A workflow for accurate metabarcoding using nanopore MinION sequencing

1. Metabarcoding has become a common approach to the rapid identification of the species composition in a mixed sample. The majority of studies use established short‐read high‐throughput sequencing platforms. The Oxford Nanopore MinION™, a portable sequencing platform, represents a low‐cost alternative allowing researchers to generate sequence data in the field. However, a major drawback is the high raw read error rate that can range from 10% to 22%. 2. To test if the MinION™ represents a viable alternative to other sequencing platforms we used rolling circle amplification (RCA) to generate full‐length consensus DNA barcodes for a bulk mock sample of 50 aquatic invertebrate species with at least 15% genetic distance to each other. By applying two different laboratory protocols, we generated two MinION™ runs that were used to build error‐corrected consensus sequences. A newly developed Python pipeline, ASHURE, was used for data processing, consensus building, clustering, and taxonomic assignment of the resulting reads. 3. Our pipeline achieved median accuracies of up to 99.3% for long concatemeric reads (>45 barcodes) and successfully identified all 50 species in the mock community. The use of RCA was integral for increasing consensus accuracy but was also the most time‐consuming step of the laboratory workflow. Most concatemeric reads were skewed towards a shorter read length range with a median read length of up to 1262bp. 4. Our study demonstrates that Nanopore sequencing can be used for metabarcoding, but exploration of other isothermal amplification procedures to improve consensus accuracy is recommended

E+E-Projekt „Integration von Naturschutzzielen in den Ökologischen Landbau am Beispiel der Hessischen Staatsdomäne Frankenhausen“ - Maßnahmen zur Entwicklung dauerhafter Landschaftsstrukturen

The Hessian state domain Frankenhausen near Kassel was converted to organic farming in July 1998 and serves as a research and demonstration farm of the univer-sity. Before that time the farm changed constantly by typical intensification of conven-tional farming: The landscape is a mirror of intense use: cleared and drained fields, canalised brooks and large fields with few structural elements. – Within the project “The Integration of Nature Conservation into Organic Farming” (supported by the German Federal Agency for Nature Conservation with funds from the Federal Envi-ronmental Ministry) and based on the analysis of the present situation using methods of landscape ecology. Measures to restore brooks, structural elements and biotopes are to be implemented. The aim is to integrate structural elements like hedgerows into the farm management

Investigating Plastid Genome Evolution in Heterotrophic Plants and the Use of Structural Changes to the Plastid Genome as Phylogenetic Markers

Photosynthesis is the hallmark of plant evolution; the vast majority of plants are autotrophic and rely entirely on this process to fix carbon. As a consequence, the plastid genome (or plastome) is highly conserved across land plants with respect to its size, synteny, and gene content. Owing to this predictable structure as well as the low rates of nucleotide substitution, the plastome is a useful source of data for addressing phylogenetic questions. I use a rare structural change to the plastome as a phylogenetic marker to address the position of Gnetales within seed plants which has been difficult to resolve. The loss of all eleven plastid ndh genes support a 'gnepine' hypothesis or the placement of Gnetales as sister to Pinaceae. About 1% of flowering plants have lost all or some of their photosynthetic ability. These so-called heterotrophic plants are capable of obtaining water and/or nutrients from a host and are divided into two distinct groups: haustorial parasites, and mycoheterotrophs. In extreme cases, fully heterotrophic plants have completely lost the ability to photosynthesize. The phylogenetic position of heterotrophic plants has been difficult to resolve due to extensive changes to plastome size, structure, and content. Broad-scale angiosperm phylogenies indicate that the haustorial parasitism has evolved at least 11 times independently and there are at least 10 independent origins of mycoheterotrophy among land plants. Using a Southern hybridization approach combined with an extensive taxon sampling, I investigated the evolution of plastome protein coding gene content in parasitic Cuscuta (Convolvulaceae) and mycoheterotrophic Ericaceae. Common patterns include the early loss of the plastid ndh genes and extreme reduction in coding content of achlorophyllous species. A most intriguing result is the apparent loss of all plastid genes, including the highly conserved ribosomal RNA genes in Cuscuta section Subulatae ('O' clade), this condition that has only been inferred among the highly specialized parasites in Rafflesiaceae. Informed by these broad surveys, I further investigated the evolution of mycoheterotrophy in Ericaceae, using a select sampling across the trophic spectrum and a next generation sequencing approach. The plastomes of all Ericaceae species are highly rearranged and lack synteny with a typical plastome. Furthermore, positive selection was found to be acting on the rps, rpl, rpo, and ndh genes in a number of photosynthetic lineages. The plastid ndh genes are variable for their presence and absence in the partially mycoheterotrophic Pyroleae. The plastomes of fully mycoheterotrophic species are highly reduced with Allotropa virgata possessing the smallest plastome among land plants observed to date. Additionally, these plastomes have been reduced to encoding a highly divergent accD open reading frame, matK, and housekeeping genes that are generally under purifying selection. However, several ribosomal proteins and matK, were found to be under positive selection.Ph.D.2018-06-08 00:00:0

Effects of Malaise trap spacing on species richness and composition of terrestrial arthropod bulk samples

The Malaise trap is a popular device for assessing diverse terrestrial arthropod communities because it collects large samples with modest effort. A number of factors influence its collection efficiency, placement being one of them. For instance, when designing larger biotic surveys using arrays of Malaise traps we need to know the optimal distance between individual traps that maximises observable species richness and community composition. We examined the influence of spacing between Malaise traps by metabarcoding samples from two field experiments at a site in Waterloo, Ontario, Canada. For one experiment, we used two trap pairs deployed at weekly increasing distances (3 m increments from 3 to 30 m). The second experiment involved a total of 10 traps set up in a row at 3 m distance intervals for three consecutive weeks. Results show that community similarity of samples decreases over distance between traps. The amount of species shared between trap pairs drops considerably at about 18 m trap-to-trap distance. This change can be observed across all major taxonomic groups and for two different habitat types (grassland and forest). Large numbers of OTUs found only once within samples cause rather large dissimilarity between distance pairs even at close proximity. This could be caused by a large number of transient species from adjacent habitats which arrive at the trap through passive transport, as well as capture of rare taxa, which end up in different traps by chance

The power of metabarcoding: Can we improve bioassessment and biodiversity surveys of stream macroinvertebrate communities?

Most stream bioassessment and biodiversity surveys are currently based on morphological identification of communities. However, DNA metabarcoding is emerging as a fast and cost-effective alternative for species identification. We compared both methods in a survey of benthic macroinvertebrate communities across 36 stream sites in northern Finland. We identified 291 taxa of which 62% were identified only by DNA metabarcoding. DNA metabarcoding produced extensive species level inventories for groups (Oligochaeta, Chironomidae, Simuliidae, Limoniidae and Limnephilidae), for which morphological identification was not feasible due to the high level of expertise needed. Metabarcoding also provided more insightful taxonomic information on the occurrence of three red-listed vulnerable or data deficient species, the discovery of two likely cryptic and potentially new species to Finland and species information of insect genera at an early larval stage that could not be separated morphologically. However, it systematically failed to reliably detect the occurrence of gastropods that were easily identified morphologically. The impact of mining on community structure could only be shown using DNA metabarcoding data which suggests that the finer taxonomic detail can improve detection of subtle impacts. Both methods generally exhibited similar strength of community-environment relationships, but DNA metabarcoding showed better performance with presence/absence data than with relative DNA sequence abundances. Our results suggest that DNA metabarcoding holds a promise for future anthropogenic impact assessments, although, in our case, the performance did not improve much from the morphological species identification. The key advantage of DNA metabarcoding lies in efficient biodiversity surveys, taxonomical studies and applications in conservation biology

The power of metabarcoding: Can we improve bioassessment and biodiversity surveys of stream macroinvertebrate communities?

Most stream bioassessment and biodiversity surveys are currently based on morphological identification of communities. However, DNA metabarcoding is emerging as a fast and cost-effective alternative for species identification. We compared both methods in a survey of benthic macroinvertebrate communities across 36 stream sites in northern Finland. We identified 291 taxa of which 62% were identified only by DNA metabarcoding. DNA metabarcoding produced extensive species level inventories for groups (Oligochaeta, Chironomidae, Simuliidae, Limoniidae and Limnephilidae), for which morphological identification was not feasible due to the high level of expertise needed. Metabarcoding also provided more insightful taxonomic information on the occurrence of three red-listed vulnerable or data deficient species, the discovery of two likely cryptic and potentially new species to Finland and species information of insect genera at an early larval stage that could not be separated morphologically. However, it systematically failed to reliably detect the occurrence of gastropods that were easily identified morphologically. The impact of mining on community structure could only be shown using DNA metabarcoding data which suggests that the finer taxonomic detail can improve detection of subtle impacts. Both methods generally exhibited similar strength of community-environment relationships, but DNA metabarcoding showed better performance with presence/absence data than with relative DNA sequence abundances. Our results suggest that DNA metabarcoding holds a promise for future anthropogenic impact assessments, although, in our case, the performance did not improve much from the morphological species identification. The key advantage of DNA metabarcoding lies in efficient biodiversity surveys, taxonomical studies and applications in conservation biology

Correction: Authentication of Herbal Supplements Using Next-Generation Sequencing.

[This corrects the article DOI: 10.1371/journal.pone.0156426.]

Authentication of Herbal Supplements Using Next-Generation Sequencing.

BACKGROUND:DNA-based testing has been gaining acceptance as a tool for authentication of a wide range of food products; however, its applicability for testing of herbal supplements remains contentious. METHODS:We utilized Sanger and Next-Generation Sequencing (NGS) for taxonomic authentication of fifteen herbal supplements representing three different producers from five medicinal plants: Echinacea purpurea, Valeriana officinalis, Ginkgo biloba, Hypericum perforatum and Trigonella foenum-graecum. Experimental design included three modifications of DNA extraction, two lysate dilutions, Internal Amplification Control, and multiple negative controls to exclude background contamination. Ginkgo supplements were also analyzed using HPLC-MS for the presence of active medicinal components. RESULTS:All supplements yielded DNA from multiple species, rendering Sanger sequencing results for rbcL and ITS2 regions either uninterpretable or non-reproducible between the experimental replicates. Overall, DNA from the manufacturer-listed medicinal plants was successfully detected in seven out of eight dry herb form supplements; however, low or poor DNA recovery due to degradation was observed in most plant extracts (none detected by Sanger; three out of seven-by NGS). NGS also revealed a diverse community of fungi, known to be associated with live plant material and/or the fermentation process used in the production of plant extracts. HPLC-MS testing demonstrated that Ginkgo supplements with degraded DNA contained ten key medicinal components. CONCLUSION:Quality control of herbal supplements should utilize a synergetic approach targeting both DNA and bioactive components, especially for standardized extracts with degraded DNA. The NGS workflow developed in this study enables reliable detection of plant and fungal DNA and can be utilized by manufacturers for quality assurance of raw plant materials, contamination control during the production process, and the final product. Interpretation of results should involve an interdisciplinary approach taking into account the processes involved in production of herbal supplements, as well as biocomplexity of plant-plant and plant-fungal biological interactions