy to identify such problematic parts of assemblies (Fig. 1a). In some cases, it is possible to manually resolve these ambiguities by using additional information not available to the assembler. Bandage facilitates this by allowing users to copy sequences directly from the graph visualization. In other cases, ambiguities cannot be resolved and the assembly cannot be completed. For these situations, Bandage provides a clear illustration of the assembly’s incompleteness and comparison of one assembly’s quality to another.

7Whole genome sequencing is frequently used to investigate evolution and transmission of bacterial pathogens and the mobile genetic elements responsible for antibiotic resistance. In this case study, multiple strains of Salmonella enterica were sequenced to determine the composition of their Salmonella genomic island (SGI), which can confer antibiotic resistance. The assembly of the SGI was complicated by sequences that were shared between the SGI, plasmids and a cloning vector used in the preparation of libraries for sequencing, making the contigs of limited use.

8With Bandage, the user could search for nodes of interest (resistance genes or known SGI sequences) and zoom in to deeply investigate those areas of the graph. By showing the graph edges, Bandage makes clear which contigs are connected, their directionality and therefore which sequences are likely to be contiguous (Fig. 1b). For many Salmonella strains, this allowed for determination that a gene of interest was or was not contained in the SGI, even when other methods were inconclusive.

916S rRNA sequences are commonly used for classification of bacteria, but this is complicated by the fact that a single bacterial genome may contain multiple distinct copies of the 16S gene. Some regions of the gene are highly conserved while others regions are variable, potentially disrupting its de novo assembly.

10In this case study, the user sequenced a bacterial isolate with whole genome shotgun sequencing and assembled the reads with Velvet v1.2.10. Bandage’s BLAST integration highlighted the 16S gene in the assembly graph. It did not assemble completely, and fragments of the gene were present in seven different nodes (Fig. 1c). Consequently, the longest available 16S sequence in the contigs file was 909 bp and only partially covered the gene.

11By illustrating graph connections and node coverage, Bandage made it clear which path represents the gene’s most abundant copy. The user was then able to extract node sequences from Bandage to manually reconstruct a 1535 bp sequence containing an entire 16S gene.

12By visualizing both nodes and edges, Bandage gives users easy, fast access to the connection information contained in assembly graphs. This is particularly useful when the assembly contains many short contigs—as is often the case when assembling short reads—and empowers users to examine and assess their assembly graphs in greater detail than when viewing contigs alone.