Cotton Genomics and Genetics 2024, Vol.15, No.2, 112-126 http://cropscipublisher.com/index.php/cgg 115 2.2.3 Oxford Nanopore sequencing Oxford Nanopore Technologies (ONT) is another third-generation sequencing platform that provides ultra-long reads, with some reads exceeding 1 million base pairs. ONT's nanopore sequencing technology involves passing a DNA or RNA molecule through a nanopore and measuring changes in electrical current to determine the sequence. This method allows for real-time sequencing and direct RNA sequencing, offering unique advantages in studying epitranscriptomics and RNA modifications (Midha et al., 2019; Athanasopoulou et al., 2021). ONT is also known for its portability, with devices like the MinION enabling sequencing in various settings outside the traditional laboratory environment (Midha et al., 2019; Lang et al., 2020). Despite its versatility, the technology still faces challenges related to accuracy and error rates, which are areas of active research and improvement. 2.3 Comparison of NGS technologies When comparing NGS technologies, several factors need to be considered, including read length, accuracy, throughput, and cost. Short-read technologies like Illumina offer high accuracy and throughput at a lower cost, making them suitable for many routine sequencing applications. However, their short read lengths can limit their ability to resolve complex genomic regions (Baptista et al., 2018; Hu et al., 2021). In contrast, long-read technologies like PacBio and ONT provide much longer reads, which are beneficial for de novo genome assembly and the detection of structural variants. PacBio offers high accuracy with its HiFi reads, while ONT provides ultra-long reads and real-time sequencing capabilities (Cui et al., 2020; Lang et al., 2020; Athanasopoulou et al., 2021). Each technology has its strengths and limitations, and the choice of platform often depends on the specific requirements of the research project. NGS technologies have transformed genomic research by providing powerful tools for sequencing and analyzing complex genomes. The continuous advancements in both short-read and long-read sequencing platforms promise to further enhance our understanding of genomics and its applications in various fields, including cotton genomics. 3 Applications of NGS in Cotton Genomics 3.1 Genome sequencing and assembly Next-generation sequencing (NGS) technologies have significantly advanced the construction of reference genomes in cotton genomics. The ability to sequence millions of reads in parallel has enabled the rapid and cost-effective generation of high-quality reference genomes. For instance, the development of Transposase Enzyme Linked Long-read Sequencing (TELL-Seq) allows for the generation of long-read-like information using short NGS reads, facilitating the construction of reference genomes with high accuracy and efficiency (Chen, 2019). Additionally, hybrid assembly strategies that combine ultra-long reads from third-generation sequencing (3GS) technologies with short reads from NGS have been shown to produce nearly gapless and high-quality reference genomes, as demonstrated in human genome studies (Ma et al., 2019). These advancements have enabled the construction of high-quality reference genomes and the assembly of novel genomes, providing a comprehensive understanding of cotton's genetic architecture. 3.1.1 Reference genome construction The construction of reference genomes is critical for understanding the genetic foundation of cotton. NGS technologies, particularly Illumina sequencing, have been instrumental in generating high-quality reference genomes. These reference genomes serve as a crucial resource for identifying genetic variations, mapping genes, and studying the evolutionary history of cotton species. The availability of a reference genome facilitates various genomic studies and supports the development of improved cotton varieties. 3.1.2de novogenome assembly de novo genome assembly involves sequencing and assembling a genome from scratch without the use of a reference. This approach is essential for studying cotton species with no existing reference genome. De novo genome assembly in cotton has been greatly enhanced by NGS technologies. The integration of short and long reads through hybrid assembly approaches has proven effective in generating complete and accurate genome assemblies. For example, the combination of NGS short reads with long reads from platforms like Nanopore has
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