1. Summary

This report includes quality check for raw sequencing data (section 2), reads mapping and assignment (section 3), and expression similarity between samples (section 4). The results of differential expression analysis are summarized in section 5 using the criteria of fold change >= 2 and FDR <= 0.05 .The top five enriched elements of each category are reported in section 6.


2. Sequencing Quality

2.1 Summary of sequencing quality

**Figure 1:** The mean quality scores of sequencing reads in each position

Figure 1: The mean quality scores of sequencing reads in each position


**Figure 2:** The average GC content of sequencing reads

Figure 2: The average GC content of sequencing reads



3. Mapping quality

3.1 Summary of mapping quality

**Figure 4:** The statistics of RNAseq mapping results

Figure 4: The statistics of RNAseq mapping results


Table 1: The summary of RNAseq mapping results
Input reads Uniquely mapped reads Mapped to multiple loci Mapped to too many loci Ratio
ER1 42442664 36824968 5081203 139080 0.9873596
ER2 36380540 31647246 4273071 120329 0.9873497
ctrl1 35405319 30682282 4273717 119971 0.9873093
ctrl2 32544619 28122469 3946112 110549 0.9853728
**Ratio* * = (Uniquely mapped + Mapped to multi ple loci)/ Input reads


4. Expression Quantification

4.1 Summary of reads assignment

**Figure 5:** The number of reads assigned to genes in uniquely mapped reads

Figure 5: The number of reads assigned to genes in uniquely mapped reads


Table 2: The summary of reads assignment to genes
Sample Assigned Percent assigned Unassigned ambiguity Unassigned no features Total uniquely mapped
ER1 30892781 93.82684 2032531 0 32925312
ER2 26333753 93.93351 1700709 0 28034462
ctrl1 25692584 93.87052 1677654 0 27370238
ctrl2 23361159 93.94516 1505644 0 24866803


4.2 Expression density of samples

**Figure 6:** Expression density distribution of each sample

Figure 6: Expression density distribution of each sample


4.3 Similarity between samples

**Figure 7:** Heatmap of expression similarity across samples

Figure 7: Heatmap of expression similarity across samples


**Figure 8:** PCA plot of samples

Figure 8: PCA plot of samples


**Figure 9:** Correlation cluster of samples

Figure 9: Correlation cluster of samples


5. Differential expression

5.1 Volcano plot

**Figure 10:** Volcano plot for all comparisons

Figure 10: Volcano plot for all comparisons


5.2 Summary of differential analysis

Table 3: DEG numbers under FC > 2 & FDR < 0.05
Comparison Number of DEGs
ER_vs_ctrl_min5_fdr0.05 373
Table 4: deseq2_ER_vs_ctrl top 10 differential expressed genes
Feature_gene_name baseMean log2FoldChange lfcSE stat pvalue padj FoldChange
ENSG00000136997 MYC 4940.9387 3.176374 0.0733927 43.27918 0 0 9.040323
ENSG00000182704 TSKU 4811.9953 2.824986 0.0745477 37.89503 0 0 7.086073
ENSG00000100219 XBP1 25329.4977 2.069243 0.0574100 36.04328 0 0 4.196664
ENSG00000173706 HEG1 1616.6958 3.408640 0.1010199 33.74227 0 0 10.619474
ENSG00000113739 STC2 8169.7740 2.588275 0.0773372 33.46742 0 0 6.013791
ENSG00000110092 CCND1 21857.9807 1.767084 0.0553333 31.93525 0 0 3.403652
ENSG00000170525 PFKFB3 5455.4875 1.974682 0.0639637 30.87191 0 0 3.930417
ENSG00000074410 CA12 9928.4744 2.190397 0.0719953 30.42417 0 0 4.564311
ENSG00000164626 KCNK5 884.9953 4.541789 0.1544625 29.40383 0 0 23.292432
ENSG00000261578 RP11-21L23.2 865.6405 3.938454 0.1380153 28.53635 0 0 15.331784


6. Funcional enrichment analysis

List of the top five significantly enriched elements


6.1 WebGestalt

6.1.1 ER_vs_ctrl

Table 5: WebGestalt_GO_BP_ER_vs_ctrl
ID Name #Gene FDR
GO:0050673 epithelial cell proliferation 23 2.96e-05
GO:0002521 leukocyte differentiation 26 3.14e-05
GO:0050679 positive regulation of epithelial cell proliferation 15 3.84e-05
GO:1902105 regulation of leukocyte differentiation 18 3.84e-05
GO:0032526 response to retinoic acid 12 6.86e-05
Table 6: WebGestalt_GO_CC_ER_vs_ctrl
ID Name #Gene FDR
GO:0045121 membrane raft 13 0.0002337
GO:0098857 membrane microdomain 13 0.0002337
GO:0098552 side of membrane 14 0.0017759
GO:0098589 membrane region 13 0.0017759
GO:0005901 caveola 6 0.0051238
Table 7: WebGestalt_GO_MF_ER_vs_ctrl
ID Name #Gene FDR
GO:0001228 transcriptional activator activity, RNA polymerase II transcription regulatory region sequence-specific binding 19 0.0000578
GO:0001077 transcriptional activator activity, RNA polymerase II core promoter proximal region sequence-specific binding 14 0.0015095
GO:0000982 transcription factor activity, RNA polymerase II core promoter proximal region sequence-specific binding 16 0.0043459
GO:0000987 core promoter proximal region sequence-specific DNA binding 16 0.0070653
GO:0001159 core promoter proximal region DNA binding 16 0.0070653
Table 8: WebGestalt_KEGG_ER_vs_ctrl
ID Name #Gene FDR
hsa05200 Pathways in cancer - Homo sapiens (human) 20 0.0043714
hsa04923 Regulation of lipolysis in adipocytes - Homo sapiens (human) 7 0.0069795
hsa05166 HTLV-I infection - Homo sapiens (human) 14 0.0130652
hsa04024 cAMP signaling pathway - Homo sapiens (human) 12 0.0130652
hsa04151 PI3K-Akt signaling pathway - Homo sapiens (human) 15 0.0480313


6.2 GSEA

6.2.1 ER_vs_ctrl

Table 9: ER_vs_ctrl_min5_fdr0.05_DESeq2_GSEA CuratedGeneSets Positive-regulated
Name Description SIZE ES NES FDR.q.val
BHAT_ESR1_TARGETS_VIA_AKT1_UP Genes bound by ESR1 [GeneID=2099] and up-regulated by estradiol [PubChem=5757] in MCF-7 cells (breast cancer) expressing constitutevly active form of AKT1 [GeneID=207]. 267 0.88191384 2.8841803 0
BHAT_ESR1_TARGETS_NOT_VIA_AKT1_UP Genes bound by ESR1 [GeneID=2099] and up-regulated by estradiol [PubChem=5757] in MCF-7 cells (breast cancer). 200 0.89294237 2.85244 0
DUTERTRE_ESTRADIOL_RESPONSE_6HR_UP Genes up-regulated in MCF7 cells (breast cancer) at 6 h of estradiol [PubChem=5757] treatment. 222 0.8737239 2.7888453 0
MASSARWEH_RESPONSE_TO_ESTRADIOL Genes rapidly up-regulated in breast cancer cell cultures by estradiol [PubChem=5757]. 59 0.9114527 2.5543926 0
MASSARWEH_TAMOXIFEN_RESISTANCE_DN Genes down-regulated in breast cancer tumors (formed by MCF-7 xenografts) resistant to tamoxifen [PubChem=5376]. 228 0.78520465 2.5366442 0
Table 10: ER_vs_ctrl_min5_fdr0.05_DESeq2_GSEA CuratedGeneSets Negative-regulated
Name Description SIZE ES NES FDR.q.val
DUTERTRE_ESTRADIOL_RESPONSE_6HR_DN Genes down-regulated in MCF7 cells (breast cancer) at 6 h of estradiol [PubChem=5757] treatment. 90 -0.8794996 -3.3900943 0
DUTERTRE_ESTRADIOL_RESPONSE_24HR_DN Genes down-regulated in MCF7 cells (breast cancer) at 24 h of estradiol [PubChem=5757] treatment. 491 -0.70555717 -3.244043 0
BHAT_ESR1_TARGETS_NOT_VIA_AKT1_DN Genes bound by ESR1 [GeneID=2099] and down-regulated by estradiol [PubChem=5757] in MCF-7 cells (breast cancer). 86 -0.7421858 -2.8064094 0
BHAT_ESR1_TARGETS_VIA_AKT1_DN Genes bound by ESR1 [GeneID=2099] and down-regulated by estradiol [PubChem=5757] in MCF-7 cells (breast cancer) expressing constitutevly active form of AKT1 [GeneID=207]. 79 -0.7437244 -2.6809692 0
FRASOR_RESPONSE_TO_ESTRADIOL_DN Genes down-regulated in MCF-7 cells (breast cancer) by estradiol (E2) [PubChem=5757]. 78 -0.62314934 -2.3264885 0.00019249396
Table 11: ER_vs_ctrl_min5_fdr0.05_DESeq2_GSEA GOGeneSets Positive-regulated
Name Description SIZE ES NES FDR.q.val
GO_PRERIBOSOME Any complex of pre-rRNAs, ribosomal proteins, and associated proteins formed during ribosome biogenesis. 60 0.845003 2.361481 0
GO_RIBOSOME_BIOGENESIS A cellular process that results in the biosynthesis of constituent macromolecules, assembly, and arrangement of constituent parts of ribosome subunits; includes transport to the sites of protein synthesis. 298 0.7148912 2.3415384 0
GO_NUCLEOLAR_PART Any constituent part of a nucleolus, a small, dense body one or more of which are present in the nucleus of eukaryotic cells. It is rich in RNA and protein, is not bounded by a limiting membrane, and is not seen during mitosis. 60 0.8183922 2.286099 0
GO_RRNA_METABOLIC_PROCESS The chemical reactions and pathways involving rRNA, ribosomal RNA, a structural constituent of ribosomes. 248 0.69911313 2.2854564 0
GO_RIBONUCLEOPROTEIN_COMPLEX_BIOGENESIS A cellular process that results in the biosynthesis of constituent macromolecules, assembly, and arrangement of constituent parts of a complex containing RNA and proteins. Includes the biosynthesis of the constituent RNA and protein molecules, and those macromolecular modifications that are involved in synthesis or assembly of the ribonucleoprotein complex. 419 0.67804366 2.27912 0
Table 12: ER_vs_ctrl_min5_fdr0.05_DESeq2_GSEA HallmarkGeneSets Positive-regulated
Name Description SIZE ES NES FDR.q.val
HALLMARK_ESTROGEN_RESPONSE_EARLY Genes defining early response to estrogen. 197 0.89685076 2.8650224 0
HALLMARK_ESTROGEN_RESPONSE_LATE Genes defining late response to estrogen. 191 0.82698476 2.641104 0
HALLMARK_MYC_TARGETS_V2 A subgroup of genes regulated by MYC - version 2 (v2). 58 0.851762 2.3625467 0
HALLMARK_MYC_TARGETS_V1 A subgroup of genes regulated by MYC - version 1 (v1). 199 0.6385056 2.05484 0
HALLMARK_UNFOLDED_PROTEIN_RESPONSE Genes up-regulated during unfolded protein response, a cellular stress response related to the endoplasmic reticulum. 110 0.6543728 1.9990724 0
Table 13: ER_vs_ctrl_min5_fdr0.05_DESeq2_GSEA HallmarkGeneSets Negative-regulated
Name Description SIZE ES NES FDR.q.val
HALLMARK_INTERFERON_ALPHA_RESPONSE Genes up-regulated in response to alpha interferon proteins. 89 -0.49427092 -1.8661766 0.0014285715
HALLMARK_INTERFERON_GAMMA_RESPONSE Genes up-regulated in response to IFNG [GeneID=3458]. 163 -0.39906272 -1.611156 0.027404075
Table 14: ER_vs_ctrl_min5_fdr0.05_DESeq2_GSEA ImmunologicGeneSets Positive-regulated
Name Description SIZE ES NES FDR.q.val
GSE6674_UNSTIM_VS_ANTI_IGM_AND_CPG_STIM_BCELL_DN Genes down-regulated in B lymphocytes: control versus anti IgM and cell anti IgM and CpG oligodeoxynucleotide 1826. 184 0.730901 2.3507802 0
GSE6674_ANTI_IGM_VS_CPG_STIM_BCELL_DN Genes down-regulated in B lymphocytes: anti IgM versus CpG oligodeoxynucleotide 1826. 177 0.7495347 2.3444552 0
GSE37532_TREG_VS_TCONV_CD4_TCELL_FROM_LN_UP Genes up-regulated in lymph node from aged mice: T reg versus T conv. 183 0.72588307 2.2985296 0
GSE21670_UNTREATED_VS_IL6_TREATED_STAT3_KO_CD4_TCELL_DN Genes down-regulated in CD4 [GeneID=920] T cells with STAT3 [GeneID=6774] knockout: medium versus IL6 [GeneID=3569]. 182 0.71945095 2.289653 0
GSE43863_TH1_VS_LY6C_INT_CXCR5POS_MEMORY_CD4_TCELL_UP Genes up-regulated in CD4 [GeneID=920] SMARTA memory T cells: Th1 versus Ly6c int CXCR5+ [GeneID=643]. 178 0.7007546 2.2173014 0
Table 15: ER_vs_ctrl_min5_fdr0.05_DESeq2_GSEA ImmunologicGeneSets Negative-regulated
Name Description SIZE ES NES FDR.q.val
GSE15750_DAY6_VS_DAY10_TRAF6KO_EFF_CD8_TCELL_UP Genes up-regulated in comparison of wild type CD8 effector T cells at day 6 versus those from mice defficient for TRAF6 [GeneID=7189] at day 10. 184 -0.5412034 -2.3076034 0
GSE39556_CD8A_DC_VS_NK_CELL_MOUSE_3H_POST_POLYIC_INJ_UP Genes up-regulated after poly(IC) injection: CD8A [GeneID=925] dendritic cells versus NK cells. 188 -0.51933783 -2.2195017 0
GSE13547_CTRL_VS_ANTI_IGM_STIM_BCELL_2H_UP Genes up-regulated in B lymphocytes: control versus stimulated by anti-IgM for 2h. 150 -0.5382146 -2.1716845 0
GSE45739_NRAS_KO_VS_WT_UNSTIM_CD4_TCELL_DN Genes down-regulated in CD4 [GeneID=920] T cells: NRAS [GeneID=4893] knockout versus wildtype. 163 -0.52058446 -2.11219 0
GSE46606_UNSTIM_VS_CD40L_IL2_IL5_DAY3_STIMULATED_BCELL_UP Genes up-regulated in at day 0 B cell wildtype versus CD40L and IL-2 IL-4 IL-5 stimulated at day 3 B cell wildtype. 160 -0.5060978 -2.0630875 0
Table 16: ER_vs_ctrl_min5_fdr0.05_DESeq2_GSEA OncogenicGeneSets Positive-regulated
Name Description SIZE ES NES FDR.q.val
MYC_UP.V1_UP Genes up-regulated in primary epithelial breast cancer cell culture over-expressing MYC [GeneID=4609] gene. 150 0.75292045 2.3476157 0
IL15_UP.V1_UP Genes up-regulated in Sez-4 cells (T lymphocyte) that were first starved of IL2 [GeneID=3558] and then stimulated with IL15 [GeneID=3600]. 141 0.69340885 2.1460347 0
IL2_UP.V1_UP Genes up-regulated in Sez-4 cells (T lymphocyte) that were first starved of IL2 [GeneID=3558] and then stimulated with IL2 [GeneID=3558]. 131 0.6811118 2.1003718 0
LTE2_UP.V1_DN Genes down-regulated in MCF-7 cells (breast cancer) positive for ESR1 [GeneID=2099] MCF-7 cells (breast cancer) and long-term adapted for estrogen-independent growth. 189 0.6297597 2.0154269 0
ERB2_UP.V1_DN Genes down-regulated in MCF-7 cells (breast cancer) positive for ESR1 [GeneID=2099] and engineered to express ligand-activatable ERBB2 [GeneID=2064]. 186 0.6292693 2.0061898 0
Table 17: ER_vs_ctrl_min5_fdr0.05_DESeq2_GSEA OncogenicGeneSets Negative-regulated
Name Description SIZE ES NES FDR.q.val
MTOR_UP.N4.V1_DN Genes down-regulated in CEM-C1 cells (T-CLL) in comparison of control vs rapamycin (sirolimus) [PubChem=6610346], an mTOR pathway inhibitor. 153 -0.53632164 -2.1790252 0
IL2_UP.V1_DN Genes down-regulated in Sez-4 cells (T lymphocyte) that were first starved of IL2 [GeneID=3558] and then stimulated with IL2 [GeneID=3558]. 114 -0.5219168 -2.0491354 0
STK33_SKM_UP Genes up-regulated in SKM-1 cells (AML) after knockdown of STK33 [GeneID=65975] by RNAi. 212 -0.44171953 -1.8461125 0.004439152
TGFB_UP.V1_DN Genes down-regulated in a panel of epithelial cell lines by TGFB1 [GeneID=7040]. 153 -0.44098106 -1.8259705 0.003966177
ERB2_UP.V1_UP Genes up-regulated in MCF-7 cells (breast cancer) positive for ESR1 [GeneID=2099] and engineered to express ligand-activatable ERBB2 [GeneID=2064]. 186 -0.43399885 -1.8058369 0.003959772


7. Results folder structure

Table 18: Result files
File Description
./RNAseq_human/RNAseq_human.count Gene expression read count table
./RNAseq_human/RNAseq_human.fpkm.tsv Gene expression abundance table
./RNAseq_human/ER_vs_ctrl_min5_fdr0.05_DESeq2.csv Differential expression analysis table
./RNAseq_human/ER_vs_ctrl_min5_fdr0.05_DESeq2_sig.csv Significantly differential expressed genes