Dataset Information

Resolving the complex Bordetella pertussis genome using barcoded nanopore sequencing.

ABSTRACT: The genome of Bordetella pertussis is complex, with high G+C content and many repeats, each longer than 1000?bp. Long-read sequencing offers the opportunity to produce single-contig B. pertussis assemblies using sequencing reads which are longer than the repetitive sections, with the potential to reveal genomic features which were previously unobservable in multi-contig assemblies produced by short-read sequencing alone. 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. This pipeline produced closed genome sequences for four strains, allowing visualization of inter-strain genomic rearrangement. Read mapping to the Tohama I reference genome suggests that the remaining strain contains an ultra-long duplicated region (almost 200?kbp), which was not resolved by our pipeline; further investigation also revealed that a second strain that was seemingly resolved by our pipeline may contain an even longer duplication, albeit in a small subset of cells. 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 characterization, we recommend hybrid assembly of long and short reads together; for comparison of genome arrangement, assembly using long reads alone is sufficient.

SUBMITTER: Ring N

PROVIDER: S-EPMC6321869 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Resolving the complex Bordetella pertussis genome using barcoded nanopore sequencing.

Ring Natalie N Abrahams Jonathan S JS Jain Miten M Olsen Hugh H Preston Andrew A Bagby Stefan S

Microbial genomics 20181121 11

The genome of Bordetella pertussis is complex, with high G+C content and many repeats, each longer than 1000 bp. Long-read sequencing offers the opportunity to produce single-contig B. pertussis assemblies using sequencing reads which are longer than the repetitive sections, with the potential to reveal genomic features which were previously unobservable in multi-contig assemblies produced by short-read sequencing alone. We used an R9.4 MinION flow cell and barcoding to sequence five B. pertussi ...[more]

PMID: 30461375

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Project description:Bordetella pertussis, the aetiological agent of whooping cough, is re-emerging globally despite widespread vaccination. B. pertussis is highly infectious and, prior to vaccination programmes, was the leading cause of infant mortality. The WHO estimated that over 600?000 deaths are prevented annually by pertussis vaccination, but B. pertussis infection was still responsible for over 63?000 deaths globally in 2013. The re-emergence of B. pertussis has been linked to strains with inactive or absent major virulence factors included in vaccines such as pertactin, pertussis toxin and filamentous haemagglutinin. Thus, the molecular surveillance of currently circulating strains is critical in understanding and controlling B. pertussis. Such information provides data on strains to inform control measures and the identification of future vaccine antigens. Current surveillance and typing methods for B. pertussis rely on the availability of clinical isolates. However, since the 1990s, the majority of pertussis cases have been diagnosed by PCR, where an isolate is not needed. The rapid decline in the availability of B. pertussis isolates impacts our ability to monitor this infection. The growing uptake of next-generation sequencing (NGS) has offered the opportunity for culture-independent genome sequencing and typing of this fastidious pathogen. Therefore, the objective of the study was to optimize respiratory sample preparation, independent of culture, in order to type B. pertussis using NGS. The study compared commercial depletion kits and specimen-processing methods using selective lysis detergents. The goal was to deplete human DNA, the major obstacle for sequencing a pathogen directly from a clinical sample. Samples spiked with a clinically relevant amount of B. pertussis were used to provide comparison between the different methods. Commercial depletion kits including the MolYsis, Qiagen Microbiome and NEBNext Kits were tested. Previously published methods, for Saponin and TritonX-100, were also trialled as a depletion. The ratio of B. pertussis to human DNA was determined by real-time PCR for ERV3 and IS481 (as markers of human and B. pertussis DNA, respectively), then samples were sequenced using the Illumina NextSeq 500 platform. The number of human and B. pertussis sequenced reads were then compared between treatments. The results showed that commercial kits reduced the human DNA present, but also reduced the concentration of target B. pertussis. However, selective lysis with Saponin treatment resulted in almost undetectable levels of human DNA, with minimal loss of target B. pertussis DNA. Sequencing read depth improved five-fold in reads to B. pertussis. Our investigation delivered a potential protocol that will enable the public health laboratory surveillance of B. pertussis in the era of culture-independent testing.

Dataset Information

Resolving the complex Bordetella pertussis genome using barcoded nanopore sequencing.

Publications

Resolving the complex Bordetella pertussis genome using barcoded nanopore sequencing.

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