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FRIDAY, APRIL 12
8:00 am Chairperson’s Opening Remarks
» Featured Presentation
8:05 Extreme PCR: Efficient Amplification in Less than One Minute
Carl Wittwer, M.D., Ph.D., Professor, Pathology, University of Utah
Although 10 min PCR was first reported in 1990, it has been hard to further decrease amplification times without losing yield. Extreme PCR combines <2 s cycles with higher primer and polymerase concentrations to enhance the efficiency of annealing and extension. Cycle times of less than 1 s for PCR products less than 100 bp result in specific, high-yield amplification as demonstrated by gels and melting curves, and real time PCR analysis reveals high efficiency.
The Evolution of PCR: Amplification in Under a Minute
8:35 SuperSelective Digital PCR Primers for the Detection of Rare Mutant Cancer Cells in Samples Containing Abundant Normal Cells
Fred Russell Kramer, Ph.D., Professor, Department of Microbiology and Molecular Genetics, Public Health Research Institute, New Jersey Medical School
Highly selective primers enable a single molecule of mutant to generate amplicons in a digital droplet (or well) without interference from more abundant wild-type molecules, even if the only difference between the mutant and the wild-type is a single-nucleotide polymorphism.
9:05 High-Throughput Planar dPCR Microfluidic Devices for High Performance Single Cell Analysis
Kevin Heyries, Ph.D., Postdoctoral Fellow & Researcher, Hansen Lab, University of British Columbia (UBC)Centre for HighThroughput Biology (CHiBi)
High density planar digital PCR arrays using surface tension based partitioning in microfluidic devices provide high throughput and high performance measurements. By integrating on chip single cell trapping, lysis, reverse transcription followed by digital PCR over 200 single cell per run, single molecule mRNA and microRNA detection is performed allowing an absolute quantification at single cell resolution.
9:35 The Use of dPCR to Measure Her-2 in Borderline-Amplified Breast Cancer Research Samples
Gabriele Zoppoli, M.D., Ph.D., Internal Medicine Resident, University of Genova & IRCCS AOU San Martino IST, Genova, IT
ERBB2 is frequently amplified in breast cancer (BC), leading to an aggressive phenotype. Since “smart” drugs target ERBB2, its precise quantification is essential for treatment decision-making. Here, we describe the use of dPCR in a set of ERBB2 “equivocal status” BC patients. We also assess TOP2A copy number, and both TOP2A and ERBB2 gene expression intensity, on the described sample set. We discuss dPCR results compared to those obtained by qRT-PCR, IHC, and aCGH methods.
10:05 Coffee Break with Exhibit and Poster Viewing
10:45 Analysis of Complex and Multi-Allelic Forms of Copy Number Variation Using Digital PCR in Nanoliter Droplets
Steven McCarroll, Ph.D., Assistant Professor, Genetics, Harvard Medical School; Director, Genetics, Stanley Center for Psychiatric Research, Broad Institute
Copy number variation is an important component of human genetic variation, but analyses to date have been largely limited to simple deletions and duplications. I will describe how, using a droplet-based approach to digital PCR, we are getting some of our first glimpses at the population genetics and disease relevance of complex and multi-allelic CNVs.
11:15 Non-Invasive Detection of Fetal Microdeletions and Microduplications
David Gerard Peters, Ph.D., Associate Professor, Department of Obstetrics, Gynecology & Reproductive Sciences, University of Pittsburgh
Current non-invasive prenatal testing (NIPT) methods are focused on the detection of common aneuploidies. However, other genomic imbalances such as microdeletions and microduplications occur at high frequency and are of major clinical significance. This presentation will cover recent developments in NIPT in the context microdeletion and microduplication detection.
11:45 High-Throughput Copy Number Counting in Single Cells – A Method for the Detection of Meiotic and Mitotic Errors
Angelika Daser, M.D., Senior Scientist, SH-Gen Research
Molecular copy number counting (MCC) in single cells is based on limiting dilution of the DNA to a concentration of less than one molecule per PCR reaction and digital PCR. As we are interested in meiotic errors we count the numbers of chromatids per chromosome by counting the numbers of positive PCR reactions representing target sequences on all chromosomes. This method is simple and applicable to monitor not only meiotic but also mitotic cell divisions and copy number changes in general.
12:15 pm Luncheon Presentation (Sponsorship Opportunity Available) or Lunch on Your Own
1:30 Chairperson’s Remarks
1:35 Enabling Single-Cell mRNAseq with Microfluidics Using the C1™ Single-Cell Auto Prep System
Kenneth Livak, Ph.D., Senior Scientific Fellow, Fluidigm Corporation
2:05 Quantitative Analysis of Single-Cell Transcriptomes
Sten Linnarsson, Ph.D., Assistant Professor, Medical Biochemistry and Biophysics,Karolinska Institute
Single-cell RNA sequencing has progressed rapidly in the last few years, to the point where it is now possible, for the first time, to obtain quantitatively accurate portraits of single-cell RNA content. I will describe the methods we use to achieve single-cell quantitative gene expression measurements, and some of the surprising discoveries that emerge from such analysis.
2:35 Future Medical Applications of Single-Cell Sequencing in Cancer
James Hicks, Ph.D., Professor, Cold Spring Harbor Lab
Advances in whole genome amplification and next-generation sequencing methods have enabled genomic analyses of single cells, and these techniques are now beginning to be used to detect genomic lesions in individual cancer cells. Sequencing of single cells is likely to improve several aspects of medicine, including the early detection of rare tumor cells, monitoring of circulating tumor cells (CTCs), measuring intratumor heterogeneity, and guiding chemotherapy. In this review we discuss the challenges and technical aspects of single-cell sequencing, with a strong focus on genomic copy number, and discuss how this information can be used to diagnose and treat cancer patients.
3:05 Genome-Wide Detection of Single Nucleotide and Copy Number Variations of a Single Human Cell
Alec R. Chapman, Graduate Researcher, Xie Group, Harvard University
We report a new amplification method: Multiple Annealing and Looping Based Amplification Cycles (MALBAC) that offer high uniformity across the genome. Sequencing MALBAC amplified DNA achieves 93% genome coverage ≥1x for a single human cell at 25x mean sequencing depth. We detected digitized copy number variations of a single cancer cell. By sequencing three kindred cells, we were able to call individual single nucleotide variations (SNVs) with no false positives reported. We directly measured the genome-wide mutation rate of a cancer cell line and found that purine-pyrimidine exchanges occurred unusually frequently among the newly acquired SNVs.
3:35 Refreshment Break with Exhibit and Poster Viewing
4:15 CLOSING PANEL: Overcoming Barriers to Adoption
Moderator: Ross Haynes, Biological Science Technician, Biochemical Science Division,National Institute of Standards and Technology (NIST)
Panelists: Lyn Chitty, Ph.D., MBBS, MRCOG, Professor, Genetics & Fetal Medicine, Institute of Child Health
Steven McCarroll, Ph.D., Assistant Professor, Genetics, Harvard Medical School; Director, Genetics, Stanley Center for Psychiatric Research, Broad Institute
Carl Wittwer, M.D., Ph.D., Professor, Pathology, University of Utah
George Karlin-Neumann, Ph.D., Director, Scientific Affairs, Digital Biology Center, Bio-Rad Laboratories
5:00 Close of Conference
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