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