Microbial diversity characterization of seawater in a pilot study using Oxford Nanopore Technologies long-read sequencing

Liem, Michael; Regensburg-Tuïnk, A.J.G.; Henkel, Christiaan V.; Spaink, Herman P.

doi:10.1101/2020.01.08.898312

Cited by 3 publications

(3 citation statements)

References 8 publications

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“…Approaches that over-come these includingsuch as blocking oligonucleotides for to remove metazoa signal [51] and the use of improved primer cocktails [52] or better DNA markers. The use of long-read high throughput methods have a real chance of providing high-resolution genomic and taxonomic diversity assessments at the species or even strain level [53]. With the inclusion of prokaryotic genetic diversity surveys, a massive increase in detection of trophic networks could be achieved.…”

Section: Discussionmentioning

confidence: 99%

Composition and Patterns of Taxa Assemblages in the Western Channel Assessed by 18S Sequencing, Microscopy and Flow Cytometry

Stern¹,

Picard²,

Clarke³

et al. 2022

Preprint

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Plankton monitoring by microscopy offers long-term ecological perspective of plankton com-munities but different detection approaches are biased uniquely. Genetic identification of marine plankton has become standard but is still not used in routine monitoring. This study assessed the diversity of plankton taxa using 18S high throughput sequencing from 2011-2012 from small-volume (~200ml) samples from the Water and Microplankton Sampler (WaMS) deployed on the Continuous Plankton Recorder platform (CPR). The 18S-HTS survey revealed a bias towards heterotrophic taxa, and phototrophs under 10µm within the photosynthetic community. In comparison with phytoplankton microscopic counts from the CPR survey and Western Channel Observatory station L4, only 8-12 taxonomic families were common to all three surveys, with a bias towards larger diatoms and dinoflagellate taxa in microscopy surveys. The WaMS survey detected a contrasting but complementary taxa set to that of microscopic surveys. Additional Quantitative PCR was carried out on the picoeukaryotic pelagophyte, Aureococcus anophagefferens, and the nanoeukaryotic potential harmful algae, Pseudo-nitzschia delicatissima, from 2011-2013. This confirmed the persistence presence of A. anophagefferens in the Western Channel and an elevated abundance of both species in 2011. Species specific seasonality were distinct from those of aggregrate phytoplankton groups.

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Section: Discussionmentioning

confidence: 99%

Composition and Patterns of Taxa Assemblages in the Western Channel Assessed by 18S Sequencing, Microscopy and Flow Cytometry

Stern¹,

Picard²,

Clarke³

et al. 2022

Preprint

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“…Finally, ONT has also demonstrated efficiency to sequence viruses from sea-water samples [53][54][55]. However, it has never been tested for the characterization of the OsHV-1 genome.…”

Section: Impact Statementmentioning

confidence: 99%

Evaluation of tangential flow filtration coupled to long-read sequencing for ostreid herpesvirus type 1 genome assembly

et al. 2022

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Whole-genome sequencing is widely used to better understand the transmission dynamics, the evolution and the emergence of new variants of viral pathogens. This can bring crucial information to stakeholders for disease management. Unfortunately, aquatic virus genomes are usually difficult to characterize because most of these viruses cannot be easily propagated in vitro. Developing methodologies for routine genome sequencing of aquatic viruses is timely given the ongoing threat of disease emergence. This is particularly true for pathogenic viruses infecting species of commercial interest that are widely exchanged between production basins or countries. For example, the ostreid herpesvirus type 1 (OsHV-1) is a Herpesvirus widely associated with mass mortality events of juvenile Pacific oyster Crassostrea gigas. Genomes of Herpesviruses are large and complex with long direct and inverted terminal repeats. In addition, OsHV-1 is unculturable. It therefore accumulates several features that make its genome sequencing and assembly challenging. To overcome these difficulties, we developed a tangential flow filtration (TFF) method to enrich OsHV-1 infective particles from infected host tissues. This virus purification allowed us to extract high molecular weight and high-quality viral DNA that was subjected to Illumina short-read and Nanopore long-read sequencing. Dedicated bioinformatic pipelines were developed to assemble complete OsHV-1 genomes with reads from both sequencing technologies. Nanopore sequencing allowed characterization of new structural variations and major viral isomers while having 99,98 % of nucleotide identity with the Illumina assembled genome. Our study shows that TFF-based purification method, coupled with Nanopore sequencing, is a promising approach to enable in field sequencing of unculturable aquatic DNA virus.

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“…All the protocols were carried out in the lab, and the authors were able to achieve up to ∼5.5 M 16S rRNA full-length sequences with exclusive barcode assignments in a single MinION run. Other groups have used MinION devices for characterizing river water [ 45 ], seawater [ 46 ], and marine sediments [ 47 ] through metagenomic sequencing. Even though these experiments were not implemented in the field, they demonstrated the possibility of obtaining higher sequencing yields ( Table 1 ).…”

Section: Portable Sequencing In Natural Environmentsmentioning

confidence: 99%

A lab in the field: applications of real-time, in situ metagenomic sequencing

Latorre‐Pérez¹,

Pascual²,

Porcar

et al. 2020

Biology Methods and Protocols

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High-throughput metagenomic sequencing is considered one of the main technologies fostering the development of microbial ecology. Widely-used second generation sequencers have enabled the analysis of extremely diverse microbial communities, the discovery of novel gene functions, and the comprehension of the metabolic interconnections established among microbial consortia. However, the high cost of the sequencers and the complexity of library preparation and sequencing protocols still hamper the application of metagenomic sequencing in a vast range of real-life applications. In this context, the emergence of portable, third-generation sequencers is becoming a popular alternative for the rapid analysis of microbial communities in particular scenarios, due to their low cost, simplicity of operation, and rapid yield of results. This review discusses the main applications of real-time, in situ metagenomic sequencing developed to date, highlighting the relevance of this technology in current challenges (such as the management of global pathogen outbreaks) and in the next future of industry and clinical diagnosis.

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Microbial diversity characterization of seawater in a pilot study using Oxford Nanopore Technologies long-read sequencing

Cited by 3 publications

References 8 publications

Composition and Patterns of Taxa Assemblages in the Western Channel Assessed by 18S Sequencing, Microscopy and Flow Cytometry

Composition and Patterns of Taxa Assemblages in the Western Channel Assessed by 18S Sequencing, Microscopy and Flow Cytometry

Evaluation of tangential flow filtration coupled to long-read sequencing for ostreid herpesvirus type 1 genome assembly

A lab in the field: applications of real-time, in situ metagenomic sequencing

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