PT  - JOURNAL ARTICLE
AU  - Brandon D. Wilson
AU  - Michael Eisenstein
AU  - H. Tom Soh
TI  - High-Fidelity Nanopore Sequencing of Ultra-Short DNA Sequences
AID  - 10.1101/552224
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 552224
4099  - http://biorxiv.org/content/early/2019/02/16/552224.short
4100  - http://biorxiv.org/content/early/2019/02/16/552224.full
AB  - 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 Oxford Nanopore Technology’s (ONT) MinION platform, 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.One Sentence Summary We introduce a simple method of accurately sequencing ultra-short (<100bp) target DNA on a nanopore sequencing platform.