Cascabel: A Scalable and Versatile Amplicon Sequence Data Analysis Pipeline Delivering Reproducible and Documented Results

Abstract

Marker gene sequencing of the rRNA operon (16S, 18S, ITS) or cytochrome c oxidase I (CO1) is a popular means to assess microbial communities of the environment, microbiomes associated with plants and animals, as well as communities of multicellular organisms via environmental DNA sequencing. Since this technique is based on sequencing a single gene, or even only parts of a single gene rather than the entire genome, the number of reads needed per sample to assess the microbial community structure is lower than that required for metagenome sequencing. This makes marker gene sequencing affordable to nearly any laboratory. Despite the relative ease and cost-efficiency of data generation, analyzing the resulting sequence data requires computational skills that may go beyond the standard repertoire of a current molecular biologist/ecologist. We have developed Cascabel, a scalable, flexible, and easy-to-use amplicon sequence data analysis pipeline, which uses Snakemake and a combination of existing and newly developed solutions for its computational steps. Cascabel takes the raw data as input and delivers a table of operational taxonomic units (OTUs) or Amplicon Sequence Variants (ASVs) in BIOM and text format and representative sequences. Cascabel is a highly versatile software that allows users to customize several steps of the pipeline, such as selecting from a set of OTU clustering methods or performing ASV analysis. In addition, we designed Cascabel to run in any linux/unix computing environment from desktop computers to computing servers making use of parallel processing if possible. The analyses and results are fully reproducible and documented in an HTML and optional pdf report. Cascabel is freely available at Github: https://github.com/AlejandroAb/CASCABEL.

Microbial Communities on Plastic Polymers in the Mediterranean Sea

“…The raw data were processed using the NIOZ in-house amplicon sequence analysis pipeline “Cascabel” ( Abdala Asbun et al, 2020 ), according to the following specifications. First, quality on the reads was assessed with FastQC 1 , then paired-end reads were merged with PEAR v0.9.10 ( Zhang et al, 2014 ) with a p value set to 0.05 (for the statistical test of a possible assembly) and a minimum overlap of 10 bases.…”

Bacteriohopanetetrol-<i>x</i>: constraining its application as a lipid biomarker for marine anammox using the water column oxygen gradient of the Benguela upwelling system

“…The prokaryotic 16S rRNA gene amplicon sequences were analyzed using the Cascabel data analysis pipeline (Abdala Asbun et al, 2020). Raw forward and reverse reads were merged using PEAR (Zhang et al, 2014).…”

Novel hydrocarbon-utilizing soil mycobacteria synthesize unique mycocerosic acids at a Sicilian everlasting fire

“…Longer-chain fatty acids and branched fatty acids like MAs have been shown to be more resistant than other biomolecules (e.g., short-chain fatty acids) to diagenetic changes in diverse studies of fossil forests, sediment cores from the Gulf of California and petroleum systems (Staccioli et al, 2002;Wenger et al, 2002;Camacho-Ibar et al, 2003). Under the right conditions, fatty acids may be preserved as bound compounds in ancient sediments through the Miocene (Ahmed et al, 2001). Indeed, studies have indicated the presence of MAs of M. tuberculosis on ancient bones from a 17 000-year-old bison and from a ca.…”