April 21, 2020  |  

Long metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity

High-throughput environmental DNA metabarcoding has revolutionized the analysis of microbial diversity, but this approach is generally restricted to amplicon sizes below 500 base pairs. These short regions contain limited phylogenetic signal, which makes it impractical to use environmental DNA in full phylogenetic inferences. However, new long-read sequencing technologies such as the Pacific Biosciences platform may provide sufficiently large sequence lengths to overcome the poor phylogenetic resolution of short amplicons. To test this idea, we amplified soil DNA and used PacBio Circular Consensus Sequencing (CCS) to obtain a ~4500 bp region of the eukaryotic rDNA operon spanning most of the small (18S) and large subunit (28S) ribosomal RNA genes. The CCS reads were first treated with a novel curation workflow that generated 650 high-quality OTUs containing the physically linked 18S and 28S regions of the long amplicons. In order to assign taxonomy to these OTUs, we developed a phylogeny-aware approach based on the 18S region that showed greater accuracy and sensitivity than similarity-based and phylogenetic placement-based methods using shorter reads. The taxonomically-annotated OTUs were then combined with available 18S and 28S reference sequences to infer a well-resolved phylogeny spanning all major groups of eukaryotes, allowing to accurately derive the evolutionary origin of environmental diversity. A total of 1019 sequences were included, of which a majority (58%) corresponded to the new long environmental CCS reads. Comparisons to the 18S-only region of our amplicons revealed that the combined 18S-28S genes globally increased the phylogenetic resolution, recovering specific groupings otherwise missing. The long-reads also allowed to directly investigate the relationships among environmental sequences themselves, which represents a key advantage over the placement of short reads on a reference phylogeny. Altogether, our results show that long amplicons can be treated in a full phylogenetic framework to provide greater taxonomic resolution and a robust evolutionary perspective to environmental DNA.

April 21, 2020  |  

Towards PacBio-based pan-eukaryote metabarcoding using full-length ITS sequences.

Development of high-throughput sequencing techniques have greatly benefited our understanding about microbial ecology; yet the methods producing short reads suffer from species-level resolution and uncertainty of identification. Here we optimize PacBio-based metabarcoding protocols covering the Internal Transcribed Spacer (ITS region) and partial Small Subunit (SSU) of the rRNA gene for species-level identification of all eukaryotes, with a specific focus on Fungi (including Glomeromycota) and Stramenopila (particularly Oomycota). Based on tests on composite soil samples and mock communities, we propose best suitable degenerate primers, ITS9munngs + ITS4ngsUni for eukaryotes and selected groups therein and discuss pros and cons of long read-based identification of eukaryotes. This article is protected by copyright. All rights reserved.

April 21, 2020  |  

Relative Performance of MinION (Oxford Nanopore Technologies) versus Sequel (Pacific Biosciences) Third-Generation Sequencing Instruments in Identification of Agricultural and Forest Fungal Pathogens.

Culture-based molecular identification methods have revolutionized detection of pathogens, yet these methods are slow and may yield inconclusive results from environmental materials. The second-generation sequencing tools have much-improved precision and sensitivity of detection, but these analyses are costly and may take several days to months. Of the third-generation sequencing techniques, the portable MinION device (Oxford Nanopore Technologies) has received much attention because of its small size and possibility of rapid analysis at reasonable cost. Here, we compare the relative performances of two third-generation sequencing instruments, MinION and Sequel (Pacific Biosciences), in identification and diagnostics of fungal and oomycete pathogens from conifer (Pinaceae) needles and potato (Solanum tuberosum) leaves and tubers. We demonstrate that the Sequel instrument is efficient for metabarcoding of complex samples, whereas MinION is not suited for this purpose due to a high error rate and multiple biases. However, we find that MinION can be utilized for rapid and accurate identification of dominant pathogenic organisms and other associated organisms from plant tissues following both amplicon-based and PCR-free metagenomics approaches. Using the metagenomics approach with shortened DNA extraction and incubation times, we performed the entire MinION workflow, from sample preparation through DNA extraction, sequencing, bioinformatics, and interpretation, in 2.5 h. We advocate the use of MinION for rapid diagnostics of pathogens and potentially other organisms, but care needs to be taken to control or account for multiple potential technical biases.IMPORTANCE Microbial pathogens cause enormous losses to agriculture and forestry, but current combined culturing- and molecular identification-based detection methods are too slow for rapid identification and application of countermeasures. Here, we develop new and rapid protocols for Oxford Nanopore MinION-based third-generation diagnostics of plant pathogens that greatly improve the speed of diagnostics. However, due to high error rate and technical biases in MinION, the Pacific BioSciences Sequel platform is more useful for in-depth amplicon-based biodiversity monitoring (metabarcoding) from complex environmental samples.Copyright © 2019 American Society for Microbiology.

April 21, 2020  |  

Full-Length Multi-Barcoding: DNA Barcoding from Single Ingredient to Complex Mixtures.

DNA barcoding has been used for decades, although it has mostly been applied to somesingle-species. Traditional Chinese medicine (TCM), which is mainly used in the form ofcombination-one type of the multi-species, identification is crucial for clinical usage.Next-generation Sequencing (NGS) has been used to address this authentication issue for the pastfew years, but conventional NGS technology is hampered in application due to its short sequencingreads and systematic errors. Here, a novel method, Full-length multi-barcoding (FLMB) vialong-read sequencing, is employed for the identification of biological compositions in herbalcompound formulas in adequate and well controlled studies. By directly sequencing the full-lengthamplicons of ITS2 and psbA-trnH through single-molecule real-time (SMRT) technology, thebiological composition of a classical prescription Sheng-Mai-San (SMS) was analyzed. At the sametime, clone-dependent Sanger sequencing was carried out as a parallel control. Further, anotherformula-Sanwei-Jili-San (SJS)-was analyzed with genes of ITS2 and CO1. All the ingredients inthe samples of SMS and SJS were successfully authenticated at the species level, and 11 exogenousspecies were also checked, some of which were considered as common contaminations in theseproducts. Methodology analysis demonstrated that this method was sensitive, accurate andreliable. FLMB, a superior but feasible approach for the identification of biological complexmixture, was established and elucidated, which shows perfect interpretation for DNA barcodingthat could lead its application in multi-species mixtures.

April 21, 2020  |  

PacBio amplicon sequencing for metabarcoding of mixed DNA samples from lichen herbarium specimens.

The detection and identification of species of fungi in the environment using molecular methods heavily depends on reliable reference sequence databases. However, these databases are largely incomplete in terms of taxon coverage, and a significant effort is required from herbaria and living fungal collections for the mass-barcoding of well-identified and well-curated fungal specimens or strains. Here, a PacBio amplicon sequencing approach is applied to recent lichen herbarium specimens for the sequencing of the fungal ITS barcode, allowing a higher throughput sample processing than Sanger sequencing, which often required the use of cloning. Out of 96 multiplexed samples, a full-length ITS sequence of the target lichenised fungal species was recovered for 85 specimens. In addition, sequences obtained for co-amplified fungi gave an interesting insight into the diversity of endolichenic fungi. Challenges encountered at both the laboratory and bioinformatic stages are discussed, and cost and quality are compared with Sanger sequencing. With increasing data output and reducing sequencing cost, PacBio amplicon sequencing is seen as a promising approach for the generation of reference sequences for lichenised fungi as well as the characterisation of lichen-associated fungal communities.

September 22, 2019  |  

PacBio metabarcoding of Fungi and other eukaryotes: errors, biases and perspectives.

Second-generation, high-throughput sequencing methods have greatly improved our understanding of the ecology of soil microorganisms, yet the short barcodes (< 500 bp) provide limited taxonomic and phylogenetic information for species discrimination and taxonomic assignment. Here, we utilized the third-generation Pacific Biosciences (PacBio) RSII and Sequel instruments to evaluate the suitability of full-length internal transcribed spacer (ITS) barcodes and longer rRNA gene amplicons for metabarcoding Fungi, Oomycetes and other eukaryotes in soil samples. Metabarcoding revealed multiple errors and biases: Taq polymerase substitution errors and mis-incorporating indels in sequencing homopolymers constitute major errors; sequence length biases occur during PCR, library preparation, loading to the sequencing instrument and quality filtering; primer-template mismatches bias the taxonomic profile when using regular and highly degenerate primers. The RSII and Sequel platforms enable the sequencing of amplicons up to 3000 bp, but the sequence quality remains slightly inferior to Illumina sequencing especially in longer amplicons. The full ITS barcode and flanking rRNA small subunit gene greatly improve taxonomic identification at the species and phylum levels, respectively. We conclude that PacBio sequencing provides a viable alternative for metabarcoding of organisms that are of relatively low diversity, require > 500-bp barcode for reliable identification or when phylogenetic approaches are intended.© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

July 19, 2019  |  

Biomonitoring for traditional herbal medicinal products using DNA metabarcoding and single molecule, real-time sequencing.

Global concerns have been paid to the potential hazard of traditional herbal medicinal products (THMPs). Substandard and counterfeit THMPs, including traditional Chinese patent medicine, health foods, dietary supplements, etc. are potential threats to public health. Recent marketplace studies using DNA barcoding have determined that the current quality control methods are not sufficient for ensuring the presence of authentic herbal ingredients and detection of contaminants/adulterants. An efficient biomonitoring method for THMPs is of great needed. Herein, metabarcoding and single-molecule, real-time (SMRT) sequencing were used to detect the multiple ingredients in Jiuwei Qianghuo Wan (JWQHW), a classical herbal prescription widely used in China for the last 800 years. Reference experimental mixtures and commercial JWQHW products from the marketplace were used to confirm the method. Successful SMRT sequencing results recovered 5416 and 4342 circular-consensus sequencing (CCS) reads belonging to the ITS2 and psbA-trnH regions. The results suggest that with the combination of metabarcoding and SMRT sequencing, it is repeatable, reliable, and sensitive enough to detect species in the THMPs, and the error in SMRT sequencing did not affect the ability to identify multiple prescribed species and several adulterants/contaminants. It has the potential for becoming a valuable tool for the biomonitoring of multi-ingredient THMPs.

July 7, 2019  |  

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.© 2017 John Wiley & Sons Ltd.

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