<i>Bordetella pertussis</i>Strain Lacking Pertactin and Pertussis Toxin

Williams, Margaret M.; Sen, Kathryn; Weigand, Michael R.; Skoff, Tami H.; Cunningham, Victoria A.; Halse, Tanya A.; Tondella, M. Lucia

doi:10.3201/eid2202.151332

Cited by 63 publications

(61 citation statements)

References 15 publications

(25 reference statements)

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“…Variation in the initial DNA concentration of samples submitted for sequencing as well as internal WGS run conditions may lead to differential read depths. Optimizing sequencing conditions to yield maximum depth may overcome the issues with identification of prn alleles by SRST2, which is particularly important in the context of increasing numbers of isolates that do not express pertactin (21). Increasing the amount of DNA processed per library or running fewer libraries per flow cell may be helpful in this regard.…”

Section: Discussionmentioning

confidence: 99%

“…These large pools of molecular data linked to clinical and epidemiological information would bolster preliminary observations regarding vaccine effectiveness and disease severity in the context of emerging strains (13,14,21).…”

Section: Discussionmentioning

confidence: 99%

“…Mainly, assembly is hindered by the high GC content and numerous long repetitive insertion sequences. Resolution of these challenges traditionally used complex bioinformatics pipelines and hybrid assemblies with lower-throughput long-read sequencing technologies, which are not as amenable for routine laboratory-based surveillance of B. pertussis strains (10,21). To address this, we propose using partially processed raw short-read sequence data to obtain the aP antigen information described above.…”

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confidence: 99%

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Short-Read Whole-Genome Sequencing for Laboratory-Based Surveillance of Bordetella pertussis

et al. 2017

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Bordetella pertussis is a Gram-negative bacterium that causes respiratory infections in humans. Ongoing molecular surveillance of B. pertussis acellular vaccine (aP) antigens is critical for understanding the interaction between evolutionary pressures, disease pathogenesis, and vaccine effectiveness. Methods currently used to characterize aP components are relatively labor-intensive and low throughput. To address this challenge, we sought to derive aP antigen genotypes from minimally processed short-read whole-genome sequencing data generated from 40 clinical B. pertussis isolates and analyzed using the SRST2 bioinformatic package. SRST2 was able to identify aP antigen genotypes for all antigens with the exception of pertactin, possibly due to low read coverage in GC-rich low-complexity regions of variation. Two main genotypes were observed in addition to a singular third genotype that contained an 84-bp deletion that was identified by SRST2 despite the issues in allele calling. This method has the potential to generate large pools of B. pertussis molecular data that can be linked to clinical and epidemiological information to facilitate research of vaccine effectiveness and disease severity in the context of emerging vaccine antigen-deficient strains.KEYWORDS Bordetella pertussis, DNA sequencing, immunization, molecular methods, surveillance studies B ordetella pertussis is a Gram-negative aerobic bacterium that causes respiratory illness in humans and is commonly referred to as whooping cough. Vaccination is the primary recommended preventative public health intervention, and since the end of the 20th century, vaccination programs in most high-income countries have incorporated acellular component vaccines (aP) as replacements for whole-cell vaccines that have been used for over 60 years, largely due to an improved safety profile (1-5). The aP is the result of decades of research into the immunological properties of B. pertussis and its virulence factors using animal models and in vitro experiments to characterize B. pertussis's mode of infection and pathophysiological effects (6-8). In the context of large and frequent B. pertussis outbreaks and decreasing vaccine effectiveness since the introduction of the aP (albeit to various degrees in different regions), additional research has been conducted to examine how vaccination may be influencing the evolution of B. pertussis and the long-term impact on the effectiveness of B. pertussis vaccines (9-14).Allele typing using select virulence factors has been useful in describing changes across larger evolutionary scales and has been a key tool for associating changes in the B. pertussis population with the introduction of aPs (15-17). The virulence factor typing

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Short-Read Whole-Genome Sequencing for Laboratory-Based Surveillance of Bordetella pertussis

et al. 2017

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“…The isolates were previously characterized in-house using a multi-locus sequence typing (MLST) scheme that includes the pertussis toxin operon and its promoter ( ptxA and ptxP ) as two of its targets [18]. Three isolates, FR3749, I979, and J365, do not produce Pt due to a 28kb deletion that includes the loss of the entire ptx / ptl operon, as confirmed by sequencing and western blot analyses [15, 16, 19]. H921 and purified Pt (Protein Express, Ohio, USA) were included as positive controls.…”

Section: Methodsmentioning

confidence: 99%

Development of a qualitative assay for screening of Bordetella pertussis isolates for pertussis toxin production

et al. 2017

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Bordetella pertussis infection has been increasing in the US, with reported cases reaching over 50,000 in 2012, a number last observed in the 1950s. Concurrently, B. pertussis lacking the pertactin protein, one of the immunogens included in the acellular vaccine formulations, has rapidly emerged since 2010, and has become the predominant circulating phenotype. Monitoring the production of the remaining acellular vaccine immunogens, such as pertussis toxin (Pt), is a critical next step. To date, methods for screening Pt have been either through genomic sequencing means or by conventional ELISAs. However, sequencing limits detection to the DNA level, missing potential disruptions in transcription or translation. Conventional ELISAs are beneficial for detecting the protein; however, they can often suffer from poor sensitivity and specificity. Here we describe a rapid, highly sensitive and specific electrochemiluminescent capture ELISA that can detect Pt production in prepared inactivated bacterial suspensions. Over 340 isolates were analyzed and analytical validation parameters, such as precision, reproducibility, and stability, were rigorously tested. Intra-plate and inter-plate variability measured at 9.8% and 11.5%, respectively. Refrigerated samples remained stable for two months and variability was unaffected (coefficient of variation was 12%). Interestingly, despite the intention of being a qualitative method, the assay was sensitive enough to detect a small, but statistically significant, difference in protein production between different pertussis promoter allelic groups of strains, ptxP1 and ptxP3. This technology has the ability to perform screening of multiple antigens at one time, thus, improving testing characteristics while minimizing costs, specimen volume, and testing time.

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“…Moreover, as Booher et al (11) described, existing SMS assemblers also fail to assemble Xanthomonas genomes. The challenge of finishing draft genomes assembled from SMS reads extends beyond Xanthomonas genomes (e.g., many genomes sequenced at the Centers for Disease Control are being finished using optical mapping) (35).…”

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Assembly of long error-prone reads using de Bruijn graphs

Lin

Yuan

Kolmogorov

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

278

220

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The recent breakthroughs in assembling long error-prone reads were based on the overlap-layout-consensus (OLC) approach and did not utilize the strengths of the alternative de Bruijn graph approach to genome assembly. Moreover, these studies often assume that applications of the de Bruijn graph approach are limited to short and accurate reads and that the OLC approach is the only practical paradigm for assembling long error-prone reads. We show how to generalize de Bruijn graphs for assembling long error-prone reads and describe the ABruijn assembler, which combines the de Bruijn graph and the OLC approaches and results in accurate genome reconstructions.de Bruijn graph | genome assembly | single-molecule sequencing T he key challenge to the success of single-molecule sequencing (SMS) technologies lies in the development of algorithms for assembling genomes from long but inaccurate reads. The pioneer in long reads technologies, Pacific Biosciences, now produces accurate assemblies from long error-prone reads (1, 2). Goodwin et al. (3) and Loman et al. (4) demonstrated that high-quality assemblies can be obtained from even less-accurate Oxford Nanopore reads. Advances in assembly of long errorprone reads recently resulted in the accurate reconstructions of various genomes (5-10). However, as illustrated in Booher et al. (11), the problem of assembling long error-prone reads is far from being resolved even in the case of relatively small bacterial genomes.Previous studies of SMS assemblies were based on the overlaplayout-consensus (OLC) approach (12) or a similar string graph approach (13), which require an all-against-all comparison of reads (14) and remain computationally challenging (see refs. 15-17 for a discussion of the pros and cons of this approach). Moreover, there is an assumption that the de Bruijn graph approach, which has dominated genome assembly for the last decade, is inapplicable to long reads. This is a misunderstanding, because the de Bruijn graph approach, as well as its variation called the A-Bruijn graph approach, was developed to assemble rather long Sanger reads (18). There is also a misunderstanding that the de Bruijn graph approach can only assemble highly accurate reads and fails when assembling long error-prone reads. Although this is true for the original de Bruijn graph approach to assembly (15-17), the A-Bruijn graph approach was originally designed to assemble inaccurate reads as long as any similarities between reads can be reliably identified. Moreover, A-Bruijn graphs have proven to be useful even for assembling mass spectra, which represent highly inaccurate fingerprints of amino acid sequences of peptides (19,20). However, although A-Bruijn graphs have proven to be useful in assembling Sanger reads and mass spectra, the question of how to apply A-Bruijn graphs for assembling long error-prone reads remains open.de Bruijn graphs are a key algorithmic technique in genome assembly (15,(21)(22)(23)(24). In addition, de Bruijn graphs have been used for sequencing by hybridization (...

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Bordetella pertussisStrain Lacking Pertactin and Pertussis Toxin

Cited by 63 publications

References 15 publications

Short-Read Whole-Genome Sequencing for Laboratory-Based Surveillance of Bordetella pertussis

Short-Read Whole-Genome Sequencing for Laboratory-Based Surveillance of Bordetella pertussis

Development of a qualitative assay for screening of Bordetella pertussis isolates for pertussis toxin production

Assembly of long error-prone reads using de Bruijn graphs

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