Three steps towards comparability and standardization among molecular methods for characterizing insect communities.

Molecular methods are currently some of the best-suited technologies for implementation in insect monitoring. However, the field is developing rapidly and lacks agreement on methodology or community standards. To apply DNA-based methods in large-scale monitoring, and to gain insight across commensurate data, we need easy-to-implement standards that improve data comparability. Here, we provide three recommendations for how to improve and harmonize efforts in biodiversity assessment and monitoring via metabarcoding: (i) we should adopt the use of synthetic spike-ins, which will act as positive controls and internal standards; (ii) we should consider using several markers through a multiplex polymerase chain reaction (PCR) approach; and (iii) we should commit to the publication and transparency of all protocol-associated metadata in a standardized fashion. For (i), we provide a ready-to-use recipe for synthetic cytochrome c oxidase spike-ins, which enable between-sample comparisons. For (ii), we propose two gene regions for the implementation of multiplex PCR approaches, thereby achieving a more comprehensive community description. For (iii), we offer guidelines for transparent and unified reporting of field, wet-laboratory and dry-laboratory procedures, as a key to making comparisons between studies. Together, we feel that these three advances will result in joint quality and calibration standards rather than the current laboratory-specific proof of concepts. This article is part of the theme issue 'Towards a toolkit for global insect biodiversity monitoring'.

Identification of a biological form in the Anopheles stephensi laboratory colony using the odorant-binding protein 1 intron I sequence.

BACKGROUND: Anopheles stephensi Listen (1901) is a major vector of malaria in Asia and has recently been found in some regions of Africa. The An. stepehnsi species complex is suspected to have three sibling species: type, intermediate, and mysorensis, each with its own vector competence to the malaria parasite and ecology. To identify the members of the species complex in our An. stephensi insectary colony, we used the morphological features of eggs and genetic markers such as AnsteObp1 (Anopheles stephensi odorant binding protein 1), mitochondrial oxidases subunit 1 and 2 (COI and COII), and nuclear internal transcribed spacer 2 locus (ITS2). METHODS: Eggs were collected from individual mosquitoes (n = 50) and counted for the number of ridges under stereomicroscope. Genomic DNA was extracted from female mosquitoes. After the amplification of partial fragments of AnsteObp1, COI, COII and ITS2 genes, the PCR products were purified and sequenced. Phylogenetic analysis was performed after aligning query sequences against the submitted sequences in GenBank using MEGA 7. RESULTS: The range of ridges number on each egg float was 12-13 that corresponds to the mysorensis form of An. stephensi. The generated COI, COII and ITS2 sequences showed 100%, 99.46% and 99.29% similarity with the sequences deposited for Chinese, Indian and Iranian strains of An. stephensi, respectively. All the generated AnsteObp1 intron I region sequences matched 100% with the sequences deposited for An. stephensi sibling species C (mysorensis form) from Iran and Afghanistan. CONCLUSIONS: This manuscript precisely describes the morphological and molecular details of the 'var mysorensis' form of An. stephensi that could be exploited in elucidating its classification as well as in differentiation from other biotypes of the same or other anopheline species. Based on our findings, we recommend AnsteObp1 as a robust genetic marker for rapid and accurate discrimination (taxonomic identification) of the An. stephensi species complex, rather than the COI, COII, and ITS2 marker, which could only be utilized for interspecies (Anopheles) differentiation.

To culture or not to culture: careful assessment of metabarcoding data is necessary when evaluating the microbiota of a modified-atmosphere-packaged vegetarian meat alternative throughout its shelf-life period.

BACKGROUND: As the increased consumption of ready-to-eat meat alternatives is a fairly recent trend, little is known about the composition and dynamics of the microbiota present on such products. Such information is nonetheless valuable in view of spoilage and food safety prevention. Even though refrigeration and modified-atmosphere-packaging (MAP) can extend the shelf-life period, microbial spoilage can still occur in these products. In the present study, the microbiota of a vegetarian alternative to poultry-based charcuterie was investigated during storage, contrasting the use of a culture-dependent method to a culture-independent metagenetic method. RESULTS: The former revealed that lactic acid bacteria (LAB) were the most abundant microbial group, specifically at the end of the shelf-life period, whereby Latilactobacillus sakei was the most abundant species. Metabarcoding analysis, in contrast, revealed that DNA of Xanthomonas was most prominently present, which likely was an artifact due to the presence of xanthan gum as an ingredient, followed by Streptococcus and Weissella. CONCLUSIONS: Taken together, these results indicated that Lb. sakei was likely the most prominent specific spoilage organisms (SSO) and, additionally, that the use of metagenetic analysis needs to be interpreted with care in this specific type of product. In order to improve the performance of metagenetics in food samples with a high DNA matrix but a low bacterial DNA load, selective depletion techniques for matrix DNA could be explored.

South Africa's contribution of insect records on the BOLD system.

South Africa is the third most biodiverse country in the world and insects represent a large part of its faunal diversity, as is seen globally. With more than 65,000 described animal species in South Africa, insects represent 44,088 species. While there are still a lot of species yet to be identified, progress may be hindered by the few insect taxonomists available in South Africa and subsequently, the time-consuming nature and costs of the methods used during species identification. DNA barcoding, on the other hand, has become a valuable tool for documenting biodiversity with the use of a small DNA fragment such as cytochrome oxidase subunit 1 (COI). This paper aims to assess South Africa's contribution to the Barcode of Life Database (BOLD) as well as highlight the regions that are under-represented on BOLD. From the 4,984,215 Insecta records on BOLD, South Africa contributed 56,392 insect records, with only 16.85% of that total identified to species level. The Gauteng Province had the most represented insect samples submitted to BOLD with 63.57% followed by Kwazulu-Natal (15.74%), and Mpumalanga (5.73%). However, the Free State, Limpopo, and the Northern Cape provinces are all under-represented on BOLD. This is evident as both the Northern Cape and Limpopo provinces contain one or more biodiversity hotspots which in turn displays the high levels of biodiversity that could still be recorded on BOLD. Improving our understanding with regards to DNA barcoding data linked to geographical regions, as well as specific insect groups, can highlight the areas in need of more research.

Automatic barcode gap discovery reveals diverse clades of Rhipicephalus spp. and Haemaphysalis spp. ticks from small mammals in 'Asir, Saudi Arabia.

BACKGROUND: The ixodid tick genera Rhipicephalus and Haemaphysalis contain several species of medical and/or veterinary importance, but their diversity in some regions of the world remains under-explored. For instance, very few modern studies have been performed on the taxonomy of these genera on the Arabian Peninsula. METHODS: In this study, we trapped small mammals in the 'Asir Mountains of south-western Saudi Arabia and collected tick specimens for morphological examination and molecular barcoding, targeting three mitochondrial loci: cox1, 16S rRNA and 12S rRNA. RESULTS: We obtained a total of 733 ticks (608 Haemaphysalis spp. and 125 Rhipicephalus spp.) from 75 small mammal hosts belonging to six species. All tick specimens were immature except for nine adults recovered from a hedgehog (Paraechinus aethiopicus). Morphologically, the Rhipicephalus ticks resembled R. camicasi, but the Haemaphysalis ticks showed differences in palp morphology compared with species previously described from Saudi Arabia. Phylogenetic analysis and automatic barcode gap discovery identified a novel clade of Rhipicephalus sp. representing most of the nymphs. This was most closely related to R. leporis, R. guilhoni and R. linnaei. The adult ticks and a small proportion of nymphs clustered with R. camicasi sequences from a previous study. Finally, the Haemaphysalis nymphs formed two distinct clades that were clearly separated from all reference sequences but closest to some African species. CONCLUSIONS: This apparent high level of tick diversity observed in a single study site of only ~ 170 km2, on a relatively small number of hosts, highlights the potential for the discovery of new tick species on the Arabian Peninsula.

Secondary predation constrains DNA-based diet reconstruction in two threatened shark species.

Increasing fishing effort, including bycatch and discard practices, are impacting marine biodiversity, particularly among slow-to-reproduce taxa such as elasmobranchs, and specifically sharks. While some fisheries involving sharks are sustainably managed, collateral mortalities continue, contributing towards > 35% of species being threatened with extinction. To effectively manage shark stocks, life-history information, including resource use and feeding ecologies is pivotal, especially among those species with wide-ranging distributions. Two cosmopolitan sharks bycaught off eastern Australia are the common blacktip shark (Carcharhinus limbatus; globally classified as Near Threatened) and great hammerhead (Sphyrna mokarran; Critically Endangered). We opportunistically sampled the digestive tracts of these two species (and also any whole prey; termed the 'Russian-doll' approach), caught in bather-protection gillnets off northern New South Wales, to investigate the capacity for DNA metabarcoding to simultaneously determine predator and prey regional feeding ecologies. While sample sizes were small, S. mokkaran fed predominantly on stingrays and skates (Myliobatiformes and Rajiformes), but also teleosts, while C. limbatus mostly consumed teleosts. Metabarcoding assays showed extensive intermixing of taxa from the digestive tracts of predators and their whole prey, likely via the predator's stomach chyme, negating the opportunity to distinguish between primary and secondary predation. This Russian-doll effect requires further investigation in DNA metabarcoding studies focussing on dietary preferences and implies that any outcomes will need to be interpreted concomitant with traditional visual approaches.

Illegal, Unreported, and Unregulated Fisheries Threatening Shark Conservation in African Waters Revealed from High Levels of Shark Mislabelling in Ghana.

Mislabelling of fish and fish products has attracted much attention over the last decades, following public awareness of the practice of substituting high-value with low-value fish in markets, restaurants, and processed seafood. In some cases, mislabelling includes illegal, unreported, and unregulated (IUU) fishing, contributing to overexploit substitute species that are undetectable when sold under wrong names. This is the first study of DNA barcoding to assess the level of mislabelling in fish marketed in Ghana, focusing on endangered shark species. Genetic identification was obtained from 650 base pair sequences within the cytochrome c oxidase I (COI) gene. All except one of 17 shark fillets analysed were wrongly labelled as compared with none of 28 samples of small commercial pelagic fish and 14 commercial shark samples purchased in Europe. Several substitute shark species in Ghana are endangered (Carcharhinussignatus and Isurusoxyrinchus) and critically endangered (Squatina aculeata). Shark products commercialized in Europe (n = 14) did not reveal mislabelling, thus specific shark mislabelling cannot be generalized. Although based on a limited number of samples and fish markets, the results that reveal trade of endangered sharks in Ghana markets encourage Ghanaian authorities to improve controls to enforce conservation measures.

The use of an integrative approach to improve accuracy of species identification and detection of new species in studies of stream fish diversity.

In this study, we made an inventory of the stream and headwater ichthyofauna of the left bank of the Itaipu Dam Reservoir, located in the lower part of the Upper Paraná River basin, using an integrative approach of molecular and morphological data. The area is located in the western portion of the Paraná state in Brazil, in an area of about 8,000 km2 highly impacted by deforestation and intensive agriculture. For taxonomic identification of species, we used an identification key combined with the DNA barcoding approach. We found 48 species belonging to six orders, 13 families, and 37 genera. The Siluriformes and Characiformes were the most representative orders (75%) and the Characidae was the most representative family (20.8%). Nine species prevailed in this region, making up to 86% of all specimens collected. The integrative approach proved to be useful by allowing the unambiguous identification of all species, including those cases in which morphological characters were not conclusive for species identification, cases of cryptic species, and species with high morphological plasticity. In addition, the integrative approach highlighted six to 13 new putative species depending on the approach considered. Our study provides a relevant contribution to the knowledge of fish diversity in a poorly studied area of the Paraná River basin. We showed that the use of an integrative approach in inventory studies improves species identification and the discovery of new, cryptic, and overlooked species, being a powerful and necessary tool to quantify biodiversity.

Can we manage fisheries with the inherent uncertainty from eDNA?

Environmental (e)DNA, as a general approach in aquatic systems, seeks to connect the presence of species' genetic material in the water and hence to infer the species' physical presence. However, fisheries managers face making decisions with risk and uncertainty when eDNA indicates a fish is present but traditional methods fail to capture the fish. In comparison with traditional methods such as nets, electrofishing and piscicides, eDNA approaches have more sources of underlying error that could give rise to false positives. This has resulted in some managers to question whether eDNA can be used to make management decisions because there is no fish in hand. As a relatively new approach, the methods and techniques have quickly evolved to improve confidence in eDNA. By evaluating an eDNA based research programmes through the pattern of the eDNA signal, assay design, experimental design, quality assurance and quality control checks, data analyses and concurrent search for fish using traditional gears, the evidence for fish presence can be evaluated to build confidence in the eDNA approach. The benefits for fisheries management from adopting an eDNA approach are numerous but include cost effectiveness, broader geographic coverage of habitat occupancy, early detection of invasive species, non-lethal stock assessments, exploration of previously inaccessible aquatic environments and discovery of new species hidden beneath the water's surface. At a time when global freshwater and marine fisheries are facing growing threats from over-harvest, pollution and climate change, we anticipate that growing confidence in eDNA will overcome the inherent uncertainty of not having a fish in hand and will empower the informed management actions necessary to protect and restore our fisheries.

Current challenges and best-practice protocols for microbiome analysis.

Analyzing the microbiome of diverse species and environments using next-generation sequencing techniques has significantly enhanced our understanding on metabolic, physiological and ecological roles of environmental microorganisms. However, the analysis of the microbiome is affected by experimental conditions (e.g. sequencing errors and genomic repeats) and computationally intensive and cumbersome downstream analysis (e.g. quality control, assembly, binning and statistical analyses). Moreover, the introduction of new sequencing technologies and protocols led to a flood of new methodologies, which also have an immediate effect on the results of the analyses. The aim of this work is to review the most important workflows for 16S rRNA sequencing and shotgun and long-read metagenomics, as well as to provide best-practice protocols on experimental design, sample processing, sequencing, assembly, binning, annotation and visualization. To simplify and standardize the computational analysis, we provide a set of best-practice workflows for 16S rRNA and metagenomic sequencing data (available at https://github.com/grimmlab/MicrobiomeBestPracticeReview).

The future of fish-based ecological assessment of European rivers: from traditional EU Water Framework Directive compliant methods to eDNA metabarcoding-based approaches.

Most of the present EU Water Framework Directive (WFD) compliant fish-based assessment methods of European rivers are multi-metric indices computed from traditional electrofishing (TEF) samples, but this method has known shortcomings, especially in large rivers. The probability of detecting rare species remains limited, which can alter the sensitivity of the indices. In recent years, environmental (e)DNA metabarcoding techniques have progressed sufficiently to allow applications in various ecological domains as well as eDNA-based ecological assessment methods. A review of the 25 current WFD-compliant methods for river fish shows that 81% of the metrics used in these methods are expressed in richness or relative abundance and thus compatible with eDNA samples. However, more than half of the member states' methods include at least one metric related to age or size structure and would have to adapt their current fish index if reliant solely on eDNA-derived information. Most trait-based metrics expressed in richness are higher when computed from eDNA than when computed from TEF samples. Comparable values are obtained only when the TEF sampling effort increases. Depending on the species trait considered, most trait-based metrics expressed in relative abundance are significantly higher for eDNA than for TEF samples or vice versa due to over-estimation of sub-surface species or under-estimation of benthic and rare species by TEF sampling, respectively. An existing predictive fish index, adapted to make it compatible with eDNA data, delivers an ecological assessment comparable with the current approved method for 22 of the 25 sites tested. Its associated uncertainty is lower than that of current fish indices. Recommendations for the development of future fish eDNA-based indices and the associated eDNA water sampling strategy are discussed.

Utility of DNA barcoding in native Oreochromis species.

The importance of Oreochromis in worldwide aquaculture and regional fisheries motivates the study of their genetic diversity in their native range. In this article, all mitochondrial cytochrome c oxidase subunit I gene (COI) sequences of Oreochromis species are retrieved from Barcode of Life Data system to quantify the available DNA barcoding information from wild individuals collected within the native ranges of the respective species. It is found that 70% of the known species in the genus still lack a COI barcode, and only 15% of the available sequences are from within the respective native ranges. Many of the available sequences have been produced from specimens acquired from aquaculture and introduced, naturalized populations, making the assessment of variation within the original native range challenging. Analyses of the wild-collected fraction of available sequences indicated the presence of cryptic lineages within Nile tilapia Oreochromis niloticus and O. schwebischi, the occurrence of potential introgressive hybridization between O. niloticus and blue tilapia O. aureus, and potential ancestral polymorphism between Karonga tilapia O. karongae and black tilapia O. placidus. This article also reports a case of misidentification of O. mweruensis as longfin tilapia O. macrochir. These results stress the importance of improving the knowledge of genetic variation within the native ranges of Oreochromis species for better-informed conservation of these natural resources.

Guidelines for the Choice of Sequences for Molecular Plant Taxonomy.

This chapter presents an overview of the major plant DNA sequences and molecular methods available for plant taxonomy. Guidelines are provided for the choice of sequences and methods to be used, based on the DNA compartment (nuclear, chloroplastic, mitochondrial), evolutionary mechanisms, and the level of taxonomic differentiation of the plants under survey.

High-Throughput Genotyping Technologies in Plant Taxonomy.

Molecular markers provide researchers with a powerful tool for variation analysis between plant genomes. They are heritable and widely distributed across the genome and for this reason have many applications in plant taxonomy and genotyping. Over the last decade, molecular marker technology has developed rapidly and is now a crucial component for genetic linkage analysis, trait mapping, diversity analysis, and association studies. This chapter focuses on molecular marker discovery, its application, and future perspectives for plant genotyping through pangenome assemblies. Included are descriptions of automated methods for genome and sequence distance estimation, genome contaminant analysis in sequence reads, genome structural variation, and SNP discovery methods.

Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity.

Analysis of genetic polymorphism is a powerful tool for epidemiological surveillance and research. Powerful inference from pathogen genetic variation, however, is often restrained by limited access to representative target DNA, especially in the study of obligate parasitic species for which ex vivo culture is resource-intensive or bias-prone. Modern sequence capture methods enable pathogen genetic variation to be analyzed directly from host/vector material but are often too complex and expensive for resource-poor settings where infectious diseases prevail. This study proposes a simple, cost-effective 'genome-wide locus sequence typing' (GLST) tool based on massive parallel amplification of information hotspots throughout the target pathogen genome. The multiplexed polymerase chain reaction amplifies hundreds of different, user-defined genetic targets in a single reaction tube, and subsequent agarose gel-based clean-up and barcoding completes library preparation at under 4 USD per sample. Our study generates a flexible GLST primer panel design workflow for Trypanosoma cruzi, the parasitic agent of Chagas disease. We successfully apply our 203-target GLST panel to direct, culture-free metagenomic extracts from triatomine vectors containing a minimum of 3.69 pg/µl T. cruzi DNA and further elaborate on method performance by sequencing GLST libraries from T. cruzi reference clones representing discrete typing units (DTUs) TcI, TcIII, TcIV, TcV and TcVI. The 780 SNP sites we identify in the sample set repeatably distinguish parasites infecting sympatric vectors and detect correlations between genetic and geographic distances at regional (< 150 km) as well as continental scales. The markers also clearly separate TcI, TcIII, TcIV and TcV + TcVI and appear to distinguish multiclonal infections within TcI. We discuss the advantages, limitations and prospects of our method across a spectrum of epidemiological research.

Development of microsatellite markers for a soricid water shrew, Chimarrogale platycephalus, and their successful use for individual identification.

The soricid water shrew Chimarrogale platycephalus is a mammalian species endemic to the Japanese Islands. The animals inhabit the islands of Honshu and Kyushu, and are considered to be extinct in Shikoku. Information on this water shrew from Honshu and Kyushu is scarce, and C. platycephalus is registered on many local governments' red lists as an endangered species. There are very few studies on their ethology, ecology or phylogenetics, due to difficulties related to the shrews being both nocturnal and aquatic: to study C. platycephalus, field research must be conducted in mountain streams at night. To overcome these challenges, we previously established a genetic analysis method using the feces of C. platycephalus, as a result of which the amount of phylogenetic and phylogeographic data has increased and our understanding of the species has improved. In this study, microsatellite markers were developed, and analyses using markers for 21 loci were performed. Moreover, to confirm the ability of these 21 microsatellite markers to differentiate individuals, all markers were tested using fecal and tissue specimens from 12 individuals reared separately in an aquarium. Using as few as 12 of these loci, individual differentiation with 100% accuracy should be achievable. The development of microsatellite markers in this study and the establishment of individual identification methods should greatly contribute to future ecological, ethological, population genetics and biogeographical research on C. platycephalus.

The Influences of Bioinformatics Tools and Reference Databases in Analyzing the Human Oral Microbial Community.

There is currently no criterion to select appropriate bioinformatics tools and reference databases for analysis of 16S rRNA amplicon data in the human oral microbiome. Our study aims to determine the influence of multiple tools and reference databases on α-diversity measurements and ß-diversity comparisons analyzing the human oral microbiome. We compared the results of taxonomical classification by Greengenes, the Human Oral Microbiome Database (HOMD), National Center for Biotechnology Information (NCBI) 16S, SILVA, and the Ribosomal Database Project (RDP) using Quantitative Insights Into Microbial Ecology (QIIME) and the Divisive Amplicon Denoising Algorithm (DADA2). There were 15 phyla present in all of the analyses, four phyla exclusive to certain databases, and different numbers of genera were identified in each database. Common genera found in the oral microbiome, such as Veillonella, Rothia, and Prevotella, are annotated by all databases; however, less common genera, such as Bulleidia and Paludibacter, are only annotated by large databases, such as Greengenes. Our results indicate that using different reference databases in 16S rRNA amplicon data analysis could lead to different taxonomic compositions, especially at genus level. There are a variety of databases available, but there are no defined criteria for data curation and validation of annotations, which can affect the accuracy and reproducibility of results, making it difficult to compare data across studies.

Optimization of the second internal transcribed spacer (ITS2) for characterizing land plants from soil.

Molecular-based taxonomy, specifically DNA barcoding, has streamlined organism identification. For land plants, the recommended 2-locus barcode of rbcL and matK is not suitable for all groups, thus the second subunit of the nuclear internal transcribed spacer (ITS2) has received attention as a possible alternative. To date, evaluations of ITS2 have mostly been limited in scope to specific plant orders/families and single source material. Prior to using ITS2 to routinely characterize land plants present in environmental samples (i.e., DNA metabarcoding), a wet lab protocol optimized for bulk sample types is needed. To address this gap, in this study we determined the broad recoverability across land plants when using published ITS2 primer pairs, and subsequently optimized the PCR reaction constituents and cycling conditions for the best two performing primer pairs (ITS2F/ITSp4 and ITSp3/ITSu4). Using these conditions, both primer pairs were used to characterize land plants present in 17 diverse soils collected from across the US. The resulting PCR amplicons were prepared into libraries and pooled for sequencing on an Illumina® MiniSeq. Our existing bioinformatics workflow was used to process raw sequencing data and taxonomically assign unique ITS2 plant sequences by comparison to GenBank. Given strict quality criteria were imposed on sequences for inclusion in data analysis, only 43.6% and 7.5% of sequences from ITS2F/ITSp4 and ITSp3/ITSu4 respectively remained for taxonomic comparisons; ~7-11% of sequences originated from fungal co-amplification. The number of orders and families recovered did differ between primer pairs, with ITS2F/ITSp4 consistently outperforming ITSp3/ITSu4 by >15%. Primer pair bias was observed in the recovery of certain taxonomic groups; ITS2F/ITSp4 preferentially recovered flowering plants and grasses, whereas ITSp3/ITSu4 recovered more moss taxa. To maximize data recovery and reduce potential bias, we advocate that studies using ITS2 to characterize land plants from environmental samples such as soil use a multiple primer pair approach.

Mitochondrial plastid DNA can cause DNA barcoding paradox in plants.

The transfer of ancestral plastid genomes into mitochondrial genomes to generate mitochondrial plastid DNA (MTPT) is known to occur in plants, but its impacts on mitochondrial genome complexity and the potential for causing a false-positive DNA barcoding paradox have been underestimated. Here, we assembled the organelle genomes of Cynanchum wilfordii and C. auriculatum, which are indigenous medicinal herbs in Korea and China, respectively. In both species, it is estimated that 35% of the ancestral plastid genomes were transferred to mitochondrial genomes over the past 10 million years and remain conserved in these genomes. Some plastid barcoding markers co-amplified the conserved MTPTs and caused a barcoding paradox, resulting in mis-authentication of botanical ingredients and/or taxonomic mis-positioning. We identified dynamic and lineage-specific MTPTs that have contributed to mitochondrial genome complexity and might cause a putative barcoding paradox across 81 plant species. We suggest that a DNA barcoding guidelines should be developed involving the use of multiple markers to help regulate economically motivated adulteration.

PEMA: a flexible Pipeline for Environmental DNA Metabarcoding Analysis of the 16S/18S ribosomal RNA, ITS, and COI marker genes.

BACKGROUND: Environmental DNA and metabarcoding allow the identification of a mixture of species and launch a new era in bio- and eco-assessment. Many steps are required to obtain taxonomically assigned matrices from raw data. For most of these, a plethora of tools are available; each tool's execution parameters need to be tailored to reflect each experiment's idiosyncrasy. Adding to this complexity, the computation capacity of high-performance computing systems is frequently required for such analyses. To address the difficulties, bioinformatic pipelines need to combine state-of-the art technologies and algorithms with an easy to get-set-use framework, allowing researchers to tune each study. Software containerization technologies ease the sharing and running of software packages across operating systems; thus, they strongly facilitate pipeline development and usage. Likewise programming languages specialized for big data pipelines incorporate features like roll-back checkpoints and on-demand partial pipeline execution. FINDINGS: PEMA is a containerized assembly of key metabarcoding analysis tools that requires low effort in setting up, running, and customizing to researchers' needs. Based on third-party tools, PEMA performs read pre-processing, (molecular) operational taxonomic unit clustering, amplicon sequence variant inference, and taxonomy assignment for 16S and 18S ribosomal RNA, as well as ITS and COI marker gene data. Owing to its simplified parameterization and checkpoint support, PEMA allows users to explore alternative algorithms for specific steps of the pipeline without the need of a complete re-execution. PEMA was evaluated against both mock communities and previously published datasets and achieved results of comparable quality. CONCLUSIONS: A high-performance computing-based approach was used to develop PEMA; however, it can be used in personal computers as well. PEMA's time-efficient performance and good results will allow it to be used for accurate environmental DNA metabarcoding analysis, thus enhancing the applicability of next-generation biodiversity assessment studies.