2014 Archived Content
Digital PCR’s ability to precisely quantitate, identify mutations and copy number variations, and to perform gene expression analysis is creating waves across the diagnostics landscape. It is well suited for single-cell analysis and shows promise to become a superior tool in the clinic due to its capacity to work with small amounts of sample. The Third Annual Digital PCR event will bring together industry visionaries and early adopters to discuss digital PCR’s capabilities, limitations, and future applications. Researchers will examine applications in cancer biomarker and rare mutation detection, non-invasive fetal DNA analysis, and infectious disease quantification, particularly in HIV. Novel digital PCR devices from startups and academic labs will be showcased. Other topics to be addressed include digital PCR integration with existing technologies, solutions for processing difficult samples as well as increasing sample throughput, and guidelines and best practices for digital detection.
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MONDAY, OCTOBER 6
8:00 am Short Course Registration and Morning Coffee
9:00 am - 12:00 pm
Digital PCR Experiment Design and Primer
Instructors: Jim Huggett, B.Sc. (Hons), Ph.D., Science Leader, Nucleic Acid Metrology, Molecular & Cell Biology, LGC
Yann Jouvenot, Ph.D., Product Manager, Digital Biology Center, Bio-Rad Laboratories
This course will introduce the concept of digital PCR (dPCR) and explain how it compares to other molecular methodologies, citing both advantages and disadvantages. Included in the discussion will be the application of dPCR to perform minority target detection, absolute quantification and measurement of copy number variation as well as the analysis of RNA using reverse transcriptase dPCR. The discussion will also include some of the specific challenges associated with performing this technique.
* Separate registration required
12:00 pm Main Conference Registration
1:25 Chairperson’s Opening Remarks
Stephen Bustin, BA(Mod), Ph.D., FSB, Anglia Ruskin University
» 1:30 KEYNOTE PRESENTATION:
Advancing Diagnostics with Digital Detection: From Quantifying Ultra-Rare Mitochondrial DNA Mutations to Profiling Tumor-Infiltrating Lymphocytes
Jason H. Bielas, Ph.D., Associate Member, Translational Research Program, Fred Hutchinson Cancer Research Center (FHCRC); Affiliate Assistant Professorship, Department of Pathology, University of Washington
In addition to precise quantification of nucleic acids, there are a number of advantages that digital PCR affords over other detection methods. I will discuss these advantages and review a number of novel digital PCR-dependent assays, which we have developed in our laboratory.
2:00 Reverse Transcription Digital PCR
Stephen Bustin, BA(Mod), Ph.D., FSB, Professor, Molecular Medicine, Postgraduate Medical Institute, Faculty of Medical Sciences, Anglia Ruskin University
Digital PCR has potential to be useful for measuring RNA in cellular gene expression studies as well as in RNA diagnostic applications. We have assessed the reliability and linearity of the RT step and conclude that there are enzyme, target and concentration-dependent issues that require urgent attention.
2:30 Digital PCR Standards
Ross Haynes, Biological Science Technician, Biochemical Science Division, National Institute of Standards and Technology (NIST)
Digital PCR has many benefits, including lack of reliance on reference materials. IRMM was the first to certify a CRM with digital PCR. AD413 was certified to have a 1:1 ratio of HMG and MON810 genes on a plasmid construct. NIST later certified SRM 2366 for concentration using digital PCR. Standards and rational behind using them will be discussed.
3:00 Refreshment Break with Exhibit and Poster Viewing
3:45 Defining Reference Standards for Digital PCR and Demonstrating Their Application in Circulating Tumor Assay Development
Jonathan Frampton, Ph.D., Global Product Manager, Horizon Diagnostics
Horizon Discovery’s patented gene editing technology (GENESIS™) has enabled the manufacture of genetically defined genomic DNA and FFPE reference standards. The reference standards used in this study include clinically relevant biomarkers (e.g. B-Raf, EGFR, K-Ras & N-Ras, etc.) and can be accurately manufactured to a specific allelic frequency (e.g. 0.01%, 0.1%, 1%, etc.). Here, we demonstrate the use of these reference standards to assess the performance of Digital PCR to detect rare mutations and copy number variants as well as their application in circulating tumor assay development.
4:00 Constellation - A High-Throughput Digital PCR Solution
Doug Roberts, Ph.D., Digital PCR Applications Scientist, Formulatrix Inc.
The benefits of digital PCR are well established; however barriers to widespread adoption remain including ease of use, high cost and low sample/assay throughput. We describe Constellation, a 96-well microplate based high-throughput digital PCR platform and performance data generated for a variety of applications.
4:15 Analysis and Visualization of Digital PCR Experiments – Applications of the dpcR Framework
Stefan Rödiger, Ph.D., Group Leader, InnoProfile, “Image-Based Assays”, Brandenburg University of Technology
Recently, we started a unified cross-platform software frame-work, designated “dpcR.” Our target user base is deliberately broad, including end users in clinics, academics, developers and educators. dpcR provides a means to understand how digital PCR works, to design and analyze experiments, and to spot potential troubles. Our framework is suitable for teaching and includes references for an elaborated methods set for dPCR statistics. The presentation will showcase customized applications of the dpcR package including a remote browser application and a standalone desktop application.
4:45 A Robust Framework for Digital PCR Data Analysis
Jo Vandesompele, Ph.D., CEO, Biogazelle; Professor, Ghent University
Digital PCR technology is maturing, but data analysis is still in its infancy. Here, we present our efforts to develop a robust mathematical framework for objective processing of this type of analog-digital data with inherent uncertainty. As proof-of-concept, we apply the framework for gene copy number analysis of circulating nucleic acids.
5:15 Welcome Reception with Exhibit and Poster Viewing
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