title: Single molecule quantitation and sequencing of rare translocations using microfluidic nested digital PCR.
abstract: Cancers are heterogeneous and genetically unstable. New methods are needed that provide the sensitivity and specificity to query single cells at the genetic loci that drive cancer progression, thereby enabling researchers to study the progression of individual tumors. Here, we report the development and application of a bead-based hemi-nested microfluidic droplet digital PCR (dPCR) technology to achieve 'quantitative' measurement and single-molecule sequencing of somatically acquired carcinogenic translocations at extremely low levels (<10(-6)) in healthy subjects. We use this technique in our healthy study population to determine the overall concentration of the t(14;18) translocation, which is strongly associated with follicular lymphoma. The nested dPCR approach improves the detection limit to 1×10(-7) or lower while maintaining the analysis efficiency and specificity. Further, the bead-based dPCR enabled us to isolate and quantify the relative amounts of the various clonal forms of t(14;18) translocation in these subjects, and the single-molecule sensitivity and resolution of dPCR led to the discovery of new clonal forms of t(14;18) that were otherwise masked by the conventional quantitative PCR measurements. In this manner, we created a quantitative map for this carcinogenic mutation in this healthy population and identified the positions on chromosomes 14 and 18 where the vast majority of these t(14;18) events occur.
authors.affiliation: School of Public Health, University of California, Berkeley, CA 94720, USA, Department of Chemistry, University of California, Berkeley, CA 94720, USA, Department of Chemistry, University of Kansas, Lawrence, KS 66045, USA, UC San Francisco/UC Berkeley Graduate Program in Bioengineering, University of California, Berkeley, CA 94720, USA, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Department of Health and Human Services, Bethesda, MD 20852, USA, Guangdong Poison Control Center, Guangzhou 510300, China and Environmental Epidemiology Division, Institute for Risk Assessment Sciences, Utrecht University, Utrecht, NL-3508, The Netherlands.
authors.forename: Joe
authors.initials: J
authors.lastname: Shuga
authors.forename: Yong
authors.initials: Y
authors.lastname: Zeng
authors.forename: Richard
authors.initials: R
authors.lastname: Novak
authors.forename: Qing
authors.initials: Q
authors.lastname: Lan
authors.forename: Xiaojiang
authors.initials: X
authors.lastname: Tang
authors.forename: Nathaniel
authors.initials: N
authors.lastname: Rothman
authors.forename: Roel
authors.initials: R
authors.lastname: Vermeulen
authors.forename: Laiyu
authors.initials: L
authors.lastname: Li
authors.forename: Alan
authors.initials: A
authors.lastname: Hubbard
authors.forename: Luoping
authors.initials: L
authors.lastname: Zhang
authors.forename: Richard A
authors.initials: RA
authors.lastname: Mathies
authors.forename: Martyn T
authors.initials: MT
authors.lastname: Smith
mesh_terms: D002460:Cell Line; D056905:Chromosome Breakpoints; D002883:Chromosomes, Human, Pair 14; D002887:Chromosomes, Human, Pair 18; D005557:Formaldehyde / Q000633:toxicity; D006801:Humans; D046210:Microfluidic Analytical Techniques; D016273:Occupational Exposure; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D014178:Translocation, Genetic
chemical_list: D005557:Formaldehyde