Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Jun 7;108(23):9530-5.
doi: 10.1073/pnas.1105422108. Epub 2011 May 17.

Detection and quantification of rare mutations with massively parallel sequencing

Isaac Kinde  1 , Jian Wu, Nick Papadopoulos, Kenneth W Kinzler, Bert Vogelstein
Affiliations

Detection and quantification of rare mutations with massively parallel sequencing

Isaac Kinde et al. Proc Natl Acad Sci U S A. .

Abstract

The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Although 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 the Safe-Sequencing System ("Safe-SeqS"), are (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 considered mutant ("supermutants") only 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.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Essential elements of Safe-SeqS. In the first step, each fragment to be analyzed is assigned a unique identification (UID) DNA sequence (green or blue bars). In the second step, the uniquely tagged fragments are amplified, producing UID families, each member of which has the same UID. A supermutant is defined as a UID family in which ≥95% of family members have the same mutation.
Fig. 2.
Fig. 2.
Safe-SeqS with endogenous UIDs plus capture. The sequences of the ends of each fragment produced by random shearing (variously colored bars) serve as the unique identifiers (UIDs). These fragments are ligated to adapters (yellow and orange bars) so they can subsequently be amplified by PCR. One uniquely identifiable fragment is produced from each strand of the double-stranded template; only one strand is shown. Fragments of interest are captured on a solid phase containing oligonucleotides complementary to the sequences of interest. Following PCR amplification to produce UID families with primers containing 5′ “grafting” sequences (black and red bars), sequencing is performed and supermutants are defined as in Fig. 1.
Fig. 3.
Fig. 3.
Safe-SeqS with exogenous UIDs. DNA (sheared or unsheared) is amplified with a set of gene-specific primers. One of the primers has a random DNA sequence (e.g., a set of 14 Ns) that forms the unique identifier (UID) (variously colored bars), located 5′ to its gene-specific sequence, and both have sequences that permit universal amplification in the next step (yellow and orange bars). Two UID assignment cycles produce two fragments—each with a different UID—from each double-stranded template molecule, as shown. Subsequent PCR with universal primers, which also contain “grafting” sequences (black and red bars), produces UID families that are directly sequenced. Supermutants are defined as in the legend to Fig. 1.
Fig. 4.
Fig. 4.
Single-base substitutions identified by conventional and Safe-SeqS analysis. The exogenous UID strategy depicted in Fig. 3 was used to produce PCR fragments from the CTNNB1 gene of three normal, unrelated individuals. Mutation numbers represent one of 87 possible single-base substitutions (3 possible substitutions/base × 29 bases analyzed). These fragments were sequenced on an Illumina GA IIx instrument and analyzed in the conventional manner (A) or with Safe-SeqS (B). Safe-SeqS results are displayed on the same scale as conventional analysis for direct comparison; the Inset is a magnified view. Note that most of the variants identified by conventional analysis are likely to represent sequencing errors, as indicated by their high frequency relative to Safe-SeqS and their consistency among unrelated samples.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Luria SE, Delbrück M. Mutations of bacteria from virus sensitivity to virus resistance. Genetics. 1943;28:491–511. - PMC - PubMed
    1. Roach JC, et al. Analysis of genetic inheritance in a family quartet by whole-genome sequencing. Science. 2010;328:636–639. - PMC - PubMed
    1. Durbin RM, et al. 1000 Genomes Project Consortium. A map of human genome variation from population-scale sequencing. Nature. 2010;467:1061–1073. - PMC - PubMed
    1. Shibata D. Mutation and epigenetic molecular clocks in cancer. Carcinogenesis. 2011;32:123–128. - PMC - PubMed
    1. McMahon MA, et al. The HBV drug entecavir - effects on HIV-1 replication and resistance. N Engl J Med. 2007;356:2614–2621. - PMC - PubMed

Publication types

LinkOut - more resources

-