Integration of Microfluidic Washing into Automated NGS Library Preparation

Next Generation Sequencing (NGS) is a critical technology that has massively increased the rate at which genetic material can be sequenced while massively reducing its cost. These benefits have enabled the use of NGS in a wide range of applications, including personalized medicine, agriculture, and archaeology. Preparing samples for NGS is currently a major bottleneck in the overall sequencing workflow, consisting of many pipetting steps and involving small quantities of many reagents which creates room for human error. One critical step in NGS sample preparation is DNA washing, which is carried out multiple times in a typical workflow to remove small DNA fragments from the sample that can negatively impact sequencing. The complexity of DNA washing and overall library preparation makes affordable automation strategies for these workflows very useful.

The goal of this research is to integrate a microfluidic system that enables DNA washing into an existing automated library preparation device. This microfluidic operates by binding a DNA sample to magnetic Solid Phase Reversible Immobilization (SPRI) beads in an input well with a high salt concentration. A magnet is then swept beneath the input well, moving the beads into a washing channel filled with isopropyl alcohol, where smaller unwanted DNA strands are removed from the beads. The beads are then moved into an elution well via magnet, where a low salt concentration causes the longer DNA strands that make up the library to elute off the beads. We have integrated this microfluidic into a library preparation device capable of preparing 8 samples at a time and demonstrated that it can wash libraries after DNA concentration has been increased via PCR amplification. This microfluidic system has been shown to reduce the concentration of unwanted short DNA strands by over 70% relative to an unwashed library while retaining over 51% of the longer DNA strands that are needed for sequencing. This research has demonstrated that this system is a viable method for automated washing of DNA libraries. Further work optimizing the movement and yield of SPRI beads through the IPA channel may lead to further improvements in library purity and yield.