Attend a Seminar grcf grcf.jhmi.edu A component of Johns Hopkins Genomics CORE SYMPOSIUM May 24th, 2016, 9:30 a.m. 1:30 p.m. Turner Concourse Genetic Resources Core Facility (GRCF) Mission To provide high quality, cost effective research services and products to investigators throughout the Johns Hopkins Scientific Community. GRCF Services Core Store The Core Store provides one-stop shopping for more than 300,000 products from 15 of the leading life science companies. In addition to its product offering the store charges no shipping and handling fees and has free delivery to three campuses East Baltimore, Bayview and Homewood. There is also convenient 24/7 access to several hundred products via the Core Store 24/7 at these locations Blalock 1026, CRB 1 B02A and the Asthma and Allergy Building 1st floor. For more information go to http://grcf.jhmi.edu/core-store/ GRCF Services Scientific Exhibitors Keynote Address Contents Each of the GRCF s Services will have an exhibit table with representatives to answer questions. Nineteen of the GRCF s Corporate Partners will have exhibit tables. Please visit their exhibits to learn about their products and services. Harry (Hal) Dietz, M.D., Institute of Genetic Medicine, Johns Hopkins University Core Coins Exciting new funding opportunity, see page 9 for details. Promotions See page 9 for promotions at the Symposium.
GRCF Services Biorepository & Cell Center facilitates basic scientific research by providing expertise and service in all mammalian cell culture, clinical blood processing and long-term cryogenic storage of biospecimens. In 2013 the GRCF Biorepository & Cell Center proudly received the international recognition of CAP (the College of American Pathologist) Accreditation. This voluntary program demands that a laboratory go well beyond acceptable quality and regulatory compliance measures to help achieve the highest standards of excellence in our services. For more information go to http://grcf.jhmi.edu/biorepository-cell-center/ The DNA Services group works together to provide solutions for all of your DNA and RNA needs. We handle basic needs like DNA isolation, plating and storage, traditional core services like Sanger sequencing, PCR support and genotyping, and the more complex needs presented by the constantly changing field of next generation sequencing. For more information on these services please go to http://grcf.jhmi.edu Scientific Exhibitors 10X Genomics Affymetrix Agilent Beckman Coulter Bio-Rad Cell Signaling Technology Corning Cellgro GE Healthcare Life Sciences Integrated DNA Technologies Illumina Kapa Biosystems New England Biolabs, Inc. Promega QIAGEN Quality Biological, Inc. Sigma-Aldrich ThermoFisher Scientific Zymo Research Corp. 2 3
Keynote Address 11:30 a.m. 12:25 p.m. Title: Found in Translation: New Insights into the Pathogenesis and Treatment of Marfan Syndrome and Related Disorders Room: Tilghman Auditorium Time: 11:30 a.m. 12:25 p.m. Sponsor: GRCF Presented by: Harry (Hal) Dietz, M.D., McKusick-Nathans Institute of Genetic Medicine, Howard Hughes Medical Institution, Johns Hopkins University Abstract: Dysregulation of TGFβ activity has been implicated in many aneurysm disease states including Marfan syndrome (MFS), caused by deficiency of the extracellular matrix protein fibrillin-1, and Loeys-Dietz syndrome, caused by mutations in many genes that encode primary effectors of TGFβ signaling. Many manifestations including aneurysm can be attenuated in mouse models using interventions that antagonize TGFβ. Multiple lines of evidence will be presented that implicate ERK activation as the primary TGFβrelated event that drives disease including the ability of ERK antagonists to achieve phenotypic rescue. Despite this progress, our understanding of how fibrillin-1 deficiency initiates altered TGFβ activity remains incomplete, as does knowledge regarding events that culminate in tissue failure or that account for the wide intrafamilial variability in the severity of vascular disease. This talk will highlight work focusing on the use of conditional provocations in model systems to develop and test both pathogenic hypotheses and novel therapeutic strategies. Specific insights regarding the cause and treatment of pregnancy-associated aortic dissection and deleterious iatrogenic gene-by-environment interactions will be discussed. An additional emphasis is the use of genetic methods to determine how nature modifies MFS, both in humans and mice. In such unbiased discovery studies, the identification of modifier loci, genes and alleles capable of protecting from the cardiovascular consequences of MFS in predisposed individuals has identified additional therapeutic targets that have already been validated in preclinical studies. Seminar Directory: 9:30 a.m. Title: A Fully Integrated Solution for DNA and RNA Analysis Room: Tilghman Auditorium Time: 9:30 a.m. 10:25 a.m. Sponsor: 10X Genomics Presented by: Ash Wilson Abstract: 10x Genomics meets the critical need for long range, structural and cellular information, with an innovative system that transforms the capability of existing short-read sequencers. With millions of uniquely addressable partitions, the Chromium System supports comprehensive genomics and high-throughput single cell transcriptomics. It enables researchers to discover previously inaccessible genomic information at unprecedented scale, including phased structural variants, phased single nucleotide variants, and dynamic gene expression of individual cells while leveraging their existing sequencing systems and workflows 4 3
Title: Introduction to Real Time PCR Room: West Room Time: 9:30 a.m. 10:25 a.m. Sponsor: ThermoFisher Scientific Presented by: Lihong Zhang, Ph.D., ThermoFisher Scientific, Field Applications Scientist-Real-time PCR Abstract: What is real time PCR? How is real-time PCR similar to traditional PCR? How does the real time PCR instrument work? Real Time PCR Chemistries: Working mechanism of Taqman chemistry and SYBR Green chemistry, Quantitative PCR method: Absolute Quantitation vs. Relative Quantitation, Applied Biosystems instrumentation and reagents. Title: Standardizing Microbiomics Removing Bias from Sample Collection through Analyse! Room: MRB-G01 Time: 9:30 a.m. 10:25 a.m. Sponsor: Zymo Research Corp. Presented by: Ryan Kemp Abstract: The rapid growth of Microbiomics has increased the demand for standard methods to improve the reproducibility and quality of the data being generated. Therefore, there is a need for standard reference materials for the development, evaluation, calibration, and validation of complex microbiomics workflows encompassing sample collection, sample preparation, and analyses. Standardization of the following methods would greatly improve the quality of data generated: (1) sample collection tools that can reliably provide a molecular snapshot at the time of collection by stabilizing the nucleic acids at ambient temperature and rendering the sample noninfectious for safe transport; (2) purification methodologies that take into consideration the biases associated with differential lysis efficiency of the organism being processed (e.g. gram-negative/positive bacteria, fungus, viruses, and associated spores); (3) analytical pipelines that reduce bias due to library preparation methods, PCR, and bioinformatics (e.g. GC content, log difference organism abundances, detection limit, and ability to distinguish closely related organisms). To address these fundamental challenges, the scientists at Zymo Research have created reference materials for the development of the most accurate and unbiased workflows from sample collection to analyses. In this seminar, we will show how these reference materials improve the quality of data generated from Microbiomic studies. Title: "Ultra-Sensitive Quantification of Genome Editing Events Using Droplet Digital PCR" Room: MRB G03 Time: 9:30 a.m. 10:25 a.m. Sponsor: Bio-Rad Presented by: Bo Song, Ph.D. Field Applications Scientist Abstract: Genome editing has become a widely used tool and allows specific modification of almost any cell's genome. However, the often low (<5%) frequency of gene edits, in particular for some primary or induced pluripotent stem cell types, hinders facile and reliable detection by gel or sequencing based methods, or requires laborious clonal isolation of edited cells. Here we introduce an ultra-sensitive, cost-efficient Droplet Digital PCR-based method for the detection of HDR and NHEJ alleles generated using the CRISPR/Cas9 and TALEN genome editing systems. 4 5
Seminar Directory 10:30 a.m. Title: Illumina Solutions for Epigenetic Studies Room: Tilghman Auditorium Time: 10:30 a.m. 11:25 a.m. Sponsor: Illumina, Inc. Presented by: Britt Flaherty, PhD Abstract: Learn how NGS enhances epigenetic studies and about the advantages of methylation sequencing and whole-genome bisulfite sequencing. Additionally, hear about TruSeq Methyl Capture EPIC panel (launching this summer), an enrichment-based bisulfite sequencing method that mirrors and expands upon the content of the MethylationEPIC chip. Now researchers can move seamlessly between array and sequencing technologies. They can take advantage of the low price point on the MethylationEPIC chip for large-scale screens and dive deep on specific samples or subsets with the TruSeq Methyl Capture EPIC panel Title: Increased efficiency of genome editing using the Alt-R CRISPR-Cas9 System Room: West Room Time: 10:30 a.m. 11:25 a.m. Sponsor: Integrated DNA Technologies Presented by: Dr. Mitch Gore Abstract: A new CRISPR genome editing system has been designed that allows for increased efficiency. This discussion will illustrate how systematic variation of crrna and tracrrna length led to the development of a crrna:tracrrna complex that shows improved gene editing in mammalian cell culture with S. pyogenes Cas9. Data will be presented that demonstrates that the shorter lengths of the synthetic Alt-R CRISPR RNAs perform better and make them more amenable to high throughput manufacturing compared to the longer, native crrna and tracrrna, resulting in improved quality and cost. In addition we will show that the higher quality RNA oligonucleotides that are used in the Alt-R system have been shown to outperform other CRISPR RNA formats for triggering CRISPR-Cas9 genome modifications. The innate immune response triggered by transfection of RNAs is also a challenge in CRISPR systems. We will present data that illustrates the activation of stress response genes such as IFIT1 (P56) and OAS2 is reduced when Alt-R CRISPR-Cas9 RNAs compared to in vitro transcribed RNAs. Title: New Techniques for Monitoring Protein Interactions in Live Cells Room: MRB-G01 Time: 10:30 a.m. 11:25 a.m. Sponsor: Promega Presented by: Dr. Kristin Riching Abstract: Measuring dynamic protein interactions in living cells remains a significant challenge. Here we present two novel approaches for monitoring intracellular protein interactions in real time, each which utilize the bright, small, and blue-shifted NanoLuc luciferase. The first approach presented is NanoBRET, a proximity based assay that relies on energy transfer from a luminescent donor to a fluorescent acceptor. Proteins whose interactions are of interest to study are expressed as a NanoLuc fusion, the energy donor, and a fluorescently-labeled HaloTag fusion, the energy acceptor. The combination of these partners result in excellent signal to background due to optimized spectral separation. The second approach, NanoBiT, is also a proximity assay, but is based upon binary complementation. NanoBiT consists of a Large BiT polypeptide (18kD) fusion and small BiT peptide fusion (~1 kda), which generate luminescence after their protein fusions interact. With these two technologies, we show the ability to monitor inhibition, induction, dimerization, conformational changes, and protease activity for a variety of proteins. Pre-developed assay include proteins in the area of epigenetics, transcriptional activation, cell signaling, and membrane receptor interactions. Both NanoBRET and NanoBiT are powerful technologies that enable real-time measurements of the dynamics of protein interactions, allowing for rapid and robust screening of biologically relevant responses in living cells or further understanding of protein function. 6 5
Title: Introducing the Kapa RNA Hyper Prep Kit and the HyperCap Sequence Capture Workflow. Room: MRB-G03 Time: 10:30 a.m. 11:25 a.m. Sponsor: KAPA Biosystems Presented by: Rachel Kasinskas, Ph.D Abstract: Kapa Biosystems originally launched the KAPA HyperPlus Library Prep Kit in response to demand for a fast, flexible, automation-friendly NGS library construction kit that did not sacrifice library quality. By utilizing our optimally formulated and evolved enzymes in a novel one-tube chemistry, KAPA HyperPlus enables higher yields of adapter-ligated library and lower amplification bias in a streamlined workflow that actually improves library quality by maintaining higher library diversity, lower duplication rates and more uniform coverage, particularly for FFPE and lower input samples. Kapa has now applied these innovations to both RNA-Seq and target enrichment applications to develop the KAPA RNA Hyper Prep Kit and the new "HyperCap" workflow that integrates KAPA HyperPlus and NimbleGen SeqCap for targeted capture. Our talk will introduce both new offerings, providing insight into our streamlined protocols and demonstrating robust performance with samples ranging from ideal to notoriously challenging. Seminar Directory 12:30 p.m. Title: Advanced CRISPR/Cas9 Genome Editing in Mammalian Systems. Room: Tilghman Auditorium Time: 12:30 p.m. 1:25 p.m. Sponsor: Sigma-Aldrich Presented by: Joseph Frangipane, Ph.D Abstract: CRISPR/Cas9 is a genomic editing system that has the potential to revolutionize biomedical research with its simple and efficient design. In this seminar, we will thoroughly examine the CRISPR system and many of its laboratory applications. Topics will include: CRISPR background and function, CRISPR activity/specificity, donor design and genome editing methodology. Specific examples of gene knockout, insertion and modification of cell lines and animal models will be presented. We will also discuss paired nickases for improved specificity as well as pooled and arrayed lentiviral CRISPR libraries. Title: Liquid Biopsies: Generating sensitive, accurate, and reproducible results from cfdna. Room: West Room Time: 12:30 p.m. 1:25 p.m. Sponsor: QIAGEN Presented by: Jennifer L. Fostel, B.S.,M.S., Senior Global Product Manager, QIAGEN Abstract: Liquid Biopsy harbors great potential to improve health care and diagnosis. Today there are two main focus areas of liquid biopsy: Non-invasive prenatal testing (NIPT) and cancer treatment, especially in tumor types where regular tissue biopsies would have great clinical value, but are simply not feasible for the patient. In both cases, nucleic acids are extracted from easily obtained whole blood or other fluid specimens. New technologies like next-generation sequencing (NGS) enable very low limits of detection from scarce amounts of these nucleic acids, including circulating cell-free tumor DNA and exosomal RNAs. Generating sensitive, accurate, and reproducible results from a cfdna sample is the goal of any liquid biopsy assay approach. This talk will cover how QIAGEN can support researchers at every step of the liquid biopsy workflow, from sample collection and stabilization to the analysis of high-quality and high-complexity NGS data. Come learn about QIAGEN s PAXgene Blood ccfdna Tubes, QIAamp Circulating Nucleic Acid kit, and mirneasy technologies for preparing high-quality Liquid Biopsy samples, our QIAseq line of NGS library prep kits optimized to meet the challenges of cfdna, and QIAGEN bioinformatics solutions that streamline the generation of actionable insights from liquid biopsy assays at any laboratory scale. 6 7
Title: Agilent NGS Target Enrichment Solutions for Exomes, Targeted Panels and Beyond. Room: MRB G-01 Time: 12:30 p.m. 1:25 p.m. Sponsor: Agilent Presented by: Josh Wang Abstract: Next Generation Sequencing (NGS) applications are widely utilized in both basic and clinical fields to study genomic aberrations, including SNPs, InDels, CNVs and fusion transcripts. With the widest selection of workflows and DNA/RNA target panels, Agilent Technologies is the leading provider of target enrichment products with two unique platforms: SureSelect and HaloPlex, used in over 600 publications. These platforms are accompanied with Agilent's superior solutions for whole genome and whole transcriptome sequencing analysis. Hybridization-based SureSelect and PCR-based HaloPlex systems to capture target regions from 1 kb to 100 Mb with single-day workflows from library preparation to sequencing Ready-to-use and custom DNA, RNA and Methylation Kits, including: Clinical Research Exome, Focused Exome, OneSeq, Premium Exome with Linked-reads, Comprehensive Cancer, disease-specific and methylation panels Novel One-Seq library to detect genome wide CNV changes together with SNP/InDel in one sequencing reaction Sensitivity to detect variant with MAF < 0.5% in FFPE cancer samples Products supporting all NGS platforms: Illumina HiSeq, MiSeq and NextSeq; Ion Proton and PGM; PacBio and Nanopore. Title: Analysis of Cellular Signaling Using Activation State-Specific Antibodies - Useful Tips to Optimize Flow Cytometric, Immunoflourescence, and Immunohistochemistry assays. Room: MRB G-03 Time: 12:30 p.m. 1:25 p.m. Sponsor: Cell Signaling Technology Presented by: Jonah Riddell, PhD, Field Application Scientist, Cell Signaling Technology Abstract: Accurate detection and quantification of protein expression and post-translational modifications are critical components to our understanding of disease-related signaling. Researchers effectively assess treatment-induced effects through the use of activation state-specific antibodies; therein quantitatively monitoring cellular signaling using cell and tissuebased applications (e.g., IHC, ICC, HCS and flow cytometry). Antibodies that specifically detect protein modifications such as phosphorylation, acetylation, ubiquitinization, methylation, and genetic abnormalities are used individually to monitor the expression of a specific protein or the activity of a specific pathway. Cell Signaling Technology can demonstrate the use of these antibodies in multiplex cell and tissue assays, which are especially important in the analysis of complex cellular signaling, as they enable the simultaneous measurement of multiple endpoints in a single cell, tube, well, or slide and can often yield more information than individual single endpoint assays. Protocols (fixation, permeabilization, and staining) are critical to obtaining reproducible and reliable results with combinatorial approaches. Dependable products that lead to solid and accurate results with limited time spent on optimization are critical to build the foundation of the rapidly expanding scientific community. 8 7
Symposium Promotions Keynote/Plenary Address Attendance Promotion: Enter for a chance to win an ipad! Here s how you can participate. 1. Attend the Keynote Address from start to finish. 2. Turn in a completed entry card* to a GRCF staff member at the end of the keynote address. 3. One entry per person. Seminar Attendance Promotion: Enter for a chance to win a GRCF Tumbler. 1. Attend any of the 12 seminars from start to finish. 2. Turn in a completed entry card* to a GRCF staff member at the end of the seminar. 3. One entry per person, per seminar. *Entry cards may be obtained on May 24th prior to the start of the Keynote Address/seminars at the registration table. Additionally, entry cards will be available prior to the Symposium at any of the GRCF Divisions located on the Blalock 10 th Floor. Core Store: There will be multiple Core Store promotions running concurrent with the Symposium. Check out http://grcf.jhmi.edu/promotions/ on March 24th to view the full listing. DNA Services: Stop by our table for a coupon for $45 off your next qpcr run OR $1000 off of your next Illumina genotyping project of 93 samples or more. Door Prizes: Be sure to sign in at the registration desk to be eligible for one of our door prizes! Exciting Funding Opportunity The GRCF is excited to announce the award of $25,000 in Core Coins as a funding mechanism to help accelerate investigation and discovery in the area of single cell genomics. Applications for the funds are due June 15, 2016- see the GRCF Biorepository & Cell Center for details or visit our website, http://grcf.jhmi.edu 8 9
Celebrating 25 Years of Sequencing Services Since the arrival of the very first fluorescently based automated sequencer in our facility in 1991, to our current offerings in the world of Next Gen and traditional sequencing, the GRCF has striven to provide researchers with the tools they need to understand basic cellular mechanisms and answer complex genetic questions. Ask us how we can help you with your current project or your next grant proposal. 10X Genomics Single Cell Platform The Single-Cell Platform is a molecular barcoding and analysis suite that delivers cell-b-cell 3 counting of mrna transcripts for many tens of thousands of cells per run. The platform supports a broad range of applications, including cancer-cell transcriptomics and cell-type identification and discovery. Because the platform works with the short read sequencers, it integrates easily into existing workflows. The 10x system is configured to be compatible with Illumina sequencers. New Lower Pricing at the GRCF HTS Center If you submit a single pool for sequencing on a single Rapid Flowcell (ie. 2 lanes), you can take advantage of lower pricing and faster turnaround times. All barcodes must be unique. Please visit our website to learn more about our updated pricing, and take advantage of the lowest cost/base on campus. http://grcf.jhmi.edu/hts/pricing In-house Sanger Sequencing Our Sanger sequencing services provide rapid turn around times and high quality at a low price, with the advantage of onsite consultation services and assistance with mutation detection projects. Drop your sample off by 4 PM, have your data by 10 AM the next business day for $5.50/sample. (Full plate pricing $5.00/sample.) Your data is archived and accessible anywhere via our secure ordering server. Pyrosequencing This Sequencing by Synthesis method is best used when you need to calculate the percentage of a particular polymorphism, whether that polymorphism is a complex set of SNPs, an indel region or a difference in methylation. Please consult us prior to planning an experiment, as appropriate assay design is required. 10 9
CORE SYMPOSIUM SEMINAR SCHEDULE Turner Concourse, May 24 th, 2016 West Room MRB-G01 MRB-G03 Tilghman Auditorium 9:30 a.m. 10:25 a.m. Thermo Fisher Scientific "Introduction to Real Time PCR" Zymo Research "Standardizing Microbiomics - Removing Bias from Sample Collection through Analyse" Bio-Rad "Ultra-Sensitive Quantification of Genome Editing Events Using Droplet Digital PCR" 10X Genomics A "Fully Integrated Solution for DNA and RNA Analysis 10:30 a.m. 11:25 a.m. Intergrated DNA Technologies "Increased efficiency of genome editing using the Alt-R CRISPR-Cas9 System Promega "New Techniques for Monitoring Protein Interations in Live Cells" KAPA Biosystems "Introducing the Kapa RNA Hyper Prep Kit and the HyperCap sequence capture workflow" Illumina "Solutions for Epigenetic Studies" Keynote Address 11:30 a.m. 12:25 p.m. Harry Dietz, M.D.. "Found in Translation: New Insights into the Pathogenesis and Treatmentof Marfan Syndrome and Related Disorders" 12:30 p.m. 1:25 p.m. QIAGEN "Liquid Biopsies:Generating sensitive, accurate, and reproducible results from cfdna" Agilent "NGS Target Enrichment Solutions for Exomes, TArgeted Panels and Beyond" Cell Signaling Technology "Analysis of Cellular Signaling Using Activation State Specific Antibodies" Sigma-Aldrich "CRISPR/Cas9 Genome Editing in Mammalian Systems " For more information visit us at grcf.jhmi.edu