Table 1.

Outline of the steps performed by Cascabel. “Script(s)” refers to Cascabel scripts in bash, java or R.

Step	Tools/Algorithms	Output
Initialize structure	Script	Project folder and file structure
Quality Control	FastQC (Andrews, 2010)	FastQC report
Merge reads	PEAR (Zhang et al., 2014)	Merged (assembled) sequences
Demultiplex	QIIME (Caporaso et al., 2010b), scripts	Sequences assigned to samples in one file and per sample
Align vs. reference	Mothur (Schloss et al., 2009)	Aligned sequences
Remove chimeras	usearch61 (Edgar, 2010), Uchime_denovo and uchime_ref (VSEARCH) (Rognes et al., 2016)	Chimera-free sequences
Remove adapters	Cutadapt (Martin, 2011)	Adapter-free sequences
Size filter	Script	Filtered sequences
Dereplicate	VSEARCH	Dereplicated sequences
Generate OTUs	Mothur (Schloss et al., 2009), prefix/suffix (Caporaso et al., 2010b), CD-HIT (Li and Godzik, 2006), SUMACLUST (Kopylova et al., 2016), Swarm (Mahé et al., 2015), UCLUST (Edgar, 2010), trie (Caporaso et al., 2010b) sortmerna (Kopylova et al., 2012)	OTU table
Pick representatives (OTUs)	Random, longest, most_abundant, first	Fasta file with representative sequences
Generate ASVs	DADA2 (Callahan et al., 2016)	ASV table
Assign taxonomy OTUs	QIIME [BLAST (Altschul et al., 1990), UCLUST, RDP (Wang et al., 2007)], blastn (BLAST+) (Camacho et al., 2009), VSEARCH	Taxonomic assignments for each OTU
Assign taxonomy ASVs	RDP	Taxonomic assignments for each ASV
Generate OTU table	QIIME, scripts	Annotated OTU table
Generate ASV table	DADA2	Annotated ASV table
Alignment	Pynast (Caporaso et al., 2010a), mafft (Katoh and Standley, 2013), infernal (Nawrocki and Eddy, 2013), clustalw (Larkin et al., 2007), muscle (Edgar, 2004)	Multiple sequence alignment
Make tree	Muscle, clustalw, raxml (Stamatakis, 2006), fasttree (Price et al., 2009)	Phylogenetic tree
Report	Scripts, Krona (Ondov et al., 2011)	HTML, pdf report, Krona charts