Taking the bait: A PCR-free enrichment strategy for nanopore sequencing applications




Planz, John V.
Hall, Courtney
Sedlazeck, Fritz
Zascavage, Roxanne R.


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Purpose: DNA methylation is a critical epigenetic modification involved in regulating gene expression. Although aberrant cytosine methylation has been implicated in diseases ranging from neurological disorders to cancer, current understanding is obscured by the inherent limitations of bisulfite conversion. Nanopore sequencing offers the ability to simultaneously ascertain genetic variation and base modifications without chemical treatment. While numerous copies of target regions may be present within a sample, this represents a small fraction of total genetic material competing for pore access. Here we evaluate RNA bait hybridization capture for enrichment of mitochondrial DNA (mtDNA) prior to nanopore sequencing. Methods: Heavy and light strands were individually captured using the Arbor Biosciences myBaits Mito panel. Elutant from the first capture served as rebaiting input with the opposite probe set. Double-stranded products subsequently generated were multiplexed and sequenced on the MinION device. Resultant data were separated by barcode and mapped to the human reference genome. Read counts were normalized for coverage comparisons and methylation was detected using Tombo. Results: Overall input and throughput were significantly lower than a typical whole genome sequencing run, however mtDNA read counts indicate successful enrichment. In addition to ensuring target regions outcompete background DNA, these techniques allowed for methylation detection within native strands. Conclusion: The significance of a PCR-free enrichment strategy for nanopore sequencing applications extends beyond mtDNA. These techniques have been used to capture challenging regions within the rat genome and could provide novel insights into the genetic and epigenetic landscape of other biomedically relevant regions.