Nanopore based DNA-sequencing delivers long reads, thereby simplifying the decipherment of bacterial communities. Since its commercial appearance, this technology has been assigned several attributes, such as its error proneness, comparatively low cost, ease-of-use, and, most notably, aforementioned long reads. The technology as a whole is under continued development. As such, benchmarks are required to conceive, test and improve analysis protocols, including those related to the understanding of the composition of microbial communities. Here we present a dataset composed of twelve different prokaryotic species split into four samples differing by nucleic acid quantification technique to assess the specificity and sensitivity of the MinION nanopore sequencer in a blind study design. Taxonomic classification was performed by standard taxonomic sequence classification tools, namely Kraken, Kraken2 and Centrifuge directly on reads. This allowed taxonomic assignments of up to 99.27% on genus level and 92.78% on species level, enabling true-positive classification of strains down to 25,000 genomes per sample. Full genomic coverage is achieved for strains abundant as low as 250,000 genomes per sample under our experimental settings. In summary, we present an evaluation of nanopore sequence processing analysis with respect to microbial community composition. It provides an open protocol and the data may serve as basis for the development and benchmarking of future data processing pipelines.

Bumble bees are important crop pollinators and provide important pollination services to their respective ecosystems. Their pollen diet and thus food preferences can be characterized through nucleic acid sequence analysis. We present ITS2 amplicon sequence data from pollen collected by bumble bees. The pollen was collected from six different bumble bee colonies that were placed in independent agricultural landscapes. We compared next-generation (Illumina), i.e., short-read, and thirdgeneration (Nanopore), i.e., MinION, sequencing techniques. MinION data were preprocessed using traditional and Nanopore specific tools for comparative analysis and were evaluated in comparison to short-read sequence data with conventional processing. Based on the results, the dietary diary of bumble bee in the studied landscapes can be identified. It is known that short reads generated by next-generation sequencers have the advantage of higher quality scores while Nanopore yields longer read lengths. We show that assignments to taxonomic units yield comparable results when querying against an ITS2-specific sequence database. Thus, lower sequence quality is compensated by longer read lengths. However, the Nanopore technology is improving in terms of data quality, much cheaper, and suitable for portable applications. With respect to the studied agricultural landscapes we found that bumble bees require higher plant diversity than only crops to fulfill their foraging requirements.

Rhodobacter sphaeroides is a purple bacterium with complex genomic architecture. Here, a draft genome is reported for R. sphaeroides strain 2.4.1 substrain H2, which was generated exclusively from Nanopore sequencing data.

Background We benchmarked sequencing technology and assembly strategies for short-read, long-read, and hybrid assemblers in respect to correctness, contiguity, and completeness of assemblies in genomes of Francisella tularensis. Benchmarking allowed in-depth analyses of genomic structures of the Francisella pathogenicity islands and insertion sequences. Five major high-throughput sequencing technologies were applied, including next-generation “short-read” and third-generation “long-read” sequencing methods. Results We focused on short-read assemblers, hybrid assemblers, and analysis of the genomic structure with particular emphasis on insertion sequences and the Francisella pathogenicity island. The A5-miseq pipeline performed best for MiSeq data, Mira for Ion Torrent data, and ABySS for HiSeq data from eight short-read assembly methods. Two approaches were applied to benchmark long-read and hybrid assembly strategies: long-read-first assembly followed by correction with short reads (Canu/Pilon, Flye/Pilon) and short-read-first assembly along with scaffolding based on long reads (Unicyler, SPAdes). Hybrid assembly can resolve large repetitive regions best with a “long-read first” approach. Conclusions Genomic structures of the Francisella pathogenicity islands frequently showed misassembly. Insertion sequences (IS) could be used to perform an evolutionary conservation analysis. A phylogenetic structure of insertion sequences and the evolution within the clades elucidated the clade structure of the highly conservative F. tularensis.

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