TreeSort Service

Overview

The idea behind TreeSort is the observation that if there is no reassortment, then the evolutionary histories of different segments should be identical. TreeSort then uses a phylogenetic tree for one segment (e.g., the HA influenza A virus segment) as an evolutionary hypothesis for another segment (e.g., the NA segment). We will refer to the first segment as the reference and the second segment as the challenge. By trying to fit the sequence alignment of the challenge segment to the reference tree, TreeSort identifies points on that tree, where this evolutionary hypothesis breaks. The “breaking” manifests in the mismatch between the divergence time on the reference tree (e.g., 1 year divergence between sister clades) and an unlikely high number of substitutions in the challenge segment that are required to explain the reference tree topology under the null hypothesis of no reassortment.

TreeSort has demonstrated very high accuracy in reassortment inference in simulations (manuscript in preparation). TreeSort can process datasets with tens of thousands of virus strains in just a few minutes and can scale to very large datasets with hundreds of thousands of strains. (This overview is from https://github.com/flu-crew/TreeSort/blob/main/README.md)

NOTE

The current version of TreeSort is meant to be used for Influenza viruses ONLY. We hope to provide an updated version in the near future that can be used with common segmented viruses.

See Also

• TreeSort Service

• TreeSort Service Tutorial (TODO)

Using the TreeSort Service

The “TreeSort” submenu under the “TOOLS & SERVICES” main menu (Viral Tools category) opens the input form.

• Note: You must be logged into BV-BRC to use this service.

Parameters

Input file

Select a FASTA file from your workspace. Note that the FASTA headers/deflines have very strict formatting requirements:

1. The segment name and sample date must be formatted like the following example (“|segment|sample date” are in yellow):

2. Only the following Influenza segment names are recognized: PB2, PB1, PA, HA, NP, NA, MP, and NS.

Output folder

The directory in your workspace where a directory will be created for the TreeSort results.

Output name

The name of the directory that will be created under “Output folder”. This name will also be used for the primary results filename (output name.tre).

Reference segment

Reassortment events are acquisitions of 1 or more novel segments relative to this (fixed) reference segment.

Segments

Select at least 2 segments to include in the analysis.

Advanced options

Match type

• Strain: Match the names (deflines in FASTAs) across the segments based on the strain name. E.g., “A/Texas/32/2021” or “A/swine/A0229832/Iowa/2021”. Works for flu A, B, C, and D, and no pre-processing is needed to standardize the names before the analysis.

• EPI_ISL_XXX: Segments are matched based on the “EPI_ISL_XXX” field (if present in deflines).

• RegEx: Provide your own custom regular expression to match the segments across the alignments.

Match RegEx

When a match type of RegEx is selected, your custom regular expression will be entered in this field.

Inference method

• local: (default)

• mincut: The mincut method:

  • Always determines the most parsimonious reassortment placement, even in ambiguous circumstances.

  • Uses the reassortment test to cut the reference phylogeny into the optimum (smallest) number of non-reassorting parts with theoretical guarantees on optimality.

  • Is more robust than the current “local” method in many instances, and does not result in “uncertain” reassortment inferences with the ‘?’ annotation.

Reference tree inference method

The tool that will be used to infer the reference tree:

• FastTree: (default)

  • Infers approximately-maximum-likelihood phylogenetic trees from alignments of nucleotide or protein sequences.

  • Can handle alignments with up to a million of sequences in a reasonable amount of time and memory.

  • https://morgannprice.github.io/fasttree/

• IQ-Tree (recommended for better accuracy)

  • A fast search algorithm (Nguyen et al., 2015) to infer phylogenetic trees by maximum likelihood.

  • https://iqtree.github.io/

Allowed deviation

Maximum deviation from the estimated substitution rate within each segment. The default is 2: The substitution rate on a particular tree branch is allowed to be twice as high or twice as low as the estimated rate. The default value was estimated from the empirical influenza A data.

P-value threshold

The cutoff p-value for the reassortment tests: the default is 0.001 (0.1 percent). You may want to decrease or increase this parameter depending on how stringent you want the analysis to be.

Clades filename

The path to an output file where clades with evidence of reassortment will be saved.

Estimate molecular clock rates for different segments

Estimate molecular clock rates for different segments, assuming equal rates.

Collapse near-zero length branches into multifurcations

Collapse near-zero length branches into multifurcations (by default, TreeSort collapses all branches shorter than 10^-7^ (1e-7) and then optimizes the multifurcations).

Buttons

• Reset: Resets the input form to default values

• Submit: Launches the classification job. A message will appear below the box to indicate that the job is now in the queue.

Output Results

Clicking the Jobs indicator at the bottom of the BV-BRC page opens the Jobs Status page, which displays all current and previous service jobs and their statuses.

Once the job has completed, you can view the results by double-clicking the job or clicking the “View” button on the green vertical Action Bar on the right-hand side of the page.

The results page consists of a header describing the job and a list of output files, which are generated by the TreeSort service and saved in your Workspace.

Result files

• TreeSort_analysis_results.html: An overview of the analysis results with links to all files generated by TreeSort, descriptions of the file types, and guidance on how to interpret the result data.

• <output name>.tre: An annotated tree file in Nexus format where output name is the text entered in the Output name field.

Segment-specific files

These files are generated for every virus segment included in the analysis, where segment name is PB2, PB1, PA, HA, NP, NA, MP, or NS.

• <segment name>-input.fasta.aln

• <segment name>-input.fasta.aln.dates.csv

• <segment name>-input.fasta.aln.rooted.tre

• <segment name>-input.fasta.tre

• <segment name>-input.fasta.aln.treetime

  • outliers.tsv

  • root_to_tip_regression.pdf

  • rtt.csv

Action Buttons

After selecting one of the output files by clicking it, a set of options becomes available in the vertical green Action Bar on the right side of the table. These include:

• Hide/Show: Toggles (hides) the right-hand side Details Pane.

• Guide Link to the corresponding User Guide

• Download: Downloads the selected item.

• View Displays the content of the file, typically as plain text or rendered html, depending on filetype.

• Delete Deletes the file.

• Rename Allows renaming of the file.

• Copy: Copies the selected items to the clipboard.

• Move Allows moving of the file to another folder.

• Edit Type Allows changing of the type of the file in terms of how BV-BRC interprets the content and uses it in other services or parts of the website. Allowable types include unspecified, contigs, nwk, reads, differential expression input data, and differential expression input metadata.

More details are available in the Action Buttons user guide.

References

1. Alexey Markin, Catherine A. Macken, Amy L. Baker, Tavis K. Anderson, “Revealing reassortment in influenza A viruses with TreeSort” bioRxiv 2024.11.15.623781; doi: https://doi.org/10.1101/2024.11.15.623781

2. GitHub: https://github.com/flu-crew/TreeSort