Ultra-long Nanopore Sequencing of Cuscuta Genome

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)].