HIR Central Lab High End Instrument

Transcription

1 HIR Central Lab High End Instrument DR KOK GAN CHAN PhD, MSc, LLM(Merit), BSc, LLB, CBiol, MIBiol. High Impact Research Coordinator PhD, MSc, LLM(Merit), BSc, LLB, CBiol, MIBiol High Impact Research Building, Level 1 ISB (Genetics & Molecular Biology), Faculty of Science, University of Malaya Kuala Lumpur kokgan@um.edu.my

2 HIR Central Labs HIR Building Ground, 1 st & 2 nd floors

3 The rationale To equip UM-HIR grantees with state-of-theart instrument To support high impact research funded by HIR To promote high quality research To enable researchers to obtain high quality data To promote UM-HIR to overseas collaborators To establish UM-HIR as nucleus for high impact research, regionally and internationally

4 HIR Priority Areas 1. Genomics 2. Proteomics 3. Analytical chemistry 4. Metabolomics 5. Bioinformatics

5 Genomics HiSeq2500, G floor Whole-Genome Sequencing: sequence a whole human genome in a day (2x100bp reads). Or, in high output mode & sequence multiple genomes in a single run (2x150bp reads) 176 Gb in ~40h including on board cluster generation and sequencing, with 90.2% of bases at or above Q30, high quality alignment and variant calling. Targeted Resequencing: individual custom panels to whole exomes Whole-Transcriptome Sequencing: gene expression profiling to deep transcriptional analysis, HiSeq offers unprecedented RNA analysis solutions. Measure abundance of individual transcripts and isoforms, discover new genes, and identify regulatory non-coding RNAs. De Novo Sequencing: de novo sequencing of any size genome; NGS of metagenomic analyses Epigenetic Regulation: Study DNA-protein interactions and gene regulation using ChIP-Seq, and quantify DNA methylation with single base resolution.

6 PacBio SMRT, G floor The PacBio RS II sequencing technology resolves single molecules in real time, allowing observation of structural and cell type variation not accessible with other technologies. De Novo assembly: extra-long read lengths that simplify and improve genome assembly. Highest N50 Fewest contigs Detect structural variation % accuracy Genome finishing at 1/10th the cost Targeted sequencing: common use of targeted sequencing is single nucleotide polymorphism (SNP) detection and validation. SMRT directly measures individual molecules, using long reads to fully characterize genetic complexity, including rare SNPs, indels, structural variants, haplotypes and phasing. Single molecule resolution allows comprehensive characterization of heterogeneous samples and identification of variation invisible to multi-molecule sequencing technologies. Benefits: Compound Mutations and Haplotype Phasing - Multi-kilobase reads facilitate the study of linked mutations hundreds, even thousands, of bases apart. Repeat Expansions - Long reads and low bias allow accurate sequencing across repeat expansions, even in low complexity regions. Full-Length Transcripts and Splice Variants - Single-molecule resolution and long reads span entire cdnas, allowing full characterization of splicing in the transcriptome. Minor Variants and Quasispecies - Single molecule sequencing simplifies the analysis of mixed populations of sequences. Exquisitely sensitive and specific. SNP Detection and Validation - Single molecule sequencing detects and validates SNPs with high accuracy by avoiding mapping errors and systematic errors.

7 Base Modification Detection: DNA base modifications are important to the understanding of biological processes such as gene expression, host-pathogen interactions, DNA damage, and DNA repair. (SMRT) Analysis software works by measuring the rate of DNA base incorporation during sequencing. This information, automatically gathered during the sequencing process, is a unique feature of our SMRT technology. Resolve strand-specific modifications - unamplified double-stranded input DNA enables detection of strand-specific modifications such as hemimethylation Single-base resolution - visualize a wide variety of base modifications in highresolution Integrated sequence and chemical modification detection - epigenetic and other base modification data are gathered in the sequencing process, as an integral part of the sequencing workflow Hypothesis-free detection - no prior knowledge of the modification is needed, allowing discovery of unknown or unexpected modification

8 Other genomics facility DNA Microarray Nanostring qpcr (Quant Studio ABI, BioRad) digital droplet PCR Pyrosequencer Luminex Oxford Nanopore: NGS of >50kb?!?! More NGS in 2013 Liquid handling robot, DNA and library prep workflow

9 Single cell analysis and NGS Microfludic single cell analysis enabling profile of multiple single cells for the expression of hundreds of genes in just a few hours, using standard reagents and easy-to-use analysis tools enable hundreds of individual cells to be tested for the expression of hundreds of genes in a few hours. Advantages: High Sample Throughput Screen up to 96 single cells across 96 assays in one array. Produce More Results Faster Produce 9,216 data points in just four hours with one array. Assay Flexibility Practice tried-and-true techniques such as TaqMan assays or other common assays.

10 Microfludic single cell analysis Enabling: Measure expression levels of genes, alleles and spliced variants Compare tissue or cell type expression levels Map transcription initiation sites Characterize alternate splicing patterns Evaluate post-transcriptional activity Discover new transcripts and gene fusions

11 Proteomics 1D & 2D gel, Western blot, ELIZA FPLC Typhoon system Isothermal Titration Calorimeter (ITC): GE Healthcare s MicroCal (ITC) allows characterization of biomolecular interactions by providing direct, label-free measurements of binding affinity and thermodynamics. In a single ITC experiment, you can determine binding affinity (K D ), stoichiometry or number of binding sites, enthalpy (ΔH), and entropy (ΔS). Thermodynamic data, specifically ΔH and ΔS, reveal the forces that drive complex formation and mechanism of action. Thermodynamic measurements provide information on conformational changes, hydrogen bonding, hydrophobic interactions, and charge-charge interactions. provide valuable information about binding interactions, molecular stability, protein folding, and enzyme kinetics. Surface Plasmon Resonance

12 Mass spectrometry instrument UHPLC GC GC-MIDI GC-MS GC-Q-TOF LC-MSMS LC-Q-TOF Orbitrap mass spec (proteomics, lipidomics, metabolomics) LC-NMR-MS

13 Orbitrap mass spec protein and metabolite identification, characterization and quantitation.

14 Chromatography, Magnetic Resonance & Mass Spectrometry Unequivocal molecular structures confirmation via exact mass of the analyzed sample and access to the sum formula ideal for sensitive and instable compounds, fully automated analysis, or provide a very convenient, quick path from LC separation to NMR.

15 UV-VIS Spectrometry: Agilent Cary 60 Luminometer: fluorescence, luminecence, visible light Atomic emission spectrometer Flow cytometer Chemostat fermenter

16 OmniLog Bacteria, Yeast & Fungi ID Microbial Community Analysis: Biolog EcoPlates: provide a sensitive and reliable index of environmental change. Measure the metabolism of 31 carbon sources per assay Each assay panel tests in triplicate Simple colorimetric readout Readable with any microplate reader

17 Phenotype MicroArrays for Microbial Cells 1. make it possible to quantitatively measure thousands of cellular phenotypes all at once. 2. Correlate genotypes with phenotypes 3. Determine a cell's metabolic and chemical sensitivity properties 4. Discover new targets for antimicrobial compounds. 5. Optimize cell lines and culture conditions in bioprocess development. 6. Characterize cell phenotypes for taxonomic or epidemiological studies Important application: Testing Cell Lines Exposed to Drugs or Other Chemicals Evaluating New Drug Candidates Toxicological Testing Testing Cell Lines with Genetic Differences Determining Functions of Important Genes (Functional Genomics) Improvement of Cell Lines Cell Cultures for Phenotypic Stability Direct Testing of Cell Lines

18 Phenotype MicroArrays for Mammalian Cells (PMM) PMM assays are cell-based assays provided in five categories and used to determine up to 1,400 metabolic and chemical sensitivity phenotypes of mammalian cells. Monitor the stability of cell lines used in research. Use PMs as a tool to understand gene function. Perform other comparisons of cell lines in basic research and drug target studies. Use PMs as a tool to test drug leads Improve Bio Processes with PMs Perform cell proliferation and chemosensitivity assays

19 Photon camera EM-CCD technology performs best in low light conditions. How low? <10 photons per pixel Luminescence imaging, single fluorescent molecule in cells expressing fusion protein, confocal calcium ion imaging Capillary electrophoresis Nanodrop Bioanalyser Qubit

20 Bioinformatics Central Bioinformatics lab High speed server and high performance cluster computers 11,000 Human Genomes 13.3 mil bacteria genomes

21 Use of HIR Central Instrument No human intervention: all booking online Nominal charges for rental esp for UM users Packages for high volume users

22 Why HIR Central Equipment?

23 Proof of Using HIR Central Instrument to increase High Impact Publication Based on true story from myself

24

25

26 On This Day in History

27 University of Malaya The Premier University in Malaysia, >100 yrs

28 Instrument Before HIR

29 Thank you Dr Kok Gan CHAN