martijn derks masoed ramuz nick alberts rico hagelaar
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Martijn Derks Masoed RamuzNick AlbertsRico Hagelaar
The development of a RNA-sequencing pipeline based on tuxedo tools
Index • Dataset • Pipeline 1 (Tophat_cuff)• Pipeline 2 (Cuff_diff)• Pipeline 3 (Summary)• Conclusions • Future prospects
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.
Tophat_cuffInput data
(FastQ)
Tophat
Cufflinks
Bamfile
Transcripts.gtf
Analysis
Transcript length
Total intron length
Configuration file
Basic for plot R
(6x )
Tophat_cuff resultsCold Drought Heat highlight Salt WT
mapped 11.01M 10.63M 11.24M 10.96M 7.41M 20.11M
unmapped 23.90M 25.18M 21.82M 19.97M 24.30M 33.64M
percentage 31.5 29.7 34.0 35.4 23.4 37.4
Tophat_cuff results
Condition # genes FPKM > 1
Cold_stress 34029 20348
Drought_stress 35060 21044
Heat_stress 33615 19079
Highlight_stress 38480 22557
Salt_stress 33778 20111
Cuff_diff (1)Control vs condition
Cuffmerge
Cuffdiff
Merged.gtf
DE-genes
transcript.gtf
Bamfile
Functions + enrichment
(5x )
Cuff_diff (2)
Get Functions
uniprot
Enrichment
David
(5x )
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
Cuff_diff results DE genes/overlap
Cuff_diff results (David) HeatColdDroughtHighlight
Salt
Summary Summary
Tophat count AT_codes
Overlap matrix
Csv maker
CV
Clustering R
Expr. intron
Conservation
GC genes vs FPKM
ID Cold Drought Heat Highlight Salt WTAT1G01010 10.5501 12.0209 6.80685 0 10.7992 6.44518AT1G01030 2.51058 2.60705 0.582286 3.71439 1.37225 2.46655AT1G01046 0 0 0 6.40264 4.73081 0AT1G01050 52.7297 75.5912 0 46.9862 0 46.5351AT1G01070 13.6023 15.9691 0 7.52686 19.3891 23.0487AT1G01073 0 0 0 0 0 0AT1G01090 80.2276 80.5032 70.2176 58.4497 67.0227 102.39AT1G01110 0.966456 1.307 0.564864 1.26781 1.88932 2.65862
CV= STDEV/ Average
HC sample Clustering
HC gene Clustering
0.15
Heatmap Clustering
HC clusters (9)
PAM clusters (10)
Transcription factorsAbscisic acid biosynthesis (stress conditions)
1. Cold, salinity and drought stresses: An overviewShilpi MahajanNarendra Tuteja2. Cold stress regulation of gene expression in plantsViswanathan Chinnusamy et al.
1
2
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
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.
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
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)
Questions
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