Biology of Composts by Unknown

Author:Unknown
Language: eng
Format: epub
ISBN: 9783030391737
Publisher: Springer International Publishing

7.4.2.2 Next-Generation Sequencing (NGS) Technologies

Although the efforts on nucleic acid sequencing started in as early as the 1960s, yet it was only in 1977 when, Sanger was able to sequence the first DNA genome, that of bacteriophage ϕX174. This classical approach employed the plus and minus technique for DNA polymerase to synthesize from a primer with the use of radiolabeled nucleotides. Subsequently, the position of nucleotides in the covered sequence could be discerned by running the products on a polyacrylamide gel and comparing between the eight lanes (Sanger 1977). This is considered as the beginning of first-generation sequencing technologies. Further, a significant improvement in the technique was made by Sanger and Nicklen (1977) through the development of a dideoxy technique that used chemical analogues of the deoxyribonucleotides (dNTPs). In the same year, Maxam and Gilbert described a new method (chemical cleavage technique) that employed the treatment of radiolabeled DNA with chemicals that break the chain at specific bases (Maxam and Gilbert 1977). In 1987, first automated capillary electrophoresis-based sequencing equipment AB370 was introduced in the market by Applied Biosystems. In 1998, AB3730xl, a higher version of this equipment was launched. These two instruments were primarily used for Human Genome projects (Collins et al. 2003). In parallel, serious research efforts continued for improvements in the sequencing technologies. Consequently, massively parallel high-throughput sequencing techniques have been developed that has in recent times revolutionized the way the genetic information is obtained. These next-generation sequencing platforms are not only quick and accurate but cost effective also. For instance, the HiSeq X® TenSystem, launched in 2014, has the capability of sequencing over 45 human genomes in a single day for approximately $1000 (Anonymous 2014). On the contrary, the first human genome mapping took more than a decade at the cost of about three billion dollars. A typical workflow of NGS platform includes random fragmentation of DNA for library preparation, ligation of adaptors (oligos bound to the 5′and 3′ end of each DNA fragment), amplification of library using clonal amplification method/PCR, and then sequencing on automated sequencer (Fig. 7.5). In this section, we discuss some of the most commonly used next-generation sequencing technologies and platforms. Examples of studies carried out using NGS platform for microbial community analysis of compost and vermicompost are provided in Table 7.1.

Fig. 7.5A typical workflow of the NGS platform

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