scholarly journals Do pseudogenes pose a problem for metabarcoding marine animal communities?

2021 ◽  
Author(s):  
Jessica Schultz ◽  
Paul Hebert
Keyword(s):  
Life History Traits ◽  
Sequence Divergence ◽  
Marine Animal ◽  
Cytochrome C Oxidase I ◽  
Diagnostic Features ◽  
Dna Metabarcoding ◽  
Reference Databases ◽  
Mitochondrial Sequences ◽  
Animal Communities ◽  
Animal Genomes

Because DNA metabarcoding typically employs sequence diversity among mitochondrial amplicons to estimate species composition, nuclear mitochondrial pseudogenes (NUMTs) can inflate diversity. This study quantifies the incidence and attributes of NUMTs derived from the 658 bp barcode region of cytochrome c oxidase I (COI) in 156 marine animal genomes. The number of NUMTs meeting four length criteria (>150 bp, >300 bp, >450 bp, >600 bp) was determined, and they were examined to ascertain if they could be recognized by their possession of indels or stop codons. In total, 389 NUMTs <100 bp were detected, with an average of 2.49 per species (range = 0–50) and a mean length of 336 bp +/- 208 bp. Among NUMTs lacking diagnostic features, 52.5% were ≤300 bp, 63.9% were ≤450 bp, and 76.2% were ≤600 bp. Studies examing 150 bp amplicons inflate the OTU count by 1.57x compared to the true species count and increase perceived intraspecific variation at COI by 1.19x (when sequence variants with >2% sequence divergence are recognized as different OTUs). There was a weak positive correlation between genome size and NUMT count but no variation among phyla, trophic groups or life history traits. While bioinformatic advances will improve NUMT detection, the best defense involves targeting long amplicons and developing reference databases that include both mitochondrial sequences and their NUMT derivatives.

scholarly journals PIMBA: a PIpeline for MetaBarcoding Analysis

2021 ◽  
Author(s):  
Renato R. M. Oliveira ◽  
Raissa L S Silva ◽  
Gisele L. Nunes ◽  
Guilherme Oliveira
Keyword(s):  
Environmental Samples ◽  
Next Generation Sequencing Data ◽  
Ecological Studies ◽  
Sequencing Data ◽  
Monitoring Method ◽  
Computational Tools ◽  
Dna Metabarcoding ◽  
Reference Databases ◽  
User Friendly ◽  
Generation Sequencing

DNA metabarcoding is an emerging monitoring method capable of assessing biodiversity from environmental samples (eDNA). Advances in computational tools have been required due to the increase of Next-Generation Sequencing data. Tools for DNA metabarcoding analysis, such as MOTHUR, QIIME, Obitools, and mBRAVE have been widely used in ecological studies. However, some difficulties are encountered when there is a need to use custom databases. Here we present PIMBA, a PIpeline for MetaBarcoding Analysis, which allows the use of customized databases, as well as other reference databases used by the softwares mentioned here. PIMBA is an open-source and user-friendly pipeline that consolidates all analyses in just three command lines.

Species in the faeces: DNA metabarcoding as a method to determine the diet of the endangered yellow-eyed penguin

2020 ◽  
Vol 47 (6) ◽  
pp. 509
Author(s):  
Melanie J. Young ◽  
Ludovic Dutoit ◽  
Fiona Robertson ◽  
Yolanda van Heezik ◽  
Philip J. Seddon ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Diet Composition ◽  
Pcr Primers ◽  
Fish Prey ◽  
Genus Level ◽  
Faecal Samples ◽  
Significant Difference ◽  
Dna Metabarcoding ◽  
Reference Databases ◽  
Diet Assessment

Abstract Context. Diet variability is a significant driver of seabird decline; however, data on seabird diet composition and trends have been affected by changes in precision and resolution owing to the evolution of different sampling methods over time. We investigated the effectiveness of applying a passive molecular diet method using faeces obtained from the endangered yellow-eyed penguin. Aims. To assess the feasibility of applying DNA metabarcoding methods to yellow-eyed penguin faeces to evaluate diet, and to compare the reliability of diet results derived from adults and chicks, and from latrine versus fresh faecal samples. Methods. We collected 313 faecal samples from yellow-eyed penguins resident on the Otago coast of New Zealand from October 2016 to August 2017. We used polymerase chain reaction (PCR) with mitochondrial 16S cephalopod and chordate primers to amplify prey DNA present in the faecal samples, and tested the completeness of our assembled reference databases based on previous diet research. Amplified prey DNA sequences were then assigned to taxa from our reference databases by using QIIME2. Key results. Mitochondrial 16S chordate PCR primers were effective at identifying 29 fish taxa, with 98.3% of amplified sequences being identified to species or genus level in 193 samples (61.7% collected). There was no significant difference in the number, occurrence or proportion of ray-finned fish prey DNA sequences derived from fresh samples or latrines. Mitochondrial 16S cephalopod PCR primers classified 1.98% of amplified DNA sequences as targets, with 96.5% of these target sequences being identified to species or genus level in 48 samples (15.3% collected), and five taxa identified. Conclusions. We recommend the collection of latrine samples to enable long-term monitoring of the diet of yellow-eyed penguins, which will optimise the trade-off between wildlife disturbance and dietary resolution. Further refinement is needed to identify cephalopod dietary components for yellow-eyed penguins, because our cephalopod primers were not as specific as those used for ray-finned fishes, amplifying a large number (&gt;98%) of non-cephalopod species. Implications. DNA metabarcoding offers a robust and comprehensive alternative to other, more intrusive, seabird diet-assessment methods, but still requires parallel studies to provide critical information on prey size, true diet composition and diet quality.

scholarly journals PLANiTS: a curated sequence reference dataset for plant ITS DNA metabarcoding

Database ◽  
2020 ◽  
Vol 2020 ◽  
Author(s):  
Elisa Banchi ◽  
Claudio G Ametrano ◽  
Samuele Greco ◽  
David Stanković ◽  
Lucia Muggia ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Its Region ◽  
Computational Effort ◽  
Its Sequences ◽  
Reference Dataset ◽  
Bioinformatic Pipeline ◽  
Taxonomic Level ◽  
Dna Metabarcoding ◽  
Reference Databases

Abstract DNA metabarcoding combines DNA barcoding with high-throughput sequencing to identify different taxa within environmental communities. The ITS has already been proposed and widely used as universal barcode marker for plants, but a comprehensive, updated and accurate reference dataset of plant ITS sequences has not been available so far. Here, we constructed reference datasets of Viridiplantae ITS1, ITS2 and entire ITS sequences including both Chlorophyta and Streptophyta. The sequences were retrieved from NCBI, and the ITS region was extracted. The sequences underwent identity check to remove misidentified records and were clustered at 99% identity to reduce redundancy and computational effort. For this step, we developed a script called ‘better clustering for QIIME’ (bc4q) to ensure that the representative sequences are chosen according to the composition of the cluster at a different taxonomic level. The three datasets obtained with the bc4q script are PLANiTS1 (100 224 sequences), PLANiTS2 (96 771 sequences) and PLANiTS (97 550 sequences), and all are pre-formatted for QIIME, being this the most used bioinformatic pipeline for metabarcoding analysis. Being curated and updated reference databases, PLANiTS1, PLANiTS2 and PLANiTS are proposed as a reliable, pivotal first step for a general standardization of plant DNA metabarcoding studies. The bc4q script is presented as a new tool useful in each research dealing with sequences clustering. Database URL: https://github.com/apallavicini/bc4q; https://github.com/apallavicini/PLANiTS.

scholarly journals Variations in terrestrial arthropod DNA metabarcoding methods recovers robust beta diversity but variable richness and site indicators

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Teresita M. Porter ◽  
Dave M. Morris ◽  
Nathan Basiliko ◽  
Mehrdad Hajibabaei ◽  
Daniel Doucet ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Beta Diversity ◽  
Ecological Integrity ◽  
Sequence Variants ◽  
Key Indicator ◽  
Pooled Dna ◽  
Dna Metabarcoding ◽  
Arthropod Fauna ◽  
Reference Databases ◽  
Arthropod Biodiversity

AbstractTerrestrial arthropod fauna have been suggested as a key indicator of ecological integrity in forest systems. Because phenotypic identification is expert-limited, a shift towards DNA metabarcoding could improve scalability and democratize the use of forest floor arthropods for biomonitoring applications. The objective of this study was to establish the level of field sampling and DNA extraction replication needed for arthropod biodiversity assessments from soil. Processing 15 individually collected soil samples recovered significantly higher median richness (488–614 sequence variants) than pooling the same number of samples (165–191 sequence variants) prior to DNA extraction, and we found no significant richness differences when using 1 or 3 pooled DNA extractions. Beta diversity was robust to changes in methodological regimes. Though our ability to identify taxa to species rank was limited, we were able to use arthropod COI metabarcodes from forest soil to assess richness, distinguish among sites, and recover site indicators based on unnamed exact sequence variants. Our results highlight the need to continue DNA barcoding local taxa during COI metabarcoding studies to help build reference databases. All together, these sampling considerations support the use of soil arthropod COI metabarcoding as a scalable method for biomonitoring.

Biological identifications through DNA barcodes: the case of the Crustacea

2007 ◽  
Vol 64 (2) ◽  
pp. 272-295 ◽  
Author(s):  
Filipe O Costa ◽  
Jeremy R deWaard ◽  
James Boutillier ◽  
Sujeevan Ratnasingham ◽  
Robert T Dooh ◽  
...  
Keyword(s):  
Sequence Variation ◽  
Sequence Divergence ◽  
Species Level ◽  
Coi Gene ◽  
Dna Barcodes ◽  
Cytochrome C Oxidase I ◽  
Sequence Profile ◽  
Amino Acid Data ◽  
Nucleotide Sequence Divergence ◽  
Diverse Groups

The ability of a 650 base pair section of the mitochondrial cytochrome c oxidase I (COI) gene to provide species-level identifications has been demonstrated for large taxonomic assemblages of animals such as insects, birds, and fishes, but not for the subphylum Crustacea, one of the most diverse groups of arthropods. In this study, we test the ability of COI to provide identifications in this group, examining two disparate levels in the taxonomic hierarchy — orders and species. The first phase of our study involved the development of a sequence profile for 23 dominant crustacean orders, based upon the analysis of 150 species, each belonging to a different family. The COI amino acid data placed these taxa into cohesive assemblages whose membership coincided with currently accepted boundaries at the order, superorder, and subclass levels. Species-level resolution was subsequently examined in an assemblage of Decapoda and in representatives of the genera Daphnia (Cladocera) and Gammarus (Amphipoda). These studies revealed that levels of nucleotide sequence divergence were from 19 to 48 times greater between congeneric species than between individuals of a species. We conclude that sequence variation in the COI barcode region will be very effective for discriminating species of Crustacea.

scholarly journals A DNA metabarcoding protocol for hyporheic freshwater meiofauna: Evaluating highly degenerate COI primers and replication strategy

2018 ◽  
Vol 2 ◽  
Author(s):  
Alexander M. Weigand ◽  
Jan-Niklas Macher
Keyword(s):  
Hyporheic Zone ◽  
Sediment Cores ◽  
Alpha Diversity ◽  
Freshwater Ecosystem ◽  
Community Similarity ◽  
Degenerate Primers ◽  
False Negatives ◽  
Replication Strategy ◽  
Dna Metabarcoding ◽  
Reference Databases

The hyporheic zone, i.e. the ecotone between surface water and the groundwater, is a rarely studied freshwater ecosystem. Hyporheic taxa are often meiofaunal (&lt;1 mm) in size and difficult to identify based on morphology. Metabarcoding approaches are promising for the study of these environments and taxa, but it is yet unclear if commonly applied metabarcoding primers and replication strategies can be used. In this study, we took sediment cores from two near natural upstream (NNU) and two ecologically improved downstream (EID) sites in the Boye catchment (Emscher River, Germany), metabarcoding their meiofaunal communities. We evaluated the usability of a commonly used, highly degenerate COI primer pair (BF2/BR2) and tested how sequencing three PCR replicates per sample and removing MOTUs present in only one out of three replicates impacts the inferred community composition. A total of 22,514 MOTUs were detected, of which only 263 were identified as Metazoa. Our results highlight the gaps in reference databases for meiofaunal taxa and the potential problems of using highly degenerate primers for studying samples containing a high number of non-metazoan taxa. Alpha diversity was higher in EID sites and showed higher community similarity when compared to NNU sites. Beta diversity analyses showed that removing MOTUs detected in only one out of three replicates per site greatly increased community similarity in samples. Sequencing three sample replicates and removing rare MOTUs is seen as a good compromise between retaining too many false-positives and introducing too many false-negatives. We conclude that metabarcoding hyporheic communities using highly degenerate COI primers can provide valuable first insights into the diversity of these ecosystems and highlight some potential application scenarios.

scholarly journals New insights into Danube’s macroinvertebrate communities from DNA metabarcoding as part of the Joint Danube Survey 4 (JDS4)

2021 ◽  
Vol 4 ◽  
Author(s):  
Arne Beermann ◽  
Dominik Buchner ◽  
Florian Leese ◽  
Till-Hendrik Macher ◽  
Miroslav Ocadlik ◽  
...  
Keyword(s):  
Bulk Sample ◽  
Taxonomic Composition ◽  
Danube River ◽  
Taxonomic Resolution ◽  
Macroinvertebrate Communities ◽  
Operational Taxonomic Units ◽  
Sampling Campaign ◽  
Dna Metabarcoding ◽  
Assessment And Monitoring ◽  
Reference Databases

The Joint Danube Survey (JDS) is a multinational effort in monitoring Danube’s water quality, including its major tributaries. The Danube river stretches over a distance of 2,800 km and flows through or borders 10 different countries to which it is of utter importance as a source of potable water and hydrodynamic power. The JDS is conducted every 6 years and provides a unique opportunity to collect comprehensive data on both abiotic parameters and organisms and to raise awareness of the importance of water as a natural resource. As part of JDS and as a biological quality element in many monitoring programs worldwide, macroinvertebrates are monitored as indicators for various environmental conditions. However, due to their diverse taxonomic composition, associated difficulties with their morphology-based identification as well as their sheer abundance, macroinvertebrates are often analysed with a low taxonomic resolution (i.e., above species level). As an alternative, DNA metabarcoding offers a promising approach to capture this species diversity more accurately. Here, we used DNA metabarcoding to investigate the macrozoobenthic diversity of 46 sites from the latest JDS sampling campaign in 2019. To analyse macroinvertebrate diversity, bulk samples were taken by kick-net sampling and analysed using two different approaches, analysing the bulk sample fixative and analysing homogenised organisms from complete bulk samples. DNA metabarcoding of the sample fixative revealed 1,146 Operational Taxonomic Units (OTUs) and 231 species compared to 833 OTUs and 333 species from homogenised sample analysis. While more dipterans, in particular Chironomidae, were detected in fixative (136 species) than homogenised bulk (90 species) analyses, the latter picked up more Trichoptera (19 vs. 2), Amphipoda (10 vs. 4) and Bivalvia species (13 vs. 5). Even though these results of a DNA-based assessment deliver new insights into species richness and composition of Danube’s macroinvertebrate communities from the Danube source to its delta already, it is evident that the majority of OTUs was not assigned to species. While filling this lack of reference sequences poses a major challenge, the JDS consortium also offers a unique opportunity to complement reference databases in a multinational effort towards a more comprehensive Danube assessment and monitoring.

scholarly journals Quantifying ecological impacts of mass extinctions with network analysis of fossil communities

2018 ◽  
Vol 115 (20) ◽  
pp. 5217-5222 ◽  
Author(s):  
A. D. Muscente ◽  
Anirudh Prabhu ◽  
Hao Zhong ◽  
Ahmed Eleish ◽  
Michael B. Meyer ◽  
...  
Keyword(s):  
Network Analysis ◽  
Ecological Impact ◽  
Ecological Impacts ◽  
Ecological Change ◽  
Marine Animal ◽  
Mass Extinctions ◽  
Community Turnover ◽  
Ecological Changes ◽  
Extinction Events ◽  
Animal Communities

Mass extinctions documented by the fossil record provide critical benchmarks for assessing changes through time in biodiversity and ecology. Efforts to compare biotic crises of the past and present, however, encounter difficulty because taxonomic and ecological changes are decoupled, and although various metrics exist for describing taxonomic turnover, no methods have yet been proposed to quantify the ecological impacts of extinction events. To address this issue, we apply a network-based approach to exploring the evolution of marine animal communities over the Phanerozoic Eon. Network analysis of fossil co-occurrence data enables us to identify nonrandom associations of interrelated paleocommunities. These associations, or evolutionary paleocommunities, dominated total diversity during successive intervals of relative community stasis. Community turnover occurred largely during mass extinctions and radiations, when ecological reorganization resulted in the decline of one association and the rise of another. Altogether, we identify five evolutionary paleocommunities at the generic and familial levels in addition to three ordinal associations that correspond to Sepkoski’s Cambrian, Paleozoic, and Modern evolutionary faunas. In this context, we quantify magnitudes of ecological change by measuring shifts in the representation of evolutionary paleocommunities over geologic time. Our work shows that the Great Ordovician Biodiversification Event had the largest effect on ecology, followed in descending order by the Permian–Triassic, Cretaceous–Paleogene, Devonian, and Triassic–Jurassic mass extinctions. Despite its taxonomic severity, the Ordovician extinction did not strongly affect co-occurrences of taxa, affirming its limited ecological impact. Network paleoecology offers promising approaches to exploring ecological consequences of extinctions and radiations.

scholarly journals Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1966 ◽  
Author(s):  
Vasco Elbrecht ◽  
Pierre Taberlet ◽  
Tony Dejean ◽  
Alice Valentini ◽  
Philippe Usseglio-Polatera ◽  
...  
Keyword(s):  
Species Level ◽  
Coi Gene ◽  
Taxonomic Resolution ◽  
Reference Database ◽  
Bias Estimation ◽  
Knowing That ◽  
Dna Metabarcoding ◽  
Local Reference ◽  
Comprehensive Survey ◽  
Reference Databases

Cytochrome c oxidase I (COI) is a powerful marker for DNA barcoding of animals, with good taxonomic resolution and a large reference database. However, when used for DNA metabarcoding, estimation of taxa abundances and species detection are limited due to primer bias caused by highly variable primer binding sites across the COI gene. Therefore, we explored the ability of the 16S ribosomal DNA gene as an alternative metabarcoding marker for species level assessments. Ten bulk samples, each containing equal amounts of tissue from 52 freshwater invertebrate taxa, were sequenced with the Illumina NextSeq 500 system. The 16S primers amplified three more insect species than the Folmer COI primers and amplified more equally, probably due to decreased primer bias. Estimation of biomass might be less biased with 16S than with COI, although variation in read abundances of two orders of magnitudes is still observed. According to these results, the marker choice depends on the scientific question. If the goal is to obtain a taxonomic identification at the species level, then COI is more appropriate due to established reference databases and known taxonomic resolution of this marker, knowing that a greater proportion of insects will be missed using COI Folmer primers. If the goal is to obtain a more comprehensive survey the 16S marker, which requires building a local reference database, or optimised degenerated COI primers could be more appropriate.

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