High-Fidelity Nanopore Sequencing of Ultra-Short DNA Targets

Nanopore sequencing offers a portable and affordable alternative to sequencing-by-synthesis methods but suffers from lower accuracy and cannot sequence ultra-short DNA. This puts applications such as molecular diagnostics based on the analysis of cell-free DNA or single-nucleotide variants (SNV) out of reach. To overcome these limitations, we report a nanopore-based sequencing strategy in which short target sequences are first circularized and then amplified via rolling-circle amplification to produce long stretches of concatemeric repeats. After sequencing on the Oxford Nanopore Technology’s MinION platform, the resulting repeat sequences can be aligned to produce a highly-accurate consensus that reduces the high error-rate present in the individual repeats. Using this approach, we demonstrate for the first time the ability to obtain unbiased and accurate nanopore data for target DNA sequences < 100 bp. Critically, this approach is sensitive enough to achieve SNV discrimination in mixtures of sequences a...