Lab Matters Summer 2019 - Page 21

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 19