MinION library preparation and sequencing

A DNA library was prepared for each diatom species using the Oxford Nanopore Technology (ONT) 1D Ligation Sequencing Kit (SQK-LSK108) and the “1D gDNA selecting for long reads” (version GLR1E_9022_v18_revT_18Oct2016) protocol with the following modifications. Pure (A260/280 = ~ 1.8, A260/230 = 2.0–2.2), unfragmented and non-size-selected P. tricornutum gDNA (6.7 μg) and T. pseudonana gDNA (5.5 μg) were used to prepare separate DNA libraries for two MinION R9.4 SpotON flow cells (FLO-MIN106). The DNA samples were repaired using the NEBNext FFPE DNA repair module (New England Biolabs cat. no. M6630) followed by a 0.45x AMPure XP bead (Beckman) clean-up in which 70 μl of resuspended beads were added to the 155 μl FFPE repair reaction and incubated at room temperature for 15 min, pelleted on a magnet and washed twice using 80% ethanol. The FFPE-repaired DNA was end-prepped using the NEBNext End repair/dA-tailing module (New England Biolabs cat. no. E7546) with extended incubation times of 30 min at 20 °C and 30 min at 65 °C. The end-prep step was followed by a 1x AMPure bead clean-up with the aforementioned modifications and extended incubation (15 min at 37 °C) of the resuspended bead pellet in nuclease-free water to improve long-molecule elution off the beads. The 1D adapter ligation was extended to 15 min at 25 °C and followed by a 0.6x AMPure bead clean-up with the previously mentioned extended incubation step and 80% ethanol washes. A total of 2 μg of prepared P. tricornutum library and 1.7 μg of prepared T. pseudonana library was loaded onto separate MinION R9.4 SpotON flow cells (FLO-MIN106), which were primed according to the specifications outlined by ONT. The “NC_48Hr_Sequencing_Run_FLO-MIN106_SQK-LSK108” sequencing script was run via MinKNOW software (v1.7.14) without live basecalling.