Oxford nanopore technologies (ONT) long read sequencing and analysis

High-molecular-weight DNA was isolated using the MagAttract kit (Qiagen # 67563). DNA was sheared to 50 kb via Megarupter (diagenode). The quality of the DNA was then assessed on a Femtopulse (Agilent) to ensure DNA fragments were > 40 kb on average. After shearing, the DNA was size selected with an SRE kit (Circulomics) to reduce the fragments size to < 20 kb. After size selection, the DNA underwent a-tailing and damage repair followed by ligation to sequencing specific adapters.

Half of the prepared library was mixed with library loading beads and motor protein and then loaded on to an ONT PromothION PROM-0002 flow-cell and allowed to sequence for 24 hr. After 24 hr, the flow-cell was treated with DNase to remove stalled DNA followed by a buffer flush. The second half of the library was then loaded and allowed to sequencing for 36 hr. The DNA was base called via Guppy 3.2 in High accuracy mode.

Long reads were aligned to the reference human genome using NGMLR (https://github.com/philres/ngmlr) and structural variants were identified using Sniffles (https://github.com/fritzsedlazeck/Sniffles) (Sedlazeck et al., 2018). The alignments and structural variants were then visualized using IGV (https://igv.org/).