A from‐benchtop‐to‐desktop workflow for validating HTS data and for taxonomic identification in diet metabarcoding studies

The main objective of this work was to develop and validate a robust and reliable “from‐benchtop‐to‐desktop” metabarcoding workflow to investigate the diet of invertebrate‐eaters. We applied our workflow to faecal DNA samples of an invertebrate‐eating fish species. A fragment of the cytochrome c oxidase I (COI) gene was amplified by combining two minibarcoding primer sets to maximize the taxonomic coverage. Amplicons were sequenced by an Illumina MiSeq platform. We developed a filtering approach based on a series of nonarbitrary thresholds established from control samples and from molecular replicates to address the elimination of cross‐contamination, PCR/sequencing errors and mistagging artefacts. This resulted in a conservative and informative metabarcoding data set. We developed a taxonomic assignment procedure that combines different approaches and that allowed the identification of ~75% of invertebrate COI variants to the species level. Moreover, based on the diversity of the variants, we introduced a semiquantitative statistic in our diet study, the minimum number of individuals, which is based on the number of distinct variants in each sample. The metabarcoding approach described in this article may guide future diet studies that aim to produce robust data sets associated with a fine and accurate identification of prey items.     

PipeCraft: Flexible open‐source toolkit for bioinformatics analysis of custom high‐throughput amplicon sequencing data

High‐throughput sequencing methods have become a routine analysis tool in environmental sciences as well as in public and private sector. These methods provide vast amount of data, which need to be analysed in several steps. Although the bioinformatics may be applied using several public tools, many analytical pipelines allow too few options for the optimal analysis for more complicated or customized designs. Here, we introduce PipeCraft, a flexible and handy bioinformatics pipeline with a user‐friendly graphical interface that links several public tools for analysing amplicon sequencing data. Users are able to customize the pipeline by selecting the most suitable tools and options to process raw sequences from Illumina, Pacific Biosciences, Ion Torrent and Roche 454 sequencing platforms. We described the design and options of PipeCraft and evaluated its performance by analysing the data sets from three different sequencing platforms. We demonstrated that PipeCraft is able to process large data sets within 24 hr. The graphical user interface and the automated links between various bioinformatics tools enable easy customization of the workflow. All analytical steps and options are recorded in log files and are easily traceable.     

Effect of marker choice and thermal cycling protocol on zooplankton DNA metabarcoding studies

DNA metabarcoding is a promising approach for rapidly surveying biodiversity and is likely to become an important tool for measuring ecosystem responses to environmental change. Metabarcoding markers need sufficient taxonomic coverage to detect groups of interest, sufficient sequence divergence to resolve species, and will ideally indicate relative abundance of taxa present. We characterized zooplankton assemblages with three different metabarcoding markers (nuclear 18S rDNA, mitochondrial COI, and mitochondrial 16S rDNA) to compare their performance in terms of taxonomic coverage, taxonomic resolution, and correspondence between morphology‐ and DNA‐based identification. COI amplicons sequenced on separate runs showed that operational taxonomic units representing >0.1% of reads per sample were highly reproducible, although slightly more taxa were detected using a lower annealing temperature. Mitochondrial COI and nuclear 18S showed similar taxonomic coverage across zooplankton phyla. However, mitochondrial COI resolved up to threefold more taxa to species compared to 18S. All markers revealed similar patterns of beta‐diversity, although different taxa were identified as the greatest contributors to these patterns for 18S. For calanoid copepod families, all markers displayed a positive relationship between biomass and sequence reads, although the relationship was typically strongest for 18S. The use of COI for metabarcoding has been questioned due to lack of conserved primer‐binding sites. However, our results show the taxonomic coverage and resolution provided by degenerate COI primers, combined with a comparatively well‐developed reference sequence database, make them valuable metabarcoding markers for biodiversity assessment.     

Analysis of trapped mosquito excreta as a noninvasive method to reveal biodiversity and arbovirus circulation

Emerging and endemic mosquito-borne viruses can be difficult to detect and monitor because they often cause asymptomatic infections in human or vertebrate animals or cause nonspecific febrile illness with a short recovery waiting period. Some of these pathogens circulate into complex cryptic cycles involving several animal species as reservoir or amplifying hosts. Detection of cases in vertebrate hosts can be complemented by entomological surveillance, but this method is not adapted to low infection rates in mosquito populations that typically occur in low or nonendemic areas. We identified West Nile virus circulation in Camargue, a wetland area in South of France, using a cost-effective xenomonitoring method based on the molecular detection of virus in excreta from trapped mosquitoes. We also succeeded at identifying the mosquito species community on several sampling sites, together with the vertebrate hosts on which they fed prior to being captured using amplicon-based metabarcoding on mosquito excreta without processing any mosquitoes. Mosquito excreta-based virus surveillance can complement standard surveillance methods because it is cost-effective and does not require personnel with a strong background in entomology. This strategy can also be used to noninvasively explore the ecological network underlying arbovirus circulation.     

Minimizing polymerase biases in metabarcoding

DNA metabarcoding is an increasingly popular method to characterize and quantify biodiversity in environmental samples. Metabarcoding approaches simultaneously amplify a short, variable genomic region, or “barcode,” from a broad taxonomic group via the polymerase chain reaction (PCR), using universal primers that anneal to flanking conserved regions. Results of these experiments are reported as occurrence data, which provide a list of taxa amplified from the sample, or relative abundance data, which measure the relative contribution of each taxon to the overall composition of amplified product. The accuracy of both occurrence and relative abundance estimates can be affected by a variety of biological and technical biases. For example, taxa with larger biomass may be better represented in environmental samples than those with smaller biomass. Here, we explore how polymerase choice, a potential source of technical bias, might influence results in metabarcoding experiments. We compared potential biases of six commercially available polymerases using a combination of mixtures of amplifiable synthetic sequences and real sedimentary DNA extracts. We find that polymerase choice can affect both occurrence and relative abundance estimates and that the main source of this bias appears to be polymerase preference for sequences with specific GC contents. We further recommend an experimental approach for metabarcoding based on results of our synthetic experiments.     

Highlighting patterns of fungal diversity and composition shaped by ocean currents using the East China Sea as a model

How ocean currents shape fungal transport, dispersal and more broadly fungal biogeography remains poorly understood. The East China Sea (ECS) is a complex and dynamic habitat with different water masses blending microbial communities. The internal transcribed spacer 2 region of fungal rDNA was analysed in water and sediment samples directly collected from the coastal (CWM), Kuroshio (KSWM), Taiwan warm (TWM) and the shelf mixed water mass (MWM), coupled with hydrographic properties measurements, to determine how ocean currents impact the fungal community composition. Almost 9k fungal operational taxonomic units (OTUs) spanning six phyla, 25 known classes, 102 orders and 694 genera were obtained. The typical terrestrial and freshwater fungal genus, Byssochlamys, was dominant in the CWM, while increasing abundance of a specific OTU affiliated with Aspergillus was revealed from coastal to open ocean water masses (TWM and KSWM). Compared with water samples, sediment harboured an increased diversity with distinct fungal communities. The proximity of the Yangtze and Qiantang estuaries homogenizes the surface water and sediment communities. A significant influence of ocean currents on community structure was found, which is believed to reduce proportionally the variation explained by environmental parameters at the scale of the total water masses. Dissolved oxygen and depth were identified as the major parameters structuring the fungal community. Our results indicate that passive fungal dispersal driven by ocean currents and river run‐off, in conjunction with the distinct hydrographic conditions of individual water masses, shapes the fungal community composition and distribution pattern in the ECS.     

基于环境DNA-宏条形码技术的水生生态系统入侵生物的早期监测与预警

外来生物入侵是继生境破坏后造成生物多样性丧失的第二大威胁因素, 已对入侵地的生态安全、经济和社会发展及人类健康等造成严重负面影响, 成为21世纪五大全球性环境问题之一。作为水产养殖、航运和水生宠物交易大国, 我国水生生态系统的生物入侵问题尤为严重。研究表明, 系统地构建并应用早期监测预警技术是防控水生生态系统生物入侵最有效的途径。和陆生生物相比, 水生生物群落的物种繁多、群落结构复杂、生物形体微小且在入侵初期群体规模极小、隐匿于水下、可用于物种鉴定的外部形态缺乏, 使得在水生生态系统中构建并应用早期监测和预警体系在技术层面更具挑战。随着高通量测序技术的快速发展, 环境DNA-宏条形码技术成为构建水生生态系统入侵生物早期监测与预警技术的首选。本文主要综述了基于环境DNA-宏条形码技术的水生生态系统入侵生物的早期监测与预警技术方法; 解析了环境DNA-宏条形码监测系统的应用现状、技术优势; 着重探讨了影响监测结果准确性的I型和II型错误及其产生原因, 并为避免两类错误提供了可行的优化/改进方案; 最后对该方法在水生入侵生物监测中的应用前景进行了展望。     

DNA metabarcoding unveils multiscale trophic variation in a widespread coastal opportunist

A thorough understanding of ecological networks relies on comprehensive information on trophic relationships among species. Since unpicking the diet of many organisms is unattainable using traditional morphology‐based approaches, the application of high‐throughput sequencing methods represents a rapid and powerful way forward. Here, we assessed the application of DNA metabarcoding with nearly universal primers for the mitochondrial marker cytochrome c oxidase I in defining the trophic ecology of adult brown shrimp, Crangon crangon, in six European estuaries. The exact trophic role of this abundant and widespread coastal benthic species is somewhat controversial, while information on geographical variation remains scant. Results revealed a highly opportunistic behaviour. Shrimp stomach contents contained hundreds of taxa (>1,000 molecular operational taxonomic units), of which 291 were identified as distinct species, belonging to 35 phyla. Only twenty ascertained species had a mean relative abundance of more than 0.5%. Predominant species included other abundant coastal and estuarine taxa, including the shore crab Carcinus maenas and the amphipod Corophium volutator. Jacobs’ selectivity index estimates based on DNA extracted from both shrimp stomachs and sediment samples were used to assess the shrimp's trophic niche indicating a generalist diet, dominated by crustaceans, polychaetes and fish. Spatial variation in diet composition, at regional and local scales, confirmed the highly flexible nature of this trophic opportunist. Furthermore, the detection of a prevalent, possibly endoparasitic fungus (Purpureocillium lilacinum) in the shrimp's stomach demonstrates the wide range of questions that can be addressed using metabarcoding, towards a more robust reconstruction of ecological networks.