README.md

A fully reproducible ancient and modern DNA pipeline in Nextflow and with cloud support..

Introduction

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, CircularMapper, or bowtie2)
• 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:

Input

• Automatic merging of complex sequencing setups (e.g. multiple lanes, sequencing configurations, library types)

Preprocessing

• 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)
• Nuclear DNA contamination estimation of human samples (angsd)

Genotyping

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

Biological Information

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

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
   

5. Start running your own ancient DNA analysis either via the command-line or GUI! Command line example:

    nextflow run nf-core/eager -profile <docker/singularity/conda> --input '*_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.

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
   • Pipeline installation
   • Adding your own system config
   • Reference genomes
3. Running the pipeline
4. Output and how to interpret the results
5. nf-core/eager code contribution checklist and guidelines
6. nf-core and Nextflow troubleshooting
7. nf-core/eager troubleshooting

Credits

This pipeline was mostly written by Alexander Peltzer (apeltzer) and James A. Fellows Yates, with contributions from Stephen Clayton, 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)

• Aida Andrades Valtueña
• Alexander Peltzer
• James A. Fellows Yates
• Judith Neukamm
• Maxime Borry
• Maxime Garcia
• Stephen Clayton
• Thiseas C. Lamnidis
• Zandra Fagernäs

Additional Contributors (alphabetical)

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

• Arielle Munters
• Hester van Schalkwyk
• Irina Velsko
• Katerine Eaton
• Luc Venturini
• Marcel Keller
• Pierre Lindenbaum
• Pontus Skoglund
• Raphael Eisenhofer
• Torsten Günter
• Kevin Lord
• Irina Velsko
• He Yu

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

Tool References

• EAGER v1, CircularMapper, DeDup* Peltzer, A., Jäger, G., Herbig, A., Seitz, A., Kniep, C., Krause, J., & Nieselt, K. (2016). EAGER: efficient ancient genome reconstruction. Genome Biology, 17(1), 1–14. https://doi.org/10.1186/s13059-016-0918-z Download: https://github.com/apeltzer/EAGER-GUI and https://github.com/apeltzer/EAGER-CLI
• FastQC download: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
• AdapterRemoval v2 Schubert, M., Lindgreen, S., & Orlando, L. (2016). AdapterRemoval v2: rapid adapter trimming, identification, and read merging. BMC Research Notes, 9, 88. https://doi.org/10.1186/s13104-016-1900-2 Download: https://github.com/MikkelSchubert/adapterremoval
• bwa Li, H., & Durbin, R. (2009). Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics , 25(14), 1754–1760. https://doi.org/10.1093/bioinformatics/btp324 Download: http://bio-bwa.sourceforge.net/bwa.shtml
• SAMtools Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., … 1000 Genome Project Data Processing Subgroup. (2009). The Sequence Alignment/Map format and SAMtools. Bioinformatics , 25(16), 2078–2079. https://doi.org/10.1093/bioinformatics/btp352 Download: http://www.htslib.org/
• DamageProfiler Judith Neukamm (Unpublished). Download: https://github.com/Integrative-Transcriptomics/DamageProfiler
• QualiMap Okonechnikov, K., Conesa, A., & García-Alcalde, F. (2016). Qualimap 2: advanced multi-sample quality control for high-throughput sequencing data. Bioinformatics , 32(2), 292–294. https://doi.org/10.1093/bioinformatics/btv566 Download: http://qualimap.bioinfo.cipf.es/
• preseq Daley, T., & Smith, A. D. (2013). Predicting the molecular complexity of sequencing libraries. Nature Methods, 10(4), 325–327. https://doi.org/10.1038/nmeth.2375. Download: http://smithlabresearch.org/software/preseq/
• PMDTools Skoglund, P., Northoff, B. H., Shunkov, M. V., Derevianko, A. P., Pääbo, S., Krause, J., & Jakobsson, M. (2014). Separating endogenous ancient DNA from modern day contamination in a Siberian Neandertal. Proceedings of the National Academy of Sciences of the United States of America, 111(6), 2229–2234. https://doi.org/10.1073/pnas.1318934111 Download: https://github.com/pontussk/PMDtools
• MultiQC Ewels, P., Magnusson, M., Lundin, S., & Käller, M. (2016). MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics , 32(19), 3047–3048. https://doi.org/10.1093/bioinformatics/btw354 Download: https://multiqc.info/
• BamUtils Jun, G., Wing, M. K., Abecasis, G. R., & Kang, H. M. (2015). An efficient and scalable analysis framework for variant extraction and refinement from population-scale DNA sequence data. Genome Research, 25(6), 918–925. https://doi.org/10.1101/gr.176552.114 Download: https://genome.sph.umich.edu/wiki/BamUtil
• FastP Chen, S., Zhou, Y., Chen, Y., & Gu, J. (2018). fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics , 34(17), i884–i890. https://doi.org/10.1093/bioinformatics/bty560 Download: https://github.com/OpenGene/fastp
• GATK 3.5 DePristo, M. A., Banks, E., Poplin, R., Garimella, K. V., Maguire, J. R., Hartl, C., … Daly, M. J. (2011). A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nature Genetics, 43(5), 491–498. https://doi.org/10.1038/ng.806 Download
• GATK 4.X - no citation available yet
• VCF2Genome - Alexander Herbig and Alex Peltzer (unpublished). Download: https://github.com/apeltzer/VCF2Genome
• MultiVCFAnalyzer Bos, K.I. et al., 2014. Pre-Columbian mycobacterial genomes reveal seals as a source of New World human tuberculosis. Nature, 514(7523), pp.494–497. Available at: http://dx.doi.org/10.1038/nature13591. Download: https://github.com/alexherbig/MultiVCFAnalyzer
• MTNucRatioCalculator Alex Peltzter (Unpublished). Download: https://github.com/apeltzer/MTNucRatioCalculator
• Sex.DetERRmine.py Lamnidis, T.C. et al., 2018. Ancient Fennoscandian genomes reveal origin and spread of Siberian ancestry in Europe. Nature communications, 9(1), p.5018. Available at: http://dx.doi.org/10.1038/s41467-018-07483-5. Download: https://github.com/TCLamnidis/Sex.DetERRmine.git
• ANGSD Korneliussen, T.S., Albrechtsen, A. & Nielsen, R., 2014. ANGSD: Analysis of Next Generation Sequencing Data. BMC bioinformatics, 15, p.356. Available at: http://dx.doi.org/10.1186/s12859-014-0356-4. Download: https://github.com/ANGSD/angsd
• bedtools Quinlan, A.R. & Hall, I.M., 2010. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics , 26(6), pp.841–842. Available at: http://dx.doi.org/10.1093/bioinformatics/btq033. Download: https://github.com/arq5x/bedtools2/releases
• MALT Download: https://software-ab.informatik.uni-tuebingen.de/download/malt/welcome.html
  • Vågene, Å.J. et al., 2018. Salmonella enterica genomes from victims of a major sixteenth-century epidemic in Mexico. Nature ecology & evolution, 2(3), pp.520–528. Available at: http://dx.doi.org/10.1038/s41559-017-0446-6.
  • Herbig, A. et al., 2016. MALT: Fast alignment and analysis of metagenomic DNA sequence data applied to the Tyrolean Iceman. bioRxiv, p.050559. Available at: http://biorxiv.org/content/early/2016/04/27/050559.
• MaltExtract Huebler, R. et al., 2019. HOPS: Automated detection and authentication of pathogen DNA in archaeological remains. bioRxiv, p.534198. Available at: https://www.biorxiv.org/content/10.1101/534198v1?rss=1. Download: https://github.com/rhuebler/MaltExtract
• Kraken2 Wood, D et al., 2019. Improved metagenomic analysis with Kraken 2. Genome Biology volume 20, Article number: 257. Available at: https://doi.org/10.1186/s13059-019-1891-0. Download: https://ccb.jhu.edu/software/kraken2/
• endorS.py Aida Andrades Valtueña (Unpublished). Download: https://github.com/aidaanva/endorS.py
• Bowtie2 Langmead, B. and Salzberg, S. L. 2012 Fast gapped-read alignment with Bowtie 2. Nature methods, 9(4), p. 357–359. doi: 10.1038/nmeth.1923.
• sequenceTools Stephan Schiffels (Unpublished). Download: https://github.com/stschiff/sequenceTools

Data References

This repository uses test data from the following studies:

• Fellows Yates, J. A. et al. (2017) ‘Central European Woolly Mammoth Population Dynamics: Insights from Late Pleistocene Mitochondrial Genomes’, Scientific reports, 7(1), p. 17714. doi: 10.1038/s41598-017-17723-1.
• Gamba, C. et al. (2014) ‘Genome flux and stasis in a five millennium transect of European prehistory’, Nature communications, 5, p. 5257. doi: 10.1038/ncomms6257.
• Star, B. et al. (2017) ‘Ancient DNA reveals the Arctic origin of Viking Age cod from Haithabu, Germany’, Proceedings of the National Academy of Sciences of the United States of America, 114(34), pp. 9152–9157. doi: 10.1073/pnas.1710186114.
• Andrades Valtueña, A. et al. (2017) ‘The Stone Age Plague and Its Persistence in Eurasia’, Current biology: CB, 27(23), pp. 3683–3691.e8. doi: 10.1016/j.cub.2017.10.025.

Citation

If you use nf-core/eager for your analysis, please cite the eager preprint as follows:

James A. Fellows Yates, Thiseas Christos Lamnidis, Maxime Borry, Aida Andrades Valtueña, Zandra Fagneräs, Stephen Clayton, Maxime U. Garcia, Judith Neukamm, Alexander Peltzer Reproducible, portable, and efficient ancient genome reconstruction with nf-core/eager bioRxiv 2020.06.11.145615; doi: https://doi.org/10.1101/2020.06.11.145615

You can cite the eager zenodo record for a specific version using the following doi: 10.5281/zenodo.3698082

You can cite the nf-core publication as follows:

The nf-core framework for community-curated bioinformatics pipelines.

Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen.

Nat Biotechnol. 2020 Feb 13. doi: 10.1038/s41587-020-0439-x.
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