Join DNA Script at ABRF 2022

Morning Eye Opener | Mar 30, 9:30am

Using Enzymatic DNA Synthesis to Build Custom Library Adaptors for Controlling Real-Time Nanopore Sequencing, presented by Tim Mercer, Ph.D., University of Queensland

Abstract: Benchtop enzymatic DNA synthesis enables the rapid creation of oligonucleotides that can be combined with real-time nanopore DNA sequencing to accelerate the iterative design and testing cycles in synthetic biology. Here, we use enzymatic DNA synthesis to build novel, custom library adaptors, termed CAPTORS, that contain functional sequence elements that can measure the qualitative and quantitative accuracy of an DNA sequencing library. We show how CAPTORS are used to prepare libraries from patient samples, with the functional information retrieved in during nanopore sequencing. We show how the analysis of CAPTORS using adaptive sequencing allows real-time evaluation of sequencing accuracy at per-read, -pore and time level during metagenome experiments in the Nevada Toluca volcano near Mexico City. CAPTORS can also analyse quantitative accuracy, improving measurements of gene expression in mRNA samples, and enabling best-in-class normalisation between samples across large patient cohorts, longitudinal patient timelines and point-of-care testing. These benefits can also be employed to improve clinical diagnosis, with CAPTORS able to model and correct systematic sequencing errors to improve the diagnosis of pathogenic BRCA1/2 variants in breast cancer. Together, this demonstrates how the enzymatic synthesis of CAPTORs can affix information within patient samples during library preparation that is later retrieved by sequencing. The information encoded within CAPTORS can act as internal ‘software’ that interfaces with external bioinformatic software for responsive analysis, and integrate patient samples into a clinical IT infrastructure.