CG920 Genomics Lecture 10 High throughput approaches Systems biology Kamil Růžička Functional genomics and proteomics of plants, Mendel Centre for Plant Genomics and Proteomics, Central European Institute of Technology (CEITEC), Masaryk University, Brno kamil.ruzicka@ceitec.muni.cz, www.ceitec.muni.cz  High throughput biology  Automation  Omics  Transcriptomics and high throughput transcriptomics  High throughput interactomics and how to read it  High throughput of anything  1000(+1) genomes, GWAS  ENCODE  Epigenenome and epitranscriptome  Little about Systems biology  Omics  Holism and modules  Gene regulation in E. coli Overview 2 3 Examples of automation in human history blacksmith manufacture assembly line robotic automation 4 High throughput sequencing gene genes genome genomes ecosystems? 5 Automation in transcriptomics qRT-PCR multichannel pipette bigger multichannel pipette pipetting robotmicroarraytranscriptome sequencing 6 insitu.fluitfly.org Fl(2)D gene in Drosophila embryos 7 emouseatlas.org KIAA1841 in mouse expressed in neurons 8 Protoplasting/cell sorting 9 http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi eFP browser 10 Yeast two-hybrid (Y2H) summary protein-protein interaction hunt 11 High throughput yeast two hybrid for various organisms (2000) (2005)(2009) 12 TAP purification affinity purification interaction hunt MALDI-TOF 13 So, far high throughput affinity purification approach slightly less popular thebiogrid.org - highly relevant for searching for interactors, but look also elsewhere! (2011) (2002) 14 Interactors of EMB2016 EMB2016 MTA-A FIP37 HAKAI Geert de Jaeger lab tandem affinity purification use databases if you have a conserved complex 15 EMB2016 interactors – RNA methylase EMB2016 MTA-B FIP37 HAKAI 37 Zhong et al. 2009 MTA-A – homolog of MTA RING finger/HAKAI was also shown to associate with splicing factors (human) 16 All guys back here when using MTA-A as bait (Immunoprecipitation) 17 EMB2016 FIP37 MTA-A/ MTA-B HAKAI Inferred protein complex 18 Inferred protein complex EMB2016 FIP37 MTA-A/ MTA-B HAKAI Flybase: EMB2016 interacts with HAKAI (no data on Biogrid) 19 20 You can order your mutant from the stock center signal.salk.edu the same for Drosophila, mouse, worm etc. 21 You can order your mutant from the stock center signal.salk.edu the same for Drosophila, mouse, worm etc.What to do if you cannot find insertion line for your gene? 22 • RNAi/amiRNA (can be also ordered) • CRISPR 23 You can order your cDNA clone from the stock center signal.salk.edu the same for yeast, Drosophila, mouse etc. 24 You can order even various constructs regarding your gene from stock centers signal.salk.edu even basic fusions (GFP, myc, TAP etc.) often ready for you 25 You can order antibodies against your protein Arabidopsis so far lagging – agrisera.com perhaps little bit. Rather commercial service. several human proteins providers: http://www.scbt.com/ www.acris-antibodies.com/ etc. - even get western and immunocytochemistry in advance 26 27 Phenoscope PHENOSCOPE: an automated largescale phenotyping platform Thisne et al. 2013 28 Phenoscope 29 Phenoscope • leaf area (camera) • photosynthesis (spectra) • weight • temperature (thermo camera) • in a dynamic manner • … • various ecotypes only, so far • commercially promising 30 GrowScreen-Root software Phenoscope – perhaps in future adaptation on other growth conditions 31 Check your phenotype online seedgenes.org – database of plant embryonic mutants (in-dept) http://rarge.psc.riken.jp/phenome/ - RIKEN Arabidopsis Phenome Information Database (kind of attempt on adult plant) 32 1000 genomes 1000 human genomes sequenced over the world wikipedia.org 33 1001 genomes - Arabidopsis http://1001genomes.org/ in both cases, much more lines already sequenced 34 How the ecotypes are collected Olivier Loudet web page 35 1001 genomes user interface several single nucleotide polymorphisms (SNP) in the selected gene Tü-WHO (DE) Caucasus Cent. Asia S. Africa 36 What could be natural variation good for? Quantitative trait loci (QTL) - nature makes genetic screen for you - QTL is analogous to gene in genetic screen 37 Genome wide association studies (GWAS) Slovak et al. 2014, Busch lab, Vienna 38 Genome wide association studies (GWAS) 163 accessions (ecotypes), several replicates (8 x 3) Slovak et al. 2014 searching for those different (say how different they might be!) 39 Genome wide association studies (GWAS) 163 accessions (ecotypes), several replicates (8 x 3) Slovak et al. 2014 searching for those different (e. g. root growth, slim root, resistant to exogenous treatment) 40 Genome wide association studies (GWAS) high p-value => SNP specifically in the “resistant” line (N-way ANOVA etc.) chromosome (locus) 41 Genome wide association studies (GWAS) =CaS cas-1 mutant has indeed shorter root Slovak et al. 2014 In contrast to human: - how to test it? 42 Genome wide association studies (GWAS) Manhattan plot by human 43 Status of cytosine methylations in various tissues can be explored in various tissues (human) Epigenetic modifications 44 How to find methylated bases in genome? Which bases are methylated? 45 How to sequence methylation of genome? bisulfite sequencing cytosine 5-methylcytosine uracil 46 What is methylation of cytosine good for? Are there other covalent modifications? 47 MeRIP – detecting adenine methylation on RNA >100 base modification detected in nucleic acids, incl. RNA N6-methyl adenosine most common in mRNA (0.5 – 5 % adenosines methylated) 48 Similar technique also adapted on DNA in C. elegans Greer et al. DNA Methylation on N6-Adenine in C. elegans, Cell 2015 49 The ENCODE project Is really only ~1 % human genome functional? The Encyclopedia of DNA Elements September 2012 1 % = gene coding regions 50 ENCODE – think big • 80 million dollars • 1,640 data sets • 147 cell types • Nature (6), Genome Biology (18), Genome Research (6 papers) 51 The ENCODE project Mainly cancer cells, lymphocytes etc. RNA transcribed regions: RNA-seq, CAGE, RNA-PET and manual annotation Protein-coding regions: mass spectrometry Transcription-factor-binding sites: ChIP-seq, DNase-seq Chromatin structure: DNase-seq, FAIRE-seq, histone ChIP-seq and MNase-seq DNA methylation sites: RRBS assay (cheaper version of bisulfite seq) 52 ENCODE - summary ~80 % genome associated with biochemical function: - enhancers, promoters - transcribed to non-coding RNA - 75 % genome transcribed, at least little bit - number of recognition sequences of DNA binding proteins doubled E. g. 75 % meaningful number? 53 ModENCODE on the way http://www.modencode.org/ Adult eclosion + several days Adult female Adult male Embryos 0-1, 0-2, 0-12, 10-12 hr etc Larvae in various instars Pupae in various stages Mated males or females etc. Drosophila tissue sources: 54 Question: where do you see the limits of high throughput biology? 55 Cons - sometimes low quality data or artifacts - occasionally data missing - biological material is quite complex - what to do with so many data? - where is the idea? 56 • next name for something between biology and chemistry? biochemistry -> proteomics molecular biology -> (functional) genomics • a real new concept? What is systems biology Inst. Plant Systems Biology, Gent, BE 57 “Multidimensional biology”  Genomics  Epigenomics  Transcriptomics  Epitranscriptomics  Translatomics / Proteomics  Metabolomics  Interactomics  Fluxomics  NeuroElectroDynamics  Phenomics  Biomics 58 Systems theory Forget about reductionism, think holistically. ὅλος [ho:'los] – greek. all, the whole, entire, complete 59 Reductionism vs. holism 60 Ludwig von Bertalanffy (1901-1972) 61 Omics-revolution shifts paradigm to large systems - Integrative bioinformatics - (Network) modeling 62 E. coli genome and proteome is small 63 Reductionism within holism Lets e.g. assume that transcription and translation is one module. 64 Conclusions – systems biology • computing capacities allow handling large data sets • fashionable • modelling whole cell processes in silico? • story frequently missing, there will be always question marks 65 Great web sites for organismal models http://www.yeastgenome.org/ http://www.pombase.org/ http://flybase.org/ http://www.wormbase.org/ http://www.arabidopsis.org/ S. cerevisiae S. pombe Drosophila C. elegans A. thaliana 66 Also nice web sites http://encodeproject.org/ http://www.thebiogrid.org/ http://www.genemania.org/ http://string-db.org/ …and many others …pay attention, if they are kept alive and curated 67 Additional literature  Venter, J.C. (2008). A life decoded: my genome, my life (London: Penguin).  Albert-László Barabási (2002) Linked. (Perseus Publishing)  http://www.youtube.com/watch?v=Z__BHVFP0Lk and further – excellent talks about systems biology from Uri Alon (Weizman Institute) – absolutely best  http://www.pnas.org/content/110/29/11952 (paper which challenges something conclusions in ENCODE) 68