CPGR Services overview 2014

Transcription

1 CPGR Services overview 2014 Your gateway to Complete Genomics & Proteomics on the African continent!

2 SAMPLE PREPARATION Standardised protocols are available for: DNA isolation from Whole blood samples (fresh and stabilized) PBMC s Mammalian & human tissue Cell lines Plant tissue Saliva FFPE samples RNA isolation from Whole blood samples Blood, serum, PBMCs Cell lines Plant tissue Mammalian & human tissue Saliva FFPE samples mirna isolation from Plant tissue Blood, serum, PBMCs Cell lines Tissue culture supernatants Mammalian & human tissue Yeast Bacteria Depletion of blood, serum, plasma for itraq experiments Saliva FFPE samples Protein isolation from Plant tissue Blood, serum, Cell lines Tissue culture supernatants Mammalian & human tissue Yeast Bacteria Depletion of blood, serum, plasma for itraq experiments Whole blood fractions Isolation of PBMCs, serum and plasma from whole blood Whole genome Amplification of small amounts of DNA for downstream applications amplification 2 P a g e

3 GENOMICS Affymetrix cartridge arrays Assay Lab workflow Bioinformatics Output acgh Copy-number variation Copy number Analytical and LOH analysis on variation data QC high-density arrays Allele Specific report (SNP 6.0) LOH Analysis Gains and losses CHIP-on- Chip DNA methylation Exon expression Large-scale (to genome-wide) profiling of DNA (e.g. promoter) binding proteins Genome-wide detection of methylation patterns in the plant and animal kingdom Exon-specific RNA profiling for human, mouse and rat on high-density GeneChip arrays Support to be discussed with bioinformaticist Support to be discussed with bioinformaticist Gene level and alternative splicing analysis Analytical data QC Analytical data QC report Analytical Data QC Annotated gene lists for differentially expressed and alternatively spliced genes Gene expression Largest variety of eukaryotic and prokaryotic arrays mirna expression mirna expression analysis on high-density arrays Genotyping Genotyping / genome-wide association studies on SNP 6.0 arrays Data Quality assessment, normalization, data filtering, outlier identification. Differentially expressed annotated genes, clustering, gene set enrichment (GSE) analysis, classification Extract expressed mirna values Extract relevant species data Segmentation Analysis of detected segments Determination of abnormalities Analytical Data QC List of differentially expressed genes Analytical Data QC List of differentially expressed mirnas MicroRNA integration with gene expression Analytical Data QC Genotyping calls Annotated gains and losses 3 P a g e

4 DMET Plus Query 1,936 known ADME markers in a single GeneChip assay CytoScan HD Ultra-high density analysis of chromosomal aberrations, CNV and LOH Genotyping Call and copy number Generation of marker lists or pathways Genotyping, LOH, Copy number Copy number Analytical data QC Phenotype Star Allele Translation Analytical Data QC Genotyping Calls LOH Annotated Gains and Losses Phenotype Association Affymetrix GeneTitan arrays Assay Lab workflow Bioinformatics Output Gene expression profiling Genome-wide analysis and genotyping applications Exploration of gene expression profiles relating to important biological phenotypes such as disease or drug response in 16-, 24- and 96-well array formats Available for human, model and applied research organisms Population genetics, targeted genotyping (human) and markerassisted breeding (model and applied organisms) Custom arrays MyGeneChip Expression array plates for human and model and applied research organisms. Axiom mydesign Genotyping array plates genomic coverage tailored for human and agricultural populations, focussing on SNPs of interest Data Quality assessment, normalization, data filtering, outlier identification Differentially expressed annotated genes, clustering, gene set enrichment (GSE) analysis, classification SNP calls, single/multiple SNP association tests performed on allele, genotype, and dominant/recessive models To be discussed with bioinformaticist Analytical Data QC Annotated Gene lists Analytical Data QC Depending on application Analytical data QC report 4 P a g e

5 Next Generation Sequencing Assay Lab workflow Bioinformatics Output Microbial Genome Sequencing Whole genome de novo sequencing Whole genome sequencing Whole exome sequencing Sample Preparation (DNA) Library construction (single and paired end) High throughput sequencing on supported platforms viz. Ion Torrent, Roche 454 Junior, Illumina MiSeq Sample Preparation (DNA) Library construction (mate pair, single and paired end) High throughput sequencing on supported platforms viz. Roche 454 GS FLX, Illumina HiSeq 2000 and SOLiD 5500xl and for smaller genomes Illumina MiSeq Sample Preparation (DNA) Library construction (mate pair, single and paired end) High throughput sequencing on supported platforms viz. Roche 454 GS FLX, Illumina HiSeq 2000 and SOLiD 5500xl Sample Preparation (DNA) Experimental design support Read QC and data filtering Alignment to reference genome Consensus calling Annotation: ORF prediction, protein domain and sequence similarity search, gene ontology, metabolic pathway classification Experimental design support Read QC and data filtering Contig and scaffold building Assembly summary Customised annotation Experimental design support Read QC and data filtering Alignment to reference genome Consensus calling SNP calling, CNV, Indels, Quality scores and base coverage Genotyping Experimental design support Read QC and data Identification of structural features Data Visualisation Detailed analysis report EMBL files complete for ENSEMBL submission Raw sequence reads All data files Visualisation plots Custom analysis to address research question Detailed Analysis Data management Annotate known and novel variants Data files Visualisation plots Custom analysis to address research question Detailed Analysis Data management Identification of rare causal 5 P a g e

6 Whole transcriptome sequencing Sample enrichment Library construction (single and paired end) High throughput sequencing supported on the platforms Roche 454 GS FLX and Illumina HiSeq 2000 and SOLiD 5500xl Sample Preparation (RNA) Library construction (single and paired end) High throughput sequencing (Roche 454 GS FLX, Illumina HiSeq 2000, SOLiD 5500xl, Illumina MiSeq (small RNAseq) and Ion Torrent platforms) filtering Alignment to reference genome Consensus calling SNP calling, CNV, Indels, Quality scores and base coverage Genotyping Reference mapping De novo transcript assembly Isoform detection Quantification of transcripts SNP discovery Pathway Analysis Genotyping variants of Mendelian disorders Data files Visualisation plots Custom analysis to address research question Detailed Analysis Data management Expression profiling Co-expression clusters Variant detection Data files Visualisation Plots Detailed Analysis Data Management qrt-pcr Assay Lab workflow Bioinformatics Output Genotyping High-throughput (large sample over low gene number) TaqMan- based genotyping assays in 384 MTPs None Genotype clustering graphs to determine allelic discrimination qrt-pcr TLDA Compatibility High-throughput quantitative geneexpression profiling Pre-designed and custom TaqMan Low-Density Arrays (TLDA) for quantitative RNA profiling (e.g. cytokine expression) ABI, Superarray, Sybr Green, Roche qbaseplus (BioGazelle) qbaseplus (BioGazelle) Relative gene expression Relative gene expression Viral titres Determine titres of baculovirus stocks through Sybr Green assay None Relative abundance 6 P a g e

7 PROTEOMICS Mass spectrometry Instrumentation Q Exactive Thermo Fisher and Maldi Tof/Tof Absciex Assay Lab workflow Bioinformatics Output PMF Peptide Mass Fingerprinting Mascot Proteins (PMF) from samples digested either in solution or in gel slices. PMF confirmation can be provided via MS MS/MS analysis database searching identified Intact mass Intact protein masses can be determined for proteins and large peptides in the range m/z = A greater dynamic range is possible if the protein forms have multiple charges Sample Fractionation Simple and complex mixture protein identification Label based relative quantification Label free relative quantification Upstream fractionation of complex protein/peptide samples Samples fractionated by variety of methods including strong cation exchange chromatography, high ph reverse phase chromatography and 1 D SDS page Purified protein identification and identification of 100s to 1000s of proteins in complex whole tissue extractions Isobaric isotope labels that can be attached to peptides prior to LC- MS allowing relative (or absolute if an internal standard is available) quantitation of up to 8 protein samples Ideally suited for protein biomarker discovery with small sample sizes MS1 signal intensity used to quantify peptides/proteins Suited to biomarker discovery with large sample size (>8) None Molecular weight of intact proteins None Simplified fractions for MS analysis Mascot and Scaffold Power analysis to determine significant fold change threshold ProteinPilot & open source workflows Power analysis to determine significant fold change threshold Masoct, Sieve, Scaffold, open source Lists of identified proteins with confidence levels Relative abundance of proteins present in the sample Relative abundance of proteins present in the sample 7 P a g e

8 Phospho-proteome analysis Specific enrichment for phosphopeptides prior to LCMS Can be applied to both qualitative and quantitative analyses Data analysis In addition to the Mascot search algorithm data can also be analysed with the advanced ProteinPilot (AB SCiex) Paragon algorithm Data are also analysed using the Scaffold combining outputs into a single result output Protein QC/QA Quality control analysis for protein and peptide products can be developed to customers specifications. Protein purification Tailor made separation solutions can be developed according to customer needs. Protein extraction A variety of validated extraction protocols exists at the CPGR and new methods can be developed according to the customer s needs ProteinPilot & open source workflow s ProteinPilot & Scaffold Identity of phosphorylated proteins Identity of proteins and in the case of quantitative studies also relative abundance None Confirmation of sample integrity None Purified protein None Protein extracts suitable for MS analysis Protein microarrays Assay Lab workflow Bioinformatics Output Extract arrays Printing of crude or partially purified biological extracts for analysis of biological activity (e.g. antibody binding to immunogenic proteins) Protein arrays Arrays of purified single proteins in recombinant or natural form for protein-protein, protein-antibody or protein-molecule interaction Protein array printing Microarray hybridization Custom printing of a variety of protein array formats on a standard high- throughput QArray2 (Genetix) contact printer Standard protein hybridizations done in an automated processing and incubation station (Tecan HS 4800) in single, dual or quad format or in flexible 1-16 array-per slide formats (automated assays done in a Tecan HydroFlex) Scanning All scanning done in 4u microarray reader compatible with most existing microarray designs and formats as well as with arrays printed in 96 MTP None Printed microarrays suitable for use in capturing assays (e.g. antibody capturing) None Printed microarrays suitable for use in capturing assays (e.g. antibody capturing) None Printed microarrays suitable for use in capturing assays (e.g. antibody capturing) None Processed microarray slides ready for image acquisition Data acquisition and quality assessment TIFF files for data extraction and analysis 8 P a g e

9 formats Pin to pin and array to array normalisation Compatibility All Cy3 or Cy5 labelled analytes and detection antibodies; colorimetric assays and read-out also available BioPlex Assay Lab workflow Bioinformatics Output Antibody None assays Compatibility Multiplex bead-based sandwich immunoassays (typically 25 data-points per assay), e.g. for cytokine profiling BioRad, Luminex, Milliplex, R&D, Widescreen Relative determination of antigens in samples 9 P a g e

10 BIOINFORMATICS Analysis Supported platforms Workflows Data-analysis platforms Experimental design Microarray, protein array, itraq Study design recommendations such as replication, sample numbers, balancing and analysis considerations None RNA expression All existing microarray applications Data Quality assessment, normalization, data filtering, outlier identification; Differentially expressed annotated genes, clustering, gene set enrichment (GSE) analysis, classification Partek Genomics Suite SNP genotyping All existing microarrayand PCRbased applications SNP calls, single/multiple SNP association tests performed on allele, genotype, and dominant/recessive models. Partek Genomics Affymetrix console Qbase Copy-number variation (CNV) All suitable array-based applications Copy number variation (CNV) Allele Specific Copy Number LOH analysis Partek Genomics Affymetrix console Open source workflows Whole Genome Whole Exome Targeted DNA Sequencing Standard Sanger and highthroughput sequencers (454, SOLID, Solexa) Large-scale raw cdna sequence analysis, sequence clean-up, sequence contigs SignalP analysis, BLAST analysis De-novo sequencing, re-sequencing, targeted re-sequencing, micro-rna, RNA-seq, ChIp-Seq, gene expression Open source workflows Torrent Suite MiSec er Biological pathway analysis Cross-platform data integration Network relationship analysis Gene Ontology (GO) analysis, OMIM links, GEO Cross literature analysis from PubMed Biomarker discovery Ingenuity Pathway Analysis (IPA), Open source workflows 10 P a g e

11 CELL CULTURE Established cell culture assays (in single tube, 24- well and 96-well formats) Established screening assays All cell culture assays suitable for further indepth studies using Whole blood cell culture PBMCs HepG2 HepaRG All formats are suitable for stimulation as well as exposure (compound screening) assays. Please contact the CPGR for further information! Cytotoxicty by LDH & MTT Flow cytometry Genome-wide gene-/exon-expression analysis mirna expression analysis Genome-wide copy-number variation analysis Genome-wide methylation analysis qrt-pcr assays Luminex biomarker profiling Mass spectrometry based protein biomarker discovery RECOMBINANT PROTEIN EXPRESSION Baculovirus Expression System High-throughput sub-cloning into transfer vector containing expression purification tags Generation of recombinant bacmid DNA Generation of high titre viral stocks via transfection of insect cells with recombinant bacmid DNA Determination of virus titre using qpcr on the ABI 7900 Optimization of protein expression in insect cells in suspension Detection of protein expression with SDS-PAGE and Western Blot analysis Large scale protein expression and affinity purification QC of pure protein by Mass Spectrometry 11 P a g e

12 HUMAN CAPITAL DEVELOPMENT Internships Course/training type Description Outcome Technology Innovation Agency (TIA) Internship National Research Foundation (NRF) Internship Programmes The TIA internship programme is aimed at postgraduate MSc students training in bioinformatics, statistics, mathematics, biology or any relevant field The programme is eligible to South African citizens The National Research Foundation (NRF) internship programme targets undergraduate students specifically South African citizens Course/training type Description Outcome Mini Internship Programme (MIP) Knowledge Transfer Programme (KTP) Personal Development Programme (PDP) The MIP is a three months full time and six months part time programme aimed at postgraduate South African students. The pre-requisite to the programme is knowledge of a technology platform The Knowledge Transfer Programme (KTP) is an initiative to bring together experts and associates facilitated by a host institution to pursue specific projects with the aim of transferring knowledge in a natural and cost effective way. It targets postgraduates, postdoctoral and researchers The objective is to enable outstanding doctoral and post-doctorate graduates to further develop their research capabilities and to gain research experience, skills development in science and technology, attract and retain young scientists and professionals, support young scientists and professionals in basic and applied research In addition to the skills and knowledge acquired, at the end of the program interns are required to produce a technical report, poster and a presentation Interns are required to produce a technical report, poster and a presentation Production of a technical report, poster and presentation of the work done, knowledge and skills acquired is required The programme produces either a publication, patent or intellectual properties depending on the project. Industry ready post doctorate candidate, publications and patents 12 P a g e

13 Other training Course/training type Description Outcome Specialised Workshops Workshops in bioinformatics led by Training in cutting-edge omics international experts and targeted at life scientists, biologists, bioinformaticians, biostatisticians, physicists and computer scientists in academia and industry A typical workshop stretches over five technologies, tools/software/pipelines, methodologies, data analysis and advanced bioinformatics applications consecutive days. Theoretical knowledge and practical skills Certification awarded Online Training Free online training offered in Ingenuity Pathway Programme (IPA), available at System requirements: Internet connection, web browser, IPA registration Runs several times a year Wet-lab Training Wet-lab training is offered to biologists in the field of Genomics and Proteomics Courses are organised in collaboration with international experts or carried out by CPGR experts A variety of core and custom modules Live or pre-recorded Internationally recognized IPA certification awarded Software Licenses offered to scientists in South Africa Detailed knowledge of genomic and proteomic workflows, and practical skills Opportunity to work with your own samples and generate raw data sets 13 P a g e

14 ABOUT THE CPGR The CPGR is based on an initiative by the South African Department of Science and Technology (DST), and funded by the Technology Innovation Agency (TIA), to boost biotech innovation and to grow an internationally competitive bio-economy in South Africa. The organisation was created in 2006 with a vision of establishing a modern, world-class facility that serves the needs of the scientific community in South Africa by providing state-of-the-art services, technical expertise and collaborative research capabilities in the high throughput genomics and proteomics sectors. CONTACT Specific Area Genomics Proteomics Bioinformatics Human Capital Development (HCD) General Contact info@cpgr.org.za info@cpgr.org.za bioinformatics@cpgr.org.za hcd@cpgr.org.za info@cpgr.org.za General Upper Level, St Peter s Mall Cnr Anzio Road & Lower Main Road Observatory Cape Town 7925 South Africa tel: fax: www: blog: 14 P a g e

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