Abstract
Cancer continues to be a leading cause of global mortality, and the current diagnostic tests for early-stage cancer detection are often invasive and expensive. This study aimed to develop an early-stage cancer diagnostic test using liquid biopsies. The study focused on using circulating DNA extracted from blood plasma to detect cancer, as dying cells release DNA into the bloodstream. However, limitations with current methods include sequencing length limitations and sequencing errors that make it difficult to distinguish true variants from sequencing artifacts.
To address these limitations, three approaches were examined in this study. The hpREADs approach was based on reduced representation sequencing and enables proofreading by comparing the sense and antisense copies of a DNA fragment physically linked in the same read, thereby reducing the background noise created by sequencing errors, facilitating the identification of genuine variants. The concatenation approach involved attaching circulating DNA fragments together to boost data generation from the ONT sequencers by creating long strands of multiple fragments, without any amplification, allowing for methylation calling, and providing insight into the DNA fragment length profile. The short library ctDNA detection approach generated multiple copies of the exact same circulating DNA molecule, facilitating the distinction of sequencing errors from true variants via generation of a consensus sequence.
Each of these approaches had bioinformatic pipelines created which facilitated in understanding the data generated as well as efficacy evaluation. These pipelines will then be used to help analyse future experiments that are undertaken using these approaches.
Overall, this study identified methylation and mutational variants related to cancer on circulating DNA extracted from liquid biopsies. However, further research is needed before the test can be used as an early-stage cancer diagnostic test. The findings of this study provide a promising direction for future research on developing a non-invasive and cost-effective diagnostic test for early-stage cancer detection.