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Scott D. Jewell, Ph.D., Professor and Director, Program for Technologies and Cores Van Andel Research Institute
Biospecimen quality is affected by preanalytical variable and the analytes from the biospecimen are the targets of that quality assessment. Purity, size, integrity and a functional assessment of nucleic acids are used to measure genomic biospecimen quality. However, a more complex measurement of quality is the assessment of the biology of the biospecimen. We investigate these questions using animal models to provide further improvements in best practices.
Randy Rasmussen, Ph.D., President and COO, BioFire Diagnostics
Stephanie Thatcher, Ph.D., Director, Systems Integration, BioFire Diagnostics
Development of molecular detection systems focuses on nucleic acid amplification and detection. This half of the problem gets the grants, the patents and the research time. Unfortunately these systems are often brought to their knees by snot, blood, poop and sputum. The hardest part of highly integrated systems is the sample prep and yet it is the part that is hardest to get people to work on. We will describe the process of learning this lesson with the FilmArray system, how we approach sample prep, and what important factors you should consider during development.
Lawrence B. Blyn, Ph.D., Director, Science and Technology, Ibis Biosciences, Abbott
We describe a sample preparation and detection system that provides for the rapid detection and identification of bacterial and Candidal nucleic acid directly in whole blood specimens from patients with suspected bloodstream infections. A lysis method and DNA purification system were designed for processing 5 ml of whole blood. PCR amplification formulations were optimized for high levels of human DNA. The system provides for rapid and sensitive molecular detection of diverse agents of these clinically important infections in approximately 6h.
Patrick Hurban, Ph.D., Vice President, Research and Development, Expression Analysis, The Quintiles Company
Formalin-fixed tissues present many sample preparation, extraction and method development challenges. It can also be challenging to understand whether certain findings stem from intrinsic genetic properties of the sample, or alternatively, are a product of how the sample was handled. Despite advancements in preservation methods, vast collections of FFPE material await analysis. Results will be presented comparing matched formalin-fixed and frozen tumor samples analyzed using a sensitive next-generation sequencing assay.
Robert D. Daber, Ph.D., Director, Research and Development and Sequencing Operations, Bio-Reference Laboratories
This presentation will discuss challenges and benefits of NGS based targeted RNA sequencing in the detection of gene fusion events, including, nucleic acid isolation, sample preparation and downstream data processing. There are a number of specific challenges related to RNA sequencing, standardized quality control metrics both before and after library prep are clearly needed.
P. Mickey Williams, Ph.D., Director, Molecular Characterization & Clinical Assay Development Laboratory (MoCha), Frederick National Laboratory for Cancer Research
NGS offers a powerful tool for assessment of molecular defects found in cancer. The utilization of NGS is becoming common practice in clinical laboratories. This complex technology requires a new level of analytical performance testing and validation. This discussion will focus on approaches used for analytical validation of the NGS clinical assay used for treatment selection in the NCI-MPACT Study.
Curt Scharfe, M.D., Ph.D., Senior Scientist, Stanford Genome Technology Center, Stanford University
Clinical molecular testing increasingly depends on the development and deployment of novel sample preparation technologies. In collaboration with physicians and clinical laboratories we are developing genomic and sequencing assays for the screening and diagnosis of cystic fibrosis, clinical viral infections, newborn and neurodevelopmental conditions and inherited cardiomyopathies. These projects have involved invention of a novel multiplex capture technology, and several innovative improvements in DNA sample preparation. Both approaches have increased speed and accuracy, while lowering costs.
Chalom Sayada, M.D., Ph.D., Co-founder & CEO, Advanced Biological Laboratories SA
Clinical environments wishing to provide genotyping services in the field of Virology or Human Genetics using Next Generation Sequencing (NGS) need robust, standardized, registered and well-validated software systems. These should be tailored to the optimized management of genomic data resulting in personalized healthcare. Dedicated downstream analysis systems help to perform an accurate, quick and simple analysis of NGS data. This begins with sample preparation and the generation of reads by any type of sequencing platform and ends with simple and easily-understandable reporting ideally suited to clinical interpretation and the connection to the local LIMS. Coupling advanced IT solutions to a well-established sequencing workflow usually helps labs with the validation of new sample prep and innovative assays, enhancing patient management.
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8:30 切合目的的生物檢體採樣與使用規範的National Cancer Institute資源
Helen Moore, Ph.D., Branch Chief, Biorepositories & Biospecimen Research Branch, Cancer Diagnosis Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute
The National Cancer Institute has led the way in developing Best Practices for Biospecimen Resources, sponsoring new research in Biospecimen Science, and building groundbreaking research biospecimen collections including postmortem biospecimens for the NIH GTEx program. New projects to build evidence-based best practices for frozen and FFPE tissues will be described.
Helen Fernandes, Ph.D., Associate Professor, Pathology and Laboratory Medicine, Weill Cornell Medical College
This presentation will discuss several important issues, such as: RNA detection in cancer tissues stored in FFPE samples, profiling microRNA expression, FFPE DNA quality control and its correlation with NGS data, and understanding pre-analytic effects on RNA gene expression.
Alexis Sauer-Budge, Ph.D., Senior Research Scientist, Fraunhofer Center for Manufacturing Innovation; Adjunct Research Assistant Professor, Biomedical Engineering, Boston University
Traditionally, bacterial pathogens in the blodd have been identified using culture-based methods that can take several days to obtain results. This can lead to physicians making treatment decisions based on an incomplete diagnosis contributing to patient morbidity. To decrease diagnosis time, we are developing a novel sample preparation device for isolating and concentrating dilute bacteria from blood. This presentation will describe the sample preparation device for methodology to isolate viable bacteria from blood which is clean enough for direct PCR or other downstream detection technologies.
Sam R. Nugen, Ph.D., Assistant Professor, Department of Food Science, University of Massachusetts, Amherst
The lack of a practical method for bacterial separation remains a hindrance for the low-cost and successful development of rapid detection methods from complex samples. We have developed T7 bacteriophage magnetic probes, where T7 bacteriophage is bound to magnetic particles. The capture efficiencies of bacteriophages on microbeads and nanoparticles for the separation of E. coli K12 were determined. The results indicated that bacteriophage magnetic particles achieved a capture efficiency of 93.7 簣 1.1% in 15 minutes.
Patricia Mueller, Ph.D., Chief, Molecular Risk Assessment Laboratory, Newborn Screening and Molecular Biology Branch, Division of Laboratory Sciences, Centers for Disease Control and Prevention (CDC)
We designed a next-generation sequencing assay using primer pairs for PCR amplification and library preparation of up to 48 samples in one Fluidigm Access Array for next-generation sequencing using the MiSeq. This assay can be scaled up using additional Access Arrays in one MiSeq run. The PCR amplification of GAA is challenging due to difficult regions in the gene. All transcribed exon sequences as well as exon/intron borders including those intron sequences containing mutations as identified in the Human Genome Mutation Database (HGMD) were targeted for amplification. Primers were designed not to overlap known HGMD mutations, and variants found in dbSNP were avoided when possible. The data was filtered at a quality score of ??30 and trimmed. We characterized reference materials including those with missense, nonsense, and splicing mutations; and small insertions and deletions. Large deletions that included exon 18 were independently characterized.
Apurva K. Srivastava, Ph.D., Principal Scientist, Frederick National Laboratory for Cancer Research Leidos Biomedical Research, Inc.
Contrary to prevalent belief, pre-analytic factors contribute to the most errors documented in clinical laboratories as compared to analytical factors where most efforts are devoted during assay validation. Dr. Srivastava will discuss the general pre-analytical variables for protein assays and how they affect accuracy in clinical laboratory tests. The focus of his presentation will be on identifying the phases of pre-analytical factors with reference to pharmacodynamic/proof-of-mechanism (POM) phospho-protein biomarkers in clinical trials. As a take home message, participants will learn that pre-analytic variables are an underappreciated area in biomarker validation process and improvements in this process will significantly impact accuracy of test results and enable laboratories to focus their quality assurance efforts.