What Else Is in Salviae officinalis folium? Comprehensive Species Identification of Plant Raw Material by DNA Metabarcoding

Planta Medica ◽  
2017 ◽  
Vol 84 (06/07) ◽  
pp. 428-433 ◽  
Author(s):  
Corinna Schmiderer ◽  
Brigitte Lukas ◽  
Joana Ruzicka ◽  
Johannes Novak
Keyword(s):  
Next Generation Sequencing ◽  
Dna Barcoding ◽  
Species Identification ◽  
Internal Transcribed Spacer ◽  
Raw Material ◽  
Next Generation ◽  
Additional Species ◽  
Internal Transcribed Spacer 2 ◽  
Dna Metabarcoding ◽  
Generation Sequencing

AbstractQuality control of drugs consists of identifying the raw material to avoid unwanted admixtures or exchange of material as well as looking for abiotic and biotic contaminations. So far, identity and microbial contamination are analyzed by separate processes and separate methods. Species identification by their DNA (“DNA barcoding”) has the potential to supplement existing methods of identification. The introduction of next-generation sequencing methods offers completely new approaches like the identification of whole communities in one analysis, termed “DNA metabarcoding”. Here we present a next-generation sequencing assessment to identify plants and fungi of two commercial sage samples (Salvia officinalis) using the standard DNA barcoding region “internal transcribed spacer” consisting of internal transcribed spacer 1 and internal transcribed spacer 2, respectively. The main species in both samples was identified as S. officinalis. The spectrum of accompanying plant and fungal species, however, was completely different between the samples. Additionally, the composition between internal transcribed spacer 1 and internal transcribed spacer 2 within the samples was different and demonstrated the influence of primer selection and therefore the need for harmonization. This next-generation sequencing approach does not result in quantitative species composition but gives deeper insight into the composition of additional species. Therefore, it would allow for a better knowledge-based risk assessment than any other method available. However, the method is only economically feasible in routine analysis if a high sample throughput can be guaranteed.

Temporal Detection Limits of Remnant Larval Bloodmeals in Nymphal Ixodes scapularis (Say, Ixodida: Ixodidae) Using Two Next-Generation Sequencing DNA Barcoding Assays

2020 ◽  
Author(s):  
Genevieve A M Lumsden ◽  
Evgeny V Zakharov ◽  
Sarah Dolynskyj ◽  
J Scott Weese ◽  
L Robbin Lindsay ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Dna Barcoding ◽  
Ixodes Scapularis ◽  
Life Stage ◽  
Next Generation ◽  
Host Detection ◽  
Host Identification ◽  
Species Specific ◽  
Generation Sequencing ◽  
Assay Detection

Abstract Using next-generation sequencing DNA barcoding, we aimed to determine: 1) if the larval bloodmeal can be detected in Ixodes scapularis nymphs and 2) the post-moult temporal window for detection of the larval bloodmeal. Subsets of 30 nymphs fed on a domestic rabbit (Oryctolagus cuniculus Linnaeus, Lagomorphia: Leporidae) as larvae were reared and frozen at 11 time points post-moult, up to 150 d. Vertebrate DNA was amplified using novel universal (UP) and species-specific primers (SSP) and sequenced for comparison against cytochrome c oxidase subunit I barcodes to infer host identification. Detectable bloodmeals decreased as time since moult increased for both assays. For the SSP assay, detection of bloodmeals decreased from 96.7% (n = 29/30) in day 0 nymphs to 3.3% (n = 1/30) and 6.7% (n = 2/30) at 4- and 5-mo post-moult, respectively. A shorter temporal detection period was achieved with the UP assay, declining from 16.7% (n = 5/30) in day 0 nymphs to 0/30 in 3-d-old nymphs. Bloodmeal detection was nonexistent for the remaining cohorts, with the exception of 1/30 nymphs at 2-mo post-moult. Host detection was significantly more likely using the SSP assay compared to the UP assay in the first three time cohorts (day 0: χ 2 = 39.1, P < 0.005; day 2: χ 2 = 19.2, P < 0.005; day 3: χ 2 = 23.3, P < 0.005). Regardless of the primer set used, the next-generation sequencing DNA barcoding assay was able to detect host DNA from a larval bloodmeal in the nymphal life stage; however, a short window with a high proportion of detection post-moult was achieved.

Next Generation Sequencing (NGS) approach applied to species identification in mixed processed seafood products

Food Control ◽  
2021 ◽  
pp. 108590
Author(s):  
Roberta Piredda ◽  
Anna Mottola ◽  
Giulia Cipriano ◽  
Roberto Carlucci ◽  
Giuseppina Ciccarese ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Species Identification ◽  
Next Generation ◽  
Seafood Products ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

scholarly journals Assessing wild bee diversity using next generation sequencing

2019 ◽  
Author(s):  
◽  
Morgan Gueuning
Keyword(s):  
Next Generation Sequencing ◽  
Dna Barcoding ◽  
Species Delimitation ◽  
Morphological Identification ◽  
Next Generation ◽  
Wild Bees ◽  
Wild Bee ◽  
Monitoring Programs ◽  
The Cost ◽  
Generation Sequencing

Wild bees are essential pollinators and therefore play a key role in both natural and agricultural ecosystems. However, bees have often been neglected in conservation studies and policies worldwide, which is surprising given their ecological importance. As a result, little is known on the conservation status of the vast majority of wild bee species in Europe, and even less worldwide. Limited surveys suggest important declines in the abundance and diversity of most wild bee communities worldwide. It is therefore urgent to implement targeted measures for the conservation of these keystone species. Once implemented, the effectiveness of these measures must be evaluated using adequate monitoring programs. To date, wild bee surveys are entirely based on morphological identification, which is both labor intensive and time consuming. Consequently, an affordable, high-throughput identification method is needed to reduce costs and improve bee monitoring. The objective of this thesis was to evaluate novel genetic techniques based on Next Generation Sequencing (NGS) methods for facilitating surveys of wild bees. NGS tools were mainly investigated for bridging two important impediments to wild bee conservation efforts, i.e., the cost of biodiversity assessment schemes and taxonomic incompleteness. With the development of NGS techniques, DNA barcoding has gained enormous momentum, enabling cost-effective, fast and accurate identifications. Before these methods can be routinely used in monitoring programs, there are however still important knowledge gaps to fill. These gaps mainly concern the detection of rare species and the acquisition of accurate quantitative data on species abundance; more generally the cost and labour effectiveness of these methods need to be evaluated. To provide a comprehensive presentation of the advantages and weaknesses of different NGS-based identification methods, we assessed three of the most promising ones, namely metabarcoding, mitogenomics and NGS barcoding. Using a regular monitoring data, we found that NGS barcoding performed best for both species’ presence/absence and abundance data, producing only few false positives and no false negatives. The other methods investigated were less reliable in term of species detection and inference of abundance data, and partly led to erroneous ecological conclusions. In terms of workload and cost, we showed that NGS techniques were more expensive than morphological identification with our dataset, although these techniques would become slightly more economical in large-scale monitoring programs. A second aim of this thesis was to provide an easy and robust genomic solution to alleviate taxonomical incompleteness, one of the major impediments to the effective conservation of many insect taxa. For conservation purposes, having stable and well-delimited species hypotheses is essential. Currently, most species are delimitated based on morphology and/or DNA barcoding. These methods are however associated with important limitations, and it is widely accepted that species delimitation should rely on multi-locus genomic markers. To overcome these limitations, ultraconserved elements (UCEs) were tested as a fast and robust approach using different species-complexes harbouring cryptic diversity, mitochondrial introgression, or mitochondrial paraphyly. Phylogenetic analyses of UCEs were highly conclusive and yielded meaningful species delimitation hypotheses in all cases. These results provide strong evidence for the potential of UCEs as a fast method for delimiting species even in cases of recently diverged lineages. Advantages and limitations of UCEs for shallow phylogenetic studies are further discussed.

scholarly journals Next‐generation DNA barcoding: using next‐generation sequencing to enhance and accelerate DNA barcode capture from single specimens

2014 ◽  
Vol 14 (5) ◽  
pp. 892-901 ◽  
Author(s):  
Shadi Shokralla ◽  
Joel F. Gibson ◽  
Hamid Nikbakht ◽  
Daniel H. Janzen ◽  
Winnie Hallwachs ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Dna Barcoding ◽  
Dna Barcode ◽  
Next Generation ◽  
Generation Sequencing

scholarly journals Application of Next-Generation Sequencing Technology Based on Single Gene Locus in Species Identification of Mixed Meat Products

2021 ◽  
Vol 2021 ◽  
pp. 1-5
Author(s):  
Xinmei Liu ◽  
Zhiyang Liu ◽  
Yiyu Cheng ◽  
Haijing Wu ◽  
Wei Shen ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
16S Rrna ◽  
Species Identification ◽  
Growth Hormone Receptor ◽  
Single Gene ◽  
Meat Products ◽  
Quantitative Detection ◽  
Next Generation ◽  
Next Generation Sequencing Technology ◽  
Generation Sequencing

Polymerase chain reaction (PCR) detection is a commonly used method for species identification of meat products. However, this method is not suitable for the analysis of meat products containing multiple mixtures. This study aimed to test whether next-generation sequencing (NGS) technology could be used as a method for the certification of mixed meat products. In this study, five kinds of common meat (pigs, cattle, sheep, chickens, and ducks) were mixed as samples with different proportions. The primers designed from mitochondrial 16S rRNA and nuclear genome gene (growth hormone receptor, GHR), respectively, were used to detect these meats. The sequencing results of NGS were analyzed using a self-designed bioinformatics program. The fragments with similar sequences were classified and compared with the database to determine their species. The results showed that all five kinds of meat components could be correctly identified using these two primers. The meat composition could be detected as low as 0.5% in the mixed samples using the NGS technology targeting GHR gene fragments, which was superior to those targeting mitochondrial 16S rRNA. However, the quantitative detection of species in the mixture was not likely to be quite accurate due to the amplification bias of PCR amplification. These results showed that the NGS technology could be applied to identify meat species in mixtures.

scholarly journals Development of a reference data set for assigning Streptococcus and Enterococcus species based on next generation sequencing of the 16S–23S rRNA region

2019 ◽  
Vol 8 (1) ◽  
Author(s):  
Maja Kosecka-Strojek ◽  
Artur J. Sabat ◽  
Viktoria Akkerboom ◽  
Anna M. D. Kooistra-Smid ◽  
Jacek Miedzobrodzki ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Sequence Analysis ◽  
16S Rrna ◽  
Species Identification ◽  
23S Rrna ◽  
Clinical Samples ◽  
Next Generation ◽  
Enterococcal Species ◽  
Reference Sequences ◽  
Generation Sequencing

Abstract Background Many members of Streptococcus and Enterococcus genera are clinically relevant opportunistic pathogens warranting accurate and rapid identification for targeted therapy. Currently, the developed method based on next generation sequencing (NGS) of the 16S–23S rRNA region proved to be a rapid, reliable and precise approach for species identification directly from polymicrobial and challenging clinical samples. The introduction of this new method to routine diagnostics is hindered by a lack of the reference sequences for the 16S–23S rRNA region for many bacterial species. The aim of this study was to develop a careful assignment for streptococcal and enterococcal species based on NGS of the 16S–23S rRNA region. Methods Thirty two strains recovered from clinical samples and 19 reference strains representing 42 streptococcal species and nine enterococcal species were subjected to bacterial identification by four Sanger-based sequencing methods targeting the genes encoding (i) 16S rRNA, (ii) sodA, (iii) tuf and (iv) rpoB; and NGS of the 16S–23S rRNA region. Results This study allowed obtainment and deposition of reference sequences of the 16S–23S rRNA region for 15 streptococcal and 3 enterococcal species followed by enrichment for 27 and 6 species, respectively, for which reference sequences were available in the databases. For Streptococcus, NGS of the 16S–23S rRNA region was as discriminative as Sanger sequencing of the tuf and rpoB genes allowing for an unambiguous identification of 93% of analyzed species. For Enterococcus, sodA, tuf and rpoB genes sequencing allowed for identification of all species, while the NGS-based method did not allow for identification of only one enterococcal species. For both genera, the sequence analysis of the 16S rRNA gene was endowed with a low identification potential and was inferior to that of other tested identification methods. Moreover, in case of phylogenetically related species the sequence analysis of only the intergenic spacer region was not sufficient enough to precisely identify Streptococcus strains at the species level. Conclusions Based on the developed reference dataset, clinically relevant streptococcal and enterococcal species can now be reliably identified by 16S–23S rRNA sequences in samples. This study will be useful for introduction of a novel diagnostic tool, NGS of the 16S–23S rRNA region, which undoubtedly is an improvement for reliable culture-independent species identification directly from polymicrobially constituted clinical samples.

Rapid detection of macroalgal seed bank on cobbles: application of DNA metabarcoding using next-generation sequencing

2019 ◽  
Vol 31 (4) ◽  
pp. 2743-2753 ◽  
Author(s):  
Shingo Akita ◽  
Yoshihito Takano ◽  
Satoshi Nagai ◽  
Hisami Kuwahara ◽  
Rumiko Kajihara ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Seed Bank ◽  
Rapid Detection ◽  
Next Generation ◽  
Dna Metabarcoding ◽  
Generation Sequencing
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