Ultra long ONT WGS for NA12884
Ultra-long reads (>100 kbp) were generated using ONT sequencing. Ultra-high molecular weight gDNA was extracted from lymphoblastoid cell lines; involving cell lysis of 3–5 × 10^7 cells in a buffer with 10 mM Tris-Cl, 0.1 M EDTA, 0.5% w/v SDS, and 20 mg/mL RNase A for 1 hour. This was followed by proteinase K treatment at 50°C for 2 hours. DNA was purified with two rounds of phenol-chloroform-isoamyl alcohol extraction followed by ethanol precipitation. Libraries were constructed using the Ultra-Long DNA Sequencing Kit (ONT, SQK-ULK001) with modifications: ~40 µg of DNA was mixed with FRA enzyme and FDB buffer, incubated for 5 minutes at room temperature, followed by heat inactivation at 75°C. This was followed by RAP enzyme incubation for 1 hour, and clean-up using Nanobind UL Library Prep Kit (Circulomics, NB-900-601-01). The library was loaded onto a primed FLO-PRO002 R9.4.1 flow cell for sequencing on the PromethION platform, with nuclease washes performed at 24 and 48 hours. Base calling was done with guppy (v6.3.7).