ROADIES: Reference-free Orthology-free Annotation-free DIscordance aware Estimation of Species tree

Quick Links

ROADIES is a fully automated pipeline to infer species trees starting from raw genome assemblies. It incorporates a unique strategy of randomly sampling segments of the input genomes to generate gene trees. This eliminates the need for predefining a set of loci, limiting the analyses to a fixed number of genes, and performing the cumbersome gene annotation and/or whole genome alignment steps. ROADIES also eliminates the need to infer orthology by leveraging existing discordance-aware methods that allow multicopy genes.

References:

Anshu Guptaa, Siavash Mirarabb and Yatish Turakhiab
Accurate, scalable, and fully automated inference of species trees from raw genome assemblies using ROADIES PNAS, 2025, vol.122(19) e2500553122 https://doi.org/10.1073/pnas.2500553122.

Documentation

ROADIES Github page

Important Notes

Module Name: roadies (see the modules page for more information)
Unusual environment variables set
- ROADIES_HOME sRNAflow installation directory
- ROADIES_BIN sRNAflow executable directory
- ROADIES_SRC sRNAflow source code directory

Interactive job

Interactive jobs should be used for debugging, graphics, or applications that cannot be run as batch jobs.

Allocate an interactive session and run the program. Sample session:

[user@biowulf]$ sinteractive --mem=20g -c 16 --gres=lscratch:50
[user@cn4278 ~]$ module load roadies    
[+] Loading singularity  4.2.2  on cn0094
[+] Loading roadies 0.1.10  ...a
[user@cn4278 ~]$ wget https://github.com/TurakhiaLab/ROADIES/archive/refs/tags/v0.1.10.tar.gz
[user@cn4278 ~]$ tar -zxf v0.1.10.tar.gz && rm -f v0.1.10.tar.gz && cd ROADIES-0.1.10
[user@cn4278 ~]$ chmod +x ./workflow/scripts/*
[user@cn4278 ~]$ mkdir -p output_files/genetrees

Download test data for 11 Drosophila genomes:

[user@cn4278 ~]$ mkdir -p test/test_data && cat test/input_genome_links.txt | xargs -I {} sh -c 'wget -O test/test_data/$(basename {}) {}'

Compile sampling executable:
[user@cn4278 ~]$ mkdir -p ./workflow/scripts/sampling/build [user@cn4278 ~]$ cd ./workflow/scripts/sampling/build [user@cn4278 ~]$ cmake .. -DZLIB_LIBRARY=/usr/local/apps/roadies/0.1.10/conda/lib/libz.so [user@cn4278 ~]$ make [user@cn4278 ~]$ cd ../../../..
Run the ROADIES pipeline:
user@cn4278 ~]$ python run_roadies.py --cores 16 --noconverge Unlocking working directory. snakemake --cores 16 --config mode=accurate config_path=config/config.yaml num_threads=4 deep_mode=Fal plete Config file config/config.yaml is extended by additional config specified via the command line. Building DAG of jobs... Using shell: /usr/bin/bash Provided cores: 16 Rules claiming more threads will be scaled down. Job stats: job count --------------- ------- all 1 filtermsa 250 lastz 11 lastz2fasta 1 mergeTrees 1 pasta 250 raxmlng 250 sequence_merge 1 sequence_select 11 total 776 Select jobs to execute... Failed to solve scheduling problem with ILP solver. Falling back to greedy solver. Run Snakemake with for debugging the problem. [Wed Aug 27 07:36:41 2025] rule sequence_select: input: test/test_data/droAna2.fa.gz output: output_files/samples/droAna2_temp.fa jobid: 9 benchmark: output_files/benchmarks/droAna2.sample.txt reason: Missing output files: output_files/samples/droAna2_temp.fa; Params have changed since last wildcards: sample=droAna2 threads: 4 resources: tmpdir=/tmp ... ./workflow/scripts/sampling/build/sampling -i test/test_data/droWil1.fa.gz -o output_files/samples/dro /usr/bin/bash: /opt/conda/envs/roadies_env/lib/libtinfo.so.6: no version information available (requir We are starting to sample test/test_data/droMoj3.fa.gz ./workflow/scripts/sampling/build/sampling -i test/test_data/droMoj3.fa.gz -o output_files/samples/dro Number of regions: 26 ID START: 181, ID END: 206 Region length: 500 Input file: test/test_data/droVir3.fa.gz Output file: output_files/samples/droVir3_temp.fa Number of resampling: 38 real 0m0.657s user 0m0.573s sys 0m0.074s Number of regions: 21 ID START: 92, ID END: 112 Region length: 500 Input file: test/test_data/droMoj3.fa.gz Output file: output_files/samples/droMoj3_temp.fa Number of resampling: 20 real 0m0.645s user 0m0.568s sys 0m0.058s [Wed Aug 27 07:36:42 2025] Finished job 16. ... [user@cn4278 ~]$ exit salloc.exe: Relinquishing job allocation 46116226 ... ASTRAL for PaRalogs and Orthologs III (ASTRAL-Pro3) *** NOW with integrated CASTLES-Pro *** Version: v1.23.3.6 #Genetrees: 52 #Duploss: 245 #Species: 11 #Rounds: 4 #Samples: 4 #Threads: 16 #NNI moves:0/42 ((((((droSec1,droSim1),(droEre2,droYak2)),droAna2),(((droMoj3,droVir3),droGri2),droWil1)),dp4),droPer1); #NNI moves:0/42 ((((((((droVir3,droMoj3),droGri2),droWil1),(dp4,droPer1)),droAna2),(droYak2,droEre2)),droSec1),droSim1); #NNI moves:0/42 ((((((((droMoj3,droVir3),droGri2),droWil1),(dp4,droPer1)),droAna2),(droSec1,droSim1)),droEre2),droYak2); #NNI moves:0/42 (((((((droSim1,droSec1),(droYak2,droEre2)),droAna2),(dp4,droPer1)),droWil1),(droMoj3,droVir3)),droGri2); Initial score: 7923 Initial tree: ((((((droSim1,droSec1),(droYak2,droEre2)),droAna2),(dp4,droPer1)),((droMoj3,droVir3),droGri2)),droWil1); *** Subsample Process *** #NNI moves:0/42 ((((((((droMoj3,droVir3),droGri2),droWil1),(dp4,droPer1)),droAna2),(droEre2,droYak2)),droSim1),droSec1); #NNI moves:0/42 ((((((((droSim1,droSec1),(droEre2,droYak2)),droAna2),(droPer1,dp4)),droWil1),droGri2),droVir3),droMoj3); #NNI moves:0/42 (((((((droYak2,droEre2),(droSec1,droSim1)),droAna2),(droPer1,dp4)),droWil1),(droMoj3,droVir3)),droGri2); #NNI moves:0/42 ((((((droSim1,droSec1),(droEre2,droYak2)),droAna2),(((droVir3,droMoj3),droGri2),droWil1)),dp4),droPer1); Current score: 7923 Current tree: ((((droVir3,droMoj3),droGri2),((((droSim1,droSec1),(droEre2,droYak2)),droAna2),(dp4,droPer1))),droWil1); Final Tree: ((((droVir3,droMoj3),droGri2),((((droSim1,droSec1),(droEre2,droYak2)),droAna2),(dp4,droPer1))),droWil1); #EqQuartets: 8625 Score: 7923 Species tree created [user@biowulf ~]$