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      Lost in plasmids: next generation sequencing and the complex genome of the tick-borne pathogen Borrelia burgdorferi

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          Abstract

          Background

          Borrelia ( B.) burgdorferi sensu lato, including the tick-transmitted agents of human Lyme borreliosis, have particularly complex genomes, consisting of a linear main chromosome and numerous linear and circular plasmids. The number and structure of plasmids is variable even in strains within a single genospecies. Genes on these plasmids are known to play essential roles in virulence and pathogenicity as well as host and vector associations. For this reason, it is essential to explore methods for rapid and reliable characterisation of molecular level changes on plasmids.

          In this study we used three strains: a low passage isolate of B. burgdorferi sensu stricto strain B31(−NRZ) and two closely related strains (PAli and PAbe) that were isolated from human patients. Sequences of these strains were compared to the previously sequenced reference strain B31 (available in GenBank) to obtain proof-of-principle information on the suitability of next generation sequencing (NGS) library construction and sequencing methods on the assembly of bacterial plasmids. We tested the effectiveness of different short read assemblers on Illumina sequences, and of long read generation methods on sequence data from Pacific Bioscience single-molecule real-time (SMRT) and nanopore (Oxford Nanopore Technologies) sequencing technology.

          Results

          Inclusion of mate pair library reads improved the assembly in some plasmids as did prior enrichment of plasmids. While cp32 plasmids remained refractory to assembly using only short reads they were effectively assembled by long read sequencing methods. The long read SMRT and nanopore sequences came, however, at the cost of indels (insertions or deletions) appearing in an unpredictable manner. Using long and short read technologies together allowed us to show that the three B. burgdorferi s.s. strains investigated here, whilst having similar plasmid structures to each other (apart from fusion of cp32 plasmids), differed significantly from the reference strain B31-GB, especially in the case of cp32 plasmids.

          Conclusion

          Short read methods are sufficient to assemble the main chromosome and many of the plasmids in B. burgdorferi. However, a combination of short and long read sequencing methods is essential for proper assembly of all plasmids including cp32 and thus, for gaining an understanding of host- or vector adaptations. An important conclusion from our work is that the evolution of Borrelia plasmids appears to be dynamic. This has important implications for the development of useful research strategies to monitor the risk of Lyme disease occurrence and how to medically manage it.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s12864-017-3804-5) contains supplementary material, which is available to authorized users.

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          Most cited references43

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          Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences

          Increased reliance on computational approaches in the life sciences has revealed grave concerns about how accessible and reproducible computation-reliant results truly are. Galaxy http://usegalaxy.org, an open web-based platform for genomic research, addresses these problems. Galaxy automatically tracks and manages data provenance and provides support for capturing the context and intent of computational methods. Galaxy Pages are interactive, web-based documents that provide users with a medium to communicate a complete computational analysis.
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            Performance comparison of benchtop high-throughput sequencing platforms.

            Three benchtop high-throughput sequencing instruments are now available. The 454 GS Junior (Roche), MiSeq (Illumina) and Ion Torrent PGM (Life Technologies) are laser-printer sized and offer modest set-up and running costs. Each instrument can generate data required for a draft bacterial genome sequence in days, making them attractive for identifying and characterizing pathogens in the clinical setting. We compared the performance of these instruments by sequencing an isolate of Escherichia coli O104:H4, which caused an outbreak of food poisoning in Germany in 2011. The MiSeq had the highest throughput per run (1.6 Gb/run, 60 Mb/h) and lowest error rates. The 454 GS Junior generated the longest reads (up to 600 bases) and most contiguous assemblies but had the lowest throughput (70 Mb/run, 9 Mb/h). Run in 100-bp mode, the Ion Torrent PGM had the highest throughput (80–100 Mb/h). Unlike the MiSeq, the Ion Torrent PGM and 454 GS Junior both produced homopolymer-associated indel errors (1.5 and 0.38 errors per 100 bases, respectively).
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              Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi.

              The genome of the bacterium Borrelia burgdorferi B31, the aetiologic agent of Lyme disease, contains a linear chromosome of 910,725 base pairs and at least 17 linear and circular plasmids with a combined size of more than 533,000 base pairs. The chromosome contains 853 genes encoding a basic set of proteins for DNA replication, transcription, translation, solute transport and energy metabolism, but, like Mycoplasma genitalium, it contains no genes for cellular biosynthetic reactions. Because B. burgdorferi and M. genitalium are distantly related eubacteria, we suggest that their limited metabolic capacities reflect convergent evolution by gene loss from more metabolically competent progenitors. Of 430 genes on 11 plasmids, most have no known biological function; 39% of plasmid genes are paralogues that form 47 gene families. The biological significance of the multiple plasmid-encoded genes is not clear, although they may be involved in antigenic variation or immune evasion.
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                Author and article information

                Contributors
                0049 913168085883 , Gabriele.margos@lgl.bayern.de
                amira.hepner@web.de
                christoph.mang103@gmx.de
                djurdjica.marosevic@gmail.com
                s.e.reynolds@bath.ac.uk
                krebs@genzentrum.lmu.de
                andreas.sing@lgl.bayern.de
                marketa.derdakova@gmail.com
                michael.a.reiter@meduniwien.ac.at
                Volker.fingerle@lgl.bayern.de
                Journal
                BMC Genomics
                BMC Genomics
                BMC Genomics
                BioMed Central (London )
                1471-2164
                30 May 2017
                30 May 2017
                2017
                : 18
                : 422
                Affiliations
                [1 ]German National Reference Centre for Borrelia (NRZ), Bavarian Health and Food Safety Authority (LGL), Veterinärstrasse 2, 85764 Oberschleissheim, Germany
                [2 ]ISNI 0000 0001 0349 2029, GRID grid.414279.d, , Bavarian Health and Food Safety Authority (LGL), ; Veterinärstrasse 2, 85764 Oberschleissheim, Germany
                [3 ]ISNI 0000 0004 1791 8889, GRID grid.418914.1, , European Programme for Public Health Microbiology Training, European Centre of Disease Prevention and Control (ECDC), ; Stockholm, Sweden
                [4 ]ISNI 0000 0001 2162 1699, GRID grid.7340.0, Department of Biology and Biochemistry, , University of Bath, ; Claverton Down, BA2 7AY Bath, UK
                [5 ]ISNI 0000 0004 1936 973X, GRID grid.5252.0, Gene Centre, Laboratory for Functional Genome Analysis, , LMU Munich, ; Feodor-Lynen-Strasse 25, 81377 Munich, Germany
                [6 ]ISNI 0000 0004 4665 5790, GRID grid.425138.9, , Institute of Zoology, Slovak Academy of Sciences, ; Bratislava, Slovakia
                [7 ]ISNI 0000 0000 9259 8492, GRID grid.22937.3d, Institut für Hygiene und Angewandte Immunologie, , Medizinische Universität Wien, ; Kinderspitalgasse 15, A-1090 Wien, Austria
                Author information
                http://orcid.org/0000-0001-5304-9615
                Article
                3804
                10.1186/s12864-017-3804-5
                5450258
                28558786
                f1289f7f-ed1a-4559-8941-91f07449c3d7
                © The Author(s). 2017

                Open AccessThis 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
                : 10 January 2017
                : 17 May 2017
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001704, European Society of Clinical Microbiology and Infectious Diseases;
                Award ID: Study Group Grant 2015
                Funded by: Bavarian Ministry of Health and Care
                Award ID: not applicable
                Funded by: Robert-Koch-Institute, Berlin, Germany
                Award ID: NRZ Funding
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2017

                Genetics
                borrelia burgdorferi,genomics,plasmids,next generation sequencing,de novo assembly
                Genetics
                borrelia burgdorferi, genomics, plasmids, next generation sequencing, de novo assembly

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