Next-generation Sequencing (NGS) is a massively parallel sequencing technology that offers high throughput, scalability, and speed. This technology helps to determine the entire sequence of nucleotides or targeted region of DNA (Deoxyribonucleotide) or RNA (Ribonucleic Acid). This technology was introduced in 2005 for commercial use, this method was initially called “massive-parallel-sequencing” because it enabled the sequencing of many DNA strands at a single time, instead of Sanger Sequencing which sequences a single strand of DNA at one time. The technology wide variety of applications in biological science, which have not been used before. This has helped in a rapid sequence of the genome, deploy sequence target regions, deeply sequence targets, utilize RNA sequencing to discover novel RNA variants, analyze epigenetic factors such as wide- DNA methylation and DNA-protein interaction, the study of the human microbiome, and identify novel pathogens.
COVID-19 AND ITS IMPACT ON THE NEXT GENERATION SEQUENCING
The emergence of SARS-CoV-2 was unpredictable. Factors such as inequitable global vaccine distribution, long-haul COVID-19 among immunocompromised patients, and possible transmission between humans and other mammals, have contributed to the rapid increase in the number of mutations. The continued emergence and spread of new variants reinforce the critical role that genomic sequencing has in the enhanced surveillance of COVID-19. Understanding the complete structure of virus NGS is really important as it gives the detailed structure of the entire genome of the virus. With the sudden emergence of COVID-19, it was important for scientists and researchers to have complete detail on the structure of the virus, as PCR (Polymerase Chain Reaction) was only able to detect the presence or absence of the virus. Thus, this COVID-19 emergence has driven the global next-generation sequencing market.
THE STEPS IN NEXT-GENERATION SEQUENCING ARE–
- Library Preparation- The sequencing library is prepared by random fragmentation of the DNA or cDNA sample, followed by 5′and a 3′adapter ligation. Alternatively, “tagmentation” combines the fragmentation and ligation reactions into a single step that greatly increases the efficiency of the library preparation process. Adapter-ligated fragments are then PCR amplified and gel purified
- Cluster Generation – For cluster generation, the library is loaded into a flow cell where fragments are captured on a lawn of surface-bound oligos complementary to the library adapters. Each fragment is then amplified into distinct, clonal clusters through bridge amplification. When cluster generation is complete, the templates are ready for sequencing.
- Sequencing – As all four reversible terminator–bound dNTPs are present during each sequencing cycle, natural competition minimizes corporation bias and greatly reduces raw error rates compared to other technologies. The result is highly accurate base-by-base sequencing that virtually eliminates sequence context–specific errors, even within repetitive sequence regions and homopolymers.
- Data Analysis – During data analysis and alignment, the newly identified sequence readers are aligned to the reference genome. Following alignment, many variations of analysis are possible, such as single nucleotide polymorphism (SNP)or insertion-deletion (indel) identification, read counting for RNA methods, phylogenetic or metagenomic analysis, and more.
In the last decades, significant policy attention has focused on the need to identify and limit emerging outbreaks that might lead to pandemics and to expand and sustain investment to build preparedness and health capacity. In this context, ultra-rapid and cost-effective methods for the reconstruction of the genomic sequences of emerging pathogens represent important tools for monitoring and countering the spread of novel human infectious diseases, as exemplified by recent experiences with SARS, MERS, Zika, and Ebola.
The application of ‘omics technologies’ to SARS-CoV-2 has been fundamental in epidemiological and other aspects of the fight against COVID-19. Different approaches, with different advantages and limitations, can be applied to the sequencing of SARS-CoV-2 genomes. Various considerations should influence the choice of approach in different clinical and research contexts. While more than 100 thousand complete SARS-CoV-2 genomes are currently available in public repositories, the integration of these data and associated metadata is, at present, problematic. Coordinated efforts are required to promote the principles of open science and data sharing to facilitate more efficient and comprehensive analyses of SARS-CoV-2 data.
In addition to sequencing being able to provide information about disease transmission, it was important for public health officials to understand the distribution in the community. Academic, clinical, and public health laboratories have been integral in the pandemic response through their sequencing efforts. Whole genome sequencing has been an important tool for obtaining insight about SARS-CoV-2 that assisted public health officials design targeted strategies to help minimize the spread and curb the reach of the virus across all communities. The utilization of NGS for SARS-CoV-2 surveillance has been a great asset for the public health and scientific communities.