Single molecule quantitation and sequencing of rare translocations using microfluidic nested digital PCR

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Issue Date
2013-06-23Author
Shuga, Joe
Zeng, Yong
Novak, Richard
Lan, Qing
Tang, Xiaojiang
Rothman, Nathaniel
Vermeulen, Roel
Li, Laiyu
Hubbard, Alan
Zhang, Luoping
Mathies, Richard A.
Smith, Martyn T.
Publisher
Oxford University Press
Type
Article
Article Version
Scholarly/refereed, publisher version
Rights
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
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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.
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Citation
Shuga, Joe, Yong Zeng, Richard Novak, Qing Lan, Xiaojiang Tang, Nathaniel Rothman, Roel Vermeulen, et al. 2013. “Single Molecule Quantitation and Sequencing of Rare Translocations Using Microfluidic Nested Digital PCR.” Nucleic Acids Research 41 (16). http://dx.doi.org/10.1093/nar/gkt613
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Except where otherwise noted, this item's license is described as: This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.