Next-Generation Sequencing: A Promising Tool for Vaccines and Other Biological Products.

Next-generation sequencing (NGS), also known as high-throughput sequencing (HTS), is a commonly used term to represent a set of DNA sequencing technologies that have been in use for almost two decades [...].

Development of Hydrolysis Probe-Based qPCR Assays for Panax ginseng and Panax quinquefolius for Detection of Adulteration in Ginseng Herbal Products.

Authentication of Panax ginseng and Panax quinquefolius products is important to be able to mitigate instances of adulteration and substitution that exist within the international supply chain of ginseng. To address this issue, species-specific hydrolysis probe qPCR assays were developed and validated for both P. ginseng and P. quinquefolius herbal dietary supplements. Performance of the probe-based assays was evaluated using analytical validation criteria, which included evaluation of: (1) specificity, in selectively identifying the target species; (2) sensitivity, in detecting the lowest amount of the target material; and (3) repeatability and reproducibility of the method in detecting the target species in raw materials on a real-time PCR platform (reliability). The species-specific probes were developed and successfully passed the validation criteria with 100% specificity, 80-120% efficiency and 100% reliability. The methods developed in this study are fit for purpose, rapid, and easy to implement in quality assurance programs; authentication of ginseng herbal supplements is possible, even with extracts where DNA is fragmented and of low quality and quantity.

Genome skimming and NMR chemical fingerprinting provide quality assurance biotechnology to validate Sarsaparilla identity and purity.

Sarsaparilla is a popular natural health product (NHP) that has been reported to be one of the most adulterated botanicals in the marketplace. Several plausible explanations are documented including economically motivated product substitution, unintentional errors due to ambiguous trade name associated with several different taxa, and wild harvesting of incorrect non-commercial plants. Unfortunately, this includes the case of an adulterant species Decalepis hamiltonii, a Red listed medicinal plant species by the International Union for Conservation of Nature (IUCN) and declared as a species with high conservation concern by the National Biodiversity Authority of India (NBA). This study provides validated genomic (genome skimming & DNA probes) and metabolomic (NMR chemical fingerprints) biotechnology solutions to prevent adulteration on both raw materials and finished products. This is also the first use of Oxford Nanopore on herbal products enabling the use of genome skimming as a tool for quality assurance within the supply chain of botanical ingredients. The validation of both genomics and metabolomics approach provided quality assurance perspective for both product identity and purity. This research enables manufactures and retailers to verify their supply chain is authentic and that consumers can enjoy safe, healthy products.

Investigating appropriate molecular and chemical methods for ingredient identity testing of plant-based protein powder dietary supplements.

Plant-based protein powders are rapidly growing in popularity, and outdated quality assurance tools expose vulnerabilities to adulteration via different methods of "protein spiking". Adequate diagnostic tools are urgently needed to be able to authenticate protein source ingredients and screen for potential adulterants. We explored the application of three diagnostic tools for ingredient identification: targeted PCR with Sanger sequencing, NGS, and LC-MS/MS. We collected 33 samples of common commercial products from the plant-based protein powder market and sought to identify botanical components using the three technologies. We found success in detection with all approaches, with at least one main protein source being identified by at least one approach in all samples. The investigation uncovered challenges to data collection or result interpretation with each technology including but not limited to amplification biases with PCR technologies, potential influence of DNA degradation, and issues with protein solubility during isolation. Ultimately, each platform demonstrated utility along with certain caveats, which epitomized the importance of orthogonality of testing.

Exploring DNA quantity and quality from raw materials to botanical extracts.

OBJECTIVES: The aim of this study was to explore the variability in DNA quality and quantity along a gradient of industrial processing of botanical ingredients from raw materials to extracts. METHODS: A data matrix was assembled for 1242 botanical ingredient samples along a gradient of industrial processing commonly used in the Natural Health Product (NHP) industry. Multivariate statistics was used to explore dependant variables for quality and quantity. The success of attaining a positive DNA test result along a gradient of industrial processing was compared among four biotechnologies: DNA barcoding, NGS, Sanger sequencing and qPCR. RESULTS: There was considerable variance in DNA quality and quantity among the samples, which could be interpreted along a gradient from raw materials with greater quantities (50-120 ng/µL) of DNA and longer DNA (400-500bp) sequences to extracts, which were characterized by lower quantities (0.1-10.0 ng/µL) and short fragments (50-150bp). CONCLUSIONS: Targeted molecular diagnostic tests for species identity can be used in the NHP industry for raw and processed samples. Non-targeted tests or the use of NGS for any identity test needs considerable research and development and must be validated before it can be used in commercial operations as these methods are subject to considerable risk of false negative and positive results. Proper use of these tools can be used to ensure ingredient authenticity, and to avert adulteration, and contamination with plants that are a health concern. Lastly these tools can be used to prevent the exploitation of rare herbal species and the harvesting of native biodiversity for commercial purposes.

Validated Identity Test Method for Ginkgo biloba NHPs Using DNA-Based Species-Specific Hydrolysis PCR Probe.

Background: There is considerable risk of adulteration of Ginkgo biloba herbal products in the natural health product (NHP) industry. Authentication of G. biloba products is challenging because of the standard, complex, analytical chemistry methods that may be too costly and not appropriate for both raw and finished products. Objective: We sought to develop and validate an alternative method to authenticate G. biloba herbal dietary supplements, based on the use of a species-specific hydrolysis PCR probe assay. Methods: A species-specific hydrolysis probe assay was developed, validated, and evaluated for the performance of the assay in accurately identifying the species of interest using the following analytical validation criteria: (1) specificity (accuracy) in identifying the target species ingredient, while not identifying other nontarget species, (2) sensitivity in detecting the smallest amount of the target material, and (3) reliability (repeatability and reproducibility) in detecting the target species in raw materials on a real-time PCR platform. Results: The species-specific hydrolysis probe assay was successfully developed for raw materials of G. biloba. The specificity of the assay was 100% to the target species. Efficiency of the assay was observed to be 99%, and the reliability of the assay was 100% for the raw/starting materials. Conclusions and Highlights: The method developed in this study is simple, rapid, and easy for supplement manufacturers to perform in their laboratories to ensure that their G. biloba supplements are authentic.

Guidelines for Validation of Qualitative Real-Time PCR Methods for Molecular Diagnostic Identification of Probiotics.

Backgroud: Probiotics have been shown to benefit human health through several mechanisms, including their role in improving the health of our gastrointestinal tracts. The health benefits of probiotics are strain specific, and therefore it is critical to include the correct strains in probiotic products when claiming specific health benefits. Several studies have reported issues concerning the accuracy of labeling of commercial probiotic products, including inaccurate taxonomy, missing species, or undeclared species. Consequently, there is a growing need to develop and validate assays to reliably verify strain identity in commercial probiotic products. PCR-based methods are the most commonly used methods for food species ingredient diagnostics because they are simple, fast, sensitive, and can be validated. Objective: The aim of this paper is to set the guidelines for validating targeted qualitative real-time PCR assays to verify the presence of specific strains in a probiotic supplement. Methods and Results: Qualitative real-time PCR assays are validated to evaluate the assay performance in terms of specificity, sensitivity, repeatability, and reproducibility in detecting target strains. Conclusions and Highlights: Setting these guidelines will facilitate and streamline the validation process for qualitative real-time PCR-based assays for probiotic identity authentication in support of quality assurance systems.

Recommendations for Validation of Real-Time PCR Methods for Molecular Diagnostic Identification of Botanicals.

Background: PCR methods are the most commonly used DNA-based identity tool in the commercial food, beverage, and natural health product markets. These methods are routinely used to identify foodborne pathogens and allergens in food. Proper validation methods for some sectors have been established, while there are none in other markets, such as botanicals. Results: A survey of the literature indicates that some validation criteria are not addressed when developing PCR tests for botanicals. Objective: We provide recommendations for qualitative real-time PCR methods for validating identity tests for botanical ingredients. Methods: These include common criteria that underpin the development and validation of rigorous tests, including (1) the aim of the validation test, (2) the applicability of different matrix variants, (3) specificity in identifying the target species ingredient, (4) sensitivity in detecting the smallest amount of the target material, (5) repeatability of methods, (6) reproducibility in detecting the target species in both raw and processed materials, (7) practicability of the test in a commercial laboratory, and (8) comparison with alternative methods. In addition, we recommend additional criteria, according to which the practicability of the test method is evaluated by transferring the method to a second laboratory and by comparison with alternative methods. Conclusions and Highlights: We hope that these recommendations encourage further publication on the validation of PCR methods for many botanical ingredients. These properly validated PCR methods can be developed on small, real-time biotechnology that can be placed directly into the supply chain ledger in support of highly transparent data systems that support QC from the farm to the fork of the consumer.

Genome sequencing and comparison of five Tilletia species to identify candidate genes for the detection of regulated species infecting wheat.

Tilletia species cause diseases on grass hosts with some causing bunt diseases on wheat (Triticum). Two of the four species infecting wheat have restricted distributions globally and are subject to quarantine regulations to prevent their spread to new areas. Tilletia indica causes Karnal bunt and is regulated by many countries while the non-regulated T. walkeri is morphologically similar and very closely related phylogenetically, but infects ryegrass (Lolium) and not wheat. Tilletia controversa causes dwarf bunt of wheat (DB) and is also regulated by some countries, while the closely related but non-regulated species, T. caries and T. laevis, both cause common bunt of wheat (CB). Historically, diagnostic methods have relied on cryptic morphology to differentiate these species in subsamples from grain shipments. Of the DNA-based methods published so far, most have focused on sequence variation among tested strains at a single gene locus. To facilitate the development of additional molecular assays for diagnostics, we generated whole genome data for multiple strains of the two regulated wheat pathogens and their closest relatives. Depending on the species, the genomes were assembled into 907 to 4633 scaffolds ranging from 24 Mb to 30 Mb with 7842 to 9952 gene models predicted. Phylogenomic analyses confirmed the placement of Tilletia in the Exobasidiomycetes and showed that T. indica and T. walkeri were in one clade whereas T. controversa, T. caries and T. laevis grouped in a separate clade. Single copy and species-specific genes were identified by orthologous group analysis. Unique species-specific genes were identified and evaluated as suitable markers to differentiate the quarantine and non-quarantine species. After further analyses and manual inspection, primers and probes for the optimum candidate genes were designed and tested in silico, for validation in future wet-lab studies.

Discovery of Negative-Sense RNA Viruses in Trees Infected with Apple Rubbery Wood Disease by Next-Generation Sequencing.

Apple rubbery wood is a disease of apple found around the world, often associated with Apple flat limb disease, and regulated in many countries. Despite its long history in apple cultivation, the disease's causal agent has remained elusive. In this study, next-generation sequencing (NGS) was used to identify and characterize several related novel viral agents from apple rubbery wood-infected plants, which have been named Apple rubbery wood virus (ARWV) 1 and 2. Additional specimens with apple rubbery wood disease tested positive by polymerase chain reaction with primers designed to ARWV 1 and 2 genomic RNA segments. In an NGS-based screening of over 100 Malus and 100 Prunus specimens from a collection of virus-infected trees, only one Malus specimen was found to be infected with ARWV not known to be infected with the disease, which strongly suggests that ARWV is not commonly found in Malus spp. or other fruit trees. The two viruses are most closely related to members of the order Bunyavirales. Three RNA segments (large, medium, and small) were characterized and the viruses likely represent a new genus under the family Phenuiviridae, with a suggested name of Rubodvirus (Rubbery wood virus).

Comparative analysis of cherry virus A genome sequences assembled from deep sequencing data.

Cherry virus A (CVA) is a ubiquitous graft-transmissible virus that mainly infects Prunus spp. Next-generation sequencing was applied to 39 tree fruit specimens infected with CVA, and 75 full and 16 partial-length CVA genome sequences were assembled. Phylogenetic analysis of these and 11 previously sequenced CVA genomes resulted in six major clusters with no observable relationship between the host and the assembled genome sequences. Recombination analysis detected four recombinants. Consistent single-nucleotide polymorphism (SNP) patterns were observed between the 75 full-length genomes and their sequence clouds, which supports a quasispecies model for CVA evolution.

Screening for plant viruses by next generation sequencing using a modified double strand RNA extraction protocol with an internal amplification control.

The majority of plant viruses contain RNA genomes. Detection of viral RNA genomes in infected plant material by next generation sequencing (NGS) is possible through the extraction and sequencing of total RNA, total RNA devoid of ribosomal RNA, small RNA interference (RNAi) molecules, or double stranded RNA (dsRNA). Plants do not typically produce high molecular weight dsRNA, therefore the presence of dsRNA makes it an attractive target for plant virus diagnostics. The sensitivity of NGS as a diagnostic method demands an effective dsRNA protocol that is both representative of the sample and minimizes sample cross contamination. We have developed a modified dsRNA extraction protocol that is more efficient compared to traditional protocols, requiring reduced amounts of starting material, that is less prone to sample cross contamination. This was accomplished by using bead based homogenization of plant material in closed, disposable 50ml tubes. To assess the quality of extraction, we also developed an internal control by designing a real-time (quantitative) PCR (qPCR) assay that targets endornaviruses present in Phaseolus vulgaris cultivar Black Turtle Soup (BTS).

Understanding the spectacular failure of DNA barcoding in willows (Salix): does this result from a trans-specific selective sweep?

Willows (Salix: Salicaceae) form a major ecological component of Holarctic floras and consequently are an obvious target for a DNA-based identification system. We surveyed two to seven plastid genome regions (~3.8 kb; ~3% of the genome) from 71 Salix species across all five subgenera, to assess their performance as DNA barcode markers. Although Salix has a relatively high level of interspecific hybridization, this may not sufficiently explain the near complete failure of barcoding that we observed: only one species had a unique barcode. We recovered 39 unique haplotypes, from more than 500 specimens, that could be partitioned into six major haplotype groups. A unique variant of group I (haplotype 1*) was shared by 53 species in three of five Salix subgenera. This unusual pattern of haplotype sharing across infrageneric taxa is suggestive of either a massive nonrandom coalescence failure (incomplete lineage sorting), or of repeated plastid capture events, possibly including a historical selective sweep of haplotype 1* across taxonomic sections. The former is unlikely as molecular dating indicates that haplotype 1* originated recently and is nested in the oldest major haplotype group in the genus. Further, we detected significant non-neutrality in the frequency spectrum of mutations in group I, but not outside group I, and demonstrated a striking absence of geographical (isolation by distance) effects in the haplotype distributions of this group. The most likely explanation for the patterns we observed involves recent repeated plastid capture events, aided by widespread hybridization and long-range seed dispersal, but primarily propelled by one or more trans-species selective sweeps.

Spatial patterns of plant diversity below-ground as revealed by DNA barcoding.

Our understanding of the spatial organization of root diversity in plant communities and of the mechanisms of community assembly has been limited by our ability to identify plants based on root tissue, especially in diverse communities. Here, we test the effectiveness of the plastid gene rbcL, a core plant DNA barcoding marker, for investigating spatial patterns of root diversity, and relate observed patterns to above-ground community structure. We collected 3800 root fragments from four randomly positioned, 1-m-deep soil profiles (two vertical transects per plot), located in an old-field community in southern Ontario, Canada, and extracted and sequenced DNA from 1531 subsampled fragments. We identified species by comparing sequences with a DNA barcode reference library developed previously for the local flora. Nearly 85% of sampled root fragments were successfully sequenced and identified as belonging to 29 plant species or species groups. Root abundance and species richness varied in horizontal space and were negatively correlated with soil depth. The relative abundance of taxa below-ground was correlated with their frequency above-ground (r = 0.73, P = 0.0001), but several species detected in root tissue were not observed in above-ground quadrats. Multivariate analyses indicated that diversity was highly structured below-ground, and associated with depth, root morphology, soil chemistry and soil texture, whereas little structure was evident above-ground. Furthermore, analyses of species co-occurrence indicates strong species segregation overall but random co-occurrence among confamilials. Our results provide insights into the role of environmental filtering and competitive interactions in the organization of plant diversity below-ground, and also demonstrate the utility of barcoding for the identification of plant roots.

Are plant species inherently harder to discriminate than animal species using DNA barcoding markers?

The ability to discriminate between species using barcoding loci has proved more difficult in plants than animals, raising the possibility that plant species boundaries are less well defined. Here, we review a selection of published barcoding data sets to compare species discrimination in plants vs. animals. Although the use of different genetic markers, analytical methods and depths of taxon sampling may complicate comparisons, our results using common metrics demonstrate that the number of species supported as monophyletic using barcoding markers is higher in animals (> 90%) than plants (~70%), even after controlling for the amount of parsimony-informative information per species. This suggests that more than a simple lack of variability limits species discrimination in plants. Both animal and plant species pairs have variable size gaps between intra- and interspecific genetic distances, but animal species tend to have larger gaps than plants, even in relatively densely sampled genera. An analysis of 12 plant genera suggests that hybridization contributes significantly to variation in genetic discontinuity in plants. Barcoding success may be improved in some plant groups by careful choice of markers and appropriate sampling; however, overall fine-scale species discrimination in plants relative to animals may be inherently more difficult because of greater levels of gene-tree paraphyly.

Multiple multilocus DNA barcodes from the plastid genome discriminate plant species equally well.

A universal barcode system for land plants would be a valuable resource, with potential utility in fields as diverse as ecology, floristics, law enforcement and industry. However, the application of plant barcoding has been constrained by a lack of consensus regarding the most variable and technically practical DNA region(s). We compared eight candidate plant barcoding regions from the plastome and one from the mitochondrial genome for how well they discriminated the monophyly of 92 species in 32 diverse genera of land plants (N = 251 samples). The plastid markers comprise portions of five coding (rpoB, rpoC1, rbcL, matK and 23S rDNA) and three non-coding (trnH-psbA, atpF-atpH, and psbK-psbI) loci. Our survey included several taxonomically complex groups, and in all cases we examined multiple populations and species. The regions differed in their ability to discriminate species, and in ease of retrieval, in terms of amplification and sequencing success. Single locus resolution ranged from 7% (23S rDNA) to 59% (trnH-psbA) of species with well-supported monophyly. Sequence recovery rates were related primarily to amplification success (85-100% for plastid loci), with matK requiring the greatest effort to achieve reasonable recovery (88% using 10 primer pairs). Several loci (matK, psbK-psbI, trnH-psbA) were problematic for generating fully bidirectional sequences. Setting aside technical issues related to amplification and sequencing, combining the more variable plastid markers provided clear benefits for resolving species, although with diminishing returns, as all combinations assessed using four to seven regions had only marginally different success rates (69-71%; values that were approached by several two- and three-region combinations). This performance plateau may indicate fundamental upper limits on the precision of species discrimination that is possible with DNA barcoding systems that include moderate numbers of plastid markers. Resolution to the contentious debate on plant barcoding should therefore involve increased attention to practical issues related to the ease of sequence recovery, global alignability, and marker redundancy in multilocus plant DNA barcoding systems.