Nebnext ultra 2 end prep enzyme mix

For Oxford nanopore sequencing, DNA of C. acuminatum, C. pamiricum and C. suecicum was used. DNA was fragmented by pipetting. The sequencing libraries were prepared from 1 μg of the partially fragmented DNA using Ligation Sequencing Kit SQK- LSK109 (Oxford Nanopore Technologies) following the manufacturer’s protocol. 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 both the FFPE and End-prep reaction buffers (New England Biolabs). The reaction was performed at 20°C for 5 min and 65°C for 5 min, followed by purification using a 1x volume of AMPure XP beads (Beckman Coulter). Subsequent steps, including adapter ligation using NEBNext Quick T4 DNA Ligase and library preparation for sequencing, were performed according to the provided protocols. Each library was loaded separately onto the FLO-MIN106 R9.4 flow cell and sequenced for 20 h. SatDNA family array searches were performed with the same algorithm employed for the assembled reads.

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).

For Oxford Nanopore sequencing, the DNA of C. acuminatum was used. The DNA was fragmented by pipetting. The sequencing libraries were prepared from 1 μg of the partially fragmented DNA using an SQK-LSK109 Ligation Sequencing Kit (Oxford Nanopore Technologies) following the manufacturer’s protocol. 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, followed by purification using a 1x volume of AMPure XP beads (Beckman Coulter). Subsequent steps, including adapter ligation using NEBNext Quick T4 DNA Ligase and library preparation for sequencing, were performed according to the provided protocols. The whole library was loaded into the FLO-MIN106 R9.4 flow cell and sequenced for 20 h.
A search for tnp2B was performed with the conserved GGCTGGGTTACC motif among the longest (> 30 kbp) ON reads of the C. acuminatum genome. For the analysis of selected ultralong ON reads, the YASS genomic similarity tool was used, which enables searches of tandem repeat organization (http://bioinfo.lifl.fr/yass/yass.php) [18 (link), 38 (link)].