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

BMC Bioinformatics, Год журнала: 2021, Номер 22(1)

Опубликована: Апрель 5, 2021

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

Язык: Английский

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A pile of pipelines: An overview of the bioinformatics software for metabarcoding data analyses

Molecular Ecology Resources, Год журнала: 2023, Номер 24(5)

Опубликована: Авг. 7, 2023

Abstract Environmental DNA (eDNA) metabarcoding has gained growing attention as a strategy for monitoring biodiversity in ecology. However, taxa identifications produced through require sophisticated processing of high‐throughput sequencing data from taxonomically informative barcodes. Various sets universal and taxon‐specific primers have been developed, extending the usability across archaea, bacteria eukaryotes. Accordingly, multitude analysis tools pipelines also developed. Often, several developed workflows are designed to process same amplicon data, making it somewhat puzzling choose one among plethora existing pipelines. each pipeline its own specific philosophy, strengths limitations, which should be considered depending on aims any study, well bioinformatics expertise user. In this review, we outline input requirements, supported operating systems particular attributes thirty‐two with goal helping users select their projects.

Язык: Английский

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MetaWorks: A flexible, scalable bioinformatic pipeline for high-throughput multi-marker biodiversity assessments

PLoS ONE, Год журнала: 2022, Номер 17(9), С. e0274260 - e0274260

Опубликована: Сен. 29, 2022

Multi-marker metabarcoding is increasingly being used to generate biodiversity information across different domains of life from microbes fungi animals such as for molecular ecology and biomonitoring applications in sectors academic research regulatory agencies industry. Current popular bioinformatic pipelines support microbial fungal marker analysis, while ad hoc methods are often process animal metabarcode markers the same study. MetaWorks provides a harmonized processing environment, pipeline, taxonomic assignment approach demultiplexed Illumina reads all biota using wide range 16S, ITS, COI. A Conda environment provided quickly gather most programs dependencies pipeline. Several workflows as: taxonomically assigning exact sequence variants, an option operational units, facilitates single-read processing. Pipelines automated Snakemake minimize user intervention facilitate scalability. All use RDP classifier provide assignments with confidence measures. We extend functionality 16S (bacteria), ITS (fungi), 28S also COI (eukaryotes), rbcL (eukaryotes, land plants, diatoms), 12S (fish, vertebrates), 18S diatoms) (fungi, plants). properly handles by trimming flanking conserved rRNA gene regions well protein coding genes providing two options removing obvious pseudogenes. can be downloaded https://github.com/terrimporter/MetaWorks quickstart instructions, pipeline details, tutorial new users found at https://terrimporter.github.io/MetaWorksSite .

Язык: Английский

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Interrogating 1000 insect genomes for NUMTs: A risk assessment for estimates of species richness

PLoS ONE, Год журнала: 2023, Номер 18(6), С. e0286620 - e0286620

Опубликована: Июнь 8, 2023

The nuclear genomes of most animal species include NUMTs, segments the mitogenome incorporated into their chromosomes. Although NUMT counts are known to vary greatly among species, there has been no comprehensive study frequency/attributes in diverse group terrestrial organisms, insects. This examines NUMTs derived from a 658 bp 5' segment cytochrome c oxidase I (COI) gene, barcode region for kingdom. assessment is important because unrecognized can elevate estimates richness obtained through DNA barcoding and approaches (eDNA, metabarcoding). investigation detected nearly 10,000 COI ≥ 100 1,002 insect (range = 0-443). Variation genome size explained 56% mitogenome-wide variation counts. orders with largest sizes possessed highest counts, was considerable component lineages. Two thirds an IPSC (indel and/or premature stop codon) allowing recognition exclusion downstream analyses. remainder as they showed 10.1% mean divergence mitochondrial homologue. extent exposure "ghost species" strongly impacted by target amplicon's length. raise apparent up 22% when amplicon examined versus doubling 150 amplicons targeted. Given these impacts, metabarcoding eDNA studies should longest possible while also avoiding use 12S/16S rDNA triple screens cannot be employed.

Язык: Английский

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Groundwater environmental DNA metabarcoding reveals hidden diversity and reflects land‐use and geology

Molecular Ecology, Год журнала: 2023, Номер 32(13), С. 3497 - 3512

Опубликована: Апрель 17, 2023

Abstract Despite being the most important source of liquid freshwater on planet, groundwater is severely threatened by climate change, agriculture, or industrial mining. It thus extensively monitored for pollutants and declines in quantity. The organisms living groundwater, however, are rarely target surveillance programmes little known about fauna inhabiting underground habitats. difficulties accessing lack expertise, apparent scarcity these challenge sampling prohibit adequate knowledge fauna. Environmental DNA (eDNA) metabarcoding provides an approach to overcome limitations but largely unexplored. Here, we sampled water 20 communal spring catchment boxes used drinking provisioning Switzerland, with a high level replication at both filtration amplification steps. We sequenced portion COI mitochondrial gene, which resulted 4917 ASVs, yet only 3% reads could be assigned species, genus, family more than 90% identity. Careful evaluation unassigned corroborated that sequences were true belonging mostly diverse eukaryotic groups, not present reference databases. Principal component analyses showed strong correlation community composition surface land‐use (agriculture vs. forest) geology (fissured rock unconsolidated sediment). While incomplete databases limit assignment taxa eDNA metabarcoding, taxonomy‐free approaches can reveal large hidden diversity couple it major drivers, revealing their imprint chemical biological properties groundwater.

Язык: Английский

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

Molecular Ecology Resources, Год журнала: 2021, Номер 22(3), С. 847 - 861

Опубликована: Сен. 8, 2021

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

Язык: Английский

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Connecting high‐throughput biodiversity inventories: Opportunities for a site‐based genomic framework for global integration and synthesis

Molecular Ecology, Год журнала: 2021, Номер 30(5), С. 1120 - 1135

Опубликована: Янв. 15, 2021

High-throughput sequencing (HTS) is increasingly being used for the characterization and monitoring of biodiversity. If applied in a structured way, across broad geographical scales, it offers potential much deeper understanding global biodiversity through integration massive quantities molecular inventory data generated independently at local, regional scales. The universality, reliability efficiency HTS can potentially facilitate seamless linking among species assemblages from different sites, hierarchical levels diversity, any taxonomic group regardless prior knowledge. However, collective international efforts are required to optimally exploit site-based synthesis, that present limited microbial domain. To contribute development an analogous strategy nonmicrobial terrestrial domain, symposium entitled "Next Generation Biodiversity Monitoring" was held November 2019 Nicosia (Cyprus). brought together evolutionary geneticists, ecologists scientists involved diverse initiatives using as core tool assessment. In this review, we summarize consensus emerged 3-day symposium. We converged on opinion effective Genomic Observatories network synthesis should be spatially led strategically united under umbrella metabarcoding approach. Subsequently, outline HTS-based collectively build integrative framework generation.

Язык: Английский

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Profile hidden Markov model sequence analysis can help remove putative pseudogenes from DNA barcoding and metabarcoding datasets

BMC Bioinformatics, Год журнала: 2021, Номер 22(1)

Опубликована: Май 19, 2021

Abstract Background Pseudogenes are non-functional copies of protein coding genes that typically follow a different molecular evolutionary path as compared to functional genes. The inclusion pseudogene sequences in DNA barcoding and metabarcoding analysis can lead misleading results. None the most widely used bioinformatic pipelines process marker gene (metabarcode) high throughput sequencing data specifically accounts for presence pseudogenes protein-coding purpose this study is develop method screen nuclear mitochondrial segments (nuMTs) large COI datasets. We do by: (1) describing nuMT characteristics from an artificial barcode dataset, (2) show impact two removal methods on perturbed community datasets with simulated nuMTs, (3) incorporate filtering step pipeline be Illumina paired-end metabarcode sequences. Open reading frame length sequence bit scores hidden Markov model (HMM) profile were detect pseudogenes. Results Our simulations showed it was more difficult identify nuMTs shorter amplicon such those full barcodes construction libraries. It also where there percentage nuMTs. Existing already remove some especially rare step, but addition up 5% even when other steps place. Conclusions alone or combined effectively out apparent There learn their frequency studies, taxonomic distribution, evolution. Thus, we encourage submission verified public databases facilitate future studies.

Язык: Английский

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Ecological network structure in response to community assembly processes over evolutionary time

Molecular Ecology, Год журнала: 2023, Номер 32(23), С. 6489 - 6506

Опубликована: Фев. 4, 2023

The dynamic structure of ecological communities results from interactions among taxa that change with shifts in species composition space and time. However, our ability to study the interplay evolutionary processes on community assembly remains relatively unexplored due difficulty measuring over long temporal scales. Here, we made use a geological chronosequence across Hawaiian Islands, representing 50 years 4.15 million ecosystem development, sample 11 arthropods their associated plant using semiquantitative DNA metabarcoding. We then examined how changed age by calculating quantitative network statistics for bipartite networks arthropod-plant associations. average number per (linkage density), ratio arthropod (vulnerability) uniformity energy flow (interaction evenness) increased significantly concert age. index specialization

Язык: Английский

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Mitochondrial Cytochrome Oxidase Subunit 1: A Promising Molecular Marker for Species Identification in Foraminifera

Frontiers in Marine Science, Год журнала: 2022, Номер 9

Опубликована: Фев. 2, 2022

Traditional morphological methods for species identification are highly time consuming, especially small organisms, such as Foraminifera, a group of shell-building microbial eukaryotes. To analyze large amounts samples more efficiently, have extended to molecular tools in the last few decades. Although wide range phyla good markers available, Foraminifera only one hypervariable marker from ribosomal region (18S) is widely used. Recently new mitochondrial cytochrome oxidase subunit 1 (COI) has been sequenced. Here we investigate whether this higher potential compared marker. We explore genetic variability both 18S and COI 22 benthic foraminiferal morphospecies (orders Miliolida Rotaliida). Using single-cell DNA, within specimens (intra) between (inter) each was assessed using next-generation sequencing. Amplification success rate twice high (151/200 specimens) than (73/200 specimens). The showed greatly decreased intra- inter-specimen six out seven selected species. phylogenetic reconstruction fails adequately cluster multiple together contrast COI. Additionally, helped recognize misclassified difficult morphologically identify level. Integrative taxonomy, combining characteristics, provides robust picture diversity. Finally, suggest use set sequences (two or more) describe showing intra-genomic additionally markers. Our findings highlight newly discovered metabarcoding purposes.

Язык: Английский

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