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Genes Aug 2022Pyrosequencing is one of the technologies widely used for quantitative methylation assessment. The protocol of pyrosequencing experiment consists of PCR amplification of...
Pyrosequencing is one of the technologies widely used for quantitative methylation assessment. The protocol of pyrosequencing experiment consists of PCR amplification of a locus of interest and subsequent sequencing via synthesis of the amplified PCR product. As the PCR in this protocol utilizes one primer set for the amplification of a template originating from both methylated and non-methylated versions of the analysed locus, the unequal amplification of one of the templates may affect the methylation level assessment by pyrosequencing. We have investigated whether the unequal amplification of one of the templates challenges the quantitative properties of the pyrosequencing technology. Our results show that the sensitivity and dynamic range of pyrosequencing can be significantly affected by unequal amplification of the methylated and non-methylated version of the locus of interest in an assay specific manner. Thus, the assessment of the effect of unequal template amplification on the performances of the specific pyrosequencing assay is necessary before using the assay either in research or especially in diagnostic settings.
Topics: High-Throughput Nucleotide Sequencing; Nucleic Acid Amplification Techniques; Polymerase Chain Reaction
PubMed: 36011328
DOI: 10.3390/genes13081418 -
Molecules (Basel, Switzerland) Feb 2018Microsatellites, or simple sequence repeats (SSRs), are one of the most informative and multi-purpose genetic markers exploited in plant functional genomics. However,... (Review)
Review
Microsatellites, or simple sequence repeats (SSRs), are one of the most informative and multi-purpose genetic markers exploited in plant functional genomics. However, the discovery of SSRs and development using traditional methods are laborious, time-consuming, and costly. Recently, the availability of high-throughput sequencing technologies has enabled researchers to identify a substantial number of microsatellites at less cost and effort than traditional approaches. Illumina is a noteworthy transcriptome sequencing technology that is currently used in SSR marker development. Although 454 pyrosequencing datasets can be used for SSR development, this type of sequencing is no longer supported. This review aims to present an overview of the next generation sequencing, with a focus on the efficient use of de novo transcriptome sequencing (RNA-Seq) and related tools for mining and development of microsatellites in plants.
Topics: Arabidopsis; Crops, Agricultural; Data Mining; Expressed Sequence Tags; Genetic Markers; High-Throughput Nucleotide Sequencing; Microsatellite Repeats; Molecular Sequence Annotation; Oryza; Polymorphism, Genetic; Software; Transcriptome
PubMed: 29438290
DOI: 10.3390/molecules23020399 -
British Journal of Haematology Mar 2013Next-generation sequencing platforms have evolved to provide an accurate and comprehensive means for the detection of molecular mutations in heterogeneous tumour... (Review)
Review
Next-generation sequencing platforms have evolved to provide an accurate and comprehensive means for the detection of molecular mutations in heterogeneous tumour specimens. Here, we review the feasibility and practicality of this novel laboratory technology. In particular, we focus on the utility of next-generation sequencing technology in characterizing haematological neoplasms and the landmark findings in key haematological malignancies. We also discuss deep-sequencing strategies to analyse the constantly increasing number of molecular markers applied for disease classification, patient stratification and individualized monitoring of minimal residual disease. Although many facets of this assay need to be taken into account, amplicon deep-sequencing has already demonstrated a promising technical performance and is being continuously developed towards routine application in diagnostic laboratories so that an impact on clinical practice can be achieved.
Topics: Hematology; High-Throughput Nucleotide Sequencing; Humans; Molecular Diagnostic Techniques; Sequence Analysis, DNA
PubMed: 23294427
DOI: 10.1111/bjh.12194 -
Revue Scientifique Et Technique... Apr 2016Viral genome sequencing has become the cornerstone of almost all aspects of virology. In particular, high-throughput, next-generation viral genome sequencing has become... (Review)
Review
Viral genome sequencing has become the cornerstone of almost all aspects of virology. In particular, high-throughput, next-generation viral genome sequencing has become an integral part of molecular epidemiological investigations into outbreaks of viral disease, such as the recent outbreaks of Middle Eastern respiratory syndrome, Ebola virus disease and Zika virus infection. Multiple institutes have acquired the expertise and necessary infrastructure to perform such investigations, as evidenced by the accumulation of thousands of novel viral sequences over progressively shorter time periods. The authors recently proposed a nomenclature comprised of five high-throughput sequencing standard categories to describe the quality of determined viral genome sequences. These five categories (standard draft, high quality, coding complete, complete and finished) cover all levels of viral genome finishing and can be applied to sequences determined by any technology platform or assembly technique.
Topics: Animals; Genome, Viral; High-Throughput Nucleotide Sequencing; Molecular Epidemiology; Terminology as Topic; Virus Diseases; Viruses
PubMed: 27217167
DOI: 10.20506/rst.35.1.2416 -
Value in Health : the Journal of the... Sep 2018
Topics: High-Throughput Nucleotide Sequencing; Humans
PubMed: 30224105
DOI: 10.1016/j.jval.2018.06.012 -
Science China. Life Sciences Feb 2013
Topics: Computational Biology; Epigenesis, Genetic; Genome, Human; High-Throughput Nucleotide Sequencing; Humans; Precision Medicine; Sequence Analysis, RNA
PubMed: 23393024
DOI: 10.1007/s11427-013-4436-x -
Biotechnology & Genetic Engineering... 2010Next-generation sequencing technologies have revolutionized our ability to identify genetic variants, either germline or somatic point mutations, that occur in cancer.... (Review)
Review
Next-generation sequencing technologies have revolutionized our ability to identify genetic variants, either germline or somatic point mutations, that occur in cancer. Parallelization and miniaturization of DNA sequencing enables massive data throughput and for the first time, large-scale, nucleotide resolution views of cancer genomes can be achieved. Systematic, large-scale sequencing surveys have revealed that the genetic spectrum of mutations in cancers appears to be highly complex with numerous low frequency bystander somatic variations, and a limited number of common, frequently mutated genes. Large sample sizes and deeper resequencing are much needed in resolving clinical and biological relevance of the mutations as well as in detecting somatic variants in heterogeneous samples and cancer cell sub-populations. However, even with the next-generation sequencing technologies, the overwhelming size of the human genome and need for very high fold coverage represents a major challenge for up-scaling cancer genome sequencing projects. Assays to target, capture, enrich or partition disease-specific regions of the genome offer immediate solutions for reducing the complexity of the sequencing libraries. Integration of targeted DNA capture assays and next-generation deep resequencing improves the ability to identify clinically and biologically relevant mutations.
Topics: DNA, Neoplasm; Genome, Human; High-Throughput Nucleotide Sequencing; Humans; Mutation; Neoplasms; Sequence Analysis, DNA
PubMed: 21415896
DOI: 10.1080/02648725.2010.10648148 -
Briefings in Bioinformatics Sep 2018Currently, there is a lack of software for detecting copy number variations and constructing copy number profile for the whole genome from single-cell DNA sequencing...
Currently, there is a lack of software for detecting copy number variations and constructing copy number profile for the whole genome from single-cell DNA sequencing data, which are often of low coverage and high technical noises. Here we introduce a new toolkit, SCNV, which features an efficient bin-free segmentation approach and provides the highest resolution possible for breakpoint detection and the subsequent copy number calling. SCNV can auto-tune parameters based on a set of normal cells from the same batch to adjust for the technical noise level of the data, facilitating its application to data gathered from different platforms and different studies.
Topics: Computational Biology; DNA Copy Number Variations; DNA, Neoplasm; Genome, Human; High-Throughput Nucleotide Sequencing; Humans; Neoplasms; Ploidies; Sequence Analysis, DNA; Single-Cell Analysis; Software
PubMed: 28159966
DOI: 10.1093/bib/bbx004 -
Pediatrics and Neonatology Feb 2023Genetic condition is one of the major etiologies causing morbidity and mortality in infants and children. More and more etiologies can be solved using next-generation...
Genetic condition is one of the major etiologies causing morbidity and mortality in infants and children. More and more etiologies can be solved using next-generation sequencing (NGS) as it develops. Currently, whole-exome sequencing (WES) and whole-genome sequencing (WGS) have been highly integrated into clinical practice. The average diagnostic yield of WES/WGS in pediatric patients with genetic condition was around 40% (range: 21%-80%), with acceptable turnaround time and cost. The higher diagnostic yield categories are deafness, ophthalmic, neurological, skeletal conditions, and inborn error of metabolism. Positive results provide benefit with those for actionable diseases, next pregnancy planning, and family members. For those in critical condition, with the emergence of sequencing technology and bioinformatics analysis tools, provisional diagnosis can be made as short as 13.5 h using ultrarapid WGS. We believe this powerful tool has changed pediatric daily practice.
Topics: Infant; Humans; Child; Genome, Human; High-Throughput Nucleotide Sequencing
PubMed: 36456424
DOI: 10.1016/j.pedneo.2022.11.002 -
Oncotarget Aug 2016Nowadays, the personalized approach to health care and cancer care in particular is becoming more and more popular and is taking an important place in the translational... (Review)
Review
Nowadays, the personalized approach to health care and cancer care in particular is becoming more and more popular and is taking an important place in the translational medicine paradigm. In some cases, detection of the patient-specific individual mutations that point to a targeted therapy has already become a routine practice for clinical oncologists. Wider panels of genetic markers are also on the market which cover a greater number of possible oncogenes including those with lower reliability of resulting medical conclusions. In light of the large availability of high-throughput technologies, it is very tempting to use complete patient-specific New Generation Sequencing (NGS) or other "omics" data for cancer treatment guidance. However, there are still no gold standard methods and protocols to evaluate them. Here we will discuss the clinical utility of each of the data types and describe a systems biology approach adapted for single patient measurements. We will try to summarize the current state of the field focusing on the clinically relevant case-studies and practical aspects of data processing.
Topics: Biomarkers, Tumor; High-Throughput Nucleotide Sequencing; Humans; Medical Oncology; Neoplasms; Pharmacogenetics; Precision Medicine; Systems Biology
PubMed: 27191992
DOI: 10.18632/oncotarget.9370