A practical guide for biologists, bioinformaticians and statisticians wishing to learn about metabarcoding analyses

Abstract

Metabarcoding analysis methods (also known as gene-based metagenomics or amplicon) are more and more used to study the diversity of species in ecosystems (microorganisms, plants, animals). DNA from an environmental sample is extracted, then a targeted fragment is amplified by PCR with a predefined couple of primers. After the addition of barcodes (unique nucleotides for each sample) and sequencing adapters, the PCR products are sequenced. After sequencing, reads are separated by sample by using the barcodes, and are assigned to taxa after a comparison with reference sequences. A lot of methods and analysis tools have been developed to obtain the most accurate view of the studied ecosystems. Preparation and sample analysis techniques depend on the ecosystem, the research issue and the sequencing technology used. We propose some advice taken from our experience, discussions and bibliographic reading so as to guide readers from the experimental planning to data analysis detailing some watchfulness at each step.