README GENERAL

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2. Enterovirus Mutation Analysis Pipeline

This pipeline analyzes mutations in enterovirus sequences, offering two modes of analysis: consensus sequence and viral population.

Prerequisites

Ensure that the following programs/packages are installed on your system before running the pipeline:

• nextflow v24.04.3
• mafft v7.520
• seqtk v1.4-r122
• libgcc-ng >= v12
• trimmomatic v0.39
• minimap2 v2.26
• lofreq v2.1.5
• bcftools >= v1.17
• samtools >= v1.17
• minMutFinder >= v1
• python3 & modules:
  • sys
  • re
  • os
  • SeqIO from Bio
  • csv
  • pandas
  • gzip
  • shutil
  • matplotlib
  • seaborn
  • plotly
  • numpy

Ensure that the paths in the nextflow.config are pointing the correct folder.

Input

• Input file: samples-mutations.csv
  • This file is expected to be generated by the enterovirus-genotyping.nf script.
  • It should contain the following columns (without headers):
    • Sample or User directory
    • Protein name (VP1)
    • Path to consensus FASTA file
    • Path to reference FASTA file

Execution

The pipeline supports two types of mutation analysis:

1. Consensus Sequence Analysis:

   • This mode compares the consensus sequence of the sample to a reference sequence.
   • Command: nextflow run mutations-nf/main.nf --file <samples-mutations.csv>

2. Viral Population Analysis:

   • This mode analyzes mutations within the viral population, considering mutation frequency and depth.
   • Command: nextflow run mutations-nf/main.nf --file <samples-mutations.csv> --viral_population "yes"
   • This mode requires that the input fastq files are provided.

Output

Outputs are stored in the results directory within each sample/user's directory.

1. Consensus Sequence Analysis Output (mutations_VP1.csv):

   • Columns:
     • Sample/User ID
     • Protein
     • Mutation type
     • Amino acid change
     • Amino acid property change
     • Nucleotide mutation

2. Viral Population Analysis Output (mutations_VP1.csv):

   • Columns:
     • Sample/User ID
     • Protein
     • Mutation type
     • Amino acid change
     • Amino acid property change
     • Nucleotide mutation
     • Mutation frequency
     • Mutation depth

3. Annotated Mutations Output (Annotated_mutations.csv):

   • If mutations are annotated, this separate file will be generated in the results directory.
   • This file will contain all the columns from the mutations_VP1.csv file, and will contain two additional columns.
     • Annotated
     • Annotated Mutation.

Example Commands

• Consensus Sequence Analysis:

  nextflow run mutations-nf/main.nf --file samples-mutations.csv

• Viral Population Analysis:

  nextflow run mutations-nf/main.nf --file samples-mutations.csv --viral_population "yes"

Notes

• Ensure the samples-mutations.csv file is correctly formatted.
• The Annotated_mutations.csv file will only be generated if mutation annotation is performed.
• The --viral_population "yes" option requires that the fastq files used to create the consensus sequence are available.