Coming of age for COI metabarcoding of whole organism community DNA: towards bioinformatic harmonisation

Abstract

Metabarcoding of DNA extracted from community samples of whole organisms (whole organism community DNA, wocDNA) is increasingly being applied to terrestrial, marine and freshwater metazoan communities to provide rapid, accurate and high resolution data for novel molecular ecology research. The growth of this field has been accompanied by considerable development that builds on microbial metabarcoding methods to develop appropriate and efficient sampling and laboratory protocols for whole organism metazoan communities. However, considerably less attention has focused on ensuring bioinformatic methods are adapted and applied comprehensively in wocDNA metabarcoding. In this study we examined over 600 papers and identified 111 studies that performed COI metabarcoding of wocDNA. We then systematically reviewed the bioinformatic methods employed by these papers to identify the state-of-the-art. Our results show that the increasing use of wocDNA COI metabarcoding for metazoan diversity is characterised by a clear absence of bioinformatic harmonisation, and the temporal trends show little change in this situation. The reviewed literature showed (i) high heterogeneity across pipelines, tasks and tools used, (ii) limited or no adaptation of bioinformatic procedures to the nature of the COI fragment, and (iii) a worrying underreporting of tasks, software and parameters. Based upon these findings we propose a set of recommendations that we think the wocDNA metabarcoding community should consider to ensure that bioinformatic methods are appropriate, comprehensive and comparable. We believe that adhering to these recommendations will improve the long-term integrative potential of wocDNA COI metabarcoding for biodiversity science.

Community metabarcoding reveals the relative role of environmental filtering and dispersal in metacommunity dynamics of soil microarthropods across a mosaic of montane forests

“…The wide implementation of harmonised field, lab and bioinformatic protocols for community metabarcoding of unexplored assemblages will increase the comparability of datasets from across the globe (Arribas et al, 2021a), providing the basis for broad-scale analyses of metacommunity patterns that would enable drawing more general conclusions on the consistency or context-dependency of ecological processes across spatial scales and fractions of biodiversity. Additionally, the ease with which all species in local communities can be characterised at the population genetic level using metabarcoding with stringent filtering raises the prospect for modelling demographic processes for each of the component species (Overcast, Emerson, & Hickerson, 2019;Overcast et al, 2021). Such an approach has the potential to elucidate historical and contemporaneous community responses to environmental heterogeneity and dispersal limitation at a much finer resolution than the summary statistics currently applied in whole-community metabarcoding.…”

“…Our ASV-level analyses were facilitated by the application of the metamate tool that utilised local and public reference sequence databases to discard non-authentic ASVs and retain only true biological sequence variants (Andújar et al, 2021). Although applying the most stringent filtering in metamate might have caused the removal of valuable rare biological haplotypes, appreciable intraspecific genetic variation (on average 2.35 ASVs per OTU) was still retrieved and produced reasonable haplotype diversity patterns as explained above.…”

“…Based on our results, we advocate stringent ASV filtering, as it can provide informative datasets without compromising the required reliability for haplotype-level metabarcoding. Even if we cannot be fully confident that all erroneous haplotypes were filtered out, as the performance of the approach depends on the completeness of the reference sequence catalogue (Andújar et al, 2021), the future incorporation of intraspecific genetic data in local reference databases will provide further confidence and the opportunities for intra-OTU analyses of ASV variation (Elbrecht et al, 2018;Zizka, Weiss, & Leese, 2020).…”

“…The development of metabarcoding provides new opportunities to accelerate studies on metacommunity structure across underexplored fractions of biodiversity and greatly increases their taxonomic resolution (Arribas, Andújar, Salces-Castellano, Emerson, & Vogler, 2021b;Bush et al, 2020;Martin et al, 2021;Zinger et al, 2019). Recent advances in field, laboratory and bioinformatic protocols for whole organism community DNA (wocDNA, Creedy et al, 2021) metabarcoding have led to improvements in both the efficiency for the generation of such high-resolution taxonomic inventories (Arribas, Andújar, Hopkins, Shepherd, & Vogler, 2016;Elbrecht, Vamos, Steinke, & Leese, 2018), and the reliability of α and β diversity estimates (Andújar et al, 2018;Creedy, Ng, & Vogler et al, 2019). New bioinformatic tools for the removal of noise generated by amplification and sequencing errors (e.g., Callahan et al, 2016;Edgar, 2016) and the filtering of spurious sequences resulting from co-amplification of nuclear mitochondrial pseudogenes (Andújar et al, 2021) allow to move beyond classical OTU (Operational Taxonomic Unit) clustering and define haplotype-level entities, or Amplicon Sequence Variants (ASVs; Callahan, McMurdie, & Holmes, 2017).…”

“…Recent advances in field, laboratory and bioinformatic protocols for whole organism community DNA (wocDNA, Creedy et al, 2021) metabarcoding have led to improvements in both the efficiency for the generation of such high-resolution taxonomic inventories (Arribas, Andújar, Hopkins, Shepherd, & Vogler, 2016;Elbrecht, Vamos, Steinke, & Leese, 2018), and the reliability of α and β diversity estimates (Andújar et al, 2018;Creedy, Ng, & Vogler et al, 2019). New bioinformatic tools for the removal of noise generated by amplification and sequencing errors (e.g., Callahan et al, 2016;Edgar, 2016) and the filtering of spurious sequences resulting from co-amplification of nuclear mitochondrial pseudogenes (Andújar et al, 2021) allow to move beyond classical OTU (Operational Taxonomic Unit) clustering and define haplotype-level entities, or Amplicon Sequence Variants (ASVs; Callahan, McMurdie, & Holmes, 2017). In contrast to OTUs, ASVs have intrinsic biological significance and offer the possibility for direct comparisons among studies that use the same marker (Callahan et al, 2017;Porter & Hajibabaei, 2020), while they potentially improve community diversity estimates (Joos et al, 2020).…”