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=====Identify differentially expressed genes between clusters===== *I took the over-dispersed genes for all samples (2014_1st_Quater_18_1N_Z1_overDispersed_expr_matrix.txt), and assigned each sample to one of the two clusters based on the k-mean analysis (2014_1st_Quater_18_1N_Z1_overDispersed_k-mean_2_clusters.txt). *Then I ran SAM to identify differentially expressed genes. The majority of the genes were differentially expressed, which is not surprising since these genes were pre-selected based on the variability, and the two clusters were well separated. [[Media:2014_1st_Quater_18_1N_Z1_overDispersed_expr_matrix_for_SAM.xlsx]] [[Image:2014_1st_Quater_18_expr_matrix_Z1_k-mean2_two-clusters-SAM-plot.png|400px]] Top ten up/down genes: {| {{table}} | align="center" style="background:#f0f0f0;"|'''Gene ID''' | align="center" style="background:#f0f0f0;"|'''Fold Change''' | align="center" style="background:#f0f0f0;"|'''q-value(%)''' | align="center" style="background:#f0f0f0;"|'''HUGO''' | align="center" style="background:#f0f0f0;"|'''Gene ID''' | align="center" style="background:#f0f0f0;"|'''Fold Change''' | align="center" style="background:#f0f0f0;"|'''q-value(%)''' | align="center" style="background:#f0f0f0;"|'''HUGO''' |- | ENSG00000157103.6||14770220922||0||SLC6A1||ENSG00000135426.9||2.56E-10||0||TESPA1 |- | ENSG00000136750.7||9609687782||0||GAD2||ENSG00000179456.9||6.60E-10||0||ZBTB18 |- | ENSG00000128683.8||9106160761||0||GAD1||ENSG00000198963.5||9.72E-10||0||RORB |- | ENSG00000077044.5||2678606715||0||DGKD||ENSG00000156140.4||1.21E-09||0||ADAMTS3 |- | ENSG00000129244.4||1642874016||0||ATP1B2||ENSG00000120738.7||1.32E-09||0||EGR1 |- | ENSG00000164512.12||1351632660||0||ANKRD55||ENSG00000060718.14||1.43E-09||0||COL11A1 |- | ENSG00000175352.6||1338510457||0||NRIP3||ENSG00000135919.8||1.52E-09||0||SERPINE2 |- | ENSG00000103460.12||1303571887||0||TOX3||ENSG00000167614.8||1.56E-09||0||TTYH1 |- | ENSG00000017427.11||938250314.6||0||IGF1||ENSG00000169507.5||1.96E-09||0||SLC38A11 |- | ENSG00000127328.17||863574011.2||0||RAB3IP||ENSG00000135636.8||2.18E-09||0||DYSF |- | |} *I then manually annotated some of the genes. Note that I used /home/kunzhang/RNAseq/SCAP/scripts/ENSG2HUGO.pl to convert a list of ENSG IDs into HUGO IDs. The top most up-regulated genes in Cluster #2 are mostly GABAergic neuron related genes, which is not surprising. The ones that are up-regulated in Cluster #1 have more diverse functions. Many are glycoproteins, related to calcium binding or cell adhesion. *I did found a few interesting transcriptional regulators, such as ZBTB18/SATB2 for Cluster #1 and TOX3 for Cluster #2. *Then I decided to focus the analysis only on known transcription factors. **Extract the gene expression matrix for all known TFs (based on the list in nrg2538). ./make_gene_set_heatmap_Hs.pl nrg2538_TF_genes.txt ../expr_matrix/2014_1st_Quater_18_1N_expr_matrix_filtered.txt **Renamed nrg2538_TF_genes_expr_matrix.txt to 2014_1st_Quater_18_1N_nrg2538_TF_genes_expr_matrix.txt, then assigned each sample to one of the two clusters(2014_1st_Quater_18_1N_Z1_overDispersed_k-mean_2_clusters.txt). **Run SAM analysis. [[Media:2014_1st_Quater_18_1N_nrg2538_TF_genes_expr_matrix_for_SAM.xlsx]] [[Image:2014_1st_Quater_18_expr_matrix_nrg2538_k-mean2_two-clusters-SAM-plot.png|400px]] **Top ten differentially expressed genes: {| {{table}} | align="center" style="background:#f0f0f0;"|'''Up in Cluster 1''' | align="center" style="background:#f0f0f0;"|''' ''' | align="center" style="background:#f0f0f0;"|''' ''' | align="center" style="background:#f0f0f0;"|'''Up in Cluser 2''' | align="center" style="background:#f0f0f0;"|''' ''' | align="center" style="background:#f0f0f0;"|''' ''' |- | Gene ID||Fold Change||q-value(%)||Gene ID||Fold Change||q-value(%) |- | RORB||9.85E-10||0||TOX3||1.07E+09||0 |- | EGR1||1.31E-09||0||RXRA||2.77E+08||0 |- | ZBTB7C||2.75E-09||0||ZNF641||9.95E+07||0.663375427 |- | HOPX||2.92E-09||0||PPARA||8.41E+07||0 |- | NEUROD2||3.60E-09||0||ETS1||7.65E+07||0 |- | STAT6||3.86E-09||0||BACH1||2.60E+01||0 |- | PLXND1||4.18E-09||0||ZNF385D||1.89E+01||0 |- | ONECUT2||4.50E-09||0||SOX6||7.97E+00||0 |- | LHX2||4.59E-09||0||TOX2||7.29E+00||0 |- | MKX||5.16E-09||0||ZNF536||6.96E+00||0 |- | |}
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