Metabarcoding with Illumina and Oxford Nanopore Technologies Provides Unique Insight into the Mycobiota of Tree Seeds
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB81455 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR13856800.fastq.gz | Fastqsanger.gz | |||
| ERR13856801.fastq.gz | Fastqsanger.gz | |||
| ERR13856802.fastq.gz | Fastqsanger.gz | |||
| ERR13856803.fastq.gz | Fastqsanger.gz | |||
| ERR13856804.fastq.gz | Fastqsanger.gz |
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Project description:Metabarcoding for Tree Seeds
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Project description:Metagenomic sequence data from defined mock communities is crucial for the assessment of sequencing platform performance and downstream analyses, including assembly, binning and taxonomic assignment. We report a comparison of shotgun metagenome sequencing and assembly metrics of a defined microbial mock community using the Oxford Nanopore Technologies (ONT) MinION, PacBio and Illumina sequencing platforms. Our synthetic microbial community BMock12 consists of 12 bacterial strains with genome sizes spanning 3.2-7.2 Mbp, 40-73% GC content, and 1.5-7.3% repeats. Size selection of both PacBio and ONT sequencing libraries prior to sequencing was essential to yield comparable relative abundances of organisms among all sequencing technologies. While the Illumina-based metagenome assembly yielded good coverage with few misassemblies, contiguity was greatly improved by both, Illumina?+?ONT and Illumina?+?PacBio hybrid assemblies but increased misassemblies, most notably in genomes with high sequence similarity to each other. Our resulting datasets allow evaluation and benchmarking of bioinformatics software on Illumina, PacBio and ONT platforms in parallel.
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Project description:Illumina MiSeq is the current standard for characterizing microbial communities in soil. The newer alternative, Oxford Nanopore Technologies MinION sequencer, is quickly gaining popularity because of the low initial cost and longer sequence reads. However, the accuracy of MinION, per base, is much lower than MiSeq (95% versus 99.9%). The effects of this difference in base-calling accuracy on taxonomic and diversity estimates remains unclear. We compared the effects of platform, primers, and bioinformatics on mock community and agricultural soil samples using short MiSeq, and short and full-length MinION 16S rRNA amplicon sequencing. For all three methods, we found that taxonomic assignments of the mock community at both the genus and species level matched expectations with minimal deviation (genus: 80.9-90.5%; species: 70.9-85.2% Bray-Curtis similarity); however, the short MiSeq with error correction (DADA2) resulted in the correct estimate of mock community species richness and much lower alpha diversity for soils. Several filtering strategies were tested to improve these estimates with varying results. The sequencing platform also had a significant influence on the relative abundances of taxa with MiSeq resulting in significantly higher abundances Actinobacteria, Chloroflexi, and Gemmatimonadetes and lower abundances of Acidobacteria, Bacteroides, Firmicutes, Proteobacteria, and Verrucomicrobia compared to the MinION platform. When comparing agricultural soils from two different sites (Fort Collins, CO and Pendleton, OR), methods varied in the taxa identified as significantly different between sites. At all taxonomic levels, the full-length MinION method had the highest similarity to the short MiSeq method with DADA2 correction with 73.2%, 69.3%, 74.1%, 79.3%, 79.4%, and 82.28% of the taxa at the phyla, class, order, family, genus, and species levels, respectively, showing similar patterns in differences between the sites. In summary, although both platforms appear suitable for 16S rRNA microbial community composition, biases for different taxa may make the comparison between studies problematic; and even with a single study (i.e., comparing sites or treatments), the sequencing platform can influence the differentially abundant taxa identified.
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Project description:As determined by a hybrid approach combining Oxford Nanopore MinION and Illumina MiniSeq sequence data, Campylobacter armoricus strain CA639 harbored a circular chromosome of 1,688,169 bp with a G+C content of 28.47% and two plasmids named pCA639-1 and pCA639-2, with lengths of 51,123 and 28,139 bp, and G+C contents of 26.5% and 28.45%, respectively.
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Project description:Rapid, accurate bacterial identification in biological samples is an important task for microbiology laboratories, for which 16S~rRNA gene Sanger sequencing of cultured isolates is frequently used. In contrast, next-generation sequencing does not require intermediate culturing steps and can be directly applied on communities, but its performance has not been extensively evaluated. We present a comparative evaluation of second (Illumina) and third (Oxford Nanopore Technologies (ONT)) generation sequencing technologies for 16S targeted genomics using a well-characterized reference sample. Different 16S gene regions were amplified and sequenced using the Illumina MiSeq, and analyzed with Mothur. Correct classification was variable, depending on the region amplified. Using a majority vote over all regions, most false positives could be eliminated at the genus level but not the species level. Alternatively, the entire 16S gene was amplified and sequenced using the ONT MinION, and analyzed with Mothur, EPI2ME, and GraphMap. Although >99\% of reads were correctly classified at the genus level, up to $\approx$40\% were misclassified at the species level. Both~technologies, therefore, allow reliable identification of bacterial genera, but can potentially misguide identification of bacterial species, and constitute viable alternatives to Sanger sequencing for rapid analysis of mixed samples without requiring any culturing steps.
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Project description:High-throughput metabarcoding studies on fungi and other eukaryotic microorganisms are rapidly becoming more frequent and more complex, requiring researchers to handle ever increasing amounts of raw sequence data. Here, we provide a flexible pipeline for pruning and analyzing fungal barcode (ITS rDNA) data generated as paired-end reads on Illumina MiSeq sequencers. The pipeline presented includes specific steps fine-tuned for ITS, that are mostly missing from pipelines developed for prokaryotes. It (1) employs state of the art programs and follows best practices in fungal high-throughput metabarcoding; (2) consists of modules and scripts easily modifiable by the user to ensure maximum flexibility with regard to specific needs of a project or future methodological developments; and (3) is straightforward to use, also in classroom settings. We provide detailed descriptions and revision techniques for each step, thus giving the user maximum control over data treatment and avoiding a black-box approach. Employing this pipeline will improve and speed up the tedious and error-prone process of cleaning fungal Illumina metabarcoding data.
| S-EPMC4113289 | biostudies-literature
Project description:Whole-genome sequencing (WGS) enables the molecular characterization of bacterial pathogens. We compared the accuracy of the Illumina and Oxford Nanopore Technologies (ONT) sequencing platforms for the determination of AMR classes and antimicrobial susceptibility testing (AST) among 181 clinical Enterobacteriaceae isolates. Sequencing reads for each isolate were uploaded to AREScloud (Ares Genetics) to determine the presence of AMR markers and the predicted WGS-AST profile. The profiles of both sequencing platforms were compared to broth microdilution (BMD) AST. Isolates were delineated by resistance to third-generation cephalosporins and carbapenems as well as the presence of AMR markers to determine clinically relevant AMR classes. The overall categorical agreement (CA) was 90% (Illumina) and 88% (ONT) across all antimicrobials, 96% for the prediction of resistance to third-generation cephalosporins for both platforms, and 94% (Illumina) and 91% (ONT) for the prediction of resistance to carbapenems. Carbapenem resistance was overestimated on ONT with a major error of 16%. Sensitivity for the detection of carbapenemases, extended-spectrum β-lactamases, and plasmid-mediated ampC genes was 98, 95, and 70% by ONT compared to the Illumina dataset as the reference. Our results highlight the potential of the ONT platform's use in clinical microbiology laboratories. When combined with robust bioinformatics methods, WGS-AST predictions may be a future approach to guide effective antimicrobial decision-making.
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Project description:Zebrafish is a widely used model organism for investigating human diseases, including hematopoietic disorders. However, a comprehensive methylation baseline for zebrafish primary hematopoietic organ, the kidney marrow (KM), is still lacking. We employed Oxford Nanopore Technologies (ONT) sequencing to profile DNA methylation in zebrafish KM by generating four KM datasets, with two groups based on the presence or absence of red blood cells. Our findings revealed that blood contamination in the KM samples reduced read quality and altered methylation patterns. Compared with whole-genome bisulfite sequencing (WGBS), the ONT-based methylation profiling can cover more CpG sites (92.4% vs 70%-80%), and exhibit less GC bias with more even genomic coverage. And the ONT methylation calling results showed a high correlation with WGBS results when using shared sites. This study establishes a comprehensive methylation profile for zebrafish KM, paving the way for further investigations into epigenetic regulation and the development of targeted therapies for hematopoietic disorders.
2023-05-19 | GSE232842 | GEO
Project description:The multi-mycotoxin occurrence for internal and superficial fungi contamination were comprehensively assessed in medicinal seeds used as food or beverage. Based on a polyphasic approach using morphological characters, β-tubulin and ITS gene blast, a total of 27 species belonging to 12 genera were identified from surface-sterilized seeds. Chaetomium globosporum was most predominant (23%), followed by Microascus trigonosporus (12%) and Alternaria alternata (9%). With respect to superficial mycobiota, thirty-four species belonging to 17 genera were detected. Aspergillus niger and Penicillium polonicum were predominant (12% and 15%, respectively). Medicinal seed samples and potential toxigenic fungi were tested for ochratoxin A (OTA) and aflatoxins (AFB1, AFB2, AFG1, AFG2) using UPLC-MS/MS. Platycladi seeds were contaminated with AFB1 (52.0 µg/kg) and tangerine seed was contaminated with OTA (92.3 µg/kg). Subsequent analysis indicated that one A. flavus strain isolated from platycladi seed was able to synthesize AFB1 (102.0 µg/kg) and AFB2 (15.3 µg/kg). Two P. polonicum strains isolated from tangerine and lychee seeds were able to synthesize OTA (4.1 µg/kg and 14.8 µg/kg, respectively). These results identify potential sources of OTA and aflatoxins in medicinal seeds and allude to the need to establish permitted limits for these mycotoxins in these seeds that are commonly consumed by humans.
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