An official website of the United States government
ABSTRACT In metabarcoding studies, Linnaean taxonomy assignments of Operational Taxonomic Units (OTUs) or Amplicon Sequence Variants (ASVs) underpin many downstream bioinformatics analyses and ecological interpretations of environmental DNA (eDNA) datasets. However, public molecular databases (i.e., SILVA, EUKARYOME, BOLD) for most microbial metazoan phyla (nematodes, tardigrades, kinorhynchs, etc.) are sparsely populated, negatively impacting our ability to assign ecologically meaningful taxonomy to these understudied groups. Additionally, the choice of bioinformatics parameters and computational algorithms can further affect the accuracy of eDNA taxonomy assignments. Here, we use twoin silicodatasets to show that taxonomy assignments using the 18S rRNA gene can be dramatically improved by curating Linnaean taxonomy strings associated with each reference sequence and closing phylogenetic gaps by improving taxon sampling. Using free‐living nematodes as a case study, we applied two commonly used taxonomy assignment algorithms (BLAST+ and the QIIME2 Naïve Bayes classifier) across six iterations of the SILVA 138 reference database to evaluate the precision and accuracy of taxonomy assignments. The BLAST+ top hit with a 90% sequence similarity cutoff often returned the highest percentage of correctly assigned taxonomy at the genus level, and the QIIME2 Naïve Bayes classifier performed similarly well when paired with a reference database containing corrected taxonomy strings. Our results highlight the urgent need for phylogenetically informed expansions of public reference databases (encompassing both genomes and common gene markers), focused on poorly sampled lineages that are now robustly recovered via eDNA metabarcoding approaches. Additional taxonomy curation efforts should be applied to popular reference databases such as SILVA, and taxon sampling could be rapidly improved by more frequent incorporation of newly published GenBank sequences linked to genus‐ and/or species‐level identifications.
more »
« less
Unambiguous identification of fungi: where do we stand and how accurate and precise is fungal DNA barcoding?
ABSTRACT True fungi (Fungi) and fungus-like organisms (e.g.Mycetozoa,Oomycota) constitute the second largest group of organisms based on global richness estimates, with around 3 million predicted species. Compared to plants and animals, fungi have simple body plans with often morphologically and ecologically obscure structures. This poses challenges for accurate and precise identifications. Here we provide a conceptual framework for the identification of fungi, encouraging the approach of integrative (polyphasic) taxonomy for species delimitation, i.e. the combination of genealogy (phylogeny), phenotype (including autecology), and reproductive biology (when feasible). This allows objective evaluation of diagnostic characters, either phenotypic or molecular or both. Verification of identifications is crucial but often neglected. Because of clade-specific evolutionary histories, there is currently no single tool for the identification of fungi, although DNA barcoding using the internal transcribed spacer (ITS) remains a first diagnosis, particularly in metabarcoding studies. Secondary DNA barcodes are increasingly implemented for groups where ITS does not provide sufficient precision. Issues of pairwise sequence similarity-based identifications and OTU clustering are discussed, and multiple sequence alignment-based phylogenetic approaches with subsequent verification are recommended as more accurate alternatives. In metabarcoding approaches, the trade-off between speed and accuracy and precision of molecular identifications must be carefully considered. Intragenomic variation of the ITS and other barcoding markers should be properly documented, as phylotype diversity is not necessarily a proxy of species richness. Important strategies to improve molecular identification of fungi are: (1) broadly document intraspecific and intragenomic variation of barcoding markers; (2) substantially expand sequence repositories, focusing on undersampled clades and missing taxa; (3) improve curation of sequence labels in primary repositories and substantially increase the number of sequences based on verified material; (4) link sequence data to digital information of voucher specimens including imagery. In parallel, technological improvements to genome sequencing offer promising alternatives to DNA barcoding in the future. Despite the prevalence of DNA-based fungal taxonomy, phenotype-based approaches remain an important strategy to catalog the global diversity of fungi and establish initial species hypotheses.
more »
« less
- Award ID(s):
- 1655980
- PAR ID:
- 10491815
- Author(s) / Creator(s):
- ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; more »
- Publisher / Repository:
- Biomed Central
- Date Published:
- Journal Name:
- IMA Fungus
- Volume:
- 11
- Issue:
- 1
- ISSN:
- 2210-6359
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract Marine zooplankton are key players in pelagic food webs, central links in ecosystem function, useful indicators of water masses, and rapid responders to environmental variation and climate change. Characterization of biodiversity of the marine zooplankton assemblage is complicated by many factors, including systematic complexity of the assemblage, with numerous rare and cryptic species, and high local-to-global ratios of species diversity. The papers in this themed article set document important advances in molecular protocols and procedures, integration with morphological taxonomic identifications, and quantitative analyses (abundance and biomass). The studies highlight several overarching conclusions and recommendations. A primary issue is the continuing need for morphological taxonomic experts, who can identify species and provide voucher specimens for reference sequence databases, which are essential for biodiversity analyses based on molecular approaches. The power of metabarcoding using multi-gene markers, including both DNA (Deoxyribonucleic Acid) and RNA (Ribonucleic Acid)templates, is demonstrated. An essential goal is the accurate identification of species across all taxonomic groups of marine zooplankton, with particular concern for detection of rare, cryptic, and invasive species. Applications of molecular approaches include analysis of trophic relationships by metabarcoding of gut contents, as well as investigation of the underlying ecological and evolutionary forces driving zooplankton diversity and structure.more » « less
-
Biodiversity genomics research requires reliable organismal identification, which can be difficult based on morphology alone. DNA-based identification using DNA barcoding can provide confirmation of species identity and resolve taxonomic issues but is rarely used in studies generating reference genomes. Here, we describe the development and implementation of DNA barcoding for the Darwin Tree of Life Project (DToL), which aims to sequence and assemble high quality reference genomes for all eukaryotic species in Britain and Ireland. We present a standardised framework for DNA barcode sequencing and data interpretation that is then adapted for diverse organismal groups. DNA barcoding data from over 12,000 DToL specimens has identified up to 20% of samples requiring additional verification, with 2% of seed plants and 3.5% of animal specimens subsequently having their names changed. We also make recommendations for future developments using new sequencing approaches and streamlined bioinformatic approaches.more » « less
-
Abstract Characterization of species diversity of zooplankton is key to understanding, assessing, and predicting the function and future of pelagic ecosystems throughout the global ocean. The marine zooplankton assemblage, including only metazoans, is highly diverse and taxonomically complex, with an estimated ~28,000 species of 41 major taxonomic groups. This review provides a comprehensive summary of DNA sequences for the barcode region of mitochondrial cytochrome oxidase I (COI) for identified specimens. The foundation of this summary is the MetaZooGene Barcode Atlas and Database (MZGdb), a new open-access data and metadata portal that is linked to NCBI GenBank and BOLD data repositories. The MZGdb provides enhanced quality control and tools for assembling COI reference sequence databases that are specific to selected taxonomic groups and/or ocean regions, with associated metadata (e.g., collection georeferencing, verification of species identification, molecular protocols), and tools for statistical analysis, mapping, and visualization. To date, over 150,000 COI sequences for ~ 5600 described species of marine metazoan plankton (including holo- and meroplankton) are available via the MZGdb portal. This review uses the MZGdb as a resource for summaries of COI barcode data and metadata for important taxonomic groups of marine zooplankton and selected regions, including the North Atlantic, Arctic, North Pacific, and Southern Oceans. The MZGdb is designed to provide a foundation for analysis of species diversity of marine zooplankton based on DNA barcoding and metabarcoding for assessment of marine ecosystems and rapid detection of the impacts of climate change.more » « less
-
Abstract Many applications in molecular ecology require the ability to match specific DNA sequences from single‐ or mixed‐species samples with a diagnostic reference library. Widely used methods for DNA barcoding and metabarcoding employ PCR and amplicon sequencing to identify taxa based on target sequences, but the target‐specific enrichment capabilities of CRISPR‐Cas systems may offer advantages in some applications. We identified 54,837 CRISPR‐Cas guide RNAs that may be useful for enriching chloroplast DNA across phylogenetically diverse plant species. We tested a subset of 17 guide RNAs in vitro to enrich plant DNA strands ranging in size from diagnostic DNA barcodes of 1,428 bp to entire chloroplast genomes of 121,284 bp. We used an Oxford Nanopore sequencer to evaluate sequencing success based on both single‐ and mixed‐species samples, which yielded mean chloroplast sequence lengths of 2,530–11,367 bp, depending on the experiment. In comparison to mixed‐species experiments, single‐species experiments yielded more on‐target sequence reads and greater mean pairwise identity between contigs and the plant species' reference genomes. But nevertheless, these mixed‐species experiments yielded sufficient data to provide ≥48‐fold increase in sequence length and better estimates of relative abundance for a commercially prepared mixture of plant species compared to DNA metabarcoding based on the chloroplasttrnL‐P6 marker. Prior work developed CRISPR‐based enrichment protocols for long‐read sequencing and our experiments pioneered its use for plant DNA barcoding and chloroplast assemblies that may have advantages over workflows that require PCR and short‐read sequencing. Future work would benefit from continuing to develop in vitro and in silico methods for CRISPR‐based analyses of mixed‐species samples, especially when the appropriate reference genomes for contig assembly cannot be known a priori.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1186/s43008-020-00033-z
