Analýza genové exprese •Genome •Transcriptome •Proteome •DNA •mRNA •Proteins • • •Transcription •Translation • • • • Genomics Transcriptomics Proteomics GP_FA7TYN03040706_inv • • • •3 billion bases •20-30,000 •genes •~100,000 •proteins •Functional Genomics • • • Rozdíly v expresi genů mohou mít velký význam •Today known that humans and chimpanzees are 98.7% identical in their genome sequences and that large differences tissue-specific differences exist in expression – particular in the brain (Enard et al. 2002) • bush-chimp Analýza genové exprese (1) Kvantifikace kandidátních transkriptů (2) Microarrays (3) RNAseq (transkriptom) Kandidátní geny - relativní kvantifikace pomocí standardů •Měření úrovně exprese (např. v různých typech tkání nebo treatement vs. non-treatement atd. •mRNA → reverzní transkripce → cDNA → PCR •housekeeping geny – slouží jako standard pro měření •stejný počet kopií ve všech buňkách •exprimované ve všech buňkách, nezávislé na experimentu • A gene which is to be used as a loading control (or internal standard) should have various features - see slide. Srovnání množství PCR produktu na elektroforéze Studium kandidátních genů - qPCR §většinou nespecifická detekce (SYBR Green, aj.) §nutné srovnání s house-keeping geny https://www.youtube.com/watch?v=y8tHiH0BzGY We can plot the Ct values for the dilutions against concentration - the result is a linear graph. It should have an excellent correlation coefficient (certainly more than 0.990). Microarray transcriptomics Microarray analysis of transcriptome (~ specific DNA hybridization) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •Target (i.e. mix of transcripts in a form of cDNA = mRNA přepsaná do DNA reverzní transkriptázou, tj. neobsahuje introny) •Probe •(i.e. synthesized oligonucleotides complementary to particular genes) The GeneAtlas System Sledování exprese genů (microarrays) •Sledování exprese mnoha (tisíce) genů najednou •Založeno na hybridizaci •Sleduje se rozdíl vůči kontrole ("heterologous hybridization") = dvoukanálový experiment http://upload.wikimedia.org/wikipedia/commons/f/f2/Cdnaarray.jpg Vyhodnocení chipu – analýza obrazu (srovnání úrovně exprese mezi kontrolou a experimentem) http://upload.wikimedia.org/wikipedia/commons/0/0e/Microarray2.gif •Komerčně dostupné pro kompletní transkriptom cca 25 druhů •(další jsou rychle vyvíjeny, i na zakázku) •desítky tisíc transkriptů Commercial or custom microarrays Agilent Technologies http://www.affymetrix.com/fa/images/logo/affymetrix.png RNA-seq Differential gene expression (DE) studies in nonmodel species without the need for prior genomic resources Figure 2 •Todd et al. 2016 Adapted from Zeng and Mortazavi, Nature Immunology 13 (2012) •RNA-Seq workflow for gene expression analysis •Fragmented mRNA •List of differentially expressed genes RNA-Seq = sekvenování transkriptomu •Využití NGS • •RNA-Seq quantification •Musí být znám referenční transkriptom •(RPKM = reads per kilobase per million reads) RNA-seq commercially available •from 245 USD/sample RNAseq - review Box Fig 1 •Alvarez et al. 2015, Todd et al. 2016 Figure 1 •Question categories: (1) the extent and structure of natural variation, e.g. SNPs, (2) organismal response to stimulus and (3) the effect of differential gene expression on phenotype • Adapted from Zeng and Mortazavi, Nature Immunology 13 (2012) •RNA-Seq workflow for gene expression analysis •Fragmented mRNA •List of differentially expressed genes •gene ontology •identifikace funkce daného genu RNA-Seq = sekvenování transkriptomu • Gene ontology (http://geneontology.org/) = functional annotation analysis •založena na databázích dostupných anotovaných genů u modelových organismů •Cellular Component - the parts of a cell or its extracellular environment •Molecular Function - the elemental activities of a gene product at the molecular level, such as binding or catalysis •Biological Process - operations or sets of molecular events with a defined beginning and end, pertinent to the functioning of integrated living units: cells, tissues, organs, and organisms. Gene ontology as a graph diag-ontology-graph.gif •Example: A set of terms under the biological process node pigmentation. Microarrays vs. RNA-seq media/image1.jpeg •Měly by dávat stejné výsledky, ale srovnávacích studií je velmi málo Příklady Figure 1 Figure 2 Figure 3 • •20721 SNPs (ddRAD) – no genetic difference at neutral loci •Only differentially expressed genes are responsible for morphological changes (zobák, zbarvení) •Mason and Taylor 2015 http://www.biomedcentral.com/content/figures/1471-2164-15-754-1-l.jpg •cca 334 miliónů sekvencí („reads“); 42 mil./sample • •210 DEGs („differentially expressed genes“) – 119 up-regulated, 91 down-regulated u žlutých jedinců • • •xanthophory •melanophory •erythrophory Konzistence výsledků •změny v expresi jdou stejným směrem u RNA-seq i RT-qPCR vybraných genů • Functional annotation clustering (= gene ontology) •up-regulated in yellow •down-regulated in yellow •xanthophory u žlutých jedinců jsou asociovány s melanogenezí •v dalším kroku je možné studovat roli jednotlivých kandidátních genů • Transcriptional profiling of inbreeding depression and genetic erosion in Scabiosa columbaria: the balance between genetic drift and selection in the genetic erosion process. scabiosa1 Scabiosa%20cretica%20(mariposa)_jpg •Case study: Joop Ouborg et al. RU eng CMYK A4 •2009 cDNA library logo Ecogmomics •roots •shoots •inbred •outbred •small pop •large pop scabiosa1 Scabiosa columbaria •Example: figure9 macro •cDNA library preparation – 454 sequencing of transcriptome logo Ecogmomics Total number of reads: 528557 Number of contigs: 40302 logo Ecogmomics •In the next phase: Annotation of these 40.000+ ESTs („expressed sequence tags“) •Automated programs available, like BLAST2GO (http://www.blast2go.de/): • just feed a file with the ESTs into the program, and turn it on…… •1 week later you will have the results, being: •Homology with known sequences •Known function •The sequences may also be searched for: •EST-associated SSR markers: MISA (http://pgrc.ipk-gatersleben.de/misa/) •SNP markers: SNP-mining software like PolyBayes (http://genome.wustl.edu/tools/software/polybayes.cgi) •Again by using search software, freeware •ALMOST HALF OF GENES (ESTs) ARE UNKNOWN !!! logo Ecogmomics cDNA library logo Ecogmomics 530.000 sequences in one run, leading to ~ 40.000 ESTs •roots •shoots •inbred •outbred •small pop •large pop scabiosa1 Scabiosa columbaria •Example: microarray5S •1. Design of quantitative RealTime-PCR methods, based on EST sequences •2. Design of a Scabiosa specific microarray QPCR samples microarray4 logo Ecogmomics •Two methods of detecting transcripts cDNA library logo Ecogmomics 15k – 30k 60-mer microarrays 530.000 sequences in one run, leading to ~ 40.000 ESTs •roots •shoots •inbred •outbred •small pop •large pop scabiosa1 Scabiosa columbaria •Experiment: transcriptional profiling of inbreeding depression •Example: