Key principles and clinical applications of next generation dna sequencing pdf

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key principles and clinical applications of next generation dna sequencing pdf

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Key Principles and Clinical Applications of “Next-Generation” DNA Sequencing

Next-generation sequencing NGS technologies represented the next step in the evolution of DNA sequencing, through the generation of thousands to millions of DNA sequences in a short time. The relatively fast emergence and success of NGS in research revolutionized the field of genomics and medical diagnosis. The traditional medicine model of diagnosis has changed to one precision medicine model, leading to a more accurate diagnosis of human diseases and allowing the selection of molecular target drugs for individual treatment.

This chapter attempts to review the main features of NGS technique concepts, data analysis, applications, advances and challenges , starting with a brief history of DNA sequencing followed by a comprehensive description of most used NGS platforms. The potential use of NGS in precision medicine is vast and a better knowledge of this technique is necessary for an efficacious implementation in the clinical workplace.

A centralized chapter describing the main NGS aspects in the clinic could help beginners, scientists, researchers and health care professionals, as they will be responsible for translating genomic data into genomic medicine. Edited by Fabio A. Marchi, Priscila D. Cirillo and Elvis C. Precision medicine is a new way of practising medicine, which has been gaining strength in recent years, is based on the individual characteristics of each patient genetic, environmental, behavioural to optimize and customize strategies for prevention, detection and therapy [ 1 , 2 ].

The molecular knowledge has contributed strongly to the advancement of precision medicine, providing specific strategies for target therapies and diagnosis of patients with cancer, Mendelian diseases and others. Statistics indicated that traditional clinical practices sometimes lead to poor health outcomes and also a waste of medical resources. As a result of the genome project, many molecular tools have been developed and allow medical and scientific groups to improve patient management based on a better understanding of disease biology, providing a more specific and accurate prevention and treatment of diseases [ 4 ].

Precision medicine redefines the way traditional medicine is practised. There is a great deal of investment nowadays in prevention using these new technologies, as opposed to old medicine based on treatment since the disease was already evident or irreversible [ 2 ]. NGS allows identifying biomarkers for early diagnosis as well as for personalized treatments. The emergence of NGS has changed the way clinical research, basic and applied science are done.

The NGS allows producing millions of data with a smaller investment [ 4 , 6 ]. Among the available NGS applications, one of them will be the resequencing of the human genome and the better genetic understanding of various human diseases. A great challenge will be the interpretation of this great number of data and its translation for the medical application [ 6 ]. One of the major near-term medical impact of the NGS revolution will be the elucidation of mechanisms of human pathogenesis, leading to improvements in the diagnosis and the selection of treatment and prevention.

This chapter encompasses revised concepts, applications, advances, limitations and the history of technological advances until the emergence of NGS technique in the era of precision medicine, starting with a brief history of DNA sequencing followed by a comprehensive description of most used NGS platforms, sequencing chemistries methodology and general workflows. Further topics will highlight the application of NGS towards routine practice, including variant detection, whole-genome sequencing WGS , whole-exome sequencing WES and multi-gene panels.

A centralized chapter describing the main NGS features in the clinic could help beginners, scientists, researchers and health care professionals, as they will be responsible for translating genomic data into genomic medicine.

His principal contribution was the understanding about the relationship between gene-enzyme, the molecular basis of genetic diseases. Although today this concept is considered outdated because of discoveries like RNA splicing, RNAi and others, its development allowed the researchers to understand how changes in DNA sequence could cause genetic disease.

This finding increased the interest of scientists to know about human DNA sequence and mutations. These special nucleotides were radiolabeled and therefore the sequence could be inferred after the disclosure of gel autoradiography [ 14 ]. Numerous modifications have been made in this technique to make the method more efficient, robust and sensitive. Among them are the substitution of nucleotide radiolabeled to fluorescence that allowed the sequencing reaction to occur in one tube [ 15 ], the development of the polymerase chain reaction [ 16 ], the separation of DNA fragments by capillary electrophoresis [ 17 ] and later the development of equipment that allowed the sequencing of more complex genomes.

To date, Sanger is still the gold-standard method in diagnostic tests and although the most recent methods have a much higher processing capacity, confirmation of some findings is made using this method.

Equipment of all generations is still being improved and released commercially. Dot: milestones; rectangle: equipments; White: first-generation sequencing; Light gray: second-generation sequencing; Dark gray: third-generation sequencing.

The second generation of DNA sequencing can be defined as the era of the parallel massive sequencing on a micro scale.

This technique differed substantially from previous ones because it did not use radio or fluorescence-labelled nucleotides and there was no need of electrophoretic run. The method is based on the action of two enzymes: ATP sulfurylase and luciferase. ATP sulfurylase converts pyrophosphate released in nucleotide incorporation into an ATP molecule that is used by luciferase substrate.

This process releases light signal in proportion to the amount of nucleotides incorporated, and the sequence can be determined according to the serial addition of nucleotides [ 19 ].

These changes and the use of microplates that compartmentalized the process and high-definition detection systems dramatically increased the amount of DNA sequenced and defined the second generation [ 20 ]. The disadvantage of this technology is related to homopolymer regions because of difficulty in interpreting the signal strength when five or more nucleotides are incorporated in a single wash cycle.

Other technologies were then developed, such as that used by Illumina which consists of binding the DNA in a flow-cell through adapters, and the parallel massive amplification occurs in clusters for each DNA strand that was originally bound in the flow-cell, called bridge-amplification. This process generates paired-ends sequences that are an advantage over other methodologies, since they improve the accuracy of mapping, mainly in repetitive regions or where DNA rearrangements or gene fusions occur.

This is one of the most accurate and with lowest error rate of sequencing methodologies used currently; however, it generally requires higher DNA concentration.

The method does not do sequencing by synthesis but by ligation of oligonucleotides fluorescence-labelled. After probe annealing and ligation, fluorescent dye is cleavage and a new probe is ligated. Multiple cycles are performed according to the read length. The template from primer n is removed and the second round of sequencing is performed with a primer complementary to the n-1 position [ 22 ].

This method shows good results; however, it is considered slow compared to the others and therefore was replaced by Ion Torrent Thermo Fisher Scientific technology. This methodology is the first to use a detection method that does not work with light signal [ 23 ]. The advantage of this technology is the speed of the process and the low cost of the equipment; however, it has the same problem about the detection of homopolymers. The second generation of the sequencing was marked by the high capacity of the sequencers in the generation of data in a single run and consequently the computational development-like bioinformatics tools to analyse them.

The cost of sequencing decreased dramatically at this stage. There are some discussions about which technology marked the beginning of the third generation [ 24 — 27 ]. In this review, we will consider the technology of single-molecule sequencing SMS , which has no need to amplify the DNA.

The process occurs in cycles where the dNTPs are incorporated and the corresponding fluorescence is captured by a CCD camera.

This process generates short readings 25 bp and it is considered slow and there is a lot of noise in the signal [ 28 ]. Despite being the first third-generation sequencing technology, its history was brief because the company Helicos Biosciences filed for Chap. ZMW allows the incorporation of each nucleotide to be monitored in real time and without interference from other light signals.

The reads are very long 40 kb and allow detecting modified bases [ 29 , 30 ]. The detection occurs due to differences in the current of ions generated by each nucleotide. The reads are incredibly long kb , and the process is extremely fast without the need for special nucleotides. In common, these technologies still have high error rates that are improving with the development of technology.

Its main use today is to aid in the assembly of complex regions of the genome where gene fusions, large deletions and insertions and repetitive regions occur. The third generation will further revolutionize precision medicine, enabling sequencing at lower cost and enabling this to occur virtually anywhere.

In recent times, NGS has made possible a better understanding of genetic diseases and became a significant technological advance in the practice of diagnostic and clinical medicine [ 32 ].

NGS allows the analysis of multiple regions of the genome in one single reaction and has been shown to be a cost-effective and an efficient tool in investigating patients with genetic diseases. Genetic data produced via NGS provides significant benefits to medical practice including accurate identification of biomarkers of disease, detecting inherited disorders and identifying genetic factors that can help predict responses to therapies [ 32 , 33 ]. However, recommendations on clinical implementation of NGS that are still in discussion and that hamper its use in the genetic clinic.

A variety of molecular diagnostic test use sequencing technology, such as single- and multi-gene panel tests, cell-free DNA for non-invasive prenatal testing, whole-exome sequencing WES , whole-genome sequencing WGS.

Considering that the use of NGS as a diagnostic tool is recent, there are challenges including when to order, on whom to order and how to interpret and communicate the results to the patient and family [ 32 ]. Therefore, it is necessary to understand the application, strength and limitations of the different approaches to recognize which one is the most suitable for your case. In the following topics, we will emphasize common applications of this technology into clinical practice.

The traditional approach still holds great value for many disorders. Single-gene testing is indicated when the clinical features for a patient are typical for a particular disorder and the association between the disorder and the specific gene is well established and has the minimal locus heterogeneity [ 34 ]. However, many genetic conditions are intractable to diagnostic evaluation, mainly because of the clinical variability and genetic locus heterogeneity, such as cardiomyopathies, epilepsy, congenital muscular dystrophy, X-linked intellectual disability and cancer susceptibility in families with atypical phenotypes [ 35 ].

The diagnostic process is exhausted, with clinical assessment followed by sequential laboratory testing, in most cases tests being negative. In cases with unidentified genetic conditions e. In diagnostic of cancer, for example, Tothill and colleagues [ 37 ] illustrate the application of these multi-gene panel by analysing samples of patients with cancers of unknown primary CUP. The clinical management of patients with CUP is hampered by the absence of a definitive site of origin and this kind of NGS analysis could help to define new therapeutic options.

In multi-gene panel tests, many genes associated with a specific phenotype are sequenced and analysed concomitantly, decreasing cost and improving efficiency of genetic diagnostic [ 37 ]. The number and which genes will be evaluated for the same or similar indications may vary significantly among different clinical laboratories and several considerations need to be taken for gene inclusion.

The majority of authors believe that only genes with a strong disease association should be included since the ability to interpret their findings is much better due to clinical evidence [ 38 ]. However, some authors consider including associated genes that have overlapping phenotypes for the purpose of differential diagnosis, or all possible genes that are remotely associated with the phenotype of interest with the objective of a better and faster diagnostic [ 34 ]. For cancer diagnostic, multi-gene panel may include high-penetrance genes as well as associated genes with a moderate increase in risk [ 35 ].

The transition from single-gene to multi-gene testing should not compromise the sensitivity of the test to identify variants, mainly at genes that are responsible for a significant proportion of the defects core genes. The sensitivity of NGS does not depend only on horizontal coverage but the vertical coverage is important as well [ 39 ].

Additional genes will increase the chance of the diagnostic, but this should not be at cost of missing mutations that would previously have been detected by single-gene testing [ 38 ]. Sanger sequencing or other available techniques can help to solve this problem for filling in low-coverage and no-coverage regions.

Whole-genome sequencing also known as WGS, full-genome sequencing, complete genome sequencing or entire genome sequencing is the process of determining the complete DNA sequence of an organism's genome at a single time. The major benefit of WGS is completed coverage of the genome, including promoters and regulatory regions.

In whole-exome sequencing WES , all coding regions are sequenced with a relatively deeper depth. For this reason, sequencing the complete coding regions exome has the power to uncover the causes of large number of rare, mostly monogenic, genetic disorders as well as predisposing variants in common diseases and cancers [ 33 ]. In , Choi and colleagues first showed the value of WES in the medical practice by making genetic diagnoses of congenital chloride diarrhoea in patients suspected of Bartter syndrome, a renal salt-wasting disease.

WES was conducted on six patients who do not show any mutations in classic genes for Bartter syndrome. Results revealed homozygous deletion in SLC26A3 gene for all patients, which provided a molecular diagnosis of congenital chloride diarrhoea that was later confirmed on clinical evaluation. This result was the first to show the value of WES in making a clinical diagnosis and several similar studies have followed [ 43 ].

There are many reasons that include poorly performing capture probes due to high GC content, sequence homology or repetitive sequences. A targeted approach, such as NGS single- or multi-gene panels, on the other hand, has higher or even complete coverage of all the specific genes by filling in the gaps with complementary technologies such as Sanger sequencing or long-range PCR.

However, all NGS tools are still prone to sequencing artefacts, and Sanger sequencing is recommended to confirm the variants detected before returning the results to the patient [ 44 ].

NGS vs. Sanger Sequencing

Next-generation sequencing NGS technologies have greatly impacted on every field of molecular research mainly because they reduce costs and increase throughput of DNA sequencing. These approaches are now widely used in research, and they are already being used in routine molecular diagnostics. However, some issues are still controversial, namely, standardization of methods, data analysis and storage, and ethical aspects. Besides providing an overview of the NGS-based approaches most frequently used to study the molecular basis of human diseases at DNA level, we discuss the principal challenges and applications of NGS in the field of human genomics. DNA sequencing is the process of determining the exact order of the nucleotides in a DNA segment, corresponding to single gene s or to a variety of molecules in the case of the whole genome, or a large part of it.

Whole-genome sequencing. National Cancer Institute website. Accessed December 20, Genome Sequencing. Genome News Network website. Updated January 15,

The sequencing of the human genome was completed in , after 13 years of international collaboration and investment of USD 3 billion. The Human Genome Project used Sanger sequencing albeit heavily optimized , the principal method of DNA sequencing since its invention in the s. Today, the demand for sequencing is growing exponentially, with large amounts of genomic DNA needing to be analyzed quickly, cheaply, and accurately. Thanks to new sequencing technologies known collectively as Next Generation Sequencing, it is now possible to sequence an entire human genome in a matter of hours. The principle behind Next Generation Sequencing NGS is similar to that of Sanger sequencing , which relies on capillary electrophoresis. The genomic strand is fragmented, and the bases in each fragment are identified by emitted signals when the fragments are ligated against a template strand. The Sanger method required separate steps for sequencing, separation by electrophoresis and detection, which made it difficult to automate the sample preparation and it was limited in throughput, scalability and resolution.

Despite opening new frontiers of genomics research, the fundamental shift away from the Sanger sequencing that next-generation technologies has created has.

Next-Generation Sequencing: Principles for Clinical Application

Molecular Pathology in Clinical Practice pp Cite as. Next-generation sequencing NGS is not a single technology, but rather several different technologies that share a common feature of massively parallel sequencing of clonally amplified or single DNA molecules in a flow cell or chip. Inherent to NGS technologies are unique sequencing chemistries that differ from the Sanger dideoxynucleotide chain termination chemistry.

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