Value Of Whole-genome Sequencing Research Articles

Limited Added Diagnostic Value of Whole Genome Sequencing in Genetic Testing of Inherited Retinal Diseases in a Swiss Patient Cohort.

The purpose of this study was to assess the added diagnostic value of whole genome sequencing (WGS) for patients with inherited retinal diseases (IRDs) who remained undiagnosed after whole exome sequencing (WES). WGS was performed for index patients in 66 families. The datasets were analyzed according to GATK's guidelines. Additionally, DeepVariant was complemented by GATK's workflow, and a novel structural variant pipeline was developed. Overall, a molecular diagnosis was established in 19/66 (28.8%) index patients. Pathogenic deletions and one deep-intronic variant contributed to the diagnostic yield in 4/19 and 1/19 index patients, respectively. The remaining diagnoses (14/19) were attributed to exonic variants that were missed during WES analysis due to bioinformatic limitations, newly described loci, or unclear pathogenicity. The added diagnostic value of WGS equals 5/66 (9.6%) for our cohort, which is comparable to previous studies. This figure would decrease further to 1/66 (1.5%) with a standardized and reliable copy number variant workflow during WES analysis. Given the higher costs and limited added value, the implementation of WGS as a first-tier assay for inherited eye disorders in a diagnostic laboratory remains untimely. Instead, progress in bioinformatic tools and communication between diagnostic and clinical teams have the potential to ameliorate diagnostic yields.

Complete genomic characterization in patients with cancer of unknown primary origin in routine diagnostics

In ∼3%-5% of patients with metastatic disease, tumor origin remains unknown despite modern imaging techniques and extensive pathology work-up. With long diagnostic delays and limited and ineffective therapy options, the clinical outcome of patients with cancer of unknown primary (CUP) remains poor. Large-scale genome sequencing studies have revealed that tumor types can be predicted based on distinct patterns of somatic variants and other genomic characteristics. Moreover, actionable genomic events are present in almost half of CUP patients. This study investigated the clinical value of whole genome sequencing (WGS) in terms of primary tumor identification and detection of actionable events, in the routine diagnostic work-up of CUP patients. A WGS-based tumor type 'cancer of unknown primary prediction algorithm' (CUPPA) was developed based on previously described principles and validated on a large pan-cancer WGS database of metastatic cancer patients (>4000 samples) and 254 independent patients, respectively. We assessed the clinical value of this prediction algorithm as part of routine WGS-based diagnostic work-up for 72 CUP patients. CUPPA correctly predicted the primary tumor type in 78% of samples in the independent validation cohort (194/254 patients). High-confidence predictions (>95% precision) were obtained for 162/254 patients (64%). When integrated in the diagnostic work-up of CUP patients, CUPPA could identify a primary tumor type for 49/72 patients (68%). Most common diagnoses included non-small-cell lung (n= 7), gastroesophageal (n= 4), pancreatic (n= 4), and colorectal cancer (n= 3). Actionable events with matched therapy options in clinical trials were identified in 47% of patients. Genome-based tumor type prediction can predict cancer diagnoses with high accuracy when integrated in the routine diagnostic work-up of patients with metastatic cancer. With identification of the primary tumor type in the majority of patients and detection of actionable events, WGS is a valuable diagnostic tool for patients with CUP.

45. Clostridioides difficile: The value of whole genome sequencing to rule out a common source healthcare associated outbreak

45. Clostridioides difficile: The value of whole genome sequencing to rule out a common source healthcare associated outbreak

WIN Symposium 2022 - Abstract Poster Presentations.

WIN Symposium 2022 - Abstract Poster Presentations.

Detailed analysis of in-hospital transmission of SARS-CoV-2 using whole genome sequencing

We present an outbreak of 56 staff and patient cases of COVID-19 over a 31 day period in a tertiary referral unit, with at least a further 29 cases identified outside of the unit and the hospital by whole genome sequencing (WGS). We document transmission from staff-to-staff, staff-to-patients and patients-to-staff and show disruption of a tertiary referral service, despite implementation of nationally recommended control measures, superior ventilation and use of PPE. We demonstrate extensive spread from the index case, despite them spending only 10 hours bed bound on the ward in strict cubicle isolation and with an initial single target low level (CT=32) PCR test. This investigation highlights critical issues including how effectively and explosively SARS-CoV-2 can spread in certain circumstances. It raises questions about infection control measures in place at the time and calls into question the premise that transmissibility can be reliably detected using lower sensitivity rapid antigen lateral flow tests. We also highlight the value of early intervention in reducing impact as well as the value of WGS in understanding outbreaks.

Feasibility of whole-genome sequencing-based tumor diagnostics in routine pathology practice.

The current increase in number and diversity of targeted anticancer agents poses challenges to the logistics and timeliness of molecular diagnostics (MolDx), resulting in underdiagnosis and treatment. Whole‐genome sequencing (WGS) may provide a sustainable solution for addressing current as well as future diagnostic challenges. The present study therefore aimed to prospectively assess feasibility, validity, and value of WGS in routine clinical practice. WGS was conducted independently of, and in parallel with, standard of care (SOC) diagnostics on routinely obtained tumor samples from 1,200 consecutive patients with metastatic cancer. Results from both tests were compared and discussed in a dedicated tumor board. From 1,200 patients, 1,302 samples were obtained, of which 1,216 contained tumor cells. WGS was successful in 70% (854/1,216) of samples with a median turnaround time of 11 days. Low tumor purity (<20%) was the main reason for not completing WGS. WGS identified 99.2% and SOC MolDx 99.7% of the total of 896 biomarkers found in genomic regions covered by both tests. Actionable biomarkers were found in 603/848 patients (71%). Of the 936 associated therapy options identified by WGS, 343 were identified with SOC MolDx (36.6%). Biomarker‐based therapy was started in 147 patients. WGS revealed 49 not previously identified pathogenic germline variants. Fresh‐frozen, instead of formalin‐fixed and paraffin‐embedded, sample logistics were easily adopted as experienced by the professionals involved. WGS for patients with metastatic cancer is well feasible in routine clinical practice, successfully yielding comprehensive genomic profiling for the vast majority of patients. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Tracking the Origin of Austrian Human Brucellosis Cases Using Whole Genome Sequencing.

Brucellosis is a zoonotic disease caused by Brucella spp. and a major concern for livestock. Most human cases are caused by B. melitensis and clinical presentation is usually a mild febrile illness. However, treatment failure is frequent and more severe complications can occur. In Austria, every human brucellosis is investigated to determine whether it was imported from endemic areas or is the sign of an undetected autochthonous transmission. For this study, 21 B. melitensis strains isolated in Austria between 2005 and 2019 were collected, 17 strains from 15 different patients and four strains from cattle. Whole genome sequencing combined with core-genome MLST analysis was used to characterize these strains. A cluster of seven isolates from 2018 (three human and four cattle isolates) was identified, with fewer than two allelic differences. They corresponded to the only Austrian B. melitensis outbreak that happened over the past 15 years. The other 12 Austrian brucellosis cases were single cases, and geographical origins were available for 8/12. Genomic data was used to locate probable geographical origins and compared with the results of the epidemiological investigations. Austrian strains were compared with 67 published B. melitensis sequences available on NCBI. The result of genomic analysis matched for 7/8 cases with documented conclusion of the epidemiological investigation. Genome analysis also pointed to the geographical origin for three of the four cases with missing epidemiological data. Strains from six cases were grouped together (<40 allelic differences) with 4/6 cases imported from the Balkans. Additional B. melitensis isolates from Serbian animals were analyzed and grouped with this branch, suggesting frequent importation from Balkan countries to Austria. Overall, this study highlights the specificities of human brucellosis in Austria. It also underlines the value of whole genome sequencing as a tool to investigate brucellosis cases, allowing to identify and investigate outbreaks but also to support epidemiological investigation of imported cases. However, the reliability of such methods depends on the number of strains for comparison, which can be challenging in low incidence countries. Increasing the availability of published sequences with documented geographical origins would help establishing genomic-based methods for investigating brucellosis cases.

The Impact of the CFTR Gene Discovery on Cystic Fibrosis Diagnosis, Counseling, and Preventive Therapy.

Discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene was the long-awaited scientific advance that dramatically improved the diagnosis and treatment of cystic fibrosis (CF). The combination of a first-tier biomarker, immunoreactive trypsinogen (IRT), and, if high, DNA analysis for CF-causing variants, has enabled regions where CF is prevalent to screen neonates and achieve diagnoses within 1–2 weeks of birth when most patients are asymptomatic. In addition, IRT/DNA (CFTR) screening protocols simultaneously contribute important genetic data to determine genotype, prognosticate, and plan preventive therapies such as CFTR modulator selection. As the genomics era proceeds with affordable biotechnologies, the potential added value of whole genome sequencing will probably enhance personalized, precision care that can begin during infancy. Issues remain, however, about the optimal size of CFTR panels in genetically diverse regions and how best to deal with incidental findings. Because prospects for a primary DNA screening test are on the horizon, the debate about detecting heterozygote carriers will likely intensify, especially as we learn more about this relatively common genotype. Perhaps, at that time, concerns about CF heterozygote carrier detection will subside, and it will become recognized as beneficial. We share new perspectives on that issue in this article.

Rapid phenotypic evolution in multidrug-resistant Klebsiella pneumoniae hospital outbreak strains.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) increasingly cause high-mortality outbreaks in hospital settings globally. Following a patient fatality at a hospital in Beijing due to a blaKPC-2-positive CRKP infection, close monitoring was put in place over the course of 14 months to characterize all blaKPC-2-positive CRKP in circulation in the hospital. Whole genome sequences were generated for 100 isolates from blaKPC-2-positive isolates from infected patients, carriers and the hospital environment. Phylogenetic analyses identified a closely related cluster of 82 sequence type 11 (ST11) isolates circulating in the hospital for at least a year prior to admission of the index patient. The majority of inferred transmissions for these isolates involved patients in intensive care units. Whilst the 82 ST11 isolates collected during the surveillance effort all had closely related chromosomes, we observed extensive diversity in their antimicrobial resistance (AMR) phenotypes. We were able to reconstruct the major genomic changes underpinning this variation in AMR profiles, including multiple gains and losses of entire plasmids and recombination events between plasmids, including transposition of blaKPC-2. We also identified specific cases where variation in plasmid copy number correlated with the level of phenotypic resistance to drugs, suggesting that the number of resistance elements carried by a strain may play a role in determining the level of AMR. Our findings highlight the epidemiological value of whole genome sequencing for investigating multi-drug-resistant hospital infections and illustrate that standard typing schemes cannot capture the extraordinarily fast genome evolution of CRKP isolates.

Genome analysis identifies a spontaneous nonsense mutation in ppsD leading to attenuation of virulence in laboratory-manipulated Mycobacterium tuberculosis

BackgroundA previous laboratory study involving wild type, mutant and devR/dosR complemented strains of Mycobacterium tuberculosis reported the attenuation phenotype of complemented strain, Comp1. This phenotype was intriguing since the parental strain H37Rv, devR mutant (Mut1) and additional complemented strains, Comp9 and Comp11, were virulent in the guinea pig model.ResultsTowards deciphering the mechanism underlying the attenuation of Comp1, a whole genome sequencing approach was undertaken. Eight Single Nucleotide Polymorphisms (SNPs) unique to the Comp1 strain were identified. Of these, 5 SNPs were non-synonymous and included a G➞A mutation resulting in a W1591Stop mutation in ppsD gene of the phthiocerol dimycocerosate (PDIM) biosynthetic cluster. Targeted sequence analysis confirmed this mutation in only Comp1 strain and not in wild type (H37Rv), devR knockout (Mut1) or other complemented (Comp9 and Comp11) bacteria. Differential expression of the PDIM locus in Comp1 bacteria was observed which was associated with a partial deficiency of PDIM, an increased sensitivity to detergent and a compromised ability to infect human THP-1 cells.ConclusionsIt is proposed that a spontaneous mutation in the ppsD gene of Comp1 underlies down-modulation of the PDIM locus which is associated with defects in permeability and infectivity as well as virulence attenuation in guinea pigs. Our study demonstrates the value of whole genome sequencing for resolving unexplainable bacterial phenotypes and recommends the assessment of PDIM status while assessing virulence properties of laboratory-manipulated strains of M. tuberculosis.

Retrospective use of whole genome sequencing to better understand an outbreak of Salmonella enterica serovar Mbandaka in New South Wales, Australia.

IntroductionSalmonella enterica serovar Mbandaka is an infrequent cause of salmonellosis in New South Wales (NSW) with an average of 17 cases reported annually. This study examined the added value of whole genome sequencing (WGS) for investigating a non-point source outbreak of Salmonella ser. Mbandaka with limited geographical spread.MethodsIn February 2016, an increase in Salmonella ser. Mbandaka was noted in New South Wales, and an investigation was initiated. A WGS study was conducted three months after the initial investigation, analysing the outbreak Salmonella ser. Mbandaka isolates along with 17 human and non-human reference strains from 2010 to 2015.ResultsWGS analysis distinguished the original outbreak cases (n = 29) into two main clusters: Cluster A (n = 11) and Cluster B (n = 6); there were also 12 sporadic cases. Reanalysis of food consumption histories of cases by WGS cluster provided additional specificity when assessing associations.DiscussionWGS has been widely acknowledged as a promising high-resolution typing tool for enteric pathogens. This study was one of the first to apply WGS to a geographically limited cluster of salmonellosis in Australia. WGS clearly distinguished the outbreak cases into distinct clusters, demonstrating its potential value for use in real time to support non-point source foodborne disease outbreaks of limited geographical spread.

Whole Genome Sequencing Demonstrates Limited Transmission within Identified Mycobacterium tuberculosis Clusters in New South Wales, Australia.

Australia has a low tuberculosis incidence rate with most cases occurring among recent immigrants. Given suboptimal cluster resolution achieved with 24-locus mycobacterium interspersed repetitive unit (MIRU-24) genotyping, the added value of whole genome sequencing was explored. MIRU-24 profiles of all Mycobacterium tuberculosis culture-confirmed tuberculosis cases diagnosed between 2009 and 2013 in New South Wales (NSW), Australia, were examined and clusters identified. The relatedness of cases within the largest MIRU-24 clusters was assessed using whole genome sequencing and phylogenetic analyses. Of 1841 culture-confirmed TB cases, 91.9% (1692/1841) had complete demographic and genotyping data. East-African Indian (474; 28.0%) and Beijing (470; 27.8%) lineage strains predominated. The overall rate of MIRU-24 clustering was 20.1% (340/1692) and was highest among Beijing lineage strains (35.7%; 168/470). One Beijing and three East-African Indian (EAI) clonal complexes were responsible for the majority of observed clusters. Whole genome sequencing of the 4 largest clusters (30 isolates) demonstrated diverse single nucleotide polymorphisms (SNPs) within identified clusters. All sequenced EAI strains and 70% of Beijing lineage strains clustered by MIRU-24 typing demonstrated distinct SNP profiles. The superior resolution provided by whole genome sequencing demonstrated limited M. tuberculosis transmission within NSW, even within identified MIRU-24 clusters. Routine whole genome sequencing could provide valuable public health guidance in low burden settings.

Whole Genome Sequence Analysis of a Large Isoniazid-Resistant Tuberculosis Outbreak in London: A Retrospective Observational Study.

BackgroundA large isoniazid-resistant tuberculosis outbreak centred on London, United Kingdom, has been ongoing since 1995. The aim of this study was to investigate the power and value of whole genome sequencing (WGS) to resolve the transmission network compared to current molecular strain typing approaches, including analysis of intra-host diversity within a specimen, across body sites, and over time, with identification of genetic factors underlying the epidemiological success of this cluster.Methods and FindingsWe sequenced 344 outbreak isolates from individual patients collected over 14 y (2 February 1998–22 June 2012). This demonstrated that 96 (27.9%) were indistinguishable, and only one differed from this major clone by more than five single nucleotide polymorphisms (SNPs). The maximum number of SNPs between any pair of isolates was nine SNPs, and the modal distance between isolates was two SNPs. WGS was able to reveal the direction of transmission of tuberculosis in 16 cases within the outbreak (4.7%), including within a multidrug-resistant cluster that carried a rare rpoB mutation associated with rifampicin resistance. Eleven longitudinal pairs of patient pulmonary isolates collected up to 48 mo apart differed from each other by between zero and four SNPs. Extrapulmonary dissemination resulted in acquisition of a SNP in two of five cases. WGS analysis of 27 individual colonies cultured from a single patient specimen revealed ten loci differed amongst them, with a maximum distance between any pair of six SNPs. A limitation of this study, as in previous studies, is that indels and SNPs in repetitive regions were not assessed due to the difficulty in reliably determining this variation.ConclusionsOur study suggests that (1) certain paradigms need to be revised, such as the 12 SNP distance as the gold standard upper threshold to identify plausible transmissions; (2) WGS technology is helpful to rule out the possibility of direct transmission when isolates are separated by a substantial number of SNPs; (3) the concept of a transmission chain or network may not be useful in institutional or household settings; (4) the practice of isolating single colonies prior to sequencing is likely to lead to an overestimation of the number of SNPs between cases resulting from direct transmission; and (5) despite appreciable genomic diversity within a host, transmission of tuberculosis rarely results in minority variants becoming dominant. Thus, whilst WGS provided some increased resolution over variable number tandem repeat (VNTR)-based clustering, it was insufficient for inferring transmission in the majority of cases.

Embryonic Lethality in Homozygous Human Her-2 Transgenic Mice Due to Disruption of the Pds5b Gene.

The development of antigen-targeted therapeutics is dependent on the preferential expression of tumor-associated antigens (TAA) at targetable levels on the tumor. Tumor-associated antigens can be generated de novo or can arise from altered expression of normal basal proteins, such as the up-regulation of human epidermal growth factor receptor 2 (Her2/ErbB2). To properly assess the development of Her2 therapeutics in an immune tolerant model, we previously generated a transgenic mouse model in which expression of the human Her2 protein was present in both the brain and mammary tissue. This mouse model has facilitated the development of Her2 targeted therapies in a clinically relevant and suitable model. While heterozygous Her2+/- mice appear to develop in a similar manner to wild type mice (Her2-/-), it has proven difficult to generate homozygous Her2+/+ mice, potentially due to embryonic lethality. In this study, we performed whole genome sequencing to determine if the integration site of the Her2 transgene was responsible for this lethality. Indeed, we report that the Her2 transgene had integrated into the Pds5b (precocious dissociation of sisters) gene on chromosome 5, as a 162 copy concatemer. Furthermore, our findings demonstrate that Her2+/+ mice, similar to Pds5b-/- mice, are embryonic lethal and confirm the necessity for Pds5b in embryonic development. This study confirms the value of whole genome sequencing in determining the integration site of transgenes to gain insight into associated phenotypes.

Clinical application of whole-genome sequencing in patients with primary immunodeficiency

This report illustrates the value of whole genome sequencing (WGS) in elucidating the genetic cause of disease in patients with primary immunodeficiency (PID). As sequencing costs decline, we predict that utilization of next generation sequencing (NGS) in the clinical setting will increase.

Genomic diversity among eastern and western topotypes of bluetongue virus serotype 16 based on whole genome sequence analysis

Bluetongue (BT) is a noncontagious, vector-borne disease of domestic and wild ruminants. The causative agent bluetongue virus (BTV) is a double stranded RNA virus belonging to genus Orbivirus within family Reoviridae. Eradication of BTV from endemic regions like India is not an easy task due to the widely distributed Culicoides spp. midge vectors, the ubiquitous distribution of vertebrate hosts and existence of a large number of serotypes of the virus (at least 26 till date). The complete genomes (19,193 base-pairs) of several strains of bluetongue virus serotype 16 (BTV-16) originated from Australia, China, Indian subcontinent, Mediterranean basin, Middle East, Africa (Nigeria) and Europe, were compared. These analysis showed that all ten genome segments of a Nigerian strain are derived from a western lineage, showing only 77% - 84% nt identity with the eastern topotype reference strain 'RSArrrr/16' (and its derivative 'RSAvvvv/16', a vaccine strain) that was originally isolated in Pakistan, 76.4% - 83% with eastern BTV-16 strain from Australia (DPP96) and 77% - 89% with a reassortant strain from India. These detailed comparisons involving global strains showed that there is a very high degree of variation (up to 24%) between BTV-16 strains from eastern and western geographical regions. These data confirm the value of whole genome sequencing for characterization of novel BTV isolates and has helped to identify representative suitable 'reference-strain' of eastern topotype (BTV-16e), western topotype (BTV-16w), as well as 'cross-topotype' reassortant strain (BTV-16r) that are generated in the field for further serological, phylogenetic and molecular epidemiology studies.

Next-Generation Sequencing of Lymphoid Cancers: From Discovery to Clinical Translation

AbstractAbstract SCI-12Since completion of the reference human genome and the introduction of next-generation sequencing (NGS) technologies, a number of key discoveries have significantly added to our understanding of the fundamental biology and mutational landscape of the mature B-cell lymphoid malignancies. By the end of 2012, most mature B-lineage lymphoid cancers will have been sequenced to a reasonable number and depth of coverage to inform on the pattern of recurrent mutations, gene fusions, and other somatic genetic events contributing to the biology. Discerning the important driver mutations from passengers and using filters to decide on what alterations to carry through to functional validation will be critical to informing on the biology in a timely manner. The value of whole genome sequencing with respect to noncoding alterations and discovery has already been realized and will become even more apparent when contrasting these data with the more economical exome-based sequencing strategies. These and related topics will be discussed by Dr. Elaine Mardis in her scientific session presentation. In 2010 we saw these technologies lead to the discovery of recurrent activating (gain-of-function) mutations in EZH2, a histone methyltransferase and member of the polycomb repressor 2 complex that is responsible for laying down repressive chromatin marks by trimethylating H3K27. These mutations were found in follicular lymphoma (FL) and in the germinal center B-cell-like subtype of diffuse large B-cell lymphoma (DLBCL). Subsequent genomic studies uncovered loss-of-function mutations in EZH2 in myeloid tumors, contrasting the very different contributions of one gene in these diverse entities. Studies by Pasqualucci, Morin, and colleagues further expanded the role of histone modification and chromatin remodeling in both FL and DLBCL by implicating recurrent mutations in CREBBP, EP300, MLL2, MEF2B, SETD2, and core histone protein genes, including HIST1H1E, HIST1H1D, HIST1H2AC, and HIST1H2BD. RNA-seq technology was used to uncover novel gene fusions in classical Hodgkin lymphoma and the related entity primary mediastinal large B-cell lymphoma that target CIITA, the master regulator of MHC class II expression. Fusion partners in some of the cases involve the ligands of PD-1 and thus establish immune escape as a key oncogenic driver in some lymphoid malignancies. The application of NGS strategies to chronic lymphocytic leukemia, hairy cell leukemia, mantle cell lymphoma, and lymphoplasmacytic lymphoma has revealed a number of recurrent somatic mutations of key genes, including NOTCH1, MYD88, XPO1, KLHL6, SF3B1, and BRAF (V600E) to name but a few. Validation in extension sets and functional studies will be required to fully understand the contribution of these genetic alterations to lymphoma biology and to know which targets are logically suited to the development of targeted therapies. Last, the deployment of these NGS discoveries into the clinical laboratory may take several forms, including targeted resequencing used for diagnosis and subclassification, assessing clonal dominance for sequencing of therapies in some lymphomas, as well as their use in minimal residual disease detection. Disclosures:No relevant conflicts of interest to declare.

Biomarkers for Detecting and Risk-Stratifying Chronic Kidney Disease

tive. At this time, the real value of whole-genome sequencing will be for unusual cases like this one, or for AML patients with intermediate-risk cytogenetic studies, for whom the decision to transplant in first remission is now being honed by an ever more complex clinical diagnostic testing algorithm. Whole-genome sequencing may well play a role in the care of these patients in the near future. Lo-Coco and Sanz also suggest that PML antibody staining could have been used as an alternative diagnostic tool for suspected APL. Although this study could be helpful in selected cases, it is not generally available in the United States as a Clinical Laboratory Improvement Amendments– approved diagnostic test. Further, it would not have resolved the molecular mechanism causing APL in a patient without a detectable t(15;17). Only whole-genome sequencing allowed us to define the precise structure of the genomic event that created the patient’s insertional PML-RARA fusion. The identification of this insertional fusion has allowed for the generation of new fluorescence in situ hybridization probes that will aid in the diagnosis of unusual APL cases like this one in the future.