Fig. 2 Adaptive sampling a) selecting regions of interest b) targeting different regions at different times during a sequencing run c) coverage of >753 COVID-related targets in a 54.1 Mb search space
If a project requires long reads or methylation analysis, it is typical to resort to whole-genome sequencing. This means generating more data than is ultimately required, increasing the experiment cost and prolonging analysis time. With adaptive sampling, many genomic regions of interest (ROI) can be targeted together, without needing additional steps during library preparation. As a strand of DNA passes through a nanopore, it is basecalled in real time, allowing the nascent sequencing read to be analysed while the strand is still passing through the pore. If the strand is likely to contain an ROI, the sequencing of that strand is allowed to continue to the end. If the strand is not likely to contain an ROI, the strand is ejected from the pore. In each case, the pore can then capture another strand and the selection process is repeated (Fig. 2a). Decisions are made for each pore individually, so it is possible to configure different pores to select for different target regions in the same sequencing run. To demonstrate this, for the first 20 hours of a sequencing run, one half of a MinION (TM) Flow Cell was set to capture the BRCA1 locus, and the other half captured BRCA2. After 20 hours of sequencing this was reversed (Fig. 2b). To demonstrate adaptive sampling on a larger panel of target regions, we captured >750 loci that have been reported to be associated with host response to COVID-19 including major immune-related genes, SNPs identified by GWAS, and genes identified by CRISPR knock-out. Sufficient coverage can be obtained from a single MinION Flow Cell for variant calling (Fig. 2c).