Ryan Koehler

High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number

Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.

Droplet Digital PCR Measurement of HER2 Copy Number Alteration in Formalin-Fixed Paraffin-Embedded Breast Carcinoma Tissue

BACKGROUND Human epidermal growth factor receptor 2 (HER2) testing is routinely performed by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH) analyses for all new cases of invasive breast carcinoma. IHC is easier to perform, but analysis can be subjective and variable. FISH offers better diagnostic accuracy and added confidence, particularly when it is used to supplement weak IHC signals, but it is more labor intensive and costly than IHC. We examined the performance of droplet digital PCR (ddPCR) as a more precise and less subjective alternative for quantifying HER2 DNA amplification. METHODS Thirty-nine cases of invasive breast carcinoma containing ≥30% tumor were classified as positive or negative for HER2 by IHC, FISH, or both. DNA templates for these cases were prepared from formalin-fixed paraffin-embedded (FFPE) tissues to determine the HER2 copy number by ddPCR. ddPCR involved emulsifying hydrolysis probe-based PCR reaction mixtures containing the ERBB2 [v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian); also known as HER2] gene and chromosome 17 centromere assays into nanoliter-sized droplets for thermal cycling and analysis. RESULTS ddPCR distinguished, through differences in the level of HER2 amplification, the 10 HER2-positive samples from the 29 HER2-negative samples with 100% concordance to HER2 status obtained by FISH and IHC analysis. ddPCR results agreed with the FISH results for the 6 cases that were equivocal by IHC analyses, confirming 2 of these samples as positive for HER2 and the other 4 as negative. CONCLUSIONS ddPCR can be used as a molecular-analysis tool to precisely measure copy number alterations in FFPE samples of heterogeneous breast tumor tissue.

A Rapid Molecular Approach for Chromosomal Phasing

Determining the chromosomal phase of pairs of sequence variants – the arrangement of specific alleles as haplotypes – is a routine challenge in molecular genetics. Here we describe Drop-Phase, a molecular method for quickly ascertaining the phase of pairs of DNA sequence variants (separated by 1-200 kb) without cloning or manual single-molecule dilution. In each Drop-Phase reaction, genomic DNA segments are isolated in tens of thousands of nanoliter-sized droplets together with allele-specific fluorescence probes, in a single reaction well. Physically linked alleles partition into the same droplets, revealing their chromosomal phase in the co-distribution of fluorophores across droplets. We demonstrated the accuracy of this method by phasing members of trios (revealing 100% concordance with inheritance information), and demonstrate a common clinical application by phasing CFTR alleles at genomic distances of 11–116 kb in the genomes of cystic fibrosis patients. Drop-Phase is rapid (requiring less than 4 hours), scalable (to hundreds of samples), and effective at long genomic distances (200 kb).

Abstract 4859: Ultra-sensitive detection of rare mutants by droplet digital PCR with conventional TaqMan assays

Molecular tests for genetic mutations play an important role in the diagnosis of cancer. Somatic mutations that drive the pathological features of most tumors have increasing promise as biomarkers for cancer prognosis and therapeutic efficacy. The detection of somatic mutations poses an analytical challenge due to the heterogeneous nature of most samples, where a gene carrying a mutation may differ from the highly abundant wild type sequence by only a single nucleotide. Although a variety of methods exist for mutation analysis, many have poor selectivity and fail to detect mutant sequence below 1 in 100 wildtype sequences. Methods that provide better discrimination and quantitation of somatic mutations are desirable. Here we present a simple strategy using droplet digital™ PCR (ddPCR™) for the detection of somatic mutations with high selectivity and sensitivity. Based on the simple principle of sample partitioning into water-in-oil microdroplets, this ddPCR method increases the abundance of a mutant DNA sequence up to 20,000 times compared to an equivalent bulk PCR reaction. Using conventional TaqMan chemistries and workflow, selectivities of up to 1/100,000 can readily be achieved in any laboratory. Here we present results on the use of ddPCR for the detection and quantitation of several clinically important mutations, including KRAS, c-KIT D816V and JAK2 from clinical samples such as bone marrow aspirates and FFPE. Results from ddPCR are compared to those of conventional approaches including allele specific real-time PCR and sequencing. This ddPCR method may play an important role in the earlier detection of cancer, monitoring the progress of disease and response to therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4859. doi:1538-7445.AM2012-4859

Abstract 3399: Detection of rare mutations in plasma by droplet digital PCR

Detection and quantitation of specific mutations in circulating plasma holds promise for earlier and less invasive diagnosis of disease. This presents significant analytical challenges, particularly as the biomarker may differ from its highly abundant wildtype by only a single nucleotide. Conventional methods have poor selectivity and fail to detect mutant sequence below 1 in 100 wildtype sequences. Compounding this, the amount of circulating nucleic acid in plasma is low. Here we present a simple strategy using droplet digital™ PCR (ddPCR™) for the detection of somatic mutations with high selectivity and sensitivity. Based on the simple principle of sample partitioning into water-in-oil microdroplets, this ddPCR method increases the abundance of a mutant DNA sequence up to 20,000 times compared to an equivalent bulk PCR reaction. Using conventional TaqMan chemistries and workflow, selectivities of up to 1/100,000 can readily be achieved in any laboratory. We evaluated ddPCR for the detection and quantitation of several clinically important mutations in the EGFR and KRAS loci from clinical samples derived from normal and tumor plasma samples. We also demonstrate the feasibility of multiplexing of Kras and EGFR assays to improve sample processing efficiency. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3399. doi:1538-7445.AM2012-3399