Implementation Of Next-generation Sequencing Research Articles

Implementation of cancer next-generation sequencing testing in a community hospital.

In the current era of personalized medicine, the field of oncology is witnessing a paradigm shift in patient care that is driving a tighter integration of genomic analysis modalities in patient care decision. This is driven by the expanding category of targeted therapies that require a broader understanding of the mutational profile of patient samples to more precisely guided personalized treatment decisions. Next-generation sequencing (NGS) has proved to be of tremendous power in detecting and characterizing a broad spectrum of activating or loss-of-function mutations across many gene targets. This power of NGS also results in significant challenges related to technical expertise, bioinformatics, computing infrastructure, laboratory practices, and integration into clinical decision-making. These challenges are particularly relevant to smaller and mid-tier hospital networks that are faced with the need to modernize their clinical practices and offer their patients access to advanced genomic technologies to improve outcomes. Adoption of such personalized medicine relies on information about a patient's cancer genome and the identification of its variants. This is best achieved using NGS. However, there are challenges to the adoption of such a complex technology and workflow, especially in smaller hospital systems. This commentary summarizes key considerations and challenges related to implementation of NGS in a community hospital setting.

New Era for Next-Generation Sequencing in Japan.

Recent progress in understanding the molecular basis of cancer—including the discovery of cancer-associated genes such as oncogenes and tumor suppressor genes—has suggested that cancer can become a treatable disease. The identification of driver oncogenes such as EGFR, ALK, ROS1, BRAF and HER2 has already been successfully translated into clinical practice for individuals with solid tumor. Next-generation sequencing (NGS) technologies have led to the ability to test for multiple cancer-related genes at once with a small amount of cells and tissues. In Japan, several hospitals have started NGS-based mutational profiling screening in patients with solid tumor in order to guide patients to relevant clinical trials. The Ministry of Health, Labor, and Welfare of Japan has also approved several cancer gene panels for use in clinical practice. However, there is an urgent need to develop a medical curriculum of clinical variant interpretation and reporting. We review recent progress in the implementation of NGS in Japan.

Genome-wide association study on ten traits of economic importance in hop (Humulus lupulus L.)

Hop is used primarily for bittering and flavoring of beer although new uses of hop have been implemented in poultry production, sugar processing and pharmaceuticals. Traditional hop breeding is a lengthy, expensive process. Modern selection methods such as marker-assisted selection are needed to greatly improve genetic gains per selection cycle as well as reduce the number of experimental lines evaluated in the field. Recent implementation of next generation sequencing enables the scanning of whole genomes for markers linked to traits of interest using an analysis called genome-wide association studies (GWAS). This study was initiated to identify markers associated with expression of 10 different traits in hop. Illumina Inc. technology sequencing of 116 genetically distinct hop cultivars from multiple regions of the world was performed. SNP marker identification was achieved using the TASSEL v3.0 UNEAK pipeline. Filtering of SNP markers resulted in a working group of 32,206 SNP markers screened across all 116 cultivars. Phenotypic data for all cultivars were collected over a 35-year period and data from four years were used. Mixed linear models analyses in TASSEL v5.0.0 were used for five of the traits while the remaining five traits were evaluated using general linear models. A total of 86 SNP markers were identified and reported as significantly associated with one of the 10 traits. Heritability for traits matched with those previously published. This is the first reported genome wide association study in hop and as such represents an important step forward in aiding marker assisted selection. Nevertheless, it remains that further validation of these SNP markers is needed prior to implementation in a breeding program.

Next-Generation Sequencing for Biodefense: Biothreat Detection, Forensics, and the Clinic.

Next-generation sequencing (NGS) is revolutionizing a variety of molecular biology fields including bioforensics, biosurveillance, and infectious disease diagnostics. For pathogen detection, the ability to sequence all nucleic acids in a sample allows near limitless multiplexability, free from a priori knowledge regarding an etiologic agent as is typically required for targeted molecular assays such as real-time PCR. Furthermore, sequencing capabilities can generate in depth genomic information, allowing detailed molecular epidemiological studies and bioforensics analysis, which is critical for source agent identification in a biothreat outbreak. However, lack of analytical specificity, inherent to NGS, presents challenges for regulated applications such as clinical diagnostics and molecular attribution. Here, we discuss NGS applications in the context of preparedness and biothreat readiness. Specifically, we investigate current and future applications of NGS technologies to affect the fields of biosurveillance, bioforensics, and clinical diagnostics with specific focus on biodefense. Overall, there are many advantages to the implementation of NGS for preparedness and readiness against biowarfare agents, from forensics to diagnostics. However, appropriate caveats must be associated with any technology. This includes NGS. While NGS is not the panacea replacing all molecular techniques, it will greatly enhance the ability to detect, characterize, and diagnose biowarfare agents, thus providing an excellent addition to the biodefense toolbox of biosurveillance, bioforensics, and biothreat diagnosis.

Strengthening laboratory surveillance of viral pathogens: Experiences and lessons learned building next-generation sequencing capacity in Ghana

ObjectiveTo demonstrate the feasibility of applying next-generation sequencing (NGS) in medium-resource reference laboratories in Africa to enhance global disease surveillance. MethodsA training program was developed to support implementation of NGS at Noguchi Memorial Institute for Medical Research (NMIMR), University of Ghana. The program was divided into two training stages, first at the Centers for Disease Control and Prevention (CDC) in Atlanta, GA, followed by on-site training at NMIMR for a larger cohort of scientists. ResultsSelf-assessment scores for topics covered during the NGS training program were higher post-training relative to pre-training. During the NGS Training II session at NMIMR, six enterovirus isolates from acute flaccid paralysis cases in Ghana were successfully sequenced by trainees, including two echovirus 6, two echovirus 11 and one echovirus 13. Another genome was an uncommon type (EV-B84), which has not been reported in Africa since its initial discovery from a Côte d’Ivoire specimen in 2003. ConclusionsThe success at NMIMR provides an example of how to approach transferring of NGS methods to international laboratories. There is great opportunity for collaboration between institutes that have genomics expertise to ensure effectiveness and long-term success of global NGS capacity building programs.

Platforms and Analytical Tools Used in Nucleic Acid Sequence-Based Microbial Genotyping Procedures.

In the decade and a half since the introduction of next-generation sequencing (NGS), the technical feasibility, cost, and overall utility of sequencing have changed dramatically, including applications for infectious disease epidemiology. Massively parallel sequencing technologies have decreased the cost of sequencing by more than 6 orders or magnitude over this time, with a corresponding increase in data generation and complexity. This review provides an overview of the basic principles, chemistry, and operational mechanics of current sequencing technologies, including both conventional Sanger and NGS approaches. As the generation of large amounts of sequence data becomes increasingly routine, the role of bioinformatics in data analysis and reporting becomes all the more critical, and the successful deployment of NGS in public health settings requires careful consideration of changing information technology, bioinformatics, workforce, and regulatory requirements. While there remain important challenges to the sustainable implementation of NGS in public health, in terms of both laboratory and bioinformatics capacity, the impact of these technologies on infectious disease surveillance and outbreak investigations has been nothing short of revolutionary. Understanding the important role that NGS plays in modern public health laboratory practice is critical, as is the need to ensure appropriate workforce, infrastructure, facilities, and funding consideration for routine NGS applications, future innovation, and rapidly scaling NGS-based infectious disease surveillance and outbreak response activities. *This article is part of a curated collection.

The added value of WES reanalysis in the field of genetic diagnosis: lessons learned from 200 exomes in the Lebanese population

BackgroundThe past few decades have witnessed a tremendous development in the field of genetics. The implementation of next generation sequencing (NGS) technologies revolutionized the field of molecular biology and made the genetic information accessible at a large scale. However, connecting a rare genetic variation to a complex phenotype remains challenging. Indeed, identifying the cause of a genetic disease requires a multidisciplinary approach, starting with the establishment of a clear phenotype with a detailed family history and ending, in some cases, with functional assays that are crucial for the validation of the pathogenicity of a mutation.MethodsTwo hundred Lebanese patients, presenting a wide spectrum of genetic disorders (neurodevelopmental, neuromuscular or metabolic disorders, etc.), sporadic or inherited, dominant or recessive, were referred, over the last three and a half years, to the Medical Genetics Unit (UGM) of Saint Joseph University (USJ). In order to identify the genetic basis of these diseases, Whole Exome Sequencing (WES), followed by a targeted analysis, was performed for each case. In order to improve the genetic diagnostic yield, WES data, generated during the first 2 years of this study, were reanalyzed for all patients who were left undiagnosed at the genetic level. Reanalysis was based on updated bioinformatics tools and novel gene discoveries.ResultsOur initial analysis allowed us to identify the specific genetic mutation causing the disease in 49.5% of the cases, in line with other international studies. Repeated WES analysis enabled us to increase the diagnostics yield to 56%.ConclusionThe present article reports the detailed results of both analysis and pinpoints the contribution of WES data reanalysis to an efficient genetic diagnosis. Lessons learned from WES reanalysis and interpretation are also shared.

Signs and Symptoms in Congenital Myopathies

Congenital myopathies (CM) represent a continuously growing group of disorders with a wide range of clinical and histopathologic presentations. The refinement and application of new technologies for genetic diagnosis have broadened our understanding of the genetic causes of CM. Our growing knowledge has revealed that there are no clear limits between each subgroup of CM, and thus the clinical overlap between genes has become more evident. The implementation of next generation sequencing has produced vast amounts of genomic data that may be difficult to interpret. With an increasing number of reports revealing variants of unknown significance, it is essential to support the genetic diagnosis with a well characterized clinical description of the patient. Phenotype-genotype correlation should be a priority at the moment of disclosing the genetic results. Thus, a detailed physical examination can provide us with subtle differences that are not only key in order to arrive at a correct diagnosis, but also in the characterization of new myopathies and candidate genes.

Clinical Validation of Targeted Solid Tumor Profiling.

Large-scale tumor profiling studies have generated massive amounts of data that have been instrumental for the detection of recurrent driver mutations in many tumor types. These driver mutations as well as the concurrent passenger mutations are now being used for a more accurate diagnosis of the tumor and prognosis for the patient. Moreover, therapeutic inhibitors toward specific mutations are already on the market and many clinical trials are ongoing to approve novel therapeutic drugs. The broad-range identification of these somatic mutations is key to this tailored personalized medicine approach, which preferentially has to be performed by a multigene multihotspot method such as massive parallel sequencing, also called next generation sequencing (NGS). The implementation of NGS in molecular diagnostics of tumor profiling however, requires a firm validation to minimize the occurrence of false positives and false negatives, thereby yielding highly accurate and robust clinical data.Here, we describe the different performance characteristics as well as quality metrics that should be analyzed for the robust diagnostic validation of tumor profiling in order to meet the requirements of international standards specific for medical laboratories, such as the ISO15189:2012 standard. These metrics include assays that assess the precision, limit of detection, accuracy, sensitivity, specificity, and robustness of the entire workflow from DNA enrichment up to the final report.

A Novel De Novo Frameshift Mutation in KAT6A Identified by Whole Exome Sequencing.

Intellectual disability is a common condition with multiple etiologies. The number of monogenic causes has increased steadily in recent years due to the implementation of next generation sequencing. Here, we describe a 2-year-old boy with global developmental delay and intellectual disability. The child had feeding difficulties since birth. He had delayed motor skills and muscular hypotonia. Brain magnetic resonance imaging revealed diffuse white matter loss and thinning of the corpus callosum. Banded karyotype and comparative genomic hybridization (CGH) array were normal. Whole exome sequencing revealed a novel de novo frameshift mutation c.3390delA (p.Lys1130Asnfs*4) in KAT6A gene (NM_006766.4). The heterozygous mutation was confirmed by Sanger sequencing in the patient and its absence in his parents. KAT6A that encodes a histone acetyltransferase has been recently found to be associated with a neurodevelopmental disorder autosomal dominant mental retardation 32 (OMIM: no. 616268). Features of this disorder are nonspecific, which makes it difficult to characterize the condition based on the clinical symptoms alone. Therefore, our findings confirm the utility of whole exome sequencing to quickly and reliably identify the etiology of such conditions.

Challenges in data storage and data management in a clinical diagnostic setting

Abstract The implementation of next-generation sequencing (NGS) in a clinical diagnostic setting opens vast opportunities through the ability to sequence all genes contributing to a certain morbidity simultaneously at a cost and speed that is superior to traditional sequencing approaches. On the other hand, the practical implementation of NGS in a clinical diagnostic setting involves a variety of new challenges, which need to be overcome. Among these are the generation, analysis and storage of unprecedented amounts of data, strict control of sequencing performance, validation of results, interpretation of detected variants and reporting. In the following sections, key aspects of data management and integration will be discussed. In particular, issues of data storage, data analysis using in-house IT infrastructure vs. data analysis employing cloud computing and the need for data integration from different sources will be covered.

Is There Value in Molecular Profiling of Soft-Tissue Sarcoma?

Soft-tissue sarcomas represent a heterogeneous group of diseases accounting for up to 1% of cancer in adults and 15% of cancer in children. Introduction of next-generation sequencing (NGS) technologies has allowed to gain additional insight into the genetic diversity and complexity of sarcomas, including the potential therapeutic implications of some genetic alterations.Two large studies have investigated the role of targeted NGS to identify actionable mutations in patients with soft-tissue sarcomas. In these two studies, actionable alterations were identified in up to 50% of patients. Retrospective data suggest that genomically guided treatments may be associated with substantial clinical benefit in sarcoma patients with advanced disease. However, prospective data are lacking. The MULTISARC is a randomized phase 3 investigating the potential of NGS implementation to improve outcome of metastatic sarcoma patients.Overall, a significant proportion of soft-tissue sarcoma bears potential targetable genomic alteration that can be identified through NGS. There is still a lack of evidence to support routine implementation of NGS for the management of sarcoma patient. The MULTISARC randomized trial which randomized patients to tumor genetic profiling or not might confirm the role of NGS to improve outcome of metastatic sarcoma patients through the identification of additional genomically guided lines of treatment.

Precision oncology: separating the wheat from the chaff

Precision oncology based on next-generation sequencing (NGS) test is growing in daily clinical practice. However, the real impact of this strategy in patients’ outcome on a large scale remains uncertain. In this review, we summarise existing literature on this topic, limitations for broad NGS implementation, bottlenecks in genomic variant interpretation and the role of molecular tumour boards.

Challenges in data storage and data management in a clinical diagnostic setting

The implementation of next-generation sequencing (NGS) in a clinical diagnostic setting opens vast opportunities through the ability to sequence all genes contributing to a certain morbidity simultaneously at a cost and speed that is superior to traditional sequencing approaches. On the other hand, the practical implementation of NGS in a clinical diagnostic setting involves a variety of new challenges, which need to be overcome. Among these are the generation, analysis and storage of unprecedented amounts of data, strict control of sequencing performance, validation of results, interpretation of detected variants and reporting. In the following sections, key aspects of data management and integration will be discussed. In particular, issues of data storage, data analysis using in-house IT infrastructure vs. data analysis employing cloud computing and the need for data integration from different sources will be covered.

Abstract 3602: Validation and implementation of next-generation sequencing in liquid biopsies by a novel NGS platform: Focus on non-small cell lung cancer

Abstract Introduction: Liquid biopsy as a novel and non-invasive test for the surveillance of cancer is rapidly growing, by next-generation sequencing (NGS) technologies with multi-gene panels. Objective: The aim of this study was to describe the validation and implementation of a cell free tumor DNA (cfDNA) from the patients with non-small cell lung cancer (NSCLC), to sequence for 19 genes and copy number variation analyses for 5 genes via a novel NGS platform; GeneReader NGS System. Materials and Methods: We performed the complete NGS workflow with modifications, and sequenced the cfDNA extracted from 100 NSCLC patients. The multi-gene panel includes 19 lung cancer related genes (AKT1, ALK, BRAF, DDR, ERBB2, ESR1, KIT, KRAS, MAP2K1, NRAS, NTRK, PDGFRA, PIK3CA, PTEN and ROS) to sequence and additional copy number variations of 5 genes (RICTOR, EGFR, MET, FGFR1 and ERBB2) that all were selected to represent the most actionable genes for the clinical oncology and molecular diagnostics. The sequencing was performed using a novel NGS platform (GeneReader NGS System, Qiagen). Results: The validation process and the implementation of genetic testing from the cfDNA by this novel NGS system have been utilized for the clinical use. After quality assessment, the data from this NGS system was bioinformatically processed and a tertiary analysis was performed using QCI-Analyze and QCI-Interpret bioinformatics tools. The data was then reviewed on alterations and those reported in different mutation databases. The clinically relevant alterations were identified and reported due to the testing indications. Overall, actionable variants in EGFR, ALK, KRAS, PIK3CA, MET, FGFR1 and ERBB2 genes were detected and reported in 62 % of all NSCLC patients while the majority were in EGFR. Conclusions: For each of the 100 samples tested thus far, at least one variant was detected showing the high sensitivity and specificity of both the multi-gene panel and this novel NGS platform. Our evaluation yielded good results for the actionable multi-gene panel selected and provides a comprehensive NGS analysis and workflow for this new platform. Citation Format: Atil Bisgin, Ozge Sonmezler, Ibrahim Boga. Validation and implementation of next-generation sequencing in liquid biopsies by a novel NGS platform: Focus on non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3602.

Whole-exome sequencing in intellectual disability; cost before and after a diagnosis.

Clinical application of whole-exome and whole-genome sequencing (WES and WGS) has led to an increasing interest in how it could drive healthcare decisions. As with any healthcare innovation, implementation of next-generation sequencing in the clinic raises questions on affordability and costing impact for society as a whole. We retrospectively analyzed medical records of 370 patients with ID who had undergone WES at various stages of their diagnostic trajectory. We collected all medical interventions performed on these patients at the University Medical Center Utrecht (UMCU), Utrecht, the Netherlands. We categorized the patients according to their WES-based preliminary diagnosis ("yes", "no", and "uncertain"), and assessed the per-patient healthcare activities and corresponding costs before (pre) and after (post) genetic diagnosis. The WES-specific diagnostic yield among the 370 patients was 35% (128 patients). Pre-WES costs were €7.225 on average. Highest average costs were observed for laboratory-based tests, including genetics, followed by consults. Compared to pre-WES costs, the post-WES costs were on average 80% lower per patient, irrespective of the WES-based diagnostic outcome. Application of WES results in a considerable reduction of healthcare costs, not just in current settings, but even more so when applied earlier in the diagnostic trajectory (genetics-first). In such context, WES may replace less cost-effective traditional technologies without compromising the diagnostic yield. Moreover, WES appears to harbor an intrinsic "end-of-trajectory" effect; regardless of the diagnosis, downstream medical interventions decrease substantially in both number and costs.

GENETICS IN ENDOCRINOLOGY: Genetic diagnosis of endocrine diseases by NGS: novel scenarios and unpredictable results and risks.

The technological advancements in genetics produced a profound impact on the research and diagnostics of non-communicable diseases. The availability of next-generation sequencing (NGS) allowed the identification of novel candidate genes but also an in-depth modification of the understanding of the architecture of several endocrine diseases. Several different NGS approaches are available allowing the sequencing of several regions of interest or the whole exome or genome (WGS, WES or targeted NGS), with highly variable costs, potentials and limitations that should be clearly known before designing the experiment. Here, we illustrate the NGS scenario, describe the advantages and limitations of the different protocols and review some of the NGS results obtained in different endocrine conditions. We finally give insights on the terminology and requirements for the implementation of NGS in research and diagnostic labs.

Genetic analysis of Charcot-Marie-Tooth disease in Denmark and the implementation of a next generation sequencing platform

Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of hereditary polyneuropathies. Variants in more than 80 different genes have been associated with the disorder. In recent years, the introduction of next generation sequencing (NGS) techniques have completely changed the genetic diagnostic approach from the analysis of a handful of genes to the analysis of all genes associated with CMT in a single run. In this study we describe the CMT diagnostics in Denmark in 1992–2012, prior to the implementation of NGS, by combining laboratory- and national registry data. We investigate the effect of implementing a targeted NGS approach of 63 genes associated with CMT in the diagnostic laboratory setting. This was performed by analyzing a cohort of 195 samples from patients previously analyzed by Sanger sequencing and quantitative analysis for the common causes of CMT without reaching a molecular diagnosis.A total of 1442 CMT analyses were performed in Denmark in the period 1992–2012; a disease-causing variant was detected in 21.6% of the cases. Interestingly, the diagnosis was genetically confirmed in significantly more women than men; 25.9% compared to18.5%. In our study cohort, we found a 5.6% increase in the diagnostic yield with the introduction of a targeted NGS approach.

Hereditary Breast and Ovarian Cancer Syndrome: Moving Beyond BRCA1 and BRCA2.

The recent implementation of next generation sequencing and multigene platforms has expanded the spectrum of hereditary breast and ovarian cancer syndrome, beyond the traditional genes BRCA1 and BRCA2. A large number of other moderate penetrance genes have now been uncovered, which also play critical roles in repairing double stranded DNA breaks through the homologous recombination pathway. This review discusses the landmark discoveries of BRCA1 and BRCA2, the homologous repair pathway and new genes discovered in hereditary breast and ovarian cancer syndrome, as well as their clinicopathologic significance and implications for genetic testing. It also highlights the new role of PARP inhibitors in the context of synthetic lethality and prophylactic surgical options.

NGS-FC: A Next-Generation Sequencing Data Format Converter

With the widespread implementation of next-generation sequencing (NGS) technologies, millions of sequences have been produced. A lot of databases were created to store and organize the high-throughput sequencing data. Numerous analysis software programs and tools have been developed over the past years. Most of them use specific formats for data representation and storage. Data interoperability becomes a crucial challenge and many tools have been developed to convert NGS data from one format to another. However, most of them were developed for specific and limited formats. Here, we present NGS-FC (Next-Generation Sequencing Format Converter), which provides a framework to support the conversion between several formats. It supports 14 formats now and provides interfaces to enable users to improve the existing converters and add new ones. Moreover, NGS-FC achieved the overall competitive performance in comparison with some existing converters in terms of RAM usage and running time. The software is written in Java and can be executed standalone. The source code and documentation are freely available at http://sysbio.suda.edu.cn/NGS-FC.