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, or preprocessed BAM inputs. It can align 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

Default 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 Steps

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)
  • Host DNA (mapped reads) stripping from input FASTQ files (for sensitive samples)

aDNA Damage manipulation

  • Damage removal/clipping for UDG+/UDG-half treatment protocols (BamUtil)
  • Damaged reads extraction and assessment (PMDTools)


  • Creation of VCF genotyping files (GATK UnifiedGenotyper, GATK HaplotypeCaller and FreeBayes)
  • Consensus sequence FASTA creation (VCF2Genome)
  • SNP Table generation (MultiVCFAnalyzer)

Biological Information

  • Mitochondrial to Nuclear read ratio calculation (MtNucRatioCalculator)
  • Statistical sex determination of human individuals (SexDetErrmine)

Metagenomic Screening

  • Taxonomic binner with alignment (MALT)
  • Taxonomic binner without alignment (Kraken2)
  • aDNA characteristic screening of taxonomically binned data from MALT (MaltExtract)

Quick Start

  1. Install nextflow (>= v19.10.0)

  2. Install one of docker, singularity or conda

  3. Download the EAGER pipeline

     nextflow pull nf-core/eager
  4. Test the pipeline using the provided test data

     nextflow run nf-core/eager -profile <docker/singularity/conda>,test --paired_end
  5. Start running your own ancient DNA analysis!

     nextflow run nf-core/eager -profile <docker/singularity/conda> --reads '*_R{1,2}.fastq.gz' --fasta '<your_reference>.fasta'
  6. Once your run has completed successfully, clean up the intermediate files.

     nextflow clean -f -k

NB. You can see an overview of the run in the MultiQC report located at ./results/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. EAGER2 Code Contribution Guidelines
  6. nf-core/nextflow Troubleshooting
  7. EAGER Troubleshooting


This pipeline was mostly written by Alexander Peltzer (apeltzer), with contributions from Stephen Clayton, James A. Fellows Yates, Thiseas C. Lamnidis, Maxime Borry, Zandra Fagernäs, Aida Andrades Valtueña and Maxime Garcia. If you want to contribute, please open an issue (or even better, a pull request!) and ask to be added to the project - everyone is welcome to contribute here!

Authors (alphabetical)

Additional Contributors (alphabetical)

Those who have provided conceptual guidance, suggestions, bug reports etc.

If you’ve contributed and you’re missing in here, please let us know and we will add you in of course!

Tool References