FROGS
FROGS is a galaxy/CLI workflow designed to produce an OTUs count matrix from high depth sequencing amplicon data.
Install / Use
/learn @geraldinepascal/FROGSREADME
Description
FROGS is a CLI workflow designed to produce and analysis an ASV count matrix from high depth sequencing amplicon data.
FROGS-wrappers allow to add FROGS on a Galaxy instance. (see https://github.com/geraldinepascal/FROGS-wrappers)
This workflow is focused on:
- User-friendliness with lots of rich graphic outputs and the integration in Galaxy thanks to FROGS-wrappers.
- Dealing with Illumina, Aviti, IonTorrent, 454, PacBio and Oxford Nanopore sequencing technology reads.
- Dealing of non overlapping pair of sequences from long amplicon like ITS, or RPB2.
- Accuracy with a clustering without global similarity threshold or a denoising process, the management of separated PCRs in the chimera removal step, and the management of multi-affiliations.
- Access to general statistics on microbial communities and differential abundance analysis.
- Prediction of functionnal annotation and metabolic pathways.
- Speed with fast algorithms parallelisation and easy to use.
- Scalability with algorithms designed to support the data growth.
Table of content
- Convenient input data
- Installation
- Memory and parallelisation advices
- Download databanks
- Troubleshooting
- License
- Copyright
- Citation
- Contact
Convenient input data
Legend for the next schemas:
.: Complete nucleic sequence
!: Region of interest
*: PCR primers
- Paired-end classical protocol: In the paired-end protocol R1 and R2 may share a nucleic region. For example the amplicons on 16S V3-V4 regions can have a length between 350 and 500nt, with 2*300pb sequencing the overlap is between 250nt and 100nt.
From: To:
rDNA .........!!!!!!................ ......!!!!!!!!!!!!!!!!!!!.....
Ampl ****!!!!!!**** ****!!!!!!!!!!!!!!!!!!!****
R1 -------------- --------------
R2 -------------- --------------
In any case, the maximum overlap between R1 and R2 can be the complete overlap.
The minimum authorized overlap between R1 and R2 is 10nt. With less, the overlap can be incorrect, it will be rejected or considered as non overlap reads.
- Single-end classical protocol:
rDNA .........!!!!!!................
Ampl ****!!!!!!****
Read --------------
- Custom protocol
rDNA .....!!!!!!!!!!!!!!............
Ampl ****!!!!!!****
Read --------------
The amplicons can have a high length variability such as ITS. The R1 and R2 can have different length.
Installation
This FROGS repository is for command line user. If you want to install FROGS on Galaxy, please refer to FROGS-wrappers.
Tools dependencies
FROGS is written in Python 3 (with external numpy and Scipy libraries), uses also home-made scripts written in PERL5 and R 4.
FROGS relies on different specific tools for each of the analysis steps.
| FROGS Tools |Dependancy | version tested | | ----------- | :--------: | -------------: | | Denoising and Remove_chimera | vsearch | 2.29.1 | | Denoising | flash (optional) | 1.2.11 | | Denoising | cutadapt (need to be >=2.8) | 2.10 | | Denoising | swarm (need to be >=2.1) | 3.1.4 | | Denoising | DADA2 | 1.22.0 | | ITSx | ITSx | 1.1.2 | | Taxonomic_affiliation | NCBI BLAST+ | 2.16 | | Taxonomic_affiliation | EMBOSS needleall | 6.6.0 | | Tree | MAFFT | 7.525 | | Tree | Fasttree | 2.1.9 | | Tree / FROGSSTAT | plotly, phangorn, rmarkdown, phyloseq, DESeq2, optparse, calibrate, formattable, DT | R 4.1.2 | | FROGSSTAT | pandoc | 2.19.2 | | FROGSFUNC | PICRUSt2 | 2.5.1 | | FROGSFUNC | ete3 | 3.1.1 |
Use PEAR as read pairs merging software in preprocess
PEAR is one of the most effective software for read pairs merging, but as its license is not free for private use, we can not distribute it in FROGS. If you work in an academic lab on a private Galaxy server, or if you have paid your license you can use PEAR in FROGS preprocess. For that you need to:
- have PEAR in your PATH or in the FROGS libexec directory. We have tested PEAR 0.9.11 version.
- use
--merge-software pearoption in the preprocess.py command line
FROGS and dependencies installation
From conda
FROGS is now available on bioconda (https://anaconda.org/bioconda/frogs).
- to create a specific environment for a specific FROGS version
conda env create --name frogs@5.1.0 --file frogs-conda-requirements.yaml
# to use FROGS, first you need to activate your environment
conda activate frogs@5.1.0
WARNING : As PICRUSt2 currently relies on a different R version, in order to use the FROGSFUNC tools, it is necessary to create a dedicated conda environment as follows:
conda env create --name frogsfunc@5.1.0 --file frogsfunc-conda-requirements.yaml
# and then activate the environment
conda activate frogsfunc@5.1.0
After that, you just have to switch from one environment to another (with conda activate frogs@5.1.0 or conda activate frogsfunc@5.1.0 depending on whether you want to use FROGSFUNC or all the other tools.
Check intallation
To check your installation you can type:
cd <conda_env_dir>/frogs@5.1.0/share/FROGS-5.1.0/test
conda activate frogs@5.1.0
sh test_frogs.sh <NB_CPU> <JAVA_MEM> <OUT_FOLDER>
"Bioinformatic" tools are performed on a small simulated dataset of one sample replicated three times. "Statistical" tools are performed on an extract of the published results of Chaillou et al, ISME 2014
This test executes the FROGS tools in command line mode. Example:
[user@computer:/home/frogs/FROGS/test/]$ sh test_frogs.sh 1 2 res
Step demultiplex Wed Jan 14 01:11:27 PM CET 2026
Step reads_processing 16S vsearch swarm fastidious Wed Jan 14 01:11:30 PM CET 2026:
Step reads_processing 16S vsearch swarm reads_processing and distance 3 Wed Jan 14 01:11:35 PM CET 2026:
Step reads_processing: dada2 keep-unmerged Wed Jan 14 01:11:39 PM CET 2026
Step reads_processing: preprocess only Wed Jan 14 01:12:04 PM CET 2026
Step remove_chimera Wed Jan 14 01:12:08 PM CET 2026
Step cluster_filters Wed Jan 14 01:12:11 PM CET 2026
Step itsx Wed Jan 14 01:12:19 PM CET 2026
Step taxonomic_affiliation Wed Jan 14 01:12:28 PM CET 2026
Step affiliation_filters: masking mode Wed Jan 14 01:1
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