Safe sequencing system

US9487829B2 · kind B2 · utility

Assignee

• THE JOHNS HOPKINS UNIVERSITY · US

Inventors

• Bert Vogelstein · Baltimore, US
• Kenneth W. Kinzler · Baltimore, US
• Nickolas Papadopoulos · Towson, US
• Isaac A. Kinde · Beaumont, US

Key dates

• Filing date: Jul 30, 2015
• Grant date: Nov 8, 2016
• Expiry date: Jul 30, 2035

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q2600/158
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

Error rates in massively parallel sequencing instruments are generally too high to allow confident identification of rare variants. An approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose, called “Safe-SeqS” for (Safe-Sequencing System) includes (i) assignment of a unique identifier (UID) to each template molecule; (ii) amplification of each uniquely tagged template molecule to create UID-families; and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID 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.