2016.05.19 KromTiD web powerpoint
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KromaTiD's directional genomic hybridization (dGH) platform uses single-stranded probes to discover, detect, and diagnose structural variants like inversions and translocations at high resolution on a cell-by-cell basis. This complements next-generation sequencing and microarrays. dGH assays can find inherited, spontaneous, and induced mutations. PinPoint FISH assays then allow targeted detection of variants identified by dGH. KromaTiD offers both research services using its techniques and collaboration with customer labs.
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Finally we’ll look at how genome editing is being applied to both basic and translational research, and in both a gene-specific and genome wide manner. For the study of disease associated genes and mutations scientists can now complement wide panels of tumour cells with genetically defined isogenic cell pairs identical in all but precise modifications in their gene of interest. The ease-of-design and efficiency of the CRISPR system is also being exploited for genome wide synthetic lethality screens, facilitating rapid drug target identification with significantly reduced risk of false negatives and off-target false positives. And again, further synergies are achieved when these approaches are combined to look for potential synthetic lethal targets in specific genomic contexts.
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AAV is a single-stranded, linear DNA virus with a a 4.7 kb genome which for the purpose of genome editing is replaced almost in entirety with the targeting vector sequence (except for the iTRs)
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Recent breakthroughs in genome editing technology have led to a rapid adoption that parallels that seen with RNAi. And like RNAi, these methods are taking the scientific world by storm, with high profile publications in fields as diverse as HIV treatment, stem cell therapy, food crop modification and drug development to name but a few.
Critically, the endogenous modification of genes enables the study of their function in a physiological context. It also overcomes some of the artefacts that can result from established techniques such as transgenesis and RNAi, which have mislead researchers with false positives or negatives. Until recently however genome editing required considerable technical expertise, and consequently was a relatively niche pursuit.
In this talk we will look at how the latest developments in genome editing tools have changed this, with improvements in both ease-of-use and targeting efficiency, as well as a concomitant reduction in costs opening up these approaches to the wider scientific community.
Rapid adoption of the CRISPR/Cas9 system has for example led to a long list of organisms and tissues in which genetic changes have been made with high efficiency. Other technologies such as recombinant adeno-associated virus (rAAV) offer further precision, stimulating the cell’s high-fidelity DNA repair pathways to insert exogenous sequence with unrivalled specificity. Targeting efficiency can be improved still further by using the technologies in combination – genome cutting induced by CRISPR can significantly enhance homologous recombination mediated by rAAV.
Despite these rapid advances, some pitfalls remain, and so we’ll discuss some of the key considerations for avoiding these, ranging from simply picking the right tool for the job to designing an experiment that maximises chances of success.
Finally we’ll look at how genome editing is being applied to both basic and translational research, and in both a gene-specific and genome wide manner. For the study of disease associated genes and mutations scientists can now complement wide panels of tumour cells with genetically defined isogenic cell pairs identical in all but precise modifications in their gene of interest. The ease-of-design and efficiency of the CRISPR system is also being exploited for genome wide synthetic lethality screens, facilitating rapid drug target identification with significantly reduced risk of false negatives and off-target false positives. And again, further synergies are achieved when these approaches are combined to look for potential synthetic lethal targets in specific genomic contexts.
Rewriting the Genome Using CRISPR and Synthetic Biology
The CRISPR/Cas9 system has emerged as one of the leading tools for modifying genomes of organisms ranging from E. coli to humans. Additionally, the simple gene targeting mechanism of CRISPR technology has been modified and adapted to other applications that include gene regulation, detection of intercellular trafficking, and pathogen detection. With a wealth of methods for introducing Cas9 and gRNAs into cells, it can be challenging to decide where to start. In this presentation, Dr Adam Clore describes the CRISPR mechanism and some of the most prominent uses for CRISPR, along with methods where IDT technologies can assist scientists in designing, testing, and executing a variety of CRISPR-mediated experiments. For more informaton, visit: http://www.idtdna.com/crispr
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We'll be discussing:
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However, CNV detection in single cells is faced with various challenges, such as incomplete genome coverage, introduction of sequence errors, GC bias and false positives.
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- The reference assembly is a model of the human genome built from many sequencing reads and is continually improved.
- Early assemblies had gaps and errors that have been improved on in newer releases. The current primary assembly is GRCh38.
- Alternate loci are now included to represent structural and haplotype variations not in the primary assembly.
- The reference assembly is important for mapping variants and interpreting genomic data.
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Dr. Chris Lowe presented on Horizon Discovery's precision genome editing platform called GENESISTM. The presentation discussed optimizing GENESISTM by combining CRISPR and rAAV technologies to improve gene targeting efficiency. Custom cell line development services are offered to modify genes of interest in various cell lines for applications such as generating disease models and studying drug sensitivity. Key considerations for successful gene editing experiments include factors like gene/cell line selection, gRNA design/activity, donor design, screening/validation approaches. Case studies demonstrated applications of engineered cell lines.
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2016.05.19 KromTiD web powerpoint
- 1. dGH™: KromaTiD’s precision cytogenetic platform for the discovery, detection, and diagnosis of driver mutations Visualizing Critical Genomic Data
- 2. An Essential Genomic Tool Metaphase 7q22CUX1 FISH Probe GREEN 5q31KIF20A FISH PROBE RED Interphase 7q22CUX1 FISH Probe GREEN 5q31KIF20A FISH PROBE RED NGS Excellent for finding SNPs, indels, some structural rearrangements Microarrays Excellent for finding SNPs, CNVs and unbalanced translocations Excellent at finding the full range of structural variants on a cell by cell basis including intra-chromosomal inversions and translocations Directional Genomic Hybridization KromaTiD dGH complements NGS & Microarrays in building a complete genomic toolbox
- 3. KromaTiD’s dGH Platform Discover; Detect; Diagnose directional Genomic Hybridization Full Genomic Profile
- 4. Directional Genomic Hybridization De Novo discovery of unknown rearrangements Normal 1 5 2 3 4 InversionDamaged 1 5 2 3 4 Normal: All DNA in proper orientation all probes bind to target chromatid Damaged: A segment of DNA is inverted Probes for inverted sequence appear on the opposite chromatid Target Chromatid Dark Chromatid Discovering small inversions and all typical rearrangements by examining chromatids not chromosomes Control
- 5. A Simple Method… Straightforward Metaphase Prep and Standard Imaging Pre-Replication G1 Chromosome 1) Selective Daughter Chromatid Degradation 2) Single-Strand dGH Probes 3) Imaging Inverted Segment Replicated Chromosome-Metaphase Configuration
- 6. A Widely Applicable Assay… Inherited (Developmental Disorders) All nucleated cells have one homolog with the same inversion Spontaneous (Cancers) A percentage of cells have recurrent inversion (the same location and size) mixed with normal cells Induced (Biodosimetry) Cells have random inversions, number, size, and the position vary Can Target Inherited, Spontaneous and Induced Mutations
- 8. Targeted Inversion Detection Inversion Normal D3FS1-HC10XX Chromosome 10 RET/PTC1 Specific 4 Probe Inversion Assay inv(10)(q11q21) dGH Targeted Assay
- 10. Translocation from painted chromatid dGH Discovery
- 11. Proprietary Chromosome Imaging Platform Broadest Assay Available • Detect sequence, location and orientation in a single test • Robust discovery and detection of DNA rearrangements • Cell by cell analysis-no pooled DNA Highest Performance • Free of repetitive sequences by design-no blocking required • Fully synthetic; consistent hybridization quality • KB Resolution of DNA rearrangements Robust and fully validatable assays for DNA rearrangements PinPoint FISH Assay Single cell analysis Routine and robust detection of inversions and translocations Translocation dGH Pinpoint FISH
- 12. Complimentary to NGS Tool Trans- location C3 to C4 Inversion C3-1 Inversion C3-2 Inversion C3-3 Inversion C3-4 C3 Deletion dGH ✓ ✓ ✓ ✓ ✓ mFISH ✓ Paired End Whole Genome Sequencing ✓ Independent re-Analysis of Sequencing Data ✓ Collaboration with Sequencing Center-of-Excellence Inversions and translocations were discovered by dGH and routinely detected in subsequent tests Results shown on next slide Multiple advanced NGS studies detected deletions, but missed all inversions and translocations with numerous false positives
- 13. Rearrangements Not Detected by NGS Visible with dGH Translocation with associated inversion Two small inversions Source location for translocation
- 14. Pinpoint FISH High resolution FISH assays using single stranded probes Designed using KromaTiD’s proprietary bioinformatics tools High Resolution By Design with KB Targets Metaphase 7q22CUX1 FISH Probe GREEN 5q31KIF20A FISH PROBE RED Interphase Performs in existing platforms and equipment Applicable in both interphase and metaphase cells 7q22CUX1 FISH Probe GREEN 5q31KIF20A FISH PROBE RED
- 15. Multi-Colored Metaphase Pinpoint FISH Assay with 10 kb Resolution Probe designed for 2 targets on Chromosome 3
- 16. Pinpoint FISH Performance Performance • High Resolution • Extraordinary signal-to-noise ratio • Excellent specificity • Very low background Easier to Use • Consistent results Flexible Design & Reliable Synthesis • Synthetic probes vs. cloned DNA • Detect smaller mutations and mutations with variable breakpoints KromaTiD synthetic FISH probes PinPoint FISH Assay Leading Competitor Pinpoint FISH Assay
- 17. Full Range of Products & Services Genome Screening for Structural Variants… • Targeted dGH assays for inversions and translocations • De novo & directed discovery from specific genomic loci to full genome …In your lab or ours • KromaTiD will perform research services from single samples to pilot projects to large libraries • KromaTiD will train your research team and provide reagents • Collaborations welcome Market validated products and services
- 18. For more information, please contact: Christopher Tompkins, PhD ctompkins@kromatid.com 303-819-5246
Editor's Notes
- KromaTiD sits in a complex market space. But, simply put advances in medicine are driven today by Genomic data and the leading tools on the market while fantastic cannot, without KromaTiD’s dGH, discover all of the causes of disease... …And this means to real patients...a fuller understanding of cancer and developmental disorders that wouldn’t be possible without dGH
- Our goal is to fit into your commercial systems, so we will use our tools to design assays that work with your existing…this is upside