DNA-seq of homo sapiens: adult female blood
Blood samples from APR cohort were aliquoted into 1.8 ml cryovials, flash frozen and stored at -80C until further processing. For sequencing, frozen aliquots were thawed, and peripheral blood mononuclear cells (PBMCs) were isolated using red blood cell lysis buffer. The purified PBMCs were counted, with approximately 60 million cells per sample proceeding to ultra-long DNA extraction using the NEB Monarch Tissue DNA Extraction Kit (#T3060L). Sequencing libraries were then prepared with the extracted DNA using the Oxford Nanopore Technologies Ultra-Long DNA Sequencing Kit (SQK-ULK114) following the manufacturer's protocol with minor modifications. Briefly, the DNA was tagmented at room temperature for 10 min followed by a 10 min incubation at 75C. Rapid sequencing adapters were then added and incubated for 30 min at room temperature. Cleanup of the adapted DNA was performed using the precipitation star and buffers provided in the kit. The final libraries were quantified and loaded onto a minimum 3 PromethION flow cells at approximately 30 ng per flow cell (Supplementary Table 2). Sequencing was performed for 96 hours per flow cell, using R10.4.1 kits, with minimum read length setting of 1 kb.