Long-read DNA Sequencing Library Prep

The sequencing libraries were prepared from 3 μg of purified HMW DNA using a Ligation Sequencing Kit SQK-LSK109 (Oxford Nanopore Technologies) as described by Vondrak et al.^{53 (link)}. Briefly, the DNA was treated with 2 μl of NEBNext FFPE DNA Repair Mix and 3 μl of NEBNext Ultra II End-prep enzyme mix in a 60 μl volume that also included 3.5 μl of FFPE and 3.5 μl of End-prep reaction buffers (New England Biolabs). The reaction was performed at 20 °C for 5 min and 65 °C for 5 min. Then, the DNA was purified using a 0.4 × volume of AMPure XP beads (Beckman Coulter). Because long DNA fragments caused clumping of the beads and were difficult to detach, the elution was performed with 3 mM TRIS–HCl (pH 8.5) and was extended up to 40 min. Subsequent steps including adapter ligation using NEBNext Quick T4 DNA Ligase and library preparation for sequencing were performed as recommended. The whole library was loaded onto FLO-MIN106 R9.4 flow cell and sequenced on MinION instrument until the number of active pores dropped below 40 (21–24 h). Basecalling of the raw reads was done using Albacore 2.3.3 (Oxford Nanopore Technologies).