Feng, Xiaodong_ Xie, Hong-Guang - Applying pharmacogenomics in therapeutics-CRC Press (2016)

Principles of Pharmacogenetic Biotechnology and Testing in Clinical Practice
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The major feature of NGS platforms is massively parallel sequencing, in which
millions of DNA fragments from a single sample are sequenced in parallel at a
microscopic level. With NGS, genetic information of an entire human genome can
now be revealed in one day. To date, several NGS platforms have been developed
and modified to provide low-cost, high-throughput sequencing. Among them, the
Life Technologies Ion Torrent and the Illumina (Solexa) sequencing are the two
most commonly used platforms in research and clinical laboratories. Different
models of these NGS platforms have been developed to meet the needs of research
and clinical diagnostics.
Although Life Technologies Ion Torrent and Illumina have distinct sequencing
technologies, they use similar methodologies for preparing sequencing libraries.
The library construction consists of a series of steps that include DNA fragmentation,
end repair, adaptor ligation, and PCR amplification (Harakalova et al. 2011)
(Figure 2.3). In principle, the final sequencing library should cover the complete
genomic view of every single starting template.
Once constructed, libraries are clonally amplified in preparation for sequencing.
The Ion Torrent method utilizes emulsion PCR to amplify single template fragments
onto microbeads, whereas the Illumina method utilizes bridge amplification to form
template clusters on a flow cell (Berglund et al. 2011).
Both platforms make use of the sequencing-by-synthesis approach to sequence
the amplified libraries, by which a new DNA strand is synthesized complementary
to a strand of sequencing libraries. Through cycles of flashing with the nucleotides
and washing off unbound ones in a sequential order, sequencing occurs when a
certain nucleotide is incorporated into the extending strand. The incorporation of
each single nucleotide into the newly synthesized strand is detected either by the Ion
Torrent semiconductor sequencer based on the induced pH change or by the Illumina
sequencer upon the released fluorescence (Figure 2.3). By these methods, millions of
DNA fragments are sequenced in parallel. Once sequencing is complete, terabytes of
genomic data will be analyzed. Sequence analysis can reveal almost endless information
on genomic variations that include SNPs, the insertion or deletion of bases,
and the detection of novel genes. Analysis can also include identification of both
somatic and germline mutations that may contribute to the diagnosis of diseases or
genetic conditions (Gogol-Doring and Chen 2012).
The applications of NGS have been enormous, allowing for rapid advances
in many fields of biological sciences and clinic practice. Various NGS assays
have been developed for genetic analyses, including whole-genome sequencing,
whole-exome sequencing, and focused assays that target only a handful of
genes. Parallel to this, different assays provide another set of tools for analyzing
epigenetic modifications of DNA (e.g., ChIP-seq, bisulfite sequencing, DNase-I
hypersensitivity site sequencing, formaldehyde-assisted isolation of regulatory
elements sequencing, and more). These methods together with NGS are routinely
used for analyzing gene expression–related processes or gene expression (RNAsequencing)
itself.
Because of relative cost-effectiveness and ease of accessibility, NGS has been
used in a broad spectrum of companion diagnostics, although more evidence
needs to be accumulated to fully evaluate this technology for clinical applications.