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Expression Quantification (I) Mario Fasold, LIFE, IZBI This - PowerPoint PPT Presentation

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Leipziger Forschungszentrum fr Medizinische Fakultt Zivilisationserkrankungen Expression Quantification (I) Mario Fasold, LIFE, IZBI This publication is supported by LIFE Leipzig Research Center for Civilization Diseases, Universitt

  1. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen Expression Quantification (I) Mario Fasold, LIFE, IZBI This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  2. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen Sequencing Technology One Illumina HiSeq 2000 run produces 2 times (paired-end) • ca. 1,2 Billion reads • ca. 120 GB FASTQ file This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  3. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen RNA-seq protocol This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  4. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen Task • Obtain some “value” (estimate) representing the true abundance of transcripts in vivo This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  5. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen Overview 1. Basic Expression Measures 2. Statistical Poisson Model 1. Single Isoform 2. Multiple Isoform 3. Statistical Inference 4. Results 3. Paired-End Sequencing 4. Negative-Binomial This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  6. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen Expression Quantification • What properties should the expression values have? • Values should be comparable • Between transcripts • Between samples (-> differential expression) This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  7. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen Problems in Expression Quantification Differences in transcript length This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  8. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen Problems in Expression Quantification Fluctuations in sequencing depth This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  9. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen Basic Measure: RPKM RPKM/FPKM = Reads/Fragments per kilobase of exon model per million mapped reads x x = the number of reads mapped onto the gene's exons 9 FPKM 10 w = total number of reads in the experiment wl l = the sum of the exons in base pairs. This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  10. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  11. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen However, there are isoforms... This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  12. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen Typical number of isoforms 7% This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  13. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  14. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen Notation n g =2 f g,2 f g,1 This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  15. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen k f k f l f l f This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  16. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen f 1 f 1 f 2 f 2 f 1 f 1 f 2 f 1 Set of all isoforms in sample Total length of isoforms f 1 f 1 f 1 f 2 f 2 f 1 f 1 f 1 f 1 f 2 This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  17. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen f 1 f 1 f 2 f 2 f 1 f 1 f 2 f 1 f 1 f 1 f 1 f 2 f 2 f 1 f 1 f 1 f 1 f 2 Expression value Probablity of a read coming from isoform f This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  18. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen Model Assumptions • Reads sampled independently (-> unique reads) • Read sampling probability uniform over all sequences (uniform coverage) • Each transcript processed independently and then sequenced This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  19. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen Model (1) • Let w be total number of reads • Given isoform f and region of length l in f • Let X be a random variable representing the number of reads falling in that region • Then X~bin(w, Θ f l) This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  20. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen Coin Toss gives Binomial Dist’n This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  21. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  22. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen Poisson Model • Each gene g handled separately • Exons only shared as a whole – isoforms either share an exon, or they don’t • Let X~Poisson( ) be a random variable representing the number of reads falling in some region of interest in g This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  23. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen Poisson Model • Each gene g handled separately • Exons only shared as a whole – isoforms either share an exon, or they don’t • Let X~Poisson( ) be a random variable representing the number of reads falling in some region of interest in g • Then for some exon j: λ = • c ij is 1 if isoform i contains exon j, 0 otherwise a i is the “sampling rate” This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

  24. Leipziger Forschungszentrum für Medizinische Fakultät Zivilisationserkrankungen Estimation of expression This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. This project was funded by means of the European Social Fund and the Free State of Saxony.

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