Clinical Applications for Next-Generation Sequencing


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Payors are looking for price points to come down so that a global adoption is financially feasible. We are facing the need to educate a wide range of healthcare specialists involved in designing, conducting, interpreting, and utilizing genetic tests: translational researchers, pathologists, geneticists, genetic counselors, biostatisticians, etc. There is a plethora of new tests in the development stage that require a massive computing resource to deal with a sheer amount of data. The storage requirements for a whole genome, depending on the coverage, range between and GB for a single person.

Added to that are the results of the variant analysis and other datasets generated as part of analysis. For a broader-scale usage, these datasets and their interpretation must be part of the patient record. This requires investment in an infrastructure to provide, gather, store, research, and clinically interpret this data on a large scale that is not currently in place.

All tests require patient consent. Because of this, a more global adoption of genetic tests is also dependent on patients agreeing to use their DNA for this purpose. The genetic testing technology and infrastructure are evolving quickly.

There are a few areas where adoption is expected first with significant testing volume: oncology, rare disease diagnosis, and pediatrics and newborn screening. Beyond these areas, there is definite potential in areas such as obesity, diabetes, and cardiac disorders. In addition to this, there is a considerable uptick in the adoption expected in the field of pharmacogenomics to determine safety, efficacy, and cost of care.

New applications of genetic testing will result in changes to current care teams and processes. It will reshape how pathologists, oncologists, geneticists, genetic counselors, biostatisticians, and bioinformaticians work together. Based on the current trajectories, it will take us well into the next 5 to 10 years to clear the hurdles characteristic of the moderate adoption phase. So, what are the top-level issues that need to be addressed once we reach a stage of full adoption?

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

Here are some of the most pressing topics:. In , there were 3,, births just in the United States. Applying routine tests on every newborn will by itself require a massively scalable and highly automated infrastructure that surpasses everything we have established in hospitals and testing labs across the nation.

Our understanding of genetically caused diseases increases with the statistical power of the underlying dataset. Once we have a highly scalable infrastructure and testing operation installed on a nationwide basis, we are in a position to generate massive datasets that can be mined for undiscovered associations. This will also allow us to verify previous findings that were made using much smaller datasets. The diagnosis and treatment of disease ultimately require that we can integrate data from multiple platforms such as imaging, biosensors, and predictive analytics—just to name a few.

For thousands of years, the smallest thing humans could detect was about as wide as a human hair. This changed with the invention of compound microscope toward the end of the s. Over time, the microscope became a cornerstone of clinical diagnosis. It has been only a little over 40 years since the invention of DNA sequencing and a little over 15 years since the completion of the Human Henome Project. However, NGS-based clinical testing is about to quickly transform both biomedical research, as well as how we apply this knowledge in the clinic. Next-generation sequencing NGS is moving along the adoption curve from early to high adoption.

Key to this progression will be several success factors. Andreas Scherer, Ph. Log in to leave a comment. This site uses Akismet to reduce spam. Learn how your comment data is processed. Top 10 U. Despite remarkable advantages of these new systems, there remains much room for development before introducing them into clinical practice 8. Mendelian or monogenic disorders result from a mutation at a single genetic locus.

RAS/RAF Targeted Gene Panel by Next Generation Sequencing, Tumor

A locus may be present on an autosome or on a sex chromosome, and it may manifest in a dominant, or a recessive or a X-linked mode. Phenylketonuria PKU , cystic fibrosis, sickle-cell anemia, and oculocutaneous albinismare examples of single-gene diseases with an autosomal recessive inheritance pattern and are associated with recessive mutations in the phenylalanine hydroxylase PAH , cystic fibrosis conductance regulator CFTR , beta hemoglobin HBB and OCA2 genes, respectively Huntington's, myotonic dystrophy, polycystic kidney, familial hypercholesterolemia, and neurofibromatosis diseases are instances of an autosomal dominant single-gene diseases.

Huntington's, myotonic dystrophy and neurofibromatosis are associated with mutations in the mutant huntingtin gene HTT and dystrophiamyotonica protein kinase DMPK , neurofibromin NF1 genes respectively. Polycystic kidney disease is associated with mutations in either the polycystic kidney disease 1 PKD1 or the polycystic kidney disease 2 PKD2 genes. Duchenne muscular dystrophy and hemophilia A are examples of single-gene diseases that exhibit an X chromosome-linked recessive pattern of inheritance.

Duchenne muscular dystrophy due to mutations in the dystrophin gene DMD X-linked dominant hypophosphatemic rickets and Rett syndrome are examples of X chromosome-linked dominant diseases and can be caused by mutations inthe phosphate-regulating endopeptidase PHEX and the methyl-CpG-binding protein 2 gene MECP2 genes, respectively Non-obstructive spermatogenic failure is one example of a Y-linked disorder that result from mutations in the ubiquitin-specific protease 9Y gene USP9Y on the Y chromosome The gold standard of molecular diagnosis in Mendelian diseases has been Sanger sequencing dideoxy method and the technique remains the choice method for clinical genetic study; the purpose of which is to confirm a suspected diagnosis and allow more accurate genetic counseling.

Frederick Sanger introduced DNA sequencing assay which was based on Sanger sequencing method also known as dideoxy method , and also Walter Gilbert developed one other sequencing technology based upon chemical modification of DNA molecule and subsequent cleavage at specific bases. The automatic sequencing instruments and associated software using the capillary sequencing technologies and Sanger sequencing methods became the main procedures for the completion of human genome project in This project greatly accelerated the development of powerful novel sequencing instrument to enhance speed and accuracy, while simultaneously reducing cost and manpower.

Not only this, X-prize also increased the development of NGS.

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Clinical Applications of Next-Generation Sequencing

The NGS technologies are different from the Sanger method in sight of massively parallel analysis, high throughput, and reduced cost In , the NGS methods, highly parallel sequencing platforms, were introduced and the next development in molecular genetics such as detection the disease-causing genes is expected NGS technology has high speed and throughput, both quantitative and qualitative sequence data, equivalent to the data from human genome project, in days.

Numerous different methods are employed in which NGS is being applied for identifying causal gene variant in the rare diseases. There are increasing numbers of reports identifying the causal variants of the diseases. In addition to gene discovery of diseases that are dominant and recessive, WES has been applied for determining somatic mutations in tumors and rare mutations with moderate effect in common disorders as well as clinical diagnoses One of the biggest challenges for clinicians is deciding between applications of targeted versus WES.

As the cost of sequencing decreases, WES appears to be a more cost-effective approach. However, there are special considerations before embarking on one over the other 17 , This means that a specific gene of interest with respect to a specific phenotype may not be covered, either completely or partially. Reasons include poorly performing capture probes due to sequence homology, repetitive sequences, or high GC content.

In addition, a targeted method has a much higher or even complete coverage of all the phenotype-specific genes by filling in the gaps with complementary methods including Sanger sequencing or long range PCR.

Clinical Applications for Next-Generation Sequencing

Moreover, a targeted testing allows for deeper coverage of these genes compared to WES, which provides more confidence in the variants detected. However, both are still disposed to sequencing artifacts, and Sanger sequencing of candidate variants is suggested in both methods before returning the results to the patients 17 , Finally, laboratories that offer targeted method can have expert knowledge of the given phenotype and may be in a better location to prioritize variants detected via NGS.

They may also be able to necessitate specific evaluations to define the significance of certain variants 17 , Currently, whole genome association surveys aim to identify the genetic basis of traits and disease susceptibilities by SNP microarrays that capture most of the common genetic variation in the human population.

However, with only a few exceptions e. There are several factors that are likely to contribute to this observation Two additional factors are rare variants and copy number variants CNVs. However, the evaluation of these variants cannot be achieved readily using present genotyping microarray technologies 19 , WGS gives a potential solution by providing the most comprehensive collection of unusual variants and structural variation for sequenced peoples Common complex diseases, in opposite to single gene disorders, are caused by the interaction of genetic and environmental factors, each one with a small effect, and a few sometimes acting individually as a necessary, although on their own insufficient, initiate the disease to occur.

For studying the genetic background of these phenotypes, principles of genetic mapping have been developed by populations rather than families Accordingly, testing of all these variants shed light on the underlying heritability and clearly identify the related key susceptibility genes. Recently, the CD-CV hypothesis has been tested following the extension of catalogues of common variants, genotyping arrays, haplotype maps and innovative and more accurate statistical methods.

Genome-wide association surveys GWAS involve the study of a comprehensive inventory of hundreds of thousands of SNPs in hundreds of thousands of cases and controls from a population to find the variants associated with a traits or disease Since , GWAS have been documented, and identified hundreds loci associated with more than common traits or diseases A few major findings emerge from this great amount of data. The detection of hundreds of loci involved in regulation of the phenotype of complex diseases and traits provides clues to detect the underlying cellular pathways, and in some cases also gives new hints concerning therapeutic methods which has recently been supported by the results of several research studies As cancer is a genetic disease caused by heritable or somatic mutations, new DNA sequencing technologies will have a significant effect on the detection, management and treatment of disease.

Numerous preliminary reports from individual studies leading to these consortia have already been published 25 , 27 , These discoveries will ultimately cause a better understanding of disease pathogenesis, bridging to a new era of molecular pathology and personalized medicine. It is easy to imagine that every patient will soon have both their constitutional and cancer genomes sequenced, the latter perhaps several times for monitor disease progression, therefore allowing a proper molecular subtyping of disease and the rational usage of molecularly guided treatments.

Several molecular pathology laboratories are now considering the sequencing platforms, methods and additional tools required for making the transition to NGS The application of NGS, mostly via WGS and WES, has made an explosion in the context and complexity of cancer genomic modifications, including point mutations, deletions or small insertions, copy number alternations and structural variations. By comparison of these changes to matched normal samples, researchers have been able to separate two categories of variants: germ line and somatic.

The whole transcriptome approach RNA-Seq can not only quantify gene expression templates, but moreover detect RNA editing, alternative splicing and fusion transcripts. In addition, epigenetic alterations, histone modifications and DNA methylation change can be investigated using other sequencing approaches including ChIP-seq and Bisulfite sequencing Bisulfite-Seq. The combination of these NGS technologies gives a high-resolution and global view of the cancer genome. Application of powerful bioinformatics equipments, researchers aim to detect the massive amount of data to improve our understanding of cancer biology and to progress personalized treatment strategy 25 , In the past few years, many NGS-based studies have been conducted to provide a comprehensive molecular diagnosis of cancers, to identify novel genetic alterations leading to oncogenesis, metastasis and cancer progression, to survey heterogeneity, evolution and tumor complexity These efforts have provided significant achievements for many diseases such as melanoma, acute myeloid leukemia AML , breast, lung, liver, kidney, ovarian, colorectal, head and neck cancers 6 , 30 - Epigenetics is definite as heritable modifications in gene activity and expression that occur without alteration in DNA sequence 38 - It is known these non-genetic changes are strictly regulated by two major epigenetic modifications: histone modifications and DNA methylation 38 - Functionally, the profiles of epigenetic modifications can serve as epigenetic markers to show expression and gene activity as well as chromatin state 41 - Epigenetic modifications are critical for packaging and illustrating the genome under the influence of physiological factors Epigenetics is one of the most rapid-growing areas of science and has now become a central aspect in biological studies of development and disease 45 - Recently, there have been quick progresses in the perception of epigenetic mechanisms, which include DNA methylation, small and non-coding RNAs, histone modifications and chromatin architecture 48 - These mechanisms, in addition to other transcriptional regulatory events, ultimately regulate gene function and expression in development and differentiation, or in reply to environmental causes 49 - Epigenetic research can help show how cells carrying identical DNA differentiate into various cell types and how they conserve differentiated cellular states Epigenetics is hence considered a relation between phenotype and genotype 38 , While epigenetics denotes to the variations of single or sets of genes, the term epigenome represents the complete epigenetic situation of a cell, and indicatives to total analyses of epigenetic markers across the whole genome It is therefore very important to map the epigenetic alteration patterns or profile the epigenome in a given cell, which afterwards can be used as epigenetic biomarkers for prognosis, diagnosis, and therapeutic development 44 , 47 , 50 , Human epigenome projects are currently active to catalog all the epigenetic markers in all major tissues across the whole genome.

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The resulting reference patterns will usher in epigenetics as an exciting new period of medical science 44 , The NGS technologies offer the potential to substantially fasten epigenomic research, including posttranslational changes of histones, the interaction among transcription factors and their direct targets, nucleosome positioning on a genome-wide scale and the diagnosis of DNA methylation maps 54 - Using methylated DNA immunoprecipitation meDIP and bisulphite methods can be studied at the methylation of DNA whereas ChIP-Seq technology, the location of transcription factors, post-translational and modifications of histones can be used to study the whole-genome level 58 , Amongst other prevalent high-throughput ways, protein-DNA interactions have been evaluated through the combination of chromatin immunoprecipitation with DNA microarray ChIP-chip.

Contrarily, ChIP-seq method prevents two advantages from the NGS platforms, first, it is not limited by the microarray content and next, it does not rely on the efficiency of probe hybridization 60 , Studies have shown that ChIP-seq had well resolution and required fewer replicates In the past few years the advent of several NGS platforms have been observed that are based on various performance of cyclic-array sequencing The notion of cyclic-array sequencing can be abbreviated as the sequencing of a dense array of DNA traits via iterative cycles of enzymatic correction and imaging-based data collection.

Although these platforms are quite different in sequencing biochemistry as well as in how the array is generated, their work flows are basically very similar All of them feasible the sequencing of millions of short sequences in a time, and are capable of sequencing a whole human genome per week at a cost fold lower than previous methods. Furthermore, NGS platforms allow the production of many kinds of sequence data: for instance, they are used to make de novo sequencing, to resequence persons when a reference genome already exists, sequence RNA to quantify expression level RNA-seq and study the regulation of genes through ChIP-Seq 63 - The emergence of NGS platforms has made various opportunities for genomic variant detection 67 - One of the advantages of exome or genome sequencing is to identify all variants within a given individual, so they can be identified variants related to the disease in question and to other diseases.

Introduction

This is also known from other genome-wide screening instruments, including microarray analysis, or from brain-wide neuroimaging, such as magnetic resonance imaging MRI scans. Furthermore, the ACMG put guidelines for clinical testing laboratories that list 56 genes in which incidentally found known pathogenic or expected pathogenic mutations should be reported to the patients However, there is an in time discussion how to best proceed with incidental findings 72 , Whereas in the research setting, these incidental findings usually do not absorb much attention, it is notable that there is growing intrigue in receiving information about incidental findings on the patient side This is also reflected by the increasing providing and popularity of direct-to-consumer genetic testing DTCGT.

DTCGT allows persons to obtain genetic tests and receive results without the interfering of a health professional. Nevertheless, even if provided in a transparent fashion, it is not easy for most individuals and even for several doctors to properly interpret the test results and risk assessment.

Clinical Applications for Next-Generation Sequencing Clinical Applications for Next-Generation Sequencing
Clinical Applications for Next-Generation Sequencing Clinical Applications for Next-Generation Sequencing
Clinical Applications for Next-Generation Sequencing Clinical Applications for Next-Generation Sequencing
Clinical Applications for Next-Generation Sequencing Clinical Applications for Next-Generation Sequencing
Clinical Applications for Next-Generation Sequencing Clinical Applications for Next-Generation Sequencing
Clinical Applications for Next-Generation Sequencing Clinical Applications for Next-Generation Sequencing
Clinical Applications for Next-Generation Sequencing Clinical Applications for Next-Generation Sequencing
Clinical Applications for Next-Generation Sequencing Clinical Applications for Next-Generation Sequencing
Clinical Applications for Next-Generation Sequencing

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