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

Joe Shuga,Martyn T Smith,Roel Vermeulen,Nathaniel Rothman,Xiaojiang Tang,Luoping Zhang,Laiyu Li,Richard A Mathies,Yong Zeng,Alan Hubbard,Qing Lan,Richard Novak

doi:10.1093/nar/gkt613

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.

Highlights

Tumor-specific somatic mutations can provide highly useful molecular biomarkers and therapeutic targets for cancer diagnosis, prognosis and treatment
We found that our standard quantitative PCR (qPCR) method was not sensitive enough to quantify and sequence t(14;18) from the clot genomic DNA (gDNA) of healthy subjects (Supplementary Figure S1); we developed a nested PCR approach (Figure 1a and c) for digital analysis of t(14;18)
The digital PCR (dPCR) methodology uses our custom-built Microfabricated emulsion generator array (MEGA) devices and a bead-based emulsion PCR assay (26) to achieve high-throughput digital quantitation and singlemolecule sequencing of t(14;18) (Figure 1d). In this methodology 2.5 nl of droplets serve as digital reaction volumes and droplets containing both single copies of t(14;18) and an IgH primer-functionalized bead yield clonal DNA beads labeled by fluorescein amidite (FAM)-labeled B-cell lymphoma-2 (BCL2) primer after thermal cycling

Summary

Introduction

Tumor-specific somatic mutations can provide highly useful molecular biomarkers and therapeutic targets for cancer diagnosis, prognosis and treatment. Central to the use of these genetic biomarkers in clinical oncology is sensitive and quantitative measurement of rare mutations in a vast excess of wild-type alleles. Discovering driver mutations that lead to carcinogenesis in a rare subset of cells is one key approach to the risk assessment, early detection and treatment of cancer (1,2). Quantification of low-level mutated sequences in cancer patients during and after treatments can provide informative data for evaluating therapy efficacy, monitoring minimal residual diseases and detecting disease relapse (4). Quantitative PCR (qPCR), a widely used approach in genetic analysis, measures the analog fluorescence signal of targets and is limited in the detection sensitivity and/or quantification accuracy owing to instrumental and experimental variation. An attractive alternative to this analog technique is digital PCR (dPCR), which provides a superior sensitivity to conventional qPCR by

Methods

Results

Conclusion