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Augur

Quick Facts

Workflow Type Applicable Kingdom Last Known Changes Command-line Compatibility Workflow Level
Phylogenetic Construction Viral PHB v2.3.0 Yes Sample-level, Set-level

Augur Workflows

Genomic Epidemiology is an important approach in the effort to understand and mitigate against disease transmission. An often-critical step in viral genomic epidemiology is the generation of phylogenetic trees to explore the genetic relationship between viruses on a local, regional, national or global scale. The Augur workflows, currently only targeted for viral pathogens, facilitate this process by generating files for the visualization of phylogenetic trees with accompanying metadata.

Two workflows are offered: Augur_Prep_PHB and Augur_PHB. These must be run sequentially, respectively, to first prepare each individual sample for running Augur, and secondly to run Augur itself on the set of samples, generating the phylogenetic tree files with accompanying metadata. The outputs from these workflows can be visualized in Auspice and UShER.

Helpful resources for epidemiological interpretation

Augur_Prep_PHB

The Augur_Prep_PHB workflow was written to prepare individual sample assemblies and their metadata for running the Augur_PHB analysis.

Augur_Prep Inputs

The Augur_Prep_PHB workflow takes assembly FASTA files and associated metadata formatted in a data table. FASTA files may be generated with one of the TheiaCoV Characterization workflows and should adhere to quality control guidelines, (e.g. QC guidelines produced by PHA4GE). The metadata can be uploaded to Terra as TSV file, formatted as in this example.

This workflow runs on the sample level.

Terra Task Name Variable Type Description Default Value Terra Status
augur_prep assembly File Assembly/consensus file (single FASTA file per sample) Required
augur_prep collection_date String Collection date of the sample Optional
augur_prep continent String Continent where sample was collected Optional
augur_prep country String Country where sample was collected Optional
augur_prep county String County (or smaller locality) where sample was collected Optional
augur_prep nextclade_clade String The Nextclade clade of the sample Optional
augur_prep pango_lineage String The Pangolin lineage of the sample Optional
augur_prep state String State (or province) where sample was collected Optional
prep_augur_metadata cpu Int Number of CPUs to allocate to the task 1 Optional
prep_augur_metadata disk_size Int Amount of storage (in GB) to allocate to the task 10 Optional
prep_augur_metadata docker String The Docker container to use for the task us-docker.pkg.dev/general-theiagen/theiagen/utility:1.1 Optional
prep_augur_metadata memory Int Amount of memory/RAM (in GB) to allocate to the task 3 Optional
prep_augur_metadata organism String The organism to be analyzed in Augur; options: "sars-cov-2", "flu", "MPXV", "rsv-a", "rsv-b" sars-cov-2 Optional
version_capture docker String The Docker container to use for the task "us-docker.pkg.dev/general-theiagen/theiagen/alpine-plus-bash:3.20.0" Optional
version_capture timezone String Set the time zone to get an accurate date of analysis (uses UTC by default) Optional

Augur_Prep Outputs

Variable Type Description
augur_metadata File TSV file of the metadata provided as input to the workflow in the proper format for Augur analysis
augur_prep_phb_analysis_date String Date of analysis
augur_prep_phb_version String Version of the Public Health Bioinformatics (PHB) repository used

Augur_PHB

Helpful Hint

You may have to generate phylogenies multiple times, running the Augur_PHB workflow, assessing results, and amending inputs to generate a final tree with sufficient diversity and high-quality data of interest.

The Augur_PHB workflow takes a set of assembly/consensus files (FASTA format) and sample metadata files (TSV format) that have been reformatted using Augur_Prep_PHB and runs Augur to generate the phylogenetic tree files with accompanying metadata. Additionally, the workflow infers pairwise SNP distances.

Augur Inputs

The Augur_PHB workflow takes in a set of SARS-CoV-2 (or any other viral pathogen) FASTA and metadata files. If running the workflow via Terra, individual samples will need to be added to a set before running the workflow. Input FASTAs should meet QA metrics. Sets of FASTAs with highly discordant quality metrics may result in the inaccurate inference of genetic relatedness. There must be some sequence diversity among the set of input assemblies. If insufficient diversity is present, it may be necessary to add a more divergent sequence to the set.

Optional Inputs

There are many optional user inputs. For SARS-CoV-2, Flu, rsv-a, rsv-b, and mpxv, default values that mimic the NextStrain builds have been preselected. To use these defaults, you must write either "sars-cov-2","flu", "rsv-a", "rsv-b", or "mpxv" for the organism variable.

For Flu - it is required to set flu_segment to either "HA" or "NA" & flu_subtype to either "H1N1" or "H3N2" or "Victoria" or "Yamagata" or "H5N1" ("H5N1" will only work with "HA") depending on your set of samples.

A Note on Optional Inputs
Default values for SARS-CoV-2
Default values for Flu
  • lat_longs_tsv = "gs://theiagen-public-files-rp/terra/flu-references/lat_longs.tsv"
  • min_num_unambig = 900
  • min_date = 2020.0
  • pivot_interval = 1
  • narrow_bandwidth = 0.1666667
  • proportion_wide = 0.0
H1N1
  • auspice_config = "gs://theiagen-public-files-rp/terra/flu-references/auspice_config_h1n1pdm.json"
  • HA
    • reference_fasta = "gs://theiagen-public-files-rp/terra/flu-references/reference_h1n1pdm_ha.gb"
    • clades_tsv = "gs://theiagen-public-files-rp/terra/flu-references/clades_h1n1pdm_ha.tsv"
  • NA
    • reference_fasta = "gs://theiagen-public-files-rp/terra/flu-references/reference_h1n1pdm_na.gb"
H3N2
  • auspice_config = "gs://theiagen-public-files-rp/terra/flu-references/auspice_config_h3n2.json"
  • HA
    • reference_fasta = "gs://theiagen-public-files-rp/terra/flu-references/reference_h3n2_ha.gb"
    • clades_tsv = "gs://theiagen-public-files-rp/terra/flu-references/clades_h3n2_ha.tsv"
  • NA
    • reference_fasta = "gs://theiagen-public-files-rp/terra/flu-references/reference_h3n2_na.gb"
Victoria
  • auspice_config = "gs://theiagen-public-files-rp/terra/flu-references/auspice_config_vic.json"
  • HA
    • reference_fasta = "gs://theiagen-public-files-rp/terra/flu-references/reference_vic_ha.gb"
    • clades_tsv = "gs://theiagen-public-files-rp/terra/flu-references/clades_vic_ha.tsv"
  • NA
    • reference_fasta = "gs://theiagen-public-files-rp/terra/flu-references/reference_vic_na.gb"
Yamagata
  • auspice_config = "gs://theiagen-public-files-rp/terra/flu-references/auspice_config_yam.json"
  • HA
    • reference_fasta = "gs://theiagen-public-files-rp/terra/flu-references/reference_yam_ha.gb"
    • clades_tsv = "gs://theiagen-public-files-rp/terra/flu-references/clades_yam_ha.tsv"
  • NA
    • reference_fasta = "gs://theiagen-public-files-rp/terra/flu-references/reference_yam_na.gb"
H5N1
  • auspice_config = "gs://theiagen-public-files-rp/terra/flu-references/auspice_config_h5n1.json"
  • HA
    • reference_fasta = "gs://theiagen-public-files-rp/terra/flu-references/reference_h5n1_ha.gb"
    • clades_tsv = "gs://theiagen-public-files-rp/terra/flu-references/h5nx-clades.tsv"
Default values for MPXV
  • min_num_unambig = 150000
  • clades_tsv = "gs://theiagen-public-files-rp/terra/augur-mpox-references/mpox_clades.tsv"
  • lat_longs_tsv = "gs://theiagen-public-files-rp/terra/flu-references/lat_longs.tsv"
  • reference_fasta = "gs://theiagen-public-files-rp/terra/augur-mpox-references/NC_063383.1.reference.fasta"
  • reference_genbank = "gs://theiagen-public-files-rp/terra/augur-mpox-references/NC_063383.1_reference.gb"
  • auspice_config = "gs://theiagen-public-files-rp/terra/augur-mpox-references/mpox_auspice_config_mpxv.json"
  • min_date = 2020.0
  • pivot_interval = 1
  • narrow_bandwidth = 0.1666667
  • proportion_wide = 0.0
Default values for RSV-A
  • min_num_unambig = 10850
  • clades_tsv = "gs://theiagen-public-files-rp/terra/rsv_references/rsv_a_clades.tsv"
  • lat_longs_tsv = "gs://theiagen-public-files-rp/terra/flu-references/lat_longs.tsv"
  • reference_fasta = "gs://theiagen-public-files-rp/terra/rsv_references/reference_rsv_a.fasta"
  • reference_genbank = ""gs://theiagen-public-files-rp/terra/rsv_references/reference_rsv_a.gb"
  • auspice_config = ""gs://theiagen-public-files-rp/terra/rsv_references/rsv_auspice_config.json"
  • min_date = 2020.0
  • pivot_interval = 1
  • narrow_bandwidth = 0.1666667
  • proportion_wide = 0.0
Default values for RSV-B
  • min_num_unambig = 10850
  • clades_tsv = "gs://theiagen-public-files-rp/terra/rsv_references/rsv_b_clades.tsv"
  • lat_longs_tsv = "gs://theiagen-public-files-rp/terra/flu-references/lat_longs.tsv"
  • reference_fasta = "gs://theiagen-public-files-rp/terra/rsv_references/reference_rsv_b.fasta"
  • reference_genbank = ""gs://theiagen-public-files-rp/terra/rsv_references/reference_rsv_b.gb"
  • auspice_config = ""gs://theiagen-public-files-rp/terra/rsv_references/rsv_auspice_config.json"
  • min_date = 2020.0
  • pivot_interval = 1
  • narrow_bandwidth = 0.1666667
  • proportion_wide = 0.0

For more information regarding these optional inputs, please view Nextrain's detailed documentation on Augur

What's required or not?

For organisms other than SARS-CoV-2 or Flu, the required variables have both the "required" and "optional" tags.

This workflow runs on the set level. Please note that for every task, runtime parameters are modifiable (cpu, disk_size, docker, and memory); most of these values have been excluded from the table below for convenience.

Terra Task Name Variable Type Description Default Value Terra Status
augur assembly_fastas Array[File] An array of the assembly files to use; use either the HA or NA segment for flu samples Required
augur build_name String Name to give to the Augur build Required
augur auspice_config File Auspice config file for customizing visualizations; takes priority over the other customization values available for augur_export Defaults are organism-specific. Please find default values for all organisms (and for Flu - their respective genome segments and subtypes) here: https://github.com/theiagen/public_health_bioinformatics/blob/main/workflows/utilities/wf_organism_parameters.wdl. For an organism without set defaults, a minimal auspice config file is provided to prevent workflow failure, "gs://theiagen-public-files-rp/terra/augur-defaults/minimal-auspice-config.json", but will not be as useful as an organism specific config file. Optional
augur clades_tsv File TSV file containing clade mutation positions in four columns Defaults are organism-specific. Please find default values for all organisms (and for Flu - their respective genome segments and subtypes) here: https://github.com/theiagen/public_health_bioinformatics/blob/main/workflows/utilities/wf_organism_parameters.wdl. For an organism without set defaults, an empty clades file is provided to prevent workflow failure, "gs://theiagen-public-files-rp/terra/augur-defaults/minimal-clades.tsv", but will not be as useful as an organism specific clades file. Optional, Required
augur distance_tree_only Boolean Create only a distance tree (skips all Augur steps after augur_tree) TRUE Optional
augur flu_segment String Required if organism = "flu". The name of the segment to be analyzed; options: "HA" or "NA" "HA" (only used if organism = "flu") Optional, Required
augur flu_subtype String Required if organism = "flu". The subtype of the flu samples being analyzed; options: "H1N1", "H3N2", "Victoria", "Yamagata", "H5N1" Optional, Required
augur lat_longs_tsv File Tab-delimited file of geographic location names with corresponding latitude and longitude values Defaults are organism-specific. Please find default values for all organisms (and for Flu - their respective genome segments and subtypes) here: https://github.com/theiagen/public_health_bioinformatics/blob/main/workflows/utilities/wf_organism_parameters.wdl. For an organism without set defaults, a minimal lat-long file is provided to prevent workflow failure, "gs://theiagen-public-files-rp/terra/augur-defaults/minimal-lat-longs.tsv", but will not be as useful as a detailed lat-longs file covering all the locations for the samples to be visualized. Optional
augur min_date Float Minimum date to begin filtering or frequencies calculations Defaults are organism-specific. Please find default values for all organisms (and for Flu - their respective genome segments and subtypes) here: https://github.com/theiagen/public_health_bioinformatics/blob/main/workflows/utilities/wf_organism_parameters.wdl. For an organism without set defaults, the default value is 0.0 Optional
augur min_num_unambig Int Minimum number of called bases in genome to pass prefilter Defaults are organism-specific. Please find default values for all organisms (and for Flu - their respective genome segments and subtypes) here: https://github.com/theiagen/public_health_bioinformatics/blob/main/workflows/utilities/wf_organism_parameters.wdl. For an organism without set defaults, the default value is 0 Optional
augur organism String Organism used to preselect default values; options: "sars-cov-2", "flu", "mpxv", "rsv-a", "rsv-b" sars-cov-2 Optional
augur reference_fasta File The reference FASTA file used to align the genomes and build the trees Defaults are organism-specific. Please find default values for all organisms (and for Flu - their respective genome segments and subtypes) here: https://github.com/theiagen/public_health_bioinformatics/blob/main/workflows/utilities/wf_organism_parameters.wdl. For an organism without set defaults, a reference fasta file must be provided otherwise the workflow fails. Optional, Required
augur reference_genbank File The GenBank .gb file for the same reference genome used for the reference_fasta Defaults are organism-specific. Please find default values for all organisms (and for Flu - their respective genome segments and subtypes) here: https://github.com/theiagen/public_health_bioinformatics/blob/main/workflows/utilities/wf_organism_parameters.wdl. For an organism without set defaults, a reference genbank file must be provided otherwise the workflow fails. Optional, Required
augur sample_metadata_tsvs Array[File] An array of the metadata files produced in Augur_Prep_PHB Optional
augur build_name_updated String Internal component, do not modify. Used for replacing spaces with underscores _ Do Not Modify
augur_align fill_gaps Boolean If true, gaps represent missing data rather than true indels and so are replaced by N after aligning. FALSE Optional
augur_ancestral infer_ambiguous Boolean If true, infer nucleotides and ambiguous sites and replace with most likely FALSE Optional
augur_ancestral inference String Calculate joint or marginal maximum likelihood ancestral sequence states; options: "joint", "marginal" joint Optional
augur_ancestral keep_ambiguous Boolean If true, do not infer nucleotides at ambiguous (N) sides FALSE Optional
augur_ancestral keep_overhangs Boolean If true, do not infer nucleotides for gaps on either side of the alignment FALSE Optional
augur_export colors_tsv File Custom color definitions, one per line in TSV format with the following fields: TRAIT_TYPE TRAIT_VALUE HEX_CODE Optional
augur_export description_md File Markdown file with description of build and/or acknowledgements Optional
augur_export include_root_sequence Boolean Export an additional JSON containing the root sequence used to identify mutations FALSE Optional
augur_export title String Title to be displayed by Auspice Optional
augur_refine branch_length_inference String Branch length mode of timetree to use; options: "auto", "joint", "marginal", "input" auto Optional
augur_refine clock_filter_iqd Int Remove tips that deviate more than n_iqd interquartile ranges from the root-to-tip vs time regression 4 Optional
augur_refine clock_rate Float Fixed clock rate to use for time tree calculations Optional
augur_refine clock_std_dev Float Standard deviation of the fixed clock_rate estimate Optional
augur_refine coalescent String Coalescent time scale in units of inverse clock rate (float), optimize as scalar ("opt") or skyline ("skyline") Optional
augur_refine covariance Boolean If true, account for covariation when estimating rates and/or rerooting TRUE Optional
augur_refine date_confidence Boolean If true, calculate confidence intervals for node dates TRUE Optional
augur_refine date_inference String Assign internal nodes to their marginally most likely dates; options: "joint", "marginal" marginal Optional
augur_refine divergence_units String Units in which sequence divergences is exported; options: "mutations" or "mutations-per-site" mutations Optional
augur_refine gen_per_year Int Number of generations per year 50 Optional
augur_refine keep_polytomies Boolean If true, don't attempt to resolve polytomies FALSE Optional
augur_refine keep_root Boolean If true, do not reroot the tree; use it as-is (overrides anything specified by root) TRUE Optional
augur_refine precision String Precision used to determine the number of grid points; options: 0 (rough) to 3 (ultra fine) auto Optional
augur_refine root String Rooting mechanism; options: "best", "least-squares", "min_dev", "oldest", etc. Optional
augur_translate genes File A file containing a list of genes to translate (from nucleotides to amino acids) Optional
augur_tree exclude_sites File File of one-based sites to exclude for raw tree building (BED format in .bed files, DRM format in tab-delimited files, or one position per line) Optional
augur_tree method String Which method to use to build the tree; options: "fasttree", "raxml", "iqtree" iqtree Optional
augur_tree override_default_args Boolean If true, override default tree builder arguments instead of augmenting them FALSE Optional
augur_tree substitution_model String The substitution model to use; only available for iqtree. Specify "auto" to run ModelTest; model options can be found here GTR Optional
augur_tree tree_builder_args String Additional tree builder arguments either augmenting or overriding the default arguments. FastTree defaults: "-nt -nosupport". RAxML defaults: "-f d -m GTRCAT -c 25 -p 235813". IQ-TREE defaults: "-ninit 2 -n 2 -me 0.05 -nt AUTO -redo" Optional
sc2_defaults nextstrain_ncov_repo_commit String The version of the from which to draw default values for SARS-CoV-2. 23d1243127e8838a61b7e5c1a72bc419bf8c5a0d Optional
organism_parameters gene_locations_bed_file File Use to provide locations of interest where average coverage will be calculated Defaults are organism-specific. Please find default values for some organisms here: https://github.com/theiagen/public_health_bioinformatics/blob/main/workflows/utilities/wf_organism_parameters.wdl. For an organism without set defaults, an empty file is provided, "gs://theiagen-public-files/terra/theiacov-files/empty.bed", but will not be as useful as an organism specific gene locations bed file. Optional
organism_parameters genome_length_input Int Use to specify the expected genome length; provided by default for all supported organisms Defaults are organism-specific. Please find default values for all organisms (and for Flu - their respective genome segments and subtypes) here: https://github.com/theiagen/public_health_bioinformatics/blob/main/workflows/utilities/wf_organism_parameters.wdl. For an organism without set defaults, the genome length input must be provided otherwise the workflow fails. Optional, Required
organism_parameters hiv_primer_version String The version of HIV primers used. Options are https://github.com/theiagen/public_health_bioinformatics/blob/main/workflows/utilities/wf_organism_parameters.wdl#L156 and https://github.com/theiagen/public_health_bioinformatics/blob/main/workflows/utilities/wf_organism_parameters.wdl#L164. This input is ignored if provided for TheiaCoV_Illumina_SE and TheiaCoV_ClearLabs v1 Optional
organism_parameters kraken_target_organism_input String The organism whose abundance the user wants to check in their reads. This should be a proper taxonomic name recognized by the Kraken database. Defaults are organism-specific. Please find default values for all organisms here: https://github.com/theiagen/public_health_bioinformatics/blob/main/workflows/utilities/wf_organism_parameters.wdl. For an organism without set defaults, the default is "". Optional
organism_parameters nextclade_dataset_name_input String NextClade organism dataset name Defaults are organism-specific. Please find default values for all organisms (and for Flu - their respective genome segments and subtypes) here: https://github.com/theiagen/public_health_bioinformatics/blob/main/workflows/utilities/wf_organism_parameters.wdl. For an organism without set defaults, the default is "NA". Optional
organism_parameters nextclade_dataset_tag_input String NextClade organism dataset tag Defaults are organism-specific. Please find default values for all organisms (and for Flu - their respective genome segments and subtypes) here: https://github.com/theiagen/public_health_bioinformatics/blob/main/workflows/utilities/wf_organism_parameters.wdl. For an organism without set defaults, the default is "NA". Optional
organism_parameters pangolin_docker_image String The Docker container to use for the task us-docker.pkg.dev/general-theiagen/staphb/pangolin:4.3.1-pdata-1.26 Optional
organism_parameters primer_bed_file File The bed file containing the primers used when sequencing was performed Defaults are organism-specific. Please find default values for all organisms here: https://github.com/theiagen/public_health_bioinformatics/blob/main/workflows/utilities/wf_organism_parameters.wdl. For an organism without set defaults, an empty primer bed file is provided, "gs://theiagen-public-files/terra/theiacov-files/empty.bed", but will not be as useful as an organism specific primer bed file. Optional
organism_parameters reference_gff_file File Reference GFF file for the organism being analyzed Defaults are organism-specific. Please find default values for all organisms here: https://github.com/theiagen/public_health_bioinformatics/blob/main/workflows/utilities/wf_organism_parameters.wdl. For an organism without set defaults, an empty gff file is provided, "gs://theiagen-public-files/terra/theiacov-files/empty.gff3", but will not be as useful as an organism specific gff file. Optional
organism_parameters vadr_max_length Int Maximum length for the fasta-trim-terminal-ambigs.pl VADR script Defaults are organism-specific. Please find default values for all organisms here: https://github.com/theiagen/public_health_bioinformatics/blob/main/workflows/utilities/wf_organism_parameters.wdl. For an organism without set defaults, the default is 0. Optional
organism_parameters vadr_mem Int Memory, in GB, allocated to this task 32 (RSV-A and RSV-B) and 8 (all other TheiaCoV organisms)
organism_parameters vadr_options String Options for the v-annotate.pl VADR script Defaults are organism-specific. Please find default values for all organisms here: https://github.com/theiagen/public_health_bioinformatics/blob/main/workflows/utilities/wf_organism_parameters.wdl. For an organism without set defaults, the default is "NA". Optional
organism_parameters vadr_skip_length Int Minimum assembly length (unambiguous) to run VADR Defaults are organism-specific. Please find default values for all organisms here: https://github.com/theiagen/public_health_bioinformatics/blob/main/workflows/utilities/wf_organism_parameters.wdl. For an organism without set defaults, the default is 0. Optional
mutation_context cpu Int CPUs requested for the mutation_context task that is specific to Mpox. 1 Optional
mutation_context disk_size Int Disk size in GB requested for the mutation_context task that is specific to Mpox. 50 Optional
mutation_context docker String Docker image used for the mutation_context task that is specific to Mpox. Do not modify. us-docker.pkg.dev/general-theiagen/theiagen/nextstrain-mpox-mutation-context:2024-06-27 Do Not Modify, Optional
mutation_context memory Int Memory size in GB requested for the mutation_context task that is specific to Mpox. 4 Optional
Workflow Tasks
Augur Workflow Tasks

The Augur_PHB workflow uses the inputs to generate a phylogenetic tree in JSON format that is compatible with phylogenetic tree visualization software.

In Augur_PHB, the tasks below are called. For the Augur subcommands, please view the Nextstrain Augur documentation for more details and explanations.

  1. cat_files - concatenate all of the input fasta files together
  2. sc2_defaults - if organism is SARS-CoV-2, establish default parameters
  3. flu_defaults - if organism is Flu, establish default parameters
  4. filter_sequences_by_length - remove any sequences that do not meet the quality threshold set by min_num_unambig
  5. tsv_join - merge the metadata files
  6. fasta_to_ids - extract a list of remaining sequences so we know which ones were dropped
  7. augur_align - perform MAFFT alignment on the sequences
  8. augur_tree - create a distance tree
  9. augur_refine - create a timetree
  10. augur_ancestral - infer ancestral sequences
  11. augur_translate - translate gene regions from nucleotides to amino acids
  12. mutation_context - if organism is MPXV, calculates the mutation fraction of G->A or C->T changes
  13. augur_clades - if clade information is provided, assign clades to nodes based on amino-acid or nucleotide signatures
  14. augur_export - export all the results in a JSON file suitable for Auspice visualization
  15. snp_dists - create a SNP matrix from the alignment
  16. reorder_matrix - reorder the SNP matrix to match the distance tree

Augur Outputs

Diversity dependent

Note that the node & branch coloring by clade or lineage assignment might be dependent on the diversity of your input dataset. This is because the clade assignment is done using the ancestrally reconstructed amino acid or nucleotide changes at the tree nodes rather than a direct sequence-to-reference mutation comparison. You may notice this happening when you get clade/lineage assignments from NextClade when running TheiaCoV workflows, but no clade/lineage assignment on the Augur Auspice tree.

To get around this issue, you can upload the Augur output file merged-metadata.tsv to Auspice that includes the correct clade/lineage assignments to allow for coloring by Clade.

Flu clade assignments

Note that for flu, the clade assignment is usually mostly done for the more recent seasonal influenza viruses. Older strains may get an "unassigned" designation for clades. Therefore, it is important to counter check with the NextClade results from TheiaCoV if the lack of clade assignment is due to analyzing older sequences or sequence related.

The auspice_input_json is intended to be uploaded to Auspice to view the phylogenetic tree. This provides a visualization of the genetic relationships between your set of samples. The metadata_merged output can also be uploaded to add context to the phylogenetic visualization. The combined_assemblies output can be uploaded to UShER to view the samples on a global tree of representative sequences from the public repositories.

The Nextstrain team hosts documentation surrounding the Augur workflow → Auspice visualization here, which details the various components of the Auspice interface: How data is exported by Augur for visualisation in Auspice.

Variable Type Description
aligned_fastas File A FASTA file of the aligned genomes
augur_fasttree_version String The fasttree version used, blank if other tree method used
augur_iqtree_model_used String The iqtree model used during augur tree, blank if iqtree not used
augur_iqtree_version String The iqtree version used during augur tree (defualt), blank if other tree method used
augur_mafft_version String The mafft version used in augur align
augur_phb_analysis_date String The date the analysis was run
augur_phb_version String The version of the Public Health Bioinformatics (PHB) repository used
augur_raxml_version String The version of raxml used during augur tree, blank if other tree method used
augur_version String Version of Augur used
auspice_input_json File JSON file used as input to Auspice
combined_assemblies File Concatenated FASTA file containing all samples
distance_tree File The distance tree created in Newick (.nwk) format
keep_list File A list of samples included in the phylogenetic tree
metadata_merged File Tab-delimited text file of the merged augur_metadata input files from all samples
snp_matrix File The SNP distance matrix for all samples used in the phylogenetic tree
time_tree File The time tree created in Newick (.nwk) format
traits_json File A JSON file containing sample traits

Mpox-specific Auspice Output JSON

If you are building a tree for Mpox samples and set the optional input parameter organism to "mpox" , an additional step will be carried out in the Augur_PHB workflow. This additional step will calculate the mutation fraction of G→A or C→T changes. These mutations have been shown to be a characteristic of APOBEC3-type editing, which indicate adaptation of the virus to circulation among humans as was observed with the 2022 clade IIb outbreak, and more recently (2024) with the clade Ib outbreak in South Kivu, Democratic Republic of the Congo.

When visualizing the output auspice_input_json file, there will be 2 new choices in the drop-down menu for "Color By":

  • G→A or C→T fraction
  • NGA/TCN context of G→A or C→T mutations.

An example Mpox tree with these "Color By" options can be viewed here: https://nextstrain.org/mpox/clade-IIb?c=GA_CT_fraction

References

When publishing work using the Augur_PHB workflow, please reference the following:

Nextstrain: Hadfield J, Megill C, Bell SM, Huddleston J, Potter B, Callender C, Sagulenko P, Bedford T, Neher RA. Nextstrain: real-time tracking of pathogen evolution. Bioinformatics. 2018 Dec 1;34(23):4121-3.

When publishing work using inferences from UShER, please reference:

UShER: Turakhia Y, Thornlow B, Hinrichs AS, De Maio N, Gozashti L, Lanfear R, Haussler D, Corbett-Detig R. Ultrafast Sample placement on Existing tRees (UShER) enables real-time phylogenetics for the SARS-CoV-2 pandemic. Nature Genetics. 2021 Jun;53(6):809-16.