Next Generation Sequencing for Diagnosis of SARS-CoV-2 Infection

Next-Generation Sequencing (NGS) is a massive parallel sequencing technology that offers ultra-high throughput, scalability, and speed. This technology is used to determine the order of nucleotides throughout the genome or target regions of DNA or RNA. NGS has revolutionized the biological sciences, allowing laboratories to perform a variety of applications and study biological systems at a level that was not previously possible. Next generation sequencing technology is used in many laboratories around the world to study genetic structure, but so far this technology has rarely been used to diagnose infectious diseases. Most next generation sequencing methods are based on the chain termination process. Thus, with the addition of deoxy-nucleotide labeled with fluorescent ends the PCR reaction and sequence reading is performed (1). This technology makes it possible to map the entire genome at an affordable cost. Currently, the COVID-19 pandemic and SARS-CoV-2, the causative agent of this disease, which has many genomic changes and causes unusual occurrences in the clinic, has increasingly attracted the attention of scientists to higher levels of genetic studies (2, 3). The next generation sequencing technique is useful in obtaining essential information about a pathogen at the beginning of an infectious outbreak and can be used as a diagnostic method for COVID-19 infection (4) and can also be useful in accurately identifying concurrent infections in COVID-19 patients. The genomic epidemiology of SARS-CoV-2 has led to the identification of several mutations in the Wuhan SARS-CoV-2 strain. During the spring of 2020, a non-synonymous mutation leading to the replacement of the D614G in Spike protein dominated the reported sequences, resulting in a higher affinity for the ACE2 receptor, enhancing viral replication. Since the summer of 2020, the emergence of major viral variants has been observed (5). These variants have been shown to be responsible for successive epidemics in different geographical areas. Cases of re-infection with SARS-CoV-2 genotypes different from genotypes that first infected patients have also been reported (6). In order to track the evolution of the virus over time, many laboratories have examined the genotype of the virus. Laboratories equipped with the ability to sequence the entire genome have reported a large number of mutations that have increased over time. However, there are significant differences between countries and in some cases, there is no database of circulating viruses (7). Analysis of SARS-CoV-2 mutations is especially important when the epitopes involved in inducing host immune responses affect the host, as they may lead to immune escape, with potential implications for vaccine (and immunotherapy) efficacy. Such an event could be evidence of an increase in transmission associated with a series of performance-related mutations for a given geographic area. SARS-CoV-2 species are defined by a set of mutations associated with the pathogenesis of the virus, and many species are now closely monitored by the World Health Organization and other public health agencies around the world (6). Variants may be directly related to lineage because they spread under the same conditions, but some species do not (e.g. B.1.1.7 - E484K is a variant, but does not conform to a particular lineage because it reproduces independently many times). A number of variants of concern (VOCs) have been categorized by the WHO, which can be recognized as the alpha variant (B.1.1.7), which has 23 mutations (13 non-synonymous mutations, four deletions, and six synonymous mutations), and more transferability and increased related mortality; beta variants (B.1.351), Gamma, Delta, and Omicron BA-1 and BA-2 with 30 mutations in Spike mentioned. Some of these mutations have recently been linked to low vaccine efficacy. Mu and Lambda can also be mentioned as variants of interest (VOIs). Co-occurring bacterial and viral infections are common, and recognizing co-infection can be helpful in applying the appropriate treatment process to overcome the disease. The next generation sequencing involves a variety of techniques, including Illumina, Ion torrent, Target enrichment, Nanopore, Metagenomics Shotgun. These techniques are considered a new approach in the diagnosis of coronaviruses (1). But it is worth noting that each of them has different advantages in the diagnosis process. For example, Shotgun Metagenomics can confirm the presence of a new pathogen that is not known, and based on this, genotypic analyzes and analysis of different variants on the new pathogen can be performed (8). Target enrichment, on the other hand, evaluates the target sample by identifying the presence of coronavirus and other key respiratory viruses in a sample using the respiratory virus panel (9). Nanopore assay, meanwhile, is a method used to correct the error by reducing the error rate of each reading by comparing multiple genome versions combined into a single combination and by analyzing readings generated from positive and negative strands. Gives. Ion torrent is another sequencing method that is a kind of semiconductor sequencing technology and has a chip that has a sensitive pH sensor and identifies the hydrogen ions released during the alignment of nucleotides for the synthesis of the genomic chain. However, the mentioned methods are different from some other aspects, one of the parameters that are different in the expressed methods is the detection threshold, which is called (LOD) (10). In Illumina method, the detection limit parameter is less than 500 copies per milliliter, and in ion torrent and Nanopore assay, the detection limit is 20 copies and 10 copies per reaction, respectively. The COVID-19 epidemic has sparked unprecedented efforts for nations. The development of effective monitoring strategies is based on sequencing the genome of the causative agent with more than 100,000 complete genomes deposited in dedicated repositories such as EpiCov, and scientists have developed this data. Studies on the evolutionary dynamics of the virus, and the identification of clinically relevant types with different techniques and equipment have been performed, and based on this, it can be concluded that the methods have different diagnostic sensitivities that depend on the purpose of the study, researchers can choose one. Pay attention to the mentioned methods. Although NGS has not been completely successful in diagnosing congenital glycosylation disorders in the past, given the enormous potential of next-generation sequencing applications, it is likely that the various next-generation sequencing techniques mentioned will soon become the first diagnostic approach in clinical laboratories, and since pandemic, we expect the next generation sequencing technology could be a promising diagnostic approach.