exomeflow 1.0.6

Production-quality Whole Exome Sequencing analysis pipeline

ExomeFlow: A Production-Quality Python WES Analysis Toolkit

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What is it?

ExomeFlow is a Python package that provides a complete, automated Whole Exome Sequencing (WES) analysis workflow — from raw FASTQ files to functionally annotated variants — in a single reproducible CLI command.

It aims to be the standard high-level pipeline for WES analysis in Python, combining GATK best-practice variant calling, hard filtering, and ANNOVAR annotation into one modular, maintainable package. It handles cohort-level processing (multiple samples), checkpointing for resumable runs, structured logging, and parallel execution out of the box.

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Table of Contents

• What is it?
• Main Features
• Pipeline Workflow
• Where to get it
• System Requirements
• Python Dependencies
• Quick Start
• Commands
• Reference Files
• Input Convention
• Output Files
• Documentation
• Getting Help
• License
• Citation

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Main Features

Here are the things ExomeFlow does well:

• One-command setup — exomeflow setup installs all system tools, downloads hg38 reference files (~13 GB) and ANNOVAR databases (~100 GB) automatically
• Automatic sample detection — scans an input directory and detects all paired-end samples from FASTQ filenames; no manifest file required
• Complete GATK best-practice workflow — fastp QC → BWA MEM alignment → coordinate sorting → duplicate marking → BQSR → HaplotypeCaller → hard filtering → ANNOVAR annotation
• Cohort processing — processes any number of samples sequentially or in parallel with --max-workers
• Checkpointing and resume — every completed step is recorded; an interrupted run resumes exactly where it left off without repeating work
• Automatic requirements check — verifies all system tools and Python packages before the pipeline starts, reporting every missing dependency at once
• Structured logging — per-sample log files plus a pipeline-wide log with INFO / WARNING / ERROR / SUCCESS levels
• GATK hard filters — applies GATK best-practice SNP and INDEL hard-filter thresholds and extracts PASS-only variants automatically
• ANNOVAR functional annotation — annotates variants against 8 databases: refGene, ClinVar, gnomAD, dbNSFP, COSMIC, ExAC, avSNP150, and dbscSNV
• Modular architecture — each pipeline step is an independent Python module; easy to extend or modify individual steps without touching the rest
• PyPI installable — pip install exomeflow; no Docker or Nextflow required

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Pipeline Workflow

Raw FASTQ
    │
    ▼
┌─────────────────────────────────────────────────────────┐
│  Step 1   fastp         Quality control & adapter trim   │
│           length ≥ 50 bp · base quality ≥ Q30            │
└──────────────────────────┬──────────────────────────────┘
                           │
                           ▼
┌─────────────────────────────────────────────────────────┐
│  Step 2   BWA MEM        Read alignment to hg38          │
│           -Y -K 100000000 · read-group tags set          │
└──────────────────────────┬──────────────────────────────┘
                           │
                           ▼
┌─────────────────────────────────────────────────────────┐
│  Step 3   GATK SortSam   Coordinate-sort BAM             │
│  Step 4   samtools       Flagstat alignment QC           │
│  Step 5   GATK MarkDuplicates   PCR duplicate removal    │
│  Step 6   GATK BuildBamIndex    BAI index                │
└──────────────────────────┬──────────────────────────────┘
                           │
                           ▼
┌─────────────────────────────────────────────────────────┐
│  Step 7   GATK BQSR      BaseRecalibrator + ApplyBQSR    │
│           Known sites: dbSNP · Mills · known indels      │
│           → recalibrated.bam  (IGV-ready)                │
└──────────────────────────┬──────────────────────────────┘
                           │
                           ▼
┌─────────────────────────────────────────────────────────┐
│  Step 8   GATK HaplotypeCaller   Variant calling         │
│           Exome intervals + padding · dbSNP annotation   │
└──────────────────────────┬──────────────────────────────┘
                           │
                    ┌──────┴──────┐
                    ▼             ▼
               SNP filters   INDEL filters
               (Step 9)       (Step 10)
                    └──────┬──────┘
                           │  MergeVcfs
                           ▼
┌─────────────────────────────────────────────────────────┐
│  Step 11  SelectVariants  Extract PASS-only variants     │
└──────────────────────────┬──────────────────────────────┘
                           │
                           ▼
┌─────────────────────────────────────────────────────────┐
│  Step 12  ANNOVAR         Functional annotation          │
│           refGene · ClinVar · gnomAD · dbNSFP · COSMIC   │
│           → multianno.vcf  +  multianno.txt              │
└─────────────────────────────────────────────────────────┘

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Where to get it

The source code is hosted on GitHub at: https://github.com/imrobintomar/exomeflow

Binary installers for the latest released version are available at the Python Package Index (PyPI).

# PyPI
pip install exomeflow

# Install latest development version from GitHub
pip install git+https://github.com/imrobintomar/exomeflow.git

The list of changes between each release can be found in the Release History.

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System Requirements

ExomeFlow calls the following external tools via the command line. They must be installed separately and available on your PATH.

Tool	Minimum Version	Install
BWA	≥ 0.7.17	conda install -c bioconda bwa
SAMtools	≥ 1.13	conda install -c bioconda samtools
GATK	≥ 4.6.0	conda install -c bioconda gatk4
fastp	≥ 0.20.1	conda install -c bioconda fastp
Perl	≥ 5.26	conda install perl
ANNOVAR	latest	Register + download from website

Tip: Run exomeflow setup after installation to automatically verify tools, download hg38 reference files, and populate ANNOVAR databases in one step.

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Python Dependencies

• typer — Builds the CLI interface
• rich — Provides coloured terminal output and structured logging
• pandas — Data handling for variant count summaries

All Python dependencies are installed automatically with pip install exomeflow.

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Quick Start

1. Install ExomeFlow

pip install exomeflow

2. Set up all dependencies and reference data

exomeflow setup \
  --refs-dir   /data/references/hg38 \
  --annovar-bin /opt/annovar \
  --annovar-db  /opt/annovar/humandb

This command will:

• Install missing Python packages
• Install system tools via conda (fastp, bwa, samtools, gatk4, perl)
• Download hg38 reference files (~13 GB) using gsutil or wget
• Download ANNOVAR annotation databases (~100 GB)

3. Prepare FASTQ files

fastq/
├── sample1_1.fastq.gz
├── sample1_2.fastq.gz
├── sample2_1.fastq.gz
└── sample2_2.fastq.gz

4. Run the pipeline

exomeflow run \
  --input-dir    fastq/ \
  --output       results/ \
  --reference    /data/references/hg38/hg38.fa \
  --dbsnp        /data/references/hg38/dbsnp.vcf.gz \
  --mills        /data/references/hg38/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz \
  --known-indels /data/references/hg38/Homo_sapiens_assembly38.known_indels.vcf.gz \
  --intervals    refs/Illumina_Exome_TargetedRegions_v1.2.hg38.bed \
  --annovar-bin  /opt/annovar \
  --annovar-db   /opt/annovar/humandb \
  --threads      32 \
  --max-workers  2

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Commands

exomeflow setup — Install dependencies and download reference data

exomeflow setup --refs-dir PATH --annovar-bin PATH --annovar-db PATH

Option	Description
--refs-dir	Directory to download hg38 reference files into
--annovar-bin	ANNOVAR installation directory (must contain annotate_variation.pl)
--annovar-db	ANNOVAR humandb directory for database downloads

exomeflow run — Execute the WES pipeline

exomeflow run [OPTIONS]

Option	Default	Description
--input-dir, -i	required	Directory containing paired FASTQ files
--output, -o	results/	Root output directory
--reference, -r	required	BWA-indexed reference FASTA (hg38.fa)
--dbsnp	required	dbSNP VCF (bgzipped + tabix-indexed)
--mills	required	Mills and 1000G gold standard indels VCF
--known-indels	required	Known indels VCF for BQSR
--intervals	(optional)	Exome capture BED file
--interval-padding	100	Base-pair padding around each target interval
--annovar-bin	required	Directory containing table_annovar.pl
--annovar-db	required	ANNOVAR humandb directory
--threads, -t	24	Threads for BWA MEM and GATK HaplotypeCaller
--fastp-threads	8	Threads for fastp
--annovar-threads	24	Threads for ANNOVAR
--max-workers	1	Number of samples to process in parallel
--java-opts	-Xmx80g	JVM options passed via JAVA_OPTS

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Reference Files

File	Source	Size
hg38.fa + BWA index	UCSC / GATK resource bundle	~10 GB
dbsnp.vcf.gz	GATK resource bundle	~10 GB
Mills_and_1000G_gold_standard.indels.hg38.vcf.gz	GATK resource bundle	~200 MB
Homo_sapiens_assembly38.known_indels.vcf.gz	GATK resource bundle	~100 MB
Exome capture BED	Your sequencing kit vendor	varies
ANNOVAR humandb (8 databases)	ANNOVAR download server	~100 GB

exomeflow setup downloads all GATK resource bundle files automatically.

Manual download:

gsutil -m cp -r gs://genomics-public-data/resources/broad/hg38/v0/ /data/refs/

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Input Convention

ExomeFlow automatically detects samples from paired-end FASTQ filenames. Files must follow the pattern:

<sample_id>_1.fastq.gz   ← Read 1
<sample_id>_2.fastq.gz   ← Read 2

The sample_id can be any string — SRR accession, patient ID, etc.

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Output Files

File	Description
Mapsam/<sample>_recalibrated.bam	Analysis-ready BAM — open in IGV
VCF/<sample>.vcf	Raw HaplotypeCaller output
VCF/<sample>_PASS.vcf	PASS-only hard-filtered variants
VCF/<sample>.annovar.hg38_multianno.vcf	Annotated VCF
VCF/<sample>.annovar.hg38_multianno.txt	Annotated tab-delimited table
filtered_fastp/<sample>_fastp.html	fastp QC report
Mapsam/<sample>_flagstat.txt	Alignment statistics
logs/analysis_<timestamp>.log	Full pipeline log
logs/<sample>_<timestamp>.log	Per-sample log

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Documentation

Full usage documentation is available in USAGE.md, including:

• Complete CLI option reference
• How to resume interrupted runs
• How to tune parallel processing
• Common errors and fixes
• Quick reference card

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Getting Help

For usage questions, please open a GitHub Issue.

Bug reports, feature requests, and general questions are all welcome.

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License

MIT

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Citation

If you use ExomeFlow in your research, please cite:

Robin Tomar. ExomeFlow: A Production-Quality Python Package for Automated Whole Exome Sequencing Analysis. AIIMS New Delhi, 2025. https://pypi.org/project/exomeflow/

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Built for the bioinformatics community · Robin Tomar, AIIMS New Delhi