Long-read sequencing
The long-range PCR target enrichments of >20 ng were
prepared for sequencing with the ONT ligation sequencing kit
(SQK-LSK109) following the manufacturer’s protocol, with adjustments for
sample type and yield. Individual sample libraries with <20 ng
were concentrated at given bead clean-up steps. For sample pooling
steps, samples were pooled in groups based on fragment size, to allow
for more accurate normalisation prior to final pooling and flow cell
loading. Prepared samples were sequenced on the MinION using the
FLO-MIN106 version 9.4.1 flow cell platform. Flow cells were run until
complete pore exhaustion, on average this was 72 hours, with minimal
refuel of flow cells performed whenever active pore percentages dropped
below 70%, achieving up to 30 billion basecall yields per flow cell and
an average coverage depth per sample of >30,000x.