Biology Reference
In-Depth Information
expensive to sequence whole genomes, as these
must first be fragmented and cloned to produce
sequencing libraries, which must then be ampli-
fied and extracted (per clone), and sequenced via
primer walking until a contiguous sequence is
produced.
Next-generation sequencing approaches
(such as the Illumina and Roche systems, as
the most common systems in current use), on
the other hand, employ techniques that sidestep
many of these difficulties. The key innovation in
both is that sequencing products are no longer
resolved and read off linear sequencing gels.
Rather, the sequencing template is immobi-
lized on a solid, 2-dimensional substrate (the
sequencing chip), and the sequencing results
“read” by taking ultra-high-resolution images of
this substrate. Each image is capable of reading
only a single base (or type of base, for 454) of
sequencing at a time, so these techniques no
longer involve irreversible termination of the
sequencing synthesis step. Rather, they either
employ reversible termination (Illumina) or
simply add only a single type of nucleotide at
a time (454). Thus, sequencing proceeds by
only one or a few base-pairs at a time, and so is
conducted in cycles. Results are read after each
cycle and built up incrementally, and finally
put together in post-sequencing processing
(details can be found on the respective websites,
Although this may appear laborious, it offers
two related key advantages. First, since the
sequencing occurs on templates immobilized on
a 2-dimensional substrate, the original template
does not have to be pure. The template is “puri-
fied” by ensuring that each region of the substrate
(sequencing chip) receives only a single tem-
plate molecule and is thus pure. Second, since
each region of the sequencing chip may have
a different template molecule, sequencing must
proceed using universal primers. This necessi-
tates a prior fragmentation and ligation step in
which the primer complementary sequence is lig-
ated to template fragments, but the result is that
sequencing reactions are now no longer depen-
dent on the template sequence, that is, the results
come close to being template-independent. Thus,
it opens the possibility of massive multiplexing,
limited essentially by the spatial resolution of the
imaging system (and storage capacity of com-
puters acquiring the data). Thus, these systems
have become characterized by extremely high-
throughput results, producing tens (and now hun-
dreds) of millions of reads per sequencing chip.
But, this has come at a cost: both systems
rely on reactions that are not 100% accurate,
and accuracy declines as the number of cycles
increases. Thus, both technologies started off
producing only very short reads (as short as 25
nucleotides), and have gradually extended this to
approximately 400-500 bp for 454 sequencing
and 100-150 bp for Illumina. The short reads,
combined with the fact that the template used
in each sequencing region can be sequenced at
most only twice (once from each end), makes it
far more challenging to use these technologies
to assemble genomes de novo, that is, without
reference to a previously sequenced genome
(e.g., Alkan et al. 2011). On the other hand, the
exceptionally large number of reads produced -
particularly by the Illumina system - does
make them extremely useful at resequencing a
known genome, or one fairly closely related to
a known genome. The large number of reads
produces very high coverage per base, resulting
in very high confidence of the sequence at each
position, even with relatively modest coverage
such as 20
×
. In addition, the high coverage
and relative template-independence of the reads
also permit the identification of heterozygous
sites. Thus, one of the first applications of these
technologies has been in SNP discovery and
associated applications in association mapping.
Application of Next-generation
Sequencing Technologies to
Salinity Tolerance Research
SNP Discovery and QTL Identification
The advantages and utility of next-generation
sequencing technologies in single nucleotide
Search WWH ::
Custom Search