AlignmentViewer: Sequence Analysis of Large Protein Families

Operation

AlignmentViewer is a web-based tool written in JavaScript with minimal system requirements. AlignmentViewer works best on Chrome regardless of operating system. AlignmentViewer is developed with the D3 library (d3js.org) to produce dynamic and interactive data visualizations, with performance (speed) for large alignments a major consideration. The tool is entirely client-based, running inside a web browser without the need for server-side computation.

Implementation

Users can access AlignmentViewer and all its features directly from alignmentviewer.org, but its serverless execution enables anyone to quickly start a local copy for online or offline use. Hyperlinks for lookup in background databases, such as Uniprot or Pfam, are made directly from the client. Alignments can be passed to AlignmentViewer also via a URL query parameter that is served by https and is properly encoded (e.g., https://alignmentviewer.org/?url=https://alignmentviewer.org/example/1bkr_A.1-108.msa.txt), enabling seamless integration from external web services via a simple link (e.g. the EVcouplings, evcouplings.org, web server (Hopf et al., 2019) offers visualization of computed alignments via a link to AlignmentViewer). The tool has been thoroughly tested with many large alignments. An alignment with, e.g., 50,000 sequences (about 13 MB of memory) loads in the Safari browser within one minute; further speedup is planned.

MSA view

Alignment details. The msa view page has summary information: number of sequences, conservation and gap counts for each position, a sequence logo, and the residues in one letter code. By default, columns with gaps in the reference sequence (first row) are omitted in order to facilitate visual focus on sequence patterns relative to a protein of interest and to avoid extremely gapped alignment views typical of many MSA presentations. The amino acids are colored using a conventional coloring scheme, adopted from Mview, based on amino acid properties.

Sequence attributes and sorting. Sequences in the alignment can be sorted using one of four different methods: (i) the original order provided by the user, (ii–iii) by % sequence identity between a particular sequence and the reference (top) sequence, relative to the first or the second (gaps not counted), and (iv) by user-provided (upload annotations tab) sequence weights or other attributes, such as alignment profile scores (e.g., HMM bit scores). Sequences can be filtered by sequence identity relative to a reference sequence or by percentage of gaps.

Pixel view (suitable for large families)

The pixel view (image view website tab) leads to an overview of the entire depth and breadth of an MSA. The amino acid letters are represented by small rectangles of pixels, retaining the amino acid type coloring (image view tab). This striking visual impression can reveal patterns of conservation and variation, especially for large alignments. This is very useful to gain an intuitive view of sequence properties, noise at the uncertain edges of a protein family, as well as subfamily distributions. The coloring scheme can be by (1) amino acid properties, (2) hydrophobicity (red to blue) or (3) mutational difference (stronger color) in a sequence relative to the reference (first row) sequence.

Stats view

The stats view tab leads to plots of statistical properties of the set of sequences in the alignment, including (i) sequence identity relative to the reference sequence, and (ii) min, max, and average of (i); and (iii) a pairwise sequence identity matrix in which each pixel represents the degree of similarity between two sequences, such that a block-diagonal structure of the matrix is indicative of distinct subfamilies, given, e.g., a tree-derived sequence order as user input. The ordering of sequences by phylogeny is (currently) not part of the tool and can be performed using external tools, e.g., Wasabi (Veidenberg et al., 2016).

Annotations and evolutionary couplings

Users can upload custom numerical attributes or labels for the sequences in the MSA (upload annotations) or evolutionary couplings between residue positions (load couplings). Adding these attributes allows users to use sequence weights, compare different measures of sequence fitness (e.g., bitscore, sequence identity, statistical energy) or visualize evolutionary coupling constraints for pairs of positions.

Sequence space

Users can view representations of the MSA sequences in two- or three-dimensional space under the “sequence space” tab. These representations are generated using the Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction algorithm (McInnes et al., 2018), whichhas been adapted for Javascript using the umap-js library (https://github.com/PAIR-code/umap-js). The alignmentviewer.org implementation uses the number of amino acid differences between pairs of sequences (the Hamming distance) as the distance metric parameter. The algorithm then iteratively calculates an embedding in two- or three-dimensional space, which is displayed in real time for the end users. UMAP hyperparameters are set to reasonable defaults, but can also be configured via the settings panel. Sequences can be colored by user provided annotations ("upload annotations" tab).