artic-mpxv-illumina-nf

A Nextflow pipeline for processing amplicon data generated by Illumina sequencing, with a focus on monkeypox virus (mpxv).

Introduction

This pipeline is based on the BCCDC-PHL/ncov2019-artic-nf pipeline, which is a fork of the connor-lab/ncov2019-artic-nf pipeline. It has been modified to support analysis of MPox virus.

flowchart TD
  ref[reference.fasta]
  primers[primer.bed]
  fastq[fastq]
  fetchHostileReference(fetchHostileReference)
  fastq --> performHostFilter
  fetchHostileReference --> performHostFilter
  performHostFilter(performHostFilter) --> readTrimming(readTrimming)
  readTrimming --> readMapping(readMapping)
  ref --> readMapping(readMapping)
  readMapping(readMapping) --> align_trim(align_trim)
  primers --> align_trim(align_trim)
  align_trim(align_trim) --> callConsensusFreebayes(callConsensusFreebayes)
  callConsensusFreebayes(callConsensusFreebayes) --> alignConsensusToReference(alignConsensusToReference)
  ref --> alignConsensusToReference
  alignConsensusToReference --> consensusAlignment[consensus.aln.fa]
  callConsensusFreebayes --> makeQCCSV
  callConsensusFreebayes --> consensus[consensus.fa]
  callConsensusFreebayes --> variants[variants.vcf]
  callConsensusFreebayes --> Squirrel(Squirrel)
  ref --> makeQCCSV
  primers --> makeQCCSV
  makeQCCSV --> qcCSV(qc.csv)
  makeQCCSV --> depthPNG(depth.png)
  Squirrel --> SquirrelReport(squirrel-report.html)

Loading

Quick-start

nextflow run artic-network/artic-mpxv-illumina-nf --help

Will print up-to-date information on all command-line parameters for the current version.

Credits / Acknowledgements

This pipeline only works due to the ongoing efforts of many people performing the often thankless job of developing and maintaining bioinformatics software, including but not limited to:

• Bwa - Heng Li, et al, citation: Li H. (2013) Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv:1303.3997v2 [q-bio.GN]. (if you use the BWA-MEM algorithm or the fastmap command, or want to cite the whole BWA package)
• Samtools - Heng Li et al, citation: Petr Danecek, James K Bonfield, Jennifer Liddle, John Marshall, Valeriu Ohan, Martin O Pollard, Andrew Whitwham, Thomas Keane, Shane A McCarthy, Robert M Davies, Heng Li. GigaScience, Volume 10, Issue 2, February 2021, giab008, https://doi.org/10.1093/gigascience/giab008
• Bcftools - Heng Li et al, citation: Petr Danecek, James K Bonfield, Jennifer Liddle, John Marshall, Valeriu Ohan, Martin O Pollard, Andrew Whitwham, Thomas Keane, Shane A McCarthy, Robert M Davies, Heng Li. GigaScience, Volume 10, Issue 2, February 2021, giab008, https://doi.org/10.1093/gigascience/giab008
• Hostile - Bede Constantinides et al, citation: Bede Constantinides, Martin Hunt, Derrick W Crook, Hostile: accurate decontamination of microbial host sequences, Bioinformatics, 2023; btad728, https://doi.org/10.1093/bioinformatics/btad728
• BBMap - Bushnell B. - sourceforge.net/projects/bbmap/
• trim_galore - Felix Krueger et al, https://github.com/FelixKrueger/TrimGalore
• ivar - Nathan Grubaugh et al, citation: Grubaugh, N.D., Gangavarapu, K., Quick, J. et al. An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar. Genome Biol 20, 8 (2019). https://doi.org/10.1186/s13059-018-1618-7
• Freebayes - Erik Garrison et al, citation: Garrison E, Marth G. Haplotype-based variant detection from short-read sequencing. arXiv preprint arXiv:1207.3907 [q-bio.GN] 2012
• MAFFT - Kazutakah Katoh et al, citation: Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013 Apr;30(4):772-80. doi: 10.1093/molbev/mst010. Epub 2013 Jan 16. PMID: 23329690; PMCID: PMC3603318.

Special thanks to the following for writing / modifying / maintaining previous versions of this pipeline:

• Matt Bull et al, https://github.com/connor-lab/ncov2019-artic-nf
• Jared Simpson, https://github.com/jts/ncov2019-artic-nf
• Dan Fornika et al, https://github.com/BCCDC-PHL/mpxv-artic-nf

Problems and Solutions

1. Error during mpxvIllumina:sequenceAnalysis:fetchHostileReference step

httpx.HTTPError: Failed to download https://objectstorage.uk-london-1.oraclecloud.com/n/lrbvkel2wjot/b/human-genome-bucket/o/human-t2t-hla.tar. Ensure you are connected to the internet, or provide a valid path to a local index

Currently human read removal is performed with hostile, which downloads an indexed human genome file on the fly. This is almost certainly a network connectivity problem.