The Next Generation Sequencing Techniques and Application in Drug Discovery and Development

The Next Generation Sequencing Techniques and Application in Drug Discovery and Development

Afzal Hussain (Maulana Azad National Institute of Technology, India)
Copyright: © 2019 |Pages: 20
DOI: 10.4018/978-1-5225-7326-5.ch011

Abstract

Next-generation sequencing or massively parallel sequencing describe DNA sequencing, RNA sequencing, or methylation sequencing, which shows its great impact on the life sciences. The recent advances of these parallel sequencing for the generation of huge amounts of data in a very short period of time as well as reducing the computing cost for the same. It plays a major role in the gene expression profiling, chromosome counting, finding out the epigenetic changes, and enabling the future of personalized medicine. Here the authors describe the NGS technologies and its application as well as applying different tools such as TopHat, Bowtie, Cufflinks, Cuffmerge, Cuffdiff for analyzing the high throughput RNA sequencing (RNA-Seq) data.
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Introduction

The human genome contains too much complexity and diversity and shows its effects on health and diseases. The journey started with the discovery of the structure of DNA. A large number of innovations in reagents and the development in the instrumentation supported the Human Genome Project initiative (Mardis, 2013; Watson & Crick, 1953). The Human genome project completion shown a need for more advanced technologies for giving the answer of complex biological questions because of the high cost and limited throughput barriers. First high-throughput sequencing platform released in the mid-2000s and dropped the cost of human genome sequencing since the human genome project and lead to the next generation sequencing. Over the past decade, next generation sequencing continued to evolve. It showed advances in the read length and reducing the cost of sequencing. The next generation sequencing technologies are more advanced than the previous technologies such as DNA microarray, NanoString, qPCR and Optical mapping. The DNA microarrays are used to identify common polymorphisms related to diseases, including cancer, cardiovascular disease and genome-wide association study (DeRisi et al., 1996; Jia et al., 2010; Keating et al., 2008; Rhodes et al., 2004; Welter et al., 2013).

Different Types of NGS Technologies

There are different Next generation sequencing are available, some of the main sequencing technologies were explained below such as

  • 1.

    Illumina sequencing

  • 2.

    Roche 454 sequencing

  • 3.

    Ion PGM from Ion Torrent sequencing

Illumina Sequecning

Solexa released the Genome analyzer in 2006 and later in 2007, it was purchased by Illumina. It adopts the technology of, sequencing by synthesis (Mardis, 2008).

It has a few steps for the sequencing

  • The input samples cleaved into short sections. The length of the sections depends on the sequencing machinery used.

  • In Illumina sequencing 100 to 150bp reads are used for the sequencing. Fragments are ligated with generic adaptors and annealed to a slide using the adaptors.

  • The PCR is used to amplify each read and creating a spot with so many copies of the same read and then separated into single strands to be sequenced.

  • The Sequencing slide is filled with nucleotides and DNA polymerase. These all the nucleotides are fluorescently labelled and also have a terminator for adding one base at a time.

  • An image is taken of the slide, At each read site, there will be a fluorescent signal which indicates the base that has been added.

  • The slide goes for the next cycle, the process is repeated, at a time only one nucleotide added and imaging in between.

  • Computers detect the base at each site in each image and construct a sequence.

  • The overall Illumina sequencing technique was shown in Figure 1.

Figure 1.

Illumina sequencing technology

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