PoolSeqFlow v1.0.0 — initial public release

Changelog

All notable changes to PoolSeqFlow will be documented in this file.

The format follows [Keep a Changelog](https://keepachangelog.com/en/1.0.0/), and this project adheres to [Semantic Versioning](https://semver.org/spec/v2.0.0.html).

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[1.0.0] - 2026-03-26 — Initial Public Release

Added

Core pipeline (Nextflow DSL2)

• End-to-end Pool-seq analysis workflow (poolseqflow.nf) with 9 modular steps
• Wrapper script (PoolSeqFlow) exposing install, run, resume, clean, and reset subcommands

Step 0 — Environment verification

• Pre-run checks for all required input files, folder structure, RGTags CSV format, and software dependencies
• Generates Reports/0_verify_environment.txt

Step 1 — Reference indexing

• Builds BWA, SAMtools (.fai), and SnpEff indices from a gzipped reference FASTA and GFF

Step 2 — Quality control and trimming

• FastQC assessment of raw reads
• Adapter trimming via Trim Galore with user-specified adapter sequences
• Automated per-cycle base-composition analysis of FastQC reports
• Intelligent hard-clipping via Cutadapt driven by A/T and G/C imbalance thresholds — no manual parameter tuning required

Step 3 — Alignment

• Paired-end alignment to the reference genome using BWA-MEM

Step 4 — BAM post-processing

• Full SAMtools-based cleanup: name-sort → fixmate → coord-sort → markdup → addreplacerg → filter → index
• Configurable alignment filter flags (samFlags.filter, samFlags.required)

Step 5 — Alignment reporting

• Per-sample alignment statistics via bamtools stats
• Coverage summaries via samtools coverage

Step 6 — Variant calling

• Multi-sample SNP and indel calling with BCFtools mpileup + call in multiallelic mode
• Outputs VCFs with per-sample AD and DP FORMAT fields

Step 7 — VCF to allele frequency tables

• Major-allele normalisation: VCF re-encoded so the major allele is always REF
• Multiallelic site support throughout variant calling and frequency conversion
• Ploidy- and pool-size-aware minimum frequency filter: $f_{\min} = 1 / (2 \times ploidy \times poolSize)$
• Depth and quality filtering
• SNP / INDEL split
• Export to tab-separated allele frequency tables

Step 8 — Variant annotation (optional)

• SnpEff-based functional annotation, toggled via params.annotate

Resume logic

• Custom filesystem-based resume strategy using symbolic links between mainDir (working directory) and projectDir (permanent storage)
• Completed steps are skipped based on presence of permanent output files — resilient to job timeouts, reboots, and work/ directory cleanups
• Supports HPC environments where compute nodes and storage are on separate filesystems

Configuration

• parameters.config for analysis parameters (mainDir, projectDir, poolSize, ploidy, adapter sequences, filter flags)
• nextflow.config for computational resources (CPUs, memory, executor)
• RGTags.csv template for sample read group metadata
• parameters.config.template for getting started

Environment

• Single conda environment (install/environment.yml) covering all dependencies
• Automated install and verification scripts (install/install.sh, install/test-install.sh)

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