Application Of Whole Genome Sequencing Research Articles

Risks for public health related to the presence of Bacillus cereus and other Bacillus spp. including Bacillus thuringiensis in foodstuffs

The Bacillus cereus group, also known as B. cereus sensu lato, is a subdivision of the Bacillus genus that consists of eight formally recognised species: B. cereus sensu stricto, B. anthracis, B. thuringiensis, B. weihenstephanensis, B. mycoides, B. pseudomycoides, B. cytotoxicus and B. toyonensis. The current taxonomy of the B. cereus group and the status of separate species mainly rely on phenotypic characteristics. Bacillus thuringiensis strains display a similar repertoire of the potential virulence genes on the chromosome as B. cereus sensu stricto strains and it has been shown that these genes can also be actively expressed in B. thuringiensis strains. Bacillus cereus and B. thuringiensis strains are usually not discriminated in clinical diagnostics or food microbiology. Thus, the actual contribution of the two species to gastrointestinal and non-gastrointestinal diseases is currently unknown. Most cases of food-borne outbreaks caused by the B. cereus group have been associated with concentrations above 105 CFU/g. However, cases of both emetic and diarrhoeal illness have been reported involving lower levels of B. cereus. The levels of B. cereus that can be considered as a risk for consumers are also valid for B. thuringiensis. There is no evidence that B. thuringiensis has the genetic determinants for the emetic toxin cereulide. The Panel has recommended the application of whole genome sequencing to provide unambiguous identification of strains used as biopesticides and the detailed characterisation of outbreak strains allowing discrimination of B. thuringiensis from B. cereus. Data gaps include: dose–response and behavioural characteristics of B. cereus group strains and specifically of B. thuringiensis. Field studies after application of B. thuringiensis biopesticides are needed to enable the establishment of pre-harvest intervals.

Whole genome sequencing as a diagnostic tool: Participant-centered consent

Summary New developments in genetics and genomics offer a constant challenge to ethical reflection. One such development is the application of whole genome sequencing (WGS) as a diagnostic tool. The use of WGS in clinical practice carries with it the promise of personalized medicine, which will likely enable more efficient care and tailored prevention. However, the complexity and sheer amount of information involved may have significant implications for the patient-doctor relationship. The roles and expectations within this relationship are affected by both the institutional dimension of the logistics of handling genomic data and the changing possibilities of returning unsolicited or new information. As the relationship between doctor and patient changes under the influence of genomic data, the question emerges how the patient's rights may be safeguarded when the role and responsibility of the clinician shift. I argue that this question necessitates a rethinking of the authority of the patient in handling the flow of information in the clinical context. Specifically, I argue that the authority of the patient in personalized medicine should be shaped from a participant-centered perspective.

Whole genome sequencing and its applications in medical genetics

Fundamental improvement was made for genome sequencing since the next‐generation sequencing (NGS) came out in the 2000s. The newer technologies make use of the power of massively‐parallel short‐read DNA sequencing, genome alignment and assembly methods to digitally and rapidly search the genomes on a revolutionary scale, which enable large‐scale whole genome sequencing (WGS) accessible and practical for researchers. Nowadays, whole genome sequencing is more and more prevalent in detecting the genetics of diseases, studying causative relations with cancers, making genome‐level comparative analysis, reconstruction of human population history, and giving clinical implications and instructions. In this review, we first give a typical pipeline of whole genome sequencing, including the lab template preparation, sequencing, genome assembling and quality control, variants calling and annotations. We compare the difference between whole genome and whole exome sequencing (WES), and explore a wide range of applications of whole genome sequencing for both mendelian diseases and complex diseases in medical genetics. We highlight the impact of whole genome sequencing in cancer studies, regulatory variant analysis, predictive medicine and precision medicine, as well as discuss the challenges of the whole genome sequencing.

Real time application of whole genome sequencing for outbreak investigation – What is an achievable turnaround time?

Whole genome sequencing (WGS) is increasingly employed in clinical settings, though few assessments of turnaround times (TAT) have been performed in real-time. In this study, WGS was used to investigate an unfolding outbreak of vancomycin resistant Enterococcus faecium (VRE) among 3 patients in the ICU of a tertiary care hospital. Including overnight culturing, a TAT of just 48.5 h for a comprehensive report was achievable using an Illumina Miseq benchtop sequencer. WGS revealed that isolates from patient 2 and 3 differed from that of patient 1 by a single nucleotide polymorphism (SNP), indicating nosocomial transmission. However, the unparalleled resolution provided by WGS suggested that nosocomial transmission involved two separate events from patient 1 to patient 2 and 3, and not a linear transmission suspected by the time line. Rapid TAT’s are achievable using WGS in the clinical setting and can provide an unprecedented level of resolution for outbreak investigations.

Novel bioinformatic developments for exome sequencing.

With the widespread adoption of next generation sequencing technologies by the genetics community and the rapid decrease in costs per base, exome sequencing has become a standard within the repertoire of genetic experiments for both research and diagnostics. Although bioinformatics now offers standard solutions for the analysis of exome sequencing data, many challenges still remain; especially the increasing scale at which exome data are now being generated has given rise to novel challenges in how to efficiently store, analyze and interpret exome data of this magnitude. In this review we discuss some of the recent developments in bioinformatics for exome sequencing and the directions that this is taking us to. With these developments, exome sequencing is paving the way for the next big challenge, the application of whole genome sequencing.

The application and research progress of whole-genome sequencing in clinical microbiology detection

Whole-genome sequencing of bacteria has recently emerged as a cost-effective and convenient approach for resolving many microbiological questions. Here, the current status of whole-genome sequencing in clinical microbiology and the main problems that exist were reviewed, focusing on six essential tasks: identifying and culturing the species of an isolate, rapid identification of pathogen properties, monitoring the emergence and spread of an epidemic outbreak, developing vaccine and monitoring its variation, metagenomics and single-cell sequencing. The authors predicted that the application of whole-genome sequencing will soon be sufficiently fast, accurate and cheap to be used in routine etiological detection, even though there is still a long way to go.(Chin J Lab Med, 2016, 39: 319-321) Key words: Microbiological techniques; Genomics; Sequence analysis; Metagenomics; Single-cell analysis

Sarcococca Blight: Use of Whole-Genome Sequencing for Fungal Plant Disease Diagnosis.

Early and accurate diagnosis of new plant pathogens is vital for the rapid implementation of effective mitigation strategies and appropriate regulatory responses. Most commonly, pathogen identification relies on morphology and DNA marker analysis. However, for new diseases, these approaches may not be sufficient for precise diagnosis. In this study, we used whole-genome sequencing (WGS) to identify the causal agent of a new disease affecting Sarcococca hookeriana (sarcococca). Blight symptoms were observed on sarcococca and adjacent Buxus sempervirens (boxwood) plants in Maryland during 2014. Symptoms on sarcococca were novel, and included twig dieback and dark lesions on leaves and stems. A Calonectria sp. was isolated from both hosts and used to fulfill Koch's postulates but morphology and marker sequence data precluded species-level identification. A 51.4-Mb WGS was generated for the two isolates and identified both as Calonectria pseudonaviculata. A single-nucleotide polymorphism at a noncoding site differentiated between the two host isolates. These results indicate that the same C. pseudonaviculata genotype has the ability to induce disease on both plant species. This study marks the first application of WGS for fungal plant pathogen diagnosis and demonstrates the power of this approach to rapidly identify causal agents of new diseases.

Genomic Epidemiology: Whole-Genome-Sequencing-Powered Surveillance and Outbreak Investigation of Foodborne Bacterial Pathogens.

As we are approaching the twentieth anniversary of PulseNet, a network of public health and regulatory laboratories that has changed the landscape of foodborne illness surveillance through molecular subtyping, public health microbiology is undergoing another transformation brought about by so-called next-generation sequencing (NGS) technologies that have made whole-genome sequencing (WGS) of foodborne bacterial pathogens a realistic and superior alternative to traditional subtyping methods. Routine, real-time, and widespread application of WGS in food safety and public health is on the horizon. Technological, operational, and policy challenges are still present and being addressed by an international and multidisciplinary community of researchers, public health practitioners, and other stakeholders.

The implications of whole-genome sequencing in the control of tuberculosis.

The availability of whole-genome sequencing (WGS) as a tool for the diagnosis and clinical management of tuberculosis (TB) offers considerable promise in the fight against this stubborn epidemic. However, like other new technologies, the best application of WGS remains to be determined, for both conceptual and technical reasons. In this review, we consider the potential value of WGS in the clinical laboratory for the detection of Mycobacterium tuberculosis and the prediction of antibiotic resistance. We also discuss issues pertaining to data generation, interpretation and dissemination, given that WGS has to date been generally performed in research labs where results are not necessarily packaged in a clinician-friendly format. Although WGS is far more accessible now than it was in the past, the transition from a research tool to study TB into a clinical test to manage this disease may require further fine-tuning. Improvements will likely come through iterative efforts that involve both the laboratories ready to move TB into the genomic era and the front-line clinical/public health staff who will be interpreting the results to inform management decisions.

Whole Exome Sequencing Identifies Novel MPL and JAK2 M utations in Triple Negative Myeloproliferative Neoplasms

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic myeloproliferative neoplasms (MPN) characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. Most of the cases are sporadic and driven by somatic mutations, although familial clustering is observed. The most common mutation affecting 50-60% of the cases is JAK2-V617F, while 25-30% of the patients carry somatic mutations in exon 9 of CALR. MPL exon 10 mutations affect ~5% of the cases. JAK2, CALR and MPL mutations are mutually exclusive and account for >90% of ET and PMF cases. In 12% of ET and 5% of PMF cases the disease drivers remain unknown. These patients are termed as triple negative. The mutational analysis for diagnostic purposes is limited to exons 14 of JAK2, exon 10 of MPL and exon 9 of CALR. The aim of this study was to identify disease causing mutation in triple negative cases of ET and PMF.To identify the somatic mutations that are potential disease drivers in triple negative MPN we performed whole exome sequencing (WES) on paired samples from the tumor and control tissue of 4 patients with ET and 4 patients with PMF. We identified somatic mutations in 3/8 analyzed cases. In two PMF cases we identified somatic mutations in genes relevant for MPN- TET2, ASXL1, CBL, SRSF2 and a mutation in MPL-S204P. We did not identify a novel recurrent mutation. In the 5 cases without somatic mutations, we looked for germline mutations in genes relevant for MPN. We identified germline mutations MPL-V285E and JAK2-G571S in one PMF case and one case of ET, respectively. SNP microarray analysis for presence of chromosomal aberrations revealed a uniparental disomy of chromosome 6p in the case with MPL -V285E mutation, suggesting clonal hematopoiesis. To determine the frequency of MPL and JAK2 mutations outside exons 10 and 14 in triple negative MPN, we performed Sanger sequencing of all coding exons of MPL in 62 patients and of JAK2 in 49 patients. We detected variants outside exon 10 of MPL in 6/62 cases (9.7%). MPL-T119I, MPL-S204F, MPL-E230G and MPL-Y591D were somatic mutations, while MPL-R321W was germline. We identified an additional patient with MPL-S204P mutation, however the control tissue was not available. JAK2 variants were found in 4/49 cases (8.1%). JAK2-G335D and JAK2-V625F were germline mutations, while for the patients with JAK2-F556V and JAK2-G571S the control tissue was unavailable. In total, we identified non-canonical MPL mutations in 8/70 (11.4%) and JAK2 mutations in 5/57 (8.8%) triple negative cases of ET and PMF. All mutations were heterozygous. The mutations in MPL and JAK2 were mutually exclusive in our patient cohort.The expression of identified MPL mutants did not induce cytokine independent growth of Ba/F3 cells, but the MPL-Y591D expressing cells showed marked hypersensitivity to TPO compared to the wild type. Using a luciferase reporter assay in JAK2-deficient gamma 2A cells, where we transiently expressed the wild type or mutant MPL cDNAs, JAK2, STAT5, STAT5 reporter Spi-Luc, and pRL-TK for transfection control, we could demonstrate that all identified MPL mutations lead to constitutive activation of JAK/STAT signaling. As the detection of activity required longer times (48h) than for the MPL-W515K (24h), we concluded that the identified mutations have a milder effect of the function of MPL. By Western immunodetection we could demonstrate that expression of JAK2-F556V and JAK2-V625F in Ba/F3-MPL cells, lead to the increased phosphorylation of STAT5 in the absence of cytokines. We also observed increased sensitivity to TPO in the Ba/F3 MPL cell lines expressing JAK2-F556V and JAK2-V625F. JAK2-V625F and JAK2-F556V are mild gain-of-function mutations, while JAK2-G335D and JAK2-G571S do not seem to alter the function on the JAK2 protein.The results of our study suggest that sequencing of all coding exons of MPL and JAK2 is recommended for the diagnostic work-up of the ET and PMF patients who do not carry other more common mutations. The lack of evidence for clonal disease in 50% of the triple negative cases and presence of germline mutations suggests that a proportion of cases are likely to be hereditary MPN-like disorders. Application of whole genome sequencing or RNA sequencing for fusion oncogene detection will likely fill in the gap of the remaining triple negative MPN cases with clonal hematopoiesis in which we did not identify a recurrent driving mutation using WES. DisclosuresGisslinger:AOP ORPHAN: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi Aventis: Consultancy; Geron: Consultancy; Janssen Cilag: Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Kralovics:AOP Orphan: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees.

Use of bacterial whole-genome sequencing to investigate local persistence and spread in bovine tuberculosis

Mycobacterium bovis is the causal agent of bovine tuberculosis, one of the most important diseases currently facing the UK cattle industry. Here, we use high-density whole genome sequencing (WGS) in a defined sub-population of M. bovis in 145 cattle across 66 herd breakdowns to gain insights into local spread and persistence. We show that despite low divergence among isolates, WGS can in principle expose contributions of under-sampled host populations to M. bovis transmission. However, we demonstrate that in our data such a signal is due to molecular type switching, which had been previously undocumented for M. bovis. Isolates from farms with a known history of direct cattle movement between them did not show a statistical signal of higher genetic similarity. Despite an overall signal of genetic isolation by distance, genetic distances also showed no apparent relationship with spatial distance among affected farms over distances <5km. Using simulations, we find that even over the brief evolutionary timescale covered by our data, Bayesian phylogeographic approaches are feasible. Applying such approaches showed that M. bovis dispersal in this system is heterogeneous but slow overall, averaging 2km/year. These results confirm that widespread application of WGS to M. bovis will bring novel and important insights into the dynamics of M. bovis spread and persistence, but that the current questions most pertinent to control will be best addressed using approaches that more directly integrate WGS with additional epidemiological data.

Abstract 4850: Identifying copy number alterations from targeted sequencing data

Abstract Identification of copy number alterations (CNAs) in tumors can assist in determining diagnosis, prognosis, or predicting response to therapy in certain cancer types. Many CNA detection tools have been developed for whole genome sequencing (WGS). These algorithms typically “call” alterations by using significant deviations of the log2 ratio of tumor to normal coverage from the expected diploid log2 ratio. Several factors, however, including reporting pertinent genomic information, hardware and software limitations, and cost, have impeded the routine application of WGS in a clinical setting. Targeted sequencing - hybrid capture and massively parallel sequencing of defined regions (targets) of the genome can overcome some of these challenges and can specifically interrogate, for example, known cancer genes, thus maximizing the “actionability” or utility of the information generated. Though more suited for large-scale clinical testing, targeted sequencing of smaller gene panels with specific clinical reference presents some significant bioinformatic challenges that hinder the application of pre-existing CNA detection tools, resulting in a heavy dependence on manual review in the clinic. In addition, varying quality of DNA, minor variations in laboratory protocols, along with batch effects, are known to confound CNA profiles and are exasperated by the decreased number of sequenced regions of targeted gene panels. These variables alter the log2 ratio, and thus, our ability to call CNAs suffers. We note, that using matched normals may mitigate some deleterious effects, however, to minimize clinical cost and throughput, we tackle these challenges with unmatched tumor-normal pairs. Similar to the closest normal approach used in SNP array analyses, we rely on the assumption that some “normal” (non-tumor) samples experience similar experimental variability that affects the tumor samples. Minimizing maximally informative cost-functions (incorporating both global and local CNA variation), we iteratively investigate different combinations of normal samples which best resemble a given cancer sample. The new set of normals which minimize the cost-function, defines a panel of normals (PON) whose target medians are used to generate the log2 ratio. Along with batch-effect reduction techniques, we then call CNAs with statistically significant thresholds at the target-level. Interestingly, we find that approximately 15 normals are typically needed to minimize CNA profile variation, as opposed to our typical approach of using all available normals (∼60). In addition, we demonstrate that our procedure can improve our sensitivity and specificity from 58±1% to 81±2% and 94±1% to 99.5±0.1%, respectively, on simulated data. We further compared our approach with other published algorithms and found a significant improvement in performance, reducing the time required to manually review CNAs and interpret copy number profiles in the clinic. Citation Format: Bernard Fendler, Ryan Abo, Samuel Hunter, Matthew Ducar, Elizabeth Garcia, Paul Van Hummelen, Neal Lindeman, Laura MacConaill. Identifying copy number alterations from targeted sequencing data. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4850. doi:10.1158/1538-7445.AM2015-4850

The Use of Non-Variant Sites to Improve the Clinical Assessment of Whole-Genome Sequence Data.

Genetic testing, which is now a routine part of clinical practice and disease management protocols, is often based on the assessment of small panels of variants or genes. On the other hand, continuous improvements in the speed and per-base costs of sequencing have now made whole exome sequencing (WES) and whole genome sequencing (WGS) viable strategies for targeted or complete genetic analysis, respectively. Standard WGS/WES data analytical workflows generally rely on calling of sequence variants respect to the reference genome sequence. However, the reference genome sequence contains a large number of sites represented by rare alleles, by known pathogenic alleles and by alleles strongly associated to disease by GWAS. It’s thus critical, for clinical applications of WGS and WES, to interpret whether non-variant sites are homozygous for the reference allele or if the corresponding genotype cannot be reliably called. Here we show that an alternative analytical approach based on the analysis of both variant and non-variant sites from WGS data allows to genotype more than 92% of sites corresponding to known SNPs compared to 6% genotyped by standard variant analysis. These include homozygous reference sites of clinical interest, thus leading to a broad and comprehensive characterization of variation necessary to an accurate evaluation of disease risk. Altogether, our findings indicate that characterization of both variant and non-variant clinically informative sites in the genome is necessary to allow an accurate clinical assessment of a personal genome. Finally, we propose a highly efficient extended VCF (eVCF) file format which allows to store genotype calls for sites of clinical interest while remaining compatible with current variant interpretation software.

A multi-country Salmonella Enteritidis phage type 14b outbreak associated with eggs from a German producer: 'near real-time' application of whole genome sequencing and food chain investigations, United Kingdom, May to September 2014.

We report an outbreak of Salmonella Enteritidis phage type 14b (PT14b) in the United Kingdom (UK) between May and September 2014 where Public Health England launched an investigation to identify the source of infection and implement control measures. During the same period, outbreaks caused by a Salmonella Enteritidis strain with a specific multilocus variable-number tandem repeat analysis (MLVA) profile occurred in other European Union Member States. Isolates from a number of persons affected by the UK outbreak, who had initially been tested by MLVA also shared this particular profile. Cases were defined as any person infected with S. Enteritidis PT14b, resident in England or Wales and without history of travel outside of this geographical area during the incubation period, reported from 1 June 2014 onwards, with a MLVA profile of 2–11–9-7–4-3–2-8–9 or a single locus variant thereof. In total, 287 cases met the definition. Food traceback investigations in the UK and other affected European countries linked the outbreaks to chicken eggs from a German company. We undertook whole genome sequencing of isolates from UK and European cases, implicated UK premises, and German eggs: isolates were highly similar. Combined with food traceback information, this confirmed that the UK outbreak was also linked to a German producer.

Identification and characterization of a novel tick-borne flavivirus subtype in goats (Capra hircus) in Spain.

In 2011, a neurological disease was reported in a herd of goats (Capra hircus) in Asturias, Spain. Initial sequencing identified the causative agent as louping ill virus (LIV). Subsequently, with the application of whole genome sequencing and phylogenetic analysis, empirical data demonstrates that the LIV-like virus detected is significantly divergent from LIV and Spanish sheep encephalitis virus (SSEV). This virus encoded an amino acid sequence motif at the site of a previously identified marker for differentiating tick-borne flaviviruses that was shared with a virus previously isolated in Ireland in 1968. The significance of these observations reflects the diversity of tick-borne flaviviruses in Europe. These data also contribute to our knowledge of the evolution of tick-borne flaviviruses and could reflect the movement of viruses throughout Europe. Based on these observations, the proposed name for this virus is Spanish goat encephalitis virus (SGEV), to distinguish it from SSEV.

Integration of whole-genome sequencing into infection control practices: the potential and the hurdles.

Microbial whole-genome sequencing (WGS) is poised to transform many of the currently used approaches in medical microbiology. Recent reports on the application of WGS to understand genetic evolution and reconstruct transmission pathways have provided valuable information that will influence infection control practices. While this technology holds great promise, obstacles to full implementation remain. Two articles in this issue of the Journal of Clinical Microbiology (S. Octavia, Q. Wang, M. M. Tanaka, S. Kaur, V. Sintchenko, and R. Lan, J Clin Microbiol 53:1063-1071, 2015, doi:10.1128/JCM.03235-14, and S. J. Salipante, D. J. SenGupta, L. A. Cummings, T. A. Land, D. R. Hoogestraat, and B. T. Cookson, J Clin Microbiol 53:1072-1079, 2015, doi:10.1128/JCM.03385-14) describe the breadth of application of WGS to the field of clinical epidemiology.

Genome sequencing defines phylogeny and spread of methicillin-resistant Staphylococcus aureus in a high transmission setting.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial infection. Whole-genome sequencing of MRSA has been used to define phylogeny and transmission in well-resourced healthcare settings, yet the greatest burden of nosocomial infection occurs in resource-restricted settings where barriers to transmission are lower. Here, we study the flux and genetic diversity of MRSA on ward and individual patient levels in a hospital where transmission was common. We repeatedly screened all patients on two intensive care units for MRSA carriage over a 3-mo period. All MRSA belonged to multilocus sequence type 239 (ST 239). We defined the population structure and charted the spread of MRSA by sequencing 79 isolates from 46 patients and five members of staff, including the first MRSA-positive screen isolates and up to two repeat isolates where available. Phylogenetic analysis identified a flux of distinct ST 239 clades over time in each intensive care unit. In total, five main clades were identified, which varied in the carriage of plasmids encoding antiseptic and antimicrobial resistance determinants. Sequence data confirmed intra- and interwards transmission events and identified individual patients who were colonized by more than one clade. One patient on each unit was the source of numerous transmission events, and deep sampling of one of these cases demonstrated colonization with a “cloud” of related MRSA variants. The application of whole-genome sequencing and analysis provides novel insights into the transmission of MRSA in under-resourced healthcare settings and has relevance to wider global health.

C-104 Personalized Medicine and Cancer Outcomes

The use of whole genome sequencing (WGS) and related genomic characterization of both the patient and the patient's tumor is becoming increasingly critical today in meeting the goal of establishing optimal information for achieving the best outcome. Mutations in certain genes have been shown to confer sensitivity to available therapies (BRAF L597 mutation in melanoma). Less than comprehensive patient and tumor genomic characterization may result in a patient being excluded from a potentially beneficial therapy or not being identified as having a drug metabolism inappropriate to adequately utilize the planned agent(s). To address 1 aspect of the increasing application of WGS in healthcare, we have studied the variation in known cancer-susceptibility genes in a general population. In this study, we profiled the nonsynonymous variation in >150 genes causally implicated in the initiation and progression of early stages of cancer using high quality WGS (>60X) from an ancestrally diverse cohort of 681 healthy individuals. Of the 2,688 allelic variants that could possibly increase susceptibility to developing cancer, there were an average of 68 such variants per individual. Most of the variants are very rare, with only 75% found in 1 or 2 individuals in the study population. Importantly, allele frequencies vary between ancestral groups, with 21 variants for which the minor allele in 1 population is the major allele in another. Specific study of a selected group of clinically important cancer genes, highlight the differences between germline variants and reported somatic mutations (PLOS ONE April2014, v.9, e94554). This data represents the first profile of germline variation in multiple cancer risk genes derived from WGS of a healthy, ancestrally diverse, single cohort adult population. As such, the data provide an important foundation for the interpretation of cancer risk when the information is obtained from a clinically obtained individual patient's WGS.

Genome analysis of Campylobacter jejuni strains isolated from a waterborne outbreak.

BackgroundWaterborne Campylobacter jejuni outbreaks are common in the Nordic countries, and PFGE (pulsed field gel electrophoresis) remains the genotyping method of choice in outbreak investigations. However, PFGE cannot assess the clonal relationship between isolates, leading to difficulties in molecular epidemiological investigations. Here, we explored the applicability of whole genome sequencing to outbreak investigation by re-analysing three C. jejuni strains (one isolated from water and two from patients) from an earlier resolved Finnish waterborne outbreak from the year 2000.ResultsOne of the patient strains had the same PFGE profile, as well as an identical overall gene synteny and three polymorphisms in comparison with the water strain. However, the other patient isolate, which showed only minor differences in the PFGE pattern relative to the water strain, harboured several polymorphisms as well as rearrangements in the integrated element CJIE2. We reconstructed the genealogy of these strains with ClonalFrame including in the analysis four C. jejuni isolated from chicken in 2012 having the same PFGE profile and sequence type as the outbreak strains. The three outbreak strains exhibited a paraphyletic relationship, implying that the drinking water from 2000 was probably contaminated with at least two different, but related, C. jejuni strains.ConclusionsOur results emphasize the capability of whole genome sequencing to unambiguously resolve the clonal relationship between isolates of C. jejuni in an outbreak situation and evaluate the diversity of the C. jejuni population.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-768) contains supplementary material, which is available to authorized users.

Practical Guidelines for Ethical and Policy Issues that Arise from the Clinical Application of Whole Genome Sequencing in Cancer Patients

ABSTRACT Aim: Our group utilizes whole genome sequencing (WGS) to identify candidate driver pathways to guide personalized treatment decisions in oncology. There is debate as to whether research grade genomic results should be provided to subjects and if so, what process should be used. We developed a working model based on respect for autonomy, non-maleficence, beneficence and justice. This model aims to address the challenges associated with reporting individual research results (IRR), incidental findings (IF) and access to raw, research grade genomic data. Methods: We created an ethics sub-committee, comprised of a bioethicist, a genetic counselor, bioinformaticians, genomic pathologists, and medical oncologists to review existing recommendations and guidelines on reporting WGS results. Results: The key element of this model is informed consent. 1) In the consent forms, the subjects are given the choice to be informed or not of both IRR and IF. 2) In case of IF, only clinically actionable genetic variants will be reported after validation by clinically approved testing and referred to genetic counseling. 3) Requests for release of the research grade raw data generated by WGS will be reviewed on a case by case basis. The data released will consist of raw base calls and not contain custom analysis or biological inferences. The reliability and validity of the data will be communicated to the participants prior to data release. 4) Subjects will be informed that due to the personalized nature of genomic information, it is not possible to guarantee anonymity of the data. 5) The project will only be responsible for reporting IFs for the duration of the research project. 6) If the data are shared with other research organizations and IFs are identified, these researchers would not be responsible for communicating these data back to the subject or their physician. 7) Disclosing IRR or IF to deceased subjects' relatives should follow the same working model developed for this program. Conclusions: WGS provides a wealth of research information, some of which can impact treatment decision-making. It is certain that IF and difficult questions regarding access to research grade data will arise in any study involving genome sequencing. We created an ethical guideline to ensure that the information is used and reported in an appropriate and responsible manner. Disclosure: All authors have declared no conflicts of interest.