nfcore/eager repository overview

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 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:

⁠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)

⁠Genotyping

• 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
   

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4. Test the pipeline using the provided test data

    nextflow run nf-core/eager -profile <docker/singularity/conda>,test
   

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5. Start running your own ancient DNA analysis!

    nextflow run nf-core/eager -profile <docker/singularity/conda> --input '*_R{1,2}.fastq.gz' --fasta '<your_reference>.fasta'
   

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6. Once your run has completed successfully, clean up the intermediate files.

    nextflow clean -f -k
   

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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. EAGER2 Code Contribution Guidelines⁠
6. nf-core/nextflow Troubleshooting⁠
7. EAGER Troubleshooting⁠

⁠Credits

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)

• 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

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