Toxicogenomics – A Drug Development Perspective - Genomic Biomarkers for Pharmaceutical Development - page 128

high density, have been constructed and allow the mapping of transcribed regions to a
very high resolution [62,63] .
● Splicing junction microarrays are designed to measure connectivity of exons through the use
of probes that span known exon junctions and can be used to detect and quantify distinct
spliced isoforms. These arrays are usually custom-designed with the goal of identifying
splicing events but require previous knowledge of splice junction positions [64-66] .
As more transcriptional information becomes available, the array-based approach will
become more complete and thus more informative. However, due to the limitations stated
above and additional drawbacks, comparing expression levels across different experiments
is often difficult and requires complicated normalization methods [67,68] . There is a general
consensus that the ultimate approach to defining transcriptional complexity will come from
the sequencing of full-length transcripts at quantitative throughput levels, which is becom-
ing feasible due to next generation sequencing (NGS) technologies.
6.3.1.2 NGS Technologies - Sequencing-based Approaches for Transcriptomics Study
The arrival of deep sequencing applications for transcriptome analyses, RNA-Seq, may
circumvent the above-mentioned disadvantages of microarray platforms. In contrast to
microarray, transcriptome sequencing studies have evolved from determining the sequence
of individual cDNA clones to more comprehensive attempts to construct cDNA sequencing
libraries representing portions of the species transcriptome [69-72] . The use of sequencing
technologies to study the transcriptome is termed RNA-Seq [73,74] . RNA-Seq uses recently
developed deep sequencing technologies. In general, a population of RNA is converted to
a library of cDNA fragments by use of adaptors attached to one or both ends. Each mol-
ecule, with or without amplification, is then sequenced in a high-throughput manner to
obtain short sequences from one or both ends. In principle, any high-throughput sequenc-
ing technology can be used for RNA-Seq. This methodology has tremendously reduced the
sequencing cost and experimental complexity, as well as improved transcript coverage, ren-
dering sequencing-based transcriptome analysis more readily available and useful to indi-
vidual laboratories. RNA-Seq technologies have demonstrated some distinct advantages
over hybridization-based approaches such as microarrays that likely will enable them to
dominate in the near future.
Currently, there are four major commercially available NGS technologies: Roche / 454,
Illumina HiSeq 2000, Applied Biosystems SOLiD, and Helicos HeliScope. Illumina's NGS
platforms have a strong presence. Their sequencing-by-synthesis approach [75-78] utilizes
fluorescently labeled reversible-terminator nucleotides on clonally amplified DNA templates
immobilized to an acrylamide coating on the surface of a glass flow cell. The Illumina Genome
Analyzer and the more recent HiSeq 2000 have been widely used for high-throughput mas-
sively parallel sequencing. In 2011, Illumina also released a lower throughput fast-turnaround
instrument, the MiSeq, aimed at smaller laboratories and the clinical diagnostics market.
Although RNA-Seq is unlikely to completely supplant hybridization-based techniques in
the near future, it offers a number of improvements over these technologies, for example:
1. unlike hybridization-based approaches, RNA-Seq does not depend on prior knowledge
of the transcriptome, and is thus capable of new discovery and could reveal the precise
boundaries of transcripts to single base precision [79] ;