Verma Mansi, Kulshrestha Samarth, Puri Ayush
Sri Venkateswara College, University of Delhi (South Campus), Benito Juarez Road, Dhaula Kuan, New Delhi, 110 021, India.
Methods Mol Biol. 2017;1525:3-33. doi: 10.1007/978-1-4939-6622-6_1.
Genome sequencing is an important step toward correlating genotypes with phenotypic characters. Sequencing technologies are important in many fields in the life sciences, including functional genomics, transcriptomics, oncology, evolutionary biology, forensic sciences, and many more. The era of sequencing has been divided into three generations. First generation sequencing involved sequencing by synthesis (Sanger sequencing) and sequencing by cleavage (Maxam-Gilbert sequencing). Sanger sequencing led to the completion of various genome sequences (including human) and provided the foundation for development of other sequencing technologies. Since then, various techniques have been developed which can overcome some of the limitations of Sanger sequencing. These techniques are collectively known as "Next-generation sequencing" (NGS), and are further classified into second and third generation technologies. Although NGS methods have many advantages in terms of speed, cost, and parallelism, the accuracy and read length of Sanger sequencing is still superior and has confined the use of NGS mainly to resequencing genomes. Consequently, there is a continuing need to develop improved real time sequencing techniques. This chapter reviews some of the options currently available and provides a generic workflow for sequencing a genome.
基因组测序是将基因型与表型特征相关联的重要一步。测序技术在生命科学的许多领域都很重要，包括功能基因组学、转录组学、肿瘤学、进化生物学、法医学等等。测序时代已被分为三代。第一代测序包括合成测序（桑格测序）和化学裂解法测序（马克萨姆-吉尔伯特测序）。桑格测序促成了各种基因组序列（包括人类基因组序列）的完成，并为其他测序技术的发展奠定了基础。从那时起，人们开发了各种技术，这些技术可以克服桑格测序的一些局限性。这些技术统称为“下一代测序”（NGS），并进一步分为第二代和第三代技术。尽管NGS方法在速度、成本和平行性方面有许多优点，但桑格测序的准确性和读长仍然更具优势，这使得NGS的应用主要局限于基因组重测序。因此，持续需要开发改进的实时测序技术。本章回顾了目前可用的一些选项，并提供了一个基因组测序的通用工作流程。
