Relating target fish DNA concentration to community composition analysis in freshwater fish via metabarcoding.

Metabarcoding has been widely accepted as a useful tool for biodiversity assessment based on eDNA. The method allows for the detection of entire groups of organisms in a single sample, making it particularly applicable in aquatic habitats. The high sensitivity of the molecular approaches is especially beneficial in detecting elusive and rare fish species, improving biodiversity assessments. Numerous biotic and abiotic factors that affect the persistence and availability of fish DNA in surface waters and therefore affecting species detectability, have been identified. However, little is known about the relationship between the total fish DNA concentration and the detectability of differential abundant species. In this study three controlled mock-community DNA samples (56 individual samples) were analyzed by (i) metabarcoding (MiSeq) of 12S rDNA (175 bp) and by (ii) total freshwater fish DNA quantification (via qPCR of 12S rDNA). We show that the fish DNA quantity affects the relative abundance of species-specific sequences and the detectability of rare species. In particular we found that samples with a concentration between 1000 pg/µL down to 10 pg/µL of total fish DNA revealed a stable relative frequency of DNA sequences obtained for a specific fish species, as well as a low variability between replicates. Additionally, we observed that even in complex mock-community DNA samples, a total fish DNA concentration of 23 pg/µL was sufficient to reliably detect all species in every replicate, including three rare species with proportions of ≤0.5 %. We also found that the DNA barcode similarity between species can affect detectability, if evenness is low. Our data suggest that the total DNA concentration of fish is an important factor to consider when analyzing and interpreting relative sequence abundance data. Therefore, the workflow proposed here will contribute to an ecologically and economically efficient application of metabarcoding in fish biodiversity assessment.

Quantitative relationships among high-throughput sequencing, cyanobacteria toxigenic genotype abundance and microcystin occurrence in bathing waters.

Toxin-producing cyanobacteria pose significant threats to human and animal health if exposed during recreational activities in bathing waters. To better safeguard public health and reduce health risks during the bathing season, an effective monitoring and management strategy is required. Molecular tools used to monitor toxigenic cyanobacteria have been evaluated on the basis of the efficiency and applicability of the method used to (i) establish an early-warning monitoring strategy for EU bathing water sites using both targeted quantitative polymerase chain reaction (qPCR) and non-targeted high-throughput sequencing (HTS) genotype analysis and (ii) to compare the toxigenic potential of cyanobacteria with actual microcystin (MC) occurrence and concentrations. For this purpose, 16 bathing water sites were monitored according to the bathing water directive (BWD) of the European Union (EU) during the bathing season of the summer of 2020 in eastern Austria. The cyanobacterial community composition was analyzed through HTS and qPCR by targeting the microcystin synthetase B gene (mcyB), which indicates MC synthesis within the genera Microcystis and Planktothrix. Within the genus Microcystis, which was identified as the primary MC producer, the mcyB genotypes formed stable subpopulations that increased linearly in correlation with the total Microcystis population. Notably, the HTS cell equivalents assigned to Microcystis and Planktothrix correlated with the corresponding qPCR estimates of genotype abundance, which serves as a confirmation of the suitability of (semi)-quantitative sequencing through HTS. In addition to the elevated trophic state, reduced transparency, increasing water temperatures, as well as cyanobacterial HTS read numbers and Microcystis cell number equivalents per mL estimated through qPCR, were associated with positive MC samples. Therefore, in combination with the monitoring of standard environmental parameters, the use of HTS and qPCR techniques is considered highly useful to ensure the timely identification of health risks to recreational users, as mandated by the BWD.

Development of a DNA Metabarcoding Method for the Identification of Insects in Food.

Insects have the potential to become an efficient and reliable food source for humans in the future and could contribute to solving problems with the current food chain. Analytical methods to verify the authenticity of foods are essential for consumer acceptance. We present a DNA metabarcoding method that enables the identification and differentiation of insects in food. The method, developed on Illumina platforms, is targeting a 200 bp mitochondrial 16S rDNA fragment, which we found to be suitable for distinguishing more than 1000 insect species. We designed a novel universal primer pair for a singleplex PCR assay. Individual DNA extracts from reference samples, DNA extracts from model foods and food products commercially available were investigated. In all of the samples investigated, the insect species were correctly identified. The developed DNA metabarcoding method has a high potential to identify and differentiate insect DNA in the context of food authentication in routine analysis.

Interlaboratory Validation of a DNA Metabarcoding Assay for Mammalian and Poultry Species to Detect Food Adulteration.

Meat species authentication in food is most commonly based on the detection of genetic variations. Official food control laboratories frequently apply single and multiplex real-time polymerase chain reaction (PCR) assays and/or DNA arrays. However, in the near future, DNA metabarcoding, the generation of PCR products for DNA barcodes, followed by massively parallel sequencing by next generation sequencing (NGS) technologies, could be an attractive alternative. DNA metabarcoding is superior to well-established methodologies since it allows simultaneous identification of a wide variety of species not only in individual foodstuffs but even in complex mixtures. We have recently published a DNA metabarcoding assay for the identification and differentiation of 15 mammalian species and six poultry species. With the aim to harmonize analytical methods for food authentication across EU Member States, the DNA metabarcoding assay has been tested in an interlaboratory ring trial including 15 laboratories. Each laboratory analyzed 16 anonymously labelled samples (eight samples, two subsamples each), comprising six DNA extract mixtures, one DNA extract from a model sausage, and one DNA extract from maize (negative control). Evaluation of data on repeatability, reproducibility, robustness, and measurement uncertainty indicated that the DNA metabarcoding method is applicable for meat species authentication in routine analysis.

Development of a DNA Metabarcoding Method for the Identification of Bivalve Species in Seafood Products.

The production of bivalve species has been increasing in the last decades. In spite of strict requirements for species declaration, incorrect labelling of bivalve products has repeatedly been detected. We present a DNA metabarcoding method allowing the identification of bivalve species belonging to the bivalve families Mytilidae (mussels), Pectinidae (scallops), and Ostreidae (oysters) in foodstuffs. The method, developed on Illumina instruments, targets a 150 bp fragment of mitochondrial 16S rDNA. We designed seven primers (three primers for mussel species, two primers for scallop species and a primer pair for oyster species) and combined them in a triplex PCR assay. In each of eleven reference samples, the bivalve species was identified correctly. In ten DNA extract mixtures, not only the main component (97.0-98.0%) but also the minor components (0.5-1.5%) were detected correctly, with only a few exceptions. The DNA metabarcoding method was found to be applicable to complex and processed foodstuffs, allowing the identification of bivalves in, e.g., marinated form, in sauces, in seafood mixes and even in instant noodle seafood. The method is highly suitable for food authentication in routine analysis, in particular in combination with a DNA metabarcoding method for mammalian and poultry species published recently.

Identification of Mammalian and Poultry Species in Food and Pet Food Samples Using 16S rDNA Metabarcoding.

The substitution of more appreciated animal species by animal species of lower commercial value is a common type of meat product adulteration. DNA metabarcoding, the combination of DNA barcoding with next-generation sequencing (NGS), plays an increasing role in food authentication. In the present study, we investigated the applicability of a DNA metabarcoding method for routine analysis of mammalian and poultry species in food and pet food products. We analyzed a total of 104 samples (25 reference samples, 56 food products and 23 pet food products) by DNA metabarcoding and by using a commercial DNA array and/or by real-time PCR. The qualitative and quantitative results obtained by the DNA metabarcoding method were in line with those obtained by PCR. Results from the independent analysis of a subset of seven reference samples in two laboratories demonstrate the robustness and reproducibility of the DNA metabarcoding method. DNA metabarcoding is particularly suitable for detecting unexpected species ignored by targeted methods such as real-time PCR and can also be an attractive alternative with respect to the expenses as indicated by current data from the cost accounting of the AGES laboratory. Our results for the commercial samples show that in addition to food products, DNA metabarcoding is particularly applicable to pet food products, which frequently contain multiple animal species and are also highly prone to adulteration as indicated by the high portion of analyzed pet food products containing undeclared species.

Apple chlorotic fruit spot viroid: a putative new pathogenic viroid on apple characterized by next-generation sequencing.

Viroid-like symptoms were observed in 2016 on apple fruits of the cultivar "Ilzer Rose" in southern Burgenland, Austria. Preliminary molecular biological investigations indicated that the symptoms were caused by a new unknown viroid. Therefore, new primers were designed, and the whole genome sequence of the viroid (354 nt) was determined by next-generation amplicon sequencing using the Illumina MiSeq® platform (San Diego, California, USA). The viroid secondary structure has a rod-like conformation and contains conserved regions (the TCR, CCR upper strand, and CCR lower strand) that are characteristic of members of the genus Apscaviroid. Based on our results and the demarcation criteria for viroids, the tentatively named "apple chlorotic fruit spot viroid" should be considered a putative new member of the genus Apscaviroid.

Development of a DNA metabarcoding method for the identification of fifteen mammalian and six poultry species in food.

Meat products are prone to adulteration by the replacement of meat from more expensive animal species with meat from cheaper sources. We present a DNA metabarcoding method allowing the identification and differentiation of 15 mammalian and six poultry species in foodstuffs. The method, developed on the MiSeq® platform, targets a mitochondrial 16S rDNA region recently found to be suitable for the differentiation of 300 mammalian species. We designed a novel primer pair for poultry and applied it in combination with the primer pair for mammalian species in a duplex assay. The applicability of the method was investigated by analysing DNA extracts from muscle, DNA extract mixtures and extracts from model sausages. Our results indicated that the species of interest can be identified, differentiated and detected down to a proportion of 0.1%. Since 96 samples can be sequenced in one run, the method has high potential for application in routine analysis.