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

Gabriele Margos, S Hepner, C Mang, D Marosevic, Stuart Reynolds, S Krebs, A Sing, M Derdakova, M A Reiter, Volker Fingerle

Research output: Contribution to journalArticle

12 Citations (Scopus)

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.
Original languageEnglish
Article number422
JournalBMC Genomics
Volume18
DOIs
Publication statusPublished - 30 May 2017

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Borrelia burgdorferi
Ticks
Plasmids
Genome
Nanopores
Lyme Disease
Technology
Virulence
Chromosomes
Borrelia burgdorferi Group
Borrelia
Nucleic Acid Databases
Libraries

Keywords

  • Borrelia burgdorferi Genomics Plasmids Next generation sequencing De novo assembly

ASJC Scopus subject areas

  • Immunology and Microbiology(all)

Cite this

Lost in plasmids: next generation sequencing and the complex genome of the tick-borne pathogen Borrelia burgdorferi. / Margos, Gabriele; Hepner, S; Mang, C; Marosevic, D; Reynolds, Stuart; Krebs, S; Sing, A; Derdakova, M; Reiter, M A; Fingerle, Volker.

In: BMC Genomics, Vol. 18, 422, 30.05.2017.

Research output: Contribution to journalArticle

Margos, G, Hepner, S, Mang, C, Marosevic, D, Reynolds, S, Krebs, S, Sing, A, Derdakova, M, Reiter, MA & Fingerle, V 2017, 'Lost in plasmids: next generation sequencing and the complex genome of the tick-borne pathogen Borrelia burgdorferi', BMC Genomics, vol. 18, 422. https://doi.org/10.1186/s12864-017-3804-5
Margos, Gabriele ; Hepner, S ; Mang, C ; Marosevic, D ; Reynolds, Stuart ; Krebs, S ; Sing, A ; Derdakova, M ; Reiter, M A ; Fingerle, Volker. / Lost in plasmids: next generation sequencing and the complex genome of the tick-borne pathogen Borrelia burgdorferi. In: BMC Genomics. 2017 ; Vol. 18.
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abstract = "BackgroundBorrelia (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.ResultsInclusion 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.ConclusionShort 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.",
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T1 - Lost in plasmids: next generation sequencing and the complex genome of the tick-borne pathogen Borrelia burgdorferi

AU - Margos, Gabriele

AU - Hepner, S

AU - Mang, C

AU - Marosevic, D

AU - Reynolds, Stuart

AU - Krebs, S

AU - Sing, A

AU - Derdakova, M

AU - Reiter, M A

AU - Fingerle, Volker

PY - 2017/5/30

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N2 - BackgroundBorrelia (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.ResultsInclusion 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.ConclusionShort 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.

AB - BackgroundBorrelia (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.ResultsInclusion 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.ConclusionShort 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.

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UR - https://doi.org/10.1186/s12864-017-3804-5

UR - https://doi.org/10.1186/s12864-017-3804-5

U2 - 10.1186/s12864-017-3804-5

DO - 10.1186/s12864-017-3804-5

M3 - Article

VL - 18

JO - BMC Genomics

JF - BMC Genomics

SN - 1471-2164

M1 - 422

ER -