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Next-Generation Sequencing Technology – the Modern Era of DNA Sequencing

During 1869, DNA (deoxyribonucleic acid) was discovered by Friedrich Miescher. Due to the belief that proteins held the genetic blueprint of life, DNA was not studied for several decades. However, after 1944, experiments by Colin MacLeod, Maclyn McCarty, and Oswald Avery revealed that purified DNA was capable of altering one strain of bacteria into a different strain. This was the first incident of DNA transforming cell properties. In 1953, Francis Crick and James Watson revealed a double-helix model of DNA, which they demonstrated by crystallised X-ray structures. The model showed DNA as two strands of nucleotides curled around each other (the strands running in opposite directions) and joined together by hydrogen bonds. Each strand was composed of four corresponding nucleotides; adenine (A), cytosine (C), guanine (G) and thymine (T) – with A on one strand always paired with T on the other strand, and C always paired with G on the other strand. They explained that this specific structure permitted each strand to reconstruct the other; a theory which became vital to the distribution of hereditary sequencing between generations. After Francis Crick and James Watson demonstrated their DNA model, the attention evolved around protein. By 1955, Frederick Sanger had completed sequencing all amino acids in insulin (a tiny protein secreted by the pancreas). This supplied the initial verification that proteins were chemical units with a specific molecular prototype rather than a random fluid-suspended substance combination. In return, Crick and Watson tried to identify how DNA influenced the arrangement of proteins within a cell. Crick developed a theory that the arrangement of nucleotides in DNA established the sequence of amino acids in proteins, which ultimately decided upon the protein’s function. He published his theory in 1958.

Genetic testing (DNA testing) is used to discover modifications in chromosome structure or DNA sequence. Genetic testing can also be to evaluate the results of genetic transformations such as to calculate protein output from biochemical analysis or to assess gene output from RNA analysis. The unique benefit of genetic testing is the capability to better understand the risk of a specific disease. Genetic testing can assist doctors to identify individuals who are prone to chronic illnesses or certain types of cancers and advise them about the management of the risk; therefore, it can facilitate in decisions about health conditions. Statistics have indicated that more than two million Americans are undiagnosed and at risk of long-term health complications. DNA sequencing is a technique that follows a procedure or technology to establish the order of nucleotides (nucleic acid) in DNA which consists of the four bases adenine, cytosine, guanine, and thymine. With the development of DNA sequencing techniques, knowledge of DNA sequences has significantly increased to benefit medical research, medical diagnosis, biotechnology, forensic biology, and virology. Rapid DNA sequencing allows for quicker and individualised medical care. Besides complete DNA sequencing of various types and species of life and several animal, plant, and microbial species the fast speed achieved with modern DNA sequencing technology has played a vital role in the sequencing of the human genome. Comparing healthy and mutated DNA sequences can diagnose different diseases (including a range of cancers), represent antibody selection, and can channel patient treatment.

Genetic diseases that can be identified by genetic testing are illnesses such as obesity, breast and ovarian cancer, bipolar disorder, celiac disease, macular degeneration, psoriasis, and Parkinson’s disease. Obesity, breast and ovarian cancer need no introduction; however, the other medical conditions need a trivial explanation. Bipolar disorder (manic depression) is a chronic mental illness that causes dramatic mood swings that affects the person’s activity level, concentration, and ability to perform daily tasks. ‘Bipolar’ describes the variation between two extreme poles of emotions – depression and manic presentations. During phases of depression the risk of self-harm and suicide is high. Celiac disease is a chronic, genetic, autoimmune disorder that leads to inflammation and damage of the small intestine of people who are prone to this disease when they consume gluten (a protein in barley, bulgur, farina, rye, and wheat). It has been established that almost everybody with celiac disease has one of two specific genes. Age-related macular degeneration is an eye disease that usually starts after the age of sixty and gradually destroys the central area of the retina (macula). It is the primary cause of vision loss. Psoriasis is a chronic autoimmune skin disease responsible for a drastic increase of skin cells which causes heaved areas of red and itchy patches with white scales on the surface of the affected areas. Parkinson’s disease is a progressive neurodegenerative disease that eventually affects mobility (balance, coordination, gait [walking], shaking, slowness, stiffness, and tremor) and eventually mental capacity. Research shows that several cases of Parkinson’s disease are purely genetic while others are a blend of ecological and genetic factors.

In healthcare, the purpose of genomic sequencing is to discover genetic variants that have been identified to influence health and disease. However, sequencing results have conflicting clinical relevance to the decision making of health care providers and therefore to the clinical outcomes of patients. Nonetheless, in clinical medicine, an important factor is that DNA sequencing is used for many purposes, including diagnosis and treatment of diseases. In general, sequencing allows healthcare practitioners to determine if a gene contains mutations or variants that are associated with a disorder. In genetic diseases it is important to demarcate fundamental genomic and genetic features to establish a final diagnosis; it is also helpful in patient counselling and management. DNA mapping is the range of methods that can be used to illustrate the positions of genes. DNA maps can confirm different levels of specifications to identify the distance between two genes; this is comparable with topological maps of cities or countries. DNA profiling is the chemical analysis of distinctive patterns of DNA sequences in the genome while DNA sequencing refers to the organisation of the nucleotide sequences of a DNA fragment. Information obtained from DNA sequencing allows researchers to recognise changes in genes, identify associations with diseases and phenotypes, and discover potential drug targets. It is used in molecular biology to study genomes and the proteins they encode. DNA sequencing is an important laboratory procedure applied to establish the particular order of the bases (A, C, G, and T) in a DNA molecule.

The DNA base sequence holds the vital cell information to accumulate protein and RNA molecules. DNA sequence information is fundamental to scientists to examine the functions of genes. When applying DNA sequencing technologies, two main methods are used. The first method is the Sanger method which is the older, classical chain termination method whereby sequencing information is clarified with the low throughput Sanger sequencing technique. The second method is by way of high throughput sequencing (HTS) technologies which are capable of sequencing multiple DNA molecules. High-throughput screening methods are mostly used in the pharmaceutical industry, typically in drugs, to test the biochemical or biological activity of a huge amount of molecules. HTS techniques deal with issues that are impossible to resolve with conventional methods. It has a healing role in cancer by blocking the reproduction of cancer cells in the presence of abnormal proteins. The techniques that can rapidly process an enormous amount of DNA molecules are jointly known as High-Throughput Sequencing (HTS) methods or Next-Generation Sequencing (NGS) technologies. Information on the sequence of a DNA segment has various applications. It can be used to discover genes; sections of DNA that code for a particular protein or phenotype. If an area of DNA has been sequenced, it can be monitored for distinctive aspects of genes. The purpose of sequencing is to inform scientists of the nature of the genetic sequence in the specific DNA segment.

For example, they can use sequence information to establish which sections of DNA contains regulatory instructions. DNA sequencing can reveal a great deal of genetic information such as to identify genes that code for proteins, to recognise disease-prone mutations and to regulate instructions that turn genes on or off. Whole Genome Sequencing is the most complete sequencing method. Whole Genome Sequencing DNA testing uses high throughput next-generation DNA sequencing technology. The present criterion for whole genome sequencing is the use of DNA obtained from blood.

Genotyping is the process of determining differences in the genetic make-up of an individual by examining the person’s DNA sequence and comparing it to another person’s DNA sequence or a reference DNA sequence to reveal what an individual has inherited from his parents. Whole Exome Sequencing (WES) is a frequently used next-generation sequencing (NGS) method to sequence all the protein-coding areas (exons) of a genome (collectively known as the exome) to discover rare or universal variants related to a disorder. It allows identification of variants in the exome of any gene, rather than only in a small amount of genes. WES is a cost-effective option instead of whole-genome sequencing. Studies report that saliva-derived DNA is used for a range of genotyping and whole exome sequencing; on condition that the amount of human DNA in each sample is adequate. The most effective way to sequence large pieces of DNA is by way of shotgun sequencing. The initial DNA is broken up into smaller pieces; each of these pieces is then sequenced individually. Shotgun sequencing is used to eliminate errors, fill in breaks or correct parts of the sequence that were initially collected defectively in clone-by-clone sequencing. As a result, the human genome is continually being developed to warrant that the genome sequence is of a supreme standard. Whole-genome shotgun sequencing of human genomic DNA has many important advantages in contrast to conventional clone-by-clone sequencing. Foremost are the discovery of huge amounts of DNA polymorphisms, more complete coverage of the genome, and improved speed and cost. Short-reads or ‘next-generation’ sequencing creates shorter reads and generates millions of reads in a reasonably short period. Next generation sequencing (NGS) is a high-throughput DNA sequencing technology that has revolutionised genomic research in that it facilitates sequence summarising from genomes to DNA-protein interactions and much more. The technology is used to verify the order of nucleotides in entire genomes or intended sections of DNA or RNA, enabling rapid sequencing of base pairs in DNA or RNA samples to study genetic variations associated with diseases. The technique can be used as a laboratory diagnostic test to sequence an entire human genome within a day. The advantage of high-throughput sequencing is that it provides superior accuracy and a wider range. High-throughput sequencing predominantly refers to sequencing techniques such as Illumina that permits the sequencing of substantial amounts of DNA at the same time. Many developed sequencing platforms referred to as next-generation sequencing (NGS) include platforms such as Illumina/Solexa platform, Ion Torrent technology, pyrosequencing, and SOLID (Sequencing by Oligonucleotide Ligation and Detection).

The Huntsman Cancer Institute at the University of Utah (Salt Lake City) has two self-regulating, but intertwined subdivisions that are situated next to each other. The one section is the High-Throughput Genomics (HTG) division and the other section is the Bioinformatic Analysis (BAS) division. The Huntsman Cancer Institute’s High-Throughput Genomics and Bioinformatics Analysis divisions provide experimental support for various genomic applications that use the Illumina sequencing platform. The High Throughput Genomics (HTG) section was created due to the need of the scientific community to have faster access to uncompleted genomic sequencing data; this section’s ‘Screening Shared Resource’ (which is managed by the Judith P. Sulzberger Columbia Genome Centre [CGC] as well as the Herbert Irving Comprehensive Cancer Centre [HICCC]) functions as a full-service facility that offers proficiency in all facets of the experimental process. Since its formation in 1995, the Judith P. Sulzberger Columbia Genome Centre provided a bond between the science/engineering and biomedical fields of the two Columbia University campuses; the Medical Centre campus in Washington Heights and the central campus in Morningside Heights. The Herbert Irving Comprehensive Cancer Centre (HICCC) is located in the United States at New York City’s Columbia University Medical Centre (it was founded in 1911 as the Institute for Cancer Research and formed part of the New York-Presbyterian Hospital Complex). NewYork-Presbyterian/Columbia University Irving Medical Centre has been extensively renewed as a delegated Comprehensive Cancer Centre by the federal government’s National Cancer Institute (NCI), the leading global sponsor for cancer research. The Centre is recognised for its contributions towards clinical care and cancer research. The National Cancer Institute Oncologists at HICCC treat approximately four thousand new cancer patients yearly.

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Lynette Barnard

I studied a Bachelor of Science degree with subjects such as Nursing, Psychology, Sociology, Anatomy, Microbiology and Physics while undergoing training as a student nurse at an academic hospital.

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