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Advantages and challenges of clinical NGS: The advantages and challenges associated Advantages and challenges of clinical NGS: The advantages and challenges with implementation of next-generation sequencing technologies in a clinical molecular diagnostic associated with implementation of next-generation sequencing technologies in a clinical laboratory have been summarized.
Source publication
The application of next-generation sequencing (NGS) to characterize cancer genomes has resulted in the discovery of numerous genetic markers. Consequently, the number of markers that warrant routine screening in molecular diagnostic laboratories, often from limited tumor material, has increased. This increased demand has been difficult to manage by...
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... summarize, NGS technologies, which represent a major revolution in genome sequencing, are able to meet the challenges associated with the increased need for routine mutation profiling of tumors. However, the high complexity of this technology and performance validation poses distinct challenges for successful adaptation in the clinical diagnostic environment (summarized in Figure 1). Increased clarity regarding the validation and implementation of NGS tests by several regulatory agencies, published reports from several clinical laboratories and technological improvements have made the implementation of NGS technologies more feasible, thus establishing them as the most preferred large-scale genome sequencing ...
Citations
... The technology's scalability, speed, and ability to detect variants at lower allele frequencies have revolutionized genetic analysis, enabling new applications in genomic and clinical research, reproductive health, and other scientific fields [18]. NGS data analysis comprises primary, secondary, and tertiary steps, with bioinformatics specialists playing a crucial role in interpreting results and extracting meaningful information from the data [19]. ...
... NGS has facilitated major discoveries in the field of genomics, including the identification of new genes, the characterization of genetic variation, and the development of new diagnostic tests (28). NGS is also being used to develop new personalized treatments for cancer and other diseases (12,29). ...
... NGS has been utilized within clinical practices to identify cancer-related genetic mutations (cancer genomics) (29). Diagnosing genetic diseases and determining the genetic variants that cause them provides information needed facilitate genetic counselling for respective patients. ...
... This technique has been successfully employed in the diagnosis of cystic fibrosis (CF), SCA and Down syndrome (DS). NGS has also provided invaluable information in cancer drug trials, by identifying the specific genetic mutations causing the tumors to develop resistance to specific treatment plans (29). Additionally, hospitals have employed NGS to identify and characterize many microorganisms, providing data that is useful in the development of new diagnostic tests and treatments for infectious diseases (30). ...
Precision medicine (PM) represents a transformative approach in
healthcare, tailoring treatments based on individual genetic and molecular profiles. This shift from a
generalized to personalized care model revolutionizes healthcare. Here, we aim to explore the evolving role
of clinical laboratories within PM in reshaping healthcare delivery and enhancing patient-specific treatments.
... The unprecedented throughput of NGS and the nature of the sequencing technology brought a whole host of challenges too, notably: highly sophisticated workflow, bioinformatics, sequencing accuracy, costly with limited access, and operational competency [3][4][5]. Getting access to large sample cohorts is another challenge. Given the widespread availability of exome and genome sequencing, samples are the new commodity. ...
Background: When it comes to developing new cancer diagnostics (Dx) and therapeutics (Rx), time is of the essence-for the patients that need treatments most, every day counts. Traditional benchmarks of Dx and Rx have delivered favorable clinical outcomes on a slower timeline than patients can afford.
... On the other hand, full DPYD gene sequencing can provide a more comprehensive assessment of genetic variability, detecting both common and rare variants. However, performing AS-PCR is generally more costeffective compared to sequencing the entire DPYD gene (21). Full gene sequencing may also introduce challenges in interpretation, as not all DPYD variants have known clinical significance. ...
Background
Fluoropyrimidine drugs are widely used in chemotherapy to treat solid tumors. However, severe toxicity has been reported in 10% to 40% of patients. The DPYD gene encodes the rate-limiting enzyme dihydropyrimidine dehydrogenase responsible for fluoropyrimidine catabolism. The DPYD variants resulting in decreased or no enzyme activity are associated with increased risk of fluoropyrimidine toxicity. This study aims to develop a pharmacogenetic test for screening DPYD variants to guide fluoropyrimidine therapy.
Methods
A multiplex allele-specific polymerase chain reaction (AS-PCR) assay, followed by capillary electrophoresis, was developed to detect 5 common DPYD variants (c.557A > G, c.1129–5923C > G, c.1679T > G, c.1905 + 1G > A, and c.2846A > T). Deidentified population samples were used for screening positive controls and optimizing assay conditions. Proficiency testing samples with known genotypes were analyzed for test validation. All variants detected were confirmed by Sanger sequencing.
Results
From the deidentified population samples, 5 samples were heterozygous for c.557A > G, 2 samples were heterozygous for c.1129–5923C > G (HapB3), and 1 sample was heterozygous for c.2846A > T. The 20 proficiency samples matched with their assigned genotypes, including 13 wild-type samples, 3 samples heterozygous for c.1679T > G, 2 samples heterozygous for c.1905 + 1G > A, and 2 samples heterozygous for c.2846A > T. One of the 3 patient samples was heterozygous for c.1129–5923C > G (HapB3). All the variants detected by the multiplex AS-PCR assay were concordant with Sanger sequencing results.
Conclusions
A robust multiplex AS-PCR assay was developed to rapidly detect 5 variants in the DPYD gene. It can be used for screening DPYD variants to identify patients with increased risk of toxicity when prescribed fluoropyrimidine therapy.
... Nevertheless, challenges persist. Currently, comprehensive next-generation sequencing (NGS) analyses are not widely accessible or as yet integrated into the standard workflow of many institutions [2]. ...
... Failure to state the authenticity of large deletions or duplications in genes (copy number variations) can lead to serious disorders. Therefore, cross-confirmatory tests are recommended to ascertain the CNV and its effect on the patient's phenotype, so that treatments can be planned accordingly 52 . ...
Clinical diagnosis of several neurodegenerative disorders based on clinical phenotype is challenging due to its heterogeneous nature and overlapping disease manifestations. Therefore, the identification of underlying genetic mechanisms is of paramount importance for better diagnosis and therapeutic regimens. With the emergence of next-generation sequencing, it becomes easier to identify all gene variants in the genome simultaneously, with a system-wide and unbiased approach. Presently various bioinformatics databases are maintained on discovered gene variants and phenotypic indications are available online. Since individuals are unique in their genome, evaluation based on their genetic makeup helps evolve the diagnosis, counselling, and treatment process at the personal level. This article aims to briefly summarize the utilization of next-generation sequencing in deciphering the genetic causes of Alzheimer’s disease and address the limitations of whole genome and exome sequencing.
Keywords:
Neurodegenerative Diseases; High-Throughput Nucleotide Sequencing; Alzheimer Disease; Exome Sequencing; Whole Genome Sequencing
... Unlike other targeted diagnostic methods such as quantitative PCR or amplicon sequencing that are looking for a few gene mutations, most MT models utilize machine learning to identify hundreds of molecular features associated with an indication 2,3,11 . While adequate positive or negative control materials can readily be generated for targeted methodologies by introducing the specific mutations or genes of interest, this is not feasible with the complex signals seen in MT disease models [12][13][14] . Control material is an essential component of any test as it serves as a known reference point for the performance of the test and helps to ensure accuracy and reproducibility. ...
Metatranscriptomics (MT), or RNA sequencing, has the potential to revolutionize the field of molecular diagnostics. Due to the complexity of MT diagnostic models, positive and negative control materials for specific disease indications can be difficult to obtain. Controls must often be sourced directly from patients. This introduces logistical burdens, assay variability, and limits high throughput clinical laboratory operations. To overcome this limitation, we developed a method for generating Synthetic Control (SC) samples, which duplicate the nucleic acid signature of complex clinical specimens and produce the desired test outcome. SCs can be easily and cost-effectively produced in large quantities (>100,000 SCs per amplification cycle), enabling high throughput diagnostic testing. Here, we report the generation of Synthetic Positive Control (SPC) samples. SPCs were validated and implemented in a clinical laboratory. The SPCs produced robust positive signals (average OC risk score of 0.997) and high levels of reproducibility (%CV of 0.2%) in a high throughput automated CLIA laboratory. SCs are a novel and useful method for the generation of high quality controls for MT-based diagnostic tests, and their adoption could herald the widespread use of MT tests in molecular diagnostics.
... Most molecular diagnostic laboratories have replaced single gene assays with massively parallel high-throughput sequencing, termed next-generation sequencing (NGS), to analyse tumour samples for clinically relevant somatic variants. [13] This technique has changed the sequencing landscape because it allows simultaneous interrogation of multiple genes in several samples with limited amounts of DNA. This improvement in diagnostics saves both time and cost and increases diagnostic yield with its massive sequencing capacity. ...
... This improvement in diagnostics saves both time and cost and increases diagnostic yield with its massive sequencing capacity. [13] It is possible to sequence a large number of genes and even whole genomes even though the turnaround time is shorter and the cost is more affordable. [14,15] The challenge is no longer sequencing, but the evaluation and interpretation of the read-out to guide diagnostic, prognostic and treatment decisions. ...
Knowledge of an underlying genetic predisposition to cancer allows the use of personalised prognostic, preventive and therapeutic strategies for the patient and carries clinical implications for family members. Despite great progress, we identified six challenging areas in the management of patients with hereditary cancer predisposition syndromes and suggest recommendations to aid in their resolution. These include the potential for finding unexpected germline variants through somatic tumour testing, optimal risk management of patients with hereditary conditions involving moderate-penetrance genes, role of polygenic risk score in an under-represented Asian population, management of variants of uncertain significance, clinical trials in patients with germline pathogenic variants and technology in genetic counselling. Addressing these barriers will aid the next step forward in precision medicine in Singapore. All stakeholders in healthcare should be empowered with genetic knowledge to fully leverage the potential of novel genomic insights and implement them to provide better care for our patients.
... Diagnosis of low-level mosaic TSC is challenging because of the inability of routine genetic testing to detect mosaic pathogenic variants in TSC1/TSC2 in the blood and subtle clinical findings associated with mosaic TSC. However, as NGS becomes increasingly available for commercial use, it is likely to play an important role in personalized medicine in future clinical practice and may enhance the detection of mosaicism (Gullapalli, 2020;Gullapalli et al., 2012;Luthra et al., 2015;Roy et al., 2016). ...
Skin findings can be critical to determine whether a patient with lymphangioleiomyomatosis (LAM), a progressive pulmonary disease that predominantly affects adult women, has sporadic disease (S-LAM) or LAM in association with tuberous sclerosis complex (TSC-LAM). Three individuals with LAM underwent evaluation for TSC-associated mucocutaneous and internal findings. We used our previously published algorithm to confirm clinical suspicion for mosaicism and guide selection of tissue specimens and genetic workup. Next-generation sequencing (NGS) of cutaneous findings was used to confirm clinical suspicion for mosaic TSC in individuals with LAM. Two individuals previously thought to have S-LAM were diagnosed with mosaic TSC-LAM upon NGS of unilateral angiofibromas in one and an unusual cutaneous hamartoma in the other. A third individual, diagnosed with TSC in childhood, was found to have a mosaic pathogenic variant in TSC2 in cutaneous tissue from a digit with macrodactyly. Accurate diagnosis of mosaic TSC-LAM may require enhanced genetic testing, and is important due to implications regarding surveillance, prognosis, and risk of transmission to offspring.
... In recent years, this technology has become more accessible and has led to extensive studies on cancers 10 . NGS results have improved our understanding of tumorigenesis pathways, providing a rational basis for drug development and individualized treatments 11 . As a result, the number of specific gene aberrations tests is increasing rapidly. ...
