omics.co.in logo omics.co.in

14. Viral Pathogen Surveillance, Consensus & Clade Phylodynamics

⚡ Pipeline 14 of 16  ·  🧬 Pathogen Genomics & Virology

Multiplexed tiled amplicon sequencing for respiratory, zoonotic, and emerging viruses, trimming primer binding sites with iVar, generating consensus FASTA sequences, and clade classification via Nextclade and Pangolin.

🔍 Ctrl + K
Pathogen • Virology Authority: ARTIC Network / Nextstrain / CDC
Production Blueprint v4.5

14. Viral Pathogen Surveillance, Consensus & Clade Phylodynamics

Multiplexed tiled amplicon sequencing for respiratory, zoonotic, and emerging viruses, trimming primer binding sites with iVar, generating consensus FASTA sequences, and clade classification via Nextclade and Pangolin.

Metric / RequirementSpecificationTarget Configuration
Core Engine / FrameworkBWA-MEM + iVar + Nextclade + FreyjaNextflow / Snakemake Compatible
Benchmark Runtime~30 – 60 minutes per batch8 CPU cores / 16 GB RAM
Standard Containerquay.io/biocontainers/ivar:1.4.2--h99489fc_1Docker • Singularity • Conda
Input → DeliverableAmplicon FASTQ → Consensus FASTA, Variant TSV, Nextclade Clade Assignment
Workflow Chain: Amplicon FASTQ → BWA-MEM → iVar trim (primer removal) → iVar consensus → Nextclade CLI → Pangolin lineage
⚡

Step 1: Align Tiled Amplicon Reads to Viral Reference Genome

BWA-MEM

Maps paired reads to viral reference (e.g. NC_045512.2).

bwa mem -t 8 /references/viral_ref.fasta viral_R1.fq.gz viral_R2.fq.gz | \
    samtools sort -@ 4 -o viral_mapped.bam -
samtools index viral_mapped.bam
Key Flags & Notes: Fast alignment preserving 5' primer mapping coordinates.
⚡

Step 2: Trim Amplicon Primer Binding Sites with iVar

iVar trim

Soft-clips tiled PCR primer sequences based on primer BED scheme to prevent artificial wild-type calls.

ivar trim -b /references/primer_scheme_v4.bed -p viral_trimmed.bam -i viral_mapped.bam -q 15 -m 30 -e
samtools sort -@ 4 viral_trimmed.bam -o viral_trimmed.sorted.bam
samtools index viral_trimmed.sorted.bam
Key Flags & Notes: -e includes reads without primers; prevents primer mismatch sequence corruption.
⚡

Step 3: Call SNVs & Generate Majority Consensus Sequence

Samtools mpileup • iVar consensus

Calculates base pileups and creates consensus FASTA with Ns at unsequenced sites (<10x depth).

samtools mpileup -aa -A -d 0 -B -Q 0 viral_trimmed.sorted.bam | \
    ivar consensus -p sample_consensus -q 20 -t 0.6 -m 10 -n N
Key Flags & Notes: -t 0.6 sets threshold for consensus calling (60% majority); -m 10 sets minimum depth.
⚡

Step 4: Global Clade & Mutation Assignment with Nextclade CLI

Nextclade CLI

Assigns WHO clade, identifies private mutations, and scores overall sequence quality.

nextclade run --input-dataset /references/nextclade_dataset/ \
              --output-csv nextclade_clade_report.csv \
              --output-tree nextclade_tree.json \
              sample_consensus.fa
Key Flags & Notes: Outputs clade classification and alignment quality scores for global surveillance.

🚀 Automated Nextflow (nf-core) Production Execution

Execute this complete workflow across any SLURM, AWS Batch, or Google Cloud HPC cluster using the verified community container pipeline:

nextflow run nf-core/viralrecon -r 2.6.0 \
  --input samplesheet.csv \
  --platform illumina \
  --protocol amplicon \
  --genome 'MN908947.3' \
  --outdir ./results_viral/ \
  -profile docker
← Return to Home Hub