Improvements in NGS technologies and bioinformatics pipelines have created opportunities for researchers to perform whole genome and transcriptome sequencing (of tissues, cells, and organisms) ( Jabeen et al., 2018), methylation sequencing, whole exome sequencing ( Kulski et al., 2014 Verma et al., 2016 Gazara et al., 2019), and chromatin immunoprecipitation followed by sequencing (ChIP-seq) ( Schmidt et al., 2010 Wani and Raza, 2019) and their subsequent in-depth analyses. Due to these advantages, NGS has become a powerful and valuable tool for research and clinical applications. The human genome was sequenced in almost 13 years using Sanger sequencing at a cost greater than one million dollars ( Naidoo et al., 2011), whereas now, it is possible to sequence the human genome within a week at the cost of 100 US dollars ( ). Moreover, NGS provides more accurate and reliable sequencing data than Sanger DNA sequencing. NGS can provide the massive parallel and high-throughput data from multiple samples per run at significantly reduced cost in comparison to Sanger ( Goodwin et al., 2016). NGS has several advantages over Sanger sequencing. Later, the emergence of NGS occurred that has opened new possibilities and challenges to biologists and clinicians in the field of genomes exploration and clinical diagnosis ( Goodwin et al., 2016 Müllauer, 2017 Raza and Ahmad, 2019). For three decades, Sanger sequencing was extensively used by the biologists. ![]() Sanger DNA sequencing allowed the analysis of single genes or parts thereof. In 1970, the first DNA sequencing called Sanger sequencing or original DNA sequencing was developed by Frederick Sanger et al. Researchers are using NGS in basic, applied, and clinical research. Next-generation sequencing (NGS) technologies are new sequencing methods for DNA and RNA sequencing ( Goodwin et al., 2016). Sandhya Verma, Rajesh Kumar Gazara, in Translational Bioinformatics in Healthcare and Medicine, 2021 1 Introduction This new way of sequencing directly from a single DNA molecule without pre-amplification of DNA fragments has been termed “third generation of NGS” technology ( Schadt, Turner, & Kasarskis, 2010). The two main SMS methods are true SMS, in development by Helicos Biosciences™ and single-molecule real-time (SMRT) sequencing from Pacific Biosciences™ (PacBio) (see Fuller et al., 2009 Pareek et al., 2011 Schadt, Turner, & Kasarskis, 2010, for detailed review). These SMS methods are not as widely available/used as the above three technologies. SMS requires much less starting DNA material (< 1 μg) compared to clonally amplified methods (3–20 μg) and results in significantly longer read lengths ( Table 1.1). More recent NGS platforms have adopted a new sequencing method, called single-molecule sequencing (SMS), which does not require prior amplification of DNA-consequently avoiding the PCR-related error reads or amplification bias toward repeat regions ( Pareek, Smoczynski, & Tretyn, 2011). Seo-Kyung Chung, in Advances in Protein Chemistry and Structural Biology, 2012 4.5 Single-molecule sequencing
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