@nextgenerationsequencing

Digital PCR: The Emergence of the Digital Age

2021-02-22T15:04:04.903Z

The Future for PCR Diagnostics

PCR is polymerase chain reaction is in vitro amplification of any given DNA or RNA sample, while digital PCR is the quantitative PCR technique that provide reproducible way of measuring amount of DNA or RNA present in particular sample. The advantage of digital PCR has high tolerance to inhibitors and it distinguish expression of alleles, and measure the cancer genes. PCR has various application such as paternity testing, detection of hereditary disease, forensic science, and DNA cloning.

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The key difference between dPCR and traditional PCR lies in the method of measuring nucleic acids amounts, with the former being a more precise method than PCR, though also more prone to error in the hands of inexperienced users.[1] A "digital" measurement quantitatively and discretely measures a certain variable, whereas an “analog” measurement extrapolates certain measurements based on measured patterns.

PCR carries out one reaction per single sample. dPCR also carries out a single reaction within a sample, however the sample is separated into a large number of partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid amounts. The method has been demonstrated as useful for studying variations in gene sequences — such as copy number variants and point mutations — and it is routinely used for clonal amplification of samples for next-generation sequencing.

The polymerase chain reaction method is used to quantify nucleic acids by amplifying a nucleic acid molecule with the enzyme DNA polymerase. Conventional PCR is based on the theory that amplification is exponential. Therefore, nucleic acids may be quantified by comparing the number of amplification cycles and amount of PCR end-product to those of a reference sample.

Digital PCR has many applications in basic research, clinical diagnostics and environmental testing. Its uses include pathogen detection and digestive health analysis;liquid biopsy for cancer monitoring, organ transplant rejection monitoring and non-invasive prenatal testing for serious genetic abnormalities;copy number variation analysis, single gene expression analysis, rare sequence detection, gene expression profiling and single-cell analysis; the detection of DNA contaminants in bioprocessing, the validation of gene edits and detection of specific methylation changes in DNA as biomarkers of cancer.

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Bioinformatics Tips Next generation Sequencing Data Processing

2021-01-14T14:49:40.379Z

Experience powerful NGS data analysis capabilities

Next generation sequencing (NGS) has created a noteworthy paradigm shift in the clinical diagnostic field. It refers to an aggregate collection of methods in which various sequencing reactions occur at the same time, bringing about vast amounts of sequencing data for a little division of the cost of Sanger sequencing.

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The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of complete DNA sequences, or genomes, of numerous types and species of life, including the human genome and other complete DNA sequences of many animal, plant, and microbial species.

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Applications of NGS

NGS technologies have many applications such as DNA-sequencing and assembly to determine an unknown genome without any preparation or search for variations among genome samples, RNA-sequencing, to analyze gene expression and to predominantly identify DNA regions of DNA binding proteins, for example, transcription factors etc.

NGS-based diagnostics implement some portion of the clinical genomic testing in which a limited set of genes are targeted and not the entire genome and exome.

DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery.

Knowledge of DNA sequences has become indispensable for basic biological research, and in numerous applied fields such as medical diagnosis, biotechnology, forensic biology, virology and biological systematics. Comparing healthy and mutated DNA sequences can diagnose different diseases including various cancers,characterize antibody repertoire, and can be used to guide patient treatment. Having a quick way to sequence DNA allows for faster and more individualized medical care to be administered, and for more organisms to be identified and cataloged.

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mRNA Treatment: Will a mRNA vaccine alter any DNA?

2021-01-06T15:13:40.424Z

Reviewing the Future Generation of Therapeutics

The mRNA treatment involves mRNA sequences that play an essential role in protein synthesis, helping direct the disease's target. It allows replacing a defective gene through substitution and integration of the correct genetic code in the genome.

RNA therapeutics are a class of medications based on ribonucleic acid (RNA). The main types are those based on messenger RNA (mRNA), antisense RNA (asRNA), RNA interference (RNAi), and RNA aptamers.

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Of the four types, mRNA-based therapy is the only type which is based on triggering synthesis of proteins within cells, making it particularly useful in vaccine development,and mRNA vaccines have been developed in 2020 for use in combating the SARS-CoV-2 global pandemic. Antisense RNA is complementary to coding mRNA and is used to trigger mRNA inactivation to prevent the mRNA from being used in protein translation. RNAi-based systems use a similar mechanism, and involve the use of both small interfering RNA (siRNA) and micro RNA (miRNA) to prevent mRNA translation. However, RNA aptamers are short, single stranded RNA molecules produced by directed evolution to bind to a variety of biomolecular targets with high affinity thereby affecting their normal in vivo activity.

RNA is synthesized from template DNA by RNA polymerase with messenger RNA (mRNA) serving as the intermediary biomolecule between DNA expression and protein translation. Because of its unique properties (such as its typically single-stranded nature and its 2' OH group) as well as its ability to adopt many different secondary/tertiary structures, bot coding and noncoding RNAs have attracted special attention in medicine. Research has begun to explore RNAs potential to be used for therapeutic benefit, and unique challenges have occurred during drug discovery and implementation of RNA therapeutics.

Applications

Cancer immunotherapy:

Recently, the new cancer immunotherapy, the combining of self-delivering RNA(sd-rxRNA) and adoptive cell transfer(ACT) therapy, was invented by RXi Pharmaceuticals and the Karolinska Institute. In this therapy, the sd-rxRNA eliminated the expression of immunosuppressive receptors and proteins in therapeutic immune cells so it improved the ability of immune cells to destroy the tumor cells.

Vaccines:

There are a few different types of IVT mRNA-based vaccine development for infectious diseases. One of the successful types is using self-amplifying IVT mRNA that has sequences of positive-stranded RNA viruses. It was originally developed for a flavivirus and it was workable with intradermal injection. One of the other ways is injecting a two-component vaccine which is containing an mRNA adjuvant and naked IVT mRNA encoding influenza hemagglutinin antigen only or in combination with neuraminidase encoding IVT mRNA.

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Genetic Gngineering using Recombinant Dna Technology

2020-12-15T17:13:20.737Z

Genetic engineering, also called genetic modification or genetic manipulation, is the direct manipulation of an organism's genes using biotechnology. It is a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species boundaries to produce improved or novel organisms.

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New DNA is obtained by either isolating and copying the genetic material of interest using recombinant DNA methods or by artificially synthesising the DNA. A construct is usually created and used to insert this DNA into the host organism.

Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.

The DNA sequences used in the construction of recombinant DNA molecules can originate from any species. For example, plant DNA may be joined to bacterial DNA, or human DNA may be joined with fungal DNA. In addition, DNA sequences that do not occur anywhere in nature may be created by the chemical synthesis of DNA, and incorporated into recombinant molecules. Using recombinant DNA technology and synthetic DNA, literally any DNA sequence may be created and introduced into any of a very wide range of living organisms.

Genetic engineering is a process that alters the genetic structure of an organism by either removing or introducing DNA. Unlike traditional animal and plant breeding, which involves doing multiple crosses and then selecting for the organism with the desired phenotype, genetic engineering takes the gene directly from one organism and delivers it to the other. This is much faster, can be used to insert any genes from any organism (even ones from different domains) and prevents other undesirable genes from also being added.

Genetic engineering, also called recombinant DNA technology, involves the group of techniques used to cut up and join together genetic material, especially DNA from different biological species, and to introduce the resulting hybrid DNA into an organism in order to form new combinations of heritable genetic material.

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Next Generation Sequencing in Clinical Medicine

2020-11-30T15:16:18.709Z

Next-generation sequencing (NGS) was developed more than a decade ago to facilitate sequencing of large amounts of genomic data. Use of NGS in clinical diagnosis is now widely accepted, with varying roles from gene panel or targeted sequencing, through whole exome sequencing (also termed clinical exome sequencing), to whole genome sequencing. NGS was first applied to clinical diagnosis when clinical exome sequencing was launched in 2012.

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Next Generation Sequencing (NGS) techniques represent the next phase in the evolution of DNA sequencing technology at dramatically reduced cost compared to traditional Sanger sequencing. A single laboratory today can sequence the entire human genome in a few days for a few thousand dollars in reagents and staff time. Routine whole exome or even whole genome sequencing of clinical patients is well within the realm of affordability for many academic institutions across the country. This paper reviews current sequencing technology methods and upcoming advancements in sequencing technology as well as challenges associated with data generation, data manipulation and data storage.

The goal of clinical NGS is the identification of point mutations and potentially larger structural changes such as translocations, rearrangements, inversions, deletions and duplications either in germline samples or in tumor samples paired with normal genomes for cancer diagnostics. While there are few NGS instruments lending to standardization of some sort, the same cannot be said for the software used to analyse the data.

Cancer is a genetic disease. Decades of research has led to this knowledge, showing that it is the accumulation of molecular alterations that is the key element of tumorigenesis, directing the acquisition of the malignant phenotype. Genes involved in oncogenesis are classified in “oncogenes,” whose activation is responsible for tumor transformation and oncosuppressors, whose inactivation leads to cellular proliferation. Mutations of oncogenes (gain of function) or oncosuppressors (loss of function) can be genetically inherited (germline), but they are mostly acquired and caused by DNA replication errors and/or exposure to carcinogens.

Microbiology

The main utility of NGS in microbiology is to replace conventional characterisation of pathogens by morphology, staining properties and metabolic criteria with a genomic definition of pathogens. The genomes of pathogens define what they are, may harbour information about drug sensitivity and inform the relationship of different pathogens with each other which can be used to trace sources of infection outbreaks.

Oncology

The fundamental premise of cancer genomics is that cancer is caused by somatically acquired mutations, and consequently it is a disease of the genome. Although capillary-based cancer sequencing has been ongoing for over a decade, these investigations were limited to relatively few samples and small numbers of candidate genes. With the advent of NGS, cancer genomes can now be systemically studied in their entirety, an endeavour ongoing via several large scale cancer genome projects.

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Next Generation Sequencing in Bioinformatics

2020-09-15T15:45:06.322Z

Next generation sequencing (NGS), massively parallel or deep sequencing are related terms that describe a DNA sequencing technology which has revolutionised genomic research. Using NGS an entire human genome can be sequenced within a single day. In contrast, the previous Sanger sequencing technology, used to decipher the human genome, required over a decade to deliver the final draft. Although in genome research NGS has mostly superseded conventional Sanger sequencing, it has not yet translated into routine clinical practice. The aim of this article is to review the potential applications of NGS in paediatrics.

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The emergence of next-generation sequencing (NGS) platforms imposes increasing demands on statistical methods and bioinformatic tools for the analysis and the management of the huge amounts of data generated by these technologies. Even at the early stages of their commercial availability, a large number of softwares already exist for analyzing NGS data. These tools can be fit into many general categories including alignment of sequence reads to a reference, base-calling and/or polymorphism detection, de novo assembly from paired or unpaired reads, structural variant detection and genome browsing. This manuscript aims to guide readers in the choice of the available computational tools that can be used to face the several steps of the data analysis workflow.

NGS Bioinformatics

The computational components of an NGS-based work flow can be conceptualized as primary, secondary, and tertiary analytics. Each of these components addresses a necessary step in the transformation of raw data into clinically actionable knowledge. Understanding the basic concepts of these analysis steps is important in assessing and addressing the informatics needs of a molecular diagnostics laboratory. Equally critical is a familiarity with the regulatory requirements addressing the bioinformatics analyses. These and other topics are covered in this review article.

Bioinformatics has become an important component in clinical laboratories generating, analyzing, maintaining, and interpreting data from molecular genetics testing. Given the rapid adoption of NGS-based clinical testing, service providers must develop informatics work flows that adhere to the rigor of clinical laboratory standards, yet are flexible to changes as the chemistry and software for analyzing sequencing data mature.

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DNA Next Generation Sequencing - Future of Genomics

2020-08-19T08:35:03.086Z

DNA sequencing is the process of determining the nucleic acid sequence - the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery.

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Knowledge of DNA sequences has become indispensable for basic biological research, and in numerous applied fields such as medical diagnosis, biotechnology, forensic biology, virology and biological systematics. Comparing healthy and mutated DNA sequences can diagnose different diseases including various cancers, characterize antibody repertoire, and can be used to guide patient treatment. Having a quick way to sequence DNA allows for faster and more individualized medical care to be administered, and for more organisms to be identified and cataloged.

Applications

DNA sequencing may be used to determine the sequence of individual genes, larger genetic regions (i.e. clusters of genes or operons), full chromosomes, or entire genomes of any organism. DNA sequencing is also the most efficient way to indirectly sequence RNA or proteins (via their open reading frames). In fact, DNA sequencing has become a key technology in many areas of biology and other sciences such as medicine, forensics, and anthropology.

Molecular biology:

Sequencing is used in molecular biology to study genomes and the proteins they encode. Information obtained using sequencing allows researchers to identify changes in genes, associations with diseases and phenotypes, and identify potential drug targets.

Evolutionary biology:

Since DNA is an informative macromolecule in terms of transmission from one generation to another, DNA sequencing is used in evolutionary biology to study how different organisms are related and how they evolved.

Metagenomics:

The field of metagenomics involves identification of organisms present in a body of water, sewage, dirt, debris filtered from the air, or swab samples from organisms. Knowing which organisms are present in a particular environment is critical to research in ecology, epidemiology, microbiology, and other fields. Sequencing enables researchers to determine which types of microbes may be present in a microbiome, for example.

Virology:

As most viruses are too small to be seen by a light microscope, sequencing is one of the main tools in virology to identify and study the virus.Viral genomes can be based in DNA or RNA. RNA viruses are more time-sensitive for genome sequencing, as they degrade faster in clinical samples.Traditional Sanger sequencing and next-generation sequencing are used to sequence viruses in basic and clinical research, as wells as for the diagnosis of emerging viral infections, molecular epidemiology of viral pathogens, and drug-resistance testing. There are more than 2.3 million unique viral sequences in GenBank. Recently, NGS has surpassed traditional Sanger as the most popular approach for generating viral genomes.

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Next Generation Sequencing: Transforms Today's Biology

2020-07-03T05:21:45.685Z

Future of Genomics

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Clinical genetics

There are numerous opportunities to use NGS in clinical practice to improve patient care, including:

The increased sensitivity of NGS allows detection of mosaic mutations Mosaic mutations are acquired as a postfertilisation event and consequently they present at variable frequency within the cells and tissues of an individual.

Microbiology

Oncology

Limitations

The main disadvantage of NGS in the clinical setting is putting in place the required infrastructure, such as computer capacity and storage, and also the personnel expertise required to comprehensively analyse and interpret the subsequent data. In addition, the volume of data needs to be managed skilfully to extract the clinically important information in a clear and robust interface. The actual sequencing cost of NGS is negligible.

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