PerkinElmer eBook - Next-Gen Sequencing - 7

Next-Gen Sequencing * The Next Next Thing in Sequencing

Dr. Stein's team is starting by focusing on DNA
sequencing. "Marrying nanopore technology with
the electrospray ionization of MS allows rapid
transfer of intact biomolecules from liquid into the
vacuum," he explains. "The technology could also
accelerate the speed and precision of analysis.
Traditional sequencing takes tens of milliseconds to
visualize an individual base, but our approach can
detect a base every microsecond or less."
The scientists faced a number of challenges, including
the need to construct their own instrumentation
and components. "We knew that building and using
a nanopore mass spectrometer would be an ambitious project," recalls Dr. Stein. "So we first wanted to
see if this was even feasible."
Not only did they find that it was possible, they also
developed a number of key improvements. "We
miniaturized the droplets sprayed into the MS instrument. Conventional MS instruments lose 99.9% or
more of the analyte molecules because they are
injected into the instrument in large, charged droplets," notes Dr. Stein. "When these droplets evaporate
and explode, they send molecules flying all over."
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"We decided to make glass nanocapillaries that
could miniaturize droplets to less than 10
nanometers, which is small enough that they can
hold only a single ion-a quantum of charge,"
he continues. "Thus, instead of the traditional
'Coulomb explosions,' we are creating ions
one-by-one by 'ion evaporation.'"
Dr. Stein believes that utilizing nanotechnology for
protein sequencing presents even more exciting
opportunities: "MS is unique in its ability to identify
all 20 amino acids. The same nanotechnology DNA
sequencing strategy also can be applied to proteins.
That is, push proteins through a nanotube, photofragment them with a laser, and use an MS detector
for accurate identification."
Gazing into the nanotechnology crystal ball, Dr.
Stein sees many futuristic applications: "Much like
a needle draws blood from a small vein in the body,
one could envision sometime in the future creating
nanotips so small that they could penetrate into a
single cell to remove a sample for analysis. Imagine
what we could learn.

Removing Toxic Transcripts
Creating high-specificity RNA-Seq libraries remains
an ongoing challenge. "It is critical to minimize the
population of undesirable transcripts (often greater
than 80% of a library) such as rRNA, globin, and
other housekeeping species, while at the same time
maintaining desirable transcripts from the original
total RNA population," advises Luke Sherlin, Ph.D.,
director of technical services, NuGEN Technologies.
The company developed a technology to do just that.
During library construction, the Insert-Dependent
Adaptor Cleavage (InDA-C) approach employs
specific enzymatic steps to deplete any unwanted
transcript sequences from the final library. This
approach contrasts with hybridization capture
methods, which can alter the original RNA populations.
"InDA-C is a flexible approach that can be easily
applied across a variety of species such as human,
mouse, rat, Drosophila, and Arabidopsis species."
The initial InDA-C step in the workflow adds forward
and reverse library adaptors during untargeted
strand selection. Next, the targeted transcripts are

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