EXCON (v2.2.0)

A Nextflow pipeline for gene family EXpansion and CONtraction analysis across multiple species using CAFE5.

Given a set of genome assemblies and annotations, EXCON builds orthogroups with OrthoFinder, fits and compares multiple CAFE models to identify gene families evolving at significantly different rates, and automatically selects the best-fitting model for downstream analysis. Optionally, GO enrichment analysis can be run on expanded and contracted gene families, and on genes grouped by chromosome.

It works with any set of species that have a genome (fasta) and annotation (gff) file. (minimum of 5 species ideally up to around 30). Maximum 100 species (normally).

GO annotation can be run in two ways:

• Run EggNOG-mapper (--run_eggnog): assigns GO terms from scratch using the EggNOG database (~45 GB). Provide --eggnog_data_dir to reuse a pre-downloaded copy and avoid re-downloading on every run.
• Supply your own GO files (--predownloaded_gofiles): if you already have gene-to-GO mappings, point to a directory of {species_id}.go.txt files and skip EggNOG entirely (see Parameters below).

Overview

The general pipeline logic is as follows:

• Downloads genome and annotation files from NCBI [NCBIGENOMEDOWNLOAD], or you provide your own.
• Unzips the files, if necessary [GUNZIP]
• Sanitises GFF annotations and extracts longest isoform [AGAT_SPKEEPLONGESTISOFORM].
• Gets the protein sequences [GFFREAD].
• Renames the genes to gene name (as some will be isoform name) RENAME_FASTA.
• Finds orthologous genes across species [ORTHOFINDER_CAFE], or accepts a pre-computed tree and orthogroups to skip this step (see --input_tree / --input_orthogroups).
• Rescales OrthoFinder branch lengths and converts to an ultrametric tree for CAFE [RESCALE_TREE], [CAFE_PREP].
• Prepares gene count input, estimates the error model, and builds an ultrametric tree [CAFE_PREP].
• Runs CAFE5 with k=1 to k=cafe_max_k (default 6) rate categories in parallel [CAFE_RUN_K].
• Compares all k runs by AIC and selects the best k [CAFE_SELECT_K].
• Re-runs CAFE5 at the best k with Poisson birth-death (-p) [CAFE_RUN_BEST].
• Compares uniform vs Poisson model at the best k by likelihood, selects the winner [CAFE_MODEL_COMPARE].
• Plots gene family expansions and contractions for the best model [CAFE_PLOT].

Optional — GO enrichment (--run_eggnog or --predownloaded_gofiles)

• Optionally downloads the EggNOG-mapper database [EGGNOG_DOWNLOAD].
• Optionally assigns GO terms to genes using EggNOG-mapper [EGGNOGMAPPER], or reads GO terms from user-supplied files.
• Optionally prepares GO gene lists from the best CAFE model results [CAFE_GO_PREP].
• Optionally runs GO enrichment in parallel, one job per species/node and direction (expansion/contraction) [CAFE_GO_RUN].

Optional — chromosome GO enrichment (--chromo_go, requires GO annotation)

• Optionally plots GO enrichment of genes by chromosome [CHROMO_GO].
• Optionally summarizes GO enrichment by chromosome [SUMMARIZE_CHROMO_GO].

Optional — genome quality statistics (--stats)

• Optionally describes genome assembly and annotation:
  • [BUSCO_BUSCO]: Completeness of the genome compared to expected gene set.
  • [QUAST]: Assembly contiguity statistics (N50 etc).
  • [AGAT_SPSTATISTICS]: Gene, exon, and intron statistics.

Installation

Nextflow pipelines require a few prerequisites. There is further documentation on the nf-core webpage here, about how to install Nextflow.

Prerequisites

• Docker or Singularity.
• Java and openJDK >= 8 (Please Note: When installing Java versions are 1.VERSION so Java 8 is Java 1.8).
• Nextflow >= v25.10.0.
• When running nextflow with this pipeline, ideally run NXF_VER=25.10.0 beforehand, to ensure functionality on this version.

Install

To install the pipeline please use the following commands but replace VERSION with a release.

wget https://github.com/Eco-Flow/excon/archive/refs/tags/VERSION.tar.gz -O - | tar -xvf -

or

curl -L https://github.com/Eco-Flow/excon/archive/refs/tags/VERSION.tar.gz --output - | tar -xvf -

This will produce a directory in the current directory called excon-VERSION which contains the pipeline.

Inputs

Required

• --input /path/to/csv/file - A singular csv file as input in one of the two formats stated below.

This csv can take 2 forms:

• A 2 field csv where each row is a unique species name followed by a Refseq genome reference ID (NOT a Genbank reference ID) i.e. data/input_small-s3.csv. The pipeline will download the relevant genome fasta file and annotation gff3 (or gff augustus) file.
• A 3 field csv where each row is a unique species name, followed by an absolute path to a genome fasta file, followed by an absolute path to an annotation gff3 (or gff augustus) file. Input can be gzipped (.gz) or not.

Please Note: The genome has to be chromosome level not contig level.

2 fields (Name,Refseq_ID):

Drosophila_yakuba,GCF_016746365.2
Drosophila_simulans,GCF_016746395.2
Drosophila_santomea,GCF_016746245.2

3 fields (Name,genome.fna,annotation.gff):

Drosophila_yakuba,data/Drosophila_yakuba/genome.fna.gz,data/Drosophila_yakuba/genomic.gff.gz
Drosophila_simulans,data/Drosophila_simulans/genome.fna.gz,data/Drosophila_simulans/genomic.gff.gz
Drosophila_santomea,data/Drosophila_santomea/genome.fna.gz,data/Drosophila_santomea/genomic.gff.gz

Note: Genomes should be chromosome-level, not contig-level. RefSeq IDs must be used (not GenBank IDs).

Parameters

Core options

Parameter	Description	Default
--input	Path to input CSV file	Required unless using --input_tree + --input_orthogroups without --run_eggnog or --stats
--outdir	Output directory	results
--groups	NCBI taxonomy group for genome download (e.g. insects, bacteria)	insects
--help	Display help message	false
--custom_config	Path to a custom Nextflow config file	null

Quality statistics (optional)

Parameter	Description	Default
--stats	Run BUSCO, QUAST and AGAT statistics on genomes	null
--busco_lineage	BUSCO lineage database (e.g. insecta_odb10)	null
--busco_mode	BUSCO mode (genome, proteins, transcriptome)	null
--busco_lineages_path	Path to local BUSCO lineage databases	null
--busco_config	Path to BUSCO config file	null

OrthoFinder options (optional)

Parameter	Description	Default
--orthofinder_v2	Use OrthoFinder v2.5.5 instead of v3.1.4. v2 produces Hierarchical Orthogroups (N0.tsv) which have lower copy-number variance and are better suited to CAFE5. v3 produces flat orthogroups (Orthogroups.tsv). Recommended for large datasets (>30 species) or when CAFE5 fails to converge.	false
--orthofinder_method	Gene tree inference method: msa or dendroblast	msa
--orthofinder_search	Sequence search program: diamond, blast, or mmseqs2	diamond
--orthofinder_msa_prog	MSA program (requires --orthofinder_method msa): mafft or muscle	mafft
--orthofinder_tree	Tree inference method (requires --orthofinder_method msa): fasttree, raxml, raxml-ng, or iqtree	fasttree

Note: -A and -T are only valid when -M msa is set. If you set --orthofinder_msa_prog or --orthofinder_tree without --orthofinder_method msa, OrthoFinder will error.

CAFE gene family evolution

Parameter	Description	Default
--skip_cafe	Skip CAFE analysis	null
--cafe_max_k	Maximum number of k rate categories to test (runs k=1 through k=N in parallel)	6
--cafe_max_differential	Maximum gene count differential for CAFE filtering on retry	50
--tree_scale_factor	Factor to multiply all OrthoFinder branch lengths by before chronos() converts the tree to a time tree for CAFE5. Lower values can cause numerical issues.	1000
--input_tree	Path to a pre-computed rooted species tree (Newick format) — skips OrthoFinder when used with --input_orthogroups	null
--input_orthogroups	Path to a pre-computed Orthogroups.tsv from a previous OrthoFinder run — skips OrthoFinder when used with --input_tree	null

Skipping OrthoFinder: OrthoFinder is the slowest step in the pipeline. If you have already run it (the results are in results/orthofinder_cafe/ortho_cafe/), you can reuse the outputs. The orthogroups file to pass depends on which version of OrthoFinder was used:

OrthoFinder v2 (--orthofinder_v2) — use N0.tsv (Hierarchical Orthogroups):

--input_tree results/orthofinder_cafe/ortho_cafe/Species_Tree/SpeciesTree_rooted_node_labels.txt \
--input_orthogroups results/orthofinder_cafe/ortho_cafe/Phylogenetic_Hierarchical_Orthogroups/N0.tsv

OrthoFinder v3 (default) — use Orthogroups.tsv (flat orthogroups):

--input_tree results/orthofinder_cafe/ortho_cafe/Species_Tree/SpeciesTree_rooted_node_labels.txt \
--input_orthogroups results/orthofinder_cafe/ortho_cafe/Orthogroups/Orthogroups.tsv

Both --input_tree and --input_orthogroups must be supplied together. If either is omitted, OrthoFinder runs normally.

Note on CAFE model selection: The pipeline runs CAFE5 with k=1 through k=cafe_max_k (default 6) rate categories in parallel, then selects the best k by AIC. It then re-runs the best k with the Poisson birth-death option (-p) and picks the final model by likelihood. Model selection results (AIC table across k values) are in results/cafe/model_comparison/. If scores cannot be parsed (e.g. on very small datasets), the pipeline defaults to the uniform model.

GO annotation (optional)

GO enrichment requires gene-to-GO mappings. Choose one of the two approaches below — they are mutually exclusive, and --run_eggnog takes priority if both are set.

Option A — Run EggNOG-mapper

Parameter	Description	Default
--run_eggnog	Run EggNOG-mapper to assign GO terms	false
--eggnog_data_dir	Path to pre-downloaded EggNOG database directory	null (downloads ~45 GB automatically)
--eggnog_target_taxa	Restrict annotations to orthologs from this taxon and its descendants (NCBI taxon ID)	null
--eggnog_tax_scope	Taxonomic scope for orthologous group assignment (e.g. 50557 for Insecta)	null
--eggnog_evalue	Maximum e-value threshold for sequence matches	null
--eggnog_score	Minimum bitscore threshold for matches	null
--eggnog_pident	Minimum percent identity (%)	null
--eggnog_query_cover	Minimum query coverage (%)	null
--eggnog_subject_cover	Minimum subject coverage (%)	null

Note: The EggNOG database is ~45 GB. We strongly recommend downloading it once and passing --eggnog_data_dir /path/to/eggnog_data to avoid re-downloading on every run.

Option B — Supply your own GO files

Parameter	Description	Default
--predownloaded_gofiles	Path to a directory of pre-computed gene-to-GO mapping files	null

The directory must contain one file per species, named {species_id}.go.txt where species_id matches the species names in your input CSV. Each file is a two-column, tab-separated file with one gene–GO pair per line:

geneA    GO:0006412
geneA    GO:0008150
geneB    GO:0003674

This lets you skip EggNOG entirely if you already have GO annotations (e.g. from a previous run, a public database, or another annotation tool).

GO enrichment analysis (optional, requires GO annotation)

Parameter	Description	Default
--chromo_go	Run GO enrichment analysis by chromosome	null
--go_cutoff	P-value cutoff for GO enrichment	0.05
--go_type	GO test type (e.g. none)	none
--go_max_plot	Maximum number of GO terms to plot	10
--go_algo	topGO algorithm and statistic (classic_fisher, weight01_t, elim_ks, weight_ks)	classic_fisher

Resource limits

Parameter	Description	Default
--max_memory	Maximum memory per job	128.GB
--max_cpus	Maximum CPUs per job	16
--max_time	Maximum runtime per job	240.h

Profiles

Profile	Description
docker	Run with Docker containers
singularity	Run with Singularity containers
conda	Run with Conda environments
test_bacteria	Test run with small bacterial genomes
test_small	Test run with small insect genomes

Profiles

This pipeline is designed to run in various modes that can be supplied as a comma separated list i.e. -profile profile1,profile2.

Container Profiles

Please select one of the following profiles when running the pipeline.

• docker - This profile uses the container software Docker when running the pipeline. This container software requires root permissions so is used when running on cloud infrastructure or your local machine (depending on permissions). Please Note: You must have Docker installed to use this profile.
• singularity - This profile uses the container software Singularity when running the pipeline. This container software does not require root permissions so is used when running on on-premise HPCs or you local machine (depending on permissions). Please Note: You must have Singularity installed to use this profile.
• apptainer - This profile uses the container software Apptainer when running the pipeline. This container software does not require root permissions so is used when running on on-premise HPCs or you local machine (depending on permissions). Please Note: You must have Apptainer installed to use this profile.

Optional Profiles

• local - This profile is used if you are running the pipeline on your local machine.
• aws_batch - This profile is used if you are running the pipeline on AWS utilising the AWS Batch functionality. Please Note: You must use the Docker profile with with AWS Batch.
• test_small - This profile is used if you want to test running the pipeline on your infrastructure, running from predownloaded go files. Please Note: You do not provide any input parameters if this profile is selected but you still provide a container profile.
• test_biomart - This profile is used if you want to test running the pipeline on your infrastructure, running from the biomart input. Please Note: You do not provide any input parameters if this profile is selected but you still provide a container profile.

Custom Configuration

If you want to run this pipeline on your institute's on-premise HPC or specific cloud infrastructure then please contact us and we will help you build and test a custom config file. This config file will be published to our configs repository.

Running the Pipeline

Please note: The -resume flag uses previously cached successful runs of the pipeline.

1. Example run the full test example data:

NXF_VER=25.04.8 # Is latest it is tested on
nextflow run main.nf -resume -profile docker,test_small

Settings in test_small: input = "input_small-s3.csv"

For the fastest run use: nextflow run main.nf -resume -profile docker,test_bacteria

2. To run on your own data (minimal run), cafe only.

# NXF_VER=25.04.8  
nextflow run main.nf -resume -profile docker --input data/input_small-s3.csv

3. To run with cafe and GO analysis

# NXF_VER=25.04.8
nextflow run main.nf -resume -profile docker --input data/input_small-s3.csv --chromo_go --go_type bonferoni --stats --run_eggnog --eggnog_data_dir /path/to/eggnogdb 

4. To run with CAFE and GO analysis using your own pre-computed GO files (skips EggNOG):

# NXF_VER=25.04.8
nextflow run main.nf -resume -profile docker --input data/input_small-s3.csv --predownloaded_gofiles /path/to/go_files/

The go_files/ directory should contain one {species_id}.go.txt per species (tab-separated gene_idGO:term, one pair per line).

5. To reuse a previous OrthoFinder run (skips the slow OrthoFinder step). Or to use tree/table from another source use:

OrthoFinder v3 (default):

# NXF_VER=25.04.8
nextflow run main.nf -resume -profile docker \
  --input data/input_small-s3.csv \
  --input_tree results/orthofinder_cafe/ortho_cafe/Species_Tree/SpeciesTree_rooted_node_labels.txt \
  --input_orthogroups results/orthofinder_cafe/ortho_cafe/Orthogroups/Orthogroups.tsv

OrthoFinder v2 (--orthofinder_v2):

# NXF_VER=25.04.8
nextflow run main.nf -resume -profile docker \
  --input data/input_small-s3.csv \
  --input_tree results/orthofinder_cafe/ortho_cafe/Species_Tree/SpeciesTree_rooted_node_labels.txt \
  --input_orthogroups results/orthofinder_cafe/ortho_cafe/Phylogenetic_Hierarchical_Orthogroups/N0.tsv

Output Structure

For a detailed description of outputs with example figures, see the Output Guide.

results/
├── cafe/
│   ├── base/                        # CAFE_PREP outputs
│   │   ├── hog_gene_counts.tsv      # Filtered gene count input to CAFE
│   │   ├── hog_filtering_report.tsv # Filtering report (only present if retry triggered)
│   │   └── SpeciesTree_rooted_ultra.txt  # Ultrametric tree used by CAFE5
│   ├── large_families/              # CAFE run on high-differential families (retry path only)
│   └── model_comparison/
│       ├── cafe_model_comparison.tsv # Uniform vs Poisson comparison at best k
│       └── best_model.txt            # "uniform" or "poisson"
├── cafe_plot/
│   └── cafe_plotter/                # Expansion/contraction plots for best model
├── cafe_go/                         # GO enrichment (one job per species/node x direction)
│   ├── CAFE_summary.txt             # Summary of expansions/contractions per branch
│   ├── *_TopGo_results_ALL.tab      # TopGO results per target
│   ├── TopGO_barplot_*.pdf          # Bar chart per target (ggplot2, full GO names)
│   ├── TopGO_dotplot_*.pdf          # Dot plot per target (fold enrichment x significance)
│   ├── TopGO_Pval_barplot_*.pdf     # Legacy barplots (base R)
│   ├── Go_summary_pos.pdf           # Summary plot across all expansions
│   ├── Go_summary_neg.pdf           # Summary plot across all contractions
│   ├── Go_summary_pos_noNode.pdf    # As above, terminal branches only
│   └── Go_summary_neg_noNode.pdf
├── chromo_go/                       # [optional] GO enrichment by chromosome
│   ├── *.pdf                        # Per-chromosome GO plots
│   └── summary/                     # Summarized results across chromosomes
├── eggnogmapper/
│   └── go_files/                    # Per-species GO annotation files
├── gffread/
│   └── *.fasta                      # Protein sequences per species
├── ncbigenomedownload/
│   ├── *.fna.gz                     # Downloaded genome assemblies
│   └── *.gff.gz                     # Downloaded annotations
├── orthofinder_cafe/
│   └── ortho_cafe/                  # OrthoFinder results including species tree
├── busco/                           # [optional, --stats] BUSCO completeness results
├── agat/                            # [optional, --stats] AGAT annotation statistics
├── quast/                           # [optional, --stats] Assembly contiguity statistics
└── pipeline_info/
    ├── execution_report_*.html      # Nextflow execution report
    ├── execution_timeline_*.html    # Per-process timeline
    ├── execution_trace_*.txt        # Per-task resource usage
    ├── pipeline_dag_*.html          # Pipeline DAG diagram
    └── software_versions.yml        # Versions of all tools used

Citation

This pipeline is published on Workflowhub using the nf-core template. If you use this pipeline in you work, the following citations are essential:

excon: Wyatt, C. (2026). Gene EXpansion and CONtraction analysis pipeline. WorkflowHub. https://doi.org/10.48546/WORKFLOWHUB.WORKFLOW.2141.4

nf-core: Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen. Nat Biotechnol. 2020 Feb 13. doi: 10.1038/s41587-020-0439-x.

If you used any of these tools within the pipeline, you must also cite:

CAFE: Fábio K Mendes, Dan Vanderpool, Ben Fulton, Matthew W Hahn, CAFE 5 models variation in evolutionary rates among gene families, Bioinformatics, 2020; btaa1022, https://doi.org/10.1093/bioinformatics/btaa1022

Orthofinder: Emms, D.M. and Kelly, S. (2019) OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biology 20:238

AGAT: Dainat J. 2022. Another Gtf/Gff Analysis Toolkit (AGAT): Resolve interoperability issues and accomplish more with your annotations. Plant and Animal Genome XXIX Conference. https://github.com/NBISweden/AGAT.

eggNOG-mapper (if used): Carlos P Cantalapiedra, Ana Hernández-Plaza, Ivica Letunic, Peer Bork, Jaime Huerta-Cepas, eggNOG-mapper v2: Functional Annotation, Orthology Assignments, and Domain Prediction at the Metagenomic Scale, Molecular Biology and Evolution, Volume 38, Issue 12, December 2021, Pages 5825–5829, https://doi.org/10.1093/molbev/msab293

A full list of tools and their versions are found in the software_versions.yml in the results/pipeline_info. So ensure to look here for any additional tools you need to cite.

For the --stats module, you would need to cite BUSCO, Quast, AGAT

Contact Us

If you need any support do not hesitate to contact us at any of:

c.wyatt [at] ucl.ac.uk

ecoflow.ucl [at] gmail.com