Fig. 2

Overview of sequencing functional principle. A The ONT sequencing process initiates by passing a DNA or RNA strand through a nanopore—a small protein opening embedded in an electrically resistant membrane, functioning akin to a biosensor. A constant voltage applied to the electrolytic solution induces an ionic current through the nanopore. As a negatively charged DNA or RNA strand traverses the nanopore, inducing a shift from negative cis to positive trans, the motor protein influences the translocation speed. The change in ionic current resulting from this charge shift corresponds to the nucleotide sequence, enabling the identification of DNA/RNA strand bases. B The PacBio sequencing process involves fragmenting target double-stranded DNA molecules and ligating them to hairpin adapters, forming a SMRTbell—a closed, single-stranded circular DNA template. These SMRTbells are loaded onto a SMRT cell equipped with Zero-Mode Waveguides (ZMWs). A single polymerase binds to hairpin adaptors situated at the ZMW's bottom, initiating replication. To identify bases, four fluorescent-labeled nucleotides with distinct emission spectrums are introduced, producing a unique light pulse when incorporated into the polymerase. This series of light pulses is recorded and utilized to interpret the sequence of bases