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Tuxedo Package vs edgeR
RNA-seq Tuxedo Package vs edgeR
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RNA-seq Reads
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DEG Directly from Cuffdiff
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Fgf21 Gene Expression (Control Gene)
Tuxedo gene sample_1 sample_2 value_1 value_2 log2(fold_change) p_value q_value significant Fgf21 WP WA yes GP GA no AP 1.172 AA
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Why We Did Not Like The Tuxedo Package?
You can not make the comparison you wish (for example compare your groups to the WP) Series of t-test You can not split the effect of the drug from the effect of the strain Every time you try to repeat the mapping, it will take very long time (3 months to map the 30 samples after burning your computer) So we decided to moved to another pipeline (Deseq or edgeR)
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Every Group Compared to WP
Fdr<0.28
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Every Group Compared to its PBS Treated one
Fdr<0.28
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Strain Effect Fdr<0.28
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edgeR Problems Our control genes are no more significantly different
Some important pathways that were highlighted with the Tuxedo package had gone The heatmaps do not look good because the impressive difference between the genes had vanished The program depletes the differences by maximum dispersion strategy
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Fgf21 Gene Expression (Control Gene)
Tuxedo gene sample_1 sample_2 value_1 value_2 log2(fold_change) p_value q_value significant Fgf21 WP WA yes GP GA no AP 1.172 AA edgeR Symbol logFC logCPM P Value FDR WA-WP GP-WP GA-WP GA-GP AP-WP AA-WP AA-AP
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What we are doing to fix the problem
First- Move to Deseq: But this will increase our problem because this program is even more sensitive than edgeR. Here is a comparison..
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Why edgeR over the Deseq and others
Number of differentially expressed genes (FDR≤0.05) using three popular algorithms for RNA-seq Venn analysis of differentially expressed genes (FDR≤0.05) using three popular algorithms for RNA-seq analysis. (A) Up-regulated by Azacitidine. (B) Down-regulated by Azacitidine “This shows that overall, the three algorithms were very similar, although edgeR was the least conservative and baySeq was the most conservative”.
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What we are doing to fix the problem
Second- Fix edgeR by managing the program setting
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Fgf21 GCN2 Atf4 WP WA AP AA Atf4 mRNA Expression Fgf21 mRNA Expression
GP GA
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Every Group Compared to its PBS Treated one
Fdr<0.28
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Strain Effect Fdr<0.28
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GO Analysis Results GCN2KO effect ATF4KO effect GCN2KO + ATF4KO
Coagulation System Oxidative Phosphorylation Hepatic Fibrosis / Hepatic Stellate Cell Activation phagosome formation Mitochondrial Dysfunction VDR/RXR Activation NF-κB Signaling Complement System IGF-1 Signaling STAT3 Pathway PXR/RXR Activation PPARα/RXRα Activation Integrin Signaling Intrinsic Prothrombin Activation Pathway G-Protein Coupled Receptor Signaling Thrombin Signaling Glutathione-mediated Detoxification ERK/MAPK Signaling ERK5 Signaling LXR/RXR Activation AMPK Signaling D-myo-inositol-5-phosphate Metabolism Fatty Acid β-oxidation I Acute Phase Response Signaling p70S6K Signaling tRNA Charging Actin Cytoskeleton Signaling Fatty Acid β-oxidation III (Unsaturated, Odd Number) mTOR Signaling Protein Ubiquitination Pathway Hepatic Cholestasis phagosome maturation Complement and coagulation cascades TR/RXR Activation Focal adhesion FXR/RXR Activation cAMP-mediated signaling Cell Cycle: G2/M DNA Damage Checkpoint Regulation NRF2-mediated Oxidative Stress Response Xenobiotic Metabolism Signaling TNFR2 Signaling Cyclins and Cell Cycle Regulation TNFR1 Signaling p38 MAPK Signaling Death Receptor Signaling PPAR Signaling p53 Signaling PPAR signaling pathway Biosynthesis of unsaturated fatty acids Glutathione metabolism Metabolism of xenobiotics by cytochrome P450 Drug metabolism Adipocytokine signaling pathway Aminoacyl-tRNA biosynthesis
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ASN/ GCN2KO ASN/ ATF4KO ASN/ both strains
Leukocyte Extravasation Signaling EIF2 Signaling AMPK Signaling Macropinocytosis Signaling Superpathway of Cholesterol Biosynthesis Acute Phase Response Signaling Actin Cytoskeleton Signaling GADD45 Signaling Integrin Signaling IL-9 Signaling Cell Cycle Control of Chromosomal Replication TR/RXR Activation NF-κB Activation Cell Cycle: G1/S Checkpoint Regulation phagosome formation Mitochondrial Dysfunction Role of JAK2 in Hormone-like Cytokine Signaling Role of JAK1 and JAK3 in γc Cytokine Signaling eNOS Signaling Cholesterol Biosynthesis I p53 Signaling IL-15 Signaling Cholesterol Biosynthesis II (via 24,25-dihydrolanosterol) Hepatic Fibrosis / Hepatic Stellate Cell Activation Oxidative Phosphorylation Cholesterol Biosynthesis III (via Desmosterol) Natural Killer Cell Signaling Branched-chain α-keto acid Dehydrogenase Complex Erythropoietin Signaling Role of Tissue Factor in Cancer Glutathione-mediated Detoxification Thrombin Signaling Xenobiotic Metabolism Signaling Superpathway of Methionine Degradation Telomerase Signaling LXR/RXR Activation autophagy Assembly of RNA Polymerase I Complex NRF2-mediated Oxidative Stress Response D-myo-inositol (1,4,5)-trisphosphate Degradation Retinoate Biosynthesis I JAK/Stat Signaling iNOS Signaling Endoplasmic Reticulum Stress Pathway ERK/MAPK Signaling Dermatan Sulfate Biosynthesis (Late Stages) Cyclins and Cell Cycle Regulation NF-κB Signaling DNA damage-induced σ Signaling Apoptosis Signaling Thrombopoietin Signaling Chondroitin Sulfate Biosynthesis (Late Stages) Role of BRCA1 in DNA Damage Response PPAR Signaling Coagulation System Heme Biosynthesis II VDR/RXR Activation Oxidative phosphorylation STAT3 Pathway Unfolded protein response Regulation of actin cytoskeleton Triacylglycerol Degradation Protein Ubiquitination Pathway Focal adhesion PXR/RXR Activation Superpathway of Inositol Phosphate Compounds Phosphatidylinositol signaling system Folate Polyglutamylation G-Protein Coupled Receptor Signaling Jak-STAT signaling pathway FXR/RXR Activation mTOR Signaling Natural killer cell mediated cytotoxicity TNFR1 Signaling Chemokine Signaling Bile Acid Biosynthesis, Neutral Pathway FGF Signaling tRNA Splicing IGF-1 Signaling Fatty Acid α-oxidation p70S6K Signaling p38 MAPK Signaling PPARα/RXRα Activation p53 signaling pathway Death Receptor Signaling Adipogenesis pathway phagosome maturation Regulation of eIF4 and p70S6K Signaling tRNA Charging mTOR signaling pathway
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GCN2KO + ASN effect (Strain+Drug effect)
ATF4KO + ASN effect (Strain+Drug effect) Strain + Drug Effect in both VDR/RXR Activation EIF2 Signaling Hepatic Fibrosis / Hepatic Stellate Cell Activation RAR Activation Regulation of eIF4 and p70S6K Signaling LXR/RXR Activation Calcium Signaling mTOR Signaling p38 MAPK Signaling Integrin Signaling Oxidative Phosphorylation NF-κB Signaling Protein Kinase A Signaling Mitochondrial Dysfunction AMPK Signaling G-Protein Coupled Receptor Signaling Superpathway of Cholesterol Biosynthesis FXR/RXR Activation Guanosine Nucleotides Degradation III Cholesterol Biosynthesis I Acute Phase Response Signaling Actin Cytoskeleton Signaling Cholesterol Biosynthesis II (via 24,25-dihydrolanosterol) Death Receptor Signaling cAMP-mediated signaling Cholesterol Biosynthesis III (via Desmosterol) phagosome maturation phagosome formation Methionine Degradation I (to Homocysteine) FGF Signaling VEGF Signaling tRNA Charging Thrombin Signaling SAPK/JNK Signaling GADD45 Signaling ERK/MAPK Signaling Wnt/Ca+ pathway Cysteine Biosynthesis III (mammalia) Chemokine Signaling TR/RXR Activation Cell Cycle: G2/M DNA Damage Checkpoint Regulation Cell Cycle: G1/S Checkpoint Regulation Regulation of Actin-based Motility by Rho Heme Biosynthesis II PPAR Signaling Superpathway of Inositol Phosphate Compounds Tryptophan Degradation III (Eukaryotic) Adipogenesis pathway Production of Nitric Oxide and Reactive Oxygen Species in Macrophages NRF2-mediated Oxidative Stress Response Xenobiotic Metabolism Signaling Natural Killer Cell Signaling Apoptosis Signaling tRNA Splicing Calcium Transport I Triacylglycerol Biosynthesis Endoplasmic Reticulum Stress Pathway Hepatic Cholestasis Superpathway of Methionine Degradation MAPK signaling pathway TNFR1 Signaling DNA damage-induced σ Signaling Erythropoietin Signaling ERK5 Signaling Focal adhesion Tryptophan Degradation to 2-amino-3-carboxymuconate Semialdehyde Chemokine signaling pathway Protein Ubiquitination Pathway Lysosome Role of JAK2 in Hormone-like Cytokine Signaling TGF-beta signaling pathway Assembly of RNA Polymerase I Complex autophagy Ceramide Degradation Retinoate Biosynthesis I Retinol Biosynthesis Role of BRCA1 in DNA Damage Response DNA Methylation and Transcriptional Repression Signaling Phosphatidylcholine Biosynthesis I p53 Signaling BER pathway Cell Cycle Control of Chromosomal Replication Unfolded protein response Cyclins and Cell Cycle Regulation Tyrosine Degradation I p70S6K Signaling PPARα/RXRα Activation Glycogen Degradation III Glutamine Degradation I Histamine Degradation Isoleucine Degradation I Myc Mediated Apoptosis Signaling Ketolysis Glutathione-mediated Detoxification Glutamate Receptor Signaling Ribosome Oxidative phosphorylation Spliceosome RNA degradation Insulin signaling pathway Ubiquitin mediated proteolysis Aminoacyl-tRNA biosynthesis
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AAR and UPR Ddit3 Asns WP WA GP GA AP AA Eif4ebp1 mRNA Expression
Asns mRNA Expression
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WP WP WA WA Atf6b GP GP Bip GA GA AP AP AA AA
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UPR WP WA GP GA AP AA Spliced XBP1 /total
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WP WP WA WA GP Tnf GP Mt2 GA GA AP AP AA AA
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GP GA AP AA WP WA Sqstm1 GP GA AP AA WP WA Redd1
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Sesn2 Sesn2 Ulk1 ER Stress ASNase Autophagy P-eIF2 ATF4 Sesn2 p53 mTOR
GP GA AP AA WP WA Sesn2 Ulk1 ER Stress ASNase P-eIF2 ATF4 Sesn2 p53 mTOR Ulk1 Autophagy
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mTOR Signalling 4EBP1 P-mTOR WP WA GP GA AP AA mTOR P-p70 WP WA GP GA
P-Tsc2 Tsc2 WP WA GP GA AP AA 4EBP1 GAPDH
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Starvation Cytokines AKT inhibit autophagy
WP WA GP Trib3 GA AP AA
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