HSV1 Reference vs Mutant Analysis

This pipeline analyzes the differences between an HSV1 reference strain (Strain 17) and a mutant strain (McKrae) using the nf-core/viralrecon pipeline. This documentation covers the setup and execution of the analysis pipeline

.
├── analysis.sh        # Main execution script
├── custom.config      # Resource configuration (Memory fixes)
├── samplesheet.csv    # Input file manifest
├── refs/              # Reference genome (HSV-1 Strain 17, Accession: NC_001806.2)
└── data/              # Input FASTQ files (McKrae Strain from Renner et al. 2021)

Setup & Data Preparation

1. Reference Genome

Used HSV-1 Strain 17 (Accession: NC_001806.2).

mkdir -p refs
cd refs
wget -O wildtype.fasta "https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?db=nuccore&id=NC_001806.2&rettype=fasta&retmode=text"
cd ..

2. Test Data (McKrae Strain)

Subsampled dataset was used (~100k reads) derived from the full shotgun sequencing run (SRR13801763). This ensures rapid testing while validating the variant calling logic.

Commands to generate the mini-dataset:

mkdir -p data
cd data

# Stream and subsample the top 400,000 lines (100k reads)
curl -L "https://ftp.sra.ebi.ac.uk/vol1/fastq/SRR138/063/SRR13801763/SRR13801763_1.fastq.gz" | zcat | head -n 400000 | gzip > McKrae_Sub_R1.fastq.gz

curl -L "https://ftp.sra.ebi.ac.uk/vol1/fastq/SRR138/063/SRR13801763/SRR13801763_2.fastq.gz" | zcat | head -n 400000 | gzip > McKrae_Sub_R2.fastq.gz
cd ..

Running the Pipeline

Prerequisites

• Nextflow
• Installation Guide
• Singularity singularity was installed using ubuntu official universe repository

sudo apt update
sudo apt install singularity-container

Execution

The analysis is controlled by analysis.sh. It uses a custom.config file to optimize memory usage for local execution (preventing Bowtie2 memory crashes).

# 1. Make executable
chmod +x analysis.sh

# 2. Run
./analysis.sh

Analysis Command Details

The analysis.sh script executes the following Nextflow command. Several packages (Assembly, Kraken2, etc.) were skipped for a faster test run as this was a test.

nextflow run nf-core/viralrecon \
    -profile singularity \
    -c custom.config \
    --input samplesheet.csv \
    --outdir ./results_test_run \
    --platform illumina \
    --protocol metagenomic \
    --genome 'refs/wildtype.fasta' \
    --skip_assembly \
    --skip_kraken2 \
    --skip_pangolin \
    --skip_nextclade \
    --skip_freyja \
    --variant_caller bcftools \
    

Results

Outputs are stored in the results_test_run/ directory.

• Quality Control: results_test_run/multiqc/multiqc_report.html
• Variants (SNPs): results_test_run/variants/bcftools/McKrae_Sub.vcf.gz
  • Contains the list of mutations distinguishing McKrae from Strain 17.
• Mapping: results_test_run/mapping/bowtie2/McKrae_Sub.bam

Troubleshooting

Memory Error: If the pipeline fails at BOWTIE2_BUILD with "Process requirement exceeds available memory", ensure custom.config is present and correctly referenced in the run command.