Detection and Quantification of Rare Mutations Using Massively Parallel Sequencing (Safe-SeqS)

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The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Though massively parallel sequencing instruments are in principle well-suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. The keys to this approach, called "Safe-SeqS" for (Safe-Sequencing System) are (i) assignment of a unique barcode to each template molecule; (ii) amplification of each barcoded template molecule using a polymerase with a low error rate; and (iii) redundant sequencing of the amplification products. PCR fragments with the same barcode are truly mutant ("super-mutants") if ≥95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.
Patent Information:
Title App Type Country Serial No. Patent No. File Date Issued Date Expire Date Patent Status
Safe Sequencing System PCT: Patent Cooperation Treaty European Patent Office 12772013.4 2697397 4/12/2012 4/5/2017   Granted
Safe Sequencing System PCT: Patent Cooperation Treaty China 201280029284.6   4/12/2012     Pending
Safe Sequencing System PCT: Patent Cooperation Treaty Australia 2012242847 2012242847 4/12/2012 5/4/2017   Granted
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Jeanine Pennington
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