Pius Brzoska

Prospective Genomic Characterization of the German Enterohemorrhagic Escherichia coli O104:H4 Outbreak by Rapid Next Generation Sequencing Technology

An ongoing outbreak of exceptionally virulent Shiga toxin (Stx)-producing Escherichia coli O104:H4 centered in Germany, has caused over 830 cases of hemolytic uremic syndrome (HUS) and 46 deaths since May 2011. Serotype O104:H4, which has not been detected in animals, has rarely been associated with HUS in the past. To prospectively elucidate the unique characteristics of this strain in the early stages of this outbreak, we applied whole genome sequencing on the Life Technologies Ion Torrent PGM™ sequencer and Optical Mapping to characterize one outbreak isolate (LB226692) and a historic O104:H4 HUS isolate from 2001 (01-09591). Reference guided draft assemblies of both strains were completed with the newly introduced PGM™ within 62 hours. The HUS-associated strains both carried genes typically found in two types of pathogenic E. coli, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). Phylogenetic analyses of 1,144 core E. coli genes indicate that the HUS-causing O104:H4 strains and the previously published sequence of the EAEC strain 55989 show a close relationship but are only distantly related to common EHEC serotypes. Though closely related, the outbreak strain differs from the 2001 strain in plasmid content and fimbrial genes. We propose a model in which EAEC 55989 and EHEC O104:H4 strains evolved from a common EHEC O104:H4 progenitor, and suggest that by stepwise gain and loss of chromosomal and plasmid-encoded virulence factors, a highly pathogenic hybrid of EAEC and EHEC emerged as the current outbreak clone. In conclusion, rapid next-generation technologies facilitated prospective whole genome characterization in the early stages of an outbreak.

Tumor Transcriptome Sequencing Reveals Allelic Expression Imbalances Associated with Copy Number Alterations

Due to growing throughput and shrinking cost, massively parallel sequencing is rapidly becoming an attractive alternative to microarrays for the genome-wide study of gene expression and copy number alterations in primary tumors. The sequencing of transcripts (RNA-Seq) should offer several advantages over microarray-based methods, including the ability to detect somatic mutations and accurately measure allele-specific expression. To investigate these advantages we have applied a novel, strand-specific RNA-Seq method to tumors and matched normal tissue from three patients with oral squamous cell carcinomas. Additionally, to better understand the genomic determinants of the gene expression changes observed, we have sequenced the tumor and normal genomes of one of these patients. We demonstrate here that our RNA-Seq method accurately measures allelic imbalance and that measurement on the genome-wide scale yields novel insights into cancer etiology. As expected, the set of genes differentially expressed in the tumors is enriched for cell adhesion and differentiation functions, but, unexpectedly, the set of allelically imbalanced genes is also enriched for these same cancer-related functions. By comparing the transcriptomic perturbations observed in one patient to his underlying normal and tumor genomes, we find that allelic imbalance in the tumor is associated with copy number mutations and that copy number mutations are, in turn, strongly associated with changes in transcript abundance. These results support a model in which allele-specific deletions and duplications drive allele-specific changes in gene expression in the developing tumor.

Comparative Analysis of Genome Sequences Covering the Seven Cronobacter Species

Background Species of Cronobacter are widespread in the environment and are occasional food-borne pathogens associated with serious neonatal diseases, including bacteraemia, meningitis, and necrotising enterocolitis. The genus is composed of seven species: C. sakazakii, C. malonaticus, C. turicensis, C. dublinensis, C. muytjensii, C. universalis, and C. condimenti. Clinical cases are associated with three species, C. malonaticus, C. turicensis and, in particular, with C. sakazakii multilocus sequence type 4. Thus, it is plausible that virulence determinants have evolved in certain lineages. Methodology/Principal Findings We generated high quality sequence drafts for eleven Cronobacter genomes representing the seven Cronobacter species, including an ST4 strain of C. sakazakii. Comparative analysis of these genomes together with the two publicly available genomes revealed Cronobacter has over 6,000 genes in one or more strains and over 2,000 genes shared by all Cronobacter. Considerable variation in the presence of traits such as type six secretion systems, metal resistance (tellurite, copper and silver), and adhesins were found. C. sakazakii is unique in the Cronobacter genus in encoding genes enabling the utilization of exogenous sialic acid which may have clinical significance. The C. sakazakii ST4 strain 701 contained additional genes as compared to other C. sakazakii but none of them were known specific virulence-related genes. Conclusions/Significance Genome comparison revealed that pair-wise DNA sequence identity varies between 89 and 97% in the seven Cronobacter species, and also suggested various degrees of divergence. Sets of universal core genes and accessory genes unique to each strain were identified. These gene sequences can be used for designing genus/species specific detection assays. Genes encoding adhesins, T6SS, and metal resistance genes as well as prophages are found in only subsets of genomes and have contributed considerably to the variation of genomic content. Differences in gene content likely contribute to differences in the clinical and environmental distribution of species and sequence types.

Accurate, rapid and high-throughput detection of strain-specific polymorphisms in Bacillus anthracis and Yersinia pestis by next-generation sequencing

BackgroundIn the event of biocrimes or infectious disease outbreaks, high-resolution genetic characterization for identifying the agent and attributing it to a specific source can be crucial for an effective response. Until recently, in-depth genetic characterization required expensive and time-consuming Sanger sequencing of a few strains, followed by genotyping of a small number of marker loci in a panel of isolates at or by gel-based approaches such as pulsed field gel electrophoresis, which by necessity ignores most of the genome. Next-generation, massively parallel sequencing (MPS) technology (specifically the Applied Biosystems sequencing by oligonucleotide ligation and detection (SOLiD™) system) is a powerful investigative tool for rapid, cost-effective and parallel microbial whole-genome characterization.ResultsTo demonstrate the utility of MPS for whole-genome typing of monomorphic pathogens, four Bacillus anthracis and four Yersinia pestis strains were sequenced in parallel. Reads were aligned to complete reference genomes, and genomic variations were identified. Resequencing of the B. anthracis Ames ancestor strain detected no false-positive single-nucleotide polymorphisms (SNPs), and mapping of reads to the Sterne strain correctly identified 98% of the 133 SNPs that are not clustered or associated with repeats. Three geographically distinct B. anthracis strains from the A branch lineage were found to have between 352 and 471 SNPs each, relative to the Ames genome, and one strain harbored a genomic amplification. Sequencing of four Y. pestis strains from the Orientalis lineage identified between 20 and 54 SNPs per strain relative to the CO92 genome, with the single Bolivian isolate having approximately twice as many SNPs as the three more closely related North American strains. Coverage plotting also revealed a common deletion in two strains and an amplification in the Bolivian strain that appear to be due to insertion element-mediated recombination events. Most private SNPs (that is, a, variant found in only one strain in this set) selected for validation by Sanger sequencing were confirmed, although rare false-positive SNPs were associated with variable nucleotide tandem repeats.ConclusionsThe high-throughput, multiplexing capability, and accuracy of this system make it suitable for rapid whole-genome typing of microbial pathogens during a forensic or epidemiological investigation. By interrogating nearly every base of the genome, rare polymorphisms can be reliably discovered, thus facilitating high-resolution strain tracking and strengthening forensic attribution.

Dominance of multidrug resistant CC271 clones in macrolide-resistant streptococcus pneumoniae in Arizona

BackgroundRates of resistance to macrolide antibiotics in Streptococcus pneumoniae are rising around the world due to the spread of mobile genetic elements harboring mef(E) and erm(B) genes and post-vaccine clonal expansion of strains that carry them.ResultsCharacterization of 592 clinical isolates collected in Arizona over a 10 year period shows 23.6% are macrolide resistant. The largest portion of the macrolide-resistant population, 52%, is dual mef(E)/erm(B)-positive. All dual-positive isolates are multidrug-resistant clonal lineages of Taiwan19F-14, mostly multilocus sequence type 320, carrying the recently described transposon Tn2010. The remainder of the macrolide resistant S. pneumoniae collection includes 31% mef(E)-positive, and 9% erm(B)-positive strains.ConclusionsThe dual-positive, multidrug-resistant S. pneumoniae clones have likely expanded by switching to non-vaccine serotypes after the heptavalent pneumococcal conjugate vaccine release, and their success limits therapy options. This upsurge could have a considerable clinical impact in Arizona.

Design and validation of a novel multiplex real-time PCR assay for Vibrio pathogen detection.

Three species--Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus--account for the majority of vibrio infections in humans. Rapid and accurate identification of Vibrio species has been problematic because phenotypic characteristics are variable within species. Additionally, biochemical identification and confirmation require 2 or more days to complete. Rapid and sensitive molecular techniques for the detection of vibrio pathogens would be useful for the surveillance and management of outbreaks. To facilitate the identification of human-pathogenic species, we designed and validated a highly sensitive, specific, and robust multiplex real-time PCR assay to identify V. cholerae, V. parahaemolyticus, and V. vulnificus using a four-dye configuration in a convenient lyophilized format. Multiple Vibrio strains were sequenced to verify candidate target TaqMan sites. Several individual assays within the multiplex contain multiple primers or probes to ensure detection of polymorphic variants. V. cholerae, V. parahaemolyticus, and V. vulnificus were detected either individually or in mixtures at ≤30 genomic copies. V. cholerae was specifically detected in the presence or absence of Vibrio mimicus. The Vibrio multiplex assay showed 100% specificity to all targets analyzed and no detection of nearest neighbor strains. Each assay exhibited 100% ± 10% efficiency. Multiplex real-time PCR can simplify pathogen detection and reduce costs per test since three species can be analyzed in a single reaction tube. Rapid and accurate detection of pathogenic vibrios in shellfish or seawater samples will improve the microbiological safety of seafood for consumers.

Molecular Technologies for Salmonella Detection

Salmonella has been associated with some of the most devastating foodborne outbreaks in recent history. Salmonella outbreaks have been linked to a variety of foods including produce [Alfalfa Sprouts2009, 2010, 2011; pistachios-2009; cantaloupes-2008, 2011 etc.], processed foods [peanuts – 2009], and prepared foods [turkey burgers2011, Banquet Pot Pies – 2007]. The contamination of commercial shell eggs with Salmonella Enteriditis in 2010 led to the recall of over a half a billion eggs, and the contamination of peanut-containing products with Salmonella Typhimurium in 2008-2009 led to one of the largest recalls in U.S. history with over 3,900 products being recalled. The Peanut Corporation of America, responsible for the Salmonella outbreak in peanuts, was forced into bankruptcy. Multiple lawsuits were filed against Wright County Egg and Hillandale Farms responsible for the Salmonella outbreak in eggs. Despite their own internal testing which showed Salmonella contamination, these facilities still shipped product. The 2008 outbreak of Salmonella in jalapeno peppers resulted in 1442 persons infected with Salmonella Saintpaul across 43 states, the District of Columbia, and Canada. Unfortunately, the tomato industry was implicated early in the investigation, which resulted in economic losses to the tomato industry in hundreds of millions of dollars. Because Salmonella is widespread in the environment (in such places as chicken houses), vegetable plants and animals (as well as meat samples, eggs etc.), rapid, reliable, and validated pathogen detection methods are needed for use in production facilities, public health labs, as well as in the regulatory and monitoring agencies. To provide comprehensive rapid food testing solutions, all components of a pathogen detection system should be addressed: sample preparation, detection and data analysis.

Abstract 4218: Validation of the Ion AmpliSeq™ Comprehensive Cancer Panel (CCP) using castPCR™ technologies.

Somatic mutation has been implicated in many aspects of cancer such as susceptibility, diagnosis, prognosis, drug response and tumor progress. Detection of somatic mutation is of wide interest in cancer research. The rapid advances in next generation sequencing (NGS) technologies have transformed cancer research. For example, the Ion AmpliSeq™ technology enables the selective amplification of 10s to 1000s of target sequences in a single multiplexed PCR and meshes seamlessly with the Ion semiconductor sequencing platform. The Ion Ampliseq™ Comprehensive Cancer Panel (CCP) provides ready-access to hundreds of genes, making it ideal for broad targeted re-sequencing studies. Alternative technologies are in demand to validate the NGS data orthogonally and screen hundreds or more cancer samples to evaluate mutation patterns, prevalence and frequencies in population. Here, we have demonstrated the utility of TaqMan® Mutation Detection Assays using our competitive allele specific TaqMan® PCR (castPCR™) technology in validation of Ion Torrent sequencing data. In this study, we applied the Ion Ampliseq™ Comprehensive Cancer Panel (CCP) panel to NCI-60 cell lines (MCF-7, MDA-MB-231, DU-145, PC-3, SK-MEL- 28) derived from breast, prostate, and skin cancers on Ion Torrent PGM sequencer. We confirmed previously reported mutations in these cell lines and identified the mutations that were not reported before, including missense and non-coding mutations. We then selected a subset of mutation targets from the cancer panel for castPCR validation including the genes of KRAS. EGFR, BRAF, NRAS, PIK3CA, PTEN, KIT, TP53, and more. Since limited sample quantity has been a challenging issue for most cancer researchers, especially for those who are interested in testing multi-targets by qPCR, we developed a preamplification method to enrich the targets of interest prior to running castPCR assays. We compared the Cq values of on-targets and off-targets from preamplified samples and non-preamplified samples, and the data indicates that our preamplification strategy not only provides roughly100 fold target enrichment but also maintains specificity during the preamplification process. For mutation detection, we showed that the mutation data from castPCR™ and from Ion Torrent PGM sequencer share high concordance for any given mutations. The detailed comparison and analysis will be presented and discussed. Our results demonstrate that castPCR™ technology is a valuable validation tool for next generation sequencing. The combination of Ion Torrent sequencing and castPCR validation empowers cancer researchers to understand the roles that somatic mutation plays in cancer. Citation Format: Kelly Li, Cora Woo, Mokang Mouanoutoua, Bonnie Moy, Emanuel Langit, Pius Brzoska, Xiaoqing You, Sejal Desai, David Keys, Junko Stevens, Benjamin Kong, Mark Shannon, Shiaw-Min Chen, David Ruff, Chieh-Yuan Li, David Joun, Iris Casuga, Robert Bennett. Validation of the Ion AmpliSeq™ Comprehensive Cancer Panel (CCP) using castPCR™ technologies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4218. doi:10.1158/1538-7445.AM2013-4218

Abstract 3071: Accurate and sensitive detection of KRAS mutations in heterogeneous cancer specimens

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The discovery of pivotal genetic alterations and the understanding of their role in cancer is leading to remarkable successes in therapeutics and patient care. Molecular diagnosis methods such as DNA sequencing and conventional genotyping of tumor biopsies have advanced research in this field, but are limited in sensitivity due to stromal contamination and by genetic heterogeneity in cancer. We have recently developed competitive allele specific TaqMan® PCR (castPCR) assays assays for detecting cancer-associated sequence variations. CastPCR not only maintains the wide dynamic range, high sensitivity and reproducibility of TaqMan® assays but also greatly improves the specificity. The technology enables detection, of as little as 1 mutant allele molecule in 10,000,000 wild type molecules. We report here sensitive and accurate detection of cancer-associated KRAS mutations within formalin-fixed paraffin-embedded (FFPE) heterogeneous cancer specimens. Eight FFPE model cell lines were initially used to validate the assays (NCI-H2009:p.G12A; SW1463:p.G12C; PANC-1:p.G12D; PSN-1: p.G12R; A549:p.G12S; SW480:p.G12V; DLD-1:p.G13D; Jurkat:Wild Type). Mutant FFPE cell line DNAs were titrated in the FFPE wild type cell line DNAs from 100% to 0.1%. Mutations were easily identified at the level of 0.1% with high reproducibility. 24 anonymous tumor tissues and 12 non-tumor tissues from FFPE specimens were also examined. No positive samples were found in non-tumor tissues. The results obtained by castPCR for the 24 tumor tissues were concordant to those previously reported by three different methods (Taqman® PCR, Taqman® PCR + PNA and Sequencing). Our results demonstrate that castPCR, as a new rare mutation detection technology, has greater sensitivity, specificity and can thereby facilitate accurate molecular diagnosis of heterogeneous cancer specimens and enable patient selection for targeted cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3071. doi:10.1158/1538-7445.AM2011-3071

Abstract 3066: Direct detection and enumeration of circulating tumor cells (CTCs) in blood by CastRT-qPCR

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Statistical data suggest that only approximately 25% of cancer patients will respond to the same treatment. Enumeration and molecular characterization of circulating tumor cells (CTCs) promise to be valuable for cancer cell diagnosis, survival prognosis, and treatment guidance. However, CTCs are very rare. There is no sensitive and specific assay available for analyzing CTCs from whole blood without extensive enrichment processes. Sample enrichment causes target cells loss, expensive and time-consuming. Here we report a highly specific competitive allele-specific TaqMan-based RT-qPCR (castRT-qPCR) for rare copies of DNA mutation detection. CastPCR can detect rare copies of mutant alleles for up to one in 10 million with a wide dynamic range of more than 6-log orders and <5 copy sensitivity. We have successfully designed and validated cast-PCR assays for more than 80 cancer-related SNPs of KRAS, EGFR, BRAF, KIT, pTEN, p53 and other oncogenes or tumor suppressor genes. CastRT-PCR was used to specifically detect and enumerate CTCs in blood samples with lung cancer cell lines spiking-in and lung cancer patient blood without prior biochemical enrichment processes. EGFR mutation (p.L858R) was detected in all 5 blood samples from late stage lung cancers. Approximately 50% of circulating lung tumor cells with positive EGFR p.L858R mutation had positive EGFR p.T790M mutation. In normal blood samples spiked in cell lines of known Kras and EGFR mutation at the level of 10 cells/mL of whole blood (close to actual patient samples), CastRT-PCR accurately detected the known mutations. Our data suggest that digital castRT-qPCR can be used to enumerate CTCs and detect cancer-related mutations in the blood, which paves the way for future individualized cancer treatment by monitoring CTC level and molecular characterization. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3066. doi:10.1158/1538-7445.AM2011-3066