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116-432: The MIQE guidelines were created due to the low quality of qPCR data submitted to academic journals at the time, which was only becoming more common as Next Generation Sequencing machinery allowed for such experiments to be run for a cheaper cost. Because the technique is utilized across all of science in multiple fields, the instruments, methods, and designs of how qPCR is used differ greatly. To help improve overall quality,

232-625: A healthy diet are also recommended. Supportive care in severe cases includes treatment to relieve symptoms , fluid therapy , oxygen support and prone positioning , and medications or devices to support other affected vital organs. More severe cases may need treatment in hospital. In those with low oxygen levels, use of the glucocorticoid dexamethasone is recommended to reduce mortality. Noninvasive ventilation and, ultimately, admission to an intensive care unit for mechanical ventilation may be required to support breathing. Extracorporeal membrane oxygenation (ECMO) has been used to address

348-506: A mobile app that allows for active marking of the MIQE checklist as each step is completed. An overview of the 10th anniversary since the publication of the MIQE guidelines was conducted in June 2020 and discussed the scientific studies that had produced better and more organized results when following the guidelines. In August 2020, an updated version of the guidelines for the digital PCR method

464-718: A molecular clock technique. Medical technicians may sequence genes (or, theoretically, full genomes) from patients to determine if there is risk of genetic diseases. This is a form of genetic testing , though some genetic tests may not involve DNA sequencing. As of 2013 DNA sequencing was increasingly used to diagnose and treat rare diseases. As more and more genes are identified that cause rare genetic diseases, molecular diagnoses for patients become more mainstream. DNA sequencing allows clinicians to identify genetic diseases, improve disease management, provide reproductive counseling, and more effective therapies. Gene sequencing panels are used to identify multiple potential genetic causes of

580-424: A précis for the broader form of the guidelines. Other researchers have been creating further versions for specific forms of qPCR that may require a supplementary or different set of items to check, including single-cell qPCR and digital PCR (dPCR). Appropriate adherence to the existing MIQE guidelines has also been overviewed in other scientific areas, including photobiomodulation and clinical biomarkers . It

696-404: A sequence alignment was done and the data obtained from it, and any data on the secondary structure of the amplified sequence. Creation of the oligonucleotides requires only two pieces of essential information: the primer sequences used and the location and details of any modifications made to the sequence. But there are several desirable pieces of data, including the identification number from

812-627: A variant of concern (VoC) or a variant of interest (VoI). Many of these variants have shared the more infectious D614G . As of May 2023, the WHO had downgraded all variants of concern to previously circulating as these were no longer detected in new infections. Sub-lineages of the Omicron variant (BA.1 – BA.5) were considered separate VoCs by the WHO until they were downgraded in March 2023 as no longer widely circulating. As of 24 September 2024 ,

928-407: 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. As most viruses are too small to be seen by

1044-826: A cDNA molecule, which can be time-consuming and labor-intensive. They are prone to errors and biases, which can affect the accuracy of the sequencing results. They are limited in their ability to detect rare or low-abundance transcripts. Advances in RNA Sequencing Technology In recent years, advances in RNA sequencing technology have addressed some of these limitations. New methods such as next-generation sequencing (NGS) and single-molecule real-timeref >(SMRT) sequencing have enabled faster, more accurate, and more cost-effective sequencing of RNA molecules. These advances have opened up new possibilities for studying gene expression, identifying new genes, and understanding

1160-493: A comprehensive international study. Such deaths include deaths due to healthcare capacity constraints and priorities, as well as reluctance to seek care (to avoid possible infection). Further research may help distinguish the proportions directly caused by COVID-19 from those caused by indirect consequences of the pandemic. In May 2022, the WHO estimated the number of excess deaths by the end of 2021 to be 14.9 million compared to 5.4 million reported COVID-19 deaths, with

1276-595: A large number of people". During the COVID-19 pandemic, as with other pandemics, the meaning of this term has been challenged. The end of a pandemic or other epidemic only rarely involves the total disappearance of a disease, and historically, much less attention has been given to defining the ends of epidemics than their beginnings. The ends of particular epidemics have been defined in a variety of ways, differing according to academic field, and differently based on location and social group. An epidemic's end can be considered

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1392-727: 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 well 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

1508-451: A median range of 0.24% to 1.49%. IFRs rise as a function of age (from 0.002% at age 10 and 0.01% at age 25, to 0.4% at age 55, 1.4% at age 65, 4.6% at age 75, and 15% at age 85). These rates vary by a factor of ≈10,000 across the age groups. For comparison, the IFR for middle-aged adults is two orders of magnitude higher than the annualised risk of a fatal automobile accident and much higher than

1624-637: A parallelized, adapter/ligation-mediated, bead-based sequencing technology and served as the first commercially available "next-generation" sequencing method, though no DNA sequencers were sold to independent laboratories. Allan Maxam and Walter Gilbert published a DNA sequencing method in 1977 based on chemical modification of DNA and subsequent cleavage at specific bases. Also known as chemical sequencing, this method allowed purified samples of double-stranded DNA to be used without further cloning. This method's use of radioactive labeling and its technical complexity discouraged extensive use after refinements in

1740-472: A particular modification, e.g., the 5mC ( 5 methyl cytosine ) common in humans, may or may not be detected. In almost all organisms, DNA is synthesized in vivo using only the 4 canonical bases; modification that occurs post replication creates other bases like 5 methyl C. However, some bacteriophage can incorporate a non standard base directly. In addition to modifications, DNA is under constant assault by environmental agents such as UV and Oxygen radicals. At

1856-508: 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. 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. The first DNA sequences were obtained in

1972-485: A random mixture of material suspended in fluid. Sanger's success in sequencing insulin spurred on x-ray crystallographers, including Watson and Crick, who by now were trying to understand how DNA directed the formation of proteins within a cell. Soon after attending a series of lectures given by Frederick Sanger in October 1954, Crick began developing a theory which argued that the arrangement of nucleotides in DNA determined

2088-824: A random sample have consistently found that total infections considerably exceed the reported case counts. Many countries, early on, had official policies to not test those with only mild symptoms. The strongest risk factors for severe illness are obesity, complications of diabetes , anxiety disorders, and the total number of conditions. During the start of the COVID-19 pandemic it was not clear whether young people were less likely to be infected, or less likely to develop symptoms and be tested. A retrospective cohort study in China found that children and adults were just as likely to be infected. Among more thorough studies, preliminary results from 9 April 2020 found that in Gangelt ,

2204-499: A result of some experiments by Oswald Avery , Colin MacLeod , and Maclyn McCarty demonstrating that purified DNA could change one strain of bacteria into another. This was the first time that DNA was shown capable of transforming the properties of cells. In 1953, James Watson and Francis Crick put forward their double-helix model of DNA, based on crystallized X-ray structures being studied by Rosalind Franklin . According to

2320-409: A series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each molecule. The fragments in the four reactions are electrophoresed side by side in denaturing acrylamide gels for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabeled DNA fragment, from which

2436-470: A significant turning point in DNA sequencing because it was achieved with no prior genetic profile knowledge of the virus. A non-radioactive method for transferring the DNA molecules of sequencing reaction mixtures onto an immobilizing matrix during electrophoresis was developed by Herbert Pohl and co-workers in the early 1980s. Followed by the commercialization of the DNA sequencer "Direct-Blotting-Electrophoresis-System GATC 1500" by GATC Biotech , which

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2552-461: A social phenomenon, not just a biological one. Time reported in March 2024 that expert opinions differ on whether or not COVID-19 is considered endemic or pandemic, and that the WHO continued to call the disease a pandemic on its website. During the initial outbreak in Wuhan , the virus and disease were commonly referred to as "coronavirus", "Wuhan coronavirus", "the coronavirus outbreak" and

2668-403: A suspected disorder. Also, DNA sequencing may be useful for determining a specific bacteria, to allow for more precise antibiotics treatments , hereby reducing the risk of creating antimicrobial resistance in bacteria populations. DNA sequencing may be used along with DNA profiling methods for forensic identification and paternity testing . DNA testing has evolved tremendously in

2784-451: A test. Conversely, deaths of people who died from underlying conditions following a positive test may be included. Countries such as Belgium include deaths from suspected cases, including those without a test, thereby increasing counts. Official death counts have been claimed to underreport the actual death toll, because excess mortality (the number of deaths in a period compared to a long-term average) data show an increase in deaths that

2900-486: Is also desirable to know the volume or mass of the sample that was processed for the qPCR. For the process of extracting the DNA/RNA, there are a number of essential guidelines. This includes a description of the extraction process done, a statement on what DNA extraction kit was used and any changes made to the directions, details on whether any DNase or RNase treatment was used, a statement on whether any contamination

3016-576: 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 . 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 and noncoding DNA (including regulatory sequences), associations with diseases and phenotypes, and identify potential drug targets. Since DNA

3132-480: 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. In February 2021, scientists reported, for the first time, the sequencing of DNA from animal remains , a mammoth in this instance, over a million years old, the oldest DNA sequenced to date. The field of metagenomics involves identification of organisms present in

3248-518: Is associated with COVID-19 and is reported in as many as 88% of cases. The disease is mainly transmitted via the respiratory route when people inhale droplets and small airborne particles (that form an aerosol ) that infected people exhale as they breathe, talk, cough, sneeze, or sing. Infected people are more likely to transmit COVID-19 when they are physically close to other non-infected individuals. However, infection can occur over longer distances, particularly indoors. SARS‑CoV‑2 belongs to

3364-611: Is discouraged altogether by the World Health Organization (WHO), due to limited evidence of its efficacy. The severity of COVID-19 varies. It may take a mild course with few or no symptoms, resembling other common upper respiratory diseases such as the common cold . In 3–4% of cases (7.4% for those over age 65) symptoms are severe enough to cause hospitalisation. Mild cases typically recover within two weeks, while those with severe or critical diseases may take three to six weeks to recover. Among those who have died,

3480-439: Is intended to provide acquired immunity against severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2), the virus that causes coronavirus disease 2019 ( COVID-19 ). Prior to the COVID-19 pandemic, an established body of knowledge existed about the structure and function of coronaviruses causing diseases like severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). This knowledge accelerated

3596-490: Is not explained by COVID-19 deaths alone. Using such data, estimates of the true number of deaths from COVID-19 worldwide have included a range from 18.2 to 33.5 million (≈27.4 million) by 18 November 2023 by The Economist , as well as over 18.5 million by 1 April 2023 by the Institute for Health Metrics and Evaluation and ≈18.2 million (earlier) deaths between 1 January 2020, and 31 December 2021, by

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3712-598: Is now implemented in Illumina 's Hi-Seq genome sequencers. In 1998, Phil Green and Brent Ewing of the University of Washington described their phred quality score for sequencer data analysis, a landmark analysis technique that gained widespread adoption, and which is still the most common metric for assessing the accuracy of a sequencing platform. Lynx Therapeutics published and marketed massively parallel signature sequencing (MPSS), in 2000. This method incorporated

3828-436: Is possible because multiple fragments are sequenced at once (giving it the name "massively parallel" sequencing) in an automated process. NGS technology has tremendously empowered researchers to look for insights into health, anthropologists to investigate human origins, and is catalyzing the " Personalized Medicine " movement. However, it has also opened the door to more room for error. There are many software tools to carry out

3944-405: Is the determination of the physical order of these bases in a molecule of DNA. However, there are many other bases that may be present in a molecule. In some viruses (specifically, bacteriophage ), cytosine may be replaced by hydroxy methyl or hydroxy methyl glucose cytosine. In mammalian DNA, variant bases with methyl groups or phosphosulfate may be found. Depending on the sequencing technique,

4060-452: Is the ratio of deaths to diagnoses. This metric can be misleading because of the delay between symptom onset and death and because testing focuses on symptomatic individuals. Based on Johns Hopkins University statistics, the global CFR was 1.02 percent (6,881,955 deaths for 676,609,955 cases) as of 10 March 2023. The number varies by region and has generally declined over time. Several variants have been named by WHO and labelled as

4176-406: Is the seventh known coronavirus to infect people, after 229E , NL63 , OC43 , HKU1 , MERS-CoV , and the original SARS-CoV . The standard method of testing for presence of SARS-CoV-2 is a nucleic acid test , which detects the presence of viral RNA fragments. As these tests detect RNA but not infectious virus, its "ability to determine duration of infectivity of patients is limited." The test

4292-460: Is then synthesized through a process called PCR ( Polymerase Chain Reaction ), which amplifies the cDNA to produce multiple copies. 3) Sequencing : The amplified cDNA is then sequenced using a technique such as Sanger sequencing or Maxam-Gilbert sequencing . Challenges and Limitations Traditional RNA sequencing methods have several limitations. For example: They require the creation of

4408-614: Is typically done on respiratory samples obtained by a nasopharyngeal swab ; however, a nasal swab or sputum sample may also be used. The WHO has published several testing protocols for the disease. Preventive measures to reduce the chances of infection include getting vaccinated, staying at home or spending more time outdoors, avoiding crowded places, keeping distance from others, wearing a mask in public, ventilating indoor spaces, managing potential exposure durations, washing hands with soap and water often and for at least twenty seconds, practising good respiratory hygiene, and avoiding touching

4524-657: 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, DNA Genographic Projects 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

4640-514: The Huanan Seafood Wholesale Market there, but it is possible that human-to-human transmission began earlier. Molecular clock analysis suggests that the first cases were likely to have been between October and November 2019. The scientific consensus is that the virus is most likely of a zoonotic origin, from bats or another closely related mammal. While other explanations such as speculations that SARS-CoV-2

4756-407: The Institute for Health Metrics and Evaluation estimating the true number of cases as of early 2022 to be in the billions. One measure that public health officials and policymakers have used to monitor the pandemic and guide decision-making is the test positivity rate ("percent positive"). According to Johns Hopkins in 2020, one benchmark for a "too high" per cent positive is 5%, which was used by

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4872-480: The MRC Centre , Cambridge , UK and published a method for "DNA sequencing with chain-terminating inhibitors" in 1977. Walter Gilbert and Allan Maxam at Harvard also developed sequencing methods, including one for "DNA sequencing by chemical degradation". In 1973, Gilbert and Maxam reported the sequence of 24 basepairs using a method known as wandering-spot analysis. Advancements in sequencing were aided by

4988-537: The University of Ghent ( Ghent , Belgium ), in 1972 and 1976. Traditional RNA sequencing methods require the creation of a cDNA molecule which must be sequenced. Traditional RNA Sequencing Methods Traditional RNA sequencing methods involve several steps: 1) Reverse Transcription : The first step is to convert the RNA molecule into a complementary DNA (cDNA) molecule using an enzyme called reverse transcriptase . 2) cDNA Synthesis : The cDNA molecule

5104-602: The basic reproduction number (R 0 ) for COVID-19 in January 2020 were between 1.4 and 2.5, but a subsequent analysis claimed that it may be about 5.7 (with a 95 per cent confidence interval of 3.8 to 8.9). In December 2021, the number of cases continued to climb due to several factors, including new COVID-19 variants. As of that 28   December, 282,790,822 individuals worldwide had been confirmed as infected. As of 14 April 2022 , over 500 million cases were confirmed globally. Most cases are unconfirmed, with

5220-474: The variants of interest as specified by the World Health Organization are BA.2.86 and JN.1, and the variants under monitoring are JN.1.7, KP.2, KP.3, KP.3.1.1, JN.1.18, LB.1, and XEC. Symptoms of COVID-19 are variable, ranging from mild symptoms to severe illness. Common symptoms include headache, loss of smell and taste , nasal congestion and runny nose , cough, muscle pain , sore throat , fever, diarrhoea , and breathing difficulties . People with

5336-429: The "Wuhan coronavirus outbreak", with the disease sometimes called "Wuhan pneumonia ". In January 2020, the WHO recommended 2019-nCoV and 2019-nCoV acute respiratory disease as interim names for the virus and disease per 2015 international guidelines against using geographical locations (e.g. Wuhan, China), animal species, or groups of people in disease and virus names in part to prevent social stigma . WHO finalised

5452-655: The ABI 370, in 1987 and by Dupont's Genesis 2000 which used a novel fluorescent labeling technique enabling all four dideoxynucleotides to be identified in a single lane. By 1990, the U.S. National Institutes of Health (NIH) had begun large-scale sequencing trials on Mycoplasma capricolum , Escherichia coli , Caenorhabditis elegans , and Saccharomyces cerevisiae at a cost of US$ 0.75 per base. Meanwhile, sequencing of human cDNA sequences called expressed sequence tags began in Craig Venter 's lab, an attempt to capture

5568-404: The MIQE guidelines were made as generalized suggestions on basic experimental procedures and forms of data that should be collected as a minimum level of reported information for other researchers to understand and use when reading the published material. Setting up a recognized and largely agreed upon set of guidelines such as these were deemed important by the scientific community especially due to

5684-627: The NGS field have been attempted to address these challenges, most of which have been small-scale efforts arising from individual labs. Most recently, a large, organized, FDA-funded effort has culminated in the BioCompute standard. On 26 October 1990, Roger Tsien , Pepi Ross, Margaret Fahnestock and Allan J Johnston filed a patent describing stepwise ("base-by-base") sequencing with removable 3' blockers on DNA arrays (blots and single DNA molecules). In 1996, Pål Nyrén and his student Mostafa Ronaghi at

5800-517: The RTPrimerDB database, the sequences from the probes , the manufacturer used to make the oligos, and how they were purified. As one of the primary segments of the guidelines, there are several essential parts on the checklist for the qPCR process itself. This includes the full set of conditions used for the reaction, the volume of both the reaction and the cDNA, the concentrations for the probes, magnesium ions, and dNTPs , what kind of polymerase

5916-659: The Royal Institute of Technology in Stockholm published their method of pyrosequencing . On 1 April 1997, Pascal Mayer and Laurent Farinelli submitted patents to the World Intellectual Property Organization describing DNA colony sequencing. The DNA sample preparation and random surface- polymerase chain reaction (PCR) arraying methods described in this patent, coupled to Roger Tsien et al.'s "base-by-base" sequencing method,

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6032-495: The Sanger methods had been made. Maxam-Gilbert sequencing requires radioactive labeling at one 5' end of the DNA and purification of the DNA fragment to be sequenced. Chemical treatment then generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). The concentration of the modifying chemicals is controlled to introduce on average one modification per DNA molecule. Thus

6148-640: The WHO in the past. As of 10 March 2023, more than 6.88   million deaths had been attributed to COVID-19. The first confirmed death was in Wuhan on 9 January 2020. These numbers vary by region and over time, influenced by testing volume, healthcare system quality, treatment options, government response, time since the initial outbreak, and population characteristics, such as age, sex, and overall health. Multiple measures are used to quantify mortality. Official death counts typically include people who died after testing positive. Such counts exclude deaths without

6264-419: The WHO, US, European Union, and Australia. On 12 November 2022, the WHO released its Global Vaccine Market Report. The report indicated that "inequitable distribution is not unique to COVID-19 vaccines"; countries that are not economically strong struggle to obtain vaccines. On 14 November 2022, the first inhalable vaccine was introduced, developed by Chinese biopharmaceutical company CanSino Biologics , in

6380-473: The author's laboratory itself or a core laboratory of the university or organization conducted the qPCR assay and an acknowledgement of any other individuals that contributed to the work. The essential requirements that samples and sample material must meet includes a description of the sample, what form of dissection was used, what processing method was done, whether the samples were frozen or fixed and how long did it take, and what sample conditions were used. It

6496-448: The broad family of viruses known as coronaviruses . It is a positive-sense single-stranded RNA (+ssRNA) virus, with a single linear RNA segment. Coronaviruses infect humans, other mammals, including livestock and companion animals, and avian species. Human coronaviruses are capable of causing illnesses ranging from the common cold to more severe diseases such as Middle East respiratory syndrome (MERS, fatality rate ≈34%). SARS-CoV-2

6612-648: The centre of a major infection cluster in Germany, 15 per cent of a population sample tested positive for antibodies . Screening for COVID-19 in pregnant women in New York City , and blood donors in the Netherlands, found rates of positive antibody tests that indicated more infections than reported. Seroprevalence -based estimates are conservative as some studies show that persons with mild symptoms do not have detectable antibodies. Initial estimates of

6728-760: The city of Shanghai, China. For the first two years of the pandemic, no specific and effective treatment or cure was available. In 2021, the European Medicines Agency's (EMA) Committee for Medicinal Products for Human Use (CHMP) approved the oral antiviral protease inhibitor , Paxlovid (nirmatrelvir plus the HIV antiviral ritonavir ), to treat adult patients. FDA later gave it an EUA. Most cases of COVID-19 are mild. In these, supportive care includes medication such as paracetamol or NSAIDs to relieve symptoms (fever, body aches, cough), adequate intake of oral fluids and rest. Good personal hygiene and

6844-479: The clinical presence of COVID-19 viral particles during the COVID-19 pandemic . The MIQE guidelines are split up into 9 different sections that make up the checklist. These include not only considerations for doing the qPCR itself, but also how the resulting data is collected, analyzed, and presented. An important part of the latter is including information relating to the analysis software used and also submitting

6960-418: The coding fraction of the human genome . In 1995, Venter, Hamilton Smith , and colleagues at The Institute for Genomic Research (TIGR) published the first complete genome of a free-living organism, the bacterium Haemophilus influenzae . The circular chromosome contains 1,830,137 bases and its publication in the journal Science marked the first published use of whole-genome shotgun sequencing, eliminating

7076-406: The computational analysis of NGS data, often compiled at online platforms such as CSI NGS Portal, each with its own algorithm. Even the parameters within one software package can change the outcome of the analysis. In addition, the large quantities of data produced by DNA sequencing have also required development of new methods and programs for sequence analysis. Several efforts to develop standards in

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7192-466: The concentration and type of reverse transcriptase used, and lastly the temperature and amount of time done for the reaction. It is also desirable to have the catalog numbers of reagents used and their manufacturers, the standard deviation for the Cq with and without the transcriptase being involved, and how the cDNA was stored. All of the basic information regarding the target is necessary here, including

7308-464: The concurrent development of recombinant DNA technology, allowing DNA samples to be isolated from sources other than viruses. The first full DNA genome to be sequenced was that of bacteriophage φX174 in 1977. Medical Research Council scientists deciphered the complete DNA sequence of the Epstein-Barr virus in 1984, finding it contained 172,282 nucleotides. Completion of the sequence marked

7424-484: The cumulative number of infected individuals (including asymptomatic and undiagnosed infections and excluding vaccinated infected individuals). It is expressed in percentage points. Other studies refer to this metric as the infection fatality risk . In November 2020, a review article in Nature reported estimates of population-weighted IFRs for various countries, excluding deaths in elderly care facilities, and found

7540-569: The development of new forensic techniques, such as DNA phenotyping , which allows investigators to predict an individual's physical characteristics based on their genetic data. In addition to its applications in forensic science, DNA sequencing has also been used in medical research and diagnosis. It has enabled scientists to identify genetic mutations and variations that are associated with certain diseases and disorders, allowing for more accurate diagnoses and targeted treatments. Moreover, DNA sequencing has also been used in conservation biology to study

7656-441: The development of various vaccine platforms during early 2020. The initial focus of SARS-CoV-2 vaccines was on preventing symptomatic and severe illness. The COVID-19 vaccines are widely credited for their role in reducing the severity and death caused by COVID-19. As of March 2023, more than 5.5 billion people had received one or more doses (11.8 billion in total) in over 197 countries. The Oxford-AstraZeneca vaccine

7772-437: The earlier methods, including Sanger sequencing . In contrast to the first generation of sequencing, NGS technology is typically characterized by being highly scalable, allowing the entire genome to be sequenced at once. Usually, this is accomplished by fragmenting the genome into small pieces, randomly sampling for a fragment, and sequencing it using one of a variety of technologies, such as those described below. An entire genome

7888-478: The early 1970s by academic researchers using laborious methods based on two-dimensional chromatography . Following the development of fluorescence -based sequencing methods with a DNA sequencer , DNA sequencing has become easier and orders of magnitude faster. 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

8004-424: The ever increasing amount of scientific work coming from developing countries with many different languages and protocols. In 2009, Stephen Bustin led an international group of scientists including Mikael Kubista to put together a set of guidelines on how to perform qPCR and what forms of data should be collected and published in the process. This also allowed editors and reviewers of scientific journals to employ

8120-513: The eyes, nose, or mouth with unwashed hands. Those diagnosed with COVID-19 or who believe they may be infected are advised by healthcare authorities to stay home except to get medical care, call ahead before visiting a healthcare provider, wear a face mask before entering the healthcare provider's office and when in any room or vehicle with another person, cover coughs and sneezes with a tissue, regularly wash hands with soap and water and avoid sharing personal household items. A COVID-19 vaccine

8236-601: The fact once their lack of proper data to pass the MIQE guidelines was noted and publicly pointed out to the journal editors. When setting up their new comparative qPCR systems titled "Dots in Boxes" in 2017, New England Biolabs stated that they had designed the data collection portion around the MIQE guidelines so that the data fit all the minimum parameter checklists in the protocols. Other scientific instrument companies have assisted in guideline compliance by purposefully tailoring their devices for them, including Bio-Rad creating

8352-461: The gene symbol, the accession database number for the sequence in question, the length of the sequence being amplified, information about the specificity screen used such as BLAST , what splicing variants exist for the sequence, and where the exon or intron for each primer is. There are several desired, but not required information pieces for this section, such as the location of the amplicon , whether any pseudogenes or homologs exist, whether

8468-530: The general public beginning in December 2020, made available through government and international programs such as COVAX , aiming to provide vaccine equity . Treatments include novel antiviral drugs and symptom control. Common mitigation measures during the public health emergency included travel restrictions , lockdowns , business restrictions and closures, workplace hazard controls , mask mandates , quarantines, testing systems, and contact tracing of

8584-400: The genetic diversity of endangered species and develop strategies for their conservation. Furthermore, the use of DNA sequencing has also raised important ethical and legal considerations. For example, there are concerns about the privacy and security of genetic data, as well as the potential for misuse or discrimination based on genetic information. As a result, there are ongoing debates about

8700-401: The guidelines involves information on how the analysis of the qPCR data was done. The essential parts of that include the program and program version used for the analysis, the method for how the Cq was determined, figuring out the outlier points in the data and how they are used or excluded and why, what results were found for the controls with no template genetic material, an explanation for why

8816-497: The guidelines when looking over a submitted paper that included qPCR data. Thus, the guidelines were set up as a sort of checklist for each step of the procedure with certain items being marked as essential (E) when submitting data for publication and others marked as just desirable (D). An additional version of the guidelines was published in September 2010 for use with fluorescence-based quantitative real-time PCR. It also acted as

8932-665: The infected. The pandemic caused severe social and economic disruption around the world, including the largest global recession since the Great Depression . Widespread supply shortages , including food shortages , were caused by supply chain disruptions and panic buying . Reduced human activity led to an unprecedented temporary decrease in pollution . Educational institutions and public areas were partially or fully closed in many jurisdictions, and many events were cancelled or postponed during 2020 and 2021. Telework became much more common for white-collar workers as

9048-432: The initial results into doubt. All of this was despite many papers directly citing Bustin's original MIQE publication, but not following through on the guideline checklist of material in their own experiments. However, some researchers have pointed out at least some success, with a number of papers being rejected by academic journals for publication due to failing to pass MIQE checklists. Other studies have been retracted after

9164-487: The issue of respiratory failure. Existing drugs such as hydroxychloroquine , lopinavir/ritonavir , and ivermectin are not recommended by US or European health authorities, as there is no good evidence they have any useful effect. The antiviral remdesivir is available in the US, Canada, Australia, and several other countries, with varying restrictions; however, it is not recommended for use with mechanical ventilation, and

9280-484: The last few decades to ultimately link a DNA print to what is under investigation. The DNA patterns in fingerprint, saliva, hair follicles, etc. uniquely separate each living organism from another. Testing DNA is a technique which can detect specific genomes in a DNA strand to produce a unique and individualized pattern. DNA sequencing may be used along with DNA profiling methods for forensic identification and paternity testing , as it has evolved significantly over

9396-576: The majority of the unreported 9.5 million deaths believed to be direct deaths due the virus, rather than indirect deaths. Some deaths were because people with other conditions could not access medical services . A December 2022 WHO study estimated excess deaths from the pandemic during 2020 and 2021, again concluding ≈14.8 million excess early deaths occurred, reaffirming and detailing their prior calculations from May as well as updating them, addressing criticisms. These numbers do not include measures like years of potential life lost and may make

9512-438: The model, DNA is composed of two strands of nucleotides coiled around each other, linked together by hydrogen bonds and running in opposite directions. Each strand is composed of four complementary nucleotides – adenine (A), cytosine (C), guanine (G) and thymine (T) – with an A on one strand always paired with T on the other, and C always paired with G. They proposed that such a structure allowed each strand to be used to reconstruct

9628-466: The most popular approach for generating viral genomes. During the 1997 avian influenza outbreak , viral sequencing determined that the influenza sub-type originated through reassortment between quail and poultry. This led to legislation in Hong Kong that prohibited selling live quail and poultry together at market. Viral sequencing can also be used to estimate when a viral outbreak began by using

9744-414: The need for initial mapping efforts. By 2001, shotgun sequencing methods had been used to produce a draft sequence of the human genome. Several new methods for DNA sequencing were developed in the mid to late 1990s and were implemented in commercial DNA sequencers by 2000. Together these were called the "next-generation" or "second-generation" sequencing (NGS) methods, in order to distinguish them from

9860-438: The need for regulations and guidelines to ensure the responsible use of DNA sequencing technology. Overall, the development of DNA sequencing technology has revolutionized the field of forensic science and has far-reaching implications for our understanding of genetics, medicine, and conservation biology. The canonical structure of DNA has four bases: thymine (T), adenine (A), cytosine (C), and guanine (G). DNA sequencing

9976-495: The official names COVID-19 and SARS-CoV-2 on 11 February 2020. Tedros Adhanom Ghebreyesus explained: CO   for corona , VI   for virus , D   for disease and 19 for when the outbreak was first identified (31 December 2019). WHO additionally uses "the COVID-19 virus" and "the virus responsible for COVID-19" in public communications. WHO named variants of concern and variants of interest using Greek letters . The initial practice of naming them according to where

10092-427: The other, an idea central to the passing on of hereditary information between generations. The foundation for sequencing proteins was first laid by the work of Frederick Sanger who by 1955 had completed the sequence of all the amino acids in insulin , a small protein secreted by the pancreas. This provided the first conclusive evidence that proteins were chemical entities with a specific molecular pattern rather than

10208-534: The outbreak a public health emergency of international concern (PHEIC) on 30 January 2020, and assessed the outbreak as having become a pandemic on 11 March. COVID-19 symptoms range from asymptomatic to deadly, but most commonly include fever, sore throat , nocturnal cough , and fatigue. Transmission of the virus is often through airborne particles . Mutations have produced many strains (variants) with varying degrees of infectivity and virulence . COVID-19 vaccines were developed rapidly and deployed to

10324-407: The pandemic 2021's leading cause of death . The time between symptom onset and death ranges from   6 to 41 days, typically about 14 days. Mortality rates increase as a function of age. People at the greatest mortality risk are the elderly and those with underlying conditions. The infection fatality ratio (IFR) is the cumulative number of deaths attributed to the disease divided by

10440-495: The pandemic evolved. Misinformation circulated through social media and mass media , and political tensions intensified . The pandemic raised issues of racial and geographic discrimination , health equity , and the balance between public health imperatives and individual rights . The WHO ended the PHEIC for COVID-19 on 5 May 2023. The disease has continued to circulate, but as of 2024, experts were uncertain as to whether it

10556-974: The past few decades to ultimately link a DNA print to what is under investigation. The DNA patterns in fingerprint, saliva, hair follicles, and other bodily fluids uniquely separate each living organism from another, making it an invaluable tool in the field of forensic science . The process of DNA testing involves detecting specific genomes in a DNA strand to produce a unique and individualized pattern, which can be used to identify individuals or determine their relationships. The advancements in DNA sequencing technology have made it possible to analyze and compare large amounts of genetic data quickly and accurately, allowing investigators to gather evidence and solve crimes more efficiently. This technology has been used in various applications, including forensic identification, paternity testing, and human identification in cases where traditional identification methods are unavailable or unreliable. The use of DNA sequencing has also led to

10672-478: The population's age structure and the age-specific pattern of infections. There have also been reviews that have compared the fatality rate of this pandemic with prior pandemics, such as MERS-CoV. For comparison the infection mortality rate of seasonal flu in the United States is 0.1%, which is 13 times lower than COVID-19. Another metric in assessing death rate is the case fatality ratio (CFR), which

10788-411: The present time, the presence of such damaged bases is not detected by most DNA sequencing methods, although PacBio has published on this. Deoxyribonucleic acid ( DNA ) was first discovered and isolated by Friedrich Miescher in 1869, but it remained under-studied for many decades because proteins, rather than DNA, were thought to hold the genetic blueprint to life. This situation changed after 1944 as

10904-488: The raw data to the relevant databases. Large portions of the guidelines include basic actions that would normally be included in experiments and publications regardless, such as an item for describing the experimental and control group differences. Other such information includes how many individual units are used in each group in the experiment. These two pieces are defined as essential for any study. This section also includes two desirable points, which are pointing out whether

11020-409: The reagents used were obtained from, what level of genetic purity was obtained, what yield was obtained, and an electrophoresis gel image for confirmation. The primary essential parts for this phase include detailing the reaction conditions in full, giving both the amount of RNA used and the total volume of the reaction, give information on the oligonucleotide used as a primer and its concentration,

11136-455: The reference genes used were chosen and why the number of them was chosen, the method used to normalize the data, how many technical replicates were included, how repeatable was the data within the assays, what methods were used to determine significance of the results, and what software was used for this part of the qualitative analysis. It is also desired to include information on the number of biological replicates and whether they matched

11252-672: The regulation of gene expression. The first method for determining DNA sequences involved a location-specific primer extension strategy established by Ray Wu at Cornell University in 1970. DNA polymerase catalysis and specific nucleotide labeling, both of which figure prominently in current sequencing schemes, were used to sequence the cohesive ends of lambda phage DNA. Between 1970 and 1973, Wu, R Padmanabhan and colleagues demonstrated that this method can be employed to determine any DNA sequence using synthetic location-specific primers. Frederick Sanger then adopted this primer-extension strategy to develop more rapid DNA sequencing methods at

11368-507: The results from the technical replicates, the reproducibility data for the concentration variants, data on the power analysis , and lastly for the researchers to submit the raw data in the RDML file format. Next Generation Sequencing DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA . It includes any method or technology that

11484-485: The results were still positive ( LOD ) along with the evidence for the LOD itself, and lastly if a multiplex is used, then the efficiency and LOD must be given for each assay done. The extra desired information includes evidence given that qPCR optimization occurred by the use of gradients, the confidence intervals to show efficiency of the qPCR, and the confidence intervals for the entire range tested. The final section of

11600-462: The risk of dying from seasonal influenza . In December 2020, a systematic review and meta-analysis estimated that population-weighted IFR was 0.5% to 1% in some countries (France, Netherlands, New Zealand, and Portugal), 1% to 2% in other countries (Australia, England, Lithuania, and Spain), and about 2.5% in Italy. This study reported that most of the differences reflected corresponding differences in

11716-496: The same infection may have different symptoms, and their symptoms may change over time. Three common clusters of symptoms have been identified: one respiratory symptom cluster with cough, sputum , shortness of breath, and fever; a musculoskeletal symptom cluster with muscle and joint pain, headache, and fatigue; a cluster of digestive symptoms with abdominal pain, vomiting, and diarrhoea. In people without prior ear, nose, and throat disorders, loss of taste combined with loss of smell

11832-464: The sequence may be inferred. This method is mostly obsolete as of 2023. COVID-19 pandemic The COVID-19 pandemic (also known as the coronavirus pandemic and COVID pandemic ), caused by severe acute respiratory syndrome coronavirus 2 ( SARS-CoV-2 ), began with an outbreak of COVID-19 in Wuhan , China, in December 2019. It spread to other areas of Asia, and then worldwide in early 2020. The World Health Organization (WHO) declared

11948-400: The sequence of amino acids in proteins, which in turn helped determine the function of a protein. He published this theory in 1958. RNA sequencing was one of the earliest forms of nucleotide sequencing. The major landmark of RNA sequencing is the sequence of the first complete gene and the complete genome of Bacteriophage MS2 , identified and published by Walter Fiers and his coworkers at

12064-411: The time from symptom onset to death has ranged from two to eight weeks. Prolonged prothrombin time and elevated C-reactive protein levels on admission to the hospital are associated with severe course of COVID-19 and with a transfer to intensive care units (ICU). Between 5% and 50% of COVID-19 patients experience long COVID , a condition characterised by long-term consequences persisting after

12180-694: The typical convalescence period of the disease. The most commonly reported clinical presentations are fatigue and memory problems, as well as malaise , headaches, shortness of breath , loss of smell, muscle weakness , low fever and cognitive dysfunction . Many countries attempted to slow or stop the spread of COVID-19 by recommending, mandating or prohibiting behaviour changes, while others relied primarily on providing information. Measures ranged from public advisories to stringent lockdowns. Outbreak control strategies are divided into elimination and mitigation. Experts differentiate between elimination strategies (known as " zero-COVID ") that aim to completely stop

12296-543: The variants were identified (e.g. Delta began as the " Indian variant") is no longer common. A more systematic naming scheme reflects the variant's PANGO lineage (e.g., Omicron 's lineage is B.1.1.529) and is used for other variants. SARS-CoV-2 is a virus closely related to bat coronaviruses , pangolin coronaviruses, and SARS-CoV . The first known outbreak (the 2019–2020 COVID-19 outbreak in mainland China ) started in Wuhan , Hubei, China, in December 2019. Many early cases were linked to people who had visited

12412-443: Was accidentally released from a laboratory have been proposed, as of 2021 these were not supported by evidence. Official "case" counts refer to the number of people who have been tested for COVID-19 and whose test has been confirmed positive according to official protocols whether or not they experienced symptomatic disease. Due to the effect of sampling bias , studies which obtain a more accurate number by extrapolating from

12528-414: Was assessed, a quantification of the amount of genetic material extracted, a description of the instruments used for the extraction, the methods used to retain RNA integrity, a statement on the RNA integrity number and quality indicator and the quantification cycle (Cq) reached, and lastly what testing was done to determine the presence or absence of inhibitors . Four desired pieces of information are where

12644-522: Was intensively used in the framework of the EU genome-sequencing programme, the complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome II. Leroy E. Hood 's laboratory at the California Institute of Technology announced the first semi-automated DNA sequencing machine in 1986. This was followed by Applied Biosystems ' marketing of the first fully automated sequencing machine,

12760-487: Was noted by Bustin in 2014 (and again by him in 2017) that there was some amount of uptake and usage of the MIQE guidelines within the scientific community, but there were still far too many published papers with qPCR experiments that lacked even the most basic of data presentation and proper confirmation of effectiveness for said data. These studies retained major reproducibility issues, where the conclusions of their evidence could not be replicated by other researchers, throwing

12876-408: Was published to account for improvement in machinery, technologies, and techniques since the original 2013 release. Additional guideline steps were added for data analysis, while also providing a more simplified checklist table for researchers to use. An RT-qPCR targeting assay was developed alongside Stephen Bustin using the MIQE guidelines for clinical biomarkers in December 2020 in order to identify

12992-434: Was set up manually or by a machine. In order to confirm the effectiveness and quality of the qPCR process that was performed, there are several actions and subsequent data that must be presented. This includes explaining the specific method of checking that the process functioned, such as using a gel, direct sequencing of the genetic material, showing a melt profile , or from digestion by restriction enzyme . If SYBR Green I

13108-457: Was still a pandemic. Pandemics and their ends are not well-defined, and whether or not one has ended differs according to the definition used. As of 24 November 2024, COVID-19 has caused 7,075,455 confirmed deaths. The COVID-19 pandemic ranks as the fifth- deadliest pandemic or epidemic in history . In epidemiology , a pandemic is defined as "an epidemic occurring over a very wide area, crossing international boundaries, and usually affecting

13224-509: Was the most widely used. According to a June 2022 study, COVID-19 vaccines prevented an additional 14.4 million to 19.8 million deaths in 185 countries and territories from 8 December 2020 to 8 December 2021. On 8 November 2022, the first recombinant protein-based COVID-19 vaccine (Novavax's booster Nuvaxovid ) was authorised for use in adults in the United Kingdom. It has subsequently received endorsement/authorisation from

13340-411: Was used and its concentration, what kit was used and its manufacturer, what additives to the reaction were used, who manufactured the qPCR machine, and what parameters were set for the thermocycling process. The only additional desired pieces of information are the chemical composition of the buffer used, who manufactured the plates and tubes used and what their catalog number is, and whether the reaction

13456-477: Was used, then the Cq of the control group with no template DNA must be given. Further essential data includes the calibration of the machine curves with the slope and y intercept noted, the efficiency of the PCR process as determined from the aforementioned slope, the correlation coefficients (r squared) for the calibration curves, the dynamic range of the linear curves, the Cq found at the lowest concentration where 95% of

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