Differential Gene Expression Analysis
Last updated: 2025-02-06
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Knit directory: CX5461_Project/
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| Rmd | 3f764ee | sayanpaul01 | 2025-02-06 | Added DGE Analysis with structured chunks |
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| Rmd | d31730d | sayanpaul01 | 2025-02-06 | Added DGE Analysis with structured chunks |
📌 Differential Gene Expression Analysis
1️⃣ Load Required Libraries
# Load necessary packages
library(edgeR)Warning: package 'edgeR' was built under R version 4.3.1Warning: package 'limma' was built under R version 4.3.1library(limma)
library(data.table)Warning: package 'data.table' was built under R version 4.3.2library(tidyverse)Warning: package 'tidyverse' was built under R version 4.3.2Warning: package 'ggplot2' was built under R version 4.3.3Warning: package 'tidyr' was built under R version 4.3.3Warning: package 'readr' was built under R version 4.3.3Warning: package 'purrr' was built under R version 4.3.1Warning: package 'dplyr' was built under R version 4.3.2Warning: package 'stringr' was built under R version 4.3.2Warning: package 'lubridate' was built under R version 4.3.1library(ggplot2)
library(dplyr)
library(scales)Warning: package 'scales' was built under R version 4.3.2library(biomaRt)Warning: package 'biomaRt' was built under R version 4.3.2library(Homo.sapiens)Warning: package 'AnnotationDbi' was built under R version 4.3.2Warning: package 'BiocGenerics' was built under R version 4.3.1Warning: package 'Biobase' was built under R version 4.3.1Warning: package 'IRanges' was built under R version 4.3.1Warning: package 'S4Vectors' was built under R version 4.3.1Warning: package 'OrganismDbi' was built under R version 4.3.1Warning: package 'GenomicFeatures' was built under R version 4.3.3Warning: package 'GenomeInfoDb' was built under R version 4.3.3Warning: package 'GenomicRanges' was built under R version 4.3.1📍 Load Data
📌 Normalize and Filter Counts
📌 Prepare DGEList Object
📌 Create Design Matrix
# ✅ Create Design Matrix
design <- model.matrix(~ 0 + Metadata_2$Drug_time)
colnames(design) <- gsub("Metadata_2\\$Drug_time", "", colnames(design))
design CX.5461_0.1_24 CX.5461_0.1_3 CX.5461_0.1_48 CX.5461_0.5_24 CX.5461_0.5_3
1 0 1 0 0 0
2 0 0 0 0 0
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4 0 0 0 0 0
5 0 0 0 0 0
6 0 0 1 0 0
7 0 0 0 0 0
8 0 0 0 0 0
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13 0 0 0 0 0
14 0 0 1 0 0
15 0 0 0 0 0
16 0 0 0 0 0
17 0 0 0 0 0
18 0 0 0 0 0
19 0 0 0 0 0
20 0 1 0 0 0
21 0 0 0 0 1
22 0 0 0 0 1
23 0 0 0 0 0
24 0 0 0 0 0
25 0 0 0 0 0
26 0 0 0 0 0
27 1 0 0 0 0
28 0 0 0 1 0
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30 0 0 0 0 0
31 0 0 0 0 0
32 0 0 0 0 0
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34 0 0 1 0 0
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37 0 0 0 0 0
38 0 0 0 0 0
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40 0 1 0 0 0
41 0 0 0 0 1
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45 0 0 0 0 0
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47 1 0 0 0 0
48 0 0 0 1 0
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55 0 0 0 0 0
56 0 0 0 0 0
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58 0 0 0 0 0
59 0 0 0 0 0
60 0 1 0 0 0
61 0 0 0 0 1
62 0 0 0 0 0
63 0 0 0 0 0
64 0 0 0 0 0
65 0 0 0 0 0
66 0 0 0 0 0
67 1 0 0 0 0
68 0 0 0 1 0
69 0 0 0 0 0
70 0 0 0 0 0
71 0 0 0 0 0
72 0 0 0 0 0
73 0 0 1 0 0
74 0 0 0 0 0
75 0 0 0 0 0
76 1 0 0 0 0
77 0 0 0 0 0
78 0 0 0 0 0
79 0 0 0 0 0
80 0 1 0 0 0
81 0 0 0 0 1
82 0 0 0 0 0
83 0 0 0 0 0
84 0 0 0 0 0
85 0 0 0 0 0
86 1 0 0 0 0
87 0 0 0 1 0
88 0 0 0 1 0
89 0 0 0 0 0
90 0 0 0 0 0
91 0 0 0 0 0
92 0 0 0 0 0
93 0 0 1 0 0
94 0 0 0 0 0
95 0 0 0 0 0
96 0 0 0 0 0
97 0 0 0 0 0
98 0 0 0 0 0
99 0 0 0 0 0
100 0 1 0 0 0
101 0 0 0 0 1
102 0 0 0 0 0
103 0 0 0 0 0
104 0 0 0 0 0
105 0 0 0 0 0
106 1 0 0 0 0
107 0 0 0 1 0
108 0 0 0 0 0
CX.5461_0.5_48 DOX_0.1_24 DOX_0.1_3 DOX_0.1_48 DOX_0.5_24 DOX_0.5_3
1 0 0 0 0 0 0
2 0 0 0 0 1 0
3 0 0 0 0 1 0
4 0 0 0 0 0 0
5 0 0 0 0 0 0
6 0 0 0 0 0 0
7 1 0 0 0 0 0
8 0 0 0 1 0 0
9 0 0 0 0 0 0
10 0 0 0 0 0 0
11 0 0 0 0 0 0
12 0 0 0 0 0 0
13 0 0 0 0 0 0
14 0 0 0 0 0 0
15 1 0 0 0 0 0
16 0 0 0 1 0 0
17 0 0 0 0 0 0
18 0 0 0 0 0 0
19 0 0 0 0 0 0
20 0 0 0 0 0 0
21 0 0 0 0 0 0
22 0 0 0 0 0 0
23 0 0 1 0 0 0
24 0 0 0 0 0 1
25 0 0 0 0 0 0
26 0 0 0 0 0 0
27 0 0 0 0 0 0
28 0 0 0 0 0 0
29 0 1 0 0 0 0
30 0 0 0 0 1 0
31 0 0 0 0 0 0
32 0 0 1 0 0 0
33 0 0 0 0 0 0
34 0 0 0 0 0 0
35 1 0 0 0 0 0
36 0 0 0 1 0 0
37 0 0 0 0 0 0
38 0 0 0 0 0 0
39 0 0 0 0 0 0
40 0 0 0 0 0 0
41 0 0 0 0 0 0
42 0 0 1 0 0 0
43 0 0 0 0 0 1
44 0 0 0 0 0 1
45 0 0 0 0 0 0
46 0 0 0 0 0 0
47 0 0 0 0 0 0
48 0 0 0 0 0 0
49 0 1 0 0 0 0
50 0 0 0 0 1 0
51 0 0 0 0 0 0
52 0 0 0 0 0 0
53 0 0 0 0 0 0
54 0 0 0 0 0 0
55 1 0 0 0 0 0
56 0 0 0 1 0 0
57 0 0 0 0 0 0
58 0 0 0 0 0 0
59 0 0 0 0 0 0
60 0 0 0 0 0 0
61 0 0 0 0 0 0
62 0 0 1 0 0 0
63 0 0 0 0 0 1
64 0 0 0 0 0 0
65 0 0 0 0 0 0
66 0 0 0 0 0 0
67 0 0 0 0 0 0
68 0 0 0 0 0 0
69 0 1 0 0 0 0
70 0 0 0 0 1 0
71 0 0 0 0 0 0
72 0 0 0 0 0 0
73 0 0 0 0 0 0
74 1 0 0 0 0 0
75 0 0 0 1 0 0
76 0 0 0 0 0 0
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78 0 0 0 0 0 0
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80 0 0 0 0 0 0
81 0 0 0 0 0 0
82 0 0 1 0 0 0
83 0 0 0 0 0 1
84 0 0 0 0 0 0
85 0 0 0 0 0 0
86 0 0 0 0 0 0
87 0 0 0 0 0 0
88 0 0 0 0 0 0
89 0 1 0 0 0 0
90 0 0 0 0 1 0
91 0 0 0 0 0 0
92 0 0 0 0 0 0
93 0 0 0 0 0 0
94 1 0 0 0 0 0
95 0 0 0 1 0 0
96 0 0 0 0 0 0
97 0 0 0 0 0 0
98 0 1 0 0 0 0
99 0 0 0 0 0 0
100 0 0 0 0 0 0
101 0 0 0 0 0 0
102 0 0 1 0 0 0
103 0 0 0 0 0 1
104 0 0 0 0 0 0
105 0 0 0 0 0 0
106 0 0 0 0 0 0
107 0 0 0 0 0 0
108 0 1 0 0 0 0
DOX_0.5_48 VEH_0.1_24 VEH_0.1_3 VEH_0.1_48 VEH_0.5_24 VEH_0.5_3 VEH_0.5_48
1 0 0 0 0 0 0 0
2 0 0 0 0 0 0 0
3 0 0 0 0 0 0 0
4 0 1 0 0 0 0 0
5 0 0 0 0 1 0 0
6 0 0 0 0 0 0 0
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51 0 1 0 0 0 0 0
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56 0 0 0 0 0 0 0
57 1 0 0 0 0 0 0
58 0 0 0 1 0 0 0
59 0 0 0 0 0 0 1
60 0 0 0 0 0 0 0
61 0 0 0 0 0 0 0
62 0 0 0 0 0 0 0
63 0 0 0 0 0 0 0
64 0 0 1 0 0 0 0
65 0 0 0 0 0 1 0
66 0 0 0 0 0 1 0
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68 0 0 0 0 0 0 0
69 0 0 0 0 0 0 0
70 0 0 0 0 0 0 0
71 0 1 0 0 0 0 0
72 0 0 0 0 1 0 0
73 0 0 0 0 0 0 0
74 0 0 0 0 0 0 0
75 0 0 0 0 0 0 0
76 0 0 0 0 0 0 0
77 1 0 0 0 0 0 0
78 0 0 0 1 0 0 0
79 0 0 0 0 0 0 1
80 0 0 0 0 0 0 0
81 0 0 0 0 0 0 0
82 0 0 0 0 0 0 0
83 0 0 0 0 0 0 0
84 0 0 1 0 0 0 0
85 0 0 0 0 0 1 0
86 0 0 0 0 0 0 0
87 0 0 0 0 0 0 0
88 0 0 0 0 0 0 0
89 0 0 0 0 0 0 0
90 0 0 0 0 0 0 0
91 0 1 0 0 0 0 0
92 0 0 0 0 1 0 0
93 0 0 0 0 0 0 0
94 0 0 0 0 0 0 0
95 0 0 0 0 0 0 0
96 1 0 0 0 0 0 0
97 0 0 0 1 0 0 0
98 0 0 0 0 0 0 0
99 0 0 0 0 0 0 1
100 0 0 0 0 0 0 0
101 0 0 0 0 0 0 0
102 0 0 0 0 0 0 0
103 0 0 0 0 0 0 0
104 0 0 1 0 0 0 0
105 0 0 0 0 0 1 0
106 0 0 0 0 0 0 0
107 0 0 0 0 0 0 0
108 0 0 0 0 0 0 0
attr(,"assign")
[1] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
attr(,"contrasts")
attr(,"contrasts")$`Metadata_2$Drug_time`
[1] "contr.treatment"📌 Voom Transformation and Linear Modeling
# ✅ Duplicate Correlation for Individual Effect
corfit <- duplicateCorrelation(object = dge$counts, design = design, block = Metadata_2$Ind)
# ✅ Voom Transformation
v <- voom(dge, design, block = Metadata_2$Ind, correlation = corfit$consensus.correlation, plot = TRUE)
| Version | Author | Date |
|---|---|---|
| d6850ef | sayanpaul01 | 2025-02-06 |
# ✅ Fit Linear Model
fit <- lmFit(v, design, block = Metadata_2$Ind, correlation = corfit$consensus.correlation)📌 Create Contrast Matrix
contrast_matrix <- makeContrasts(
CX_0.1_3vsVEH_0.1_3 = CX.5461_0.1_3 - VEH_0.1_3,
CX_0.1_24vsVEH_0.1_24 = CX.5461_0.1_24 - VEH_0.1_24,
CX_0.1_48vsVEH_0.1_48 = CX.5461_0.1_48 - VEH_0.1_48,
CX_0.5_3vsVEH_0.5_3 = CX.5461_0.5_3 - VEH_0.5_3,
CX_0.5_24vsVEH_0.5_24 = CX.5461_0.5_24 - VEH_0.5_24,
CX_0.5_48vsVEH_0.5_48 = CX.5461_0.5_48 - VEH_0.5_48,
DOX_0.1_3vsVEH_0.1_3 = DOX_0.1_3 - VEH_0.1_3,
DOX_0.1_24vsVEH_0.1_24 = DOX_0.1_24 - VEH_0.1_24,
DOX_0.1_48vsVEH_0.1_48 = DOX_0.1_48 - VEH_0.1_48,
DOX_0.5_3vsVEH_0.5_3 = DOX_0.5_3 - VEH_0.5_3,
DOX_0.5_24vsVEH_0.5_24 = DOX_0.5_24 - VEH_0.5_24,
DOX_0.5_48vsVEH_0.5_48 = DOX_0.5_48 - VEH_0.5_48,
levels = design
)📌 Fit Model and Generate Results
# ✅ Apply Contrasts
fit2 <- contrasts.fit(fit, contrast_matrix)
fit2 <- eBayes(fit2)
# ✅ Summary of Results
results_summary <- decideTests(fit2, adjust.method = "BH", p.value = 0.05)
summary(results_summary) CX_0.1_3vsVEH_0.1_3 CX_0.1_24vsVEH_0.1_24 CX_0.1_48vsVEH_0.1_48
Down 0 204 294
NotSig 14278 14074 13961
Up 1 1 24
CX_0.5_3vsVEH_0.5_3 CX_0.5_24vsVEH_0.5_24 CX_0.5_48vsVEH_0.5_48
Down 1 259 337
NotSig 14277 14001 13909
Up 1 19 33
DOX_0.1_3vsVEH_0.1_3 DOX_0.1_24vsVEH_0.1_24 DOX_0.1_48vsVEH_0.1_48
Down 1 2528 1696
NotSig 14277 9215 10818
Up 1 2536 1765
DOX_0.5_3vsVEH_0.5_3 DOX_0.5_24vsVEH_0.5_24 DOX_0.5_48vsVEH_0.5_48
Down 476 5067 5177
NotSig 13746 4451 4151
Up 57 4761 4951
sessionInfo()R version 4.3.0 (2023-04-21 ucrt)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 11 x64 (build 22631)
Matrix products: default
locale:
[1] LC_COLLATE=English_United States.utf8
[2] LC_CTYPE=English_United States.utf8
[3] LC_MONETARY=English_United States.utf8
[4] LC_NUMERIC=C
[5] LC_TIME=English_United States.utf8
time zone: America/Chicago
tzcode source: internal
attached base packages:
[1] stats4 stats graphics grDevices utils datasets methods
[8] base
other attached packages:
[1] Homo.sapiens_1.3.1
[2] TxDb.Hsapiens.UCSC.hg19.knownGene_3.2.2
[3] org.Hs.eg.db_3.18.0
[4] GO.db_3.18.0
[5] OrganismDbi_1.44.0
[6] GenomicFeatures_1.54.4
[7] GenomicRanges_1.54.1
[8] GenomeInfoDb_1.38.8
[9] AnnotationDbi_1.64.1
[10] IRanges_2.36.0
[11] S4Vectors_0.40.1
[12] Biobase_2.62.0
[13] BiocGenerics_0.48.1
[14] biomaRt_2.58.2
[15] scales_1.3.0
[16] lubridate_1.9.3
[17] forcats_1.0.0
[18] stringr_1.5.1
[19] dplyr_1.1.4
[20] purrr_1.0.2
[21] readr_2.1.5
[22] tidyr_1.3.1
[23] tibble_3.2.1
[24] ggplot2_3.5.1
[25] tidyverse_2.0.0
[26] data.table_1.14.10
[27] edgeR_4.0.1
[28] limma_3.58.1
[29] workflowr_1.7.1
loaded via a namespace (and not attached):
[1] DBI_1.2.3 bitops_1.0-7
[3] RBGL_1.78.0 rlang_1.1.3
[5] magrittr_2.0.3 git2r_0.35.0
[7] matrixStats_1.4.1 compiler_4.3.0
[9] RSQLite_2.3.3 getPass_0.2-4
[11] png_0.1-8 callr_3.7.6
[13] vctrs_0.6.5 pkgconfig_2.0.3
[15] crayon_1.5.3 fastmap_1.1.1
[17] dbplyr_2.5.0 XVector_0.42.0
[19] Rsamtools_2.18.0 promises_1.3.0
[21] rmarkdown_2.29 tzdb_0.4.0
[23] graph_1.80.0 ps_1.8.1
[25] bit_4.0.5 xfun_0.50
[27] zlibbioc_1.48.0 cachem_1.0.8
[29] jsonlite_1.8.9 progress_1.2.3
[31] blob_1.2.4 later_1.3.2
[33] DelayedArray_0.28.0 BiocParallel_1.36.0
[35] parallel_4.3.0 prettyunits_1.2.0
[37] R6_2.5.1 bslib_0.8.0
[39] stringi_1.8.3 rtracklayer_1.62.0
[41] jquerylib_0.1.4 SummarizedExperiment_1.32.0
[43] Rcpp_1.0.12 knitr_1.49
[45] Matrix_1.6-1.1 httpuv_1.6.15
[47] timechange_0.3.0 tidyselect_1.2.1
[49] abind_1.4-8 rstudioapi_0.17.1
[51] yaml_2.3.10 codetools_0.2-20
[53] curl_6.0.1 processx_3.8.5
[55] lattice_0.22-5 withr_3.0.2
[57] KEGGREST_1.42.0 evaluate_1.0.3
[59] BiocFileCache_2.10.2 xml2_1.3.6
[61] Biostrings_2.70.1 BiocManager_1.30.25
[63] pillar_1.10.1 filelock_1.0.3
[65] MatrixGenerics_1.14.0 whisker_0.4.1
[67] generics_0.1.3 rprojroot_2.0.4
[69] RCurl_1.98-1.13 hms_1.1.3
[71] munsell_0.5.1 glue_1.7.0
[73] tools_4.3.0 BiocIO_1.12.0
[75] locfit_1.5-9.8 GenomicAlignments_1.38.2
[77] fs_1.6.3 XML_3.99-0.17
[79] grid_4.3.0 colorspace_2.1-0
[81] GenomeInfoDbData_1.2.11 restfulr_0.0.15
[83] cli_3.6.1 rappdirs_0.3.3
[85] S4Arrays_1.2.1 gtable_0.3.6
[87] sass_0.4.9 digest_0.6.34
[89] SparseArray_1.2.4 rjson_0.2.23
[91] memoise_2.0.1 htmltools_0.5.8.1
[93] lifecycle_1.0.4 httr_1.4.7
[95] statmod_1.5.0 bit64_4.0.5