Cotton Genomics and Genetics 2024, Vol.15, No.2, 112-126 http://cropscipublisher.com/index.php/cgg 113 Various NGS methodologies, such as sequencing by synthesis, ion semiconductor sequencing, and nanopore sequencing, offer unique strengths and are continually evolving to overcome existing limitations (Satam et al., 2023). These technologies have been instrumental in advancing our understanding of complex genomes, including those of polyploid crops like cotton, by providing detailed insights into genetic diversity, gene function, and evolutionary processes (Rexach et al., 2019). In cotton genomics, NGS has opened new avenues for exploring genetic diversity, identifying key genes and regulatory networks, and accelerating breeding programs through marker-assisted selection and genomic selection. This study summarizes the current advancements in cotton genome research and highlights the major challenges and opportunities posed by the complexity of the cotton genome. It discusses the progress in cotton genomics driven by NGS, including the identification of genetic markers, understanding fiber biogenesis, and gaining insights into the evolutionary history of Gossypium species. The study evaluates the potential of NGS-enabled breeding strategies to enhance cotton yield, quality, and environmental adaptability, thereby contributing to sustainable agricultural practices. This study aims to identify the key roles of NGS technologies in advancing cotton genomics, providing insights for future research and agricultural innovation. 2 Overview of Next-Generation Sequencing Technologies Next-Generation Sequencing (NGS) technologies have revolutionized the field of genomics by providing unprecedented depth, accuracy, and throughput in sequencing. These technologies have evolved significantly since the days of Sanger sequencing, offering a wide array of applications and efficiencies that were previously unattainable. 2.1 History and development of NGS The development of NGS technologies marked a significant leap from the traditional Sanger sequencing method, which was the gold standard for many years. The advent of NGS in the early 2000s introduced high-throughput sequencing capabilities, drastically reducing the time and cost associated with sequencing large genomes. NGS technologies enable the simultaneous sequencing of millions of DNA fragments, providing comprehensive insights into genome structure, genetic variations, and gene expression profiles. The continuous evolution of these technologies has led to significant breakthroughs in various fields, including personalized medicine, evolutionary biology, and agricultural genomics. The first generation of NGS technologies, often referred to as second-generation sequencing, includes platforms like Illumina and Ion Torrent, which are characterized by their short-read lengths and high accuracy (Kumar er al., 2019; Hu et al., 2021). The early 2010s saw the emergence of third-generation sequencing (TGS) technologies, such as Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), which offer long-read sequencing capabilities, further enhancing the ability to resolve complex genomic regions and structural variations (Midha et al., 2019; Athanasopoulou et al., 2021). 2.2 Types of NGS technologies NGS technologies are diverse and can be broadly categorized based on their sequencing methodologies and read lengths. The most prominent NGS platforms include Illumina sequencing, Pacific Biosciences (PacBio) sequencing, and Oxford Nanopore sequencing (Figure 1), each offering unique advantages and limitations. 2.2.1 Illumina sequencing Illumina sequencing, also known as sequencing by synthesis (SBS), a short-read technology, is one of the most widely used NGS platforms. It employs a sequencing-by-synthesis approach, where fluorescently labeled nucleotides are incorporated into a growing DNA strand and detected in real-time. This method provides high accuracy and throughput, making it suitable for a wide range of applications, including whole-genome sequencing, RNA sequencing, and targeted sequencing (Hu et al., 2021; Kumar et al., 2019). However, the short read lengths (typically 150-300 base pairs) can pose challenges in sequencing repetitive regions and complex genomes (Baptista et al., 2018). 2.2.2 PacBio sequencing Pacific Biosciences (PacBio) sequencing, or Single Molecule Real-Time (SMRT) sequencing, a third-generation technology, offers long-read sequencing capabilities through its Single Molecule Real-Time (SMRT) sequencing
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