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The development of a RNA-sequencing pipeline based on tuxedo tools. Martijn Derks Masoed Ramuz Nick Alberts Rico Hagelaar. Index . Dataset Pipeline 1 ( Tophat_cuff ) Pipeline 2 ( Cuff_diff ) Pipeline 3 (Summary) Conclusions Future prospects. Dataset.

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Martijn derks masoed ramuz nick alberts rico hagelaar

The development of a RNA-sequencing pipeline

based on tuxedo tools

Martijn Derks

Masoed Ramuz

Nick Alberts

Rico Hagelaar


Index
Index

  • Dataset

  • Pipeline 1 (Tophat_cuff)

  • Pipeline 2 (Cuff_diff)

  • Pipeline 3 (Summary)

  • Conclusions

  • Future prospects


Dataset
Dataset

  • Arabidopsis thaliana (advanced)

  • Six conditions:

    • Cold stress

    • Drought stress

    • Heat stress

    • Highlight stress

    • Salt stress

    • Control

      Ganet al. 2011. Multiple reference genomes and transcriptomes for Arabidopsis thaliana. Nature. 477, P 419–423.


Tophat cuff

Configuration file

Input data (FastQ)

Tophat_cuff

Tophat

(6x )

Bamfile

Cufflinks

Transcripts.gtf

Analysis

Total intron length

Basic for plot R

Transcript length


Tophat cuff results
Tophat_cuff results


Tophat cuff results1
Tophat_cuff results


Cuff diff 1
Cuff_diff (1)

(5x )

Control vs condition

Cuffmerge

transcript.gtf

Merged.gtf

Cuffdiff

Bamfile

DE-genes

Functions + enrichment


Cuff diff 2
Cuff_diff (2)

(5x )

uniprot

Get Functions

David

Enrichment


Cuff diff results uniprot
Cuff_diff results (Uniprot)

  • XLOC_005119 XLOC_005119 Hsp70b 1:5502205-5504535 WT_controlheat_stress OK 1.88554 4668.1 11.2736 -4.26394 2.00852e-05 0.00596

  • 568 yesQ9S9N1 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 DE genes/overlap


Cuff diff results david
Cuff_diff results (David)

Cold

Drought

Heat

Salt

Highlight


Summary
Summary

Summary

Tophat count

AT_codes

Csv maker

GC genes vs FPKM

CV

Expr. intron

Conservation

Overlap matrix

Clustering R

CV= STDEV/ Average




Heatmap clustering
Heatmap Clustering




Transcription factors
Transcription factors

Abscisic acid biosynthesis (stress conditions)

1

2

1. Cold, salinity and drought stresses: An overview

ShilpiMahajan

NarendraTuteja

2. Cold stress regulation of gene expression in plants

ViswanathanChinnusamy et al.


Conserved genes in arabidopsis
Conserved genes in Arabidopsis

  • Abiotic stress genes which also occur in Arabidopsis were retrieved from Oryzasativa (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
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. pp471-487.

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
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
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)



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