Techniques in sequencing

Determination of the nucleotide composition and order in a gene or genome is a critical technology in current molecular diagnostics. DNA sequencing can reveal the information of genes, and it can also reveal mutations and the genes' relatedness. Furthermore, it can also indicate the cause of diseases. The first DNA sequencing method developed was chain termination sequencing which uses the dideoxynucleotide procedure. It is commonly used to sequence DNA fragments containing one to a few genes as well as entire genomes from many different organisms. However, interest in sequencing large numbers of DNA molecules in less time and at a lower cost has driven the recent development of new sequencing technologies that can process thousands to millions of sequences concurrently. Many second-generation (next-generation) and third-generation sequencing techniques have been developed, including pyrosequencing, massively parallel sequencing, sequencing using reversible chain terminators, sequencing by ligation, and direct single-molecule sequencing. The development of these high throughput methods has dramatically improved the accuracy and speed of clinical diagnosis and thus has revolutionized the field of genomics and clinical diagnosis. The genomics sequencing information obtained by next-generation sequencing (NGS) and third-generation sequencing coupled with advanced bioinformatics technology can translate the potential of NGS into clinical applications for healthcare providers and patients. Currently, disease diagnostics, pathogen detection, and genetic mutations are some of the growing clinical areas where NGS is widely used. As such, the principles and applications of these high throughput techniques and bioinformatics analysis are discussed in this chapter.