W6 - Magnetic Based Mixing to Improve Elution Performance of On Chip DNA Sequencing Library Preparation

Introduction: : Emerging pathogen detection has become a critical part of environmental surveillance due to the recent SARS-CoV2 pandemic. Wastewater provides a robust sample of the environment as bacteria and viruses collect in central locations. Taxonomic profiles of wastewater can be obtained by isolating DNA, concentrating it, preparing the genomic library for sequencing, and sequencing. Improvements in sequencing technology now allow for in-field sequencing with the emergence of Oxford Nanopore-based long-read sequencing. In-field sequencing reduces transportation time from site to lab and limits sample degradation. Many locations where emerging pathogens are predicted to originate are often in low resource environments, with little access to laboratory equipment making it difficult to prepare the library for sequencing (a critical and labor-intensive process). Here, we present a device that would allow for streamlined library preparation “on chip,” reducing the need for laboratory equipment with a small portable platform.

Materials and

Methods: : Library preparation devices were fabricated from 3 mm thick acrylic using laser cutting. Wells were cut directly from the sheet, and connecting channels were engraved. The device was sealed on both sides with PCR tape (Figure A). DNA bound to AMPure XP Beads was loaded into the chip. Wells were separated by silicone oil (5,000 cSt viscosity), which prevented reagent mixing and restricted hydrophilic compound migration (Figure A, B). A cylindrical N52 magnet was used to pellet and transport beads between wells (Figure A). After an initial DNA wash, elution into the end-repair well was performed. To improve elution efficiency, an electromagnet-based mixer was used. When powered, the electromagnet lifted the beads; when off, the beads dropped. This cycling caused exterior beads to shear off into suspension. After 10 minutes of magnet cycling, complete bead resuspension was achieved. The well was then heated to 50 °C for 10 minutes to elute DNA from the resuspended beads (Figure C). The eluted DNA was repaired using NEBNext FFPE DNA Repair Mix (Figure B). The DNA was subsequently rebound to AMPure XP Beads and passed through another wash. The sample was eluted using the same electromagnet-based mixing method into a ligation well, where DNA ligation was carried out using adapter, buffer, and long fragment buffer from Oxford Nanopore Technologies (Figure B). A final wash was performed, and DNA was eluted off the beads using the magnetic mixer. The resulting sample was ready for sequencing (Figure B).

Results, Conclusions, and Discussions:: Initial results revealed that elution of DNA into nuclease free water was inhibited by the lack of mixing in the well resulting in major DNA losses across the chip. To address this problem, pipette mixing was used initially; however, this resulted in a 70% loss of DNA by the end of the first wash step leaving little to no DNA for sequenci ng. This optimization study focused on the binding, washing, and elution of DNA for the first lane only because 70% of DNA was lost in the first lane. Transportation of enough DNA for sequencing from the start to the finish of the library preparation device was difficult to accomplish without some active mixing technique. Our magnetic-based active mixer has shown promise in accomplishing this task by increasing the yield of the elution by 45%. Although yield is currently still underperforming compared to traditional in-tube methods, whole libraires have been sequenced with success on chip with comparable outputs to the in-tube method. Further investigation into improving elution yield is still required to improve enzymatic reaction efficiency across the whole chip.

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