Analysis of Pilin Antigenic Variation inNeisseria meningitidisby Next-Generation Sequencing

ABSTRACTMany pathogenic microbes evade host immune surveillance by varying the surface antigens, a process termed antigenic variation. While the process of pilin antigenic variation has been extensively studied in the human pathogenNeisseria gonorrhoeae(gonococcus [Gc]), relatively few studies of pilin antigenic variation have been conducted withNeisseria meningitidis(meningococcus [Mc]). Mc is usually a commensal organism that colonizes the human nasopharynx, but when it translocates to the bloodstream or meninges, it results in the severe and often deadly meningococcal disease. The type IV pili of Mc isolates play a critical role in host surface adherence, and its major pilin component (PilE) can undergo antigenic variation. In this study, Roche 454 pyrosequencing was used to examine the pilin antigenic variation of Mc strain 8013, as well as 8013recA,recX,recQ,rep, andrecJmutants, Gc orthologues which have been shown to play a role in pilin antigenic variation. This study confirms that the McrecA,rep, andrecJgenes are essential for pilin antigenic variation. While the McrecQandrecXgene products contribute to normal frequencies of antigenic variation, the loss of these factors does not alter the types of pilin variants produced. Overall, this study shows that the mechanisms of pilin antigenic variation are conserved between Gc and Mc.IMPORTANCEAntigenic variation is a strategy used by many pathogens to escape host immune surveillance and establish persistent infections. This study successfully applies next-generation sequencing to study pilin antigenic variation in the human pathogenNeisseria meningitidis. This assay provides an affordable and efficient solution for quantifying antigenic variation frequency in mutant strains and for defining the recombination products of the process. We determined that there is a nonuniformity of silent donor copies used during meningococcus antigenic variation, and by the analysis of selected mutants deficient for specific recombination pathways, we show for the first time that the processes are conserved betweenN. meningitidisandNeisseria gonorrhoeae.

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Draft Genome Sequence of Saccharomyces cerevisiae Strain Awamori Number 101, Commonly Used to Make Awamori, a Traditional Spirit, in Okinawa, Japan

Microbiology Resource Announcements ◽

10.1128/mra.01414-20 ◽

2021 ◽

Vol 10 (25) ◽

Author(s):

Masatoshi Tsukahara ◽

Kotaro Ise ◽

Maiko Nezuo ◽

Haruna Azuma ◽

Takeshi Akao ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Next Generation Sequencing ◽

Genome Sequence ◽

Draft Genome ◽

Draft Genome Sequence ◽

Next Generation ◽

Industrial Strain ◽

Content Type ◽

Short Reads ◽

Generation Sequencing

We report here the draft genome sequence for Saccharomyces cerevisiae strain Awamori number 101, an industrial strain used for producing awamori, a distilled alcohol beverage. It was constructed by assembling the short reads obtained by next-generation sequencing. The 315 contigs constitute an 11.5-Mbp genome sequence coding 6,185 predicted proteins.

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Next-Generation Sequencing and Bioinformatics Protocol for Malaria Drug Resistance Marker Surveillance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02474-17 ◽

2018 ◽

Vol 62 (4) ◽

pp. e02474-17 ◽

Cited By ~ 20

Author(s):

Eldin Talundzic ◽

Shashidhar Ravishankar ◽

Julia Kelley ◽

Dhruviben Patel ◽

Mateusz Plucinski ◽

...

Keyword(s):

Drug Resistance ◽

Next Generation Sequencing ◽

Deep Sequencing ◽

Artemisinin Resistance ◽

The United States ◽

Imported Malaria ◽

Chloroquine Resistance ◽

Next Generation ◽

Content Type ◽

Generation Sequencing

ABSTRACT The recent advances in next-generation sequencing technologies provide a new and effective way of tracking malaria drug-resistant parasites. To take advantage of this technology, an end-to-end Illumina targeted amplicon deep sequencing (TADS) and bioinformatics pipeline for molecular surveillance of drug resistance in P. falciparum, called malaria resistance surveillance (MaRS), was developed. TADS relies on PCR enriching genomic regions, specifically target genes of interest, prior to deep sequencing. MaRS enables researchers to simultaneously collect data on allele frequencies of multiple full-length P. falciparum drug resistance genes (crt, mdr1, k13, dhfr, dhps, and the cytochrome b gene), as well as the mitochondrial genome. Information is captured at the individual patient level for both known and potential new single nucleotide polymorphisms associated with drug resistance. The MaRS pipeline was validated using 245 imported malaria cases that were reported to the Centers for Disease Control and Prevention (CDC). The chloroquine resistance crt CVIET genotype (mutations underlined) was observed in 42% of samples, the highly pyrimethamine-resistant dhps IRN triple mutant in 92% of samples, and the sulfadoxine resistance dhps mutation SGEAA in 26% of samples. The mdr1 NFSND genotype was found in 40% of samples. With the exception of two cases imported from Cambodia, no artemisinin resistance k13 alleles were identified, and 99% of patients carried parasites susceptible to atovaquone-proguanil. Our goal is to implement MaRS at the CDC for routine surveillance of imported malaria cases in the United States and to aid in the adoption of this system at participating state public health laboratories, as well as by global partners.

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Bloody coli: a Gene Cocktail in Escherichia coli O104:H4

mBio ◽

10.1128/mbio.00066-13 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 13

Author(s):

Fernando Baquero ◽

Raquel Tobes

Keyword(s):

Escherichia Coli ◽

Next Generation Sequencing ◽

Genetic Material ◽

Next Generation ◽

Content Type ◽

Pathogenicity Genes ◽

Sequencing Technologies ◽

Population Sizes ◽

Sequential Acquisition ◽

Generation Sequencing

ABSTRACT A recent study published in mBio [Y. H. Grad et al., mBio 4(1):e00452-12, 2013] indicates that a rapid introgressive evolution has occurred in Escherichia coli O104:H4 by sequential acquisition of foreign genetic material involving pathogenicity traits. O104 genetic promiscuity cannot be readily explained by high population sizes. However, extensive interactions leading to cumulative assemblies of pathogenicity genes might be assured by small K-strategist populations exploiting particular intestinal niches. Next-generation sequencing technologies will be critical to detect particular “gene cocktails” as potentially pathogenic ensembles and to predict the risk of future outbreaks.

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Improved Detection by Next-Generation Sequencing of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates

Journal of Clinical Microbiology ◽

10.1128/jcm.01179-15 ◽

2015 ◽

Vol 53 (12) ◽

pp. 3779-3783 ◽

Cited By ~ 16

Author(s):

Nontuthuko E. Maningi ◽

Luke T. Daum ◽

John D. Rodriguez ◽

Matsie Mphahlele ◽

Remco P. H. Peters ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Next Generation Sequencing ◽

Sanger Sequencing ◽

Detection Sensitivity ◽

Next Generation ◽

Resistance Mutations ◽

Coding Region ◽

Content Type ◽

Mdr Tb ◽

Generation Sequencing

The technical limitations of common tests used for detecting pyrazinamide (PZA) resistance inMycobacterium tuberculosisisolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragment (comprising thepncAcoding region plus the promoter) was sequenced using Ion Torrent next-generation sequencing (NGS) to detect associated PZA resistance mutations in 88 recultured MDR-TB isolates from an archived series collected in 2001. These 88 isolates were previously Sanger sequenced, with 55 (61%) designated as carrying the wild-typepncAgene and 33 (37%) showing mutations. PZA susceptibility of the isolates was also determined using the Bactec 460 TB system and the Wayne test. In this study, isolates were recultured and susceptibility testing was performed in Bactec 960 MGIT. Concordance between NGS and MGIT results was 93% (n= 88), and concordance values between the Bactec 460, the Wayne test, orpncAgene Sanger sequencing and NGS results were 82% (n= 88), 83% (n= 88), and 89% (n= 88), respectively. NGS confirmed the majority ofpncAmutations detected by Sanger sequencing but revealed several new and mixed-strain mutations that resolved discordancy in other phenotypic results. Importantly, in 53% (18/34) of these isolates,pncAmutations were located in the 151 to 360 region and warrant further exploration. In these isolates, with their known resistance to rifampin, NGS ofpncAimproved PZA resistance detection sensitivity to 97% and specificity to 94% using NGS as the gold standard and helped to resolve discordant results from conventional methodologies.

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Propionibacterium acnes: Disease-Causing Agent or Common Contaminant? Detection in Diverse Patient Samples by Next-Generation Sequencing

Journal of Clinical Microbiology ◽

10.1128/jcm.02723-15 ◽

2016 ◽

Vol 54 (4) ◽

pp. 980-987 ◽

Cited By ~ 50

Author(s):

Sarah Mollerup ◽

Jens Friis-Nielsen ◽

Lasse Vinner ◽

Thomas Arn Hansen ◽

Stine Raith Richter ◽

...

Keyword(s):

Next Generation Sequencing ◽

Propionibacterium Acnes ◽

Opportunistic Pathogen ◽

Next Generation Sequencing Data ◽

Clinical Samples ◽

Next Generation ◽

Sequencing Data ◽

Tissue Samples ◽

Content Type ◽

Generation Sequencing

Propionibacterium acnesis the most abundant bacterium on human skin, particularly in sebaceous areas.P. acnesis suggested to be an opportunistic pathogen involved in the development of diverse medical conditions but is also a proven contaminant of human clinical samples and surgical wounds. Its significance as a pathogen is consequently a matter of debate. In the present study, we investigated the presence ofP. acnesDNA in 250 next-generation sequencing data sets generated from 180 samples of 20 different sample types, mostly of cancerous origin. The samples were subjected to either microbial enrichment, involving nuclease treatment to reduce the amount of host nucleic acids, or shotgun sequencing. We detected high proportions ofP. acnesDNA in enriched samples, particularly skin tissue-derived and other tissue samples, with the levels being higher in enriched samples than in shotgun-sequenced samples.P. acnesreads were detected in most samples analyzed, though the proportions in most shotgun-sequenced samples were low. Our results show thatP. acnescan be detected in practically all sample types when molecular methods, such as next-generation sequencing, are employed. The possibility of contamination from the patient or other sources, including laboratory reagents or environment, should therefore always be considered carefully whenP. acnesis detected in clinical samples. We advocate that detection ofP. acnesalways be accompanied by experiments validating the association between this bacterium and any clinical condition.

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Arenavirus Coinfections Are Common in Snakes with Boid Inclusion Body Disease

Journal of Virology ◽

10.1128/jvi.01112-15 ◽

2015 ◽

Vol 89 (16) ◽

pp. 8657-8660 ◽

Cited By ~ 33

Author(s):

J. Hepojoki ◽

P. Salmenperä ◽

T. Sironen ◽

U. Hetzel ◽

Y. Korzyukov ◽

...

Keyword(s):

Next Generation Sequencing ◽

Inclusion Body ◽

New Genus ◽

De Novo ◽

Sequence Assembly ◽

Next Generation ◽

Content Type ◽

The Family ◽

Inclusion Body Disease ◽

Generation Sequencing

Recently, novel arenaviruses were found in snakes with boid inclusion body disease (BIBD); these form the new genusReptarenaviruswithin the familyArenaviridae. We used next-generation sequencing andde novosequence assembly to investigate reptarenavirus isolates from our previous study. Four of the six isolates and all of the samples from snakes with BIBD contained at least two reptarenavirus species. The viruses sequenced comprise four novel reptarenavirus species and a representative of a new arenavirus genus.

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Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis of the Penicillium chrysogenum Velvet Protein PcVelA Identifies Methyltransferase PcLlmA as a Novel Downstream Regulator of Fungal Development

mSphere ◽

10.1128/msphere.00149-16 ◽

2016 ◽

Vol 1 (4) ◽

Cited By ~ 7

Author(s):

Kordula Becker ◽

Sandra Ziemons ◽

Katharina Lentz ◽

Michael Freitag ◽

Ulrich Kück

Keyword(s):

Next Generation Sequencing ◽

Secondary Metabolism ◽

Filamentous Fungi ◽

Chromatin Immunoprecipitation ◽

Next Generation ◽

Content Type ◽

Major Interest ◽

Lactam Antibiotic ◽

Velvet Complex ◽

Generation Sequencing

ABSTRACT Filamentous fungi are of major interest for biotechnological and pharmaceutical applications. This is due mainly to their ability to produce a wide variety of secondary metabolites, many of which are relevant as antibiotics. One of the most prominent examples is penicillin, a β-lactam antibiotic that is produced on the industrial scale by fermentation of P. chrysogenum. In recent years, the multisubunit protein complex velvet has been identified as one of the key regulators of fungal secondary metabolism and development. However, until recently, only a little has been known about how velvet mediates regulation at the molecular level. To address this issue, we performed ChIP-seq (chromatin immunoprecipitation in combination with next-generation sequencing) on and follow-up analysis of PcVelA, the core component of the velvet complex in P. chrysogenum. We demonstrate direct involvement of velvet in transcriptional control and present the putative methyltransferase PcLlmA as a new downstream factor and interaction partner of PcVelA. Penicillium chrysogenum is the sole industrial producer of the β-lactam antibiotic penicillin, which is the most commonly used drug for treating bacterial infections. In P. chrysogenum and other filamentous fungi, secondary metabolism and morphogenesis are controlled by the highly conserved multisubunit velvet complex. Here we present the first chromatin immunoprecipitation next-generation sequencing (ChIP-seq) analysis of a fungal velvet protein, providing experimental evidence that a velvet homologue in P. chrysogenum (PcVelA) acts as a direct transcriptional regulator at the DNA level in addition to functioning as a regulator at the protein level in P. chrysogenum, which was previously described. We identified many target genes that are related to processes known to be dependent on PcVelA, e.g., secondary metabolism as well as asexual and sexual development. We also identified seven PcVelA target genes that encode putative methyltransferases. Yeast two-hybrid and bimolecular fluorescence complementation analyses showed that one of the putative methyltransferases, PcLlmA, directly interacts with PcVelA. Furthermore, functional characterization of PcLlmA demonstrated that this protein is involved in the regulation of conidiosporogenesis, pellet formation, and hyphal morphology, all traits with major biotechnological relevance. IMPORTANCE Filamentous fungi are of major interest for biotechnological and pharmaceutical applications. This is due mainly to their ability to produce a wide variety of secondary metabolites, many of which are relevant as antibiotics. One of the most prominent examples is penicillin, a β-lactam antibiotic that is produced on the industrial scale by fermentation of P. chrysogenum. In recent years, the multisubunit protein complex velvet has been identified as one of the key regulators of fungal secondary metabolism and development. However, until recently, only a little has been known about how velvet mediates regulation at the molecular level. To address this issue, we performed ChIP-seq (chromatin immunoprecipitation in combination with next-generation sequencing) on and follow-up analysis of PcVelA, the core component of the velvet complex in P. chrysogenum. We demonstrate direct involvement of velvet in transcriptional control and present the putative methyltransferase PcLlmA as a new downstream factor and interaction partner of PcVelA.

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Next-Generation Sequencing Confirms Presumed Nosocomial Transmission of Livestock-Associated Methicillin-Resistant Staphylococcus aureus in the Netherlands

Applied and Environmental Microbiology ◽

10.1128/aem.00773-16 ◽

2016 ◽

Vol 82 (14) ◽

pp. 4081-4089 ◽

Cited By ~ 17

Author(s):

Thijs Bosch ◽

Sandra Witteveen ◽

Anja Haenen ◽

Fabian Landman ◽

Leo M. Schouls

Keyword(s):

Health Care ◽

Staphylococcus Aureus ◽

Next Generation Sequencing ◽

The Netherlands ◽

Next Generation ◽

Methicillin Resistant ◽

Content Type ◽

Dutch Health ◽

Dutch Health Care ◽

Generation Sequencing

ABSTRACTLivestock-associated methicillin-resistantStaphylococcus aureus(LA-MRSA) was detected in 2003 and rapidly became the predominant MRSA clade in the Netherlands. Studies have shown that transmissions are difficult to identify, since this MRSA variant represents a genetically homogenous clade when current typing techniques are used. Here, next-generation sequencing was performed on 206 LA-MRSA isolates to assess the capability of LA-MRSA to be transmitted between humans. The usefulness of single nucleotide variants (SNVs), the composition of the SCCmecregion, and the presence of plasmids to identify transmission of LA-MRSA were assessed. In total, 30 presumed putative nosocomial transmission events and 2 LA-MRSA outbreaks were studied; in most cases, SNV analysis revealed that the isolates of the index patient and the contact(s) clustered closely together. In three presumed events, the isolates did not cluster together, indicating that transmission was unlikely. The composition of the SCCmecregion corroborated these findings. However, plasmid identification did not support our SNV analysis, since different plasmids were present in several cases where SNV and SCCmecanalysis suggested that transmission was likely. Next-generation sequencing shows that transmission of LA-MRSA does occur in Dutch health care settings. Transmission was identified based on SNV analysis combined with epidemiological data and in the context of epidemiologically related and unrelated isolates. Analysis of the SCCmecregion provided limited, albeit useful, information to corroborate conclusions on transmissions, but plasmid identification did not.IMPORTANCEIn 2003, a variant of methicillin-resistantStaphylococcus aureus(MRSA) isolated from pigs was also found in pig farmers in France and the Netherlands. Soon thereafter, this livestock-associated MRSA (LA-MRSA) was identified in many other countries. Transmission of LA-MRSA between humans, particularly in the health care setting, is regarded to occur sporadically. Moreover, studies that describe LA-MRSA transmission used molecular characterization of isolates with limited discriminatory power, making the validity of the conclusion that transmission occurred questionable. In our study, we sequenced the complete genomes of 206 LA-MRSA isolates, obtained from more than 30 presumed LA-MRSA transmission events. Analysis of the data showed that transmission of LA-MRSA between humans had indeed occurred in more than 90% of these events. We conclude that transmission of LA-MRSA between humans does occur in Dutch health care settings; therefore, a decision to discontinue the search and destroy policy for LA-MRSA should be taken with caution.

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Rapid Quantification of Mutant Fitness in Diverse Bacteria by Sequencing Randomly Bar-Coded Transposons

mBio ◽

10.1128/mbio.00306-15 ◽

2015 ◽

Vol 6 (3) ◽

Cited By ~ 167

Author(s):

Kelly M. Wetmore ◽

Morgan N. Price ◽

Robert J. Waters ◽

Jacob S. Lamson ◽

Jennifer He ◽

...

Keyword(s):

Next Generation Sequencing ◽

Biological Significance ◽

Transposon Mutagenesis ◽

Next Generation ◽

Experimental Conditions ◽

Content Type ◽

Bar Code ◽

Genome Wide ◽

Powerful Approach ◽

Generation Sequencing

ABSTRACT Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with any transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative d-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. IMPORTANCE A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are too laborious to be applied to hundreds of experimental conditions across multiple bacteria. Here, we describe an approach, random bar code transposon-site sequencing (RB-TnSeq), which greatly simplifies the measurement of gene fitness by using bar code sequencing (BarSeq) to monitor the abundance of mutants. We performed 387 genome-wide fitness assays across five bacteria and identified phenotypes for over 5,000 genes. RB-TnSeq can be applied to diverse bacteria and is a powerful tool to annotate uncharacterized genes using phenotype data.

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Next-Generation Sequencing of Haematococcus lacustris Reveals an Extremely Large 1.35-Megabase Chloroplast Genome

Genome Announcements ◽

10.1128/genomea.00181-18 ◽

2018 ◽

Vol 6 (12) ◽

Cited By ~ 16

Author(s):

Nicholas Bauman ◽

Srividya Akella ◽

Elizabeth Hann ◽

Robert Morey ◽

Ariel S. Schwartz ◽

...

Keyword(s):

Next Generation Sequencing ◽

Chloroplast Genome ◽

Next Generation ◽

Content Type ◽

Haematococcus Lacustris ◽

Long Read ◽

Generation Sequencing

ABSTRACT Haematococcus lacustris is an industrially relevant microalga that is used for the production of the carotenoid astaxanthin. Here, we report the use of PacBio long-read sequencing to assemble the chloroplast genome of H. lacustris strain UTEX:2505. At 1.35 Mb, this is the largest assembled chloroplast of any plant or alga known to date.

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