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bioRxiv · 10.64898/2026.07.06.736710

Cas12a-Targeted Multiplexed Nanopore Sequencing

Rueegg, A. B.·Gehrold, R.·Agathos, K.·Chun, S.·Baur, A.·Pelczar, P.
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Abstract

Targeted long read sequencing (LRS) of native genomic DNA (gDNA) using Oxford Nanopore Technologies (ONT) is an economically and computationally accessible method for sequencing selected genomic regions without the limitations associated with amplification-based approaches. At present, efficiency, multiplexing, and scalability remain key challenges for existing targeted LRS. We have developed Cas12a-Targeted Multiplexed Nanopore Sequencing (CTM-nSeq), which combines Cas12a-targeting, DNA fragment enrichment, and optimized adapter ligation using T7 DNA ligase. Unlike previously established protocols, CTM-nSeq is compatible with the latest ONT flow cell chemistry. Performing CTM-nSeq on a single sample with an R10.4 MinION flow cell routinely yields hundreds of on-target reads. Furthermore, CTM-nSeq enables targeting of multiple loci and is the first targeted ONT sequencing method, allowing reliable, barcode-assisted multiplexing. CTM-nSeq is an efficient and accessible method for sequencing native gDNA and analysing DNA methylation, repeat expansions, and sequence integrity. As such, CTM-nSeq has a wide range of analytical and diagnostic applications. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/736710v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1c7c9a1org.highwire.dtl.DTLVardef@1867539org.highwire.dtl.DTLVardef@7a10bdorg.highwire.dtl.DTLVardef@1e10c70_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstract illustrating CTM-nSeq workflow and advantages, showing the key steps for sample preparation: Cas12a cleavage, DNA fragment size enrichment, T7 DNA ligase mediated ligation and ONT-based sequencing. Key advantages include increased specificity, optimized cohesive end ligation, barcode-based multiplexing, and over 50-fold coverage.

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Rueegg, A. B., Gehrold, R., Agathos, K., Chun, S., Baur, A., Pelczar, P.. 2026-07-07. Cas12a-Targeted Multiplexed Nanopore Sequencing. https://doi.org/10.64898/2026.07.06.736710

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