Best practices in metabarcoding of fungi: From experimental design to results

Molecular Ecology, Год журнала: 2022, Номер 31(10), С. 2769 - 2795

Опубликована: Апрель 8, 2022

The development of high-throughput sequencing (HTS) technologies has greatly improved our capacity to identify fungi and unveil their ecological roles across a variety ecosystems. Here we provide an overview current best practices in metabarcoding analysis fungal communities, from experimental design through molecular computational analyses. By reanalysing published data sets, demonstrate that operational taxonomic units (OTUs) outperform amplified sequence variants (ASVs) recovering diversity, finding is particularly evident for long markers. Additionally, the full-length ITS region allows more accurate placement other eukaryotes compared ITS2 subregion. Finally, show specific methods compositional analyses reliable estimates shifts community structure. We conclude are especially promising integrating into full microbiome broader ecosystem functioning context, recovery novel lineages ancient organisms as well barcoding old specimens including type material.

Язык: Английский

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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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DNA barcoding of fungal specimens using PacBio long‐read high‐throughput sequencing

Molecular Ecology Resources, Год журнала: 2022, Номер 22(8), С. 2871 - 2879

Опубликована: Июнь 6, 2022

Abstract Molecular methods are increasingly used to identify species that lack conspicuous macro‐ or micromorphological characters. Taxonomic and ecological research teams barcode large numbers of collected voucher specimens annually. In this study we assessed the efficiency long‐read high throughput sequencing (HTS) as opposed traditionally Sanger method for taxonomic identification multiple vouchered fungal specimens. We also evaluated whether can provide reference information about intraindividual gene polymorphism. developed a workflow based on test set 423 basidiomycete (representing 195 species), PacBio HTS method, ribosomal rRNA operon internal transcribed spacer (ITS) 28S (LSU) markers. The had higher success rate than at comparable cost. Species reads was usually straightforward, because dominant operational unit (OTU) typically represented targeted organism. enabled us detect widespread polymorphism within ITS marker. conclude multiplex DNA barcoding LSU markers using is useful tool amounts competitive price. Furthermore, accurately recovers various alleles paralogues, which crucial delimitation population‐level studies.

Язык: Английский

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A Genome-Wide Analysis of Nuclear Mitochondrial DNA Sequences (NUMTs) in Chrysomelidae Species (Coleoptera)

Insects, Год журнала: 2025, Номер 16(2), С. 150 - 150

Опубликована: Фев. 2, 2025

Nuclear mitochondrial DNA sequences (NUMTs) are fragments in the nuclear genome, and their unclear distribution Chrysomelidae species hinders selection of accurate molecular markers for identification phylogenetic analysis. Our study presents a genome-wide survey NUMTs 32 species. Filtering strategies based on sequence length open reading frame (ORF) features were employed to identify protein-coding genes (PCGs) minimally affected by NUMTs. Phylogenetic relationships inferred from both PCG datasets COX1 dataset containing results show that chromosomally specific, species-specific, widely distributed. ATP8, COX1, ND1, ND4 identified as relatively reliable markers. analysis is influenced other factors such type saturation. A total 66 independent gene integration events estimated across species, mostly distinct lineages. These findings suggest reflect key evolutionary processes flow lineage diversification. Their prevalence emphasizes need refined analysis, while also highlighting importance understanding contribution species’ history.

Язык: Английский

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Procladius (Diptera, Chironomidae) of Europe and a global view

Zootaxa, Год журнала: 2025, Номер 5591(1), С. 1 - 127

Опубликована: Фев. 25, 2025

A project initiated in 1991 to untangle species-taxonomy of European Procladius (Chironomidae) has been accomplished. Increasing amount material, loans and especially the development barcodes BIN-system BOLD, made finalization possible after about 33 years. An iterative process based on detailed studies male morphology barcode clusters, BINs, resulted identification 27 species present Europe, most them also Asia (China, Japan, Mongolia Russia) North America (Canada United States). One hundred morphological characters were adopted for which 30 important ones used construct a key an additional helpdesk. The contains three each separation as this is frequently needed reliable identification. ratio GspR, outer length gonostylus versus margin gonostylus, proved be character All but two have BINs. one BIN contained only species. exception that previously was divided into BINs containing morphologically distinct Intraspecific divergence within ranged from 0‒3.3% interspecific 2.0‒8.8%. Four new are presented. These P. exilis Brodin, species, gemma saeticubitus tenebricosus Brodin & Hellberg, other 23 presented follows with synonyms brackets: appropinquatus (Lundström, 1916) [P. ruris Roback, 1971], bellus (Loew, 1866) [Tanypus rufovittatus van der Wulp, 1874, latifrons Kieffer, 1922, leucocoma profundorum 1923], breviatus Remmert, 1953, choreus (Meigen, 1804) [Chironomus incomptus Walker, 1856], clavus 1971, crassinervis (Zetterstedt, 1838) pectinatus 1909, bifasciatus Goetghebuer, 1936, cinereus abetus culiciformis (Linnaeus, 1767) sagittalis Trichotanypus scapularis 1924, freemani Sublette, 1964 part], dentus ferrugineus (Kieffer, 1918) [Trichotanypus parvulus 1918, fulvus rugulosus Saether 2010], fimbriatus Wülker, 1959, flavifrons Edwards, 1929, floralis 1915, frigidus (Holmgren, 1869) gretis imicola 1922 bathyphilus nietus islandicus (Goetghebuer, 1931) fuscus Brundin, 1949, vesus longistilus suecicus 1949], lugens 1915 macrotrichus lugubris 1850) barbatus johnsoni 1980], nudipennis 1947, pruinosus 1924), signatus nigriventris denticulatus simplicistilus Freeman, 1948, tatrensis Gowin, 1944. In addition, 12 not found Europe briefly described it indicated where they appear species-key. Species reported 133 countries or autonomies worldwide. As many extreme cold places northern hemisphere, mean annual temperature ‒10 C more. Altitude records at 4 730 m above sea level Himalayas. Larvae known omnivorous, although predation might more beneficial growth. Synonyms dubious names reduce number valid (accepted) according Catalogue Life Systema Dipterorum 34% After inclusion four study others worldwide land 69.

Язык: Английский

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Research on HMM-Based Efficient Stock Price Prediction

Mobile Information Systems, Год журнала: 2022, Номер 2022, С. 1 - 8

Опубликована: Март 7, 2022

Stock market is one of the most important parts investment market. Compared with other industries, stock not only has a higher rate return on but also risk, and price prediction always been close concern investors. Therefore, research methods how to reduce error become hot topic for many scholars at home abroad. In recent years, development computer technology such as machine learning econometric method makes more reliable. Due hidden Markov nature price, this paper proposes based model (HMM). To be specific, since data have continuity in time series, it necessary extend discrete HMM continuous HMM, then put forward up down trend HMM. The first-order extended second-order predicted by combining fluctuation range. As result, proposed HMM-based simulated Hang Seng Index (HSI), earliest indexes Hong Kong. evaluation results six months HSI show that value very actual outperforms three benchmarks terms RMSE, MAE, R2.

Язык: Английский

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Mitochondrial cytochrome c oxidase subunit I (COI) metabarcoding of Foraminifera communities using taxon-specific primers

PeerJ, Год журнала: 2022, Номер 10, С. e13952 - e13952

Опубликована: Сен. 5, 2022

Foraminifera are a species-rich phylum of rhizarian protists that highly abundant in most marine environments. Molecular methods such as metabarcoding have revealed high, yet undescribed diversity Foraminifera. However, so far only one molecular marker, the 18S ribosomal RNA, was available for studies on Primers allow amplification foraminiferal mitochondrial cytochrome oxidase I (COI) and identification species were recently published. Here we test performance these primers whole communities, compare their to degenerate LerayXT primers, which amplify same COI region wide range eukaryotes. We applied 48 samples taken along three transects spanning North Sea beach Netherlands from dunes low tide level, analysed both sediment meiofauna samples, contained taxa between 42 µm 1 mm body size obtained by decantation sand samples. used single-cell (Girard et al., 2022) generate reference library containing 32 Foraminifera, this taxonomically annotate our community data. Our analyses show do not while Foraminifera- specific, with about 90% reads assigned amplifying all major groups, i.e., monothalamids, Globothalamea, Tubothalamea. identified 176 ASVs found change composition high dominance single-chambered monothalamid results highlight can be powerful tool assessing Foraminiferal communities.

Язык: Английский

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