Nanopore of Bsal
Batrachochytrium salamandrivorans zoosporangia and zoospores were cultured in tryptone-gelatin hydrolysate-lactose (TgFl) broth in cell culture flasks at 18C. 200ml of 6 days old cultures were harvested and centrifugated at 1700g for 10 mins at 4C. Cell pellet was washed with ice cold water and snap frozen in liquid nitrogen. Highmolecular weight DNA for Nanopore sequencing was obtained by a customized cetyltrimethylammonium bromide (CTAB) extraction procedure 1,2 with the modification of using RNase A (T3018, NEB) instead of RNase T1. Briefly, cell pellet was ground with a mortar and pestle in liquid nitrogen with 2g of sand, followed by lysis with CTAB, two-step purification with phenol/chloroform/isoamyl alcohol and precipitation with isopropanol. Care was taken to avoid DNA shearing (cut off tips, no heating of samples). DNA concentration was checked using the Qubit BR assay (Invitrogen) and DNA size range profile was checked by Tapestation gDNA screentape (Agilent).   Two independent sequencing libraries were constructed, one with long unfragmented DNA, one with DNA fragmented to 12kb with a gTube (520079, Covaris). DNA ends were FFPE repaired and end-prepped/dA-tailed using the NEBNext FFPE DNA Repair kit (M6630, NEB) and the NEBNext Ultra II End-Repair/dA-tailing Module (E7546, NEB) followed by AMPure XP bead clean-up (A63882, Beckman Coulter). Adapters were ligated using the Genomic DNA by Ligation kit (SQK-LSK109, Oxford Nanopore Technologies) and NEBNext Quick T4 DNA Ligase (E6056, NEB) followed by AMPure XP bead clean-up. The two libraries were successively loaded onto a single PromethION (FLO-PRO002, type R9.4.1)flow cell. The unfragmented library was loaded first. Basecalling was achieved using the Guppy Basecalling Software v. 3.2.8+bd67289.