Application Of Whole Genome Sequencing Research Articles

Use of whole-genome sequencing to distinguish relapse from reinfection in a completed tuberculosis clinical trial

BackgroundRIFAQUIN was a tuberculosis chemotherapy trial in southern Africa including regimens with high-dose rifapentine with moxifloxacin. Here, the application of whole-genome sequencing (WGS) is evaluated within RIFAQUIN for identifying new infections in treated patients as either relapses or reinfections. WGS is further compared with mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) typing. This is the first report of WGS being used to evaluate new infections in a completed clinical trial for which all treatment and epidemiological data are available for analysis.MethodsDNA from 36 paired samples of Mycobacterium tuberculosis cultured from patients before and after treatment was typed using 24-loci MIRU-VNTR, in silico spoligotyping and WGS. Following WGS, the sequences were mapped against the reference strain H37Rv, the single-nucleotide polymorphism (SNP) differences between pairs were identified, and a phylogenetic reconstruction was performed.ResultsWGS indicated that 32 of the paired samples had a very low number of SNP differences (0–5; likely relapses). One pair had an intermediate number of SNP differences, and was likely the result of a mixed infection with a pre-treatment minor genotype that was highly related to the post-treatment genotype; this was reclassified as a relapse, in contrast to the MIRU-VNTR result. The remaining three pairs had very high SNP differences (>750; likely reinfections).ConclusionsWGS and MIRU-VNTR both similarly differentiated relapses and reinfections, but WGS provided significant extra information. The low proportion of reinfections seen suggests that in standard chemotherapy trials with up to 24 months of follow-up, typing the strains brings little benefit to an analysis of the trial outcome in terms of differentiating relapse and reinfection. However, there is a benefit to using WGS as compared to MIRU-VNTR in terms of the additional genotype information obtained, in particular for defining the presence of mixed infections and the potential to identify known and novel drug-resistance markers.

Identifying the clonal relationship model of multifocal papillary thyroid carcinoma by whole genome sequencing

PurposeTo evaluate the application of whole genome sequencing (WGS) in determining the inter-foci clonal relationship of multifocal papillary thyroid carcinoma (mPTC). MethodsAfter reviewing PTC patient profiles, 8 cancer foci and germline control samples from 3 mPTC patients were analyzed by WGS. Single nucleotide variations (SNVs), copy number variation (CNV), structural variation and mutational signature were examined. ResultsThe multifocality rate of PTC was 35.1% and mPTC were shown to have larger primary tumor diameter, higher rate of lymph node metastasis and less number of accompanying non-cancerous lesions than single PTC in one or both gender groups. Out of the 8 cancer foci, 5 foci were identified as clonal-independent model with the rest 3 foci as clonal-derived model according to exonic SNVs spectrum. Non-exonic mutations provided compelling evidence at the genome-wide level for the classification. Specific CNV and 12 mutational signatures were also identified. ConclusionsWGS could be an impressive tool in clonal relationship determination of PTC by providing a panoramic view of genome-wide somatic mutations. The substantial sequencing data provided additional information that could help studying the mechanism of carcinogenesis.

Whole-Genome Sequencing in Epidemiology of Campylobacter jejuni Infections.

This review describes the current state of knowledge regarding the application of whole-genome sequencing (WGS) in the epidemiology of Campylobacter jejuni, the leading cause of bacterial gastroenteritis worldwide. We describe how WGS has increased our understanding of the evolutionary and epidemiological dynamics of this pathogen and how WGS has the potential to improve surveillance and outbreak detection. We have identified hurdles to the full implementation of WGS in public health settings. Despite these challenges, we think that ample evidence is available to support the benefits of integrating WGS into the routine monitoring of C. jejuni infections and outbreak investigations.

Whole genome sequence study of cannabis dependence in two independent cohorts.

Recent advances in genome wide sequencing techniques and analytical methods allow for more comprehensive examinations of the genome than microarray-based genome-wide association studies (GWAS). The present report provides the first application of whole genome sequencing (WGS) to identify low frequency variants involved in cannabis dependence across two independent cohorts. The present study used low-coverage whole genome sequence data to conduct set-based association and enrichment analyses of low frequency variation in protein-coding regions as well as regulatory regions in relation to cannabis dependence. Two cohorts were studied: a population-based Native American tribal community consisting of 697 participants nested within large multi-generational pedigrees and a family-based sample of 1832 predominantly European ancestry participants largely nested within nuclear families. Participants in both samples were assessed for Diagnostic and Statistical Manual of Mental Disorders-IV (DSM-IV) lifetime cannabis dependence, with 168 and 241 participants receiving a positive diagnosis in each sample, respectively. Sequence kernel association tests identified one protein-coding region, C1orf110 and one regulatory region in the MEF2B gene that achieved significance in a meta-analysis of both samples. A regulatory region within the PCCB gene, a gene previously associated with schizophrenia, exhibited a suggestive association. Finally, a significant enrichment of regions within or near genes with multiple splice variants or involved in cell adhesion or potassium channel activity were associated with cannabis dependence. This initial study demonstrates the potential utility of low pass whole genome sequencing for identifying genetic variants involved in the etiology of cannabis use disorders.

Commentary on clinical utility of whole genome sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

3The present study is commentary and aims to evaluate the practical application of Whole Genome Sequencing (WGS) and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) in clinical microbiology. The MALDI-TOF MS method has been replaced cultural and biochemical tests for species identification in most laboratories worldwide. Moreover, WGS has elevated the discriminatory power by detection of single nucleotide polymorphisms (SNPs), and helped with the identification of microorganisms that were misidentified by previous culture and biochemical tests. Moreover, WGS can be used in epidemiological studies (e.g. clinical outbreaks) by tracking of phylogenetic maps and analyzing SNP distance; antimicrobial resistance studies such as the study of antimicrobial resistance genes, e.g. CTX-M, NDM, KPC, or OXA; and the study of recombination of plasmid or insertion sequence (IS) elements to elucidate the mechanism of antimicrobial resistance. MALDI-TOF technology is used for bacterial or fungal species identification in most laboratories. VITEK MS (bioMerieux) and Biotyper (Bruker Daltonics) are examples of commercially available MALDI-TOF MS systems. The technique has been used for direct identification of organisms from blood culture bottles, markedly shortening the identification time. Beta-lactamases or carbapenemases have been detected by analyzing specific protein peaks using MALDI-TOF MS. Shiga toxin-producing Escherichia coli, Salmonella serotype, and Vibrio phenotypes can also be detected.

Recent Technology of Clinical Microbiology; Whole Genome Sequencing (WGS) and Matrix-Assisted, Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS)

The hottest issue in the field of clinical microbiology in latest years is the application of whole genome sequencing (WGS) to the clinical diagnostics and therapeutics through the methodology of Next generation sequencing. The application of WGS includes species identification, epidemiological study and study of antimicrobial resistance and so on. The single nucleotide polymorphism (SNP) based analysis increases the accuracy and sensitivity of species identification by marked discriminatory power, and the ability of tracking of transmission process enables infection control including outbreaks. We can clarify the antimicrobial resistance mechanism of extended spectrum betalactamases or carbapenemases and elucidate the mechanism of horizontal transfer of mobile genomic islands. In case of setting up a new PCR method, the target and the primer could be chosen by using WGS databases. The MALDI-TOF method is used in nearly all laboratories, identifying bacterial, fungal and mycobacterial species. Direct identification in blood culture bottle is possible in bacteremia patients, and Beta-lactamases or Carbapenemases can be detected. In addition, it can be applied in Shiga toxin E. coli, Salmonella serotypes or C. difficile ribotypes.

The Hidden Genomics of Chlamydia trachomatis.

The application of whole-genome sequencing has moved us on from sequencing single genomes to defining unravelling population structures in different niches, and at the -species, -serotype or even -genus level, and in local, national and global settings. This has been instrumental in cataloguing and revealing a huge a range of diversity in this bacterium, when at first we thought there was little. Genomics has challenged assumptions, added insight, as well as confusion and glimpses of truths. What is clear is that at a time when we start to realise the extent and nature of the diversity contained within a genus or a species like this, the huge depth of knowledge communities have developed, through cell biology, as well as the new found molecular approaches will be more precious than ever to link genotype to phenotype. Here we detail the technological developments and insights we have seen during the relatively short time since we began to see the hidden genome of Chlamydia trachomatis.

Bacterial Whole Genome Sequencing in Investigation of Salmonella Oranienburg Infection Outbreak, Missouri, USA

Background: In April 2015, the Missouri Department of Health and Senior Services identified a cluster of four Salmonella enterica serovar Oranienburg infections among residents of the St. Louis metro area. Clinical isolates from those patients shared an indistinguishable pulsed-field gel electrophoresis (PFGE) pattern. Methods and Findings: The ensuing outbreak investigation included epidemiological, laboratory, and environmental investigations. Clinical and environmental isolates were subtyped by PFGE pattern and whole genome sequencing (WGS). Eggs from a single poultry farm and processing facility were identified as the likely source of this outbreak based on the epidemiologic data, highly related Salmonella isolates from the farm and processing facility, and clinical isolates from patients. The egg farm operations were suspended, and the producer issued a voluntary recall of all eggs produced up to the time of the recall. Conclusions: WGS is a valuable tool for Salmonella Oranienburg infection outbreak investigation. Early prospective application of WGS enhances several aspects of the outbreak investigation. A thorough analysis of combined epidemiological, traceback, and advanced molecular laboratory data is needed for the proper management of foodborne outbreak investigation.

The Effect of Nucleic Acid Extraction Platforms and Sample Storage on the Integrity of Viral RNA for Use in Whole Genome Sequencing

Extraction of viral RNA and the storage of sample material are extremely important factors in the detection and whole genome sequencing (WGS) of viral pathogens. Although PCR-based detection methods focus on small amplicons, viral WGS applications require RNA of high quality and integrity for adequate sequence coverage and depth. This study examined the fitness of one manual and four automated RNA extraction platforms commonly used in diagnostic laboratories for use in metagenomic sequencing, how the practice of storing sample material in Qiagen buffer AVL before extraction affected the integrity of viral RNA and its suitability for use in amplicon-based WGS methods, and how the addition of Triton X-100 to buffer AVL affected the capability of the extraction platforms and the integrity of viral RNA in stored samples. This study found that the EZ1 platform gave the best performance of the automated platforms and gave comparable results to the frequently used manual Qiagen extraction protocol when extracted viral RNA was used in metagenomics sequencing. To maintain high levels of viral RNA integrity suitable for amplicon-based WGS, nucleic acid should be extracted from samples immediately, because even short storage periods in buffer AVL have a severe effect on integrity, and the addition of Triton X-100 had little effect on the quality of viral material for WGS.

Successful Application of Whole Genome Sequencing in a Medical Genetics Clinic.

A pilot program was initiated using whole genome sequencing (WGS) to diagnose suspected genetic disorders in the Genetics Clinic at Children's Hospital of Wisconsin. Twenty-two patients underwent WGS between 2010 and 2013. Initially, we obtained a 14% (3/22) diagnosis rate over 2 years; with subsequent reanalysis, this increased to 36% (8/22). Disease causing variants were identified in SKIV2L, CECR1, DGKE, PYCR2, RYR1, PDGFRB, EFTUD2, and BCS1L. In 75% (6/8) of diagnosed cases, the diagnosis affected treatment and/or medical surveillance. Additionally, one case demonstrated a homozygous A18V variant in VLDLR that appears to be associated with a previously undescribed phenotype.

Exploiting Bacterial Whole-Genome Sequencing Data for Evaluation of Diagnostic Assays: Campylobacter Species Identification as a Case Study.

The application of whole-genome sequencing (WGS) to problems in clinical microbiology has had a major impact on the field. Clinical laboratories are now using WGS for pathogen identification, antimicrobial susceptibility testing, and epidemiological typing. WGS data also represent a valuable resource for the development and evaluation of molecular diagnostic assays, which continue to play an important role in clinical microbiology. To demonstrate this application of WGS, this study used publicly available genomic data to evaluate a duplex real-time PCR (RT-PCR) assay that targets mapA and ceuE for the detection of Campylobacter jejuni and Campylobacter coli, leading global causes of bacterial gastroenteritis. In silico analyses of mapA and ceuE primer and probe sequences from 1,713 genetically diverse C. jejuni and C. coli genomes, supported by RT-PCR testing, indicated that the assay was robust, with 1,707 (99.7%) isolates correctly identified. The high specificity of the mapA-ceuE assay was the result of interspecies diversity and intraspecies conservation of the target genes in C. jejuni and C. coli. Rare instances of a lack of specificity among C. coli isolates were due to introgression in mapA or sequence diversity in ceuE. The results of this study illustrate how WGS can be exploited to evaluate molecular diagnostic assays by using publicly available data, online databases, and open-source software.

A Whole-Genome Analysis Framework for Effective Identification of Pathogenic Regulatory Variants in Mendelian Disease

The interpretation of non-coding variants still constitutes a major challenge in the application of whole-genome sequencing in Mendelian disease, especially for single-nucleotide and other small non-coding variants. Here we present Genomiser, an analysis framework that is able not only to score the relevance of variation in the non-coding genome, but also to associate regulatory variants to specific Mendelian diseases. Genomiser scores variants through either existing methods such as CADD or a bespoke machine learning method and combines these with allele frequency, regulatory sequences, chromosomal topological domains, and phenotypic relevance todiscover variants associated to specific Mendelian disorders. Overall, Genomiser is able to identify causal regulatory variants as thetop candidate in 77% of simulated whole genomes, allowing effective detection and discovery of regulatory variants in Mendelian disease.

Abstract 2631: Restrictions on access to systemic therapy limit the application of whole genome sequencing in clinical care

Abstract Background: Whole genome analyses have the potential to identify the full landscape of activating and inactivating genomic abnormalities at work within cancers, and can thus be used to provide rationales for selection of treatment agents or clinical trials in a broad range of patients. Patients & Methods: Eligible patients (pts) with metastatic cancers were recruited within a general oncology practice across the province of B.C., Canada. Each pt underwent a fresh tumor biopsy and a blood sample and had comprehensive DNA (80X) and RNA sequencing. In-depth bioinformatic analyses were preformed to identify genomic changes that may be cancer “drivers” or therapeutically actionable targets. Aberrant pathways were matched to drug databases and manual literature reviews undertaken to identify drugs or clinical trials of potential utility for the individual pt. Results: Between July 2012 - Oct 2015: 380 pts (358 adult + 22 peds) consented; 227 have completed whole DNA and RNA sequencing and analysis to date (remainder ongoing). For this analysis, data is available on 160 pts. Genome bioinformaticians assessed the genomic data to be potentially druggable in all cases. Medical Oncologists assessed this data to be directly clinically actionable in 135 (84%); the difference being that clinicians did not agree that some putative “druggable” drivers (such as p53) or pathways with no current drugs available (MMP, AURA, WNT) were “actionable”. Of the 135 cases, 58 (43%) pts received therapy based directly on this genomic information; 6 on a phase 1 clinical trial. The most common reasons for 77 pts defined as actionable but who have not received genomically-informed therapy were: drug only available on a clinical trial but trial not available to pt - 22 (26%); drug approved but not available off label - 18 (23%); pt presently on first-line therapy that is working - 14 (18%); or death/too unwell 13 (17%). The limited availability of clinical trials was primarily because of highly restrictive trials entry criteria, primarily limiting patients to one primary tumour type or narrowly defined biomarker entry criteria. The most commonly mutated cancer genes identified by the genome analysts were: p53 as the predominant driver in 42%; APC in 16%; KRAS and PI3KCA mutations in 14%. Going forward it is essential to distinguish what driver mutations might be clinically actionable from those that are still only theoretically druggable; and similarly learn to distinguish which targets are actionable but not truly drivers. Conclusions: Genomic DNA and RNA sequencing data were found to be clinically actionable in 84% of pts with advanced cancers in a population cancer care setting. However, the ability to act on this information is limited by the restrictive nature of clinical trials and the lack of accessibility of off-label drugs despite an identified biomarker. As genome sequencing becomes integrated into cancer management these drug access issues need to be addressed. Citation Format: Janessa J. Laskin, Yaoqing Shen, Daniel Renouf, Martin Jones, Howard Lim, Alexandra Fok, Cheryl Ho, Balvir Deol, Karen A. Gelmon, Stephen Chia, Richard Moore, Andrew Mungall, Stephen Yip, Steven Jones, Marco Marra. Restrictions on access to systemic therapy limit the application of whole genome sequencing in clinical care. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2631.

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.