FROM THE BENCH
often than not, poor library preparation
is at fault. Under-tagmentation results in
fragment lengths above the recommended
size and can lead to suboptimal cluster
densities. Over-tagmentation, contrarily,
can lead to reduced library yield, shorter
read lengths and coverage dropout (e.g.,
high cluster density resulting in fewer
clusters passing the filter).
Common WGS wet lab practices that
contribute to tagmentation errors include
using too much/little DNA/enzyme
and incorrect tagmentation time (too
long or short). To alleviate these issues,
automated WGS library preparation evenly
distributes sample DNA and reagents and
maintains accurate tagmentation times,
as samples are not repeatedly transported
between workbenches and PCR machines.
Automated liquid handlers also deliver
accurate volumes of enzymes/reagents,
which ensures sample DNA is cleaved and
tagged correctly each time.
Post-PCR Clean-Up
After tagged sample DNA is
PCR-amplified, these samples must
undergo purification and fragment size
selection to produce high-quality data.
These two important steps rely on the
use of AMPure XP beads, which are
paramagnetic and remain in solution
until a magnetic force is applied. Each
bead is coated with carboxyl molecules
that reversibly bind DNA in the presence
of polyethylene glycol (PEG) and salt,
giving them their purification and size
selection ability. Initially, 0.5X AMPure
beads are added to the amplified samples.
Then, samples are vortexed rapidly to
ensure a fully homogenized solution. At
this step, negatively-charged DNA forms
hydrogen bonds with the carboxyl groups
on the bead surface. The bead/DNA
solution is then separated from impurities
and unwanted fragment sizes using a
magnetic plate stand and contaminants
are washed away with ethanol (80%).
Molecular Microbiology/Epidemiology Lab Group (from l to r):
Haley Flores, Allen Hord, Dr. Laura Lane, Jessica Threatt and
Megan Davis. Photo: SC PHL
Summary & Next Steps
At the end of the ethanol washes, AMPure
beads undergo a drying step to remove
excess ethanol. Once the ethanol has
evaporated, resuspension buffer is added
and beads/DNA are re-suspended in
solution. Since the elution buffer does
not contain PEG or salts, DNA separates
from the carboxyl molecules on the beads
and the beads are sequestered by the
magnetic stand. The purified DNA can
then be transferred to a fresh 96-well
plate and quantified. The ability to rapidly and inexpensively
generate bacterial WGS data makes
answering public health-related
questions, such as whether an infectious
bacterial isolate belongs to a local or
national outbreak, relatively easy. The
massive influx of samples for sequencing,
however, requires PHLs to rethink
how they approach day-to-day wet lab
techniques so they can provide the most
rapid and accurate outbreak response and
confirmatory testing.
During manual WGS post-PCR clean-up,
many challenges can arise as several
steps are time- and quantity-sensitive.
For example, if suboptimal amounts of
AMPure beads are added, fragment sizes
may be too small or long, which affects
clustering densities and sequencing
run quality. Additionally, more quality
issues arise when time-sensitive steps
are not executed correctly. For instance,
if enough time is not allotted for input
DNA to interact with the AMPure beads,
DNA quantities may be too low and/or
the wrong DNA fragment size may be
selected. The automated liquid handler
can address all these issues, as a post-PCR
clean-up procedure that accurately
measures quantities and times for each
step, providing high-quality DNA that can
be loaded onto a WGS sequencer for data
analysis and outbreak surveillance. To address these concerns and advance
the PHL’s programmatic goals, automated
WGS provides several solutions that
improve sample preparation quality
and sequencing data output. Whereas
all procedures in current WGS wet labs
are hands-on—limiting the number
of samples processed at once and
increasing room for human error—
samples processed using automated WGS
methodologies can be managed in greater
numbers (up to 96 samples at a time) with
a large decrease in error.
Although automation of WGS does not
prevent all technical errors that may arise
(e.g., poor DNA quantification or expired
reagents) it does provide a means to
correct the most common WGS wet lab
errors which, in turn, increases testing
efficiency and the ability for PHLs to
quickly screen for and link foodborne
outbreaks at both the local and national
levels. n
Analyzing DNA quality of bacterial samples. Photo: SC PHL
PublicHealthLabs
@APHL
APHL.org
Summer 2019 LAB MATTERS
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