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NGS data analysis Overview
- 1. Computational Biology and Genomics Facility, Indian Veterinary Research Institute Overview of NGS data analysis
- 2. Computational Biology and Genomics Facility, Indian Veterinary Research Institute Sequencing 1st Generation Sanger's Chain termination method (Irreversible method) 2nd Generation 3rd Generation Chemical Method (Maxam and Gilbert) Slab gel electrophoresis Capillary gel electrophoresis Sequencing by synthesis Sequencing by ligation Single Molecule Real Time Sequencing Pacific Bio RSII Pacific Bio Sequel Illumina (HiSeq, MiSeq,NextSeq) 454(GS-FLX) - Pyrosequencing Ion torrent, Ion Proton and Ion S5 SOLiD Sequencing Bridge PCR Emulsion PCR No amplification step 4th Generation Nanopore No amplification step Next generation sequencing platforms
- 3. Computational Biology and Genomics Facility, Indian Veterinary Research Institute Data analysis workflow Primary analysis Tertiary analysis • Image acquisition / semiconductor based detection • Base calling and • Quality metrics Secondary analysis • Sequence alignment • Sequence Stats • Consensus Calling • Sequence assembly • Application specific analysis • DNA-Seq • Re-Sequencing • De-novo Sequencing • ChiP Sequencing • RNA-Seq • Epigenetics Basics of RNA - Seq data analysis • RNA - Seq vs Microarray
- 4. Computational Biology and Genomics Facility, Indian Veterinary Research Institute Data formats Sequence formats Other sequence Visualisation formats Sequence alignment formats Data Processing FASTA FASTQ SRF SFF SCARF AB1 GCG IG EMBL SAM BAM CRAM WIG BED GFF GTF Data Formats
- 5. Computational Biology and Genomics Facility, Indian Veterinary Research Institute Data (Fastq) FASTQC Prinseqlite Trimming/filtering Data Filtering
- 6. Computational Biology and Genomics Facility, Indian Veterinary Research Institute Quality filtered/trimmed RNA-Seq Short reads FPKM based strategy Calculate transcript abundances (Cufflinks) Reference Genome (Y/N) Mapping to the reference (GMAP, Tophat,Bowtie,etc.) Y N De novo Transcriptome assembly (Trinity) Mapping and detection of DEGs (RSEM) Count based strategy Generate count data (RSEM) Detection of DEGs (cuffdiff2) Detection of DEGs (DESeq, edgeR, EBSeq) Identification of differentially expressed genes
- 7. Computational Biology and Genomics Facility, Indian Veterinary Research Institute Quality filtered/trimmed RNA-Seq Short reads FPKM based strategy Calculate transcript abundances (Cufflinks) Reference Genome Mapping to the reference (GMAP) Detection of DEGs (cuffdiff2) Downloading the reference genome and gtf from UCSC genome browser Identification of DEGs using Cufflinks
- 8. Computational Biology and Genomics Facility, Indian Veterinary Research Institute Quality filtered/trimmed RNA-Seq Short reads Calculate transcript abundances (RSEM) Reference Genome Mapping to the reference (Bowtie) Detection of DEGs (DESeq, edgeR,EBSeq) Downloading the reference genome and GTF from Ensembl genome browser Count based strategy Identification of DEGs using RSEM
- 9. Computational Biology and Genomics Facility, Indian Veterinary Research Institute Commonly differentially expressed genes DAVID, AmiGO2, g:Profiler, PROSITE, PRINTS, Pfam, ProDom, SMART, TIGRFAMs, PIR superfamily, SUPERFAMILY, Gene3D, PANTHER, BLAST2GO and HAMAP Differentially expressed genes identified by Cuffdiff, EBSeq,DESeq2 and edgeR Functional annotation Tools Functional annotation of DEGs
- 10. Computational Biology and Genomics Facility, Indian Veterinary Research Institute Comparative analysis A. 48 h B. 120 h C. Temporal analysis Signalling by NGF Integrin alphaIIb beta3 signaling GRB2:SOS provides linkage to MAPK signaling for Intergrins Signaling by ERBB4 Downstream Signaling Events Of B Cell Receptor (BCR) Signaling by FGFR in disease Toll Like Receptor TLR1:TLR2 Cascade Semaphorin interactions PI3K events in ERBB2 signaling Signaling by SCF−KIT Signaling by ERBB2 Sema3A PAK dependent Axon repulsion PI3K/AKT activation Golgi Cisternae Pericentriolar Stack Reorganization Signaling by FGFR PI−3K cascade NGF signalling via TRKA from the plasma membrane Downstream signal transduction Downstream signaling of activated FGFR Signaling by PDGF PI3K events in ERBB4 signaling Constitutive PI3K/AKT Signaling in Cancer PI3K/AKT Signaling in Cancer PIP3 activates AKT signaling GAB1 signalosome Role of LAT2/NTAL/LAB on calcium mobilization DAP12 signaling Signal regulatory protein (SIRP) family interactions Signaling by the B Cell Receptor (BCR) Response to elevated platelet cytosolic Ca2+ Platelet activation, signaling and aggregation Signaling by EGFR Antiviral mechanism by IFN−stimulated genes p75NTR signals via NF−kB p75 NTR receptor−mediated signalling TRIF−mediated programmed cell death TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling RIG−I/MDA5 mediated induction of IFN−alpha/beta pathways TRAF6 mediated IRF7 activation Toll Like Receptor 2 (TLR2) Cascade Advanced glycosylation endproduct receptor signaling NF−kB is activated and signals survival Cell death signalling via NRAGE, NRIF and NADE ZBP1(DAI) mediated induction of type I IFNs Toll Like Receptor TLR6:TLR2 Cascade Negative regulators of RIG−I/MDA5 signaling Toll Like Receptor 10 (TLR10) Cascade Activation of IRF3/IRF7 mediated by TBK1/IKK epsilon TRAF6 mediated induction of TAK1 complex TAK1 activates NFkB by phosphorylation and activation of IKKs complex Nucleotide−binding domain, leucine rich repeat containing receptor (NLR) signaling pathways RIP−mediated NFkB activation via ZBP1 Interleukin−1 signaling Toll Like Receptor 7/8 (TLR7/8) Cascade Toll Like Receptor 9 (TLR9) Cascade Signaling by Leptin Cellular Senescence Cytosolic sensors of pathogen−associated DNA Nuclear Events (kinase and transcription factor activation) ERK/MAPK targets Toll Like Receptor 5 (TLR5) Cascade p130Cas linkage to MAPK signaling for integrins CREB phosphorylation through the activation of Ras Thrombin signalling through proteinase activated receptors (PARs) MAP kinase activation in TLR cascade Translocation of ZAP−70 to Immunological synapse Cell−Cell communication MyD88 dependent cascade initiated on endosome MyD88 cascade initiated on plasma membrane Platelet Aggregation (Plug Formation) Activation of NMDA receptor upon glutamate binding and postsynaptic events Signalling to RAS Recycling pathway of L1 Post NMDA receptor activation events MyD88:Mal cascade initiated on plasma membrane FCERI mediated MAPK activation Axon guidance Developmental Biology Immune System Signaling by Interleukins SHC1 events in ERBB2 signaling FCGR activation Interferon alpha/beta signaling Interleukin−3, 5 and GM−CSF signaling Cytokine Signaling in Immune system NCAM signaling for neurite out−growth Activation of the AP−1 family of transcription factors Activated TLR4 signalling Frs2−mediated activation IKK complex recruitment mediated by RIP1 Interleukin−2 signaling Toll Like Receptor 4 (TLR4) Cascade Cellular responses to stress DEx/H−box helicases activate type I IFN and inflammatory cytokines production Senescence−Associated Secretory Phenotype (SASP) MAPK targets/ Nuclear events mediated by MAP kinases TRAF6 mediated NF−kB activation Signaling by EGFR in Cancer DAP12 interactions PECAM1 interactions Fc epsilon receptor (FCERI) signaling Extracellular matrix organization Platelet degranulation Hemostasis Costimulation by the CD28 family Integrin cell surface interactions Regulation of KIT signaling PD−1 signaling Immunoregulatory interactions between a Lymphoid and a non−Lymphoid cell MyD88−independent cascade Phospholipase C−mediated cascade FCERI mediated Ca+2 mobilization GPVI−mediated activation cascade PLCG1 events in ERBB2 signaling Signalling to ERKs Role of phospholipids in phagocytosis Regulation of actin dynamics for phagocytic cup formation Innate Immune System Growth hormone receptor signaling PLC−gamma1 signalling EGFR interacts with phospholipase C−gamma Downstream TCR signaling CD28 co−stimulation Signaling by FGFR1 mutants TCR signaling TRIF−mediated TLR3/TLR4 signaling Interferon gamma signaling Interferon Signaling ISG15 antiviral mechanism Toll Like Receptor 3 (TLR3) CascadePhosphorylation of CD3 and TCR zeta chains TRAF6 mediated induction of NFkB and MAP kinases upon TLR7/8 or 9 activation DAG and IP3 signaling Signaling by FGFR1 fusion mutants Generation of second messenger molecules Signaling by FGFR mutants Toll−Like Receptors CascadesFcgamma receptor (FCGR) dependent phagocytosis Cell surface interactions at the vascular wall Platelet sensitization by LDL Antigen Activates B Cell Receptor Leading to Generation of Second Messengers Prolonged ERK activation events Netrin−1 signaling Adaptive Immune System MyD88:Mal cascade initiated on plasma membrane Platelet activation, signaling and aggregation Platelet Aggregation (Plug Formation) Platelet degranulation TRAF6 mediated induction of NFkB and MAP kinases upon TLR7/8 or 9 activation Response to elevated platelet cytosolic Ca2+ Interferon gamma signaling Cytokine Signaling in Immune system Interferon Signaling ISG15 antiviral mechanism Antigen Activates B Cell Receptor Leading to Generation of Second Messengers Regulation of innate immune responses to cytosolic DNA mediated induction pathways cascade domain, leucine rich repeat containing receptor (NLR) signaling pathways TLR3/TLR4 signaling Toll Like Receptor 3 (TLR3) Cascade MAP kinase activation in TLR cascade MyD88 dependent cascade initiated on endosome Activation of IRF3/IRF7 mediated by TBK1/IKK epsilon Cascades Toll Like Receptor 9 (TLR9) Cascade Antiviral mechanism genes IKK complex recruitment mediated by RIP1 Regulation of KIT signaling programmed cell death Interferon alpha/beta signaling Activated TLR4 signalling Cytosolic sensors of DNA FCERI mediated Ca+2 mobilization TCR signaling Fc epsilon receptor (FCERI) signaling Fcgamma receptor (FCGR) dependent phagocytosis FCERI mediated MAPK activation Role of phospholipids in phagocytosis DAP12 signaling signaling Generation of second messenger molecules activated TAK1 mediates p38 MAPK activation Toll Like Receptor 4 (TLR4) Cascade Toll Like Receptor 2 (TLR2) Cascade signaling Toll Like Receptor TLR6:TLR2 Cascade Cell surface interactions at the vascular wall Integrin cell surface interactions Integrin alphaIIb beta3 signaling Signalling to RAS Regulation of actin dynamics for phagocytic cup formation Signal regulatory protein (SIRP) family interactions FCGR activation p130Cas linkage to MAPK signaling for integrins Signalling to ERKs GRB2:SOS provides linkage to MAPK signaling for Intergrins g NCAM signaling for Signaling by PECAM1 interactions Negative regulators signaling Toll Like Receptor TLR1:TLR2 Cascade Signaling by Interleukins TRAF6 mediated induction of TAK1 complex signaling Costimulation by the CD28 family Toll Like Receptor 7/8 (TLR7/8) Cascade Class I MHC mediated antigen processing & presentation Natural killer cell mediated cytotoxicity Chemokine receptors bind chemokines Systemic lupus erythematosus Measles Creation of C4 and C2 activators Cytosolic DNA−sensing pathway GPCR ligand binding Rheumatoid arthritis Type I diabetes mellitus Fc gamma R−mediated phagocytosis Allograft rejection Intestinal immune network for IgA production Class A/1 (Rhodopsin−like receptors) Cell adhesion molecules (CAMs) Phagosome G alpha (i) signalling events Chemokine signaling pathway Influenza A Antigen processing−Cross presentation Immune System Phagosomal maturation (early endosomal stage) Herpes simplex infection Autoimmune thyroid disease Prion diseases Peptide ligand−binding receptors Diabetes pathways NOD−like receptor signaling pathway Initial triggering of complement Complement cascade Adaptive Immune System Innate Immune System Jak−STAT signaling pathway Classical antibody−mediated complement activation RIG−I−like receptor signaling pathway Toll−like receptor signaling pathway C. Temporal analysis Comparative analysis A. 48 h B. 120 h C. Temporal analysis Signalling by NGF Integrin alphaIIb beta3 signaling GRB2:SOS provides linkage to MAPK signaling for Intergrins Signaling by ERBB4 Downstream Signaling Events Of B Cell Receptor (BCR) Signaling by FGFR in disease Toll Like Receptor TLR1:TLR2 Cascade Semaphorin interactions PI3K events in ERBB2 signaling Signaling by SCF−KIT Signaling by ERBB2 Sema3A PAK dependent Axon repulsion PI3K/AKT activation Golgi Cisternae Pericentriolar Stack Reorganization Signaling by FGFR PI−3K cascade NGF signalling via TRKA from the plasma membrane Downstream signal transduction Downstream signaling of activated FGFR Signaling by PDGF PI3K events in ERBB4 signaling Constitutive PI3K/AKT Signaling in Cancer PI3K/AKT Signaling in Cancer PIP3 activates AKT signaling GAB1 signalosome Role of LAT2/NTAL/LAB on calcium mobilization DAP12 signaling Signal regulatory protein (SIRP) family interactions Signaling by the B Cell Receptor (BCR) Response to elevated platelet cytosolic Ca2+ Platelet activation, signaling and aggregation Signaling by EGFR Antiviral mechanism by IFN−stimulated genes p75NTR signals via NF−kB p75 NTR receptor−mediated signalling TRIF−mediated programmed cell death TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling RIG−I/MDA5 mediated induction of IFN−alpha/beta pathways TRAF6 mediated IRF7 activation Toll Like Receptor 2 (TLR2) Cascade Advanced glycosylation endproduct receptor signaling NF−kB is activated and signals survival Cell death signalling via NRAGE, NRIF and NADE ZBP1(DAI) mediated induction of type I IFNs Toll Like Receptor TLR6:TLR2 Cascade Negative regulators of RIG−I/MDA5 signaling Toll Like Receptor 10 (TLR10) Cascade Activation of IRF3/IRF7 mediated by TBK1/IKK epsilon TRAF6 mediated induction of TAK1 complex TAK1 activates NFkB by phosphorylation and activation of IKKs complex Nucleotide−binding domain, leucine rich repeat containing receptor (NLR) signaling pathways RIP−mediated NFkB activation via ZBP1 Interleukin−1 signaling Toll Like Receptor 7/8 (TLR7/8) Cascade Toll Like Receptor 9 (TLR9) Cascade Signaling by Leptin Cellular Senescence Cytosolic sensors of pathogen−associated DNA Nuclear Events (kinase and transcription factor activation) ERK/MAPK targets Toll Like Receptor 5 (TLR5) Cascade p130Cas linkage to MAPK signaling for integrins CREB phosphorylation through the activation of Ras Thrombin signalling through proteinase activated receptors (PARs) MAP kinase activation in TLR cascade Translocation of ZAP−70 to Immunological synapse Cell−Cell communication MyD88 dependent cascade initiated on endosome MyD88 cascade initiated on plasma membrane Platelet Aggregation (Plug Formation) Activation of NMDA receptor upon glutamate binding and postsynaptic events Signalling to RAS Recycling pathway of L1 Post NMDA receptor activation events MyD88:Mal cascade initiated on plasma membrane FCERI mediated MAPK activation Axon guidance Developmental Biology Immune System Signaling by Interleukins SHC1 events in ERBB2 signaling FCGR activation Interferon alpha/beta signaling Interleukin−3, 5 and GM−CSF signaling Cytokine Signaling in Immune system NCAM signaling for neurite out−growth Activation of the AP−1 family of transcription factors Activated TLR4 signalling Frs2−mediated activation IKK complex recruitment mediated by RIP1 Interleukin−2 signaling Toll Like Receptor 4 (TLR4) Cascade Cellular responses to stress DEx/H−box helicases activate type I IFN and inflammatory cytokines production Senescence−Associated Secretory Phenotype (SASP) MAPK targets/ Nuclear events mediated by MAP kinases TRAF6 mediated NF−kB activation Signaling by EGFR in Cancer DAP12 interactions PECAM1 interactions Fc epsilon receptor (FCERI) signaling Extracellular matrix organization Platelet degranulation Hemostasis Costimulation by the CD28 family Integrin cell surface interactions Regulation of KIT signaling PD−1 signaling Immunoregulatory interactions between a Lymphoid and a non−Lymphoid cell MyD88−independent cascade Phospholipase C−mediated cascade FCERI mediated Ca+2 mobilization GPVI−mediated activation cascade PLCG1 events in ERBB2 signaling Signalling to ERKs Role of phospholipids in phagocytosis Regulation of actin dynamics for phagocytic cup formation Innate Immune System Growth hormone receptor signaling PLC−gamma1 signalling EGFR interacts with phospholipase C−gamma Downstream TCR signaling CD28 co−stimulation Signaling by FGFR1 mutants TCR signaling TRIF−mediated TLR3/TLR4 signaling Interferon gamma signaling Interferon Signaling ISG15 antiviral mechanism Toll Like Receptor 3 (TLR3) CascadePhosphorylation of CD3 and TCR zeta chains TRAF6 mediated induction of NFkB and MAP kinases upon TLR7/8 or 9 activation DAG and IP3 signaling Signaling by FGFR1 fusion mutants Generation of second messenger molecules Signaling by FGFR mutants Toll−Like Receptors CascadesFcgamma receptor (FCGR) dependent phagocytosis Cell surface interactions at the vascular wall Platelet sensitization by LDL Antigen Activates B Cell Receptor Leading to Generation of Second Messengers Prolonged ERK activation events Netrin−1 signaling Adaptive Immune System MyD88:Mal cascade initiated on plasma membrane Platelet activation, signaling and aggregation Platelet Aggregation (Plug Formation) Platelet degranulation TRAF6 mediated induction of NFkB and MAP kinases upon TLR7/8 or 9 activation Response to elevated platelet cytosolic Ca2+ Interferon gamma signaling Cytokine Signaling in Immune system Interferon Signaling ISG15 antiviral mechanism Antigen Activates B Cell Receptor Leading to Generation of Second Messengers Regulation of innate immune responses to cytosolic DNA mediated induction pathways cascade domain, leucine rich repeat containing receptor (NLR) signaling pathways TLR3/TLR4 signaling Toll Like Receptor 3 (TLR3) Cascade MAP kinase activation in TLR cascade MyD88 dependent cascade initiated on endosome Activation of IRF3/IRF7 mediated by TBK1/IKK epsilon Cascades Toll Like Receptor 9 (TLR9) Cascade Antiviral mechanism genes IKK complex recruitment mediated by RIP1 Regulation of KIT signaling programmed cell death Interferon alpha/beta signaling Activated TLR4 signalling Cytosolic sensors of DNA FCERI mediated Ca+2 mobilization TCR signaling Fc epsilon receptor (FCERI) signaling Fcgamma receptor (FCGR) dependent phagocytosis FCERI mediated MAPK activation Role of phospholipids in phagocytosis DAP12 signaling signaling Generation of second messenger molecules activated TAK1 mediates p38 MAPK activation Toll Like Receptor 4 (TLR4) Cascade Toll Like Receptor 2 (TLR2) Cascade signaling Toll Like Receptor TLR6:TLR2 Cascade Cell surface interactions at the vascular wall Integrin cell surface interactions Integrin alphaIIb beta3 signaling Signalling to RAS Regulation of actin dynamics for phagocytic cup formation Signal regulatory protein (SIRP) family interactions FCGR activation p130Cas linkage to MAPK signaling for integrins Signalling to ERKs GRB2:SOS provides linkage to MAPK signaling for Intergrins g NCAM signaling for Signaling by PECAM1 interactions Negative regulators signaling Toll Like Receptor TLR1:TLR2 Cascade Signaling by Interleukins TRAF6 mediated induction of TAK1 complex signaling Costimulation by the CD28 family Toll Like Receptor 7/8 (TLR7/8) Cascade Class I MHC mediated antigen processing & presentation Natural killer cell mediated cytotoxicity Chemokine receptors bind chemokines Systemic lupus erythematosus Measles Creation of C4 and C2 activators Cytosolic DNA−sensing pathway GPCR ligand binding Rheumatoid arthritis Type I diabetes mellitus Fc gamma R−mediated phagocytosis Allograft rejection Intestinal immune network for IgA production Class A/1 (Rhodopsin−like receptors) Cell adhesion molecules (CAMs) Phagosome G alpha (i) signalling events Chemokine signaling pathway Influenza A Antigen processing−Cross presentation Immune System Phagosomal maturation (early endosomal stage) Herpes simplex infection Autoimmune thyroid disease Prion diseases Peptide ligand−binding receptors Diabetes pathways NOD−like receptor signaling pathway Initial triggering of complement Complement cascade Adaptive Immune System Innate Immune System Jak−STAT signaling pathway Classical antibody−mediated complement activation RIG−I−like receptor signaling pathway Toll−like receptor signaling pathway C. Temporal analysis 2. Gene enrichment analysis - ClueGo analysis
- 11. Computational Biology and Genomics Facility, Indian Veterinary Research Institute C. Temporal analysis Signalling by NGF Integrin alphaIIb beta3 signaling GRB2:SOS provides linkage to MAPK signaling for Intergrins Signaling by ERBB4 Downstream Signaling Events Of B Cell Receptor (BCR) Signaling by FGFR in disease Toll Like Receptor TLR1:TLR2 Cascade Semaphorin interactions PI3K events in ERBB2 signaling Signaling by SCF−KIT Signaling by ERBB2 Sema3A PAK dependent Axon repulsion PI3K/AKT activation Golgi Cisternae Pericentriolar Stack Reorganization Signaling by FGFR PI−3K cascade NGF signalling via TRKA from the plasma membrane Downstream signal transduction Downstream signaling of activated FGFR Signaling by PDGF PI3K events in ERBB4 signaling Constitutive PI3K/AKT Signaling in Cancer PI3K/AKT Signaling in Cancer PIP3 activates AKT signaling GAB1 signalosome Role of LAT2/NTAL/LAB on calcium mobilization DAP12 signaling Signal regulatory protein (SIRP) family interactions Signaling by the B Cell Receptor (BCR) Response to elevated platelet cytosolic Ca2+ Platelet activation, signaling and aggregation Signaling by EGFR Antiviral mechanism by IFN−stimulated genes p75NTR signals via NF−kB p75 NTR receptor−mediated signalling TRIF−mediated programmed cell death TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling RIG−I/MDA5 mediated induction of IFN−alpha/beta pathways TRAF6 mediated IRF7 activation Toll Like Receptor 2 (TLR2) Cascade Advanced glycosylation endproduct receptor signaling NF−kB is activated and signals survival Cell death signalling via NRAGE, NRIF and NADE ZBP1(DAI) mediated induction of type I IFNs Toll Like Receptor TLR6:TLR2 Cascade Negative regulators of RIG−I/MDA5 signaling Toll Like Receptor 10 (TLR10) Cascade Activation of IRF3/IRF7 mediated by TBK1/IKK epsilon TRAF6 mediated induction of TAK1 complex TAK1 activates NFkB by phosphorylation and activation of IKKs complex Nucleotide−binding domain, leucine rich repeat containing receptor (NLR) signaling pathways RIP−mediated NFkB activation via ZBP1 Interleukin−1 signaling Toll Like Receptor 7/8 (TLR7/8) Cascade Toll Like Receptor 9 (TLR9) Cascade Signaling by Leptin Cellular Senescence Cytosolic sensors of pathogen−associated DNA Nuclear Events (kinase and transcription factor activation) ERK/MAPK targets Toll Like Receptor 5 (TLR5) Cascade p130Cas linkage to MAPK signaling for integrins CREB phosphorylation through the activation of Ras Thrombin signalling through proteinase activated receptors (PARs) MAP kinase activation in TLR cascade Translocation of ZAP−70 to Immunological synapse Cell−Cell communication MyD88 dependent cascade initiated on endosome MyD88 cascade initiated on plasma membrane Platelet Aggregation (Plug Formation) Activation of NMDA receptor upon glutamate binding and postsynaptic events Signalling to RAS Recycling pathway of L1 Post NMDA receptor activation events MyD88:Mal cascade initiated on plasma membrane FCERI mediated MAPK activation Axon guidance Developmental Biology Immune System Signaling by Interleukins SHC1 events in ERBB2 signaling FCGR activation Interferon alpha/beta signaling Interleukin−3, 5 and GM−CSF signaling Cytokine Signaling in Immune system NCAM signaling for neurite out−growth Activation of the AP−1 family of transcription factors Activated TLR4 signalling Frs2−mediated activation IKK complex recruitment mediated by RIP1 Interleukin−2 signaling Toll Like Receptor 4 (TLR4) Cascade Cellular responses to stress DEx/H−box helicases activate type I IFN and inflammatory cytokines production Senescence−Associated Secretory Phenotype (SASP) MAPK targets/ Nuclear events mediated by MAP kinases TRAF6 mediated NF−kB activation Signaling by EGFR in Cancer DAP12 interactions PECAM1 interactions Fc epsilon receptor (FCERI) signaling Extracellular matrix organization Platelet degranulation Hemostasis Costimulation by the CD28 family Integrin cell surface interactions Regulation of KIT signaling PD−1 signaling Immunoregulatory interactions between a Lymphoid and a non−Lymphoid cell MyD88−independent cascade Phospholipase C−mediated cascade FCERI mediated Ca+2 mobilization GPVI−mediated activation cascade PLCG1 events in ERBB2 signaling Signalling to ERKs Role of phospholipids in phagocytosis Regulation of actin dynamics for phagocytic cup formation Innate Immune System Growth hormone receptor signaling PLC−gamma1 signalling EGFR interacts with phospholipase C−gamma Downstream TCR signaling CD28 co−stimulation Signaling by FGFR1 mutants TCR signaling TRIF−mediated TLR3/TLR4 signaling Interferon gamma signaling Interferon Signaling ISG15 antiviral mechanism Toll Like Receptor 3 (TLR3) CascadePhosphorylation of CD3 and TCR zeta chains TRAF6 mediated induction of NFkB and MAP kinases upon TLR7/8 or 9 activation DAG and IP3 signaling Signaling by FGFR1 fusion mutants Generation of second messenger molecules Signaling by FGFR mutants Toll−Like Receptors CascadesFcgamma receptor (FCGR) dependent phagocytosis Cell surface interactions at the vascular wall Platelet sensitization by LDL Antigen Activates B Cell Receptor Leading to Generation of Second Messengers Prolonged ERK activation events Netrin−1 signaling Adaptive Immune System C. Temporal RIG-I/MDA5 pathways, NF-kB activation, ISG15 antiviral signaling, 48 h p.i.
- 12. Computational Biology and Genomics Facility, Indian Veterinary Research Institute Comparative analysis A. 48 h B. 120 h C. Temporal analysis Signalling by NGF Integrin alphaIIb beta3 signaling GRB2:SOS provides linkage to MAPK signaling for Intergrins Signaling by ERBB4 Downstream Signaling Events Of B Cell Receptor (BCR) Signaling by FGFR in disease Toll Like Receptor TLR1:TLR2 Cascade Semaphorin interactions PI3K events in ERBB2 signaling Signaling by SCF−KIT Signaling by ERBB2 Sema3A PAK dependent Axon repulsion PI3K/AKT activation Golgi Cisternae Pericentriolar Stack Reorganization Signaling by FGFR PI−3K cascade NGF signalling via TRKA from the plasma membrane Downstream signal transduction Downstream signaling of activated FGFR Signaling by PDGF PI3K events in ERBB4 signaling Constitutive PI3K/AKT Signaling in Cancer PI3K/AKT Signaling in Cancer PIP3 activates AKT signaling GAB1 signalosome Role of LAT2/NTAL/LAB on calcium mobilization DAP12 signaling Signal regulatory protein (SIRP) family interactions Signaling by the B Cell Receptor (BCR) Response to elevated platelet cytosolic Ca2+ Platelet activation, signaling and aggregation Signaling by EGFR Antiviral mechanism by IFN−stimulated genes p75NTR signals via NF−kB p75 NTR receptor−mediated signalling TRIF−mediated programmed cell death TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling RIG−I/MDA5 mediated induction of IFN−alpha/beta pathways TRAF6 mediated IRF7 activation Toll Like Receptor 2 (TLR2) Cascade Advanced glycosylation endproduct receptor signaling NF−kB is activated and signals survival Cell death signalling via NRAGE, NRIF and NADE ZBP1(DAI) mediated induction of type I IFNs Toll Like Receptor TLR6:TLR2 Cascade Negative regulators of RIG−I/MDA5 signaling Toll Like Receptor 10 (TLR10) Cascade Activation of IRF3/IRF7 mediated by TBK1/IKK epsilon TRAF6 mediated induction of TAK1 complex TAK1 activates NFkB by phosphorylation and activation of IKKs complex Nucleotide−binding domain, leucine rich repeat containing receptor (NLR) signaling pathways RIP−mediated NFkB activation via ZBP1 Interleukin−1 signaling Toll Like Receptor 7/8 (TLR7/8) Cascade Toll Like Receptor 9 (TLR9) Cascade Signaling by Leptin Cellular Senescence Cytosolic sensors of pathogen−associated DNA Nuclear Events (kinase and transcription factor activation) ERK/MAPK targets Toll Like Receptor 5 (TLR5) Cascade p130Cas linkage to MAPK signaling for integrins CREB phosphorylation through the activation of Ras Thrombin signalling through proteinase activated receptors (PARs) MAP kinase activation in TLR cascade Translocation of ZAP−70 to Immunological synapse Cell−Cell communication MyD88 dependent cascade initiated on endosome MyD88 cascade initiated on plasma membrane Platelet Aggregation (Plug Formation) Activation of NMDA receptor upon glutamate binding and postsynaptic events Signalling to RAS Recycling pathway of L1 Post NMDA receptor activation events MyD88:Mal cascade initiated on plasma membrane FCERI mediated MAPK activation Axon guidance Developmental Biology Immune System Signaling by Interleukins SHC1 events in ERBB2 signaling FCGR activation Interferon alpha/beta signaling Interleukin−3, 5 and GM−CSF signaling Cytokine Signaling in Immune system NCAM signaling for neurite out−growth Activation of the AP−1 family of transcription factors Activated TLR4 signalling Frs2−mediated activation IKK complex recruitment mediated by RIP1 Interleukin−2 signaling Toll Like Receptor 4 (TLR4) Cascade Cellular responses to stress DEx/H−box helicases activate type I IFN and inflammatory cytokines production Senescence−Associated Secretory Phenotype (SASP) MAPK targets/ Nuclear events mediated by MAP kinases TRAF6 mediated NF−kB activation Signaling by EGFR in Cancer DAP12 interactions PECAM1 interactions Fc epsilon receptor (FCERI) signaling Extracellular matrix organization Platelet degranulation Hemostasis Costimulation by the CD28 family Integrin cell surface interactions Regulation of KIT signaling PD−1 signaling Immunoregulatory interactions between a Lymphoid and a non−Lymphoid cell MyD88−independent cascade Phospholipase C−mediated cascade FCERI mediated Ca+2 mobilization GPVI−mediated activation cascade PLCG1 events in ERBB2 signaling Signalling to ERKs Role of phospholipids in phagocytosis Regulation of actin dynamics for phagocytic cup formation Innate Immune System Growth hormone receptor signaling PLC−gamma1 signalling EGFR interacts with phospholipase C−gamma Downstream TCR signaling CD28 co−stimulation Signaling by FGFR1 mutants TCR signaling TRIF−mediated TLR3/TLR4 signaling Interferon gamma signaling Interferon Signaling ISG15 antiviral mechanism Toll Like Receptor 3 (TLR3) CascadePhosphorylation of CD3 and TCR zeta chains TRAF6 mediated induction of NFkB and MAP kinases upon TLR7/8 or 9 activation DAG and IP3 signaling Signaling by FGFR1 fusion mutants Generation of second messenger molecules Signaling by FGFR mutants Toll−Like Receptors CascadesFcgamma receptor (FCGR) dependent phagocytosis Cell surface interactions at the vascular wall Platelet sensitization by LDL Antigen Activates B Cell Receptor Leading to Generation of Second Messengers Prolonged ERK activation events Netrin−1 signaling Adaptive Immune System MyD88:Mal cascade initiated on plasma membrane Platelet activation, signaling and aggregation Platelet Aggregation (Plug Formation) Platelet degranulation TRAF6 mediated induction of NFkB and MAP kinases upon TLR7/8 or 9 activation Response to elevated platelet cytosolic Ca2+ Interferon gamma signaling Cytokine Signaling in Immune system Interferon Signaling ISG15 antiviral mechanism Antigen Activates B Cell Receptor Leading to Generation of Second Messengers Regulation of innate immune responses to cytosolic DNA mediated induction pathways cascade domain, leucine rich repeat containing receptor (NLR) signaling pathways TLR3/TLR4 signaling Toll Like Receptor 3 (TLR3) Cascade MAP kinase activation in TLR cascade MyD88 dependent cascade initiated on endosome Activation of IRF3/IRF7 mediated by TBK1/IKK epsilon Cascades Toll Like Receptor 9 (TLR9) Cascade Antiviral mechanism genes IKK complex recruitment mediated by RIP1 Regulation of KIT signaling programmed cell death Interferon alpha/beta signaling Activated TLR4 signalling Cytosolic sensors of DNA FCERI mediated Ca+2 mobilization TCR signaling Fc epsilon receptor (FCERI) signaling Fcgamma receptor (FCGR) dependent phagocytosis FCERI mediated MAPK activation Role of phospholipids in phagocytosis DAP12 signaling signaling Generation of second messenger molecules activated TAK1 mediates p38 MAPK activation Toll Like Receptor 4 (TLR4) Cascade Toll Like Receptor 2 (TLR2) Cascade signaling Toll Like Receptor TLR6:TLR2 Cascade Cell surface interactions at the vascular wall Integrin cell surface interactions Integrin alphaIIb beta3 signaling Signalling to RAS Regulation of actin dynamics for phagocytic cup formation Signal regulatory protein (SIRP) family interactions FCGR activation p130Cas linkage to MAPK signaling for integrins Signalling to ERKs GRB2:SOS provides linkage to MAPK signaling for Intergrins g NCAM signaling for Signaling by PECAM1 interactions Negative regulators signaling Toll Like Receptor TLR1:TLR2 Cascade Signaling by Interleukins TRAF6 mediated induction of TAK1 complex signaling Costimulation by the CD28 family Toll Like Receptor 7/8 (TLR7/8) Cascade Class I MHC mediated antigen processing & presentation Natural killer cell mediated cytotoxicity Chemokine receptors bind chemokines Systemic lupus erythematosus Measles Creation of C4 and C2 activators Cytosolic DNA−sensing pathway GPCR ligand binding Rheumatoid arthritis Type I diabetes mellitus Fc gamma R−mediated phagocytosis Allograft rejection Intestinal immune network for IgA production Class A/1 (Rhodopsin−like receptors) Cell adhesion molecules (CAMs) Phagosome G alpha (i) signalling events Chemokine signaling pathway Influenza A Antigen processing−Cross presentation Immune System Phagosomal maturation (early endosomal stage) Herpes simplex infection Autoimmune thyroid disease Prion diseases Peptide ligand−binding receptors Diabetes pathways NOD−like receptor signaling pathway Initial triggering of complement Complement cascade Adaptive Immune System Innate Immune System Jak−STAT signaling pathway Classical antibody−mediated complement activation RIG−I−like receptor signaling pathway Toll−like receptor signaling pathway C. Temporal analysis Comparative analysis A. 48 h B. 120 h C. Temporal analysis Signalling by NGF Integrin alphaIIb beta3 signaling GRB2:SOS provides linkage to MAPK signaling for Intergrins Signaling by ERBB4 Downstream Signaling Events Of B Cell Receptor (BCR) Signaling by FGFR in disease Toll Like Receptor TLR1:TLR2 Cascade Semaphorin interactions PI3K events in ERBB2 signaling Signaling by SCF−KIT Signaling by ERBB2 Sema3A PAK dependent Axon repulsion PI3K/AKT activation Golgi Cisternae Pericentriolar Stack Reorganization Signaling by FGFR PI−3K cascade NGF signalling via TRKA from the plasma membrane Downstream signal transduction Downstream signaling of activated FGFR Signaling by PDGF PI3K events in ERBB4 signaling Constitutive PI3K/AKT Signaling in Cancer PI3K/AKT Signaling in Cancer PIP3 activates AKT signaling GAB1 signalosome Role of LAT2/NTAL/LAB on calcium mobilization DAP12 signaling Signal regulatory protein (SIRP) family interactions Signaling by the B Cell Receptor (BCR) Response to elevated platelet cytosolic Ca2+ Platelet activation, signaling and aggregation Signaling by EGFR Antiviral mechanism by IFN−stimulated genes p75NTR signals via NF−kB p75 NTR receptor−mediated signalling TRIF−mediated programmed cell death TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling RIG−I/MDA5 mediated induction of IFN−alpha/beta pathways TRAF6 mediated IRF7 activation Toll Like Receptor 2 (TLR2) Cascade Advanced glycosylation endproduct receptor signaling NF−kB is activated and signals survival Cell death signalling via NRAGE, NRIF and NADE ZBP1(DAI) mediated induction of type I IFNs Toll Like Receptor TLR6:TLR2 Cascade Negative regulators of RIG−I/MDA5 signaling Toll Like Receptor 10 (TLR10) Cascade Activation of IRF3/IRF7 mediated by TBK1/IKK epsilon TRAF6 mediated induction of TAK1 complex TAK1 activates NFkB by phosphorylation and activation of IKKs complex Nucleotide−binding domain, leucine rich repeat containing receptor (NLR) signaling pathways RIP−mediated NFkB activation via ZBP1 Interleukin−1 signaling Toll Like Receptor 7/8 (TLR7/8) Cascade Toll Like Receptor 9 (TLR9) Cascade Signaling by Leptin Cellular Senescence Cytosolic sensors of pathogen−associated DNA Nuclear Events (kinase and transcription factor activation) ERK/MAPK targets Toll Like Receptor 5 (TLR5) Cascade p130Cas linkage to MAPK signaling for integrins CREB phosphorylation through the activation of Ras Thrombin signalling through proteinase activated receptors (PARs) MAP kinase activation in TLR cascade Translocation of ZAP−70 to Immunological synapse Cell−Cell communication MyD88 dependent cascade initiated on endosome MyD88 cascade initiated on plasma membrane Platelet Aggregation (Plug Formation) Activation of NMDA receptor upon glutamate binding and postsynaptic events Signalling to RAS Recycling pathway of L1 Post NMDA receptor activation events MyD88:Mal cascade initiated on plasma membrane FCERI mediated MAPK activation Axon guidance Developmental Biology Immune System Signaling by Interleukins SHC1 events in ERBB2 signaling FCGR activation Interferon alpha/beta signaling Interleukin−3, 5 and GM−CSF signaling Cytokine Signaling in Immune system NCAM signaling for neurite out−growth Activation of the AP−1 family of transcription factors Activated TLR4 signalling Frs2−mediated activation IKK complex recruitment mediated by RIP1 Interleukin−2 signaling Toll Like Receptor 4 (TLR4) Cascade Cellular responses to stress DEx/H−box helicases activate type I IFN and inflammatory cytokines production Senescence−Associated Secretory Phenotype (SASP) MAPK targets/ Nuclear events mediated by MAP kinases TRAF6 mediated NF−kB activation Signaling by EGFR in Cancer DAP12 interactions PECAM1 interactions Fc epsilon receptor (FCERI) signaling Extracellular matrix organization Platelet degranulation Hemostasis Costimulation by the CD28 family Integrin cell surface interactions Regulation of KIT signaling PD−1 signaling Immunoregulatory interactions between a Lymphoid and a non−Lymphoid cell MyD88−independent cascade Phospholipase C−mediated cascade FCERI mediated Ca+2 mobilization GPVI−mediated activation cascade PLCG1 events in ERBB2 signaling Signalling to ERKs Role of phospholipids in phagocytosis Regulation of actin dynamics for phagocytic cup formation Innate Immune System Growth hormone receptor signaling PLC−gamma1 signalling EGFR interacts with phospholipase C−gamma Downstream TCR signaling CD28 co−stimulation Signaling by FGFR1 mutants TCR signaling TRIF−mediated TLR3/TLR4 signaling Interferon gamma signaling Interferon Signaling ISG15 antiviral mechanism Toll Like Receptor 3 (TLR3) CascadePhosphorylation of CD3 and TCR zeta chains TRAF6 mediated induction of NFkB and MAP kinases upon TLR7/8 or 9 activation DAG and IP3 signaling Signaling by FGFR1 fusion mutants Generation of second messenger molecules Signaling by FGFR mutants Toll−Like Receptors CascadesFcgamma receptor (FCGR) dependent phagocytosis Cell surface interactions at the vascular wall Platelet sensitization by LDL Antigen Activates B Cell Receptor Leading to Generation of Second Messengers Prolonged ERK activation events Netrin−1 signaling Adaptive Immune System MyD88:Mal cascade initiated on plasma membrane Platelet activation, signaling and aggregation Platelet Aggregation (Plug Formation) Platelet degranulation TRAF6 mediated induction of NFkB and MAP kinases upon TLR7/8 or 9 activation Response to elevated platelet cytosolic Ca2+ Interferon gamma signaling Cytokine Signaling in Immune system Interferon Signaling ISG15 antiviral mechanism Antigen Activates B Cell Receptor Leading to Generation of Second Messengers Regulation of innate immune responses to cytosolic DNA mediated induction pathways cascade domain, leucine rich repeat containing receptor (NLR) signaling pathways TLR3/TLR4 signaling Toll Like Receptor 3 (TLR3) Cascade MAP kinase activation in TLR cascade MyD88 dependent cascade initiated on endosome Activation of IRF3/IRF7 mediated by TBK1/IKK epsilon Cascades Toll Like Receptor 9 (TLR9) Cascade Antiviral mechanism genes IKK complex recruitment mediated by RIP1 Regulation of KIT signaling programmed cell death Interferon alpha/beta signaling Activated TLR4 signalling Cytosolic sensors of DNA FCERI mediated Ca+2 mobilization TCR signaling Fc epsilon receptor (FCERI) signaling Fcgamma receptor (FCGR) dependent phagocytosis FCERI mediated MAPK activation Role of phospholipids in phagocytosis DAP12 signaling signaling Generation of second messenger molecules activated TAK1 mediates p38 MAPK activation Toll Like Receptor 4 (TLR4) Cascade Toll Like Receptor 2 (TLR2) Cascade signaling Toll Like Receptor TLR6:TLR2 Cascade Cell surface interactions at the vascular wall Integrin cell surface interactions Integrin alphaIIb beta3 signaling Signalling to RAS Regulation of actin dynamics for phagocytic cup formation Signal regulatory protein (SIRP) family interactions FCGR activation p130Cas linkage to MAPK signaling for integrins Signalling to ERKs GRB2:SOS provides linkage to MAPK signaling for Intergrins g NCAM signaling for Signaling by PECAM1 interactions Negative regulators signaling Toll Like Receptor TLR1:TLR2 Cascade Signaling by Interleukins TRAF6 mediated induction of TAK1 complex signaling Costimulation by the CD28 family Toll Like Receptor 7/8 (TLR7/8) Cascade Class I MHC mediated antigen processing & presentation Natural killer cell mediated cytotoxicity Chemokine receptors bind chemokines Systemic lupus erythematosus Measles Creation of C4 and C2 activators Cytosolic DNA−sensing pathway GPCR ligand binding Rheumatoid arthritis Type I diabetes mellitus Fc gamma R−mediated phagocytosis Allograft rejection Intestinal immune network for IgA production Class A/1 (Rhodopsin−like receptors) Cell adhesion molecules (CAMs) Phagosome G alpha (i) signalling events Chemokine signaling pathway Influenza A Antigen processing−Cross presentation Immune System Phagosomal maturation (early endosomal stage) Herpes simplex infection Autoimmune thyroid disease Prion diseases Peptide ligand−binding receptors Diabetes pathways NOD−like receptor signaling pathway Initial triggering of complement Complement cascade Adaptive Immune System Innate Immune System Jak−STAT signaling pathway Classical antibody−mediated complement activation RIG−I−like receptor signaling pathway Toll−like receptor signaling pathway C. Temporal analysis
- 13. Computational Biology and Genomics Facility, Indian Veterinary Research Institute Class I MHC mediated antigen processing & presentation Natural killer cell mediated cytotoxicity Chemokine receptors bind chemokines Systemic lupus erythematosus Measles Creation of C4 and C2 activators Cytosolic DNA−sensing pathway GPCR ligand binding Rheumatoid arthritis Type I diabetes mellitus Fc gamma R−mediated phagocytosis Allograft rejection Intestinal immune network for IgA production Class A/1 (Rhodopsin−like receptors) Cell adhesion molecules (CAMs) Phagosome G alpha (i) signalling events Chemokine signaling pathway Influenza A Antigen processing−Cross presentation Immune System Phagosomal maturation (early endosomal stage) Herpes simplex infection Autoimmune thyroid disease Prion diseases Peptide ligand−binding receptors Diabetes pathways NOD−like receptor signaling pathway Initial triggering of complement Complement cascade Adaptive Immune System Innate Immune System Jak−STAT signaling pathway Classical antibody−mediated complement activation RIG−I−like receptor signaling pathway Toll−like receptor signaling pathway TLR signaling pathway, RIG signaling pathway, chemokine signaling pathway, Class-I MHC presentation and processing, and classical antibody mediated complement activation Temporal analysis
- 14. Computational Biology and Genomics Facility, Indian Veterinary Research Institute Commonly differentially expressed genes - Orthologs in humans Representing the network in the Cytoscape Protein - protein interactions among the DEGs Filtering the genes on degree and fold change BioGRID intreractions - well defined in Humans Protein - protein interactions among the filtered DEGs Protein - Protein Interaction network (Cytoscape)
- 15. Computational Biology and Genomics Facility, Indian Veterinary Research Institute CDH2 PHKG2 ANXA1 TUBB4B LGALS1 ACSL3 SH3BP4 FKBP4 GSN YES1 FLOT2 TPM1 RRADSRXN1 RPL36 TPM2 MYH10 CD4 VIM PF4 IGF2BP2 FN1 TUBB2B GAPDH EVL LTA EIF2AK2 HP HSP90AA1 CD63 CD8A SLC3A2 ENO2 VKORC1 PROCR CCR5 VCAM1 CORO1B RPS28 ACTA1 RPS3A ACTA2 VAMP8 RPL10A DBN1 ARRB2 LCK PLD2 F2R CSF1R SYK ERBB3 EGR1 CAT PTPN6 CTSB KIT KEAP1 PPARG CCND1 SPI1 SIAH2 TAGLN IKBKE MAPRE1 STX1A SNAPIN ISG15 LEF1OTUB2 HDAC11 SMAD7 CALM2 PTPRC GADD45BAPOE ALDOC PRC1 CBX5 PML CCNE1 MAPK11 CCND2 CHEK2 E2F1 CA2 USP42 MAP2K3 DSTN IFIT5 LAMB1 SORD ME1 IFIT3 GCLM UBD PSTPIP1 EPN1 CDK1 TRIM25SERPINH1 AIFM1 USP21 HERC5 MX1 NQO1 EEF1G SEC61B MCM5 CD2 HSPD1 PPP2CB GADD45A AMOT OFD1 RRM2 BAG3 RCN2 MAP1B SPP1 AURKA PLK3 DNAJB1 THRA SRC SF3B14 HSPA5 ARRB1 NGFR DNM1 CEBPG UCHL1 BATF ATF3 COA1 MLLT3 CDK5 F5 MITF ECT2 CDC25B TRIB3 SIRT1 FLNC HSPA2 HSPA1L USP50 HR FER BTK PLAU ENC1 STAT5A PLAT KRT18 SSBP1 HSPA8 HMGA1 CHEK1 HMGB1 ANXA4 H2AFX RGS2 KANK2 SFPQ DBP CAV1 HES1 HSPA1A CDK18 MED26 FOSL1 LDOC1 JUN BATF2 FOS ZBTB16 ERLIN2 IKZF1 CKS2 DYNC1I1 SHBG EIF2AK2 CVIMA2M PPP2R2B A. 48 h C. Temporal analysisISG15 was found to be highly connected interacting with IFIT3, IFIT5, TRIM25, MX1, HERC5 and USP2 48 h p.i.
- 16. Computational Biology and Genomics Facility, Indian Veterinary Research Institute Extracting the 5000 bp upstream of the 5'UTR from Ensembl Representing the data in GENE-E Predicting the motifs using MEME Predicting TFs that co-regulate DEHC genes by submitting the meme.txt output to Tom Tom Selecting the TFs based on specific criteria (researcher's purview) Identification of Transcription factors that co-regulate the DEGs
- 17. Computational Biology and Genomics Facility, Indian Veterinary Research Institute relative row min row max ETV6 KLF16 Foxj3 FOXC1 ONECUT3 ONECUT1 HNF4A Foxk1 Meis2 Meis3 DPRX FOXO3 NFKB1 NFKB2 NKX2-8 TFAP2C Tcfap2a T ZNF524 EBF1 BCL6B E2F7 NFATC1 CREB3L1 RFX3 TEAD1 TEAD3 TEAD4 VDR Vdr FOXK1 HOXC11 HOXC12 HOXD12 Hic1 KLF14 PRDM1 RHOXF1 TBX1 HOXD11 Hoxc10 KLF13 SP8 IRF3 Klf12 SPDEF ZNF306 ZNF410 GLIS3 Nkx3-1 NKX3-1 GLI2 GLIS2 ZBTB7C ZBTB7B NKX2-3 Rarg PAX2 PAX5 PITX1 TBX19 NR2F6 EGR3 Zfp740 SP4 Nr2f6 NR2F1 RARA SP3 SP1 ZNF740 RARG Annotation SHBG CD44 EIF2AK2 CTTN IFIT5 RGS16 CCL2 S100A9 LDHA SDC1 FOS PFKP VCAN CD9 ID3 A2M AK4 MT2A ERBB3 IRF3 LMNA BIRC5 C2 SFXN3 ADAM15 DNAJB1 CTNND1 IL8 PICALM CDC20 ACVRL1 CDKN1A ELF3 FGR BUB1B RSU1 IRF9 COA1 PACSIN1 ISG15 RBPMS PLAU CCL5 LYN PAK1 PTRF IFIT3 HSPA1L STOM UBE2C PRC1 ANXA2 TLR4 IRAK3 GSN GAS7 MAPT PLAUR CALU TRIB3 DYNC1I1 NEK2 TUBA1C TJP2 SERPINH1 DAB2 PLD2 MGST3 MMP2 PCBD1 F3 NUF2 ATP6V1B2 PRKCDBP VIM UCHL1 PML CALD1 CDC25C TLR9 CD55 MAP1B CDCA8 ATP6V1F TPI1 GATA3 ANXA5 MET PGK1 UCHL3 USP18 CLU CLK1 BCL2L1 HCK FKBP1A ENO1 GK NEK6 ALAS1 TNF CAV1 AK1 GRN HGF GADD45G PPP2R2B NXF1 CEP55 THBS1 CDH2 FN1 ACVR2A CSF1R TLR2 NPL SDC2 relative row min row max KLF16 FOXC1 Foxj3 HNF4A ESRRA ESRRG Esrra ESRRB JDP2 Jdp2 CREB3L1 RFX3 IRF3 PRDM1 T SRF Meis2 Meis3 ETS1 RUNX2 NR2E1 Nr2e1 FOXK1 Foxk1 PAX9 NKX2-8 HOXD11 PAX1 CENPB EBF1 FOXO3 GLI2 GLIS2 MAFF PAX2 PAX5 PITX1 RHOXF1 TBX19 Nkx3-1 NKX2-3 NKX3-2 NKX3-1 ZBTB7C ZBTB7B ALX3 Ascl2 RUNX3 Rhox11 NHLH1 VSX1 Tcf21 E2F7 NFATC1 ZNF410 ZNF306 HOXC11 HOXD12 HOXC12 Vdr TEAD1 TEAD3 ONECUT1 KLF14 AR HIC2 SP8 RARG SP3 Hic1 SP4 ZIC3 Zic3 TBX20 ZIC1 ZNF740 ZNF524 Zfp740 TBX1 ONECUT2 ONECUT3 SP1 Annotation VCAM1 VAMP8 ANXA4 CD2 CBX5 MLLT3 CDK18 EGR1 GAPDH KIT EPN1 MAP1B JUN APOE TUBB2B NGFR PPP2CB EIF2AK2 ENC1 ACTA1 RGS2 USP21 ANXA1 LCK ECT2 YES1 CD4 TPM1 MITF ERLIN2 PLK3 GADD45A GCLM CDK1 PROCR CCR5 SSBP1 ISG15 SMAD1 PML HSP90AA1 CCND1 RPL36 UCHL1 HMGB1 THRA SORD LTA MAP2K3 SF3B14 KRT18 EEF1G TAGLN PTPN6 H2AFX HSPA1L FOSL1 CAT RPL10A ALDOC SEC61B IFIT5 NQO1 FKBP4 UBD USP50 VKORC1 PSTPIP1 PF4 FN1 COA1 SERPINH1 CDH2 CAV1 PPARG PHKG2 SIRT1 ENO2 ZBTB16 TRIB3 GADD45B MCM5 PLD2 MYH10 ACTA2 STX1A PLAU SPI1 TRIM25 BATF RRAD BTK KEAP1 CSF1R TPM2 OTUB2 SPP1 CD8A SLC3A2 HSPD1 CKS2 DNM1 PLAT DNAJB1 ARRB2 EVL HP STAT5A CEBPG PRC1 ACSL3 MAPRE1 CHEK2 GSN FOS SYK CHEK1 ERBB3 HSPA8 IFIT3 CD63 SFPQ SNAPIN AURKA CDC25B VIM Transcription Factors (DEHC genes) - 48 h Transcription Factors (DEHC genes) - 120 hA. B. 4. TF Identification
- 18. Computational biology and genomics facility a b a b a b a b a b a b Confirmation through real-time PCR
- 19. Computational Biology and Genomics Facility, Indian Veterinary Research Institute Extracting the 3'UTR of DEHC genes from Ensembl Running miRanda to identify potential miRNA binding sites to the 3'UTR of DEHC genes Selecting miRNA based on specific criteria (researcher's purview) Constructing a network using Cytoscape Extracting miRNA specific to Bos taurus Identification of miRNAs target sites among the DEGs
- 20. Computational Biology and Genomics Facility, Indian Veterinary Research Institute Deep Sequencing Short reads mirDeep2.pl DESeq2 for differential expression Mapping to the reference (mapper.pl) Indexing using Bowtie Collapsed reads.fa & Collapsed reads vs Genome.arf Identifies novel and known miRNAs mirDeep2.plQuantifier.pl Predicting novel and known miRNAs - Differential Expression
- 21. Computational Biology and Genomics Facility, Indian Veterinary Research Institute PNG Configuration file circos.conf Track Data File • histogram.txt • scatter.txt • heatmap.txt • links1.txt • links2.txt Additional Configuration files ideogram.conf Post Process Illustrator inkscape Photoshop Gimp Image Magick SVG ticks.conf Circos Representing data using Circos
- 22. Computational Biology and Genomics Facility, Indian Veterinary Research Institute Indexing the reference in Bowtie2 Bowtie2 mapping Inspecting the Index Extracting the mapped reads from the bam file Converting SAM to BAM and sorting the BAM Assembling the contigs using MacVector 14.0.6 Submission of the sequence to the genbank using Sequin Viral genome assembly
- 23. Computational Biology and Genomics Facility, Indian Veterinary Research Institute Quality filtered/trimmed RNA-Seq Short reads Calculate transcript abundances (RSEM) Reference Genome of the Virus Mapping to the reference (Bowtie) Downloading the reference genome from NCBI and manually preparing the GTF Count based strategy Viral transcript quantification
- 24. Computational Biology and Genomics Facility, Indian Veterinary Research Institute Microarray data analysis - using GeneSpring
- 25. Computational Biology and Genomics Facility, Indian Veterinary Research Institute Predicted Immune signaling pathway triggered by Sungri/96 vaccine virus Cell Membrane CD46 Nucleus ISGs IRF-3/7/9TLR-7 Adapter-TRIM14/21 IKBKE IRF-3/7/9 ISG-15 SLAM Possible other Receptors ISGylation HERC5 Pin1Ubiquitination IFN-α/IFN-β X P" ISRE IRF-3/7/9 P" N"C" (Zhou et al., 2014; Yang et al.,2009) ISGylated IRF3 leads to increased stability and sustained activity (Shi et al.,2009) Anti viral state (Schneider et al., 2014)Non-structural proteins (C and V proteins) of paramyxoviruses antagonize IFN function (Nanda S.K and Baron M.D., 2006) IFIT3 & IFIT5 OAS1X, OAS1Y & OAS1Z RSAD2 Mx1 & Mx2 IFITM2 & IFITM2