C-WAP is a bash-based bioinformatics pipeline for the analysis of either long-read (ONT or PacBio) or short-read (Illumina) whole genome sequencing data of DNA extracted from wastewater. It was developed for SARS-CoV2 and its variants.

C-WAP was developed by the United States Food and Drug Administration, Center for Food Safety and Applied Nutrition.

The CFSAN Wastewater Analysis Pipeline uses a reference-based alignment to create a matrix of SNPs for a given set of samples and estimate the perentage of SC2 variants in the sample

The process includes the following:

1. Designating a reference and NGS data in fastq format
2. Alignment of reads to the reference via Bowtie2
3. Taxonomy check via kraken2
4. Processing of alignment results via samtools
5. Detection of variant positions with ivar
6. Determine composition of variants via kallisto, linear regression, kraken2/bracken and freyja
7. Generate an html and pdf formatted summary of results

• NextFlow v21.0+
• kraken2 v2.1.2
• bracken
• samtools v1.13
• bcftools
• ivar
• bowtie2
• minimap2 v2.22
• edirect
• pangolin
• Freyja
• kallisto
• wkhtmltopdf
• ghostscript

The configuration file prepareEnvironment.sh enumerates the dependencies the other scripts assume that all referred executables are available in the search path.

Download and save

The driver script is startWorkflow.nf and a standard execution with paired end illumina reads would be:
startWorkflow.nf --platform i --primers path/to/bed --in path/to/fastq/ --out path/to/outputDir

C-WAP produces a number of files from the various processing steps.

sorted.stats - Samtools stats output from aligned but untrimmed reads
kallisto.out - Python-parsed summary of the kallisto lineage abundance estimates
deconvolution.output - Output of linear deconvolution method for estimating variant composition
linearDeconvolution_abundance.csv - Linear deconvolution estimates of variant composition
freyja.demix - Lineage abundance estimate generated by Freyja
kallisto_abundance.tsv - Kallisto estimates of variant composition
k2-majorCovid.out - Covid-specific kraken2 output with major lineages identified, against majorCovid DB
k2-majorCovid_bracken.out - Bracken lineage abundance estimates, against majorCovid DB
k2-allCovid.out - Covid-specific kraken2 output, against allcovid DB
k2-allCovid_bracken.out - Bracken lineage abundance estimates, against allCovid DB
pangolin_lineage_report.csv - Pangolin lineage prediction for the consensus sequence
consensus.fa - consensus fasta file generated by ivar
calls.vcf.gz - Variant call file generated by bcftools
pos-coverage-quality.tsv - QC metrics on coverage and quality obtained from the pileup file
rawVarCalls.tsv - Variant calls generated by iVar, vcg equivalent of samtools
k2-std.out - kraken2 output with standard database
report - standalone directory containing html and pdf summary report

Variant composition analyses should be interpreted with caution where they should be treated as suspect if there are substantial gaps in coverage across the reference genome and/or a lack of sequencing depth. The linear deconvolution and kraken2/bracken covid method are internally developed methods and under testing and validation.

This work is currently unpublished. If you are making use of this package, we would appreciate if you gave credit to our repository.

See the LICENSE.txt file included in the C-WAP Pipeline distribution.