The development of a RNA-sequencing pipeline
based on tuxedo tools
Martijn Derks
Masoed Ramuz
Nick Alberts
Rico Hagelaar
Index
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•
•
•
•
•
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_cuff
Input data
(FastQ)
Configuration
file
Tophat
(6x )
Bamfile
Cufflinks
Transcripts.gtf
Analysis
Total intron
length
Transcript
length
Basic for plot R
Tophat_cuff results
Cold
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)
(5x )
Control vs
condition
transcript.gtf
Cuffmerge
Merged.gtf
Bamfile
Cuffdiff
DE-genes
Functions +
enrichment
Cuff_diff (2)
uniprot
Get Functions
David
Enrichment
(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) Drought
Cold
Heat
Salt
Highlight
Summary
Summary
ID
Cold
Drought Heat
Highlight Salt
WT
AT1G01010
10.5501
12.0209 6.80685
6.44518
Tophat
count
AT_codes
Csv maker 0 10.7992
GC genes
vs FPKM
AT1G01030 2.51058 2.60705 0.582286 3.71439 1.37225 2.46655
AT1G01046
0
0
0 6.40264 4.73081
0
AT1G01050 52.7297 75.5912
0 CV46.9862
0 46.5351
Expr.
intron
AT1G01070 13.6023 15.9691
0 7.52686 19.3891 23.0487
AT1G01073
0
0
0
0
0
0
AT1G01090 80.2276
70.2176
58.4497
67.0227 102.39
Conservation
Overlap 80.5032
matrix
Clustering
R
AT1G01110 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 factors
Abscisic acid biosynthesis (stress conditions)
1
2
1. Cold, salinity and drought stresses:
An overview
Shilpi Mahajan
Narendra Tuteja
2. Cold stress regulation of gene
expression in plants
Viswanathan Chinnusamy et al.
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
• 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.
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.
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