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Ion semiconductor sequencing. Bridging PCR. Emulsion PCR. Differences between sequencing. Sequencing pipelines and timelines. Third generation sequencing. Advantages of NGS compared with Sanger sequencing. High throughput output Improved resolution Cost effective. Disadvantages of NGS compared with Sanger sequencing. Shorter read lengths Decreased raw accuracy in areas of homology, repeat expansions, large indels, copy number variants CNVs or other structural variants.

Overview of NGS methodology. Library sample preparation : random fragmentation of DNA followed by ligation of common adaptor sequences Sample input: DNA molecules from blood, bone marrow, buccal swab, saliva, formalin fixed paraffin embedded tissue, etc. Data output and analysis. Demultiplexing: pooled patient samples sample libraries that were sequenced simultaneously are then separated by barcodes specific to each patient sample indices Reads are physically clustered together based on sequence similarity, and forward and reverse reads are aligned paired Alignment: Resequencing: sequence reads aligned to reference sequence the reference genome of one individual De novo: sequence reads aligned to each other Interpretation: pathogenic, likely pathogenic, variant of uncertain significance, likely benign, benign J Mol Diagn ; , Genet Med ; Platforms for NGS.

Sequencing mechanism: Ionic current is passed through nanopore proteins Target DNA is then passed through a nanopore and each base alters the current Sequencing occurs in real time as the DNA molecule passes through the nanopore Hairpin library structure allows forward and reverse strands to be sequenced e.

Whole Exome Sequencing

Computer science. Overview: raw sequencing data is translated into individual sequences reads , each read is mapped to its targeted region in the genome and this alignment identifies differences between the sample DNA and the standardized reference Base calling and Demultiplexing: raw format data fluorescence intensity, electrical impulse, etc.

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Clinical applications. Oncology testing: Often utilizes specific gene panels to identify somatic variants or sequence variants specific to hematologic or solid malignancies Assists in diagnosis Offers prognostic information Impacts treatment options targeted therapies BRAF VE mutation is treated with BRAF inhibitor e. Microbiology : Monitoring disease outbreaks by genotyping pathogens Clin Microbiol Infect ; Pharmacogenomics : Link genomic variations to phenotypic differences in drug response or drug-drug interactions Cold Spring Harb Perspect Med ;9:a This Review discusses bioinformatics tools that have been devised to handle the numerous characteristic features of these long-range data types, with applications in genome assembly, genetic variant detection, haplotype phasing, transcriptomics and epigenomics.

Despite the remarkable throughput of next-generation sequencing technologies, standard techniques are limited by the difficulty in distinguishing sequencing errors from genuine low-frequency DNA variants within heterogeneous cellular or molecular populations. This Review discusses sequencing methodologies and bioinformatic strategies that have been devised for the reliable detection of rare mutations and describes various important applications in diverse fields including cancer, ageing and metagenomics.

Although cancer genome sequencing is becoming routine in cancer research, cancer transcriptome profiling through methods such as RNA sequencing RNA-seq provides information not only on mutations but also on their functional cellular consequences. This Review discusses how technical and analytical advances in cancer transcriptomics have provided various clinically valuable insights into gene expression signatures, driver gene prioritization, cancer microenvironments, immuno-oncology and prognostic biomarkers.

Next-generation sequencing has the potential to support public health surveillance systems to improve the early detection of emerging infectious diseases. This Review delineates the role of genomics in rapid outbreak response and the challenges that need to be tackled for genomics-informed pathogen surveillance to become a global reality.

Ancient genomes can inform our understanding of the history of human adaptation through the direct tracking of changes in genetic variant frequency across different geographical locations and time periods. The authors review recent ancient DNA analyses of human, archaic hominin, pathogen, and domesticated animal and plant genomes, as well as the insights gained regarding past human evolution and behaviour. Technical errors can hamper the interpretation of next-generation sequencing NGS data, which poses a major challenge for the clinical application of this technology.

This Review discusses how reference standards circumvent this issue by calibrating NGS measurements and evaluating diagnostic performance of NGS-based genetic tests. Genetic variation of the human Y chromosome plays a key part in studies of human evolution, population history, genealogy, forensics and male medical genetics.

Clinical Applications of Next-Generation Sequencing

This Review outlines how next-generation sequencing has contributed to recent progress in these fields. Next-generation sequencing technologies have enabled the comprehensive characterization of human and mouse genomes, including at the transcriptional level. This article reviews the degree of conservation of human and mouse transcriptomes, along with the challenges of identifying when the mouse is a suitable model of human physiology. A genome sequence is only useful once the information encoded in it can be deciphered. In this Review, Mudge and Harrow describe the latest approaches to higher eukaryote gene annotation, including making the best use of complex transcriptome data sets, integrating evidence for functionality and extending annotations to encompass regulatory features.

Mutation is the source of genetic diversity on which natural selection acts, therefore understanding the rates of mutations is crucial for understanding evolutionary trajectories. In this Opinion article, the authors discuss how emerging experimental mutation-rate data from genome-wide sequencing studies, combined with population-genetic theory, can provide unifying explanations for the diversity in mutation rates between species and across genomic locations.

Clinical Applications for Next-Generation Sequencing | D&R - Kültür, Sanat ve Eğlence Dünyası

Opinion 14 Oct Nature Reviews Genetics. Precision medicine is a strategy for tailoring clinical decision making to the underlying genetic causes of disease. This Review describes how, despite the straightforward overall principles of precision medicine, adopting it responsibly into clinical practice will require many technical and conceptual hurdles to be overcome.

Such challenges include optimized sequencing strategies, clinically focused bioinformatics pipelines and reliable metrics for the disease causality of genetic variants. Marine vertebrates are key contributors to global biodiversity and human food supply. In this Review, the authors discuss how comparative genomics studies in marine vertebrates have provided insight into major evolutionary transitions between the land and sea, as well as intra-species adaptation to diverse types of aquatic environments. They also highlight applications in species management and conservation.

Advances in DNA sequencing technologies have led to vast increases in the diversity of sequencing-based applications and in the amount of data generated. This Review discusses the current state-of-the-art technologies in both short-read and long-read DNA sequencing, their underlying mechanisms, relative strengths and limitations, and emerging applications.

Clinical applications of next generation sequencing in cancer: from panels, to exomes, to genomes

This Review describes the widespread potential applications of RNA-seq in clinical medicine, such as detecting disease-associated mutations and gene expression disruptions, as well as characteristic non-coding RNAs, circulating extracellular RNAs or pathogen RNAs. The authors also highlight the challenges in adopting RNA-seq routinely into clinical practice. Genomic analyses of cancer genomes have largely focused on mutations in protein-coding regions, but the functional importance of alterations to non-coding regions is becoming increasingly appreciated through whole-genome sequencing.

This Review discusses our current understanding of non-coding sequence variants in cancer — both somatic mutations and germline variants, and their interplay — including their identification, computational and experimental evidence for functional impact, and their diverse mechanisms of action for dysregulating coding genes and non-coding RNAs. Single-cell genome sequencing can provide detailed insights into the composition of single genomes that are not readily apparent when studying bulk cell populations.

This Review discusses the considerable technical challenges of amplifying and interrogating genomes from single cells, emerging innovative solutions and various applications in microbiology and human disease, in particular in cancer.

The phenotypic heterogeneity of intellectual disability ID disorders has hampered studies of the underlying genetics, but major progress has been achieved by recent applications of next-generation sequencing. This Review discusses our latest understanding of ID genetics, including the identification of de novo and inherited mutations of various types, strategies for assigning disease causality to the mutations, emerging pathological mechanisms and future research directions.

The wealth of existing and emerging DNA-sequencing data provides an opportunity for a comprehensive understanding of human genetic variation, including the discovery of disease-causing variants. This Review describes how the limitations of current reference-genome assemblies confound the characterization of genetic variation and how this can be mitigated by important advances in algorithms and sequencing technology that facilitate the de novo assembly of genomes.

Sequencing genomes of ancient specimens, including human ancestors, can provide rich insights into evolutionary histories. However, ancient DNA samples are frequently degraded, damaged and contaminated with ancient and modern DNA from various sources. This Review describes the methodological and bioinformatic advances that allow these challenges to be overcome in order to process and sequence ancient samples for genome reconstruction, as well as recent progress in characterizing ancient epigenomes.

High-throughput DNA sequencing technologies are providing an ever-expanding wealth of genome sequence data, including detailed information on human genetic variation. However, such data typically lack haplotype information that is, the cis -connectivity of variants along individual chromosomes. This Review describes diverse recent experimental methods by which genetic variants can be resolved into haplotypes, accompanying computational methods and important applications of these methods in genomics and biomedical science.

Various small molecules, including numerous anticancer agents, act by targeting DNA or protein components of chromatin. This Review describes how various complementary technologies use high-throughput sequencing to delineate drug responses, from identifying the genomic binding sites of drugs or their targets, to the ensuing changes to chromatin states and gene expression.

Clinical Applications for Next-Generation Sequencing

These insights should facilitate the rational use of these therapies. The resolution of epigenomic profiling has been vastly augmented with the adoption of new approaches to interrogate varied features of the epigenome. This Review describes these techniques and outlines the ways in which these genome-wide tools can be used to examine the epigenome. This Review describes how whole-genome sequencing of pooled DNA from many individuals Pool-seq is an economical alternative to sequencing the genomes of individuals separately.

The authors outline the strengths and pitfalls of Pool-seq, and provide example applications across diverse species and biological questions. Forward genetic screens have a long history of uncovering the genetic mutations underlying phenotypes of interest. This Review describes how next-generation sequencing technology can be integrated into forward genetic screens not only to enhance their efficiency but also to allow them to be carried out using expanded repertoires of species, populations and experimental strategies.

Single-cell sequencing of uncultivated microbial species is rapidly providing a wealth of new information. Here, the authors provide an update on recent progress in capturing novel genomes, large-scale environmental studies and research relating to human health, as well as recent methodological improvements and remaining technical challenges.

Progress 5 Aug Nature Reviews Genetics.

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Whole-genome assemblies of humans and non-human primates are yielding data on the evolutionary origins of the human genome, as well as insights into genetic similarities and differences between species used as models for disease-related research. This Review discusses current knowledge and opportunities for comparative primate genomics created by recent advances in genome sequencing technologies. There continues to be active debate about the timings, locations and details of various events in human population history. This Review describes how whole-genome sequencing of modern and ancient humans has complemented more traditional methods to provide valuable historical insights.

Ribosome profiling is a recently developed technique that uses deep sequencing to study translation in vivo. This approach has provided new insights into the identities and amounts of proteins produced by cells, as well as into the mechanism of protein synthesis itself. Innovation 28 Jan Nature Reviews Genetics. Next-generation sequencing for variant identification is now becoming widespread, although pipelines have not yet been optimized. In this Perspective article, the authors discuss ways to minimize erroneous variant calls, in particular, by using replicates.

Opinion 10 Dec Nature Reviews Genetics. Bacterial whole-genome sequencing is showing promise in clinical applications. Here, the authors present their opinions on what the main bioinformatic challenges are in transferring bacterial whole-genome sequencing to medical diagnostics. Opinion 26 Nov Nature Reviews Genetics. Technologies that are based on next-generation sequencing are increasingly being used to study individual cells.

The authors discuss the application of this approach to single-cell genomics and transcriptomics, and explore the implications for both basic research and medicine. Next-generation sequencing is now poised for the discovery of genetic variants involved in common and rare diseases. Here, the authors present considerations for the workflow of these studies in order to identify true associations of disease and mutation. This Review discusses the considerations for designing cancer genome-sequencing studies to fulfil different study aims, such as detecting recurrent mutations or assessing clonal evolution. For example, the cohort type and depth of sequencing can influence the downstream analysis. Clinical sequencing tests that focus on genes linked to specific diseases or phenotypes are increasingly widely being used. This article discusses how disease-targeting tests retain several advantages despite moves towards the clinical application of whole-genome or exome sequencing.

Opinion 12 Mar Nature Reviews Genetics. We asked five experts their opinions on issues that arise from new clinical tests that are based on next-generation sequencing.