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      Annotated bacterial chromosomes from frame-shift-corrected long-read metagenomic data

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          Abstract

          Background

          Short-read sequencing technologies have long been the work-horse of microbiome analysis. Continuing technological advances are making the application of long-read sequencing to metagenomic samples increasingly feasible.

          Results

          We demonstrate that whole bacterial chromosomes can be obtained from an enriched community, by application of MinION sequencing to a sample from an EBPR bioreactor, producing 6 Gb of sequence that assembles into multiple closed bacterial chromosomes. We provide a simple pipeline for processing such data, which includes a new approach to correcting erroneous frame-shifts.

          Conclusions

          Advances in long-read sequencing technology and corresponding algorithms will allow the routine extraction of whole chromosomes from environmental samples, providing a more detailed picture of individual members of a microbiome.

          Electronic supplementary material

          The online version of this article (10.1186/s40168-019-0665-y) contains supplementary material, which is available to authorized users.

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          Most cited references10

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          Completing bacterial genome assemblies with multiplex MinION sequencing

          Illumina sequencing platforms have enabled widespread bacterial whole genome sequencing. While Illumina data is appropriate for many analyses, its short read length limits its ability to resolve genomic structure. This has major implications for tracking the spread of mobile genetic elements, including those which carry antimicrobial resistance determinants. Fully resolving a bacterial genome requires long-read sequencing such as those generated by Oxford Nanopore Technologies (ONT) platforms. Here we describe our use of the ONT MinION to sequence 12 isolates of Klebsiella pneumoniae on a single flow cell. We assembled each genome using a combination of ONT reads and previously available Illumina reads, and little to no manual intervention was needed to achieve fully resolved assemblies using the Unicycler hybrid assembler. Assembling only ONT reads with Canu was less effective, resulting in fewer resolved genomes and higher error rates even following error correction with Nanopolish. We demonstrate that multiplexed ONT sequencing is a valuable tool for high-throughput bacterial genome finishing. Specifically, we advocate the use of Illumina sequencing as a first analysis step, followed by ONT reads as needed to resolve genomic structure.
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            METAXA2: improved identification and taxonomic classification of small and large subunit rRNA in metagenomic data.

            The ribosomal rRNA genes are widely used as genetic markers for taxonomic identification of microbes. Particularly the small subunit (SSU; 16S/18S) rRNA gene is frequently used for species- or genus-level identification, but also the large subunit (LSU; 23S/28S) rRNA gene is employed in taxonomic assignment. The METAXA software tool is a popular utility for extracting partial rRNA sequences from large sequencing data sets and assigning them to an archaeal, bacterial, nuclear eukaryote, mitochondrial or chloroplast origin. This study describes a comprehensive update to METAXA - METAXA2 - that extends the capabilities of the tool, introducing support for the LSU rRNA gene, a greatly improved classifier allowing classification down to genus or species level, as well as enhanced support for short-read (100 bp) and paired-end sequences, among other changes. The performance of METAXA2 was compared to other commonly used taxonomic classifiers, showing that METAXA2 often outperforms previous methods in terms of making correct predictions while maintaining a low misclassification rate. METAXA2 is freely available from http://microbiology.se/software/metaxa2/.
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              GenBank

              GenBank (R) is a comprehensive database that contains publicly available DNA sequences for more than 205 000 named organisms, obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects. Most submissions are made using the Web-based BankIt or standalone Sequin programs and accession numbers are assigned by GenBank staff upon receipt. Daily data exchange with the EMBL Data Library in Europe and the DNA Data Bank of Japan ensures worldwide coverage. GenBank is accessible through NCBI's retrieval system, Entrez, which integrates data from the major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and the biomedical journal literature via PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bimonthly releases and daily updates of the GenBank database are available by FTP. To access GenBank and its related retrieval and analysis services, go to the NCBI Homepage at .
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                Author and article information

                Contributors
                AKrithika@ntu.edu.sg
                caner.bagci@uni-tuebingen.de
                irina.bessarab@nus.edu.sg
                sina.beier@uni-tuebingen.de
                buchfink@gmail.com
                anna.gorska@uni-tuebingen.de
                glqiu@ntu.edu.sg
                daniel.huson@uni-tuebingen.de
                lsirbhw@nus.edu.sg
                Journal
                Microbiome
                Microbiome
                Microbiome
                BioMed Central (London )
                2049-2618
                16 April 2019
                16 April 2019
                2019
                : 7
                : 61
                Affiliations
                [1 ]ISNI 0000 0001 2224 0361, GRID grid.59025.3b, Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, ; 60 Nanyang Drive, SBS-01N-27, Singapore, 637551 Singapore
                [2 ]ISNI 0000 0001 2190 1447, GRID grid.10392.39, Department of Computer Science, University of Tübingen, Sand 14, ; Tübingen, 72076 Germany
                [3 ]ISNI 0000 0001 2190 1447, GRID grid.10392.39, International Max Planck Research School From Molecules to Organisms, Max Planck Institute for Developmental Biology and Eberhard Karls University Tübingen, ; Max-Planck-Ring 5, Tübingen, 72076 Germany
                [4 ]ISNI 0000 0001 2180 6431, GRID grid.4280.e, Singapore Centre for Environmental Life Sciences Engineering, National University of Singapore, ; 28 Medical Drive, Singapore, 117456 Singapore
                [5 ]ISNI 0000 0001 1014 8330, GRID grid.419495.4, Max-Planck-Institute for Developmental Biology, ; Max-Planck-Ring 5, Tübingen, 72076 Germany
                [6 ]ISNI 0000 0001 2180 6431, GRID grid.4280.e, Life Sciences Institute, National University of Singapore, ; 28 Medical Drive, Singapore, 117456 Singapore
                Author information
                http://orcid.org/0000-0002-2961-604X
                Article
                665
                10.1186/s40168-019-0665-y
                6469205
                30992083
                706c6f4c-039a-419d-8603-a174d18ad377
                © The Author(s) 2019

                Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 4 January 2019
                : 11 March 2019
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001381, National Research Foundation Singapore;
                Award ID: 0
                Funded by: Deutsche Forschungsgemeinschaft
                Award ID: INST~37/935-1 FUGG
                Funded by: Deutsche Forschungsgemeinschaft
                Award ID: HU 566/12-1
                Categories
                Short Report
                Custom metadata
                © The Author(s) 2019

                microbiome,long-read sequencing,microbial genomics,sequence assembly,frame-shifts,algorithms,software

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