Dataset Information

Identification of plastic-associated species in the Mediterranean Sea using DNA metabarcoding with Nanopore MinION.

ABSTRACT: Plastic debris in the ocean form a new ecosystem, termed 'plastisphere', which hosts a variety of marine organisms. Recent studies implemented DNA metabarcoding to characterize the taxonomic composition of the plastisphere in different areas of the world. In this study, we used a modified metabarcoding approach which was based on longer barcode sequences for the characterization of the plastisphere biota. We compared the microbiome of polyethylene food bags after 1 month at sea to the free-living biome in two proximal but environmentally different locations on the Mediterranean coast of Israel. We targeted the full 1.5 kb-long 16S rRNA gene for bacteria and 0.4-0.8 kb-long regions within the 18S rRNA, ITS, tufA and COI loci for eukaryotes. The taxonomic barcodes were sequenced using Oxford Nanopore Technology with multiplexing on a single MinION flow cell. We identified between 1249 and 2141 species in each of the plastic samples, of which 61 species (34 bacteria and 27 eukaryotes) were categorized as plastic-specific, including species that belong to known hydrocarbon-degrading genera. In addition to a large prokaryotes repertoire, our results, supported by scanning electron microscopy, depict a surprisingly high biodiversity of eukaryotes within the plastisphere with a dominant presence of diatoms as well as other protists, algae and fungi.

SUBMITTER: Davidov K

PROVIDER: S-EPMC7568539 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Identification of plastic-associated species in the Mediterranean Sea using DNA metabarcoding with Nanopore MinION.

Davidov Keren K Iankelevich-Kounio Evgenia E Yakovenko Iryna I Koucherov Yuri Y Rubin-Blum Maxim M Oren Matan M

Scientific reports 20201016 1

Plastic debris in the ocean form a new ecosystem, termed 'plastisphere', which hosts a variety of marine organisms. Recent studies implemented DNA metabarcoding to characterize the taxonomic composition of the plastisphere in different areas of the world. In this study, we used a modified metabarcoding approach which was based on longer barcode sequences for the characterization of the plastisphere biota. We compared the microbiome of polyethylene food bags after 1 month at sea to the free-livin ...[more]

PMID: 33067509

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Project description:Differentiation between mitis group streptococci (MGS) bacteria in routine laboratory tests has become important for obtaining accurate epidemiological information on the characteristics of MGS and understanding their clinical significance. The most reliable method of MGS species identification is multilocus sequence analysis (MLSA) with seven house-keeping genes; however, because this method is time-consuming, it is deemed unsuitable for use in most clinical laboratories. In this study, we established a scheme for identifying 12 species of MGS (S. pneumoniae, S. pseudopneumoniae, S. mitis, S. oralis, S. peroris, S. infantis, S. australis, S. parasanguinis, S. sinensis, S. sanguinis, S. gordonii, and S. cristatus) using the MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, UK) with the taxonomic aligner "What's in My Pot?" (WIMP; Oxford Nanopore's cloud-based analysis platform) and Kraken2 pipeline with the custom database adjusted for MGS species identification. The identities of the species in reference genomes (n = 514), clinical isolates (n = 31), and reference strains (n = 4) were confirmed via MLSA. The nanopore simulation reads were generated from reference genomes, and the optimal cut-off values for MGS species identification were determined. For 31 clinical isolates (S. pneumoniae = 8, S. mitis = 17 and S. oralis = 6) and 4 reference strains (S. pneumoniae = 1, S. mitis = 1, S. oralis = 1, and S. pseudopneumoniae = 1), a sequence library was constructed via a Rapid Barcoding Sequencing Kit for multiplex and real-time MinION sequencing. The optimal cut-off values for the identification of MGS species for analysis by WIMP and Kraken2 pipeline were determined. The workflow using Kraken2 pipeline with a custom database identified all 12 species of MGS, and WIMP identified 8 MGS bacteria except S. infantis, S. australis, S. peroris, and S. sinensis. The results obtained by MinION with WIMP and Kraken2 pipeline were consistent with the MGS species identified by MLSA analysis. The practical advantage of whole genome analysis using the MinION nanopore sequencer is that it can aid in MGS surveillance. We concluded that MinION sequencing with the taxonomic aligner enables accurate MGS species identification and could contribute to further epidemiological surveys.

Project description:Rapid, cost-effective identification of genetic variants in small candidate genomic regions remains a challenge, particularly for less well equipped or lower throughput laboratories. The application of Oxford Nanopore Technologies' MinION sequencer has the potential to fulfil this requirement. We demonstrate a proof of concept for a multiplexing assay that pools PCR amplicons for MinION sequencing to enable sequencing of multiple templates from multiple individuals, which could be applied to gene-targeted diagnostics. A combined strategy of barcoding and sample pooling was developed for simultaneous multiplex MinION sequencing of 100 PCR amplicons. The amplicons are family-specific, spanning a total of 30 loci in DNA isolated from 82 human neurodevelopmental cases and family members. The target regions were chosen for further interrogation because a potentially disease-causative variant had been identified in affected individuals following Illumina exome sequencing. The pooled MinION sequences were deconvoluted by aligning to custom references using the minimap2 aligner software. Our multiplexing approach produced an interpretable and expected sequence from 29 of the 30 targeted genetic loci. The sequence variant which was not correctly resolved in the MinION sequence was adjacent to a five nucleotide homopolymer. It is already known that homopolymers present a resolution problem with the MinION approach. Interestingly despite equimolar quantities of PCR amplicon pooled for sequencing, significant variation in the depth of coverage (127×-19,626×; mean = 8321×, std err = 452.99) was observed. We observed independent relationships between depth of coverage and target length, and depth of coverage and GC content. These relationships demonstrate biases of the MinION sequencer for longer templates and those with lower GC content. We demonstrate an efficient approach for variant discovery or confirmation from short DNA templates using the MinION sequencing device. With less than 130 × depth of coverage required for accurate genotyping, the methodology described here allows for rapid highly multiplexed targeted sequencing of large numbers of samples in a minimally equipped laboratory with a potential cost as much 200 × less than that from Sanger sequencing.

Dataset Information

Identification of plastic-associated species in the Mediterranean Sea using DNA metabarcoding with Nanopore MinION.

Publications

Identification of plastic-associated species in the Mediterranean Sea using DNA metabarcoding with Nanopore MinION.

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