1 Design of the experiment
- E12.5 placentae were dissected from 3 litters coming from crosses of a mother OgtY841A-het x OgtY841A-homo male.
- The head of the embryo was used to assign it to the 4 possible genotypes coming from this cross.
- Among the 4 genotypes, the het females represent the control for females, the wt males represent the control for males.
- 6 placentae for each genotype were sequenced, trying to have the 6 biological replicates coming from at least 2 different litters.
| SRR | sample | genotype | litter | batch | biol_rep | tech_rep | run | library_layout | read_length | sex |
|---|---|---|---|---|---|---|---|---|---|---|
| none | P1 | F_HET | 6 | 1 | 1 | 1 | 1 | PAIRED - | 40 | F |
| none | P2 | F_HET | 7 | 1 | 2 | 1 | 1 | PAIRED - | 40 | F |
| none | P3 | F_HET | 8 | 1 | 3 | 1 | 1 | PAIRED - | 40 | F |
| none | P13 | F_HET | 6 | 1 | 4 | 1 | 1 | PAIRED - | 40 | F |
| none | P15 | F_HET | 8 | 1 | 5 | 1 | 1 | PAIRED - | 40 | F |
| none | P24 | F_HET | 7 | 1 | 6 | 1 | 1 | PAIRED - | 40 | F |
| none | P4 | F_HOMO | 6 | 1 | 1 | 1 | 1 | PAIRED - | 40 | F |
| none | P5 | F_HOMO | 7 | 1 | 2 | 1 | 1 | PAIRED - | 40 | F |
| none | P6 | F_HOMO | 8 | 1 | 3 | 1 | 1 | PAIRED - | 40 | F |
| none | P16 | F_HOMO | 7 | 1 | 4 | 1 | 1 | PAIRED - | 40 | F |
| none | P17 | F_HOMO | 7 | 1 | 5 | 1 | 1 | PAIRED - | 40 | F |
| none | P18 | F_HOMO | 8 | 1 | 6 | 1 | 1 | PAIRED - | 40 | F |
| none | P7 | M_HEMI | 6 | 1 | 1 | 1 | 1 | PAIRED - | 40 | M |
| none | P8 | M_HEMI | 6 | 1 | 2 | 1 | 1 | PAIRED - | 40 | M |
| none | P9 | M_HEMI | 7 | 1 | 3 | 1 | 1 | PAIRED - | 40 | M |
| none | P19 | M_HEMI | 6 | 1 | 4 | 1 | 1 | PAIRED - | 40 | M |
| none | P20 | M_HEMI | 6 | 1 | 5 | 1 | 1 | PAIRED - | 40 | M |
| none | P21 | M_HEMI | 7 | 1 | 6 | 1 | 1 | PAIRED - | 40 | M |
| none | P10 | M_WT | 6 | 1 | 1 | 1 | 1 | PAIRED - | 40 | M |
| none | P11 | M_WT | 7 | 1 | 2 | 1 | 1 | PAIRED - | 40 | M |
| none | P12 | M_WT | 8 | 1 | 3 | 1 | 1 | PAIRED - | 40 | M |
| none | P14 | M_WT | 7 | 1 | 4 | 1 | 1 | PAIRED - | 40 | M |
| none | P22 | M_WT | 6 | 1 | 5 | 1 | 1 | PAIRED - | 40 | M |
| none | P23 | M_WT | 6 | 1 | 6 | 1 | 1 | PAIRED - | 40 | M |
2 Clustering
2.1 Heatmap for quality assessment
After applying, in order, log2(norm counts + 1), rlog and Variance Stabilizing Transformation, I plot heatmaps for top highly expressed genes to check for eventual big sample heterogeneity present in the dataset.
- Sample P1 has a problem, I will remove it from the dataset.
- rlog transformation is not appropriate with this dataset.
2.2 PCA
Clustering based on genotype does not appear in the PCA. log2 transformation makes the sex clustering more evident.
3 Differential Expression Analysis - all Female vs all Male samples
The threshold used for a dot to be colored in red in the MA-plots is: p-value adjusted < 0.05, to be labeled is color, baseMean > 10 and log2FC > 0.2.
4 Differential Expression Analysis - genotypes comparison
The threshold used for a dot to be colored in red in the MA-plots is: p-value adjusted < 0.05, to be labeled is color, baseMean > 10 and log2FC > 0.2.
sessionInfo()## R version 4.1.3 (2022-03-10)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 20.04.6 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_US.UTF-8 LC_NUMERIC=C
## [3] LC_TIME=it_IT.UTF-8 LC_COLLATE=en_US.UTF-8
## [5] LC_MONETARY=it_IT.UTF-8 LC_MESSAGES=en_US.UTF-8
## [7] LC_PAPER=it_IT.UTF-8 LC_NAME=C
## [9] LC_ADDRESS=C LC_TELEPHONE=C
## [11] LC_MEASUREMENT=it_IT.UTF-8 LC_IDENTIFICATION=C
##
## attached base packages:
## [1] stats4 stats graphics grDevices utils datasets methods
## [8] base
##
## other attached packages:
## [1] enrichplot_1.14.2 org.Mm.eg.db_3.14.0
## [3] AnnotationDbi_1.56.2 clusterProfiler_4.2.2
## [5] data.table_1.14.6 gridExtra_2.3
## [7] gplots_3.1.3 ggpubr_0.5.0
## [9] pheatmap_1.0.12 vsn_3.62.0
## [11] reshape2_1.4.4 ggrepel_0.9.2
## [13] ggplot2_3.4.0 tximport_1.22.0
## [15] DESeq2_1.34.0 SummarizedExperiment_1.24.0
## [17] Biobase_2.54.0 MatrixGenerics_1.6.0
## [19] matrixStats_0.62.0 GenomicRanges_1.46.1
## [21] GenomeInfoDb_1.30.1 IRanges_2.28.0
## [23] S4Vectors_0.32.4 BiocGenerics_0.40.0
##
## loaded via a namespace (and not attached):
## [1] shadowtext_0.1.2 backports_1.4.1 fastmatch_1.1-3
## [4] plyr_1.8.8 igraph_1.3.5 lazyeval_0.2.2
## [7] splines_4.1.3 BiocParallel_1.28.3 digest_0.6.30
## [10] invgamma_1.1 yulab.utils_0.0.5 htmltools_0.5.3
## [13] GOSemSim_2.20.0 viridis_0.6.2 GO.db_3.14.0
## [16] SQUAREM_2021.1 fansi_1.0.3 magrittr_2.0.3
## [19] memoise_2.0.1 limma_3.50.3 Biostrings_2.62.0
## [22] annotate_1.72.0 graphlayouts_0.8.3 colorspace_2.0-3
## [25] blob_1.2.3 xfun_0.35 dplyr_1.0.10
## [28] crayon_1.5.2 RCurl_1.98-1.9 jsonlite_1.8.3
## [31] scatterpie_0.1.8 genefilter_1.76.0 ape_5.6-2
## [34] survival_3.2-13 glue_1.6.2 polyclip_1.10-4
## [37] gtable_0.3.1 zlibbioc_1.40.0 XVector_0.34.0
## [40] DelayedArray_0.20.0 car_3.1-1 abind_1.4-5
## [43] scales_1.2.1 DOSE_3.20.1 DBI_1.1.3
## [46] rstatix_0.7.1 Rcpp_1.0.9 viridisLite_0.4.1
## [49] xtable_1.8-4 tidytree_0.4.1 gridGraphics_0.5-1
## [52] bit_4.0.5 preprocessCore_1.56.0 truncnorm_1.0-8
## [55] httr_1.4.4 fgsea_1.20.0 RColorBrewer_1.1-3
## [58] pkgconfig_2.0.3 XML_3.99-0.12 farver_2.1.1
## [61] sass_0.4.2 locfit_1.5-9.6 utf8_1.2.2
## [64] labeling_0.4.2 ggplotify_0.1.0 tidyselect_1.2.0
## [67] rlang_1.0.6 munsell_0.5.0 tools_4.1.3
## [70] cachem_1.0.6 downloader_0.4 cli_3.4.1
## [73] generics_0.1.3 RSQLite_2.2.18 broom_1.0.1
## [76] evaluate_0.18 stringr_1.4.1 fastmap_1.1.0
## [79] yaml_2.3.6 ggtree_3.2.1 knitr_1.40
## [82] bit64_4.0.5 tidygraph_1.2.2 caTools_1.18.2
## [85] purrr_0.3.5 KEGGREST_1.34.0 ggraph_2.1.0
## [88] nlme_3.1-155 aplot_0.1.8 DO.db_2.9
## [91] compiler_4.1.3 rstudioapi_0.14 png_0.1-7
## [94] affyio_1.64.0 ggsignif_0.6.4 treeio_1.18.1
## [97] tibble_3.1.8 tweenr_2.0.2 geneplotter_1.72.0
## [100] bslib_0.4.1 stringi_1.7.8 highr_0.9
## [103] lattice_0.20-45 Matrix_1.5-3 vctrs_0.5.1
## [106] pillar_1.8.1 lifecycle_1.0.3 BiocManager_1.30.22
## [109] jquerylib_0.1.4 irlba_2.3.5.1 bitops_1.0-7
## [112] patchwork_1.1.2 qvalue_2.26.0 R6_2.5.1
## [115] affy_1.72.0 KernSmooth_2.23-20 codetools_0.2-18
## [118] MASS_7.3-55 gtools_3.9.3 assertthat_0.2.1
## [121] withr_2.5.0 GenomeInfoDbData_1.2.7 parallel_4.1.3
## [124] grid_4.1.3 prettydoc_0.4.1 ggfun_0.0.8
## [127] tidyr_1.2.1 rmarkdown_2.18 ashr_2.2-54
## [130] carData_3.0-5 mixsqp_0.3-48 ggforce_0.4.1