Best practice recommendations for sample preservation in metabarcoding studies: a case study on diatom environmental samples

Abstract

The development of DNA metabarcoding and High-Throughput Sequencing for diatoms is nowadays offering an interesting approach to assess their communities in freshwater and marine ecosystems. In the context of the implementation of these genomic methods to environmental monitoring, protocol constraints are moving from scientific to operational applications, requiring operational guidelines and standards. In particular, the first steps of the diatom metabarcoding process, which consist of sampling and storage, have been addressed in various ways in scientific and pilot studies. The objective of this study was to compare three currently applied preservation protocols through different storage durations (ranging from one day to one year) for phytobenthos and phytoplankton samples intended for diatom DNA metabarcoding analysis. The experimental design included four freshwater and two marine samples from sites of diverse ecological characteristics. The impact of the preservation and storage was assessed through diatom metabarcoding endpoints: DNA quality and quantity, diversity and richness, community composition and ecological index values (for freshwater samples). The yield and quality of extracted DNA only decreased for freshwater phytobenthos samples preserved with ethanol. Diatom diversity was not affected and their taxonomic composition predominantly reflects the site origin. Only rare taxa (below 100 reads) differed among methods and durations. Thus, importance of preservation method choice is important for low-density species (rare, invasive, threatened or toxic species). However, for biomonitoring purposes, freshwater ecological index values were not affected whatever the preservation method and duration considered (including ethanol preservation), reflecting the site ecological status. This study proved that diatom metabarcoding is robust enough to replace or complement the current approach based on morphotaxonomy, paving the way to new possibilities for biomonitoring. Thus, accompanied by operational standards, the method will be ready to be confidently deployed and prescribed in future regulatory monitoring.