Source: https://github.com/markziemann/SurveyEnrichmentMethods
suppressPackageStartupMessages({
library("getDEE2")
library("DESeq2")
library("clusterProfiler")
library("org.Hs.eg.db")
library("mitch")
library("kableExtra")
library("eulerr")
})In Kaumadi et al 2021 we showed that major problems plague functional enrichment analysis in many journal articles.
Here I will do my best to reproduce and replicate the findings of some studies. These were selected on the following criteria:
Included in the 2019 group that was studies in Kaumadi et al 2021.
Human study
RNA-seq gene expression
Performed DAVID gene set analysis
Provided gene list
From this subset, four studies were selected
| Study | Analytical issues |
|---|---|
| PMC6349697 | Background |
| PMC6425008 | Background, FDR |
| PMC6463127 | Background, FDR |
| PMC6535219 | Background |
In this document I will be seeing if I can reproduce the same findings by using the same tool with the gene list provided in the article. Also I will correct the method wherever possible. This means using the correct background gene list, using FDR control and performing separate analysis of up and down-regulated gene lists.
The study examined in this document is PMC6463127: Chai et al, 2019.
The focus of this study is to compare the RNA expression profiles of parathyroid adenomas with normal parathyroid gland tissue. They found 247 differentially expressed genes; 45 up-regulated, 202 down-regulated.
They used DAVID for GO/KEGG analysis. They did not perform FDR correction of enrichment results.
Here are the upregulated and downregulated GO terms and the downregulated KEGG pathways. (Presuming there were no significantly upregulated KEGG pathways.)
orig1 <- read.table("PMC6463127_orig_up.tsv",header=TRUE,sep="\t")
orig1 %>% kbl(caption="upregulated GO terms shown in the article") %>% kable_paper("hover", full_width = F)| GOtype | Category | GO_Term_ID | GO_Term_Count | pvalue |
|---|---|---|---|---|
| BP | GO:0006351 | transcription, DNA-templated | 14 | <0.001 |
| BP | GO:0050684 | regulation of mRNA processing | 3 | <0.001 |
| BP | GO:0006325 | chromatin organization | 3 | 0.004 |
| BP | GO:0045893 | positive regulation of transcription, DNA-templated | 6 | 0.005 |
| BP | GO:0018026 | peptidyl-lysine monomethylation | 2 | 0.017 |
| BP | GO:0006355 | regulation of transcription, DNA-templated | 8 | 0.038 |
| CC | GO:0005654 | nucleoplasm | 19 | <0.001 |
| CC | GO:0005634 | nucleus | 26 | <0.001 |
| MF | GO:0003676 | nucleic acid binding | 10 | <0.001 |
| MF | GO:0003682 | chromatin binding | 6 | 0.002 |
| MF | GO:0003713 | transcription coactivator activity | 5 | 0.002 |
| MF | GO:0000166 | nucleotide binding | 5 | 0.008 |
| MF | GO:0003677 | DNA binding | 10 | 0.012 |
| MF | GO:0003690 | double-stranded DNA binding | 3 | 0.015 |
| MF | GO:0003729 | mRNA binding | 3 | 0.034 |
| MF | GO:0016279 | protein-lysine N-methyltransferase activity | 2 | 0.039 |
orig2 <- read.table("PMC6463127_orig_dn.tsv",header=TRUE,sep="\t")
orig2 %>% kbl(caption="down-regulated GO terms shown in the article") %>% kable_paper("hover", full_width = F)| GOtype | Category | GO_Term_ID | GO_Term_Count | p.value |
|---|---|---|---|---|
| BP | GO:0002474 | antigen processing and presentation of peptide antigen via MHC class I | 5 | <0.001 |
| BP | GO:0061077 | chaperone-mediated protein folding | 5 | <0.001 |
| BP | GO:0006465 | signal peptide processing | 4 | 0.002 |
| BP | GO:0006506 | GPI anchor biosynthetic process | 4 | 0.003 |
| BP | GO:0044829 | positive regulation by host of viral genome replication | 3 | 0.003 |
| CC | GO:0070062 | extracellular exosome | 79 | <0.001 |
| CC | GO:0016020 | membrane | 64 | <0.001 |
| CC | GO:0005789 | endoplasmic reticulum membrane | 36 | <0.001 |
| CC | GO:0005783 | endoplasmic reticulum | 31 | <0.001 |
| CC | GO:0042470 | melanosome | 12 | <0.001 |
| MF | GO:0044822 | poly(A) RNA binding | 23 | 0.003 |
| MF | GO:0005515 | protein binding | 110 | 0.004 |
| MF | GO:0001540 | beta-amyloid binding | 4 | 0.005 |
| MF | GO:0019904 | protein domain specific binding | 8 | 0.006 |
| MF | GO:0051087 | chaperone binding | 5 | 0.01 |
orig3 <- read.table("PMC6463127_orig_keggdn.tsv",header=TRUE,sep="\t")
orig3 %>% kbl(caption="down-regulated KEGG pathways shown in the article") %>% kable_paper("hover", full_width = F)| Term_ID | Term | Count | p.value | Genes |
|---|---|---|---|---|
| hsa04141 | Protein processing in endoplasmic reticulum | 15 | <0.001 | SEC63, DNAJA1, EIF2AK1, XBP1, UGGT2, CANX, SEC61G, STT3A, PDIA6, HYOU1, SEC13, RPN1, PDIA3, CALR, BCAP31 |
| hsa03060 | Protein export | 5 | <0.001 | SEC63, SPCS2, SEC61G, SEC11A, SEC11C |
| hsa03013 | RNA transport | 8 | 0.008 | STRAP, XPO1, EIF3E, SUMO1, EIF2S3, NUP205, SEC13, RGPD2 |
| hsa00563 | Glycosylphosphatidylinositol (GPI)-anchor biosynthesis | 3 | 0.043 | PIGU, PIGG, PIGP |
| hsa00240 | Pyrimidine metabolism | 5 | 0.049 | CTPS2, NT5C3A, NME7, POLR2H, DPYD |
So let’s see if I can reproduce their result using DAVID without a background gene list. The study provided the differentially regulated gene lists in Table S2
Here are the significant GO terms for the up and down directions, along with the downregulated KEGG pathways.
repl1up <- read.table("PMC6463127_repl_up.tsv",header=TRUE,sep="\t")
repl1up %>% kbl(caption="Upregulated GO terms obtained using article gene list without background") %>% kable_paper("hover", full_width = F)| Category | Term | Count | X. | PValue | List.Total | Pop.Hits | Pop.Total | FoldEnrichment | FDR |
|---|---|---|---|---|---|---|---|---|---|
| GOTERM_BP_DIRECT | GO:0006351~transcription, DNA-templated | 14 | 30.4348 | 0.0001 | 37 | 1955 | 16792 | 3.250 | 0.012 |
| GOTERM_BP_DIRECT | GO:0050684~regulation of mRNA processing | 3 | 6.5217 | 0.0002 | 37 | 9 | 16792 | 151.279 | 0.012 |
| GOTERM_BP_DIRECT | GO:0006325~chromatin organization | 3 | 6.5217 | 0.0038 | 37 | 43 | 16792 | 31.663 | 0.176 |
| GOTERM_BP_DIRECT | GO:0045893~positive regulation of transcription, DNA-templated | 6 | 13.0435 | 0.0046 | 37 | 515 | 16792 | 5.287 | 0.176 |
| GOTERM_BP_DIRECT | GO:0018026~peptidyl-lysine monomethylation | 2 | 4.3478 | 0.0170 | 37 | 8 | 16792 | 113.459 | 0.524 |
| GOTERM_BP_DIRECT | GO:0006355~regulation of transcription, DNA-templated | 8 | 17.3913 | 0.0380 | 37 | 1504 | 16792 | 2.414 | 0.976 |
| GOTERM_BP_DIRECT | GO:0022604~regulation of cell morphogenesis | 2 | 4.3478 | 0.0523 | 37 | 25 | 16792 | 36.307 | 1.000 |
| GOTERM_BP_DIRECT | GO:0006357~regulation of transcription from RNA polymerase II promoter | 4 | 8.6957 | 0.0679 | 37 | 441 | 16792 | 4.116 | 1.000 |
| GOTERM_BP_DIRECT | GO:0009411~response to UV | 2 | 4.3478 | 0.0863 | 37 | 42 | 16792 | 21.611 | 1.000 |
| GOTERM_CC_DIRECT | GO:0005654~nucleoplasm | 19 | 41.3043 | 0.0000 | 42 | 2784 | 18224 | 2.961 | 0.001 |
| GOTERM_CC_DIRECT | GO:0005634~nucleus | 26 | 56.5217 | 0.0000 | 42 | 5415 | 18224 | 2.083 | 0.001 |
| GOTERM_CC_DIRECT | GO:0005813~centrosome | 4 | 8.6957 | 0.0704 | 42 | 426 | 18224 | 4.074 | 1.000 |
| GOTERM_CC_DIRECT | GO:0016607~nuclear speck | 3 | 6.5217 | 0.0750 | 42 | 201 | 18224 | 6.476 | 1.000 |
| GOTERM_MF_DIRECT | GO:0003676~nucleic acid binding | 10 | 21.7391 | 0.0003 | 40 | 985 | 16881 | 4.285 | 0.026 |
| GOTERM_MF_DIRECT | GO:0003682~chromatin binding | 6 | 13.0435 | 0.0020 | 40 | 391 | 16881 | 6.476 | 0.067 |
| GOTERM_MF_DIRECT | GO:0003713~transcription coactivator activity | 5 | 10.8696 | 0.0025 | 40 | 248 | 16881 | 8.509 | 0.067 |
| GOTERM_MF_DIRECT | GO:0000166~nucleotide binding | 5 | 10.8696 | 0.0083 | 40 | 348 | 16881 | 6.064 | 0.165 |
| GOTERM_MF_DIRECT | GO:0003677~DNA binding | 10 | 21.7391 | 0.0124 | 40 | 1674 | 16881 | 2.521 | 0.198 |
| GOTERM_MF_DIRECT | GO:0003690~double-stranded DNA binding | 3 | 6.5217 | 0.0150 | 40 | 81 | 16881 | 15.631 | 0.200 |
| GOTERM_MF_DIRECT | GO:0003729~mRNA binding | 3 | 6.5217 | 0.0337 | 40 | 125 | 16881 | 10.129 | 0.385 |
| GOTERM_MF_DIRECT | GO:0016279~protein-lysine N-methyltransferase activity | 2 | 4.3478 | 0.0386 | 40 | 17 | 16881 | 49.650 | 0.386 |
| GOTERM_MF_DIRECT | GO:0003700~transcription factor activity, sequence-specific DNA binding | 6 | 13.0435 | 0.0687 | 40 | 961 | 16881 | 2.635 | 0.611 |
| GOTERM_MF_DIRECT | GO:0018024~histone-lysine N-methyltransferase activity | 2 | 4.3478 | 0.0863 | 40 | 39 | 16881 | 21.642 | 0.691 |
repl1dn <- read.table("PMC6463127_repl_dn.tsv",header=TRUE,sep="\t")
repl1dn %>% kbl(caption="Downregulated GO terms obtained using article gene list without background") %>% kable_paper("hover", full_width = F)| Category | Term | Count | X. | PValue | List.Total | Pop.Hits | Pop.Total | Fold.Enrichment | FDR |
|---|---|---|---|---|---|---|---|---|---|
| GOTERM_BP_DIRECT | GO:0002474~antigen processing and presentation of peptide antigen via MHC class I | 5 | 2.4875622 | 0.0002874 | 182 | 30 | 16792 | 15.377289 | 0.3282420 |
| GOTERM_BP_DIRECT | GO:0061077~chaperone-mediated protein folding | 5 | 2.4875622 | 0.0006532 | 182 | 37 | 16792 | 12.468072 | 0.3729583 |
| GOTERM_BP_DIRECT | GO:0006465~signal peptide processing | 4 | 1.9900498 | 0.0023792 | 182 | 25 | 16792 | 14.762198 | 0.7080755 |
| GOTERM_BP_DIRECT | GO:0006506~GPI anchor biosynthetic process | 4 | 1.9900498 | 0.0026683 | 182 | 26 | 16792 | 14.194421 | 0.7080755 |
| GOTERM_BP_DIRECT | GO:0044829~positive regulation by host of viral genome replication | 3 | 1.4925373 | 0.0031002 | 182 | 8 | 16792 | 34.598901 | 0.7080755 |
| GOTERM_BP_DIRECT | GO:0036498~IRE1-mediated unfolded protein response | 5 | 2.4875622 | 0.0037456 | 182 | 59 | 16792 | 7.818961 | 0.7129117 |
| GOTERM_BP_DIRECT | GO:0002576~platelet degranulation | 6 | 2.9850746 | 0.0051479 | 182 | 103 | 16792 | 5.374587 | 0.8398473 |
| GOTERM_BP_DIRECT | GO:0006888~ER to Golgi vesicle-mediated transport | 7 | 3.4825871 | 0.0078089 | 182 | 160 | 16792 | 4.036538 | 0.9599597 |
| GOTERM_BP_DIRECT | GO:0034975~protein folding in endoplasmic reticulum | 3 | 1.4925373 | 0.0083356 | 182 | 13 | 16792 | 21.291631 | 0.9599597 |
| GOTERM_BP_DIRECT | GO:0018279~protein N-linked glycosylation via asparagine | 4 | 1.9900498 | 0.0090825 | 182 | 40 | 16792 | 9.226374 | 0.9599597 |
| GOTERM_BP_DIRECT | GO:0098609~cell-cell adhesion | 9 | 4.4776119 | 0.0092465 | 182 | 271 | 16792 | 3.064109 | 0.9599597 |
| GOTERM_BP_DIRECT | GO:0050900~leukocyte migration | 6 | 2.9850746 | 0.0103602 | 182 | 122 | 16792 | 4.537561 | 0.9704459 |
| GOTERM_BP_DIRECT | GO:0008543~fibroblast growth factor receptor signaling pathway | 5 | 2.4875622 | 0.0118969 | 182 | 82 | 16792 | 5.625838 | 0.9704459 |
| GOTERM_BP_DIRECT | GO:0006890~retrograde vesicle-mediated transport, Golgi to ER | 5 | 2.4875622 | 0.0118969 | 182 | 82 | 16792 | 5.625838 | 0.9704459 |
| GOTERM_BP_DIRECT | GO:0006457~protein folding | 7 | 3.4825871 | 0.0134351 | 182 | 180 | 16792 | 3.588034 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0006413~translational initiation | 6 | 2.9850746 | 0.0164310 | 182 | 137 | 16792 | 4.040748 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0006614~SRP-dependent cotranslational protein targeting to membrane | 5 | 2.4875622 | 0.0187913 | 182 | 94 | 16792 | 4.907646 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0006509~membrane protein ectodomain proteolysis | 3 | 1.4925373 | 0.0231706 | 182 | 22 | 16792 | 12.581419 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0030433~ER-associated ubiquitin-dependent protein catabolic process | 4 | 1.9900498 | 0.0269179 | 182 | 60 | 16792 | 6.150916 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0048208~COPII vesicle coating | 4 | 1.9900498 | 0.0280917 | 182 | 61 | 16792 | 6.050081 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:1903298~negative regulation of hypoxia-induced intrinsic apoptotic signaling pathway | 2 | 0.9950249 | 0.0319914 | 182 | 3 | 16792 | 61.509158 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0019083~viral transcription | 5 | 2.4875622 | 0.0330295 | 182 | 112 | 16792 | 4.118917 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0031647~regulation of protein stability | 4 | 1.9900498 | 0.0398571 | 182 | 70 | 16792 | 5.272214 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0000184~nuclear-transcribed mRNA catabolic process, nonsense-mediated decay | 5 | 2.4875622 | 0.0398897 | 182 | 119 | 16792 | 3.876628 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0006611~protein export from nucleus | 3 | 1.4925373 | 0.0412609 | 182 | 30 | 16792 | 9.226374 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0048205~COPI coating of Golgi vesicle | 2 | 0.9950249 | 0.0424274 | 182 | 4 | 16792 | 46.131868 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0006886~intracellular protein transport | 7 | 3.4825871 | 0.0429208 | 182 | 236 | 16792 | 2.736636 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0016032~viral process | 8 | 3.9800995 | 0.0436464 | 182 | 299 | 16792 | 2.468595 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0007030~Golgi organization | 4 | 1.9900498 | 0.0457686 | 182 | 74 | 16792 | 4.987229 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0034976~response to endoplasmic reticulum stress | 4 | 1.9900498 | 0.0473106 | 182 | 75 | 16792 | 4.920733 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0045454~cell redox homeostasis | 4 | 1.9900498 | 0.0504707 | 182 | 77 | 16792 | 4.792921 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0001843~neural tube closure | 4 | 1.9900498 | 0.0504707 | 182 | 77 | 16792 | 4.792921 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0035459~cargo loading into vesicle | 2 | 0.9950249 | 0.0527515 | 182 | 5 | 16792 | 36.905495 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0045053~protein retention in Golgi apparatus | 2 | 0.9950249 | 0.0527515 | 182 | 5 | 16792 | 36.905495 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0050821~protein stabilization | 5 | 2.4875622 | 0.0596755 | 182 | 136 | 16792 | 3.392049 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0060323~head morphogenesis | 2 | 0.9950249 | 0.0629648 | 182 | 6 | 16792 | 30.754579 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:1990000~amyloid fibril formation | 2 | 0.9950249 | 0.0629648 | 182 | 6 | 16792 | 30.754579 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0042985~negative regulation of amyloid precursor protein biosynthetic process | 2 | 0.9950249 | 0.0629648 | 182 | 6 | 16792 | 30.754579 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0003158~endothelium development | 2 | 0.9950249 | 0.0629648 | 182 | 6 | 16792 | 30.754579 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0015031~protein transport | 9 | 4.4776119 | 0.0646049 | 182 | 395 | 16792 | 2.102212 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0051262~protein tetramerization | 3 | 1.4925373 | 0.0690317 | 182 | 40 | 16792 | 6.919780 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0060907~positive regulation of macrophage cytokine production | 2 | 0.9950249 | 0.0730687 | 182 | 7 | 16792 | 26.361068 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0006986~response to unfolded protein | 3 | 1.4925373 | 0.0751574 | 182 | 42 | 16792 | 6.590267 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0006874~cellular calcium ion homeostasis | 4 | 1.9900498 | 0.0792352 | 182 | 93 | 16792 | 3.968333 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:1903071~positive regulation of ER-associated ubiquitin-dependent protein catabolic process | 2 | 0.9950249 | 0.0830642 | 182 | 8 | 16792 | 23.065934 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0006623~protein targeting to vacuole | 2 | 0.9950249 | 0.0830642 | 182 | 8 | 16792 | 23.065934 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0030968~endoplasmic reticulum unfolded protein response | 3 | 1.4925373 | 0.0846538 | 182 | 45 | 16792 | 6.150916 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0051028~mRNA transport | 3 | 1.4925373 | 0.0911758 | 182 | 47 | 16792 | 5.889175 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0022417~protein maturation by protein folding | 2 | 0.9950249 | 0.0929525 | 182 | 9 | 16792 | 20.503052 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0045047~protein targeting to ER | 2 | 0.9950249 | 0.0929525 | 182 | 9 | 16792 | 20.503052 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0043030~regulation of macrophage activation | 2 | 0.9950249 | 0.0929525 | 182 | 9 | 16792 | 20.503052 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0036500~ATF6-mediated unfolded protein response | 2 | 0.9950249 | 0.0929525 | 182 | 9 | 16792 | 20.503052 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:2000146~negative regulation of cell motility | 2 | 0.9950249 | 0.0929525 | 182 | 9 | 16792 | 20.503052 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0030148~sphingolipid biosynthetic process | 3 | 1.4925373 | 0.0944905 | 182 | 48 | 16792 | 5.766483 | 1.0000000 |
| GOTERM_CC_DIRECT | GO:0070062~extracellular exosome | 80 | 39.8009950 | 0.0000000 | 193 | 2811 | 18224 | 2.687296 | 0.0000000 |
| GOTERM_CC_DIRECT | GO:0016020~membrane | 64 | 31.8407960 | 0.0000000 | 193 | 2200 | 18224 | 2.746905 | 0.0000000 |
| GOTERM_CC_DIRECT | GO:0005789~endoplasmic reticulum membrane | 37 | 18.4079602 | 0.0000000 | 193 | 862 | 18224 | 4.053040 | 0.0000000 |
| GOTERM_CC_DIRECT | GO:0005783~endoplasmic reticulum | 32 | 15.9203980 | 0.0000000 | 193 | 828 | 18224 | 3.649270 | 0.0000001 |
| GOTERM_CC_DIRECT | GO:0042470~melanosome | 12 | 5.9701493 | 0.0000000 | 193 | 101 | 18224 | 11.218796 | 0.0000005 |
| GOTERM_CC_DIRECT | GO:0005790~smooth endoplasmic reticulum | 6 | 2.9850746 | 0.0000068 | 193 | 26 | 18224 | 21.790355 | 0.0003081 |
| GOTERM_CC_DIRECT | GO:0016021~integral component of membrane | 79 | 39.3034826 | 0.0001564 | 193 | 5163 | 18224 | 1.444812 | 0.0060990 |
| GOTERM_CC_DIRECT | GO:0071556~integral component of lumenal side of endoplasmic reticulum membrane | 5 | 2.4875622 | 0.0002307 | 193 | 29 | 18224 | 16.280150 | 0.0078743 |
| GOTERM_CC_DIRECT | GO:0005925~focal adhesion | 14 | 6.9651741 | 0.0002668 | 193 | 391 | 18224 | 3.380942 | 0.0080918 |
| GOTERM_CC_DIRECT | GO:0005794~Golgi apparatus | 21 | 10.4477612 | 0.0008047 | 193 | 863 | 18224 | 2.297708 | 0.0219676 |
| GOTERM_CC_DIRECT | GO:0005829~cytosol | 52 | 25.8706468 | 0.0024966 | 193 | 3315 | 18224 | 1.481174 | 0.0619600 |
| GOTERM_CC_DIRECT | GO:0005788~endoplasmic reticulum lumen | 8 | 3.9800995 | 0.0043038 | 193 | 192 | 18224 | 3.934370 | 0.0952707 |
| GOTERM_CC_DIRECT | GO:0033116~endoplasmic reticulum-Golgi intermediate compartment membrane | 5 | 2.4875622 | 0.0046363 | 193 | 64 | 18224 | 7.376943 | 0.0952707 |
| GOTERM_CC_DIRECT | GO:0030176~integral component of endoplasmic reticulum membrane | 6 | 2.9850746 | 0.0048857 | 193 | 104 | 18224 | 5.447589 | 0.0952707 |
| GOTERM_CC_DIRECT | GO:0034663~endoplasmic reticulum chaperone complex | 3 | 1.4925373 | 0.0057051 | 193 | 11 | 18224 | 25.752237 | 0.0981527 |
| GOTERM_CC_DIRECT | GO:0005793~endoplasmic reticulum-Golgi intermediate compartment | 5 | 2.4875622 | 0.0057525 | 193 | 68 | 18224 | 6.943005 | 0.0981527 |
| GOTERM_CC_DIRECT | GO:0005765~lysosomal membrane | 9 | 4.4776119 | 0.0086876 | 193 | 274 | 18224 | 3.101547 | 0.1395126 |
| GOTERM_CC_DIRECT | GO:0000139~Golgi membrane | 14 | 6.9651741 | 0.0100020 | 193 | 591 | 18224 | 2.236799 | 0.1516968 |
| GOTERM_CC_DIRECT | GO:0016324~apical plasma membrane | 9 | 4.4776119 | 0.0121764 | 193 | 291 | 18224 | 2.920357 | 0.1698042 |
| GOTERM_CC_DIRECT | GO:0043202~lysosomal lumen | 5 | 2.4875622 | 0.0124640 | 193 | 85 | 18224 | 5.554404 | 0.1698042 |
| GOTERM_CC_DIRECT | GO:0005739~mitochondrion | 24 | 11.9402985 | 0.0130619 | 193 | 1331 | 18224 | 1.702627 | 0.1698042 |
| GOTERM_CC_DIRECT | GO:0005615~extracellular space | 24 | 11.9402985 | 0.0148719 | 193 | 1347 | 18224 | 1.682403 | 0.1845465 |
| GOTERM_CC_DIRECT | GO:0012507~ER to Golgi transport vesicle membrane | 4 | 1.9900498 | 0.0174343 | 193 | 52 | 18224 | 7.263452 | 0.2069375 |
| GOTERM_CC_DIRECT | GO:0030660~Golgi-associated vesicle membrane | 3 | 1.4925373 | 0.0203314 | 193 | 21 | 18224 | 13.489267 | 0.2220194 |
| GOTERM_CC_DIRECT | GO:0000792~heterochromatin | 3 | 1.4925373 | 0.0203314 | 193 | 21 | 18224 | 13.489267 | 0.2220194 |
| GOTERM_CC_DIRECT | GO:0005771~multivesicular body | 3 | 1.4925373 | 0.0222116 | 193 | 22 | 18224 | 12.876119 | 0.2277143 |
| GOTERM_CC_DIRECT | GO:0043209~myelin sheath | 6 | 2.9850746 | 0.0225212 | 193 | 152 | 18224 | 3.727298 | 0.2277143 |
| GOTERM_CC_DIRECT | GO:0009986~cell surface | 12 | 5.9701493 | 0.0288991 | 193 | 542 | 18224 | 2.090587 | 0.2817660 |
| GOTERM_CC_DIRECT | GO:0005764~lysosome | 7 | 3.4825871 | 0.0327285 | 193 | 226 | 18224 | 2.924664 | 0.3048380 |
| GOTERM_CC_DIRECT | GO:0043231~intracellular membrane-bounded organelle | 12 | 5.9701493 | 0.0346087 | 193 | 558 | 18224 | 2.030642 | 0.3048380 |
| GOTERM_CC_DIRECT | GO:0005769~early endosome | 7 | 3.4825871 | 0.0346153 | 193 | 229 | 18224 | 2.886350 | 0.3048380 |
| GOTERM_CC_DIRECT | GO:0031012~extracellular matrix | 8 | 3.9800995 | 0.0378166 | 193 | 296 | 18224 | 2.552023 | 0.3226225 |
| GOTERM_CC_DIRECT | GO:0016323~basolateral plasma membrane | 6 | 2.9850746 | 0.0420509 | 193 | 180 | 18224 | 3.147496 | 0.3423692 |
| GOTERM_CC_DIRECT | GO:0005887~integral component of plasma membrane | 23 | 11.4427861 | 0.0426394 | 193 | 1415 | 18224 | 1.534821 | 0.3423692 |
| GOTERM_CC_DIRECT | GO:0045177~apical part of cell | 4 | 1.9900498 | 0.0447568 | 193 | 75 | 18224 | 5.035993 | 0.3491032 |
| GOTERM_CC_DIRECT | GO:0032580~Golgi cisterna membrane | 4 | 1.9900498 | 0.0462471 | 193 | 76 | 18224 | 4.969730 | 0.3507072 |
| GOTERM_CC_DIRECT | GO:0000932~cytoplasmic mRNA processing body | 4 | 1.9900498 | 0.0493005 | 193 | 78 | 18224 | 4.842301 | 0.3637579 |
| GOTERM_CC_DIRECT | GO:0005913~cell-cell adherens junction | 8 | 3.9800995 | 0.0554097 | 193 | 323 | 18224 | 2.338697 | 0.3811091 |
| GOTERM_CC_DIRECT | GO:0019898~extrinsic component of membrane | 4 | 1.9900498 | 0.0556946 | 193 | 82 | 18224 | 4.606091 | 0.3811091 |
| GOTERM_CC_DIRECT | GO:0005802~trans-Golgi network | 5 | 2.4875622 | 0.0558402 | 193 | 136 | 18224 | 3.471503 | 0.3811091 |
| GOTERM_CC_DIRECT | GO:0043234~protein complex | 9 | 4.4776119 | 0.0705348 | 193 | 412 | 18224 | 2.062679 | 0.4645540 |
| GOTERM_CC_DIRECT | GO:0030137~COPI-coated vesicle | 2 | 0.9950249 | 0.0714698 | 193 | 7 | 18224 | 26.978534 | 0.4645540 |
| GOTERM_CC_DIRECT | GO:0009897~external side of plasma membrane | 6 | 2.9850746 | 0.0750769 | 193 | 213 | 18224 | 2.659855 | 0.4766513 |
| GOTERM_CC_DIRECT | GO:0014704~intercalated disc | 3 | 1.4925373 | 0.0814291 | 193 | 45 | 18224 | 6.294991 | 0.5052305 |
| GOTERM_CC_DIRECT | GO:0044853~plasma membrane raft | 2 | 0.9950249 | 0.0909399 | 193 | 9 | 18224 | 20.983304 | 0.5517021 |
| GOTERM_CC_DIRECT | GO:1903561~extracellular vesicle | 3 | 1.4925373 | 0.0974370 | 193 | 50 | 18224 | 5.665492 | 0.5729490 |
| GOTERM_MF_DIRECT | GO:0051082~unfolded protein binding | 7 | 3.4825871 | 0.0010319 | 177 | 110 | 16881 | 6.069183 | 0.3694082 |
| GOTERM_MF_DIRECT | GO:0005515~protein binding | 111 | 55.2238806 | 0.0031570 | 177 | 8785 | 16881 | 1.205053 | 0.4049901 |
| GOTERM_MF_DIRECT | GO:0044822~poly(A) RNA binding | 23 | 11.4427861 | 0.0033938 | 177 | 1129 | 16881 | 1.942937 | 0.4049901 |
| GOTERM_MF_DIRECT | GO:0001540~beta-amyloid binding | 4 | 1.9900498 | 0.0052581 | 177 | 34 | 16881 | 11.220339 | 0.4471162 |
| GOTERM_MF_DIRECT | GO:0019904~protein domain specific binding | 8 | 3.9800995 | 0.0062446 | 177 | 208 | 16881 | 3.668188 | 0.4471162 |
| GOTERM_MF_DIRECT | GO:0051087~chaperone binding | 5 | 2.4875622 | 0.0101924 | 177 | 81 | 16881 | 5.887215 | 0.5021017 |
| GOTERM_MF_DIRECT | GO:0031625~ubiquitin protein ligase binding | 9 | 4.4776119 | 0.0105277 | 177 | 287 | 16881 | 2.990787 | 0.5021017 |
| GOTERM_MF_DIRECT | GO:0098641~cadherin binding involved in cell-cell adhesion | 9 | 4.4776119 | 0.0112201 | 177 | 290 | 16881 | 2.959848 | 0.5021017 |
| GOTERM_MF_DIRECT | GO:0004576~oligosaccharyl transferase activity | 2 | 0.9950249 | 0.0410596 | 177 | 4 | 16881 | 47.686441 | 1.0000000 |
| GOTERM_MF_DIRECT | GO:0005524~ATP binding | 23 | 11.4427861 | 0.0625561 | 177 | 1495 | 16881 | 1.467275 | 1.0000000 |
| GOTERM_MF_DIRECT | GO:1990247~N6-methyladenosine-containing RNA binding | 2 | 0.9950249 | 0.0707502 | 177 | 7 | 16881 | 27.249395 | 1.0000000 |
| GOTERM_MF_DIRECT | GO:0005049~nuclear export signal receptor activity | 2 | 0.9950249 | 0.0804425 | 177 | 8 | 16881 | 23.843220 | 1.0000000 |
| GOTERM_MF_DIRECT | GO:0004579~dolichyl-diphosphooligosaccharide-protein glycotransferase activity | 2 | 0.9950249 | 0.0900343 | 177 | 9 | 16881 | 21.193974 | 1.0000000 |
repl1kegg <- read.table("PMC6463127_repl_kegg.tsv",header=TRUE,sep="\t")
repl1kegg %>% kbl(caption="Downregulated KEGG pathways obtained using article gene list without background") %>% kable_paper("hover", full_width = F)| Category | Term | Count | X. | PValue | List.Total | Pop.Hits | Pop.Total | Fold.Enrichment | FDR |
|---|---|---|---|---|---|---|---|---|---|
| KEGG_PATHWAY | hsa04141:Protein processing in endoplasmic reticulum | 15 | 7.462687 | 0.0000000 | 94 | 169 | 6879 | 6.495342 | 0.0000055 |
| KEGG_PATHWAY | hsa03060:Protein export | 5 | 2.487562 | 0.0002277 | 94 | 23 | 6879 | 15.908881 | 0.0133206 |
| KEGG_PATHWAY | hsa03013:RNA transport | 8 | 3.980100 | 0.0084305 | 94 | 172 | 6879 | 3.403761 | 0.3287904 |
| KEGG_PATHWAY | hsa00563:Glycosylphosphatidylinositol(GPI)-anchor biosynthesis | 3 | 1.492537 | 0.0443501 | 94 | 25 | 6879 | 8.781702 | 1.0000000 |
| KEGG_PATHWAY | hsa00240:Pyrimidine metabolism | 5 | 2.487562 | 0.0473992 | 94 | 101 | 6879 | 3.622814 | 1.0000000 |
| KEGG_PATHWAY | hsa04612:Antigen processing and presentation | 4 | 1.990050 | 0.0831330 | 94 | 76 | 6879 | 3.851624 | 1.0000000 |
There is a supplement with all expressed genes (Supplementary Table S2) which I will use as a background. To get this to work, I will convert the gene symbols to Ensembl identifiers. There were only ~8000 expressed genes which is a bit low.
bgg <- readLines("PMC6463127_bgg.txt")
head(bgg)## [1] "UBE2Q2" "ATRX" "TCOF1" "NSRP1" "OPA1" "ITGA8"length(bgg)## [1] 8090geneinfo <- read.table("http://dee2.io/data/hsapiens/hsa_gene_info.tsv",header=TRUE)
bg <- geneinfo[which(geneinfo$GeneSymbol %in% bgg),"GeneID"]
bg <- bg[which(!is.na(bg))]
bg <- unique(bg)
length(bg)## [1] 8261writeLines(bg,"PMC6463127_bge.txt")Then I ran DAVID again using the correct background. Here is the result of the upregulated genes.
repl2up <- read.table("PMC6463127_repl_up2.tsv",header=TRUE,sep="\t")
repl2up %>% kbl(caption="Upregulated GO terms obtained using article gene list with a background") %>% kable_paper("hover", full_width = F)| Category | Term | Count | X. | PValue | List.Total | Pop.Hits | Pop.Total | Fold.Enrichment | FDR |
|---|---|---|---|---|---|---|---|---|---|
| GOTERM_BP_DIRECT | GO:0006351~transcription, DNA-templated | 14 | 30.434783 | 0.0003474 | 37 | 849 | 6549 | 2.918728 | 0.0538469 |
| GOTERM_BP_DIRECT | GO:0050684~regulation of mRNA processing | 3 | 6.521739 | 0.0010324 | 37 | 9 | 6549 | 59.000000 | 0.0800125 |
| GOTERM_BP_DIRECT | GO:0006325~chromatin organization | 3 | 6.521739 | 0.0057779 | 37 | 21 | 6549 | 25.285714 | 0.2985260 |
| GOTERM_BP_DIRECT | GO:0045893~positive regulation of transcription, DNA-templated | 6 | 13.043478 | 0.0091219 | 37 | 238 | 6549 | 4.462185 | 0.3534740 |
| GOTERM_BP_DIRECT | GO:0018026~peptidyl-lysine monomethylation | 2 | 4.347826 | 0.0325444 | 37 | 6 | 6549 | 59.000000 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0006355~regulation of transcription, DNA-templated | 8 | 17.391304 | 0.0512985 | 37 | 627 | 6549 | 2.258373 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0022604~regulation of cell morphogenesis | 2 | 4.347826 | 0.0895553 | 37 | 17 | 6549 | 20.823529 | 1.0000000 |
| GOTERM_BP_DIRECT | GO:0060070~canonical Wnt signaling pathway | 2 | 4.347826 | 0.0995640 | 37 | 19 | 6549 | 18.631579 | 1.0000000 |
| GOTERM_CC_DIRECT | GO:0005634~nucleus | 26 | 56.521739 | 0.0005510 | 41 | 2500 | 6954 | 1.763942 | 0.0391184 |
| GOTERM_CC_DIRECT | GO:0005654~nucleoplasm | 19 | 41.304348 | 0.0013439 | 41 | 1569 | 6954 | 2.053910 | 0.0477071 |
| GOTERM_MF_DIRECT | GO:0003676~nucleic acid binding | 10 | 21.739130 | 0.0004760 | 40 | 407 | 6600 | 4.054054 | 0.0395119 |
| GOTERM_MF_DIRECT | GO:0003682~chromatin binding | 6 | 13.043478 | 0.0080209 | 40 | 214 | 6600 | 4.626168 | 0.3004610 |
| GOTERM_MF_DIRECT | GO:0003713~transcription coactivator activity | 5 | 10.869565 | 0.0108600 | 40 | 148 | 6600 | 5.574324 | 0.3004610 |
| GOTERM_MF_DIRECT | GO:0000166~nucleotide binding | 5 | 10.869565 | 0.0201690 | 40 | 178 | 6600 | 4.634831 | 0.3479144 |
| GOTERM_MF_DIRECT | GO:0003677~DNA binding | 10 | 21.739130 | 0.0209587 | 40 | 715 | 6600 | 2.307692 | 0.3479144 |
| GOTERM_MF_DIRECT | GO:0016279~protein-lysine N-methyltransferase activity | 2 | 4.347826 | 0.0292071 | 40 | 5 | 6600 | 66.000000 | 0.3836217 |
| GOTERM_MF_DIRECT | GO:0003690~double-stranded DNA binding | 3 | 6.521739 | 0.0323536 | 40 | 48 | 6600 | 10.312500 | 0.3836217 |
| GOTERM_MF_DIRECT | GO:0003729~mRNA binding | 3 | 6.521739 | 0.0788914 | 40 | 79 | 6600 | 6.265823 | 0.7593817 |
| GOTERM_MF_DIRECT | GO:0003700~transcription factor activity, sequence-specific DNA binding | 6 | 13.043478 | 0.0823426 | 40 | 397 | 6600 | 2.493703 | 0.7593817 |
| GOTERM_MF_DIRECT | GO:0030145~manganese ion binding | 2 | 4.347826 | 0.0959542 | 40 | 17 | 6600 | 19.411765 | 0.7964195 |
repl2dn <- read.table("PMC6463127_repl_dn2.tsv",header=TRUE,sep="\t")
repl2dn %>% kbl(caption="Downregulated GO terms obtained using article gene list with a background") %>% kable_paper("hover", full_width = F)| Category | Term | Count | X. | PValue | List.Total | Pop.Hits | Pop.Total | Fold.Enrichment | FDR |
|---|---|---|---|---|---|---|---|---|---|
| GOTERM_BP_DIRECT | GO:0061077~chaperone-mediated protein folding | 5 | 2.48756218905473 | 0.001516300935472 | 180 | 19 | 6549 | 9.57456140350877 | 1 |
| GOTERM_BP_DIRECT | GO:0006465~signal peptide processing | 4 | 1.99004975124378 | 0.002091265338259 | 180 | 10 | 6549 | 14.5533333333333 | 1 |
| GOTERM_BP_DIRECT | GO:0002474~antigen processing and presentation of peptide antigen via MHC class I | 5 | 2.48756218905473 | 0.002684221501672 | 180 | 22 | 6549 | 8.26893939393939 | 1 |
| GOTERM_BP_DIRECT | GO:0006506~GPI anchor biosynthetic process | 4 | 1.99004975124378 | 0.003682368176522 | 180 | 12 | 6549 | 12.1277777777778 | 1 |
| GOTERM_BP_DIRECT | GO:0002576~platelet degranulation | 6 | 2.98507462686567 | 0.009544239799516 | 180 | 48 | 6549 | 4.54791666666667 | 1 |
| GOTERM_BP_DIRECT | GO:0018279~protein N-linked glycosylation via asparagine | 4 | 1.99004975124378 | 0.010294624191692 | 180 | 17 | 6549 | 8.56078431372549 | 1 |
| GOTERM_BP_DIRECT | GO:0044829~positive regulation by host of viral genome replication | 3 | 1.49253731343284 | 0.010365483557096 | 180 | 6 | 6549 | 18.1916666666667 | 1 |
| GOTERM_BP_DIRECT | GO:0036498~IRE1-mediated unfolded protein response | 5 | 2.48756218905473 | 0.01189211624059 | 180 | 33 | 6549 | 5.51262626262626 | 1 |
| GOTERM_BP_DIRECT | GO:0034975~protein folding in endoplasmic reticulum | 3 | 1.49253731343284 | 0.014252421785821 | 180 | 7 | 6549 | 15.5928571428571 | 1 |
| GOTERM_BP_DIRECT | GO:0050900~leukocyte migration | 6 | 2.98507462686567 | 0.015466993846307 | 180 | 54 | 6549 | 4.04259259259259 | 1 |
| GOTERM_BP_DIRECT | GO:0006509~membrane protein ectodomain proteolysis | 3 | 1.49253731343284 | 0.028940194386034 | 180 | 10 | 6549 | 10.915 | 1 |
| GOTERM_BP_DIRECT | GO:0008543~fibroblast growth factor receptor signaling pathway | 5 | 2.48756218905473 | 0.03366821498868 | 180 | 45 | 6549 | 4.04259259259259 | 1 |
| GOTERM_BP_DIRECT | GO:0006888~ER to Golgi vesicle-mediated transport | 7 | 3.48258706467662 | 0.043182440703185 | 180 | 94 | 6549 | 2.70939716312057 | 1 |
| GOTERM_BP_DIRECT | GO:0030148~sphingolipid biosynthetic process | 3 | 1.49253731343284 | 0.047551493070601 | 180 | 13 | 6549 | 8.39615384615385 | 1 |
| GOTERM_BP_DIRECT | GO:0006898~receptor-mediated endocytosis | 5 | 2.48756218905473 | 0.049851420767272 | 180 | 51 | 6549 | 3.56699346405229 | 1 |
| GOTERM_BP_DIRECT | GO:0006486~protein glycosylation | 4 | 1.99004975124378 | 0.051565964241758 | 180 | 31 | 6549 | 4.69462365591398 | 1 |
| GOTERM_BP_DIRECT | GO:0046135~pyrimidine nucleoside catabolic process | 2 | 0.99502487562189 | 0.053921833370183 | 180 | 2 | 6549 | 36.3833333333333 | 1 |
| GOTERM_BP_DIRECT | GO:1903298~negative regulation of hypoxia-induced intrinsic apoptotic signaling pathway | 2 | 0.99502487562189 | 0.053921833370183 | 180 | 2 | 6549 | 36.3833333333333 | 1 |
| GOTERM_BP_DIRECT | GO:0006611~protein export from nucleus | 3 | 1.49253731343284 | 0.061783100473787 | 180 | 15 | 6549 | 7.27666666666667 | 1 |
| GOTERM_BP_DIRECT | GO:0030433~ER-associated ubiquitin-dependent protein catabolic process | 4 | 1.99004975124378 | 0.06473405193018 | 180 | 34 | 6549 | 4.28039215686275 | 1 |
| GOTERM_BP_DIRECT | GO:0048208~COPII vesicle coating | 4 | 1.99004975124378 | 0.06473405193018 | 180 | 34 | 6549 | 4.28039215686275 | 1 |
| GOTERM_BP_DIRECT | GO:0006890~retrograde vesicle-mediated transport, Golgi to ER | 5 | 2.48756218905473 | 0.0660802396374 | 180 | 56 | 6549 | 3.2485119047619 | 1 |
| GOTERM_BP_DIRECT | GO:0006457~protein folding | 7 | 3.48258706467662 | 0.072115803486644 | 180 | 107 | 6549 | 2.38021806853583 | 1 |
| GOTERM_BP_DIRECT | GO:0007283~spermatogenesis | 8 | 3.98009950248756 | 0.076014012970454 | 180 | 135 | 6549 | 2.15604938271605 | 1 |
| GOTERM_BP_DIRECT | GO:0060907~positive regulation of macrophage cytokine production | 2 | 0.99502487562189 | 0.079788335863835 | 180 | 3 | 6549 | 24.2555555555556 | 1 |
| GOTERM_BP_DIRECT | GO:0048205~COPI coating of Golgi vesicle | 2 | 0.99502487562189 | 0.079788335863835 | 180 | 3 | 6549 | 24.2555555555556 | 1 |
| GOTERM_BP_DIRECT | GO:0060323~head morphogenesis | 2 | 0.99502487562189 | 0.079788335863835 | 180 | 3 | 6549 | 24.2555555555556 | 1 |
| GOTERM_BP_DIRECT | GO:0042985~negative regulation of amyloid precursor protein biosynthetic process | 2 | 0.99502487562189 | 0.079788335863835 | 180 | 3 | 6549 | 24.2555555555556 | 1 |
| GOTERM_BP_DIRECT | GO:1990000~amyloid fibril formation | 2 | 0.99502487562189 | 0.079788335863835 | 180 | 3 | 6549 | 24.2555555555556 | 1 |
| GOTERM_BP_DIRECT | GO:0001843~neural tube closure | 4 | 1.99004975124378 | 0.084425030786951 | 180 | 38 | 6549 | 3.82982456140351 | 1 |
| GOTERM_BP_DIRECT | GO:0031647~regulation of protein stability | 4 | 1.99004975124378 | 0.089704619119023 | 180 | 39 | 6549 | 3.73162393162393 | 1 |
| GOTERM_BP_DIRECT | GO:0006874~cellular calcium ion homeostasis | 4 | 1.99004975124378 | 0.089704619119023 | 180 | 39 | 6549 | 3.73162393162393 | 1 |
| GOTERM_BP_DIRECT | GO:0006810~transport | 7 | 3.48258706467662 | 0.094363198891799 | 180 | 115 | 6549 | 2.21463768115942 | 1 |
| GOTERM_BP_DIRECT | GO:0010951~negative regulation of endopeptidase activity | 4 | 1.99004975124378 | 0.095119139763482 | 180 | 40 | 6549 | 3.63833333333333 | 1 |
| GOTERM_BP_DIRECT | GO:0055114~oxidation-reduction process | 10 | 4.97512437810945 | 0.096297131545988 | 180 | 200 | 6549 | 1.81916666666667 | 1 |
| Category | Term | Count | % | PValue | List Total | Pop Hits | Pop Total | Fold Enrichment | FDR |
| GOTERM_CC_DIRECT | GO:0005789~endoplasmic reticulum membrane | 36 | 17.910447761194 | 5.18600202099025E-13 | 191 | 315 | 6954 | 4.16095736724009 | 1.46763857194024E-10 |
| GOTERM_CC_DIRECT | GO:0070062~extracellular exosome | 79 | 39.3034825870647 | 5.83737688084654E-12 | 191 | 1369 | 6954 | 2.10099472615392 | 8.25988828639785E-10 |
| GOTERM_CC_DIRECT | GO:0016020~membrane | 64 | 31.8407960199005 | 3.82161832432916E-09 | 191 | 1124 | 6954 | 2.0730748448883 | 3.60505995261718E-07 |
| GOTERM_CC_DIRECT | GO:0016021~integral component of membrane | 78 | 38.8059701492537 | 6.16342444655258E-09 | 191 | 1541 | 6954 | 1.84286398646422 | 4.36062279593595E-07 |
| GOTERM_CC_DIRECT | GO:0005783~endoplasmic reticulum | 31 | 15.4228855721393 | 2.58620942152547E-08 | 191 | 357 | 6954 | 3.16151172510889 | 1.46379453258342E-06 |
| GOTERM_CC_DIRECT | GO:0042470~melanosome | 12 | 5.97014925373134 | 1.98794384552135E-07 | 191 | 55 | 6954 | 7.9436458829129 | 9.37646847137571E-06 |
| GOTERM_CC_DIRECT | GO:0005790~smooth endoplasmic reticulum | 6 | 2.98507462686567 | 6.84157771985112E-05 | 191 | 17 | 6954 | 12.8500153988297 | 0.002765952135311 |
| GOTERM_CC_DIRECT | GO:0005887~integral component of plasma membrane | 23 | 11.4427860696517 | 0.000616529701599 | 191 | 379 | 6954 | 2.20947934078382 | 0.021809738194058 |
| GOTERM_CC_DIRECT | GO:0071556~integral component of lumenal side of endoplasmic reticulum membrane | 5 | 2.48756218905473 | 0.00151655120228 | 191 | 19 | 6954 | 9.58115183246073 | 0.045266698723216 |
| GOTERM_CC_DIRECT | GO:0005615~extracellular space | 24 | 11.9402985074627 | 0.00159952999022 | 191 | 434 | 6954 | 2.0133664680194 | 0.045266698723216 |
| GOTERM_CC_DIRECT | GO:0005788~endoplasmic reticulum lumen | 8 | 3.98009950248756 | 0.002028783742832 | 191 | 66 | 6954 | 4.41313660161828 | 0.052195072656502 |
| GOTERM_CC_DIRECT | GO:0033116~endoplasmic reticulum-Golgi intermediate compartment membrane | 5 | 2.48756218905473 | 0.005038697420568 | 191 | 26 | 6954 | 7.00161095449054 | 0.118829280835062 |
| GOTERM_CC_DIRECT | GO:0005794~Golgi apparatus | 21 | 10.4477611940299 | 0.008967844208267 | 191 | 414 | 6954 | 1.84680173002504 | 0.188779515460879 |
| GOTERM_CC_DIRECT | GO:0016324~apical plasma membrane | 9 | 4.47761194029851 | 0.010214170577938 | 191 | 110 | 6954 | 2.97886720609234 | 0.188779515460879 |
| GOTERM_CC_DIRECT | GO:0030660~Golgi-associated vesicle membrane | 3 | 1.49253731343284 | 0.010360970919022 | 191 | 6 | 6954 | 18.2041884816754 | 0.188779515460879 |
| GOTERM_CC_DIRECT | GO:0005793~endoplasmic reticulum-Golgi intermediate compartment | 5 | 2.48756218905473 | 0.010673046810509 | 191 | 32 | 6954 | 5.68880890052356 | 0.188779515460879 |
| GOTERM_CC_DIRECT | GO:0034663~endoplasmic reticulum chaperone complex | 3 | 1.49253731343284 | 0.018656204213232 | 191 | 8 | 6954 | 13.6531413612565 | 0.300504043611152 |
| GOTERM_CC_DIRECT | GO:0000139~Golgi membrane | 14 | 6.96517412935323 | 0.019113331395762 | 191 | 250 | 6954 | 2.03886910994764 | 0.300504043611152 |
| GOTERM_CC_DIRECT | GO:0009986~cell surface | 12 | 5.97014925373134 | 0.022225286658766 | 191 | 202 | 6954 | 2.16287387901094 | 0.318098424365239 |
| GOTERM_CC_DIRECT | GO:0005925~focal adhesion | 14 | 6.96517412935323 | 0.022750880621066 | 191 | 256 | 6954 | 1.99108311518325 | 0.318098424365239 |
| GOTERM_CC_DIRECT | GO:0005765~lysosomal membrane | 9 | 4.47761194029851 | 0.023604476719682 | 191 | 128 | 6954 | 2.5599640052356 | 0.318098424365239 |
| GOTERM_CC_DIRECT | GO:0043202~lysosomal lumen | 5 | 2.48756218905473 | 0.026922203942072 | 191 | 42 | 6954 | 4.33433059087509 | 0.341098862933122 |
| GOTERM_CC_DIRECT | GO:0009897~external side of plasma membrane | 6 | 2.98507462686567 | 0.028285532364902 | 191 | 63 | 6954 | 3.46746447270007 | 0.341098862933122 |
| GOTERM_CC_DIRECT | GO:0005771~multivesicular body | 3 | 1.49253731343284 | 0.028927112050866 | 191 | 10 | 6954 | 10.9225130890052 | 0.341098862933122 |
| GOTERM_CC_DIRECT | GO:0030176~integral component of endoplasmic reticulum membrane | 5 | 2.48756218905473 | 0.03131526984236 | 191 | 44 | 6954 | 4.13731556401713 | 0.35448885461551 |
| GOTERM_CC_DIRECT | GO:0032580~Golgi cisterna membrane | 4 | 1.99004975124378 | 0.03289908091304 | 191 | 26 | 6954 | 5.60128876359243 | 0.358093842245784 |
| GOTERM_CC_DIRECT | GO:0000792~heterochromatin | 3 | 1.49253731343284 | 0.047529470029043 | 191 | 13 | 6954 | 8.40193314538864 | 0.478885341810709 |
| GOTERM_CC_DIRECT | GO:0016323~basolateral plasma membrane | 6 | 2.98507462686567 | 0.048735221298123 | 191 | 73 | 6954 | 2.99246933945349 | 0.478885341810709 |
| GOTERM_CC_DIRECT | GO:0005764~lysosome | 7 | 3.48258706467662 | 0.049073056227953 | 191 | 97 | 6954 | 2.62740864683975 | 0.478885341810709 |
| GOTERM_CC_DIRECT | GO:0031090~organelle membrane | 3 | 1.49253731343284 | 0.054475923680904 | 191 | 14 | 6954 | 7.80179506357517 | 0.513889546723193 |
| GOTERM_CC_DIRECT | GO:0005886~plasma membrane | 50 | 24.8756218905473 | 0.073754428250327 | 191 | 1477 | 6954 | 1.23251106849529 | 0.656669092817492 |
| GOTERM_CC_DIRECT | GO:0012507~ER to Golgi transport vesicle membrane | 4 | 1.99004975124378 | 0.074252335583603 | 191 | 36 | 6954 | 4.04537521815009 | 0.656669092817492 |
| GOTERM_CC_DIRECT | GO:0005769~early endosome | 7 | 3.48258706467662 | 0.097358105059117 | 191 | 116 | 6954 | 2.19705723054703 | 0.83491950702212 |
| Category | Term | Count | % | PValue | List Total | Pop Hits | Pop Total | Fold Enrichment | FDR |
| GOTERM_MF_DIRECT | GO:0001540~beta-amyloid binding | 4 | 1.99004975124378 | 0.004239029699521 | 175 | 13 | 6600 | 11.6043956043956 | 1 |
| GOTERM_MF_DIRECT | GO:0051082~unfolded protein binding | 7 | 3.48258706467662 | 0.010013545360428 | 175 | 70 | 6600 | 3.77142857142857 | 1 |
| GOTERM_MF_DIRECT | GO:0051087~chaperone binding | 5 | 2.48756218905473 | 0.022127904591892 | 175 | 41 | 6600 | 4.5993031358885 | 1 |
| GOTERM_MF_DIRECT | GO:0019904~protein domain specific binding | 8 | 3.98009950248756 | 0.037273359782583 | 175 | 119 | 6600 | 2.53541416566627 | 1 |
| GOTERM_MF_DIRECT | GO:0031625~ubiquitin protein ligase binding | 9 | 4.47761194029851 | 0.041344973173309 | 175 | 148 | 6600 | 2.29343629343629 | 1 |
| GOTERM_MF_DIRECT | GO:0030246~carbohydrate binding | 5 | 2.48756218905473 | 0.041992305737711 | 175 | 50 | 6600 | 3.77142857142857 | 1 |
| GOTERM_MF_DIRECT | GO:0008236~serine-type peptidase activity | 3 | 1.49253731343284 | 0.065110296796912 | 175 | 16 | 6600 | 7.07142857142857 | 1 |
repl2kegg <- read.table("PMC6463127_repl_kegg2.tsv",header=TRUE,sep="\t")
repl2kegg %>% kbl(caption="Downregulated KEGG pathways obtained using article gene list with a background") %>% kable_paper("hover", full_width = F)| Category | Term | Count | X. | PValue | Genes | List.Total | Pop.Hits | Pop.Total | Fold.Enrichment | Bonferroni | Benjamini | FDR |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| KEGG_PATHWAY | hsa04141:Protein processing in endoplasmic reticulum | 15 | 7.462687 | 0.0000000 | PDIA3, BCAP31, XBP1, SEC13, EIF2AK1, RPN1, PDIA6, DNAJA1, SEC61G, CANX, STT3A, HYOU1, CALR, UGGT2, SEC63 | 94 | 169 | 6879 | 6.495342 | 0.0000055 | 0.0000055 | 0.0000055 |
| KEGG_PATHWAY | hsa03060:Protein export | 5 | 2.487562 | 0.0002277 | SPCS2, SEC61G, SEC11A, SEC11C, SEC63 | 94 | 23 | 6879 | 15.908881 | 0.0262925 | 0.0133206 | 0.0133206 |
| KEGG_PATHWAY | hsa03013:RNA transport | 8 | 3.980100 | 0.0084305 | SEC13, NUP205, EIF2S3, XPO1, SUMO1, STRAP, EIF3E, RGPD2 | 94 | 172 | 6879 | 3.403761 | 0.6286286 | 0.3287904 | 0.3287904 |
| KEGG_PATHWAY | hsa00563:Glycosylphosphatidylinositol(GPI)-anchor biosynthesis | 3 | 1.492537 | 0.0443501 | PIGU, PIGP, PIGG | 94 | 25 | 6879 | 8.781702 | 0.9950459 | 1.0000000 | 1.0000000 |
| KEGG_PATHWAY | hsa00240:Pyrimidine metabolism | 5 | 2.487562 | 0.0473992 | NT5C3A, NME7, DPYD, CTPS2, POLR2H | 94 | 101 | 6879 | 3.622814 | 0.9965914 | 1.0000000 | 1.0000000 |
| KEGG_PATHWAY | hsa04612:Antigen processing and presentation | 4 | 1.990050 | 0.0831330 | PDIA3, CD74, CANX, CALR | 94 | 76 | 6879 | 3.851624 | 0.9999611 | 1.0000000 | 1.0000000 |
Here I’m using the enrichGO function to analyse the data. The algorithm is slightly different and the gene sets might be a different version. First GO biological processes.
# from supp table 2
upg <- readLines("PMC6463127_up.txt")
dng <- readLines("PMC6463127_dn.txt")
upe <- geneinfo[which(geneinfo$GeneSymbol %in% upg),"GeneID"]
dne <- geneinfo[which(geneinfo$GeneSymbol %in% dng),"GeneID"]
# up
go_up <- enrichGO(gene = upe, keyType = "ENSEMBL", universe = bg,
OrgDb = org.Hs.eg.db, ont = "ALL", pAdjustMethod = "BH", pvalueCutoff = 0.05,
qvalueCutoff = 1, readable = TRUE)
go_up <- data.frame(go_up)
nrow(go_up)## [1] 9go_up <- subset(go_up,qvalue<0.05)
nrow(go_up)## [1] 9go_up[,c(1,3:7)] %>% kbl(caption="Upregulated GO terms obtained using clusterProfiler with the correct background") %>% kable_paper("hover", full_width = F)| ONTOLOGY | Description | GeneRatio | BgRatio | pvalue | p.adjust | |
|---|---|---|---|---|---|---|
| GO:0000137 | CC | Golgi cis cisterna | 3/43 | 19/7347 | 0.0001695 | 0.0169537 |
| GO:0018024 | MF | histone-lysine N-methyltransferase activity | 3/41 | 26/7074 | 0.0004284 | 0.0192526 |
| GO:0016278 | MF | lysine N-methyltransferase activity | 3/41 | 29/7074 | 0.0005948 | 0.0192526 |
| GO:0016279 | MF | protein-lysine N-methyltransferase activity | 3/41 | 29/7074 | 0.0005948 | 0.0192526 |
| GO:0042054 | MF | histone methyltransferase activity | 3/41 | 30/7074 | 0.0006582 | 0.0192526 |
| GO:0000977 | MF | RNA polymerase II transcription regulatory region sequence-specific DNA binding | 9/41 | 486/7074 | 0.0015283 | 0.0339253 |
| GO:0008276 | MF | protein methyltransferase activity | 3/41 | 43/7074 | 0.0018989 | 0.0339253 |
| GO:0008170 | MF | N-methyltransferase activity | 3/41 | 44/7074 | 0.0020297 | 0.0339253 |
| GO:0000987 | MF | cis-regulatory region sequence-specific DNA binding | 8/41 | 429/7074 | 0.0027810 | 0.0406717 |
# dn
go_dn <- enrichGO(gene = dne, keyType = "ENSEMBL", universe = bg,
OrgDb = org.Hs.eg.db, ont = "ALL", pAdjustMethod = "BH", pvalueCutoff = 0.05,
qvalueCutoff = 1, readable = TRUE)
go_dn <- data.frame(go_dn)
nrow(go_dn)## [1] 50go_dn <- subset(go_dn,qvalue<0.05)
nrow(go_dn)## [1] 50go_dn[,c(1,3:7)] %>% kbl(caption="Downregulated GO terms obtained using clusterProfiler with the correct background") %>% kable_paper("hover", full_width = F)| ONTOLOGY | Description | GeneRatio | BgRatio | pvalue | p.adjust | |
|---|---|---|---|---|---|---|
| GO:0006643 | BP | membrane lipid metabolic process | 12/195 | 72/7108 | 0.0000005 | 0.0007070 |
| GO:0034976 | BP | response to endoplasmic reticulum stress | 17/195 | 151/7108 | 0.0000007 | 0.0007070 |
| GO:1901135 | BP | carbohydrate derivative metabolic process | 31/195 | 437/7108 | 0.0000008 | 0.0007070 |
| GO:0006664 | BP | glycolipid metabolic process | 7/195 | 35/7108 | 0.0000368 | 0.0190877 |
| GO:1903509 | BP | liposaccharide metabolic process | 7/195 | 35/7108 | 0.0000368 | 0.0190877 |
| GO:0002576 | BP | platelet degranulation | 9/195 | 62/7108 | 0.0000422 | 0.0190877 |
| GO:0035966 | BP | response to topologically incorrect protein | 12/195 | 117/7108 | 0.0000797 | 0.0308871 |
| GO:0055080 | BP | cation homeostasis | 19/195 | 266/7108 | 0.0001167 | 0.0395827 |
| GO:0098771 | BP | inorganic ion homeostasis | 19/195 | 270/7108 | 0.0001421 | 0.0428423 |
| GO:0042175 | CC | nuclear outer membrane-endoplasmic reticulum membrane network | 50/198 | 454/7347 | 0.0000000 | 0.0000000 |
| GO:0005789 | CC | endoplasmic reticulum membrane | 49/198 | 446/7347 | 0.0000000 | 0.0000000 |
| GO:0098827 | CC | endoplasmic reticulum subcompartment | 49/198 | 448/7347 | 0.0000000 | 0.0000000 |
| GO:0140534 | CC | endoplasmic reticulum protein-containing complex | 14/198 | 74/7347 | 0.0000000 | 0.0000007 |
| GO:0042470 | CC | melanosome | 12/198 | 59/7347 | 0.0000000 | 0.0000023 |
| GO:0048770 | CC | pigment granule | 12/198 | 59/7347 | 0.0000000 | 0.0000023 |
| GO:0030667 | CC | secretory granule membrane | 16/198 | 130/7347 | 0.0000003 | 0.0000176 |
| GO:0030176 | CC | integral component of endoplasmic reticulum membrane | 12/198 | 80/7347 | 0.0000013 | 0.0000569 |
| GO:0031227 | CC | intrinsic component of endoplasmic reticulum membrane | 12/198 | 84/7347 | 0.0000022 | 0.0000862 |
| GO:0030659 | CC | cytoplasmic vesicle membrane | 24/198 | 320/7347 | 0.0000046 | 0.0001641 |
| GO:0016324 | CC | apical plasma membrane | 14/198 | 125/7347 | 0.0000059 | 0.0001935 |
| GO:0031301 | CC | integral component of organelle membrane | 16/198 | 164/7347 | 0.0000078 | 0.0002144 |
| GO:0012506 | CC | vesicle membrane | 24/198 | 330/7347 | 0.0000078 | 0.0002144 |
| GO:0031300 | CC | intrinsic component of organelle membrane | 16/198 | 173/7347 | 0.0000154 | 0.0003933 |
| GO:0098576 | CC | lumenal side of membrane | 6/198 | 23/7347 | 0.0000244 | 0.0005834 |
| GO:0045177 | CC | apical part of cell | 15/198 | 161/7347 | 0.0000262 | 0.0005868 |
| GO:0005793 | CC | endoplasmic reticulum-Golgi intermediate compartment | 9/198 | 63/7347 | 0.0000420 | 0.0008842 |
| GO:0030141 | CC | secretory granule | 24/198 | 378/7347 | 0.0000727 | 0.0014454 |
| GO:0005798 | CC | Golgi-associated vesicle | 7/198 | 42/7347 | 0.0001125 | 0.0019690 |
| GO:0071556 | CC | integral component of lumenal side of endoplasmic reticulum membrane | 5/198 | 19/7347 | 0.0001155 | 0.0019690 |
| GO:0098553 | CC | lumenal side of endoplasmic reticulum membrane | 5/198 | 19/7347 | 0.0001155 | 0.0019690 |
| GO:0033116 | CC | endoplasmic reticulum-Golgi intermediate compartment membrane | 6/198 | 31/7347 | 0.0001489 | 0.0024236 |
| GO:0031226 | CC | intrinsic component of plasma membrane | 27/198 | 486/7347 | 0.0002406 | 0.0037456 |
| GO:0005887 | CC | integral component of plasma membrane | 26/198 | 464/7347 | 0.0002769 | 0.0041297 |
| GO:0005788 | CC | endoplasmic reticulum lumen | 11/198 | 122/7347 | 0.0004247 | 0.0060822 |
| GO:0005768 | CC | endosome | 24/198 | 431/7347 | 0.0005287 | 0.0072802 |
| GO:0005790 | CC | smooth endoplasmic reticulum | 4/198 | 16/7347 | 0.0007221 | 0.0093833 |
| GO:0099503 | CC | secretory vesicle | 24/198 | 441/7347 | 0.0007339 | 0.0093833 |
| GO:0098552 | CC | side of membrane | 14/198 | 206/7347 | 0.0012922 | 0.0159514 |
| GO:0000139 | CC | Golgi membrane | 19/198 | 335/7347 | 0.0016595 | 0.0198039 |
| GO:0098554 | CC | cytoplasmic side of endoplasmic reticulum membrane | 3/198 | 10/7347 | 0.0020114 | 0.0232284 |
| GO:0098590 | CC | plasma membrane region | 23/198 | 453/7347 | 0.0023749 | 0.0265688 |
| GO:0042827 | CC | platelet dense granule | 3/198 | 12/7347 | 0.0035435 | 0.0373112 |
| GO:0044322 | CC | endoplasmic reticulum quality control compartment | 3/198 | 12/7347 | 0.0035435 | 0.0373112 |
| GO:0030660 | CC | Golgi-associated vesicle membrane | 4/198 | 25/7347 | 0.0041515 | 0.0424641 |
| GO:0098791 | CC | Golgi apparatus subcompartment | 20/198 | 395/7347 | 0.0047031 | 0.0467693 |
| GO:0000323 | CC | lytic vacuole | 17/198 | 318/7347 | 0.0052886 | 0.0498238 |
| GO:0005764 | CC | lysosome | 17/198 | 318/7347 | 0.0052886 | 0.0498238 |
| GO:1904680 | MF | peptide transmembrane transporter activity | 5/189 | 16/7074 | 0.0000444 | 0.0159612 |
| GO:0042887 | MF | amide transmembrane transporter activity | 5/189 | 18/7074 | 0.0000833 | 0.0159612 |
| GO:0008509 | MF | anion transmembrane transporter activity | 11/189 | 113/7074 | 0.0002002 | 0.0255593 |
Now KEGG analysis but need to convert to entrez first
dn_entrez <- unlist(mget(dne, org.Hs.egENSEMBL2EG, ifnotfound = NA))
bg_entrez <- unlist(mget(bg, org.Hs.egENSEMBL2EG, ifnotfound = NA))
kegg <- enrichKEGG(gene = dn_entrez, universe = bg_entrez,
organism = "hsa", pAdjustMethod = "BH", pvalueCutoff = 0.05,
qvalueCutoff = 1)## Reading KEGG annotation online:
##
## Reading KEGG annotation online:kegg <- as.data.frame(kegg)
kegg[,c(2:7)] %>% kbl(caption="Clusterprofiler KEGG results obtained using the correct background") %>% kable_paper("hover", full_width = F)| Description | GeneRatio | BgRatio | pvalue | p.adjust | qvalue | |
|---|---|---|---|---|---|---|
| hsa04141 | Protein processing in endoplasmic reticulum | 15/107 | 112/3405 | 0.0000013 | 0.0001956 | 0.0001918 |
| hsa03060 | Protein export | 5/107 | 17/3405 | 0.0001278 | 0.0093268 | 0.0091453 |
In the article, they obtained 45 upregulated and 78 downregulated GO terms, along with 5 downregulated KEGG pathways.
The authors don’t specifically draw any conclusions from the enrichment analysis, but let the reader come to their own conclusions on the basis of the results tables presented.
Anyway, we can look at whether the top results were confirmed with a corrected method.
I repeated the analysis without a background and received the same upregulated GO terms as the article (16 terms with p<0.05), but of these, only 5 gave an FDR<0.05.
When a correct background gene list was used, only 2 GOs were upregulated with FDR<0.05. Basically, there were no significantly upregulated GO BP or MF terms when using a correct background and FDR control. Using a clusterProfiler script, I got 9 upregulated GOs, 8 of which were different to the DAVID results.
sessionInfo()## R version 4.1.2 (2021-11-01)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 20.04.3 LTS
##
## Matrix products: default
## BLAS: /usr/lib/x86_64-linux-gnu/blas/libblas.so.3.9.0
## LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.9.0
##
## locale:
## [1] LC_CTYPE=en_AU.UTF-8 LC_NUMERIC=C
## [3] LC_TIME=en_AU.UTF-8 LC_COLLATE=en_AU.UTF-8
## [5] LC_MONETARY=en_AU.UTF-8 LC_MESSAGES=en_AU.UTF-8
## [7] LC_PAPER=en_AU.UTF-8 LC_NAME=C
## [9] LC_ADDRESS=C LC_TELEPHONE=C
## [11] LC_MEASUREMENT=en_AU.UTF-8 LC_IDENTIFICATION=C
##
## attached base packages:
## [1] parallel stats4 stats graphics grDevices utils datasets
## [8] methods base
##
## other attached packages:
## [1] eulerr_6.1.1 kableExtra_1.3.4
## [3] mitch_1.4.1 org.Hs.eg.db_3.13.0
## [5] AnnotationDbi_1.54.1 clusterProfiler_4.0.5
## [7] DESeq2_1.32.0 SummarizedExperiment_1.22.0
## [9] Biobase_2.52.0 MatrixGenerics_1.4.3
## [11] matrixStats_0.61.0 GenomicRanges_1.44.0
## [13] GenomeInfoDb_1.28.4 IRanges_2.26.0
## [15] S4Vectors_0.30.2 BiocGenerics_0.38.0
## [17] getDEE2_1.2.0
##
## loaded via a namespace (and not attached):
## [1] shadowtext_0.0.9 fastmatch_1.1-3 systemfonts_1.0.3
## [4] plyr_1.8.6 igraph_1.2.8 lazyeval_0.2.2
## [7] splines_4.1.2 BiocParallel_1.26.2 ggplot2_3.3.5
## [10] digest_0.6.28 yulab.utils_0.0.4 htmltools_0.5.2
## [13] GOSemSim_2.18.1 viridis_0.6.2 GO.db_3.13.0
## [16] fansi_0.5.0 magrittr_2.0.1 memoise_2.0.0
## [19] Biostrings_2.60.2 annotate_1.70.0 graphlayouts_0.7.1
## [22] svglite_2.0.0 enrichplot_1.12.3 colorspace_2.0-2
## [25] rvest_1.0.2 blob_1.2.2 ggrepel_0.9.1
## [28] xfun_0.28 dplyr_1.0.7 crayon_1.4.2
## [31] RCurl_1.98-1.5 jsonlite_1.7.2 scatterpie_0.1.7
## [34] genefilter_1.74.1 survival_3.2-13 ape_5.5
## [37] glue_1.5.0 polyclip_1.10-0 gtable_0.3.0
## [40] zlibbioc_1.38.0 XVector_0.32.0 webshot_0.5.2
## [43] htm2txt_2.1.1 DelayedArray_0.18.0 scales_1.1.1
## [46] DOSE_3.18.3 DBI_1.1.1 GGally_2.1.2
## [49] Rcpp_1.0.7 viridisLite_0.4.0 xtable_1.8-4
## [52] gridGraphics_0.5-1 tidytree_0.3.6 bit_4.0.4
## [55] htmlwidgets_1.5.4 httr_1.4.2 fgsea_1.18.0
## [58] gplots_3.1.1 RColorBrewer_1.1-2 ellipsis_0.3.2
## [61] pkgconfig_2.0.3 reshape_0.8.8 XML_3.99-0.8
## [64] farver_2.1.0 sass_0.4.0 locfit_1.5-9.4
## [67] utf8_1.2.2 ggplotify_0.1.0 tidyselect_1.1.1
## [70] rlang_0.4.12 reshape2_1.4.4 later_1.3.0
## [73] munsell_0.5.0 tools_4.1.2 cachem_1.0.6
## [76] downloader_0.4 generics_0.1.1 RSQLite_2.2.8
## [79] evaluate_0.14 stringr_1.4.0 fastmap_1.1.0
## [82] yaml_2.2.1 ggtree_3.0.4 knitr_1.36
## [85] bit64_4.0.5 tidygraph_1.2.0 caTools_1.18.2
## [88] purrr_0.3.4 KEGGREST_1.32.0 ggraph_2.0.5
## [91] nlme_3.1-153 mime_0.12 aplot_0.1.1
## [94] xml2_1.3.2 DO.db_2.9 rstudioapi_0.13
## [97] compiler_4.1.2 beeswarm_0.4.0 png_0.1-7
## [100] treeio_1.16.2 tibble_3.1.6 tweenr_1.0.2
## [103] geneplotter_1.70.0 bslib_0.3.1 stringi_1.7.5
## [106] highr_0.9 lattice_0.20-45 Matrix_1.3-4
## [109] vctrs_0.3.8 pillar_1.6.4 lifecycle_1.0.1
## [112] jquerylib_0.1.4 data.table_1.14.2 cowplot_1.1.1
## [115] bitops_1.0-7 httpuv_1.6.3 patchwork_1.1.1
## [118] qvalue_2.24.0 R6_2.5.1 promises_1.2.0.1
## [121] KernSmooth_2.23-20 echarts4r_0.4.2 gridExtra_2.3
## [124] gtools_3.9.2 MASS_7.3-54 assertthat_0.2.1
## [127] GenomeInfoDbData_1.2.6 grid_4.1.2 ggfun_0.0.4
## [130] tidyr_1.1.4 rmarkdown_2.11 ggforce_0.3.3
## [133] shiny_1.7.1