Random-Primed cfDNA Sequencing for Low-Frequency Cancer Markers

The patent segments the detection process into multiple independent components: (1) fragmentation of cfDNA into smaller pieces, (2) random priming with multiple primers targeting different regions, (3) exponential enrichment through multiple rounds of PCR amplification, and (4) high-depth sequencing. This segmentation allows each component to be optimized independently, achieving ultra-sensitive detection while maintaining reliability through the cumulative effect of multiple filtering stages.

The patent introduces random primers as intermediaries that bind to fragmented cfDNA sequences. These primers serve as mediators between the cfDNA sample and the sequencing process, enabling selective amplification and enrichment of tumor-derived DNA fragments. The random priming approach allows the system to capture diverse cfDNA sequences without requiring prior knowledge of specific tumor mutations, thereby improving early detection sensitivity while maintaining specificity through subsequent high-depth sequencing validation.

The patent performs preliminary fragmentation and random priming of cfDNA before the main sequencing process. This preliminary action prepares the DNA sample by creating numerous small fragments with accessible binding sites, enabling subsequent exponential enrichment. By performing these preparatory steps beforehand, the system can achieve ultra-high detection sensitivity (0.01% frequency) without requiring prohibitively high sequencing depth, thus maintaining productivity.

The patent dramatically changes the enrichment parameter by implementing multiple rounds of PCR amplification with random primers. This exponential enrichment process increases the relative abundance of tumor-derived DNA fragments from trace levels to detectable levels. By changing the enrichment parameter rather than simply increasing sequencing depth, the system achieves 0.01% detection frequency while maintaining reasonable sequencing throughput and cost efficiency.

The patent employs random primers with universal binding characteristics that can recognize and amplify diverse cfDNA sequences regardless of their origin or mutation type. This universal approach allows a single assay design to detect multiple cancer types and various genetic alterations (mutations, indels, rearrangements) without requiring cancer-specific or mutation-specific primers. The randomness of the priming approach provides multi-functionality, enabling comprehensive cancer detection while maintaining assay simplicity.

The random priming system allows the cfDNA sample itself to determine which sequences are amplified and detected, rather than requiring pre-selection of target regions based on known cancer associations. The fragmentation and random priming process naturally enriches for tumor-derived sequences based on their inherent characteristics, enabling the assay to adapt to different cancer types and mutations without requiring complex pre-programming or customization for each cancer type.