Beyond taxonomy: Validating functional inference approaches in the
context of fish-farm impact assessments
Abstract
Characterization of microbial assemblages via environmental DNA
metabarcoding is increasingly being used in routine monitoring programs
due to its sensitivity and cost-effectiveness. Several programs have
been developed recently which infer functional profiles from 16S rRNA
gene data using hidden-state prediction (HSP) algorithms. These might
offer an economic and scalable alter-native to shotgun metagenomics. To
date, HSP-based methods have seen limited use for benthic marine surveys
and their performance in these environments remains unevaluated. In this
study, 16S rRNA metabarcoding was applied to sediment samples collected
at 0 and ≥ 1200 m from Norwegian salmon farms, and three metabolic
inference approaches (PAPRICA, PICRUSt2 and TAX4FUN2) evaluated against
metagenomics and environmental data. While metabarcoding and
metagenomics recovered a comparable functional diversity, the taxonomic
composition differed be-tween approaches, with genera richness up to 20×
higher for metabarcoding. Comparisons between the sensitivity (highest
true positive rates) and specificity (lowest true negative rates) of
HSP-based programs in detecting functions found in metagenomics data
ranged, respectively, from 0.52 and 0.60 to 0.76 and 0.79. However,
little correlation was observed between the relative abundance of their
specific functions. Functional beta-diversity of HSP-based data was
strongly associated with that of metagenomics (r ≥ 0.86 for PAPRICA and
TAX4FUN2) and responded similarly to the impact of fish farm activities.
Our results demonstrate that although HSP-based metabarcoding approaches
provide a slightly different functional profile than metagenomics,
partly due to recovering a distinct community, they represent a
cost-effective and valuable tool for characterizing and assessing the
effects of fish farming on benthic ecosystems.