4.12 Summary
Before proceeding to differential expression analysis, confirm all QC checks pass:
| Check | Expected outcome | This dataset |
|---|---|---|
| Size factors ≈ 1.0 across samples | Library sizes are balanced | ⚠️ C3 = 2.324 — corrected by normalisation |
| Boxplots of normalised counts overlap | No extreme outlier samples | ✅ All samples overlap after normalisation |
| Correlation heatmap: within-group distances < between-group | Replicates are reproducible | ✅ Clean block structure |
| PCA PC1 separates conditions | Condition is the dominant source of variance | ✅ PC1 = 85.4%, perfect separation |
| No isolated samples in PCA or heatmap | No technical outliers | ⚠️ C3 offset on PC2 — consistent, not alarming |
Overall the dataset passes QC. C3 shows a higher sequencing depth and mild transcriptional offset from C1/C2, visible consistently across all QC plots. This is within acceptable range for biological replicates and does not compromise the downstream analysis. We proceed to differential expression.
⭐ Important: If any check fails, investigate the cause before running DESeq2. Proceeding with outlier samples or confounded designs will compromise all downstream results
# ── Export DDS for downstream analysis ────────────────────────────────────────
results_dir <- file.path(git_root, "results", "rds")
dir.create(results_dir, recursive = TRUE, showWarnings = FALSE)
dds_path <- file.path(results_dir, "dds_ecoli_MG1655.rds")
saveRDS(dds, file = dds_path)cat("✅ DDS saved to:", dds_path, "\n")
cat(" Dimensions :", nrow(dds), "genes ×", ncol(dds), "samples\n")
cat(" Conditions :", paste(levels(dds$condition), collapse = " vs "), "\n")## R version 4.4.1 (2024-06-14)
## Platform: aarch64-apple-darwin20
## Running under: macOS Sonoma 14.3
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## Matrix products: default
## BLAS: /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRblas.0.dylib
## LAPACK: /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRlapack.dylib; LAPACK version 3.12.0
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## locale:
## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
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## time zone: Europe/Copenhagen
## tzcode source: internal
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## attached base packages:
## [1] stats4 stats graphics grDevices utils datasets methods
## [8] base
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## other attached packages:
## [1] heatmaply_1.6.0 viridis_0.6.5
## [3] viridisLite_0.4.3 KEGGREST_1.46.0
## [5] fgsea_1.32.4 mulea_1.1.1
## [7] plotly_4.12.0 DT_0.34.0
## [9] kableExtra_1.4.0 knitr_1.51
## [11] factoextra_2.0.0 pheatmap_1.0.13
## [13] RColorBrewer_1.1-3 ggpubr_0.6.3
## [15] DESeq2_1.46.0 SummarizedExperiment_1.36.0
## [17] Biobase_2.66.0 MatrixGenerics_1.18.1
## [19] matrixStats_1.5.0 GenomicRanges_1.58.0
## [21] GenomeInfoDb_1.42.3 IRanges_2.40.1
## [23] S4Vectors_0.44.0 BiocGenerics_0.52.0
## [25] reshape2_1.4.5 lubridate_1.9.5
## [27] forcats_1.0.1 stringr_1.6.0
## [29] dplyr_1.2.1 purrr_1.2.2
## [31] readr_2.2.0 tidyr_1.3.2
## [33] tibble_3.3.1 ggplot2_4.0.3
## [35] tidyverse_2.0.0
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## loaded via a namespace (and not attached):
## [1] gridExtra_2.3 rlang_1.2.0 magrittr_2.0.5
## [4] otel_0.2.0 compiler_4.4.1 png_0.1-9
## [7] systemfonts_1.3.2 vctrs_0.7.3 pkgconfig_2.0.3
## [10] crayon_1.5.3 fastmap_1.2.0 backports_1.5.1
## [13] XVector_0.46.0 labeling_0.4.3 ca_0.71.1
## [16] rmarkdown_2.31 tzdb_0.5.0 UCSC.utils_1.2.0
## [19] ragg_1.5.2 xfun_0.57 zlibbioc_1.52.0
## [22] cachem_1.1.0 jsonlite_2.0.0 DelayedArray_0.32.0
## [25] BiocParallel_1.40.2 broom_1.0.12 parallel_4.4.1
## [28] R6_2.6.1 bslib_0.10.0 stringi_1.8.7
## [31] car_3.1-5 numDeriv_2016.8-1.1 jquerylib_0.1.4
## [34] iterators_1.0.14 assertthat_0.2.1 Rcpp_1.1.1-1.1
## [37] bookdown_0.46 Matrix_1.7-5 timechange_0.4.0
## [40] tidyselect_1.2.1 rstudioapi_0.18.0 abind_1.4-8
## [43] yaml_2.3.12 TSP_1.2.7 codetools_0.2-20
## [46] lattice_0.22-9 plyr_1.8.9 withr_3.0.2
## [49] S7_0.2.2 coda_0.19-4.1 evaluate_1.0.5
## [52] xml2_1.5.2 Biostrings_2.74.1 pillar_1.11.1
## [55] carData_3.0-6 foreach_1.5.2 generics_0.1.4
## [58] emdbook_1.3.14 hms_1.1.4 scales_1.4.0
## [61] glue_1.8.1 lazyeval_0.2.3 tools_4.4.1
## [64] apeglm_1.28.0 dendextend_1.19.1 data.table_1.18.2.1
## [67] webshot_0.5.5 locfit_1.5-9.12 ggsignif_0.6.4
## [70] registry_0.5-1 mvtnorm_1.3-6 fastmatch_1.1-8
## [73] cowplot_1.2.0 grid_4.4.1 bbmle_1.0.25.1
## [76] seriation_1.5.8 crosstalk_1.2.2 bdsmatrix_1.3-7
## [79] colorspace_2.1-2 GenomeInfoDbData_1.2.13 Formula_1.2-5
## [82] cli_3.6.6 textshaping_1.0.5 S4Arrays_1.6.0
## [85] svglite_2.2.2 gtable_0.3.6 rstatix_0.7.3
## [88] sass_0.4.10 digest_0.6.39 SparseArray_1.6.2
## [91] ggrepel_0.9.8 htmlwidgets_1.6.4 farver_2.1.2
## [94] htmltools_0.5.9 lifecycle_1.0.5 httr_1.4.8
## [97] MASS_7.3-65