Development of a method for preparing circular DNA molecules for sequencing and diagnostics

Abstract

Whole genome sequencing (WGS), targeted sequencing and other next generation sequencing (NGS) approaches have revolutionized genome research and its applications, like cancer diagnostics. However, considerable challenges remain, such as limited DNA sample material, and error prone workflow steps like PCR. This thesis develops a method for circular DNA preparation to provide solutions to these problems. The aim of this thesis was to prepare circular single-stranded DNA (cssDNA) molecules from genomic double-stranded DNA (dsDNA) for unlimited amplification by rolling circle amplification (RCA). This could be utilized for various applications, including sequencing library preparation, amplification of any dsDNA for sequence analyses, and long-term DNA data storage. The method is based on creating circular DNA molecules by ligation of Y-shaped adapter sequences to template dsDNA and allowing the binding of Bending Bridge and Nicking Loop oligomers to form the circular molecule. After circularization, the molecules are amplified by RCA. RCA produces long linear concatemeric single-stranded DNA (ssDNA) copies, which are then digested by a specific nicking enzyme that digests a recognition sequence of the Nicking Loop part to produce multiple monomers. As a result of this thesis, a functioning protocol was developed for circularizing dsDNA sequences and amplifying them by RCA, enabling unlimited DNA production and avoiding PCR-related complications. This is particularly beneficial in circulating tumor DNA (ctDNA) sequencing where sample amounts are limited. The method improves diagnostic accuracy and reliability by permitting analyses with minimal distortions to the original DNA. This method can be applied to various sequencing library preparation technologies, such as targeted sequencing and fragmentomics, and can be used on multiple sequencing platforms. Additionally, this method opens possibilities for DNA data storage, as circular DNA offers durable and stable nucleic acid-based storage form for digital data. The data stored in DNA can be retrieved by sequencing the DNA using NGS platforms, followed by decoding the resulting sequences back into binary code.