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 ultrashort DNA. This puts applications such as molecular diagnostics based on the analysis of cell-free DNA or single-nucleotide variants (SNVs) out...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2019-05, Vol.91 (10), p.6783-6789
Main Authors: Wilson, Brandon D, Eisenstein, Michael, Soh, H. Tom
Format: Article
Language:English
Subjects:
Accuracy
Amplification
Analytical chemistry
Chemistry
Deoxyribonucleic acid
DNA
DNA sequencing
Nucleotide sequence
Porosity
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Summary:Nanopore sequencing offers a portable and affordable alternative to sequencing-by-synthesis methods but suffers from lower accuracy and cannot sequence ultrashort DNA. This puts applications such as molecular diagnostics based on the analysis of cell-free DNA or single-nucleotide variants (SNVs) 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 Technologies 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
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.9b00856