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Abstract
The ability to quantitatively survey the global behavior of transcriptomes has been
a key milestone in the field of systems biology, enabled by the advent of DNA microarrays.
While this approach has literally transformed our vision and approach to cellular
physiology, microarray technology has always been limited by the requirement to decide,
a priori, what regions of the genome to examine. While very high density tiling arrays
have reduced this limitation for simpler organisms, it remains an obstacle for larger,
more complex, eukaryotic genomes. The recent development of "next-generation" massively
parallel sequencing (MPS) technologies by companies such as Roche (454 GS FLX), Illumina
(Genome Analyzer II), and ABI (AB SOLiD) has completely transformed the way in which
quantitative transcriptomics can be done. These new technologies have reduced both
the cost-per-reaction and time required by orders of magnitude, making the use of
sequencing a cost-effective option for many experimental approaches. One such method
that has recently been developed uses MPS technology to directly survey the RNA content
of cells, without requiring any of the traditional cloning associated with EST sequencing.
This approach, called "RNA-seq", can generate quantitative expression scores that
are comparable to microarrays, with the added benefit that the entire transcriptome
is surveyed without the requirement of a priori knowledge of transcribed regions.
The important advantage of this technique is that not only can quantitative expression
measures be made, but transcript structures including alternatively spliced transcript
isoforms, can also be identified. This article discusses the experimental approach
for both sample preparation and data analysis for the technique of RNA-seq.