CZ032_ONT
The Ultra Long (UL) DNA sequencing library preparation for Oxford Nanopore Technologies (ONT) platforms is a specialized methodology designed to maximize read length by preserving the integrity of extremely high molecular weight (eHMW) DNA. The protocol begins with a gentle extraction of intact genomic DNA, often using solid-phase reversible immobilization (SPRI) beads or other enzyme-based methods to avoid mechanical shearing. The resulting eHMW DNA is then quantified using fluorometry, as standard spectrophotometry is unreliable. For library construction, the standard ligation sequencing kit is used but with critical modifications: the DNA input is never vortexed or pipetted vigorously, and wide-bore tips are employed throughout. A primary step involves using a remarkably low amount of the DNA damage repair buffer, and the adapter ligation step is performed under high-salt conditions to promote efficient end-binding for very long fragments. Crucially, the library is never subjected to a final clean-up step that could preferentially recover shorter fragments; instead, it is loaded directly onto the flow cell. This meticulous, gentle approach minimizes fragmentation, enabling the sequencing of fragments hundreds of kilobases to over 4 megabases in length, with a read N50 typically exceeding 100 kb.