title: Ultrasensitive Detection of Multiplexed Somatic Mutations Using MALDI-TOF Mass Spectrometry.
abstract: Multiplex detection of low-frequency mutations is becoming a necessary diagnostic tool for clinical laboratories interested in noninvasive prognosis and prediction. Challenges include the detection of minor alleles among abundant wild-type alleles, the heterogeneous nature of tumors, and the limited amount of available tissue. A method that can reliably detect minor variants <1% in a multiplexed reaction using a platform amenable to a variety of throughputs would meet these requirements. We developed a novel approach, UltraSEEK, for high-throughput, multiplexed, ultrasensitive mutation detection and used it for detection of mutant sequence mixtures as low as 0.1% minor allele frequency. The process consisted of multiplex PCR, followed by mutation-specific, single-base extension using chain terminators labeled with a moiety for solid phase capture. The captured and enriched products were then identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. For verification, we successfully analyzed ultralow fractions of mutations in a set of characterized cell lines, and included a direct comparison to droplet digital PCR. Finally, we verified the specificity in a set of 122 paired tumor and circulating cell-free DNA samples from melanoma patients. Our results show that the UltraSEEK chemistry is a particularly powerful approach for the detection of somatic variants, with the potential to be an invaluable resource to investigators in saving time and material without compromising analytical sensitivity and accuracy.
authors.affiliation: Agena Bioscience, San Diego, California.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Mosko
authors.affiliation: Agena Bioscience, San Diego, California.
authors.forename: Aleksey A
authors.initials: AA
authors.lastname: Nakorchevsky
authors.affiliation: Agena Bioscience, San Diego, California.
authors.forename: Eunice
authors.initials: E
authors.lastname: Flores
authors.affiliation: Agena Bioscience, San Diego, California.
authors.forename: Heath
authors.initials: H
authors.lastname: Metzler
authors.affiliation: Sequenom, San Diego, California.
authors.forename: Mathias
authors.initials: M
authors.lastname: Ehrich
authors.affiliation: Sequenom, San Diego, California.
authors.forename: Dirk J
authors.initials: DJ
authors.lastname: van den Boom
authors.affiliation: AstraZeneca, Cambridge, United Kingdom.
authors.forename: James L
authors.initials: JL
authors.lastname: Sherwood
authors.affiliation: Agena Bioscience, San Diego, California. Electronic address: anders.nygren@agenabio.com.
authors.forename: Anders O H
authors.initials: AO
authors.lastname: Nygren
mesh_terms: D000483:Alleles; D045744:Cell Line, Tumor; D004247:DNA / Q000097:blood* / Q000235:genetics; D005787:Gene Frequency / Q000235:genetics; D006801:Humans; D057230:Limit of Detection; D008545:Melanoma / Q000175:diagnosis* / Q000235:genetics*; D060885:Multiplex Polymerase Chain Reaction; D009154:Mutation / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D004247:DNA