The identification and isolation of genes underlying quantitative trait loci (QTLs) associated with agronomic traits in crops have been recently accelerated thanks to next-generation sequencing (NGS)-based technologies combined with plant genetics. With NGS, various revisited genetic approaches, which benefited from higher marker density, have been elaborated. These approaches improved resolution in QTL position and assisted in determining functional causative variations in genes. Examples of QTLs/genes associated with agronomic traits in crops and identified using different strategies based on whole-genome sequencing (WGS)/whole-genome resequencing (WGR) or RNA-seq are presented and discussed in this review. More specifically, we summarize and illustrate how NGS boosted bulk-segregant analysis (BSA), expression profiling, and the construction of polymorphism databases to facilitate the detection of QTLs and causative genes.

• Keywords
• bulk-segregant analysis, candidate gene, crops, genetics, next-generation sequencing, quantitative trait loci,
• MeSH
• Phenotype MeSH
• Genetic Association Studies MeSH
• Polymorphism, Single Nucleotide MeSH
• Quantitative Trait Loci * MeSH
• Chromosome Mapping MeSH
• High-Throughput Nucleotide Sequencing * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

Molecular assays for translocation detection in different tumors have gradually been incorporated into routine diagnostics. However, conventional methods such as fluorescence in situ hybridization (FISH) and reverse transcriptase-PCR come with several drawbacks. Next-generation sequencing (NGS) can provide in-depth detection of numerous gene alterations. The anchored multiplex PCR assay proved to be a fast and easy-to-analyze approach for routine diagnostics laboratories. Next-generation sequencing-based anchored multiplex PCR technique (Archer FusionPlex Panels) is beneficial in both diagnosis for patient care and in identification of a novel fusion breakpoint in tumors. NGS is useful in identifying targetable molecular changes (point mutations, fusion genes, etc.) in tumors that can serve as a rationale for inclusion of patients with advanced disease in ongoing clinical trials and allow for better risk stratification.

• Keywords
• Fusion genes, next generation sequencing (NGS), somatic mutations, targeted therapy,
• MeSH
• In Situ Hybridization, Fluorescence MeSH
• Humans MeSH
• Multiplex Polymerase Chain Reaction MeSH
• Neoplasms * diagnosis   genetics   MeSH
• Translocation, Genetic MeSH
• High-Throughput Nucleotide Sequencing * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

PURPOSE: Preimplantation genetic testing for monogenic disorders (PGT-M) allows early diagnosis in embryos conceived in vitro. PGT-M helps to prevent known genetic disorders in affected families and ensures that pathogenic variants in the male or female partner are not passed on to offspring. The trend in genetic testing of embryos is to provide a comprehensive platform that enables robust and reliable testing for the causal pathogenic variant(s), as well as chromosomal abnormalities that commonly occur in embryos. In this study, we describe PGT protocol that allows direct mutation testing, haplotyping, and aneuploidy screening. METHODS: Described PGT protocol called OneGene PGT allows direct mutation testing, haplotyping, and aneuploidy screening using next-generation sequencing (NGS). Whole genome amplification product is combined with multiplex PCR used for SNP enrichment. Dedicated bioinformatic tool enables mapping, genotype calling, and haplotyping of informative SNP markers. A commercial software was used for aneuploidy calling. RESULTS: OneGenePGT has been implemented for seven of the most common monogenic disorders, representing approximately 30% of all PGT-M indications at our IVF centre. The technique has been thoroughly validated, focusing on direct pathogenic variant testing, haplotype identification, and chromosome abnormality detection. Validation results show full concordance with Sanger sequencing and karyomapping, which were used as reference methods. CONCLUSION: OneGene PGT is a comprehensive, robust, and cost-effective method that can be established for any gene of interest. The technique is particularly suitable for common monogenic diseases, which can be performed based on a universal laboratory protocol without the need for set-up or pre-testing.

• Keywords
• Aneuploidy, Monogenic disorders, Next-generation sequencing, Preimplantation genetic testing,
• MeSH
• Aneuploidy MeSH
• Blastocyst pathology   MeSH
• Genetic Testing methods   MeSH
• Humans MeSH
• Mutation genetics   MeSH
• Preimplantation Diagnosis * methods   MeSH
• Pregnancy MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Pregnancy MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

Genetic causes of canine mast cell tumours (MCTs), except for mutations in the KIT gene detected in some MCTs, are generally unknown. We used whole exome sequencing to reveal mutation spectra in canine MCTs. We detected somatic mutations in 87 genes including 10 genes recognized as human cancer drivers. Besides KIT, 14 other genes were recurrently mutated. Subsequently, we performed next generation sequencing of a panel of 50 selected genes in additional MCT samples. In this group, the most frequently altered gene was GNB1 showing a recurrent dinucleotide substitution at position of Gly116 in 30% of the MCT samples (n = 6/20) and Ile80 substitution accompanied by a splice region mutation in one case. We extended the study by analysis of the above mentioned GNB1 regions in additional MCT samples by Sanger sequencing, and assessed the overall prevalence of GNB1 mutations to 17.3% (n = 14/81), which is similar to the prevalence of KIT alterations. Our results indicate that GNB1 mutations are probably involved in canine MCT pathogenesis in both cutaneous and subcutaneous MCT cases. As opposed to KIT alterations, the presence of GNB1 mutations did not negatively affect survival times, and our data even showed a trend towards positive prognosis. If our results are confirmed in a larger number of MCTs, an extension of molecular testing of canine MCTs by GNB1 analysis would help to refine the molecular stratification of MCTs, and become useful for targeted treatment strategies.

• Keywords
• GNB1, KIT, cancer, dog, mast cell tumour, mutation, next generation sequencing, whole exome sequencing,
• MeSH
• Mast-Cell Sarcoma genetics   pathology   veterinary   MeSH
• Mast Cells pathology   MeSH
• Mutation MeSH
• Dog Diseases genetics   pathology   MeSH
• GTP-Binding Protein beta Subunits genetics   MeSH
• Proto-Oncogene Proteins c-kit genetics   MeSH
• Dogs MeSH
• High-Throughput Nucleotide Sequencing veterinary   MeSH
• Animals MeSH
• Check Tag
• Dogs MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• GTP-Binding Protein beta Subunits MeSH
• Proto-Oncogene Proteins c-kit MeSH

INTRODUCTION: Driver mutations in Philadelphia chromosome-negative myeloproliferative neoplasms are well known. In the past, whole-genome sequencing identified nondriver mutations in other genes, potentially contributing to evolution of malignant clones. METHODS: Next-generation sequencing was used to assess the presence of any mutations in 14 candidate genes at the point of diagnosis and the resultant impact on the clinical course of the disease. RESULTS: The study analysed 63 patients with myelofibrosis (MF). Nondriver mutations were detected in 44% of them. The most frequently affected genes were ASXL1 (27%), TET2 (11%) and SF3B1 (6%). The frequency of such mutations was highest in primary MF (59%) and lowest in the prefibrotic phase of primary MF (21%). Patients with prognostically unfavourable sequence variants in genes had significantly worse overall survival (53 vs 71 months; HR = 2.77; 95% CI 1.17-6.56; P = .017). CONCLUSION: In our study, multivariate analysis proved DIPSS to be the only significant factor to predict patient survival. DIPSS contains all of the important clinical and laboratory factors except genetic changes. Stratification of patients according to DIPSS is still beneficial although there are newer and improved scoring systems like GIPSS or MIPSS70. Assessing subclonal mutations in candidate genes during diagnosis may aid in the identification of high-risk MF patients and is therefore relevant for making a prediction for overall survival more accurate.

• Keywords
• myelofibrosis, next-generation sequencing, prognostic stratification,
• MeSH
• Dioxygenases genetics   MeSH
• DNA-Binding Proteins genetics   MeSH
• Adult MeSH
• Phosphoproteins genetics   MeSH
• Middle Aged MeSH
• Humans MeSH
• Mutation MeSH
• Primary Myelofibrosis genetics   MeSH
• Repressor Proteins genetics   MeSH
• Aged MeSH
• RNA Splicing Factors genetics   MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• ASXL1 protein, human MeSH Browser
• Dioxygenases MeSH
• DNA-Binding Proteins MeSH
• Phosphoproteins MeSH
• Repressor Proteins MeSH
• RNA Splicing Factors MeSH
• SF3B1 protein, human MeSH Browser
• TET2 protein, human MeSH Browser

The aims of the study were: i) to compare circulating tumor DNA (ctDNA) yields obtained by different manual extraction procedures, ii) to evaluate the addition of various carrier molecules into the plasma to improve ctDNA extraction recovery, and iii) to use next generation sequencing (NGS) technology to analyze KRAS, BRAF, and NRAS somatic mutations in ctDNA from patients with metastatic colorectal cancer. Venous blood was obtained from patients who suffered from metastatic colorectal carcinoma. For plasma ctDNA extraction, the following carriers were tested: carrier RNA, polyadenylic acid, glycogen, linear acrylamide, yeast tRNA, salmon sperm DNA, and herring sperm DNA. Each extract was characterized by quantitative real-time PCR and next generation sequencing. The addition of polyadenylic acid had a significant positive effect on the amount of ctDNA eluted. The sequencing data revealed five cases of ctDNA mutated in KRAS and one patient with a BRAF mutation. An agreement of 86% was found between tumor tissues and ctDNA. Testing somatic mutations in ctDNA seems to be a promising tool to monitor dynamically changing genotypes of tumor cells circulating in the body. The optimized process of ctDNA extraction should help to obtain more reliable sequencing data in patients with metastatic colorectal cancer.

• Keywords
• Carrier, Circulating tumor DNA, Extraction, Next generation sequencing, Real-time PCR,
• MeSH
• DNA, Neoplasm blood   genetics   isolation & purification   MeSH
• Colorectal Neoplasms blood   diagnosis   genetics   pathology   MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Middle Aged MeSH
• Humans MeSH
• Neoplasm Metastasis MeSH
• DNA Mutational Analysis MeSH
• Prognosis MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Carrier Proteins blood   MeSH
• High-Throughput Nucleotide Sequencing * MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Neoplasm MeSH
• Carrier Proteins MeSH

BACKGROUND: Primary ciliary dyskinesia (PCD) is a multigenic autosomal recessive condition affecting respiratory tract and other organs where ciliary motility is required. The extent of its genetic heterogeneity is remarkable. The aim of the study was to develop a cost-effective pipeline for genetic diagnostics using a combination of Sanger and next generation sequencing (NGS). MATERIALS AND METHODS: Data and samples of 33 families with 38 affected subjects with PCD diagnosed in childhood were collected over the territory of the Czech Republic. A panel of 18 PCD causative or candidate genes was implemented into an Illumina TruSeq Custom Amplicon NGS assay, and three ancestral mutations in SPAG1 were screened by conventional Sanger sequencing, which was also used for the confirmation of the NGS results and for the analysis of familial segregation. RESULTS: The causative gene was DNAH5 in 11/33 (33%) probands, SPAG1 in 8/33 (24%), and DNAI1, CCDC40, LRRC6 in one family each. If the high proportion of subjects with bi-allelic ancestral mutations in SPAG1 is corroborated in other Caucasian populations, a simple Sanger sequencing test for these three mutations may serve as an effective pre-screening step, being followed by an NGS panel for other, much larger, PCD genes. CONCLUSIONS: We present a combination of Sanger sequencing with an NGS panel for known and candidate PCD genes, implemented in a moderate-size national collection of patients. This strategy has proven to be cost-effective, rapid and reliable, and was able to detect the causative gene in two thirds of our PCD patients.

• Keywords
• SPAG1 gene, next generation sequencing, targeted panel,
• MeSH
• Alleles MeSH
• Antigens, Surface genetics   MeSH
• Child MeSH
• Kartagener Syndrome diagnosis   genetics   MeSH
• Infant MeSH
• Humans MeSH
• Adolescent MeSH
• Mutation * MeSH
• Child, Preschool MeSH
• GTP-Binding Proteins genetics   MeSH
• High-Throughput Nucleotide Sequencing * MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic MeSH
• Names of Substances
• Antigens, Surface MeSH
• GTP-Binding Proteins MeSH
• SPAG1 protein, human MeSH Browser

AIM: The primary goal was to determine the yield of next-generation sequencing (NGS) epilepsy gene panels used for epilepsy etiology diagnosing using a multidisciplinary approach and to demonstrate the importance of genotype-phenotype correlations. The secondary goal was to evaluate the application of precision medicine in selected patients. METHODS: This single-center retrospective study included a total of 175 patients (95 males and 80 females) aged 0-19 years. They were examined between 2015 and 2020 using an NGS epilepsy gene panel (270 genes). A bioinformatic analysis was performed including copy number variation identification. Thorough genotype-phenotype correlation was performed. RESULTS: Out of 175 patients, described pathogenic variants or novel variants with clear pathogenic impact were identified in 30 patients (17.14%). Genotype-phenotype correlations and parental DNA analysis were performed, and genetic diagnosis was confirmed on the basis of the results in another 16 out of 175 patients (9.14%). The diagnostic yield of our study increased from 30 to 46 patients (by 53.33%) by the precise genotype-phenotype correlation. INTERPRETATION: We emphasize a complex genotype-phenotype correlation and a multidisciplinary approach in evaluating the results of the NGS epilepsy gene panel, which enables the most accurate genetic diagnosis and correct interpretation of results.

• Keywords
• Children, Epilepsy, Genetic testing, Next-generation sequencing, Precise medicine,
• MeSH
• Epilepsy * diagnosis   genetics   MeSH
• Phenotype MeSH
• Genetic Association Studies MeSH
• Genetic Testing methods   MeSH
• Humans MeSH
• Mutation MeSH
• Retrospective Studies MeSH
• DNA Copy Number Variations * MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Neuromuscular diseases (NMDs) are a clinically and genetically heterogeneous group of diseases. Currently, 608 genes associated with different types of NMD have been identified. Most of these diseases are rare with a very low prevalence. Advance in the identification of genes associated with NMD can be attributed to technological development in an area of next generation sequencing (NGS) and the affordability of this methodical approach. NGS applications can be divided into analysis of (a) a selected set of genes, (b) an exom, and (c) a genome. The identification of pathogenic variants leads to a significant shift in the understanding of the etiopathogenesis of the disease, allows the prediction of the course of the disease, or its targeted treatment, which may be specific for individual types of NMD or even for particular pathogenic sequence variants.

• Keywords
• NGS, diagnosis, diagnostics, neuromuscular diseases,
• MeSH
• Humans MeSH
• Neuromuscular Diseases * diagnosis   genetics   MeSH
• High-Throughput Nucleotide Sequencing * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Molecular genetic methods have evolved dramatically in the last decade. In particular, sequencing of the new generation - NGS has become a financially and technically available examination. Thus, it begins to be used in the clinical practice of many specializations, including oncology. The proper use of these methods is a way to personalized oncology - treatment of the patient based on the occurrence of specific genetic aberrations, which are confirmed in his cancer, regardless of the histopathological type of tumor. This principle is completely new in oncology and raises number of questions and problems. The interpretation of the results of molecular genetic examinations is very complex and demanding, and therefore new multidisciplinary teams, so-called molecular tumor boards, are being created. The worldwide standardization of these boards is currently underway. Recommendations regarding the indication of NGS examinations in oncology patients are also being set.At the European level in the form of ESMO recommendations and at the national level also. Personalized oncology is the future of this field, which will lead to the best treatment response and minimize side effects.

• Keywords
• NGS, lung cancer, molecular tumor board, next generation sequencing, personalized therapy, precision oncology,
• MeSH
• Precision Medicine MeSH
• Humans MeSH
• Neoplasms * genetics   MeSH
• Oncologists * MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH