1. Martijn Derks Masoed Ramuz Nick Alberts Rico Hagelaar The development of a RNA-sequencing pipeline based on tuxedo tools

2. Index Dataset Pipeline 1 (Tophat_cuff) Pipeline 2 (Cuff_diff) Pipeline 3 (Summary) Conclusions Future prospects

3. Dataset Arabidopsis thaliana (advanced) Six conditions: Cold stress Drought stress Heat stress Highlight stress Salt stress Control Gan et al. 2011. Multiple reference genomes and transcriptomes for Arabidopsis thaliana. Nature. 477, P 419–423.

4. Tophat_cuff Input data (FastQ) Tophat Cufflinks Bamfile Transcripts.gtf Analysis Transcript length Total intron length Configuration file Basic for plot R (6x )

5. Tophat_cuff results ColdDroughtHeathighlightSaltWT mapped11.01M10.63M11.24M10.96M7.41M20.11M unmapped23.90M25.18M21.82M19.97M24.30M33.64M percentage31.529.734.035.423.437.4

6. Tophat_cuff results Condition# genesFPKM > 1 Cold_stress3402920348 Drought_stress3506021044 Heat_stress3361519079 Highlight_stress3848022557 Salt_stress3377820111

7. Cuff_diff (1) Control vs condition Cuffmerge Cuffdiff Merged.gtf DE-genes transcript.gtf Bamfile Functions + enrichment (5x )

8. Cuff_diff (2) Get Functions uniprot Enrichment David (5x )

9. Cuff_diff results (Uniprot) XLOC_005119 XLOC_005119 Hsp70b 1:5502205-5504535 WT_control heat_stress OK 1.88554 4668.1 11.2736 -4.26394 2.00852e-05 0.00596 568 yes Q9S9N1 Heat shock 70 kDa protein 5 (Heat shock protein 70-5) (AtHsp70-5) (Heat shock protein 70b) FUNCTION: In cooperation with other chaperones, Hsp70s stabilize preexistent proteins against aggregation and mediate the folding of newly translated polypeptides in the cytosol as well as within organelles. These chaperones participate in all these processes through their ability to recognize nonnative conformations of other proteins. They bind extended peptide segments with a net hydrophobic character exposed by polypeptides during translation and membrane translocation, or following stress-induced damage (By similarity). Cytopla sm. ATP binding; cell wall; chloroplast; plasma membrane; response to heat; response to virus GO:0005524; GO:0005618; GO:0009507; GO:0005886; GO:0009408; GO: 0009615

10. Cuff_diff results DE genes/overlap

11. Cuff_diff results (David) HeatColdDrought Highlight Salt

12. Summary Tophat countAT_codes Overlap matrix Csv maker CV Clustering R Expr. intron Conservation GC genes vs FPKM IDColdDroughtHeatHighlightSaltWT AT1G0101010.550112.02096.80685010.79926.44518 AT1G010302.510582.607050.5822863.714391.372252.46655 AT1G010460006.402644.730810 AT1G0105052.729775.5912046.9862046.5351 AT1G0107013.602315.969107.5268619.389123.0487 AT1G01073000000 AT1G0109080.227680.503270.217658.449767.0227102.39 AT1G011100.9664561.3070.5648641.267811.889322.65862 CV= STDEV/ Average

13. HC sample Clustering

14. HC gene Clustering 0.15

15. Heatmap Clustering

16. HC clusters (9)

17. PAM clusters (10)

18. Transcription factors Abscisic acid biosynthesis (stress conditions) 1. Cold, salinity and drought stresses: An overview Shilpi Mahajan Narendra Tuteja 2. Cold stress regulation of gene expression in plants Viswanathan ChinnusamyViswanathan Chinnusamy et al. 1 2

19. Conserved genes in Arabidopsis Abiotic stress genes which also occur in Arabidopsis were retrieved from Oryza sativa (Rabbani et al). These genes were compared with the DE stress genes found in the results. Three genes were found in the salt, cold and drought conditions. Rabbani, M.A. Maruyama, K. Abe, H. Khan, M. A. Katsura, K. Ito, Yoshiwara, K. Seki, M. Shinozaki, K. Yamaguchi-Shinozaki, K. 2003. Monitoring Expression Profiles of Rice Genes under Cold, Drought, and High-Salinity Stresses and Abscisic Acid Application Using cDNA Microarray and RNA Gel-Blot Analyses. Plant Physiology vol. 133. No 4. Pp 1755-1767

20. Literature overlap Seki, M. Narusaka, M. Ishida, J. Nanjo, T. Fujita, M. Oono, Y. Kamiya, A. Nakajima, M. Enju, A. Sakurai, T. Satou, M. Akiyama, K. Taji, T. Yamaguchi-Shinozaki, K. Carninci, P. Kawai, J. Hayashizaki, Y. Shinozaki, K. 2002. Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. V 31. I 3. pp 279-292. Baniwal, K. S. Bharti, K. Yu Chan, K. Fauth, M. Ganguli, A. Kotak, S. Mishra, S. K. Nover, L. Port, M. Scharf, K. Tripp, J. Weber, C. Zielinski, D. Koskull-Doring, P. 2004. Heat stress response in plants: a complex game with chaperones and more than twenty heat stress transcription factors. J Biosci. V 29. I 4. pp 471-487. Bartels, D. Nelson, D. 1994. Approaches to improve stress tolerance using molecular genetics. Plant, Cell and Environment. V 17. pp 659-667. Wang, W. Vinocur, B. Shoseyov, O. Altman, A. 2004. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. V 9. I 5. pp. 244-252. Results of the GO enrichment are backed up by the literature, with the exception of high light stress The crosstalk between drought, cold and salt stress was confirmed by the literature with a greater emphasis on drought and salt stress.

21. Conclusions Working pipeline for (Paired + Unpaired) RNAseq analysis DE genes + Gene Enrichment detection Cluster analysis CV genes Differential expressed genes identified (stress conditions vs. WT) Correlation Transcript length with FPKM Not found in Intron/GC percentage Clusters of Co-expressed genes Assumption of co-regulated genes

22. Future perspectives Use different IDs (TAIR IDs are not suitable) Transcription factors to cluster genes (similar regulatory elements? ) Conservation other plant species (synteny) Validation different dataset (organisms, paired end)

23. Questions