Nanopore sequencing of control and in vitro methylated DNA for methylation detection method development
Datasets of control unmodified DNA and DNA methylated in vitro were prepared using the PCR barcoding kit (SQK-PBK004, Oxford Nanopore Technologies). Fragmented DNA (approx. 200 ng) was ligated to the Barcode adapters (BCA) from the sequencing kit (SQK-PBK004), then amplified using the Rapid Barcode primers (LWB, SQK-PBK004) and LongAmp Taq DNA polymerase (New England Biolabs) and treated by exonuclease I (New England Biolabs).  DNA aliquots (approx. 1 µg) were modified using 160 µM S-adenosyl methionine and methyltransferases  M.EcoRI (40 U), M.BamHI (12 U), or M.HhaI (25 U) (New England Biolabs) for 1 hour at 37°C. The methyltransferase reactions were then stopped at 65°C for 20 min. The modification by M.TaqI (10 U) was carried out at 65°C for 1 hour. DNA was purified on AMPure XP beads and eluted into 10 mM Tris.Cl pH 8.0, 50 mM NaCl. Methylated and control barcoded DNAs were pooled in equal ratio. Rapid adapters (RAP, SQK-PBK004) were attached to the ends of pooled DNAs (approx. 400 ng). The library preparation, flow cell priming, and loading were completed as described in the protocol (SQK-PBK004, version PBK_9073_v1_revA_23May2018). Nanopore sequencing was carried out in a MinION Mk1B device using a FLO-MIN106 (R9.4.1, revD) flow cell (Oxford Nanopore Technologies).