Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

Resolving the complex Bordetella pertussis genome using barcoded nanopore sequencing

View ORCID ProfileNatalie Ring, View ORCID ProfileJonathan Abrahams, View ORCID ProfileMiten Jain, Hugh Olsen, View ORCID ProfileAndrew Preston, View ORCID ProfileStefan Bagby
doi: https://doi.org/10.1101/381640
Natalie Ring
1Department of Biology and Biochemistry, University of Bath, Bath, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Natalie Ring
Jonathan Abrahams
1Department of Biology and Biochemistry, University of Bath, Bath, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Jonathan Abrahams
Miten Jain
2Nanopore Group, University of California Santa Cruz, California, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Miten Jain
Hugh Olsen
2Nanopore Group, University of California Santa Cruz, California, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Andrew Preston
1Department of Biology and Biochemistry, University of Bath, Bath, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Andrew Preston
Stefan Bagby
1Department of Biology and Biochemistry, University of Bath, Bath, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Stefan Bagby
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Supplementary material
  • Preview PDF
Loading

ABSTRACT

The genome of Bordetella pertussis is complex, with high GC content and many repeats, each longer than 1,000 bp. Short-read DNA sequencing is unable to resolve the structure of the genome; however, long-read sequencing offers the opportunity to produce single-contig B. pertussis assemblies using sequencing reads which are longer than the repetitive sections. We used an R9.4 MinION flow cell and barcoding to sequence five B. pertussis strains in a single sequencing run. We then trialled combinations of the many nanopore-user-community-built long-read analysis tools to establish the current optimal assembly pipeline for B. pertussis genome sequences. Our best long-read-only assemblies were produced by Canu read correction followed by assembly with Flye and polishing with Nanopolish, whilst the best hybrids (using nanopore and Illumina reads together) were produced by Canu correction followed by Unicycler. This pipeline produced closed genome sequences for four strains, revealing inter-strain genomic rearrangement. However, read mapping to the Tohama I reference genome suggests that the remaining strain contains an ultra-long duplicated region (over 100 kbp), which was not resolved by our pipeline. We have therefore demonstrated the ability to resolve the structure of several B. pertussis strains per single barcoded nanopore flow cell, but the genomes with highest complexity (e.g. very large duplicated regions) remain only partially resolved using the standard library preparation and will require an alternative library preparation method. For full strain characterisation, we recommend hybrid assembly of long and short reads together; for comparison of genome arrangement, assembly using long reads alone is sufficient.

DATA SUMMARY

  1. Final sequence read files (fastq) for all 5 strains have been deposited in the SRA, BioProject PRJNA478201, accession numbers SAMN09500966, SAMN09500967, SAMN09500968, SAMN09500969, SAMN09500970

  2. A full list of accession numbers for Illumina sequence reads is available in Table S1

  3. Assembly tests, basecalled read sets and reference materials are available from figshare: https://figshare.com/projects/Resolving_the_complex_Bordetella_pertussis_genome_using_barcoded_nanopore_sequencing/31313

  4. Genome sequences for B. pertussis strains UK36, UK38, UK39, UK48 and UK76 have been deposited in GenBank; accession numbers: CP031289, CP031112, CP031113, QRAX00000000, CP031114

  5. Source code and full commands used are available from Github: https://github.com/nataliering/Resolving-the-complex-Bordetella-pertussis-genome-using-barcoded-nanopore-sequencing

IMPACT STATEMENT Over the past two decades, whole genome sequencing has allowed us to understand microbial pathogenicity and evolution on an unprecedented level. However, repetitive regions, like those found throughout the B. pertussis genome, have confounded our ability to resolve complex genomes using short-read sequencing technologies alone. To produce closed B. pertussis genome sequences it is necessary to use a sequencing technology which can generate reads longer than these problematic genomic regions. Using barcoded nanopore sequencing, we show that multiple B. pertussis genomes can be resolved per flow cell. Use of our assembly pipeline to resolve further B. pertussis genomes will advance understanding of how genome-level differences affect the phenotypes of strains which appear monomorphic at nucleotide-level.

This work expands the recently emergent theme that even the most complex genomes can be resolved with sufficiently long sequencing reads. Additionally, we utilise a more widely accessible alternative sequencing platform to the Pacific Biosciences platform already used by large research centres such as the CDC. Our optimisation process, moreover, shows that the analysis tools favoured by the sequencing community do not necessarily produce the most accurate assemblies for all organisms; pipeline optimisation may therefore be beneficial in studies of unusually complex genomes.

  • ABBREVIATIONS

    BUSCO
    Benchmarking Universal Single-Copy Orthologs
    CLIMB
    Cloud Infrastructure for Microbial Bioinformatics
    FFPE
    Formalin-Fixed, Paraffin-Embedded
    FHA
    filamentous haemagglutinin
    IS
    insertion sequence
    MRC
    Medical Research Council
    ONT
    Oxford Nanopore Technologies
    Prn
    Pertactin
    Pt
    Pertussis toxin
    SPRI
    Solid Phase Reversible Immobilization
  • Copyright 
    The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
    Back to top
    PreviousNext
    Posted August 03, 2018.
    Download PDF

    Supplementary Material

    Email

    Thank you for your interest in spreading the word about bioRxiv.

    NOTE: Your email address is requested solely to identify you as the sender of this article.

    Enter multiple addresses on separate lines or separate them with commas.
    Resolving the complex Bordetella pertussis genome using barcoded nanopore sequencing
    (Your Name) has forwarded a page to you from bioRxiv
    (Your Name) thought you would like to see this page from the bioRxiv website.
    CAPTCHA
    This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
    Share
    Resolving the complex Bordetella pertussis genome using barcoded nanopore sequencing
    Natalie Ring, Jonathan Abrahams, Miten Jain, Hugh Olsen, Andrew Preston, Stefan Bagby
    bioRxiv 381640; doi: https://doi.org/10.1101/381640
    Reddit logo Twitter logo Facebook logo LinkedIn logo Mendeley logo
    Citation Tools
    Resolving the complex Bordetella pertussis genome using barcoded nanopore sequencing
    Natalie Ring, Jonathan Abrahams, Miten Jain, Hugh Olsen, Andrew Preston, Stefan Bagby
    bioRxiv 381640; doi: https://doi.org/10.1101/381640

    Citation Manager Formats

    • BibTeX
    • Bookends
    • EasyBib
    • EndNote (tagged)
    • EndNote 8 (xml)
    • Medlars
    • Mendeley
    • Papers
    • RefWorks Tagged
    • Ref Manager
    • RIS
    • Zotero
    • Tweet Widget
    • Facebook Like
    • Google Plus One

    Subject Area

    • Microbiology
    Subject Areas
    All Articles
    • Animal Behavior and Cognition (4851)
    • Biochemistry (10792)
    • Bioengineering (8040)
    • Bioinformatics (27286)
    • Biophysics (13983)
    • Cancer Biology (11120)
    • Cell Biology (16049)
    • Clinical Trials (138)
    • Developmental Biology (8778)
    • Ecology (13279)
    • Epidemiology (2067)
    • Evolutionary Biology (17354)
    • Genetics (11687)
    • Genomics (15915)
    • Immunology (11028)
    • Microbiology (26070)
    • Molecular Biology (10637)
    • Neuroscience (56533)
    • Paleontology (417)
    • Pathology (1732)
    • Pharmacology and Toxicology (3003)
    • Physiology (4544)
    • Plant Biology (9628)
    • Scientific Communication and Education (1615)
    • Synthetic Biology (2685)
    • Systems Biology (6975)
    • Zoology (1508)