nf-core pipeline · adna, ancient-dna-analysis, ancientdna
nf-core/eager
A reproducible Nextflow pipeline for processing, quality-controlling and analysing ancient genomic sequencing data.
What nf-core/eager is
nf-core/eager is a scalable bioinformatics pipeline designed for the analysis of next-generation sequencing (NGS) data, tailored specifically to ancient DNA (aDNA). It processes palaeogenomic data from modern and degraded samples across a wide variety of contexts, including human, animal, plant, microbial, and microbiome sequencing studies.
Ancient DNA analysis presents distinct computational challenges due to high levels of degradation, short sequence fragments, post-mortem cytosine-to-thymine (C-to-T) damage, and modern environmental contamination. Originally developed by Alexander Peltzer, James A. Fellows Yates, and collaborators across the nf-core community, nf-core/eager automates standard aDNA processing procedures into a unified, portable workflow.
The pipeline handles complex sequencing arrangements and delivers comprehensive quality-control assessments, aDNA damage quantification, reference mapping, contamination estimation, and genotyping formats suited for downstream population genetics and pathogen discovery.
What the pipeline does
By default, nf-core/eager indexes the reference genome using BWA, SAMtools, and Picard. It runs FastQC on sequencing reads and performs adapter removal and paired-end read merging via AdapterRemoval. Reads are then aligned against a reference using bwa aln, bwa mem, CircularMapper, or Bowtie2. Following post-mapping processing and conversion to BAM format with SAMtools, PCR duplicates are removed with DeDup or Picard MarkDuplicates. Ancient DNA deamination patterns are visualised using DamageProfiler or mapDamage, sequencing depth and library complexity are assessed using Qualimap and preseq, and overall run metrics are compiled by MultiQC.
Optional modules extend processing across preprocessing, damage manipulation, biological profiling, and screening. Users can strip host DNA, remove poly-G tails or trim reads via fastp, clip terminal damage for UDG protocols using BamUtil, isolate damaged reads using PMDTools, and assess nuclear contamination or calculate genotype likelihoods with ANGSD. Additional tools enable sex determination (Sex.DetERRmine), mitochondrial-to-nuclear ratio calculation, variant calling (GATK, FreeBayes, pileupCaller for EIGENSTRAT files, and MultiVCFAnalyzer), as well as metagenomic screening through MALT, MaltExtract, Kraken2, and BBDuk.
Key facts
- Latest release
- 2.5.3 (2025-03-18)
- Pipeline engine
- Nextflow (>=20.07.1 and <=22.10.6 for version 2.*)
- Supported inputs
- Raw FASTQ files, preprocessed BAM files, reference FASTA
- Primary outputs
- BAM files, VCFs, EIGENSTRAT files, consensus FASTAs, MultiQC report
- Execution environments
- Docker, Singularity, Podman, Shifter, Charliecloud, Conda
What you can use it for
- Palaeogenomic Variant Calling
- Reconstruct ancient human or animal genomes from degraded reads using bwa aln, clip post-mortem damage using BamUtil, and generate VCF or EIGENSTRAT genotyping files for population genetics.
- Ancient DNA Damage Authentication
- Verify the authenticity of historical or archaeological specimens by profiling characteristic terminal C-to-T deamination patterns using DamageProfiler or mapDamage.
- Metagenomic Pathogen Screening
- Screen ancient microbiome samples or infected historical tissues for taxonomic abundance and pathogen-specific damage profiles using Kraken2, MALT, and MaltExtract.
- Biological Sex and Contamination Profiling
- Determine human chromosomal sex using Sex.DetERRmine and estimate nuclear DNA contamination levels via ANGSD.
Run it here
The research agent runs nf-core/eager with Nextflow on its own machine — up to 32 cores, 256 GiB of memory and 24 hours per run, which covers the test profile and real single-sample runs. It builds the samplesheet, checks it against the pipeline's schema and reads the results back. When a run is too big for one machine, it gives you the exact command for your own cluster instead.
Strengths and limits
Good at
- Integrates specialised aDNA authentication steps, including deamination profiling, damage read extraction, and contamination estimation.
- Automates the merging of complex sequencing configurations spanning different lanes, libraries, and sequencing types.
- Supports circular genome mapping and multiple alignment algorithms tailored to degraded short-read data.
- Produces standard population genetics outputs directly, including EIGENSTRAT and MultiVCFAnalyzer tables.
Watch out for
- Version 2.* releases are constrained to run only on Nextflow versions up to 22.10.6.
- Metagenomic alignment tools such as MALT require substantial computing resources and dedicated reference databases.
Questions
Can I run nf-core/eager without my own cluster?
The research agent runs nf-core/eager with Nextflow on its own machine — up to 32 cores, 256 GiB of memory and 24 hours per run, which covers the test profile and real single-sample runs. It builds the samplesheet, checks it against the pipeline's schema and reads the results back. When a run is too big for one machine, it gives you the exact command for your own cluster instead.
What licence is nf-core/eager under?
MIT, like every nf-core pipeline.
What inputs does nf-core/eager accept?
The pipeline accepts raw sequencing reads in FASTQ format (single-end or paired-end) as well as preprocessed BAM alignment files, along with a reference genome in FASTA format.
Which aligners can be used for mapping short ancient DNA fragments?
Users can align reads using bwa aln, bwa mem, CircularMapper (for circular references like mitochondrial DNA or bacterial plasmids), or Bowtie2.
How does the pipeline handle post-mortem ancient DNA damage during variant calling?
It provides modules such as BamUtil to trim damage ends from UDG-treated libraries, PMDTools to extract or filter damaged reads, and specialised variant caller setups (such as pileupCaller for EIGENSTRAT) to mitigate reference bias and deamination artefacts.
Can nf-core/eager screen for ancient microbial pathogens?
Yes. It supports metagenomic screening via Kraken2 or MALT, and can authenticate ancient pathogen candidates using MaltExtract.
How to cite it
Fellows Yates JA, Lamnidis TC, Borry M, Valtueña Andrades A, Fagernäs Z, Clayton S, Garcia MU, Neukamm J, Peltzer A. 2021. Reproducible, portable, and efficient ancient genome reconstruction with nf-core/eager. PeerJ 9:e10947. DOI: 10.7717/peerj.10947.
Written from nf-core/eager's own README. Statements citing a figure that does not appear there are removed before publication. Licence and running details come from our registry.