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The Increasing Role of Next Generation Sequencing Methods in Mutation Analysis

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A special issue of Life (ISSN 2075-1729). This special issue belongs to the section "Genetics and Genomics".

Deadline for manuscript submissions: closed (30 June 2021) | Viewed by 41745

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Special Issue Editor

Dr. István Balogh

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Guest Editor

Division of Clinical Genetics, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
Interests: genetic testing; rare diseases; monogenic diseases; next-generation sequencing

Special Issue Information

Dear colleagues,

The implementation of next-generation DNA sequencing techniques has revolutionized molecular diagnostics. In the middle of an era where we are moving towards a more simplified workflow of mutation analysis, a lot of issues arise. There is a need for standardized wet lab and in silico analysis methods. In the case of exome and genome sequencing-based diagnosis, we have to deal with incidental findings and also with the evidence-based pharmacogenetic data that might be included in the personal healthcare database of the patient. The classification of detected mutations is also inevitable. Mutation-specific treatment is more and more widespread. Altogether, these improvements open up possibilities not only for personalized genome-based medicine, but also for predictive medicine as well.

Molecular genetic diagnostics involves both germline and somatic mutation analysis; therefore, this Special Issue of the journal Life, covering the topic "The Increasing Role of Next Generation Sequencing Methods in Mutation Analysis" is open for research papers and reviews from both major fields of genetic testing, i.e., the analysis of inherited disorders and cancers.

Dr. István Balogh
Guest Editor

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Keywords

next-generation sequencing
molecular genetic diagnostics
monogenic disorders
mutation analysis
predictive medicine

Published Papers (13 papers)

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Research

Jump to: Review

5 pages, 193 KiB

Open AccessCommunication

Current Status of Next-Generation Sequencing-Based Cancer Genome Profiling Tests in Japan and Prospects for Liquid Biopsy

by Yumi Yoshii, Shunsuke Okazaki and Masayuki Takeda

Life 2021, 11(8), 796; https://doi.org/10.3390/life11080796 - 06 Aug 2021

Cited by 10 | Viewed by 2492

Abstract

Next-generation sequencing-based comprehensive genome profiling (CGP) testing, OncoGuide NCC Oncopanel System, and FoundationOne CDx Cancer Genomic Profile have been covered by the Japanese national health insurance system since June 2019. Because CGP was initially developed to enroll patients into an early-phase clinical trial for solid tumors, its approved indications have been limited to patients who have completed the standard chemotherapy treatment. Approximately 14,000 cases have been registered with the Center for Cancer Genomics and Advanced Therapeutics as of March 2021. Measuring the drug access rate is not enough due to patients’ deteriorating condition during CGP analysis and due to the limited number of ongoing clinical trials available, although tumor-agnostic therapies, such as the use of pembrolizumab in high microsatellite-instable solid tumors and in conditions with a high tumor mutational burden (≥10 mut/Mb) as well as the use of entrectinib and larotrectinib in NTRK fusion-positive tumors have been approved in Japan. Moreover, since this analysis is performed using DNA derived from tumor tissue, it is difficult to perform CGP in cases in which an insufficient amount of tissue exists. Thus, noninvasive blood-based assays have been developed, and CGP panels using circulating tumor DNA from blood were approved in March 2021. However, cost, timing, and the number of tests allowed by the health system have not yet been determined. Therefore, in this review, we outline the current status and issues of CGP testing using tumor tissues as well as the expectations and limitations of liquid biopsy for use in Japanese clinical practice. Full article

(This article belongs to the Special Issue The Increasing Role of Next Generation Sequencing Methods in Mutation Analysis)

20 pages, 520 KiB

Open AccessArticle

A Comprehensive Analysis of Hungarian MODY Patients—Part II: Glucokinase MODY Is the Most Prevalent Subtype Responsible for about 70% of Confirmed Cases

by Zsolt Gaál, Zsuzsanna Szűcs, Irén Kántor, Andrea Luczay, Péter Tóth-Heyn, Orsolya Benn, Enikő Felszeghy, Zsuzsanna Karádi, László Madar and István Balogh

Life 2021, 11(8), 771; https://doi.org/10.3390/life11080771 - 30 Jul 2021

Cited by 4 | Viewed by 2299

Abstract

MODY2 is caused by heterozygous inactivating mutations in the glucokinase (GCK) gene that result in persistent, stable and mild fasting hyperglycaemia (5.6–8.0 mmol/L, glycosylated haemoglobin range of 5.6–7.3%). Patients with GCK mutations usually do not require any drug treatment, except during pregnancy. The GCK gene is considered to be responsible for about 20% of all MODY cases, transcription factors for 67% and other genes for 13% of the cases. Based on our findings, GCK and HNF1A mutations together are responsible for about 90% of the cases in Hungary, this ratio being higher than the 70% reported in the literature. More than 70% of these patients have a mutation in the GCK gene, this means that GCK-MODY is the most prevalent form of MODY in Hungary. In the 91 index patients and their 72 family members examined, we have identified a total of 65 different pathogenic (18) and likely pathogenic (47) GCK mutations of which 28 were novel. In two families, de novo GCK mutations were detected. About 30% of the GCK-MODY patients examined were receiving unnecessary OAD or insulin therapy at the time of requesting their genetic testing, therefore the importance of having a molecular genetic diagnosis can lead to a major improvement in their quality of life. Full article

(This article belongs to the Special Issue The Increasing Role of Next Generation Sequencing Methods in Mutation Analysis)

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13 pages, 434 KiB

Open AccessArticle

A Comprehensive Analysis of Hungarian MODY Patients—Part I: Gene Panel Sequencing Reveals Pathogenic Mutations in HNF1A, HNF1B, HNF4A, ABCC8 and INS Genes

Life 2021, 11(8), 755; https://doi.org/10.3390/life11080755 - 27 Jul 2021

Cited by 7 | Viewed by 2194

Abstract

Maturity-onset diabetes of the young (MODY) has about a dozen known causal genes to date, the most common ones being HNF1A, HNF4A, HNF1B and GCK. The phenotype of this clinically and genetically heterogeneous form of diabetes depends on the gene in which the patient has the mutation. We have tested 450 Hungarian index patients with suspected MODY diagnosis with Sanger sequencing and next-generation sequencing and found a roughly 30% positivity rate. More than 70% of disease-causing mutations were found in the GCK gene, about 20% in the HNF1A gene and less than 10% in other MODY-causing genes. We found 8 pathogenic and 9 likely pathogenic mutations in the HNF1A gene in a total of 48 patients and family members. In the case of HNF1A-MODY, the recommended first-line treatment is low dose sulfonylurea but according to our data, the majority of our patients had been on unnecessary insulin therapy at the time of requesting their genetic testing. Our data highlights the importance of genetic testing in the diagnosis of MODY and the establishment of the MODY subtype in order to choose the most appropriate treatment. Full article

(This article belongs to the Special Issue The Increasing Role of Next Generation Sequencing Methods in Mutation Analysis)

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16 pages, 2873 KiB

Open AccessArticle

Cross-Validation of Next-Generation Sequencing Technologies for Diagnosis of Chromosomal Mosaicism and Segmental Aneuploidies in Preimplantation Embryos Model

by Anil Biricik, Ettore Cotroneo, Maria Giulia Minasi, Pier Francesco Greco, Sara Bono, Matteo Surdo, Federica Lecciso, Mariateresa Sessa, Francesco Fiorentino, Francesca Spinella and Ermanno Greco

Life 2021, 11(4), 340; https://doi.org/10.3390/life11040340 - 12 Apr 2021

Cited by 8 | Viewed by 2963

Abstract

Detection of mosaic embryos is crucial to offer more possibilities of success to women undergoing in vitro fertilization (IVF) treatment. Next Generation Sequencing (NGS)-based preimplantation genetic testing are increasingly used for this purpose since their higher capability to detect chromosomal mosaicism in human embryos. In the recent years, new NGS systems were released, however their performance for chromosomal mosaicism are variable. We performed a cross-validation analysis of two different NGS platforms in order to assess the feasibility of these techniques and provide standard parameters for the detection of such aneuploidies. The study evaluated the performance of Miseq^TM Veriseq (Illumina, San Diego, CA, USA) and Ion Torrent Personal Genome Machine PGM^TM ReproSeq (Thermo Fisher, Waltham, MA, USA) for the detection of whole and segmental mosaic aneuploidies. Reconstructed samples with known percentage of mosaicism were analyzed with both platforms and sensitivity and specificity were determined. Both platforms had high level of specificity and sensitivity with a Limit Of Detection (LOD) at ≥30% of mosaicism and a showed a ≥5.0 Mb resolution for segmental abnormalities. Our findings demonstrated that NGS methodologies are capable of accurately detecting chromosomal mosaicism and segmental aneuploidies. The knowledge of LOD for each NGS platform has the potential to reduce false-negative and false-positive diagnoses when applied to detect chromosomal mosaicism in a clinical setting. Full article

(This article belongs to the Special Issue The Increasing Role of Next Generation Sequencing Methods in Mutation Analysis)

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12 pages, 867 KiB

Open AccessArticle

Cross-Disorder Analysis of De Novo Variants Increases the Power of Prioritising Candidate Genes

by Kuokuo Li, Zhengbao Ling, Tengfei Luo, Guihu Zhao, Qiao Zhou, Xiaomeng Wang, Kun Xia, Jinchen Li and Bin Li

Life 2021, 11(3), 233; https://doi.org/10.3390/life11030233 - 12 Mar 2021

Cited by 1 | Viewed by 1790

Abstract

De novo variants (DNVs) are critical to the treatment of neurodevelopmental disorders (NDDs). However, effectively identifying candidate genes in small cohorts is challenging in most NDDs because of high genetic heterogeneity. We hypothesised that integrating DNVs from multiple NDDs with genetic similarity can significantly increase the possibility of prioritising the candidate gene. We catalogued 66,186 coding DNVs in 50,028 individuals with nine types of NDDs in cohorts with sizes spanning from 118 to 31,260 from Gene4Denovo database to validate this hypothesis. Interestingly, we found that integrated DNVs can effectively increase the number of prioritised candidate genes for each disorder. We identified 654 candidate genes including 481 shared candidate genes carrying putative functional variants in at least two disorders. Notably, 13.51% (65/481) of shared candidate genes were prioritised only via integrated analysis including 44.62% (29/65) genes validated in recent large cohort studies. Moreover, we estimated that more novel candidate genes will be prioritised with the increase in cohort size, in particular for some disorders with high putative functional DNVs per individual. In conclusion, integrated DNVs may increase the power of prioritising candidate genes, which is important for NDDs with small cohort size. Full article

(This article belongs to the Special Issue The Increasing Role of Next Generation Sequencing Methods in Mutation Analysis)

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10 pages, 235 KiB

Open AccessArticle

Whole-Genome Sequencing in Diagnostics of Selected Slovenian Undiagnosed Patients with Rare Disorders

by Gaber Bergant, Aleš Maver and Borut Peterlin

Life 2021, 11(3), 205; https://doi.org/10.3390/life11030205 - 05 Mar 2021

Cited by 5 | Viewed by 2247

Abstract

Several patients with rare genetic disorders remain undiagnosed following comprehensive diagnostic testing using whole-exome sequencing (WES). In these patients, pathogenic genetic variants may reside in intronic or regulatory regions or they may emerge through mutational mechanisms not detected by WES. For this reason, we implemented whole-genome sequencing (WGS) in routine clinical diagnostics of patients with undiagnosed genetic disorders and report on the outcome in 30 patients. Criteria for consideration included (1) negative WES, (2) a high likelihood of a genetic cause for the disorders, (3) positive family history, (4) detection of large blocks of homozygosity or (5) detection of a single pathogenic variant in a gene associated with recessive conditions. We successfully discovered a causative genetic variant in 6 cases, a retrotranspositional event in the APC gene, non-coding variants in the intronic region of the OTC gene and the promotor region of the UFM1 gene, repeat expansion in the RFC1 gene and a single exon duplication in the CNGB3 gene. We also discovered one coding variant, an indel, which was missed by variant caller during WES data analysis. Our study demonstrates the impact of WGS in the group of patients with undiagnosed genetic diseases after WES in the clinical setting and the diversity of mutational mechanisms discovered, which would remain undetected using other methods. Full article

(This article belongs to the Special Issue The Increasing Role of Next Generation Sequencing Methods in Mutation Analysis)

23 pages, 2059 KiB

Open AccessArticle

Resolving Differential Diagnostic Problems in von Willebrand Disease, in Fibrinogen Disorders, in Prekallikrein Deficiency and in Hereditary Hemorrhagic Telangiectasia by Next-Generation Sequencing

by Réka Gindele, Adrienne Kerényi, Judit Kállai, György Pfliegler, Ágota Schlammadinger, István Szegedi, Tamás Major, Zsuzsanna Szabó, Zsuzsa Bagoly, Csongor Kiss, János Kappelmayer and Zsuzsanna Bereczky

Life 2021, 11(3), 202; https://doi.org/10.3390/life11030202 - 05 Mar 2021

Cited by 3 | Viewed by 2874

Abstract

Diagnosis of rare bleeding disorders is challenging and there are several differential diagnostics issues. Next-generation sequencing (NGS) is a useful tool to overcome these problems. The aim of this study was to demonstrate the usefulness of molecular genetic investigations by summarizing the diagnostic work on cases with certain bleeding disorders. Here we report only those, in whom NGS was indicated due to uncertainty of diagnosis or if genetic confirmation of initial diagnosis was required. Based on clinical and/or laboratory suspicion of von Willebrand disease (vWD, n = 63), hypo-or dysfibrinogenemia (n = 27), hereditary hemorrhagic telangiectasia (HHT, n = 10) and unexplained activated partial thromboplastin time (APTT) prolongation (n = 1), NGS using Illumina platform was performed. Gene panel covered 14 genes (ACVRL1, ENG, MADH4, GDF2, RASA1, F5, F8, FGA, FGB, FGG, KLKB1, ADAMTS13, GP1BA and VWF) selected on the basis of laboratory results. We identified forty-seven mutations, n = 29 (6 novel) in vWD, n = 4 mutations leading to hemophilia A, n = 10 (2 novel) in fibrinogen disorders, n = 2 novel mutations in HHT phenotype and two mutations (1 novel) leading to prekallikrein deficiency. By reporting well-characterized cases using standardized, advanced laboratory methods we add new pieces of data to the continuously developing “bleeding disorders databases”, which are excellent supports for clinical patient management. Full article

(This article belongs to the Special Issue The Increasing Role of Next Generation Sequencing Methods in Mutation Analysis)

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14 pages, 7247 KiB

Open AccessArticle

A Rapid PCR-Free Next-Generation Sequencing Method for the Detection of Copy Number Variations in Prenatal Samples

by Xiya Zhou, Xiangbin Chen, Yulin Jiang, Qingwei Qi, Na Hao, Chengkun Liu, Mengnan Xu, David S. Cram and Juntao Liu

Life 2021, 11(2), 98; https://doi.org/10.3390/life11020098 - 28 Jan 2021

Cited by 8 | Viewed by 2469

Abstract

Next-generation sequencing (NGS) is emerging as a new method for the detection of clinically significant copy number variants (CNVs). In this study, we developed and validated rapid CNV-sequencing (rCNV-seq) for clinical application in prenatal diagnosis. Low-pass whole-genome sequencing was performed on PCR libraries prepared from amniocyte genomic DNA. From 10–40 ng of input DNA, PCR-free libraries consistently produced sequencing data with high unique read mapping ratios, low read redundancy, low coefficient of variation for all chromosomes and high genomic coverage. In validation studies, reliable and accurate CNV detection using PCR-free-based rCNV-seq was demonstrated for a range of common trisomies and sex chromosome aneuploidies as well as microdeletion and duplication syndromes. In reproducibility studies, CNV copy number and genomic intervals closely matched those defined by chromosome microarray analysis. Clinical testing of genomic DNA samples from 217 women referred for prenatal diagnosis identified eight samples (3.7%) with known chromosome disorders. We conclude that PCR-free-based rCNV-seq is a sensitive, specific, reproducible and efficient method that can be used in any NGS-based diagnostic laboratory for detection of clinically significant CNVs. Full article

(This article belongs to the Special Issue The Increasing Role of Next Generation Sequencing Methods in Mutation Analysis)

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9 pages, 765 KiB

Open AccessArticle

The Effect of Synonymous Single-Nucleotide Polymorphisms on an Atypical Cystic Fibrosis Clinical Presentation

by Giovana B. Bampi, Anabela S. Ramalho, Leonardo A. Santos, Johannes Wagner, Lieven Dupont, Harry Cuppens, Kris De Boeck and Zoya Ignatova

Life 2021, 11(1), 14; https://doi.org/10.3390/life11010014 - 27 Dec 2020

Cited by 5 | Viewed by 2443

Abstract

Synonymous single nucleotide polymorphisms (sSNPs), which change a nucleotide, but not the encoded amino acid, are perceived as neutral to protein function and thus, classified as benign. We report a patient who was diagnosed with cystic fibrosis (CF) at an advanced age and presented very mild CF symptoms. The sequencing of the whole cystic fibrosis transmembrane conductance regulator (CFTR) gene locus revealed that the patient lacks known CF-causing mutations. We found a homozygous sSNP (c.1584G>A) at the end of exon 11 in the CFTR gene. Using sensitive molecular methods, we report that the c.1584G>A sSNP causes cognate exon skipping and retention of a sequence from the downstream intron, both of which, however, occur at a relatively low frequency. In addition, we found two other sSNPs (c.2562T>G (p.Thr854=) and c.4389G>A (p.Gln1463=)), for which the patient is also homozygous. These two sSNPs stabilize the CFTR protein expression, compensating, at least in part, for the c.1584G>A-triggered inefficient splicing. Our data highlight the importance of considering sSNPs when assessing the effect(s) of complex CFTR alleles. sSNPs may epistatically modulate mRNA and protein expression levels and consequently influence disease phenotype and progression. Full article

(This article belongs to the Special Issue The Increasing Role of Next Generation Sequencing Methods in Mutation Analysis)

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18 pages, 264 KiB

Open AccessArticle

Clinical Evaluation of a Custom Gene Panel as a Tool for Precision Male Infertility Diagnosis by Next-Generation Sequencing

by Rossella Cannarella, Vincenza Precone, Giulia Guerri, Gian Maria Busetto, Gian Carlo Di Renzo, Sandro Gerli, Elena Manara, Astrit Dautaj, Matteo Bertelli and Aldo Eugenio Calogero

Life 2020, 10(10), 242; https://doi.org/10.3390/life10100242 - 15 Oct 2020

Cited by 12 | Viewed by 2757

Abstract

Background: Up to 15% of couples are infertile and male factor infertility accounts for approximately 50% of these cases. Male infertility is a multifactorial pathological condition. The genetic of male infertility is very complex and at least 2000 genes are involved in its etiology. Genetic testing by next-generation sequencing (NGS) technologies can be relevant for its diagnostic value in male infertile patients. Therefore, the aim of this study was to implement the diagnostic offer with the use of an NGS panel for the identification of genetic variants. Methods: We developed an NGS gene panel that we used in 22 male infertile patients. The panel consisted of 110 genes exploring the genetic causes of male infertility; namely spermatogenesis failure due to single-gene mutations, central hypogonadism, androgen insensitivity syndrome, congenital hypopituitarism, and primary ciliary dyskinesia. Results: NGS and a subsequent sequencing of the positive pathogenic or likely pathogenic variants, 5 patients (23%) were found to have a molecular defect. In particular, pathogenic variants were identified in TEX11, CCDC39, CHD7, and NR5A1 genes. Moreover, 14 variants of unknown significance and 7 novel variants were found that require further functional studies and family segregation. Conclusion: This extended NGS-based diagnostic approach may represent a useful tool for the diagnosis of male infertility. The development of a custom-made gene panel by NGS seems capable of reducing the proportion of male idiopathic infertility. Full article

(This article belongs to the Special Issue The Increasing Role of Next Generation Sequencing Methods in Mutation Analysis)

Review

Jump to: Research

12 pages, 1737 KiB

Open AccessReview

Liquid Biopsy, ctDNA Diagnosis through NGS

by Chen Lin, Xuzhu Liu, Bingyi Zheng, Rongqin Ke and Chi-Meng Tzeng

Life 2021, 11(9), 890; https://doi.org/10.3390/life11090890 - 28 Aug 2021

Cited by 37 | Viewed by 6103

Abstract

Liquid biopsy with circulating tumor DNA (ctDNA) profiling by next-generation sequencing holds great promise to revolutionize clinical oncology. It relies on the basis that ctDNA represents the real-time status of the tumor genome which contains information of genetic alterations. Compared to tissue biopsy, liquid biopsy possesses great advantages such as a less demanding procedure, minimal invasion, ease of frequent sampling, and less sampling bias. Next-generation sequencing (NGS) methods have come to a point that both the cost and performance are suitable for clinical diagnosis. Thus, profiling ctDNA by NGS technologies is becoming more and more popular since it can be applied in the whole process of cancer diagnosis and management. Further developments of liquid biopsy ctDNA testing will be beneficial for cancer patients, paving the way for precision medicine. In conclusion, profiling ctDNA with NGS for cancer diagnosis is both biologically sound and technically convenient. Full article

(This article belongs to the Special Issue The Increasing Role of Next Generation Sequencing Methods in Mutation Analysis)

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17 pages, 708 KiB

Open AccessReview

Next-Generation Sequencing Technologies and Neurogenetic Diseases

by Hui Sun, Xiao-Rong Shen, Zi-Bing Fang, Zong-Zhi Jiang, Xiao-Jing Wei, Zi-Yi Wang and Xue-Fan Yu

Life 2021, 11(4), 361; https://doi.org/10.3390/life11040361 - 19 Apr 2021

Cited by 11 | Viewed by 5937

Abstract

Next-generation sequencing (NGS) technology has led to great advances in understanding the causes of Mendelian and complex neurological diseases. Owing to the complexity of genetic diseases, the genetic factors contributing to many rare and common neurological diseases remain poorly understood. Selecting the correct genetic test based on cost-effectiveness, coverage area, and sequencing range can improve diagnosis, treatments, and prevention. Whole-exome sequencing and whole-genome sequencing are suitable methods for finding new mutations, and gene panels are suitable for exploring the roles of specific genes in neurogenetic diseases. Here, we provide an overview of the classifications, applications, advantages, and limitations of NGS in research on neurological diseases. We further provide examples of NGS-based explorations and insights of the genetic causes of neurogenetic diseases, including Charcot–Marie–Tooth disease, spinocerebellar ataxias, epilepsy, and multiple sclerosis. In addition, we focus on issues related to NGS-based analyses, including interpretations of variants of uncertain significance, de novo mutations, congenital genetic diseases with complex phenotypes, and single-molecule real-time approaches. Full article

(This article belongs to the Special Issue The Increasing Role of Next Generation Sequencing Methods in Mutation Analysis)

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21 pages, 1119 KiB

Open AccessReview

The Application of Next-Generation Sequencing to Define Factors Related to Oral Cancer and Discover Novel Biomarkers

by Soyeon Kim, Joo Won Lee and Young-Seok Park

Life 2020, 10(10), 228; https://doi.org/10.3390/life10100228 - 02 Oct 2020

Cited by 21 | Viewed by 3769

Abstract

Despite the introduction of next-generation sequencing in the realm of DNA sequencing technology, it is not often used in the investigation of oral squamous cell carcinoma (OSCC). Oral cancer is one of the most frequently occurring malignancies in some parts of the world and has a high mortality rate. Patients with this malignancy are likely to have a poor prognosis and may suffer from severe facial deformity or mastication problems even after successful treatment. Therefore, a thorough understanding of this malignancy is essential to prevent and treat it. This review sought to highlight the contributions of next-generation sequencing (NGS) in unveiling the genetic alterations and differential expressions of miRNAs involved in OSCC progression. By applying an appropriate eligibility criterion, we selected relevant studies for review. Frequently identified mutations in genes such as TP53, NOTCH1, and PIK3CA are discussed. The findings of existing miRNAs (e.g., miR-21) as well as novel discoveries pertaining to OSCC are also covered. Lastly, we briefly mention the latest findings in targeted gene therapy and the potential use of miRNAs as biomarkers. Our goal is to encourage researchers to further adopt NGS in their studies and give an overview of the latest findings of OSCC treatment. Full article

(This article belongs to the Special Issue The Increasing Role of Next Generation Sequencing Methods in Mutation Analysis)

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