High-coverage DNA sequence and modification profiling of targeted genomic elements using Nanopore-based Cas12a Targeted Ligation and Enrichment Sequencing (nCasTLES).

Third-generation sequencing technologies, like nanopore sequencing, allow for long-read sequencing and direct examination of nucleic acid modifications at a low cost. However, nanopore sequencing is limited by low throughput, making targeted sequencing necessary to examine particular genomic elements. The established method is nanopore Cas9-targeted sequencing (nCATS), which uses blunt-end cleavage of phosphorylated DNA to render targeted DNA sites as the sole ligation-capable ends for sequencing adapter attachment.

While nCATS boosts on-target sequencing yield, it falls short in overall sequencing output and experiences faster flow cell degradation, leading to a higher cost per sequencing due to non-sequencing DNA.

We introduce a modified strategy, based on creating defined base overhangs with Cas12a/Cpf1 as ligation substrates for biotinylated oligos followed by bead enrichment, called nanopore Cas-12a Targeted Ligation-Enrichment Sequencing, or nCasTLES. nCasTLES eliminates off-target DNA via bead washes instead of rendering it inert. By removing the inert off-target DNA, nCasTLES libraries can be combined with other sequencing libraries in a single run, achieving the same on-target DNA sequencing as nCATS while boosting the overall yield of useful data and slowing down the flow cell degradation.

We demonstrate the potential of nCasTLES to study methylation dynamics at a frequently-methylated gene promoter. We also use the Cas12a cleavage on an integrated lentiviral vector to evaluate clonality in a transfected population and examine the integration state and transgene effects in selected clones. Moreover, we showcase nCasTLES' advantage in flow cell throughput by mixing nCasTLES libraries with whole-genome sequencing (WGS) libraries to characterize genetic and modified base information, such as clonal copy number variation analysis or BrdU incorporation, along with targeted sequencing.

This method enables highly targeted genomic investigation combined with full throughput of off-target reads.