Tip:
Highlight text to annotate it
X
This presentation is about Next
Generation sequencing platforms.
Oftentimes, people that do research have questions about
cost-benefit analysis and the various aspects that go into
deciding whether or not to buy a specific type
of sequencing platform.
In the analysis given in this presentation, several aspects
will be elaborated on.
Questions and buzz words often come up when people try to
answer the question, what's new in Next Gen sequencing?
One particular example, because it is new, is the
MiSeq desktop sequencer.
This is new so people are very interested.
But is it really a good purchase?
So one of the most striking changes recently is the
introduction of benchtop instruments such as the 454 TM
Junior Ion Torrent and MiSeq with a low end specification,
an output of 10 megabases and upwards
to around 500 megabases.
The developments in this direction are primarily with
one eye on the human and animal
health diagnostic markets.
Eukaryotic whole genome sequencing is unlikely to be
cost effective in the interim and presents huge problems in
terms of how we handle, store, and interpret this
information.
Consequently, targeted sequencing of amplicons
spanning specific regions or the genome are much more
attractive, and are already being routinely performed
using Next Gen technology.
While applications in human genetics and cancer are
obvious applications, sequencing can meet many other
needs in diagnostics, not least in relation to
infectious diseases.
And all of the three main companies are putting efforts
into sequencing solution in this field.
Up until now epidemiological studies of infectious agents
have been conducted on the larger instruments, such as
Pacific Biosiences RS in the 2010 paper, looking into the
origin of Haitian cholera outbreak but Chin, et. al.
More recently, German e-coli outbreak strains were
sequenced by [? Rascal ?] et al using the same instrument.
Bruce [INAUDIBLE]
et al sequenced two islets of the 454 TM titanium FLX.
However, news of both Ion Torrent and Luminous MiSeq TM
sequences also being used to produce real time sequences
that will make publicly available from the German
outbreak overshadowed this.
The next slide shows a quick time line of how the Next
Generation sequencing technologies evolved.
In the late 1970s, The Sanger sequencing technology is based
on the use of DDNTPs.
DDNTPs are nucleotides that have an extra
hydroxyl group detached.
So therefore, they're called the dideoxy NTPs.
Each of these dideoxy NTPs are differently labeled with a
fluorescent label.
So therefore, the principle of the reaction is that during
primary elongation and chain termination each of the four
dideoxy nucleotides will terminate the chain at a
different length.
So that leads to a different population of molecules that
will then run through a capillary gel at a different
defined position.
The four nucleotides are then sequentially read by a
detector and chromatogram is saved as a data file.
Similarly to Sanger sequencing, in the technology
that was developed at Illumina, also fluorescently
labeled are used to read the signal.
However, with Illumina sequencing technology the
reads are read in real time.
Just as before, fragment libraries produced in which
adaptors are allocated to
opposite ends of the fragments.
Linkers that are covalently attached to the surface of the
flow cell capture the library and the bridge is formed
between these.
Isothermal amplification is then initiated from double
stranded ends of the bridge.
Displacement and re-annealing to the surface, and
amplification of the locally bond sequence thus forms a
cluster of clonally expanded sequences.
Finally, opening of the bridge allows the release of one end
in preparation for the sequencing.
Both the Illumina and solid chemistry differ from 454
chemistry in that they incorporate fluorescent dyes
as markers of nucleotide extension.
Illumina employs novel approach to clonal expansion
termed bridge amplification.
This is performed on the surface of the flow cell.