Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 64,969 bioRxiv papers from 287,914 authors.
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. These can be sequenced on the MinION platform from Oxford Nanopore Technologies (ONT), and the resulting repeat sequences 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 of < 100 bp. Critically, this approach is sensitive enough to achieve SNV discrimination in mixtures of sequences and even enables quantitative detection of specific variants present at ratios of < 10%. Our method is simple, cost-effective, and only requires well-established processes. It therefore expands the utility of nanopore sequencing for molecular diagnostics and other applications, especially in resource-limited settings.
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