Putting COI Metabarcoding in Context: The Utility of Exact Sequence Variants (ESVs) in Biodiversity Analysis

Porter, Teresita M.; Hajibabaei, Mehrdad

doi:10.3389/fevo.2020.00248

Cited by 41 publications

(46 citation statements)

References 118 publications

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“…A recently debated matter concerns the treatment of reads by denoising procedures or by clustering techniques [ 7 ]. Both methods are often presented as alternative approaches to the same process (e.g., [ 7 – 11 ]). However, both are philosophically and analytically different [ 12 ].…”

Section: Introductionmentioning

confidence: 99%

To denoise or to cluster, that is not the question: optimizing pipelines for COI metabarcoding and metaphylogeography

et al. 2021

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Background The recent blooming of metabarcoding applications to biodiversity studies comes with some relevant methodological debates. One such issue concerns the treatment of reads by denoising or by clustering methods, which have been wrongly presented as alternatives. It has also been suggested that denoised sequence variants should replace clusters as the basic unit of metabarcoding analyses, missing the fact that sequence clusters are a proxy for species-level entities, the basic unit in biodiversity studies. We argue here that methods developed and tested for ribosomal markers have been uncritically applied to highly variable markers such as cytochrome oxidase I (COI) without conceptual or operational (e.g., parameter setting) adjustment. COI has a naturally high intraspecies variability that should be assessed and reported, as it is a source of highly valuable information. We contend that denoising and clustering are not alternatives. Rather, they are complementary and both should be used together in COI metabarcoding pipelines. Results Using a COI dataset from benthic marine communities, we compared two denoising procedures (based on the UNOISE3 and the DADA2 algorithms), set suitable parameters for denoising and clustering, and applied these steps in different orders. Our results indicated that the UNOISE3 algorithm preserved a higher intra-cluster variability. We introduce the program DnoisE to implement the UNOISE3 algorithm taking into account the natural variability (measured as entropy) of each codon position in protein-coding genes. This correction increased the number of sequences retained by 88%. The order of the steps (denoising and clustering) had little influence on the final outcome. Conclusions We highlight the need for combining denoising and clustering, with adequate choice of stringency parameters, in COI metabarcoding. We present a program that uses the coding properties of this marker to improve the denoising step. We recommend researchers to report their results in terms of both denoised sequences (a proxy for haplotypes) and clusters formed (a proxy for species), and to avoid collapsing the sequences of the latter into a single representative. This will allow studies at the cluster (ideally equating species-level diversity) and at the intra-cluster level, and will ease additivity and comparability between studies.

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Section: Introductionmentioning

confidence: 99%

To denoise or to cluster, that is not the question: optimizing pipelines for COI metabarcoding and metaphylogeography

et al. 2021

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“…While 97% identity is considered the default threshold for delineating taxonomic units from DNA barcodes (Alberdi et al, 2018;Porter & Hajibabaei, 2020), our analyses demonstrate that a more stringent 98% or 99% identification threshold not only increases the number of insects that can successfully be differentiated, but also reduces the amplicon length required to do so. This is particularly notable for implementing metabarcoding on production scale HTS platforms such as the Illumina NovaSeq, which offer the lowest cost per sample but require much shorter amplicons due to their typical read lengths of only 2 × 150 bp (Piper et al, 2019).…”

Section: Discussionmentioning

confidence: 88%

“…This is particularly notable for implementing metabarcoding on production scale HTS platforms such as the Illumina NovaSeq, which offer the lowest cost per sample but require much shorter amplicons due to their typical read lengths of only 2 × 150 bp (Piper et al, 2019). While use of more stringent identification thresholds has been constrained by the common practice of clustering metabarcoding reads to resolve sequencing errors (Porter & Hajibabaei, 2020), recent denoising algorithms provide single nucleotide resolution that can be leveraged for more accurate and reproducible taxonomic assignment (Callahan et al, 2017;Porter & Hajibabaei, 2020). Nevertheless, the metric of identification success used within our study (proportion of clusters containing only a single species) does not consider the actual availability of reference sequences to match unknown taxa against, and false negatives may be introduced when using these more stringent thresholds if intraspecific diversity isn't sufficiently represented in the reference database.…”

Section: Discussionmentioning

confidence: 99%

Computational Evaluation of DNA Metabarcoding for Universal Diagnostics of Invasive Insect Pests

Piper

Cogan

Cunningham

et al. 2021

Preprint

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Appropriate design and selection of PCR primers plays a critical role in determining the sensitivity and specificity of a metabarcoding assay. Despite several studies applying metabarcoding to insect pest surveillance, the diagnostic performance of the short "mini-barcodes" required by high-throughput sequencing platforms has not been established across the broader taxonomic diversity of invasive insects. We address this by computationally evaluating the diagnostic sensitivity and predicted amplification bias for 68 published and novel cytochrome c oxidase subunit 1 (COI) primers on a curated database of 110,676 insect species, including 2,625 registered on global invasive species lists. We find that mini-barcodes between 125-257 bp can provide comparable resolution to the full-length barcode for both invasive insect pests and the broader Insecta, conditional upon the subregion of COI targeted and the genetic similarity threshold used to identify species. Taxa that could not be identified by any barcode lengths were phylogenetically clustered within "problem groups", many arising through taxonomic inconsistencies rather than insufficient diagnostic information within the barcode itself. Substantial variation in predicted PCR bias was seen across published primers, with those including 4-5 degenerate nucleotide bases showing almost no mismatch to major insect orders. While not completely universal, a single COI mini-barcode can successfully differentiate the majority of pest and non-pest insects from their congenerics, even at the small amplicon size imposed by 2 x 150 bp sequencing. We provide a ranked summary of high-performing primers and discuss the bioinformatic steps required to curate reliable reference databases for metabarcoding studies.

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“…A general obstacle in interpreting and communicating studies based on massive parallel sequencing of genetic markers (DNA metabarcoding) or shotgun sequencing of metagenomes, has been-and still is-a large fraction of terminal taxa (OTUs) that usually cannot be assigned to formally described species. The UNITE and BOLD concepts have greatly benefited the communication of terminal taxa (OTUs) in metabarcoding datasets of fungi and animals, respectively (e.g., [61,62]), by providing a possibility of connecting OTUs to unambiguous SHs or BINs via sequence comparison. For fungi, the UNITE TH system enables the construction of taxon-level traits based on metadata associated with sequence accessions in taxonomic and molecular identification studies [63].…”

Section: Th Paradigm and Metagenomicsmentioning

confidence: 99%

The Taxon Hypothesis Paradigm—On the Unambiguous Detection and Communication of Taxa

et al. 2020

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Here, we describe the taxon hypothesis (TH) paradigm, which covers the construction, identification, and communication of taxa as datasets. Defining taxa as datasets of individuals and their traits will make taxon identification and most importantly communication of taxa precise and reproducible. This will allow datasets with standardized and atomized traits to be used digitally in identification pipelines and communicated through persistent identifiers. Such datasets are particularly useful in the context of formally undescribed or even physically undiscovered species if data such as sequences from samples of environmental DNA (eDNA) are available. Implementing the TH paradigm will to some extent remove the impediment to hastily discover and formally describe all extant species in that the TH paradigm allows discovery and communication of new species and other taxa also in the absence of formal descriptions. The TH datasets can be connected to a taxonomic backbone providing access to the vast information associated with the tree of life. In parallel to the description of the TH paradigm, we demonstrate how it is implemented in the UNITE digital taxon communication system. UNITE TH datasets include rich data on individuals and their rDNA ITS sequences. These datasets are equipped with digital object identifiers (DOI) that serve to fix their identity in our communication. All datasets are also connected to a GBIF taxonomic backbone. Researchers processing their eDNA samples using UNITE datasets will, thus, be able to publish their findings as taxon occurrences in the GBIF data portal. UNITE species hypothesis (species level THs) datasets are increasingly utilized in taxon identification pipelines and even formally undescribed species can be identified and communicated by using UNITE. The TH paradigm seeks to achieve unambiguous, unique, and traceable communication of taxa and their properties at any level of the tree of life. It offers a rapid way to discover and communicate undescribed species in identification pipelines and data portals before they are lost to the sixth mass extinction.

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Putting COI Metabarcoding in Context: The Utility of Exact Sequence Variants (ESVs) in Biodiversity Analysis

Cited by 41 publications

References 118 publications

To denoise or to cluster, that is not the question: optimizing pipelines for COI metabarcoding and metaphylogeography

To denoise or to cluster, that is not the question: optimizing pipelines for COI metabarcoding and metaphylogeography

Computational Evaluation of DNA Metabarcoding for Universal Diagnostics of Invasive Insect Pests

The Taxon Hypothesis Paradigm—On the Unambiguous Detection and Communication of Taxa

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