nf-core/eager is a bioinformatics best-practice analysis pipeline for NGS sequencing based ancient DNA (aDNA) data analysis.

The pipeline uses Nextflow, a bioinformatics workflow tool. It pre-processes raw data from FASTQ inputs, aligns the reads and performs extensive general NGS and aDNA specific quality-control on the results. It comes with docker, singularity or conda containers making installation trivial and results highly reproducible.

Pipeline steps

By default the pipeline currently performs the following:

  • Create reference genome indices for mapping (bwa, samtools, and picard)
  • Sequencing quality control (FastQC)
  • Sequencing adapter removal and for paired end data merging (AdapterRemoval)
  • Read mapping to reference using (bwa aln, bwa mem or CircularMapper)
  • Post-mapping processing, statistics and conversion to bam (samtools)
  • Ancient DNA C-to-T damage pattern visualisation (DamageProfiler)
  • PCR duplicate removal (DeDup or MarkDuplicates)
  • Post-mapping statistics and BAM quality control (Qualimap)
  • Library Complexity Estimation (preseq)
  • Overall pipeline statistics summaries (MultiQC)

Additional functionality contained by the pipeline currently includes:

  • Illumina two-coloured sequencer poly-G tail removal (fastp)
  • Automatic conversion of unmapped reads to FASTQ (samtools)
  • Damage removal/clipping for UDG+/UDG-half treatment protocols (BamUtil)
  • Damage reads extraction and assessment (PMDTools)

Quick Start

  1. Install nextflow

  2. Install one of docker, singularity or conda

  3. Download the EAGER pipeline

nextflow pull nf-core/eager
  1. Test the pipeline using the provided test data
nextflow run nf-core/eager -profile <docker/singularity/conda>,test --pairedEnd
  1. Start running your own ancient DNA analysis!
nextflow run nf-core/eager -profile <docker/singularity/conda> --reads'*_R{1,2}.fastq.gz' --fasta '<REFERENCE>.fasta'

NB. You can see an overview of the run in the MultiQC report located at <OUTPUT_DIR>/MultiQC/multiqc_report.html

Modifications to the default pipeline are easily made using various options as described in the documentation.


The nf-core/eager pipeline comes with documentation about the pipeline, found in the docs/ directory or on the main homepage of the nf-core project:

  1. Nextflow Installation
  2. Pipeline configuration
  3. Running the pipeline
  4. Output and how to interpret the results
  5. Troubleshooting
  6. EAGER Troubleshooting


This pipeline was written by Alexander Peltzer (apeltzer), with major contributions from Stephen Clayton, ideas and documentation from James A. Fellows Yates, Raphael Eisenhofer, Maxime Borry and Judith Neukamm. If you want to contribute, please open an issue and ask to be added to the project - happy to do so and everyone is welcome to contribute here!


If you’ve contributed and you’re missing in here, please let me know and I’ll add you in.

Tool References