PHYling Phylogenomics — Nextflow Pipeline

DSL2 Nextflow pipeline for multi-locus ML phylogenomics using BUSCO single-copy orthologs. Wraps PHYling, PhyKIT, ModelTest-NG, IQ-TREE 3, and RAxML-NG into two named workflows:

Workflow	--mode	Input	Data type	RAxML-NG bootstraps
CDS_TREE	cds_tree	.fa per taxon	DNA / CDS	500
PROTEIN_TREE	protein_tree	.fa.gz per taxon	Amino acid	100

Both workflows run AIC and BIC partition schemes through IQ-TREE and RAxML-NG in parallel, producing four independently-supported trees per markerset.

---

Pipeline steps

INPUT: one sequence file per taxon
         ↓ (per markerset, in parallel)
  phyling align       BUSCO marker search + MSA
  phyling filter      Remove loci below occupancy threshold (default 80% of taxa)
  phyling tree        Per-gene FastTree (exploratory gene trees)
         ↓
  phykit create_concat   Concatenate filtered MSAs; write partition file
  sed AUTO→DNA/PROT       Fix data-type for modeltest-ng
         ↓
  modeltest-ng        Partitioned model selection → AIC and BIC partition files
         ↓          (AIC and BIC run in parallel from here)
  iqtree3 MF+MERGE    Partition merging + ML tree
  iqtree3 UFBoot      Bootstrap (-B 1000 --alrt 1000 --bnni --wbtl)
  raxml-ng --parse    Produce binary alignment; extract thread recommendation
  raxml-ng --all      ML search + bootstraps

---

Quick start

Run directly from GitHub (no clone needed)

nextflow run stajichlab/phyling-phylogenomics \
  -profile slurm,ucr_hpcc \
  --mode protein_tree \
  --input /path/to/pep \
  --prefix my_project \
  --markerset fungi_odb12,mucoromycota_odb12 \
  --outdir results/pep

Clone and run locally

git clone https://github.com/stajichlab/phyling-phylogenomics
cd phyling-phylogenomics

nextflow run main.nf \
  -profile local \
  --mode cds_tree \
  --input /path/to/cds \
  --prefix my_project \
  --markerset fungi_odb12 \
  --outdir results/test

---

Requirements

Option A — pixi (recommended)

# Install pixi if not already present
curl -fsSL https://pixi.sh/install.sh | bash

# Install all pipeline tools
pixi install

Tools installed: nextflow, modeltest-ng, iqtree ≥2.3 (provides iqtree3), raxml-ng from bioconda; phykit and phyling from PyPI.

Option B — environment modules (HPC)

Module names expected by -profile slurm: phyling, phykit, modeltest-ng, iqtree, raxml-ng

---

Execution profiles

Profiles can be combined with commas: -profile slurm,ucr_hpcc

Profile	Executor	Environment	Use when
slurm	SLURM	module load per tool	HPC with environment modules
ucr_hpcc	—	—	UCR HPCC queue names (combine with slurm or pixi_slurm)
pixi	local	pixi env	local workstation with pixi
pixi_slurm	SLURM	pixi env	HPC without modules; combine with a site profile
local	local	tools in PATH	testing

Using on a different cluster

Queue names are cluster-specific. Create a minimal site config and pass it with -c:

// my_cluster.config
process {
    withName: 'PHYLING_ALIGN'    { queue = 'bigmem' }
    withName: 'MODELTEST_NG|IQTREE_MF|IQTREE_BOOTSTRAP|RAXMLNG_ALL' { queue = 'compute' }
    withName: 'PHYLING_FILTER|PHYLING_TREE|PHYKIT_CONCAT|RAXMLNG_PARSE' { queue = 'short' }
}

nextflow run stajichlab/phyling-phylogenomics \
  -profile slurm -c my_cluster.config \
  --mode protein_tree --input pep/ --prefix my_project \
  --markerset fungi_odb12

---

All parameters

Parameter	Default	Description
--mode	protein_tree	Workflow: cds_tree or protein_tree
--input	(required)	Directory of .fa (CDS) or .fa.gz (protein) files
--prefix	(required)	Base name for output files, e.g. my_project_v1
--markerset	fungi_odb12,mucoromycota_odb12	Comma-separated BUSCO lineage names
--outdir	results	Directory for published outputs
--publish_mode	copy	publishDir mode: copy, link, or symlink
--top_n_to_keep	80	Number of top markers to retain (TOP_N_TOVERR in phyling filter -n)
--rcluster	10	IQ-TREE partition merging aggressiveness
--bs_count	1000	IQ-TREE UFBoot replicates (-B)
--alrt_count	1000	IQ-TREE SH-aLRT replicates (--alrt)
--pars_trees	10	RAxML-NG parsimony starting trees
--bs_trees_cds	500	RAxML-NG bootstrap replicates for CDS mode
--bs_trees_pep	100	RAxML-NG bootstrap replicates for protein mode

Common BUSCO markersets

Markerset	Scope
fungi_odb10 / fungi_odb12	All Fungi (ODB10 / ODB12)
mucoromycota_odb10 / mucoromycota_odb12	Mucoromycota-specific
basidiomycota_odb10	Basidiomycota
ascomycota_odb10	Ascomycota

Multiple markersets run as independent parallel branches.

---

Example commands

Protein tree — SLURM + modules (UCR HPCC)

nextflow run stajichlab/phyling-phylogenomics \
  -profile slurm,ucr_hpcc \
  --mode protein_tree \
  --input pep/ \
  --prefix my_project_v1 \
  --markerset fungi_odb12,mucoromycota_odb12 \
  --outdir results/pep

CDS tree — SLURM + modules (UCR HPCC)

nextflow run stajichlab/phyling-phylogenomics \
  -profile slurm,ucr_hpcc \
  --mode cds_tree \
  --input cds/ \
  --prefix my_project_v1 \
  --markerset fungi_odb12,mucoromycota_odb12 \
  --outdir results/cds

Resume an interrupted run

nextflow run stajichlab/phyling-phylogenomics -resume \
  -profile slurm,ucr_hpcc \
  --mode protein_tree \
  --input pep/ \
  --prefix my_project_v1 \
  --markerset fungi_odb12,mucoromycota_odb12 \
  --outdir results/pep

---

Output structure

results/
└── {cds|pep}/
    ├── align/{markerset}/          phyling align output
    ├── filter/{markerset}/         filtered .mfa files (one per locus)
    ├── tree/{markerset}/           per-gene FastTree trees
    ├── buildtree/{markerset}/
    │   ├── *.fa                    concatenated alignment
    │   ├── *.partition             partition file (data-type fixed)
    │   ├── *.part.aic / *.part.bic ModelTest-NG AIC and BIC schemes
    │   ├── *.aic.bs.treefile       IQ-TREE AIC bootstrap consensus
    │   ├── *.bic.bs.treefile       IQ-TREE BIC bootstrap consensus
    │   ├── *.aic.raxml.support     RAxML-NG AIC support tree
    │   └── *.bic.raxml.support     RAxML-NG BIC support tree
    └── pipeline_info/
        ├── timeline.html
        ├── report.html
        └── dag.svg

Tree files (.treefile, .raxml.support) are Newick format — open in FigTree, iTOL, or ggtree.

---

Interpreting support values

Measure	Flag	Strong support threshold
UFBoot2 (ultrafast bootstrap)	-B 1000	≥ 95
SH-aLRT	--alrt 1000	≥ 80
RAxML-NG bootstrap	--bs-trees	≥ 70

UFBoot values are systematically higher than standard bootstrap — do not compare directly. A clade is robustly supported when all three exceed their thresholds.

---

Repository structure

phyling-phylogenomics/
├── main.nf                  entry point
├── nextflow.config          params + profiles
├── pixi.toml                conda/PyPI environment
├── conf/
│   ├── base.config          per-process resource labels
│   └── ucr_hpcc.config      UCR HPCC queue assignments (site example)
├── workflows/
│   ├── cds_tree.nf
│   └── protein_tree.nf
└── modules/local/
    ├── phyling/{align,filter,tree}/
    ├── phykit/concat/
    ├── modeltestng/
    ├── iqtree/{mf,bootstrap}/
    └── raxmlng/{parse,all}/

---

Citation

If you use this pipeline, please cite the underlying tools:

• PHYling: Tsai et al. (2026) G3 jkag062 https://doi.org/10.1093/g3journal/jkag062
• PhyKIT: Steenwyk et al. (2021) Bioinformatics 37:2325–2328 https://doi.org/10.1093/bioinformatics/btab096
• ModelTest-NG: Darriba et al. (2020) Mol Biol Evol 37:291–294
• IQ-TREE 3: Minh et al. (2020) Mol Biol Evol 37:1530–1534
• RAxML-NG: Kozlov et al. (2019) Bioinformatics 35:4453–4455
• BUSCO: Manni et al. (2021) Mol Biol Evol 38:4647–4654