ANOMALY: AmplicoN wOrkflow for Microbial community AnaLYsis

Abstract

Bioinformatic tools for amplicon sequencing data analysis are continuously and rapidly evolving, thus integrating We present an R workflow for 16S and ITS amplicons based sequencing. It is mainly based on the Dada2 and Phyloseq R packages. This workflow is based on several scripts in order to perform an analysis from fastq sequence files to final statistical analysis. The objective was to automate bioinformatic analyses to ensure reproducibility between projects trying to be versatile and simple to integrate new bioinformatic tools or statistical techniques.ANOMALY use Amplicon Sequence Variant (ASV from Dada2 package) as taxonomic unit, allowing an easy and relevant sequence tracking between different environments and/or projects. Decontam package is included for an accurate and consistent detection of contaminant ASV and taxonomic assignment step relies on IDTAXA method. Our workflow is able to merge and check annotations from two taxonomic databases to unravel misannotation, discordance or inconsistency. The well known Phyloseq package provides the most common graphical representation, with additional statistics to assess significant impact of tested factors on microbial communities. The workflow incorporate multiple differential analyses (DESeq2 etc...) to reveal thin community contrast between conditions. Finally we are able to combine those results for cross-validation and thinner interpretation.ANOMALY is a simple and customizable R workflow, that uses ASVs level for community characterization and integrates all assets of the up-to-date methods such as better sequence tracking, decontamination, merged taxonomic annotation, statistical tests, and cross-validated differential analysis