Comparative Evaluation of Whole-Capsid NGS and Traditional GPLN Methods in Polio Surveillance

The repository implements the Illumina whole-capsid approach after culture and provides comparable outputs to conventional GPLN.

Program summary

This repository contains one end to end pipeline that can reproduce the analyses reported in the manuscript. It supports the following components under one roof:

• Inputs
  • Paired end FASTQ from cultured isolates prepared for whole capsid sequencing on Illumina MiSeq 2x150.
• Quality control and trimming
  • fastp for adapter and quality trimming with default Q30 sliding window and minimum length 50.
• Mapping and primer trimming
  • BWA MEM mapping to references for WPV1 and Sabin strains.
  • BAMClipper to remove primer sequences when amplicon primers are used.
  • Picard MarkDuplicates to mark PCR duplicates.
• Coverage and consensus
  • BEDTools genomecov to compute depth profiles.
  • FreeBayes variant calling followed by BCFtools consensus with a depth mask. Default minimum depth 10.
• Antigenic site analysis
  • Translate VP1, VP2, VP3. Summarize amino acid changes in canonical antigenic sites Ag1, Ag2, Ag3. Output tidy TSV reports per sample.
• Phylogeny and context
  • Fetch selected context references from GenBank.
  • MAFFT alignment of masked consensuses plus context.
  • IQ-TREE ML tree with 1000 ultrafast bootstraps. ModelFinder can be toggled on.
• Statistics
  • Optional scripts to compute McNemar test and paired t test when you provide detection tables and per site identities from Sanger vs NGS.
• Optional de novo assembly
  • SPAdes assembly and contig QC, BLAST for sanity checking and for identifying best reference when needed.

The workflow is implemented in Snakemake and can be run on a laptop or a small server. No clinical data are included.

Requirements

• Python 3.11 or newer
• Option A: pip and virtualenv
• Option B: conda or mamba
• Snakemake 7 or newer for the full pipeline
• System tools installed through conda: fastp, bwa, samtools, bcftools, freebayes, bedtools, picard, bamclipper, mafft, iqtree, spades, seqkit, entrez-direct

NCBI usage note

Set a contact email once per shell for E-utilities. Optional API key improves rate limits.

export NCBI_EMAIL="[email protected]"
export NCBI_API_KEY="xxxxxxxxxxxxxxxxxxxxxxxxxxxx"   # optional

Quick verification

python -m venv .venv
source .venv/bin/activate
python -m pip install -r env/requirements.txt
python analysis/scripts/example_qc_plot.py --in data-example/example_counts.tsv --out results-example/example_plot.png

One command run

export NCBI_EMAIL="[email protected]"
conda env create -f env/environment.yml
conda activate polio-capsid-env
snakemake -s workflow/Snakefile -c 4 --printshellcmds

Configuration

Edit config/config.yaml. Minimal example:

pairs:
  - sample: PV1_001
    r1: data-private/PV1_001_R1.fastq.gz
    r2: data-private/PV1_001_R2.fastq.gz

references:
  wpv1: KY941935.1
  sabin1: V01150.1
  sabin2: AY184220.1
  sabin3: AY184221.1

context_acc:
  - KY941935.1
  - V01150.1
  - AY184220.1
  - AY184221.1

params:
  threads: 4
  min_len: 50
  min_qual: 20
  min_depth_consensus: 10
  use_model_finder: false
  iqtree_model: GTR+G+I
  bootstrap: 1000

Outputs

• results/consensus/<sample>.fa - masked consensus per sample
• results/consensus/all_consensus.fasta - combined consensuses
• results/aln/wg_alignment.fasta - alignment used for phylogeny
• results/iqtree/wg.treefile - ML tree
• results/coverage/<sample>.depth.txt - per base depth
• results/mutations/<sample>_ag_sites.tsv - amino acid changes in Ag1, Ag2, Ag3
• Optional de novo outputs under results/spades/<sample>/ with QC tables and BLAST hits

How to cite

• Software: Haider SA. Polio whole-capsid post-culture NGS analysis pipeline. Version 1.0.0. 2025. GitHub repository.

References

• Chen S, Zhou Y, Chen Y, et al. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018.
• Li H. Aligning sequence reads with BWA-MEM. 2013. arXiv:1303.3997.
• Danecek P, et al. Twelve years of SAMtools and BCFtools. GigaScience. 2021.
• Garrison E, Marth G. Haplotype-based variant detection from short-read sequencing. arXiv:1207.3907. FreeBayes.
• Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010.
• Picard Toolkit. Broad Institute. 2019.
• Au CH, Ho DN, Kwong A, et al. BAMClipper. Sci Rep. 2017.
• Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7. Mol Biol Evol. 2013.
• Minh BQ, et al. IQ-TREE 2. Mol Biol Evol. 2020.
• Bankevich A, et al. SPAdes. J Comput Biol. 2012.
• Cock PJ, et al. Biopython. Bioinformatics. 2009.
• Kans J. Entrez Programming Utilities Help. NCBI.
• Köster J, Rahmann S. Snakemake. Bioinformatics. 2012.
• Minor PD, Ferguson M, Evans DM, et al. Antigenic structure of polioviruses. J Gen Virol. 1986.
• Hogle J, Chow M, Filman D. Three-dimensional structure of poliovirus at 2.9 Å resolution. Science. 1985.

Contributing

See CONTRIBUTING.md. Open an issue for questions. Do not commit restricted data.

License

MIT. See LICENSE for details.