PoolSeqFlow

A Nextflow pipeline for allele frequency analysis from pooled Illumina sequencing data

📖 Read the documentation →

Platform note: PoolSeqFlow is developed and tested on Linux and macOS. Windows is not supported — the resume logic relies on symbolic links and Unix-style paths that are not compatible with native Windows filesystems.

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Overview

PoolSeqFlow takes raw FASTQ files and a reference genome and gives back allele frequency tables. It automates quality control, adapter trimming with composition-aware clipping, alignment, BAM post-processing, variant calling and VCF-to-frequency conversion, with optional annotation.

Pool-seq sequences many individuals together, so the unit of analysis is not a genotype but a frequency — and that difference runs through every stage. Multiallelic sites are preserved rather than collapsed, VCFs are re-encoded so the most-read allele is the reference, and the minimum credible frequency is derived from your pool size and ploidy instead of a fixed cutoff. See When to use PoolSeqFlow for what the design assumes about your data.

Raw FASTQ reads
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[Step 0] Verify environment, parameters and folder structure
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[Step 1] Build reference dictionaries (BWA, SAMtools, SnpEff)
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[Step 2] QC & trimming (FastQC → Trim Galore → composition-aware clipping)
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[Step 3] Alignment (BWA-MEM)
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[Step 4] BAM cleanup (name-sort → fixmate → coord-sort → markdup → addRG → filter → index)
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[Step 5] Alignment, coverage & depth reports; each sample's depth ceiling
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[Step 6] Depth capping, then variant calling (BCFtools mpileup + call)
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[Step 7] VCF → allele frequency tables   [Step 8] Annotation (SnpEff, optional)

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Quick start

Requires Linux or macOS and conda. Every bioinformatics tool is installed for you into an isolated environment, pinned to an exact build.

# 1. Download the latest release
curl -LO https://github.com/ozankiratli/PoolSeqFlow/releases/latest/download/PoolSeqFlow.tar.gz
tar -xzf PoolSeqFlow.tar.gz
cd PoolSeqFlow-*/

# 2. Build and verify the environment
./PoolSeqFlow install

# 3. Populate a project directory
mkdir -p /path/to/project && cd /path/to/project
PoolSeqFlow init
#    then edit parameters.config: mainDir, storageDir, readPattern,
#    referenceFile, poolSize, ploidy

# 4. Run — this is also the resume command
PoolSeqFlow run

Your project directory needs a Data/ folder of FASTQs, a reference genome (gzipped or not), and a metadata.csv. init writes metadata.csv.example for you to start from — the file is not only metadata, it decides which FASTQ pairs count as one pool, the order your result columns come out in, and each pool's detection limit.

Full walkthrough: Install and Quick Start.

Configure through parameters.config only. PoolSeqFlow does not accept command-line parameter overrides. A run is therefore fully described by a file you can version, diff and publish. It also avoids a silent failure: Nextflow delivers --param values as strings, so --annotate false sets the string "false", which Groovy evaluates as true.

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Commands

Command	Description
PoolSeqFlow install	Create the conda environment, install the pipeline, then verify both
PoolSeqFlow init	Populate the current directory as a project
PoolSeqFlow init_multi	The same, for a project running several parameter sets over one set of reads
PoolSeqFlow check install	Verify an installation — the tools and helpers it is built to run
PoolSeqFlow check project	Verify a project — its configuration, and the commands it names
PoolSeqFlow run	Start — or resume — the pipeline
PoolSeqFlow dryrun	Create the directory tree a run would write, empty, before any compute is spent
PoolSeqFlow dryclean	Remove that preview
PoolSeqFlow migrate_config	Carry an older parameters.config onto the current template
PoolSeqFlow clean	Remove Nextflow work directories
PoolSeqFlow reset	Remove all progress and start fresh (typed confirmation required)
PoolSeqFlow analysis <command>	The analysis layer — see below
PoolSeqFlow version	Print the installed version
PoolSeqFlow cite	Print how to cite this copy, and which DOI to use
PoolSeqFlow list	List the pipelines and conda environments installed on this machine
PoolSeqFlow uninstall	Remove one installed version — environment and pipeline together, after confirmation
PoolSeqFlow uninstall_all	Remove every PoolSeqFlow environment and installation, after confirmation

./PoolSeqFlow install is the only command you type with a ./. Until it has run there is no PoolSeqFlow on your PATH, so that first one is called from inside the folder you extracted. It installs the command, and everything after it — including init, which you run from your own project directory — is called by name from wherever you are.

analysis takes install, check, modules, version, cite, uninstall, or the name of a module to run. analysis modules available|install|list|uninstall manages the modules themselves, which are published separately from the pipeline. The analysis layer ships with the pipeline and is enabled separately with PoolSeqFlow analysis install, which creates the conda environment that carries R.

There is no -resume flag. Every step checks whether its outputs already exist in permanent storage and skips itself if they do, so run both starts and resumes — and that survives job timeouts, reboots and work/ cleanups. Why →

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Documentation

When to use it	What pooling buys and costs, and what the pipeline assumes about your design
Design decisions	Why configuration is a file, why resume is filesystem-based, what each choice costs
The filter chain	All nine filters in order, what each removes, and how to tune them
Interpreting results	The frequency table format, and the mistakes that are easy to make reading it
Configuration	Every parameter, sorted by whether it changes your results
Metadata	metadata.csv, and why RG_Sample decides what counts as a pool
Pipeline steps	Steps 0–8 in detail
Upgrading	Your parameters.config is never touched by an update — read this first
Troubleshooting	Errors by symptom

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Citation

Your installed copy prints its own citation, with the version filled in:

PoolSeqFlow cite

Cite the version you actually ran, not the newest one. Zenodo issues a separate DOI for every release, and results depend on which release produced them — filters, defaults and parameter names have all changed between versions. Step 0 records the release that produced a project's results in .poolseqflow_version, named again in the header of the readable Output/run_parameters.txt, and refuses to run under a different one — so a project belongs to one release and there is never a question of which to cite.

10.5281/zenodo.19245611 is the all-versions DOI: it always resolves to the newest release. Use it to refer to the software in general, and a version DOI when reporting results. Details →

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License

Apache 2.0 — see LICENSE. The tools PoolSeqFlow invokes carry their own licenses.

Analysis modules under analysis/modules/ carry their own, declared in each manifest.json and printed in the report of every analysis. basicstats, association and mds are GPL-3.0-or-later; verify belongs to the frame and is Apache 2.0.

Contact

Ozan L. Z. Kiratli · @ozankiratli · Issues