Table 2.

Collection of bioinformatics methods used for SARS-CoV-2 wastewater sequencing data (WWS data) analysis. This overview comprises tools that were developed for the general analysis of sequencing data, adapted for WWS data, and specifically developed for WWS. The overview is not exhaustive but provides a starting point for most representative and popular bioinformatics tools for WWS data analytics

	Software	Source
Data QC, primer trimming trimming and adapter removal, paired-end reads merging	BBMap (BBDuk) [20, 27, 53, 61, 90, 166]	https://github.com/BioInfoTools/BBMap
	FastQC [64, 167, 168]	https://github.com/s-andrews/FastQC
	MultiQC [31, 63, 169, 170]	https://github.com/MultiQC/MultiQC
	iVAR [19, 20, 39, 56, 57, 65, 67, 90, 167, 169–173]	https://github.com/andersen-lab/ivar
	fastp [31, 169–171, 174]	https://github.com/OpenGene/fastp
	PRINSEQ-lite [175]	https://github.com/uwb-linux/prinseq
	Trim Galore [56, 61]	https://github.com/FelixKrueger/TrimGalore
	Trimmomatic [48, 55, 62–64, 67, 176]	https://github.com/usadellab/Trimmomatic
	cutadapt [27, 54, 58, 167, 176–178]	https://github.com/marcelm/cutadapt
	Primerclip [62, 166]	https://github.com/swiftbiosciences/primerclip
	BBTools [20, 27, 53, 54, 90, 166, 167]	https://github.com/kbaseapps/BBTools
	BAMClipper [179]	https://github.com/tommyau/bamclipper
Read mapping	BWA-MEM [20, 53, 55, 57, 58, 61, 62, 64, 65, 67, 68, 90, 166, 167, 169, 173, 174]	https://github.com/lh3/bwa
	BWA-MEM2	https://github.com/bwa-mem2/bwa-mem2
	Minimap2 [19, 27, 31, 58, 154, 172, 178, 180]	https://github.com/lh3/minimap2
	Bowtie2 [48, 71, 171, 177, 181]	https://github.com/BenLangmead/bowtie2
SNV calling	iVAR [19, 20, 39, 57, 58, 65, 69, 171, 178, 182]	https://github.com/andersen-lab/ivar
	LoFreq [20, 39, 53, 61, 64, 90, 169, 173]	https://github.com/CSB5/lofreq
	FreeBayes [62, 172]	https://github.com/freebayes/freebayes
	BCFTools [53, 55, 62, 64]	https://github.com/samtools/bcftools
	V-PIPE [27, 61]	https://github.com/cbg-ethz/V-pipe
	Nanopolish [11]	https://github.com/jts/nanopolish
	VarScan [56, 68, 170, 183]	https://github.com/dkoboldt/varscan
	GATK [166]	https://github.com/broadinstitute/gatk
	SAMtools [54, 56, 61, 183]	https://github.com/samtools/
	Medaka [184]	https://github.com/nanoporetech/medaka
Relative abundance estimation (LRA), allele frequency estimation (AF), lineage detection (LD)	Freyja (LRA) [19]	https://github.com/andersen-lab/Freyja
	QualD (LD) [20]	https://gitlab.com/treangenlab/quaid
	SAM Refiner (AF) [27, 61, 177]	https://github.com/degregory/SAM_Refiner
	Kallisto-VLQ (LRA) [67]	https://github.com/baymlab/wastewater_analysis
	iVAR (AF) [57, 182]	https://github.com/andersen-lab/ivar
	VaQuERo (LRA) [53]	https://github.com/fabou-uobaf/VaQuERo
	Cojac (LD) [52, 56]	https://github.com/cbg-ethz/cojac
	Lineagespot (AF) [172]	https://github.com/BiodataAnalysisGroup/lineagespot
	LCS (LRA) [185]	https://github.com/rvalieris/LCS
De novo assembly	SOAPdenovo	https://github.com/aquaskyline/SOAPdenovo2
	Velvet	https://github.com/dzerbino/velvet
	ABySS	https://github.com/bcgsc/abyss
	IBDA	https://github.com/loneknightpy/idba
	SPAdes [186]	https://github.com/ablab/spades
	Edena	https://github.com/ddhz/edena
	Vicuna	https://www.broadinstitute.org/viral-genomics/vicuna
	Haploflow	https://github.com/hzi-bifo/Haploflow
	Flye	https://github.com/fenderglass/Flye
Lineage and clade assignment, Phylogeny	IQ-Tree2 [11, 19, 178]	https://github.com/Cibiv/IQ-TREE
	Pangolin [53, 65, 171, 175, 187–189]	https://github.com/stevenlovegrove/Pangolin
	NextClade [58, 168, 173, 188]	https://github.com/nextstrain/nextclade
	UShER [170, 173]	https://github.com/yatisht/usher
Pipelines	COVID-19 VIral Epidemiology Workflow (C-VIEW) [19]	https://github.com/ucsd-ccbb/C-VIEW
	CFSAN Wastewater Analysis Pipeline (C-WAP) [190]	https://github.com/CFSAN-Biostatistics/C-WAP
	PiGx SARS-CoV-2 Wastewater Sequencing Pipeline [169]	https://github.com/BIMSBbioinfo/pigx_sars-cov-2
	VLQ-n [67]	https://github.com/rki-mf1/vlq-nf