Agriculture Reference
In-Depth Information
New-generation sequencing
technologies
reads (Pop and Salzberg 2008), and the use of these
technologies in wheat should be explored.
It seems most likely that the wheat genome ulti-
mately will be sequenced using a combination of
these new-generation sequencing technologies.
One possible cost-effi cient scenario would be to
produce a low-redundancy draft of the wheat
genome using GS FLX Titanium sequencing of
pooled BAC clones originating from the physical
map-based MTPs. This could be achieved in about
20 runs per diploid genome. Alternatively, DNA
isolated from fl ow-sorted chromosomes or chro-
mosome arms could be shotgun-sequenced by GS
FLX Titanium. The resulting 2X draft, which
would allow the assembly of most of the genic
regions, could then be used as template for the
assembly of short Illumina or SOLiD reads gener-
ated at 20X. Sequencing each genome at 20X using
the Illumina or SOLiD technology would require
50 to 60 runs per genome. The total cost for
sequencing the hexaploid wheat genome in this
fashion would be, at most, $2 million. Add a few
million dollars for the bioinformatics, and the
entire wheat genome, or at least the genic portion
of it, could be completed with these new-genera-
tion sequencing technologies for a fraction of the
cost of sequencing the entire genome or even the
gene space using Sanger technology. As sequence
technologies continue to develop at a fast pace—
one of the latest developments is real-time sequenc-
ing from single molecules that can produce read
lengths of a few kilobases (Eid et al., 2009)—it is
expected that it will be possible to produce a high-
quality draft sequence of the wheat genome for this
price tag or less in the coming years.
A series of different sequencing technologies has
been developed in recent years, and more are under
development. Currently, the three leading systems
are the Roche Diagnostics Corporation (Indianap-
olis, Indiana) GS FLX system (previously 454),
the Illumina (San Diego, California) Genome
Analyzer (previously Solexa), and the Applied
Biosystems (Foster City, California) SOLiD DNA
sequencer. The sequencing technologies used by
the three companies differ, but for all platforms the
massive numbers of sequencing reactions can be
conducted in parallel, producing several hundred
megabases or even a few gigabases of sequence data
per run for a few thousand dollars.
Before discussing if and how these technologies
can be applied for sequencing the wheat genome,
it is important to compare advantages and disad-
vantages of the different instruments. All three
instruments have the capability to run paired-end
reads, which will aid in the assembly process.
Roche GS FLX Titanium produces approximately
1.2 million reads of 400 bp (500 Mb total data).
The Illumina Genome Analyzer produces some 40
million reads of 35 bp, or a total of 1.4 Gb per run.
For paired-end reads on the ABI SOLiD, read
lengths are 25 bp and the total output is in the
range 1.5-2 Gb of sequence data. Sequence accu-
racy is similar for the three instruments. While
these technologies allow a genome to be sequenced
inexpensively, new strategies will need to be devel-
oped that will allow these small reads to be assem-
bled correctly. Wicker et al. (2006) demonstrated
that 100-bp reads, generated from four barley BAC
clones at 10X redundancy using the 454 pyrose-
quencing technology, could be assembled quite
easily for regions that were single-copy in the BAC
clones. The effi ciency with which pooled BAC
clones can be assembled can be further enhanced
by bar-coding individual BACs or BAC subpools.
Currently, the Illumina Genome Analyzer and
ABI SOLiD instruments are used mainly to
resequence species for which a reference genome
is already available. However, software is being
development for the
de novo
assembly of short
FUTURE PERSPECTIVES
Forward genetics—starting from a trait and iso-
lating the gene(s) that control that trait—is now
possible in wheat, although map-based cloning
remains highly labor-intensive. This process will
be greatly facilitated in the coming years by the
availability of a physical map of the wheat genome
to which the sequence of the gene space or even
the entire wheat genome has been anchored.
