Oluwadamilola Fateru

A Novel Exonuclease Time-of-Flight (XToF) Device for High-Accuracy DNA Sequencing to Identify Mutations in Cancer Patients:

In this study, we aim to address the limitations of next-generation sequencing (NGS) for identifying mutations in cancer patients. NGS techniques suffer from high error rates, short read lengths, GC bias, complex data analysis, and high costs. To overcome these challenges, we propose the use of a novel exonuclease-time-of-flight device (XToF device) developed by our research group. This device will enable rapid, amplification-free DNA sequencing with long reads, enhancing the accuracy of mutation identification in cancer patients.

Next-generation sequencing (NGS) has been widely used for mutational profiling for precision therapy in cancer patients, but its limitations impact its efficacy. NGS is prone to errors during DNA library preparation, amplification, and base calling, leading to inaccuracies in generated sequences. Additionally, short read lengths hinder the accurate sequencing and assembly of long DNA fragments or regions with repetitive sequences. Structural variations and haplotypes are also challenging to resolve with short reads. Furthermore, NGS generates vast amounts of data, necessitating resource-intensive handling, analysis, and specialized expertise. The costs associated with NGS equipment, maintenance, and per-sample analysis make it less accessible for smaller research laboratories or institutions with limited resources.

To address these limitations, we have developed the exonuclease-time-of-flight device (XToF device), which offers several advantages for DNA sequencing. The XToF device is a nanofluidic device fabricated in thermoplastics. It incorporates a nanoscale enzymatic reactor where an exonuclease enzyme (Lambda exonuclease) is immobilized using EDC/NHS coupling chemistry. This immobilization process ensures the covalent attachment of the enzyme in the bioreactor. The device operates by introducing double-stranded DNA (dsDNA) molecules into an input channel and electrokinetically driving them towards the bioreactor. Upon reaching the bioreactor, the 5' end of the dsDNA molecule enters the active site of the immobilized enzyme, forming a complex. To initiate digestion of the complexed DNA molecule into deoxyribonucleotides (dNMPs), a Mg2+ cofactor is introduced through a cofactor channel. The released dNMPs are then driven electrokinetically into the nano-flight tube, which contains two in-plane nanopores. As the dNMPs travel through the nanopores, a current amplitude is generated, allowing for their identification based on the time-of-flight, representing the time it takes for the dNMPs to travel from one pore to another.

My objective in this research is to understand the enzymology aspects of the XToF device by investigating the enzyme's processivity, clipping rate, and how factors such as salt concentrations and buffer composition influence the enzyme's activity and stability. By optimizing these parameters, we aim to enhance the performance and accuracy of the XToF device for mutation identification in cancer patients. The XToF device represents a promising alternative to NGS for DNA sequencing in cancer patients. Its rapid, amplification-free, and highly accurate sequencing capabilities, coupled with long read lengths, address the limitations associated with NGS. Through ongoing research focused on enzyme optimization, we aim to further improve the device's performance and establish its potential as a valuable tool for precision therapy in cancer patients.