Biases in bulk: DNA metabarcoding of marine communities and the
methodology involved
Abstract
With the growing anthropogenic pressure on marine ecosystems, the need
for efficient monitoring of biodiversity grows stronger. DNA
metabarcoding of bulk samples is increasingly implemented in ecosystem
assessments and is more cost-efficient and less time-consuming than
monitoring based on morphology. However, before raw sequences are
obtained from bulk samples, a profound number of methodological choices
must be made. Here, we critically review the recent methods used for
metabarcoding of marine bulk samples (including benthic, plankton and
diet samples) and indicate how potential biases can be introduced
throughout sampling, pre-processing, DNA extraction, marker and primer
selection, PCR amplification and sequencing. From a total of 64 studies
evaluated, our recommendations for best practices include to (a)
consider DESS as a fixative instead of ethanol, (b) use the DNeasy
PowerSoil kit for any samples containing traces of sediment, (c) not
limit the marker selection to COI only, but preferably include multiple
markers for higher taxonomic resolution, (d) avoid touchdown PCR
profiles, (e) use a fixed annealing temperature for each primer pair
when comparing across studies or institutes, (f) use a minimum of 3 PCR
replicates and (g) include both negative and positive controls. Although
the implementation of DNA metabarcoding still faces several technical
complexities, we foresee wide-ranging advances in the near future,
including improved bioinformatics for taxonomic assignment, sequencing
of longer fragments, and the use of whole-genome information. Despite
the bulk of biases involved in metabarcoding of bulk samples, it is
clear that DNA metabarcoding provides a valuable tool in ecosystem
assessments.